Biotin

Joshi SN, Fathalla M, Koul R, Al. Maney M, Bayoumi R. Biotin responsive seizures and encephalopathy due to biotinidase deficiency. Neurol India [serial online] 2010 [cited 2011 Oct 13]; 58: 323-4. Available from: http://www. neurologyindia. com/text. asp? 2010/58/2/ ...

The spatial distribution of synaptic inputs on the dendritic tree of a neuron can have significant influence on neuronal function. Consequently, accurate anatomical reconstructions of neuron morphology and synaptic localization are critical when modeling and predicting physiological responses of individual neurons. Historically, generation of three-dimensional (3D) neuronal reconstructions together with comprehensive mapping of synaptic inputs has been an extensive task requiring manual identification of putative synaptic contacts directly from tissue samples or digital images. Recent developments in neuronal tracing software applications have improved the speed and accuracy of 3D reconstructions, but localization of synaptic sites through the use of pre-and/or post-synaptic markers has remained largely a manual process. To address this problem, we have developed an algorithm, based on 3D distance measurements between putative pre-synaptic terminals and the post-synaptic dendrite, to automate synaptic contact detection on dendrites of individually labeled neurons from 3D immunofluorescence image sets. In this study, the algorithm is implemented with custom routines in Matlab, and its effectiveness is evaluated through analysis of primary sensory afferent terminals on motor neurons. Optimization of algorithm parameters enabled automated identification of synaptic contacts that matched those identified by manual inspection with low incidence of error. Substantial time savings and the elimination of variability in contact detection introduced by different users are significant advantages of this method.

Although the precise mechanisms explaining loss of, and failure to regain, function after spinal cord injury are unknown, there is increasing interest in the role of "secondary cell death." One prevalent theme in cell loss in other regions of the CNS involves apoptosis executed by the intracellular caspase proteases. A recent study demonstrated that spinal cord injury rapidly increased the activation of caspase-3. Our previous studies demonstrated peak apoptosis in three of four cellular compartments 3 days after controlled contusion in the rat. We have extended these analyses to include enzyme and substrate studies of caspase subfamilies both in rostral and in caudal adjacent segments compared to the lesion site. Although presumed activation of programmed proenzyme is considered the mechanism for enhanced caspases, our novel analyses were designed to detect upregulation of gene expression. We surveyed traumatically injured spinal cord for caspase family messages with a modified differential mRNA display approach and found that the caspase-3 (CASP3) message was present and upregulated severalfold after injury. Our results clearly demonstrate that cell death in the spinal cord occurs after posttranslational activation of caspases that follow, at least for caspase-3, initial upregulation of CASP3 mRNA levels.

Fast immobilization of probe beads by dielectrophoresis-controlled adhesion in a versatile microfluidic platform for affinity assay The use of probe beads for lab-on-chip affinity assays is very interesting from a practical point of view. It is easier to handle and trap beads than molecules in microfluidic systems. We present a method for the immobilization of probe beads at defined areas on a chip using dielectrophoresis (DEP)-controlled adhesion. The method is fast, i.e., it takes between 10 and 120 s-depending on the protocol-to functionalize a chip surface at defined areas. The method is versatile, i.e., it works for beads with different types of probe molecule coatings. The immobilization is irreversible, i.e., the retained beads are able to withstand high flow velocities in a flow-through device even after the DEP voltage is turned off, thus allowing the use of conventional high-conductivity analyte buffers in the following assay procedure. We demonstrate the on-chip immobilization of fluorescent beads coated with biotin, protein A, and goat-antimouse immunoglobulin G (IgG). The number of immobilized beads at an electrode array can be determined from their fluorescence signal. Further, we use this method to demonstrate the detection of streptavidin and mouse IgG. Finally, we demonstrate the feasibility of the parallel detection of different analyte molecules on the same chip.

A mu-capture enzyme-linked immunosorbent assay (ELISA) for detection of serum immunoglobulin M (IgM) antibodies to Borrelia burgdorferi by using biotinylated purified B. burgdorferi flagella was developed. The diagnostic performance of the mu-capture ELISA was compared with that of a conventional indirect ELISA. Sera from untreated patients with erythema migrans (n = 50), neuroborreliosis (n = 100), and acrodermatitis chronica atrophicans (ACA; n = 48) were investigated. The cutoff of the ELISAs was adjusted to a diagnostic specificity of 98% on the basis of examination of 200 serum specimens from healthy controls. The mu-capture ELISA increased the diagnostic sensitivity in patients with erythema migrans from 32 to 48% (P less than 0.01) and in patients with neuroborreliosis from 37 to 57% (P less than 0.001). Because of an increased signal/noise ratio, the mu-capture ELISA yielded a significantly better quantitative discrimination of individual positive measurements from the cutof...

Cranial visceral afferents travel via the solitary tract (ST) to contact neurons within the ST nucleus (NTS) and activate homeostatic reflexes. Hypothalamic projections from the paraventricular nucleus (PVN) release oxytocin (OT) to modulate visceral afferent communication with NTS neurons. However, the cellular mechanisms through which OT acts are poorly understood. Here, we electrophysiologically identified second-order NTS neurons in horizontal brainstem slices by their low-jitter, ST-evoked glutamatergic EPSCs. OT increased the frequency of miniature EPSCs in half of the NTS second-order neurons (13/24) but did not alter event kinetics or amplitudes. These actions were blocked by a selective OT receptor antagonist. OT increased the amplitude of ST-evoked EPSCs with no effect on event kinetics. Variance-mean analysis of ST-evoked EPSCs indicated OT selectively increased the release probability of glutamate from the ST afferent terminals. In OT-sensitive neurons, OT evoked an inwa...

The apical dendrites of pyramidal neurons in the cerebral cortex form vertical bundles whose distribution and density vary across species and areas. To understand their relationships with cortical columns, we labeled retrogradely neurons from the white matter underlying the visual cortex with 1,1%-dioctadecyl-3,3,3%,3%-tetramethylindocarbocyanine perchlorate (DiI) at P3 and P10 and with biotinylated dextran amine at P30. We also mapped the distribution of apical dendrites in tangential sections, immunostained for microtubule-associated proteins (MAP2). Their composition and distribution were studied with Neurolucida and NeuroExplorer software. The apical dendrites of pyramidal neurons formed different bundle types: at P3 we found bundles formed (a) by neurons located in cortical plate; (b) by layer V neurons; and (c) by upper layer V neurons and cortical plate neurons. At P10, the amount of supragranular neurons participating in the bundles increased. The inter-dendritic and inter-bundle distances increased with age. These findings confirm that dendritic bundles are present in the rat visual cortex early in development and are formed by neurons belonging to different cortical layers. The existence of different types of bundles relative to the layer of location of their parent neurons suggests that they are heterogeneous from each other in nature and in the pattern of connectivity.

Six novel docetaxel analogues that possess a N-(7-nitrobenz-2-oxa-1,3-diazo-4-yl)amido-6-caproyl chain in position 7 or 3' (11 and 16a), a N-(7-nitrobenz-2-oxa-l,3-diazo-4-yl)amido-3-propanoyl group at 3' (16b) and a 5'-biotinylamido-6-caproyl chain in position 7, 10 or 3', respectively, have been synthesized. These compounds exhibit activity against microtubule disassembly similar to that of docetaxel but show discrepant activities on living ceils. Although addition of microtubules to 11, 16a and b enhance their fluorescence, no shift of the emission maxima was observed. The fluorescent docetaxel derivatives show a specific labeling of mierotubules in living cells, demonstrating that the mierotubule cytoskeleton constitutes their main subeellular localization.

The malaria parasite Plasmodium falciparum infects human erythrocytes, and it induces an increased rate of uptake into the infected cell of a range of solutes, including essential nutrients required for parasite development. Several models have been proposed for the mechanism(s) underlying parasite-induced solute uptake, each differing with respect to the site of entry into infected cells. We show that a biotin derivative that is excluded from non-infected erythrocytes gains access to infected erythrocytes via a pathway that is inhibited by compounds shown previously to block the pathways responsible for the increased uptake of solutes. The derivative was found to bind erythrocyte cytoskeletal proteins and to hemoglobin, providing evidence that the novel pathways are in the erythrocyte membrane and allow direct access of solutes to the erythrocyte cytosol. The derivative inhibited its own uptake and blocked the parasite-induced transport of other solutes. In whole-cell patch-clamp analyses, biotinylation of infected erythrocytes caused significant decrease in a parasite-induced outward rectifying conductance. In vitro, biotinylation of trophozoite-stage parasitized erythrocytes delayed parasite development. Treatment of infected cells in the final developmental stage abrogated the parasite's ability to complete development. The data are consistent with the novel pathways playing an important role in parasite growth.

To investigate the behavioral mechanism of chemotaxis in Caenorhabditis elegans, we recorded the instantaneous position, speed, and turning rate of single worms as a function of time during chemotaxis in gradients of the attractants ammonium chloride or biotin. Analysis of turning rate showed that each worm track could be divided into periods of smooth swimming (runs) and periods of frequent turning (pirouettes). The initiation of pirouettes was correlated with the rate of change of concentration (dC/dt) but not with absolute concentration. Pirouettes were most likely to occur when a worm was heading down the gradient (dC/dt Ͻ 0) and least likely to occur when a worm was heading up the gradient (dC/dt Ͼ 0). Further analysis revealed that the average direction of movement after a pirouette was up the gradient. These observations suggest that chemotaxis is produced by a series of pirouettes that reorient the animal to the gradient. We tested this idea by imposing the correlation between pirouettes and dC/dt on a stochastic point model of worm motion. The model exhibited chemotaxis behavior in a radial gradient and also in a novel planar gradient. Thus, the pirouette model of C. elegans chemotaxis is sufficient and general.

This paper describes a sequential staining procedure for double immunoenzymatic staining of pairs of antigens in frozen tissue sections and cell smears using monoclonal antibodies. This technique involves performance of an indirect immunoperoxidase sandwich (including development of the enzyme reaction) followed by an unlabelled immuno-alkaline phosphatase sandwich (the APAAP method). The two enzyme labels are revealed using DAB/H202 for peroxidase and naphthol AS-MX plus fast blue or fast red for alkaline phosphatase. When compared with a hapten-sandwich/biotin-avidin system, the sequential staining procedure proved to be simpler and more sensitive and was also more suitable for double immunoenzymatic staining when monoclonal antibodies were only available in small amounts. The sequential staining procedure is particularly useful for the identification of antigens distributed in different cell populations or in different sites (e.g., nucleus and cytoplasm or cell surface) of the same cell. In contrast, this method does not appear to be very suitable for demonstrating two antigens located in the same site (e.g., surface membrane) of the same cell for which purpose double immunofluorescence remains the first choice.

Activated platelets bind numerous adhesive and procoagulant proteins by receptor-mediated processes. Although there is little evidence to suggest that these processes are heterogeneous in platelets, we previously found that platelets co-stimulated with collagen and thrombin express functional alpha-granule factor V only on a subpopulation of cells. Here we show that these cells, referred to as 'COAT-platelets', bind additional alpha-granule proteins, including fibrinogen, von Willebrand factor, thrombospondin, fibronectin and alpha2-antiplasmin. These proteins are all transglutaminase substrates, and inhibitors of transglutaminase prevent the production of COAT-platelets. A synthetic transglutaminase substrate (CP15) also binds to COAT-platelets, and analysis by high performance liquid chromatography/mass spectrometry shows that a product is formed with a relative molecular mass (Mr) equal to CP15 plus 176. Serotonin, an abundant component of platelet-dense granules, has an ...

Approximately 50% of human spinal cord injuries (SCI) are at the cervical level, resulting in impairments in motor function of the upper extremity. Even modest recovery of upper extremity function could have an enormous impact on quality of life for quadriplegics. Thus, there is a critical need to develop experimental models for cervical SCI and techniques to assess deficits and recovery of forelimb motor function. Here, we analyze forelimb and forepaw motor function in rats after a lateral hemisection at C5 and assessed the relationship between the functional impairments and the extent of damage to one descending motor system, the corticospinal tract (CST). Female Sprague-Dawley rats were trained on various behavioral tasks that require the forelimb, including a task that measures gripping ability by the hand (as measured by a grip strength meter, GSM), a food reaching task, and horizontal rope walking. After 8 weeks of post-injury testing, the distribution of the CST was evaluated by injecting BDA into the sensorimotor cortex either ipsi-or contralateral to the cervical lesion. Complete unilateral hemisection injuries eliminated the ability to grip and caused severe impairments in food retrieval by the forepaw ipsilateral to the lesion. There was no indication of recovery in either task. In cases in which hemisections spared white matter near the midline, there was some recovery of forelimb motor function over time. Assessment of rope climbing ability revealed permanent impairments in forelimb use and deficits in hindlimb use and trunk stability. Sensory testing using a dynamic plantar aesthesiometer revealed that there was no increase in touch sensitivity in the affected forelimb. For the cases in which both histological and behavioral data were available, spared forelimb motor function was greatest in rats in which there was sparing of the dorsal CST. D

In recent years, techniques have been developed to study and manipulate single molecules of DNA and other biopolymers. In one such technique, the magnetic trap, a single DNA molecule is bound at one end to a glass surface and at the other to a magnetic microbead. Small magnets, whose position and rotation can be controlled, pull on and rotate the microbead. This provides a simple method to stretch and twist the molecule. The system allows one to apply and measure forces ranging from 10(-3) to >100 picoNewtons (pN). In contrast to other techniques, the force measurement is absolute and does not require calibration of the sensor. This protocol describes a procedure for building and using a magnetic trap. It gives a method for constructing a microchamber suitable for magnetic tweezers studies, including antibody coating and passivation. It also describes a series of simple steps to achieve end-labeling of DNA anchoring fragments. One anchoring fragment is biotin-labeled and the othe...

The efficiency of hybridization analysis with oligonucleotide microarrays depends heavily on the method of detection. Conventional methods based on labeling nucleic acids with fluorescent, chemiluminescent, enzyme, or radioactive reporters suffer from a number of serious drawbacks which demand development of new detection techniques. Here, we report two new approaches for detection of hybridization with oligonucleotide microarrays employing magnetic beads as active labels. In the first method streptavidin-coated magnetic beads are used to discover biotin-labeled DNA molecules hybridized with arrayed oligonucleotide probes. In the second method biotin-labeled DNA molecules are bound first to the surface of magnetic beads and then hybridized with arrayed complementary strands on bead-array contacts. Using a simple low-power microscope with a dark-field illumination and a pair of complementary primers as a model hybridization system we evaluated sensitivity, speed, and cost of the new detection method and compared its performance with the detection techniques employing enzyme and fluorescent labels. It was shown that the detection of microarrayhybridized DNA with magnetic beads combines low cost with high speed and enhanced assay sensitivity, opening a new way to routine hybridization assays which do not require precise measurements of DNA concentration.

Over a 10-month period, 24 randomly selected riding horses were fed various amounts of biotin. Statistically significant improvements in growth rates and hardness of hooves were produced by biotin supplementation. Greater growth rates and hardness were achieved at a daily dose of 15 mg than at 7.5 mg. Increased hoof hardness was greatest in the hoof quarters and toe. No ring formation occurred in hooves of horses fed biotin intermittently.

A comparison of the binding properties of avidin, streptavidin, neutrAvidin, and antibiotin antibody to a biotinylated lipid bilayer was studied using second-harmonic generation. Protein binding assays were performed on a planar supported lipid bilayer of 1,2dioleoyl-sn-glycero-3-phosphocholine (DOPC) containing 4 mol % biotinylated-cap-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (biotincap-DOPE). The equilibrium binding affinities of these biotin−protein interactions were determined, revealing the relative energetic contributions for each protein to the biotinylated lipid ligand. The results show that the binding affinities of avidin, streptavidin, and neutrAvidin for biotin were all strengthened by protein−protein interactions but that the stronger protein−protein interactions observed for streptavidin and neutrAvidin make their binding more energetically favorable. It was also shown that neutrAvidin has the highest degree of nonspecific adsorption to a pure DOPC bilayer, compared to avidin and streptavidin. In addition, the biotin-binding affinity of the antibiotin antibody was found to be of the same order of magnitude as that of avidin, streptavidin, and neutrAvidin. These findings provide important new insights into these biotin-bound protein complexes commonly used in several bioanalytical applications.

Aim: To study the impact of assimilable nitrogen, biotin and their interaction on growth, fermentation rate and volatile formation by Saccharomyces. Methods and Results: Fermentations of synthetic grape juice media were conducted in a factorial design with yeast assimilable nitrogen (YAN) (60 or 250 mg l )1 ) and biotin (0, 1 or 10 lg l )1 ) as variables. All media contained 240 g l )1 glucose + fructose (1 : 1) and were fermented using biotin-depleted Saccharomyces cerevisiae strains EC1118 or UCD 522. Both strains exhibited weak growth and sluggish fermentation rates without biotin. Increased nitrogen concentration resulted in higher maximum fermentation rates, while adjusting biotin from 1 to 10 lg l )1 had no effect. Nitrogen · biotin interactions influenced fermentation time, production of higher alcohols and hydrogen sulfide (H 2 S). Maximum H 2 S production occurred in the medium containing 60 mg l )1 YAN and 1 lg l )1 biotin. Conclusions: Nitrogen · biotin interactions affect fermentation time and volatile production by Saccharomyces depending on strain. Biotin concentrations sufficient to complete fermentation may affect the organoleptic impact of wine. Significance and Impact of the Study: This study demonstrates the necessity to consider nutrient interactions when diagnosing problem fermentations.

Previous anatomical studies in rabbits and rats have shown that the superior vestibular nucleus (SVN), medial vestibular nucleus (MVN) and inferior vestibular nucleus (IVN) project to the parabrachial nucleus (PBN) and Kölliker -Fuse (KF) nucleus. Adult male albino rabbits and Long -Evans rats received iontophoretic injections of biotinylated dextran amine, Phaseolus vulgaris leucoagglutinin, Fluoro-Gold or tetramethylrhodamine dextran amine into either the vestibular nuclei or the PBN and KF nuclei. The results were similar in both rats and rabbits. Injections of retrograde tracers into the vestibular nuclei produced retrogradely labeled neurons bilaterally in caudal third of the medial, external medial, and external lateral PBN in both species, with more variable labeling in KF. Rats also had consistent bilateral (predominantly contralateral) labeling in the ventrolateral PBN. The most prominent labeling was produced from injections that included the SVN, with fewer labeled neurons observed from injections in the caudal MVN and the IVN. Anterograde transport of BDA from injections into the PBN and KF nuclei of rabbits revealed prominent projections to the SVN, dorsal aspect of the rostral MVN, caudal MVN, pars beta of the LVN and IVN. These connections appear to contain a component that is reciprocal to the vestibulo-parabrachial pathway and a nonreciprocal component to regions connected with the vestibulocerebellum and vestibulo-motor reflex pathways. These connections support the concept that a synthesis of autonomic, vestibular and limbic information is an integral property of pathways related to balance control in both the brain stem and forebrain. It is suggested that these projections may contribute broadly to both performance tradeoffs in vestibularrelated pathways during variations in the behavioral context and affective state and the close association between anxiety and balance function. D

We report a family affected to the fourth generation by uncombable hair syndrome. This syndrome is characterized by unruly, dry, blond hair with a tangled appearance. The family pedigree strongly supports the hypothesis of autosomal dominant inheritance; some members of the family had, apart from uncombable hair, minor signs of atopy and ectodermal dysplasia, such as abnormalities of the nails. The diagnosis was confirmed by means of extensive scanning electron microscopy. A trial with oral biotin 5 mg/day was started on two young patients with excellent results as regards the hair appearance, although scanning electron microscopy did not show structural changes in the hair. After a 2-year-period of follow-up, hair normality was maintained without biotin, while nail fragility still required biotin supplementation for control.

Hair loss in women is a very common clinical complaint, and is usually associated with severe emotional distress. In this article, the authors review the most common clinical causes of hair loss in women, and emphasize the role of hormonal changes in the regulation of hair loss and hair growth.

Biotinylated dextran amine (BDA) is a highly ef fi cient and powerful marker for bidirectional tracing of nerve pathways in a wide variety of species at the light and electron microscopic level. The BDA tracttracing method can readily be combined with other anterograde or retrograde tracers for multiple neuroanatomical labeling studies to map the neuronal connectivity, or with immunocytochemistry for neurotransmitters and their receptors to reveal details of synaptic specializations within the multisynaptic neuronal circuits. Here, we describe an experimental protocol for anterograde and retrograde tracing using BDA. By applying BDA 10 kDa as an anterograde tracer, we demonstrate the existence of a direct bilateral nigro-trigeminal pathway in the rat.

Standardized purified diets limited to required nutrients are needed for nutritional and toxicological studies. In the present study, we formulated a biotinand cellulose-free diet of reproducible mineral composition (diet A), based on diet AIN-76, and fed it to weanling Long-Evans rats for 3 wk. Inductively coupled argon plasma atomic emission spectrometry was used to determine Ca, Cu, Fe, K, Mg, Mn, Na, P and Zn in liver, duodenum, kidney, spleen and femur. Results were compared with those obtained with rats fed biotin-and/or cellulose-supplemented variations of diet A, diet AIN-76 and diet NIH-31 (an open-formula stock diet). Weanling rats grew slowly and steadily on purified diet A. Growth rates increased when diet A was sup plemented with biotin and cellulosa In general, differences among tissue mineral levels in rats fed diet NIH-31 and those fed diet AIN-76 were more pronounced than those among groups fed our purified diets. Values for hemoglobin and hematocrit were significantly lower in rats fed all purified diets than in those fed diet NIH-31. Diets A + biotin, A + cellulose and A + cellulose + biotin appear satisfactory as reference diets for measuring mineral interactions at near-requirement levels as well as effects of fiber on mineral utilization or for studies on vitamins whose endogenous synthesis may be influenced by dietary fiber.

The biotin switch assay has recently been proposed as the eligible method to identify different S-nitrosated proteins in biological matrices. However, notwithstanding its wide application, a thorough validation of this method is still lacking. In particular, it has been suggested that ascorbate concentrations higher than 1mM (as proposed in the original method) are needed since ascorbate reaction with S-nitrosothiols is slow. But the selectivity of ascorbate as a cleaving agent of the S-N bond under these conditions has not been well characterized. Our data indicate that ascorbate is able to reduce disulfide bridges of DTNB, cystine, cystinylglycine, glutathione disulfide, protein mixed disulfides and biotin-HPDP with pH and concentration dependent rates. Additionally, we tested the effect of indirect sunlight on ascorbate-mediated cleavage of both disulfides and S-nitrosothiols.

Amine modification of filamentous virions (phage particles) is widely used in phage display technology to couple small groups such as biotin or fluorescent dyes to the major coat protein pVIII. We have developed a generalized kinetic model for protein amine modification and applied it to the modification of pVIII with biotin and the near-infrared fluorophor Alexa Fluor 680. Empirically optimized kinetic parameters for the two modification reactions allow the modification level to be predicted for a wide range of virions and modifying reagent concentrations. Virions with 0.03 biotins per pVIII subunit have 50% of the maximal binding capacity for a streptavidin conjugate.

The specificity and sensitivity of immunotluorescence microscopy and ELISA tests were compared both with and without the use of biotinylated anti-species antibody and streptavidin. conjugated with either fluorescein isothiocyanate or horse radish peroxidase. In the biotin-streptavidin system monoclonal and polyclonal antibodies against yellow fever virus had titres between ten-and fifty-fold higher and background readings, particularly in ELISA tests, were noticeably reduced. Plaque variants of yellow fever virus, selected for their apparent loss of antigenic determinants against some monoclonal antibodies in conventional fluorescence tests, were found to possess the antigenic determinants when tested with the more sensitive system. biotin streptavidin flavivirus detection 0166-0934/85/%03.30

Esophageal sensory afferent inputs terminate principally in the central subnucleus of the tractus solitarius (cNTS). Neurons of the cNTS comprise two major neurochemical subpopulations. One contains neurons that are nitric oxide synthase (NOS) immunoreactive (-IR) while the other comprises neurons that are tyrosine hydroxylase (TH)-IR. We have shown recently that TH-IR neurons are involved in esophageal-distention induced gastric relaxation. We used whole cell patch clamp techniques in rat brainstem slices combined with immunohistochemical and morphological reconstructions to characterize cNTS neurons. Postrecording reconstruction of cNTS neurons revealed two morphological neuronal subtypes; one group of cells (41 out of 131 neurons, i.e., 31%) had a multipolar soma, while the other group (87 out of 131 neurons, i.e., 66%) had a bipolar soma. Of the 43 cells in which we conducted a neurochemical examination, 15 displayed TH-IR (9 with bipolar morphology, 6 with multipolar morphology) while the remaining 28 neurons did not display TH-IR (18 with bipolar morphology, 10 with multipolar morphology). Even though the range of electrophysiological properties varied significantly, morphological or neurochemical distinctions did not reveal characteristics peculiar to the subgroups. Spontaneous excitatory postsynaptic currents (sEPSC) recorded in cNTS neurons had a frequency of 1.5 ± 0.15 events s -1 and an amplitude of 27 ± 1.2 pA (Vh = -50 mV) and were abolished by pretreatment with 30 μM AP-5 and 10 μM CNQX, indicating the involvement of both NMDA and non-NMDA receptors. Some cNTS neurons also received a GABAergic input that was abolished by perfusion with 30-50 μM bicuculline. In conclusion, our data show that despite the heterogeneity of morphological and neurochemical membrane properties, the electrophysiological characteristics of cNTS neurons are not a distinguishing feature.

Four biotinylated tri and tetrasaccharide fragments of plant and invertebrate N-glycans were synthesized using methyl tert-butyl phenyl (MBP) thioglycosides donors in order to evaluate their involvement in cross-allergies as cross-reactive carbohydrate determinants (CCDs). Various levels of reactivity to anti-bee and anti-HRP antibodies and with sera from allergic patients were observed when the conjugates were coated on streptavidin microplates. The results showed the potential utility of these xylosylated and fucosylated oligosaccharide fragments in determining CCD antibody epitopes.

Single-molecule fluorescence resonance energy transfer (smFRET) is one of the most general and adaptable single-molecule techniques. Despite the explosive growth in the application of smFRET to answer biological questions in the last decade, the technique has been practiced mostly by biophysicists. We provide a practical guide to using smFRET, focusing on the study of immobilized molecules that allow measurements of single-molecule reaction trajectories from 1 ms to many minutes. We discuss issues a biologist must consider to conduct successful smFRET experiments, including experimental design, sample preparation, single-molecule detection and data analysis. We also describe how a smFRET-capable instrument can be built at a reasonable cost with off-the-shelf components and operated reliably using well-established protocols and freely available software.

The two most common models for the evolution of metabolism are the patchwork evolution model, where enzymes are thought to diverge from broad to narrow substrate specificity, and the retrograde evolution model, according to which enzymes evolve in response to substrate depletion. Analysis of the distribution of homologous enzyme pairs in the metabolic network can shed light on the respective importance of the two models. We here investigate the evolution of the metabolism in E. coli viewed as a single network using EcoCyc.

The interaction of biotinylated human interleukin 5 ([BTIhILS) with immobilized receptor was measured with a real-time biosensor, and these results were used as a basis for configuring an ELISA for screening antagonists of hIL5-receptor binding. The recombinant proteins used, hIL5 and shIL5Ra-Fc (chimeric fusion receptor constructed by linking the soluble component of the hIL5 receptor a subunit to the constant domain (Fc) of immunoglobulin G ) , were produced by the expression of cloned vectors in Drosophila schneider (S2) cells. Initial attempts to develop a screening assay by direct immobilization of soluble IL5 receptor to microtiter plates proved unsatisafactory and led to use of the Fc chimera attached by oriented immobilization via protein A. Hence, shIL5Ra-Fc was bound to protein A covalently immobilized on a carboxymethyl dextran (CM-5) biosensor chip. Specific binding was demonstrated of [BTIhILS to protein A/shILSRa-Fc receptor complex. The binding was high affinity ( K~p p = 6 nM), reversible and saturable. The affinity of [BTJhILS was similar to that determined with the biosensor assay for unmodified hIL5. The observed kinetics of the interactions of Fc chimera with protein A (slow dissociation) and of [BTIhILS with immobilized Fc chimera (faster dissociation) were favorable for subsequently establishing a microtiter plate based ELISA assay. In the latter, Fc chimera was immobilized to the plate via protein A as in the biosensor experiment. Binding of [BTIhILS to immobilized Fc chimera in the ELISA was concentration dependent and was competed by both hIL5 and shIL5Ra. The results demonstrate a novel use of real-time biosensor technology as a macromolecular interaction analysis tool to help develop other assays using immobilized ligands, including high throughput screening assays to identify antagonist leads. The biosensor technology also offers a means to evaluate the mechanism of action of lead compounds by competition binding, as well as by direct binding if the leads are large enough or can be immobilized to the sensor surface.

In this article, recent progress related to the use of different types of polypeptide fusion handles or 'tags' for the purification of recombinant proteins are critically discussed. In addition, novel aspects of the molecular cassette concept are elaborated, together with areas of potential application of these fundamental principles in molecular recognition. As evident from this review, the use of these concepts provides a powerful strategy for the high throughput isolation and purification of recombinant proteins and their derived domains, generated from functional genomic or zeomic studies, as part of the bioprocess technology leading to their commercial development, and in the study of molecular recognition phenomena per se. In addition, similar concepts can be exploited for high sensitivity analysis and detection, for the characterisation of protein bait/ prey interactions at the molecular level, and for the immobilisation and directed orientation of proteins for use as biocatalysts/biosensors.

The formation and recall of sensory, motor, and cognitive representations require coordinated fast communication among multiple cortical areas. Interareal projections are mainly mediated by glutamatergic pyramidal cell projections; only few long-range GABAergic connections have been reported. Using in vivo recording and labeling of single cells and retrograde axonal tracing, we demonstrate novel long-range GABAergic projection neurons in the rat hippocampus: (1) somatostatin- and predominantly mGluR1alpha-positive neurons in stratum oriens project to the subiculum, other cortical areas, and the medial septum; (2) neurons in stratum oriens, including somatostatin-negative ones; and (3) trilaminar cells project to the subiculum and/or other cortical areas but not the septum. These three populations strongly increase their firing during sharp wave-associated ripple oscillations, communicating this network state to the septotemporal system. Finally, a large population of somatostatin-ne...

Diphenyl 1-(N-peptidylamino)alkanephosphonate appears to react specifically with one chymase while esters are highly reactive, specific, and aqueously leaving other chymases untouched. Perforin-depenstable irreversible inhibitors which can be used to dent lysis mediated by cytotoxic lymphocyte granprobe the functions of many serine proteases, includules was inactivated by Z-Ala-Ala-Ala P (OPh) 2 , Z-Met Ping the lymphocyte granzymes. We synthesized 16 (OPh-4-Cl) 2 , Z-Leu-Phe P (OPh) 2 , and Bi-Aca-Aca-Phepeptide phosphonates with Ala, Met, Phe, or Val P1 Leu-Phe P (OPh) 2 . The biochemical properties and amino acid residues, including two biotinylated debiological efficacy of these inhibitors make them rivatives for future functional and biochemical charsuitable for cellular and physiological studies of acterization of granzymes. The reactivity of the ingranzyme function. ᭧ 1997 Academic Press hibitors was characterized with human leukocyte Key Words: Granzymes; lymphocyte serine proteases; elastase (HLE), porcine pancreatic elastase (PPE), phosphonate inhibitors; cytolysis; chymase; elastase; bovine chymotrypsin, and the granzymes of natural chymotrypsin. killer (NK) cells, which include a number of proteolytic activities (Asp-ase, Met-ase, etc.) that cleave peptide substrates with these residues in the P1 position. The reactivity and specificity of the phospho-Serine proteases are enzymes that have important nates depended on the length and sequence of the physiological roles including digestion, blood coagulapeptidyl moiety and on the leaving group. Z-Ala-Alation, fibrinolysis, complement activation, and peptide Ala P (OPh) 2 was a good inhibitor of HLE and PPE hormone activation (1). Pathophysiological proteolysis, (k obsd /[I] Å 38 and 30 M 01 s 01 , respectively) and had catalyzed by serine proteases, can result in diseases little reactivity with chymotrypsin. Z-Phe-Pro-Phesuch as pulmonary emphysema, adult respiratory dis-P (OPh) 2 was a good inhibitor of chymotrypsin (k obsd / tress syndrome, pancreatitis and inflammation (2). Cy-[I] Å 17,000 M 01 s 01 ) and had little reactivity with the totoxic T lymphocytes (CTL) 2 and natural killer (NK) elastases. The leaving group of Z-Met P (OPh-4-Cl) 2 made it a more effective chymotrypsin inhibitor than Z-Met P (OPh) 2 (k obsd /[I] values of 142 and 30 M 01 s 01 , 2 acted with a fraction of the Met-ase, chymase, and ylformamide; FAB, fast atom bombardment; Hepes, N-2-hydroxyethyl-

The effect of biotin binding on the thermal stability of streptavidin (STV) and avidin (AVD) was evaluated using differential scanning calorimetry. Biotin binding increases the midpoint of temperature T m of thermally induced denaturation of STV and AVD in phosphate buffer from 75 and 83°C to 112 and 117°C at full biotin saturation, respectively. This thermostability is the highest reported for proteins coming from either mesophilic or thermophilic organisms. In both proteins, biotin also increases the calorimetric enthalpy and the cooperativity of the unfolding. Thermal stability of STV was also evaluated in the presence of high concentrations of urea or guanidinium hydrochloride (GuHCl). In 6 M GuHCl, STV remains as a tetramer and the T m of the STV-biotin complex is centered at 108°C, a few degrees below the value obtained in phosphate buffer. On the contrary, STV under fully saturating condition remains mainly in its dimeric form in 8 M urea and the thermogram shows two endotherms. The main endotherm at a lower temperature has been ascribed to the dimeric liganded state with a T m of 87°C, and the higher temperature endotherm to the tetrameric liganded form with a T m of 106°C. As the thermostability of unliganded protein in the presence of urea is unchanged upon binding we related the extremely high thermal stability of this protein to both an increase in structural ordering and compactness with the preservation of the tetramer integrity.

There is strong evidence that commissural interneurons, neurons with axons that extend to the contralateral side of the spinal cord, play an important role in the coordination of left/right alternation during locomotion. In this study we investigated the projections of commissural interneurons to motor neurons and other commissural interneurons on the other side of the spinal cord in neonatal rats. To establish whether there are direct contacts between axons of commissural interneurons and motor neurons, we carried out two series of experiments. In the first experiment we injected biotinylated dextran amine (BDA) into the lateral motor column to retrogradely label commissural interneurons that may have direct projections to motor neurons. Stained neurons were recovered in the ventromedial areas of the contralateral gray matter in substantial numbers. In the second experiment BDA was injected into the ventromedial gray matter on one side of the lumbar spinal cord, whereas motor neurons were simultaneously labeled on the opposite side by applying biocytin onto the ventral roots. BDA injections into the ventromedial gray matter labeled a strong axon bundle that arose from the site of injection, crossed the midline in the ventral commissure, and extensively arborized in the contralateral ventral gray matter. Many of these axons made close appositions with dendrites and somata of motor neurons and also with commissural interneurons retrogradely labeled with BDA. The results suggest that commissural interneurons may establish monosynaptic contacts with motor neurons on the opposite side of the spinal cord. Our findings also indicate that direct reciprocal connections between commissural interneurons on the two sides of the spinal cord may also exist.

Morphological features and functional implications of projections of the parabrachial nucleus to the central nucleus of the amygdala were investigated in the rat. The anatomical study was based on injections of the tracers horseradish peroxidase and biotinylated dextran amine. An extremely dense concentration of labeled fibers was found in the lateral and lateral capsular subdivisions of the central nucleus of the amygdala, originating mainly from the external lateral and ventral lateral subnuclei of the parabrachial nucleus. The parabrachial fibers exhibited the morphological characteristic of forming dense pericellular terminal arborizations. The functional implications of this pathway in cardiovascular functions were verified using Fos protein induction in response to hypotension induced by continuous intravenous administration of hydralazine-hydrochloride. In this paradigm, Fos immunoreactivity was found to be confined to the lateral and lateral capsular subdivisions of the central nucleus of the amygdala. Double immunostaining methods were used to visualize, at the electron microscopic level, terminals labeled by biotinylated dextran amine and Fos cell labeling. With this approach, we were able to confirm that Fos-immunoreactive neurons in the central nucleus of the amygdala receive axosomatic terminals from the parabrachial nucleus. The present findings point out that parabrachial inputs to the central nucleus of the amygdala play a relevant role in regulating cardiovascular function.

Diffusion weighted imaging (DWI) and tractography allow the non-invasive study of anatomical brain connectivity. However, a gold standard for validating tractography of complex connections is lacking. Using the porcine brain as a highly gyrated brain model, we quantitatively and qualitatively assessed the anatomical validity and reproducibility of in vitro multi-fiber probabilistic tractography against two invasive tracers: the histochemically detectable biotinylated dextran amine and manganese enhanced magnetic resonance ...

The rostral ventromedial medulla (RVMM) is a sympathoexcitatory area. However, little is known about its efferent projections. In this study, biotinylated dextran amine (BDA) or Phaseolus vulgaris leucoagglutinin (PHA-L) were used to investigate the medullary and spinal cord projections from pressor sites in RVMM. Initially, RVMM was systematically explored in urethane-anesthetized rats using microinjection of L-glutamate for sites that elicited increases in arterial pressure. A pressor area was identified that included the rostral magnocellular reticular and rostral lateral paragigantocellular reticular nuclei. In the second series of experiments, BDA or PHA-L was iontophoretically injected into RVMM pressor sites. Anterograde labeling was observed throughout the brainstem and spinal cord, bilaterally, but with an ipsilateral predominance. Dense labeling was observed within the nucleus of the solitary tract (NTS); the greatest density of labeling was observed in the caudal dorsolateral, medial, and ventrolateral subnuclei. Additionally, light to moderately dense labeling was found within the nucleus substantia gelatinosus and commissural nucleus. In the nucleus ambiguus/ventrolateral medullary (Amb/VLM) region, the density of labeling was greatest in caudal regions. Within Amb, most of the labeling was localized to its external formation. Anterograde labeling was also found throughout the spinal cord. In the thoracolumbar segments, dense axonal labeling was observed within the dorsolateral funiculus. These labeled axons innervated the intermediolateral nucleus and the central autonomic area. Taken together, these data suggest that RVMM neurons elicit increases in sympathetic activity by likely providing a direct excitatory input to spinal sympathetic preganglionic neurons, and by a direct inhibitory input to medullary cardioinhibitory and depressor areas.

The dynamics of G protein heterotrimer complex formation and disassembly in response to nucleotide binding and receptor activation govern the rate of responses to external stimuli. We use a novel flow cytometry approach to study the effects of lipid modification, isoform specificity, lipid environment, and receptor stimulation on the affinity and kinetics of G protein subunit binding. Fluoresceinlabeled myristoylated GR i1 (F-R i1 ) was used as the ligand bound to G γ in competition binding studies with differently modified GR subunit isoforms. In detergent solutions, the binding affinity of GR i to γ was 2 orders of magnitude higher than for GR o and GR s (IC 50 of 0.2 nM vs 17 and 27 nM, respectively), while in reconstituted bovine brain lipid vesicles, binding was slightly weaker. The effects of receptor on the G protein complex were assessed in R 2A AR receptor expressing CHO cell membranes into which purified γ subunits and F-R i1 were reconstituted. These cell membrane studies led to the following observations: (1) binding of R subunit to the γ was not enhanced by receptor in the presence or absence of agonist, indicating that γ contributed essentially all of the binding energy for R i1 interaction with the membrane; (2) activation of the receptor facilitated GTPγS-stimulated detachment of F-R i1 from γ and the membrane. Thus flow cytometry permits quantiatitive and real-time assessments of protein-protein interactions in complex membrane environments.