Proper functioning of each secretory and endocytic compartment relies on its unique pH micro-environment that is known to be dictated by the rates of V-ATPase-mediated H+ pumping and its leakage back to the cytoplasm via an elusive “H+... more
Proper functioning of each secretory and endocytic compartment relies on its unique pH micro-environment that is known to be dictated by the rates of V-ATPase-mediated H+ pumping and its leakage back to the cytoplasm via an elusive “H+ leak” pathway. Here, we show that this proton leak across Golgi membranes is mediated by the AE2a (SLC4A2a)-mediated bicarbonate-chloride exchange, as it is strictly dependent on bicarbonate import (in exchange for chloride export) and the expression level of the Golgi-localized AE2a anion exchanger. In the acidic Golgi lumen, imported bicarbonate anions and protons then facilitate a common buffering reaction that yields carbon dioxide and water before their egress back to the cytoplasm via diffusion or water channels. The flattened morphology of the Golgi cisternae helps this process, as their high surface-volume ratio is optimal for water and gas exchange. Interestingly, this net acid efflux pathway is often upregulated in cancers and established ca...
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A complementary metal-oxide-semiconductor (CMOS) chip biosensor was developed for cell viability monitoring based on an array of capacitance sensors utilizing a ring oscillator. The chip was packaged in a low temperature co-fired ceramic... more
A complementary metal-oxide-semiconductor (CMOS) chip biosensor was developed for cell viability monitoring based on an array of capacitance sensors utilizing a ring oscillator. The chip was packaged in a low temperature co-fired ceramic (LTCC) module with a flip chip bonding technique. A microcontroller operates the chip, while the whole measurement system was controlled by PC. The developed biosensor was applied for measurement of the proliferation stage of adherent cells where the sensor response depends on the ratio between healthy, viable and multiplying cells, which adhere onto the chip surface, and necrotic or apoptotic cells, which detach from the chip surface. This change in cellular adhesion caused a change in the effective permittivity in the vicinity of the sensor element, which was sensed as a change in oscillation frequency of the ring oscillator. The sensor was tested with human lung epithelial cells (BEAS-2B) during cell addition, proliferation and migration, and fin...
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Immunoglobulin G (IgG) is a major effector molecule of the human immune response, and aberrations in IgG glycosylation are linked to various diseases. However, the molecular mechanisms underlying protein glycosylation are still poorly... more
Immunoglobulin G (IgG) is a major effector molecule of the human immune response, and aberrations in IgG glycosylation are linked to various diseases. However, the molecular mechanisms underlying protein glycosylation are still poorly understood. We present a data-driven approach to infer reactions in the IgG glycosylation pathway using large-scale mass-spectrometry measurements. Gaussian graphical models are used to construct association networks from four cohorts. We find that glycan pairs with high partial correlations represent enzymatic reactions in the known glycosylation pathway, and then predict new biochemical reactions using a rule-based approach. Validation is performed using data from a GWAS and results from three in vitro experiments. We show that one predicted reaction is enzymatically feasible and that one rejected reaction does not occur in vitro. Moreover, in contrast to previous knowledge, enzymes involved in our predictions colocalize in the Golgi of two cell line...
Research Interests: Algorithms, Computational Biology, Mass Spectrometry, Proteomics, Multidisciplinary, and 14 moreHumans, Female, Male, Young Adult, Glycosylation, High Pressure Liquid Chromatography, Aged, Middle Aged, Caco-2 cells, Adult, Glycosyltransferases, Nature Communications, immunoglobulin G, and Cohort Studies
Cell viability monitoring is an important part of biosafety evaluation for the detection of toxic effects on cells caused by nanomaterials, preferably by label-free, noninvasive, fast, and cost effective methods. These requirements can be... more
Cell viability monitoring is an important part of biosafety evaluation for the detection of toxic effects on cells caused by nanomaterials, preferably by label-free, noninvasive, fast, and cost effective methods. These requirements can be met by monitoring cell viability with a capacitance-sensing integrated circuit (IC) microchip. The capacitance provides a measurement of the surface attachment of adherent cells as an indication of their health status. However, the moist, warm, and corrosive biological environment requires reliable packaging of the sensor chip. In this work, a second generation of low temperature co-fired ceramic (LTCC) technology was combined with flip-chip bonding to provide a durable package compatible with cell culture. The LTCC-packaged sensor chip was integrated with a printed circuit board, data acquisition device, and measurement-controlling software. The packaged sensor chip functioned well in the presence of cell medium and cells, with output voltages dep...
Band 3 multigene family consists of several distinct but structurally related polypeptides which are probably involved in the transport of anions across the plasma membrane of both erythrocytes and nonerythroid cells. A novel member of... more
Band 3 multigene family consists of several distinct but structurally related polypeptides which are probably involved in the transport of anions across the plasma membrane of both erythrocytes and nonerythroid cells. A novel member of this family of polypeptides that resides in the Golgi complex was identified with antibodies to Band 3. The Golgi antigen had a larger molecular size and was antigenically distinct from Band 3 in the amino-terminal domain. It was expressed most prominently in cells that secrete large amounts of sulfated proteins and proteoglycans. This polypeptide may participate in sulfate transport across Golgi membranes.
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Currently available methods for detection of early-stage colorectal cancer are reliant on faecal occult blood (FOB) tests. Bleeding, however, is not specific for colorectal neoplasia. Enzymatically detected or peanut agglutinin... more
Currently available methods for detection of early-stage colorectal cancer are reliant on faecal occult blood (FOB) tests. Bleeding, however, is not specific for colorectal neoplasia. Enzymatically detected or peanut agglutinin (PNA)-detectable galactose-beta1-3-N-acetyl-galactosamine residues found in rectal mucus have been used to detect colorectal cancer. The sensitivity and specificity of the PNA rectal mucus test were compared with those of an immunological test for faecal occult blood (Hemolex) in 199 symptomatic patients referred for colorectal investigations. All patients also underwent a colonoscopy. SDS-PAGE and PNA-overlay were used to characterize PNA-binding proteins in normal and malignant colorectal tissue. The PNA test had a similar sensitivity to that of Hemolex for colorectal carcinoma (83% vs. 72%), adenomas (55% vs. 50%), inflammatory bowel disease (52% vs. 48%) and hyperplastic polyps (48% vs. 25%). The sensitivity of the PNA test and Hemolex for colorectal neoplasia was 69% vs. 59% and specificity 68% vs. 86% (p=0.002). SDS-PAGE and PNA-overlay showed some commonly expressed PNA-binding proteins in both normal mucosa and colorectal cancer and a higher and even selective expression of 160 kD PNA-binding protein in colorectal cancer. A single PNA test in its present form is as sensitive an indicator of colorectal neoplasia as Hemolex completed over three days, but lacks specificity. The 160 kD cancer-associated antigen we have identified is under further characterization for development of a more specific PNA test.
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Human and rat spermatozoa were stained for different carbonic anhydrase (CA) isoenzymes using specific antisera to human CA I, II and VI in conjunction with the immunofluorescence technique. The spermatozoa of both species were found to... more
Human and rat spermatozoa were stained for different carbonic anhydrase (CA) isoenzymes using specific antisera to human CA I, II and VI in conjunction with the immunofluorescence technique. The spermatozoa of both species were found to contain only CA II, which was located principally in the postacrosomal region of the human spermatozoa and in the acrosomal cap region of the rat spermatozoa. The presence of CA II could be confirmed by immunoblotting, which revealed a 29 K polypeptide in both the human and rat spermatozoa. No CA I or VI-specific fluorescence could be detected in the spermatozoa of either species. The immunoblottings were also negative. The results show mammalian spermatozoa to contain the high activity carbonic anhydrase isoenzyme II. Its presence is probably linked to hydration of CO2 produced by active energy metabolism and thereby to the maintaining of an adequate intraspermatozoal bicarbonate concentration as required for the maintenance of sperm motility.
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Hyaluronan content is a powerful prognostic factor in many cancer types, but the molecular basis of its synthesis in cancer still remains unclear. Hyaluronan synthesis requires the transport of hyaluronan synthases (HAS1-3) from Golgi to... more
Hyaluronan content is a powerful prognostic factor in many cancer types, but the molecular basis of its synthesis in cancer still remains unclear. Hyaluronan synthesis requires the transport of hyaluronan synthases (HAS1-3) from Golgi to plasma membrane (PM), where the enzymes are activated. For the very first time, the present study demonstrated a rapid recycling of HAS3 between PM and endosomes, controlled by the cytosolic levels of the HAS substrates UDP-GlcUA and UDP-GlcNAc. Depletion of UDP-GlcNAc or UDP-GlcUA shifted the balance towards HAS3 endocytosis, and inhibition of hyaluronan synthesis. In contrast, UDP-GlcNAc surplus suppressed endocytosis and lysosomal decay of HAS3, favoring its retention in PM, stimulating hyaluronan synthesis, and HAS3 shedding in extracellular vesicles. The concentration of UDP-GlcNAc also controlled the level of O-GlcNAc modification of HAS3. Increasing O-GlcNAcylation reproduced the effects of UDP-GlcNAc surplus on HAS3 trafficking, while its suppression showed the opposite effects, indicating that O-GlcNAc signaling is associated to UDP-GlcNAc supply. Importantly, a similar correlation existed between the expression of GFAT1 (the rate limiting enzyme in UDP-GlcNAc synthesis) and hyaluronan content in early and deep human melanomas, suggesting the association of UDP-sugar metabolism in initiation of melanomagenesis. In general, changes in glucose metabolism, realized through UDP-sugar contents and O-GlcNAc signaling, are important in HAS3 trafficking, hyaluronan synthesis, and correlates with melanoma progression.
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In vitro degradation of LH receptor after occupancy by hCG was studied. Rat ovarian membranes labeled with [125I]iodo-hCG were incubated at 37 C; as a result, 30-40% of the radioactivity initially bound was rendered soluble in the medium.... more
In vitro degradation of LH receptor after occupancy by hCG was studied. Rat ovarian membranes labeled with [125I]iodo-hCG were incubated at 37 C; as a result, 30-40% of the radioactivity initially bound was rendered soluble in the medium. The molecular complexes in the medium and in incubated membranes solubilized with 1% Triton X-100 were then cross-linked with glutaraldehyde and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Particulate receptor-[125I]iodo-hCG complex exhibiting an apparent mol wt of 125,000 was cleaved during incubation into two distinct components (mol wt, 96,000 and 74,000) which appeared in the medium. Using tritiated hCG (beta-subunit labeled) instead of radioiodinated hCG (alpha-subunit labeled), these same two components were also observed, indicating that they both contain intact hCG (alpha and beta) as a part of their structure. In addition to the hormone (mol wt, 48,000), these two components contain receptor fragments with mol wt of 64,000 or 38,000, demonstrated directly by labeling the particulate receptor itself with periodate-tritiated borohydride before tagging with unlabeled hCG and in vitro incubation. These receptor fragments were purified from the medium by hCG-directed immunoaffinity chromatography and detached from the hormone by pH treatment. The intact receptor extracted from the membranes with detergent and purified identically in the absence of proteolysis migrated as a 90,000 mol wt polypeptide. These results demonstrate that after hormone occupancy, proteolytic cleavage of the 90,000 mol wt receptor polypeptide occurs at two specific sites. Thiol-blocking agents selectively prevented the appearance of the larger component (hCG coupled to 64,000 mol wt receptor fragment), while metal-chelating agents markedly decreased the appearance of the smaller component (hCG coupled to 38,000 mol wt receptor fragment) in the medium. Identical observations, obtained upon incubation of plasma membranes purified by sucrose density gradient centrifugation, suggest that plasma membrane enzymes are involved.
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ABSTRACT
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Glycosylation of proteins and lipids takes place in the Golgi apparatus by the consecutive actions of functionally distinct glycosidases and glycosyltransferases. Current evidence indicates that they function as enzyme homomers and/or... more
Glycosylation of proteins and lipids takes place in the Golgi apparatus by the consecutive actions of functionally distinct glycosidases and glycosyltransferases. Current evidence indicates that they function as enzyme homomers and/or heteromers in the living cell. Here we investigate their organizational interplay and show that glycosyltransferase homomers are assembled in the endoplasmic reticulum. Upon transport to the Golgi, the majority of homomers are disassembled to allow the formation of enzyme heteromers between sequentially acting medial-Golgi enzymes GnT-I and GnT-II or trans-Golgi enzymes GalT-I and ST6Gal-I. This transition is driven by the acidic Golgi environment, as it was markedly inhibited by raising Golgi luminal pH with chloroquine. Our FRAP (fluorescence recovery after photobleaching) measurements showed that the complexes remain mobile Golgi membrane constituents that can relocate to the endoplasmic reticulum or to the scattered Golgi mini-stacks upon brefeldin...
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Lysyl hydroxylase (LH), an enzyme required early during collagen biosynthesis, appears to be exceptional among proteins that are thought to be residents of the endoplasmic reticulum (ER). It is a homodimer and does not contain either of... more
Lysyl hydroxylase (LH), an enzyme required early during collagen biosynthesis, appears to be exceptional among proteins that are thought to be residents of the endoplasmic reticulum (ER). It is a homodimer and does not contain either of the two previously characterized ER-specific retention motifs (KDEL or the double lysine motif) in its primary structure. We now show that LH, nevertheless, resides in the lumen of the ER. In immunofluorescence experiments, LH co-localizes with a KDEL-containing protein, protein disulfide isomerase (PDI), and also co-sediments with it after fractionation of subcellular organelles by sucrose density gradient centrifugation. In addition, LH seems to be stress-inducible. In one respect, however, LH differs from PDI and other known luminal proteins in the organelle. It is found in situ only in association with the ER membranes. Our cell fractionation and Triton X-114 phase separation experiments suggest that it binds to the membranes via weak electrostat...
Research Interests: Membrane Proteins, Immunohistochemistry, Biological Chemistry, Biological Sciences, Antibodies, and 15 moreHumans, Biological, Endoplasmic Reticulum, Tunicamycin, Peptides, Skin, CHEMICAL SCIENCES, Molecular weight, Amino Acid Sequence, Subcellular Fractions, Isomerases, Potassium Chloride, Molecular Sequence Data, fibroblasts, and Medical and Health Sciences
Many tumour-specific antigens in gastrointestinal cancers have carbohydrate immuno-determinants. These epitopes can be identified by lectins and monoclonal antibodies. By using fluorescein-isothiocyanate (FITC)-conjugated peanut... more
Many tumour-specific antigens in gastrointestinal cancers have carbohydrate immuno-determinants. These epitopes can be identified by lectins and monoclonal antibodies. By using fluorescein-isothiocyanate (FITC)-conjugated peanut agglutinin (PNA) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) we have investigated glycoproteins carrying altered carbohydrate epitopes in normal and carcinomatous human colorectal mucosa. In normal mucosa PNA stained goblet cell glycoconjugates in the supranuclear (Golgi) distribution. After neuraminidase pretreatment PNA stained actual mucin goblet itself at all levels of the crypts. Colorectal carcinomas displayed a strong and direct binding of PNA to apical cell membranes of carcinomatous cells and intraluminal secretions. Analysis of the glycoproteins by SDS-PAGE and PNA-labelling revealed four carcinoma-associated glycoproteins (26kD, 32kD, 35kD and 50kD). In addition, four glycoproteins (29kD, 30kD, 33kD and 36kD) common to...
Research Interests: Apoptosis, Nature, Cell Division, Adipose tissue, Humans, and 15 moreIntestinal Mucosa, Lectins, Glycoproteins, Plant Lectins, Neuraminidase, Cancer cells, Molecular weight, Gel electrophoresis, Polyacrylamide Gel Electrophoresis, Cancers, Arachis Hypogaea, Epitopes, Binding Site, Mucins, and Colonic Neoplasms
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Tyrosine sulfation of proteins is an important post-translational modification shown to play a role in many membrane-associated or extracellular processes such as virus entry, blood clotting, antibody-mediated immune response,... more
Tyrosine sulfation of proteins is an important post-translational modification shown to play a role in many membrane-associated or extracellular processes such as virus entry, blood clotting, antibody-mediated immune response, inflammation and egg fecundation. The sole two human enzymes that transfer sulfate moieties from 3'-phospho-adenosine-5'-phospho-sulfate onto tyrosine residues, TPST1 and TPST2, are anchored to the membranes of the trans-Golgi compartment with the catalytic domain oriented to the lumen. In contrast to the relatively well studied organization of medial Golgi enzymes, the organization of trans-Golgi transferases remains elusive. Although tyrosylprotein sulfotransferases are known to exist as homodimers in the Golgi membranes, this organization level may represent only a small piece of a puzzle that is linked to the entire picture. Here we report the formation of TPST1/TPST2 heterodimers and a novel interaction between either TPST1 or TPST2 and the α-2,6-sialyltransferase, indicating a higher organization level of tyrosylprotein sulfotransferases that may serve for substrate selectivity and/or effective organization of multiple post-translational modification of proteins.
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Hydroxysteroid dehydrogenase (17HSD) type 2e Yciently catalyzes the conversion of the high activity 17‚-hydroxy forms of sex steroids into less potent 17-ketosteroids. In the present study in situ hybridization was utilized to analyze the... more
Hydroxysteroid dehydrogenase (17HSD) type 2e Yciently catalyzes the conversion of the high activity 17‚-hydroxy forms of sex steroids into less potent 17-ketosteroids. In the present study in situ hybridization was utilized to analyze the cellular localization of 17HSD type 2 expression in adult male and female mice. The data indicate that 17HSD type 2 mRNA is expressed in several epithelial
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... Further experiments, in young, senescent, and neoplastic cell lines are warranted for quantifying the ratio of cdc25to weel expres-sion in subsequent ... MUNMUN CHAKRABORTY, DIPTENDU CHATTERJEE, SAKARI KELLOKUMPU, HOWARD RASMUSSEN,... more
... Further experiments, in young, senescent, and neoplastic cell lines are warranted for quantifying the ratio of cdc25to weel expres-sion in subsequent ... MUNMUN CHAKRABORTY, DIPTENDU CHATTERJEE, SAKARI KELLOKUMPU, HOWARD RASMUSSEN, ROLAND BARON* ...
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Currently available methods for detection of early-stage colorectal cancer are reliant on faecal occult blood (FOB) tests. Bleeding, however, is not specific for colorectal neoplasia. Enzymatically detected or peanut agglutinin... more
Currently available methods for detection of early-stage colorectal cancer are reliant on faecal occult blood (FOB) tests. Bleeding, however, is not specific for colorectal neoplasia. Enzymatically detected or peanut agglutinin (PNA)-detectable galactose-beta1-3-N-acetyl-galactosamine residues found in rectal mucus have been used to detect colorectal cancer. The sensitivity and specificity of the PNA rectal mucus test were compared with those of an immunological test for faecal occult blood (Hemolex) in 199 symptomatic patients referred for colorectal investigations. All patients also underwent a colonoscopy. SDS-PAGE and PNA-overlay were used to characterize PNA-binding proteins in normal and malignant colorectal tissue. The PNA test had a similar sensitivity to that of Hemolex for colorectal carcinoma (83% vs. 72%), adenomas (55% vs. 50%), inflammatory bowel disease (52% vs. 48%) and hyperplastic polyps (48% vs. 25%). The sensitivity of the PNA test and Hemolex for colorectal neoplasia was 69% vs. 59% and specificity 68% vs. 86% (p=0.002). SDS-PAGE and PNA-overlay showed some commonly expressed PNA-binding proteins in both normal mucosa and colorectal cancer and a higher and even selective expression of 160 kD PNA-binding protein in colorectal cancer. A single PNA test in its present form is as sensitive an indicator of colorectal neoplasia as Hemolex completed over three days, but lacks specificity. The 160 kD cancer-associated antigen we have identified is under further characterization for development of a more specific PNA test.
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A ligand blotting technique was developed to identify the lutropin receptor after size separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. The separated proteins were transferred to a... more
A ligand blotting technique was developed to identify the lutropin receptor after size separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. The separated proteins were transferred to a nitrocellulose sheet, which was subsequently incubated with 125I-labeled human choriogonadotropin (125I-hCG), and subjected to autoradiography. An Mr 90,000 band was specifically and intensely labeled with 125I-hCG. The band was not observed, when the hormone incubation was performed in the presence of an excess of unlabeled hCG or human lutropin. The presence of rat follitropin or rat prolactin did not, however, abolish the labeling. No specific labeling was found when down-regulated ovarian tissue or rat liver was used as starting material. The Mr 90,000 band disappeared when the protein samples were treated with reducing agent, showing that integrity of receptor disulfide bonds is essential for the hormone-receptor interaction. In addition, a receptor-positive murine Leydig tumor cell line produced an Mr 90,000-92,000 band in ligand blotting, thus demonstrating the similarity between rat and murine lutropin receptors. These results provide strong evidence that the lutropin receptor is an Mr 90,000 protein.
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SUMMARY Na+-independent Cl−/HCO3− exchangers (AE1, AE2, AE3) are generally known as ubiquitous, multispanning plasma membrane proteins that regulate intracellular pH and transepithelial acid–base balance in animal tissues. However,... more
SUMMARY Na+-independent Cl−/HCO3− exchangers (AE1, AE2, AE3) are generally known as ubiquitous, multispanning plasma membrane proteins that regulate intracellular pH and transepithelial acid–base balance in animal tissues. However, previous immunological evidence has suggested that anion exchanger (AE) proteins may also be present in intracellular membranes, including membranes of the Golgi complex and mitochondria. Here we provide several lines of evidence to show that an AE protein is indeed a resident of the Golgi membranes and that this protein corresponds to the full-length AE2a isoform in fibroblasts. First, both the N- and C-terminal antibodies to AE2 (but not to AE1) detected an AE protein in the Golgi membranes. Golgi localization of this AE2 antigen was evident also in cycloheximide-treated cells, indicating that it is a true Golgi-resident protein. Second, our Northern blotting and RT-PCR analyses demonstrated the presence of only the full-length AE2a mRNA in cells that s...
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Research Interests: Catalysis, Biological Chemistry, Magnetic Resonance Spectroscopy, Gene expression, Biological Sciences, and 15 moreHumans, Circular Dichroism, Escherichia coli, Animals, Biological, Polymerase Chain Reaction, Endoplasmic Reticulum, CHEMICAL SCIENCES, Cysteine, Protein Conformation, Amino Acid Sequence, Protein Denaturation, Molecular Sequence Data, binding sites, and Medical and Health Sciences
Research Interests: Catalysis, Biological Chemistry, Biological Sciences, Cercopithecus aethiops, Humans, and 13 moreSequence alignment, Animals, Biological, Endoplasmic Reticulum, CHEMICAL SCIENCES, Substrate Specificity, Amino Acid Sequence, Protein Binding, Molecular Sequence Data, protein disulfide isomerase (PDI), binding sites, conserved sequence , and Medical and Health Sciences
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The in vivo uptake of human chorionic gonadotropin (hCG) in the rat testes and mode of the early testosterone response were studied after a single intravenous injection of varying doses of hCG. The uptake of hCG by the testes was parallel... more
The in vivo uptake of human chorionic gonadotropin (hCG) in the rat testes and mode of the early testosterone response were studied after a single intravenous injection of varying doses of hCG. The uptake of hCG by the testes was parallel up to 6 h with all hormone doses, decreased thereafter to low level by 24 h with low hormone doses but continued to increase up to 24 h with the highest dose of hCG. The clearance rate of hCG from the blood was independent of the hormone dose used. Serum testosterone peaked gradually earlier when the hCG dose increased, and the highest hCG dose caused a slightly biphasic response with maxima at 1 and 12 h, the former peak being more pronounced. These results suggest that the in vivo uptake of hCG in the testes is modulated by the hormone dose used and that the mode of early serum testosterone response to varying hCG doses is dose dependent.
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Research Interests: Electron Microscopy, Stem Cells, Protein Structure and Function, Cell fusion, Animals, and 14 moreVesicular Stomatitis Virus, Endoplasmic Reticulum, Cell Fractionation, P-glycoprotein, Muscles, Functional impairment, Glycosylation, Clinical Sciences, Rats, Muscle development, G protein, Golgi Apparatus, Cell Surface Markers, and Biochemistry and cell biology
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The type VI variant of Ehlers-Danlos syndrome (EDS) is a heritable connective tissue disorder caused by a deficiency in the activity of lysyl hydroxylase, an enzyme required for the post-translational processing of collagens. We have... more
The type VI variant of Ehlers-Danlos syndrome (EDS) is a heritable connective tissue disorder caused by a deficiency in the activity of lysyl hydroxylase, an enzyme required for the post-translational processing of collagens. We have characterized a novel type of mutation in a young female patient with type VI EDS, in which cells possess only 12% of the lysyl hydroxylase activity that is detected in unaffected cells. The syndrome was found to be caused by a homozygous insertion of two thymidines at the 5' splice site consensus sequence of intron 9 in the lysyl hydroxylase gene. The insertion interfered with normal splicing of the primary RNA transcript and resulted in an inframe deletion of the 132 nucleotides coded by exon 9 from the lysyl hydroxylase mRNA. In addition, the mutation caused a marked reduction in the steady-state level of the truncated mRNA, which was less than 15% of the level found in unaffected cells. The mutation also reduced the amount of the enzyme protein produced, which was estimated to be about 20% of that in control cells. However, the mutation did not affect the stability of the abnormally spliced mRNA nor the normal localization of the enzyme protein in the endoplasmic reticulum. According to our results, the reduction in enzymatic activity observed in this patient is caused by low levels of both lysyl hydroxylase mRNA and enzyme protein. The primary cellular defect associated with this mutation, therefore, appears to be at the level of nuclear mRNA metabolism even though the mutation did not create a premature translation termination codon.
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Lysyl hydroxylase (LH) is a peripheral membrane protein in the lumen of the endoplasmic reticulum (ER) that catalyses hydroxylation of lysine residues in collagenous sequences. Previously, we have mapped its primary ER localization motif... more
Lysyl hydroxylase (LH) is a peripheral membrane protein in the lumen of the endoplasmic reticulum (ER) that catalyses hydroxylation of lysine residues in collagenous sequences. Previously, we have mapped its primary ER localization motif within a 40-amino acid segment at its C-terminus. Here, we have characterized this localization mechanism in more detail, and our results indicate that this segment confers ER residency in a KDEL-receptor-independent manner, and without any apparent recycling of the enzyme between the Golgi apparatus and the ER. In addition, we show that a rather long peptide region, rather than a specific peptide sequence per se, is required for efficient retention of a reporter protein in the ER. Accordingly, the minimal retention motif was found to require the last 32 C-terminal amino acids, and sequential substitution of all five charged residues within this critical segment interfered only marginally with the retention or association of the enzyme with the ER m...
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Most organelles within the exocytic and endocytic pathways typically acidify their interiors, a phenomenon that is known to be crucial for their optimal functioning in eukaryotic cells. This review highlights recent advances in our... more
Most organelles within the exocytic and endocytic pathways typically acidify their interiors, a phenomenon that is known to be crucial for their optimal functioning in eukaryotic cells. This review highlights recent advances in our understanding of how Golgi acidity is maintained and regulated, and how its misregulation contributes to organelle dysfunction and disease. Both its biosynthetic products (glycans) and protein-sorting events are highly sensitive to changes in Golgi luminal pH and are affected in certain human disease states such as cancers and cutis laxa. Other potential disease states that are caused by, or are associated with, Golgi pH misregulation will also be discussed.
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Most organelles within the exocytic and endocytic pathways typically acidify their interiors, a phenomenon that is known to be crucial for their optimal functioning in eukaryotic cells. This review highlights recent advances in our... more
Most organelles within the exocytic and endocytic pathways typically acidify their interiors, a phenomenon that is known to be crucial for their optimal functioning in eukaryotic cells. This review highlights recent advances in our understanding of how Golgi acidity is maintained and regulated, and how its misregulation contributes to organelle dysfunction and disease. Both its biosynthetic products (glycans) and protein-sorting events are highly sensitive to changes in Golgi luminal pH and are affected in certain human disease states such as cancers and cutis laxa. Other potential disease states that are caused by, or are associated with, Golgi pH misregulation will also be discussed.
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Research Interests: Chromatography, Flow Cytometry, Biological Chemistry, Fluorescence Resonance Energy Transfer, Biological Sciences, and 10 moreCercopithecus aethiops, Humans, Animals, Glycosylation, CHEMICAL SCIENCES, HeLa cells, Neoplasms, Golgi Apparatus, Hydrogen-Ion Concentration, and Medical and Health Sciences
In vertebrates, hyaluronan is produced in plasma membrane from cytosolic UDP-sugar substrates by HAS1-3 isoenzymes that transfer N-Acetyl glucosamine (GlcNAc) and glucuronic acid (GlcUA) in alternative positions in the growing... more
In vertebrates, hyaluronan is produced in plasma membrane from cytosolic UDP-sugar substrates by HAS1-3 isoenzymes that transfer N-Acetyl glucosamine (GlcNAc) and glucuronic acid (GlcUA) in alternative positions in the growing polysaccharide chain during its simultaneous extrusion into the extracellular space. It has been shown that HAS2 immunoprecipitates contain functional HAS2 homomers and also heteromers with HAS3 (Karousou et al. 2010 J. Biol. Chem. 285 23647-23654). Here we have systematically screened in live cells potential interactions among the HAS isoenzymes using fluorescence resonance energy transfer (FRET) and flow cytometric quantification. We show that all HAS isoenzymes form homomeric and also heteromeric complexes with each other. The same complexes were detected both in Golgi apparatus and plasma membrane by using FRET microscopy and the acceptor photobleaching method. Proximity ligation assays with HAS antibodies confirmed the presence of HAS1-HAS2, HAS2-HAS2, an...
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ABSTRACT Incorporating complementary metal oxide semiconductor (CMOS) chips that can perform signal processing, control, information readout, and direct sensing into microfluidic systems adds powerful capabilities to lab on a chip (LOC)... more
ABSTRACT Incorporating complementary metal oxide semiconductor (CMOS) chips that can perform signal processing, control, information readout, and direct sensing into microfluidic systems adds powerful capabilities to lab on a chip (LOC) devices. For example, on-chip sensors allow system miniaturization, amplifiers placed directly under the sensors provide high signal to noise ratios (SNRs), and signal processing circuitry reduces the amount of data that must be communicated off-chip. Packaging such chips to expose the sensors on the surface to a fluid environment while protecting the input/output region at the periphery has been challenging, however. We present a new packaging method based on forming an epoxy handle wafer around the chip, photolithographic patterning of metal and polymer films for interconnection and passivation, and bonding to PDMS microfluidics. Such packaged chips last for months in the incubator and can be sterilized and re-used. We will show two examples of cell-based sensing with these systems using chips produced in a commercially-available CMOS technology: monitoring the cytotoxicity of nanomaterials through capacitance changes and recording action potentials from electrogenic cells. Adherent cells normally spread out on surfaces, while stressed cells contract and apoptosis leads to detachment. A chip was produced consisting of an array of fully differential capacitance sensors and readout circuitry. Cells (kidney, Cercopithecus aethiops) were cultured on the chip surface to confluence and then exposed to cytotoxic TiO2 nanowires. Cell viability was evaluated with both the chip and a commercial cytotoxicity kit. Preliminary results indicate that viability can be monitored by capacitance measurements. In the second example, a cluster of cardiomyocytes was cultured on the surface of a different chip having an array of electrodes connected to on-chip amplifiers. Electrical recordings showed strong action potentials from the cluster, corresponding in time with the beating of the clump. The signal amplitude decreased with distance to the electrodes, as expected.