The glycerol facilitator is known as the only example of a transport protein that catalyzes facil... more The glycerol facilitator is known as the only example of a transport protein that catalyzes facilitated diffusion across the Escherichia coli inner membrane. Here we show that the gene encoding the facilitator, glpF, is the first gene in an operon with glpK, encoding glycerol kinase, at 88 min of the E. coli chromosome. The operon is transcribed counterclockwise. We cloned the glpF gene, demonstrated that it complemented a chromosomal glycerol transport-minus mutation, and identified the gene product. The GlpF protein appeared in the membrane fraction of plasmid-bearing strains and had an apparent Mr of 25,000.
The ongoing emergence of human infections originating from wildlife highlights the need for bette... more The ongoing emergence of human infections originating from wildlife highlights the need for better knowledge of the microbial community in wildlife species where traditional diagnostic approaches are limited. Here we evaluate the microbial biota in healthy mule deer (Odocoileus hemionus) by analyses of lymph node meta-transcriptomes. cDNA libraries from five individuals and two pools of samples were prepared from retropharyngeal lymph node RNA enriched for polyadenylated RNA and sequenced using Roche-454 Life Sciences technology. Protein-coding and 16S ribosomal RNA (rRNA) sequences were taxonomically profiled using protein and rRNA specific databases. Representatives of all bacterial phyla were detected in the seven libraries based on protein-coding transcripts indicating that viable microbiota were present in lymph nodes. Residents of skin and rumen, and those ubiquitous in mule deer habitat dominated classifiable bacterial species. Based on detection of both rRNA and protein-codi...
Assessment of the microbial diversity residing in arthropod vectors of medical importance is cruc... more Assessment of the microbial diversity residing in arthropod vectors of medical importance is crucial for monitoring endemic infections, for surveillance of newly emerging zoonotic pathogens, and for unraveling the associated bacteria within its host. The tick Ixodes ricinus is recognized as the primary European vector of disease-causing bacteria in humans. Despite I. ricinus being of great public health relevance, its microbial communities remain largely unexplored to date. Here we evaluate the pathogen-load and the microbiome in single adult I. ricinus by using 454- and Illumina-based metagenomic approaches. Genomic DNA-derived sequences were taxonomically profiled using a computational approach based on the BWA algorithm, allowing for the identification of known tick-borne pathogens at the strain level and the putative tick core microbiome. Additionally, we assessed and compared the bacterial taxonomic profile in nymphal and adult I. ricinus pools collected from two distinct geogr...
The Tasmanian devil (Sarcophilus harrisii) is threatened with extinction because of a contagious ... more The Tasmanian devil (Sarcophilus harrisii) is threatened with extinction because of a contagious cancer known as Devil Facial Tumor Disease. The inability to mount an immune response and to reject these tumors might be caused by a lack of genetic diversity within a dwindling population. Here we report a whole-genome analysis of two animals originating from extreme northwest and southeast Tasmania, the maximal geographic spread, together with the genome from a tumor taken from one of them. A 3.3-Gb de novo assembly of the sequence data from two complementary next-generation sequencing platforms was used to identify 1 million polymorphic genomic positions, roughly one-quarter of the number observed between two genetically distant human genomes. Analysis of 14 complete mitochondrial genomes from current and museum specimens, as well as mitochondrial and nuclear SNP markers in 175 animals, suggests that the observed low genetic diversity in today's population preceded the Devil Faci...
The glpFK operon maps near minute 88 on the linkage map of Escherichia coli K-12 with glpF promot... more The glpFK operon maps near minute 88 on the linkage map of Escherichia coli K-12 with glpF promoter proximal. The glpF gene encodes a cytoplasmic membrane protein which facilitates the diffusion of glycerol into the cell. The glpK gene encodes glycerol kinase. In the present work, the nucleotide sequence of the 5'-end of the operon, including the control region, the glpF gene, and part of the glpK gene, was determined. The facilitator was predicted to contain 281 amino acids with a calculated molecular weight of 29,780. It is a highly hydrophobic protein with a minimum of six potential transmembrane alpha helices. The transcription start site for the glpFK operon was located 71 base pairs upstream from the proposed translation start codon for glpF. Preceding the transcription start site were sequences similar to the -10 and -35 consensus sequences for bacterial promoters. Binding sites for the cAMP-cAMP receptor protein (CRP) complex and the glp repressor were identified by DNas...
Overproduction of chimeric proteins containing the HMG2/1 peptide, which comprises the seven tran... more Overproduction of chimeric proteins containing the HMG2/1 peptide, which comprises the seven transmembrane domains of Saccharomyces cerevisiae 3-hydroxy-3-methylglutaryl-CoA reductase isozymes 1 and 2, has previously been observed to induce the proliferation of internal endoplasmic reticulum-like membranes. In order to exploit this amplified membrane surface area for the accommodation of heterologous microsomal proteins, we fused sequences coding for human cytochrome P4501A1 (CYP1A1) to sequences encoding the HMG2/1 peptide and expressed the hybrid genes in yeast. The heterologous hybrid proteins were targeted into strongly proliferated membranes, as shown by electron microscopic and immunofluorescent analysis. Fusion proteins comprising the whole CYP1A1 polypeptide (HMG2/1-CYP1A1) exhibited 7-ethoxyresorufin-O-deethylase activity, whereas fusion proteins lacking the N-terminal 56 amino acids of CYP1A1 (HMG2/1-delta CYP1A1) were inactive and appeared to be unable to incorporate protoheme. Similar amounts of heterologous protein were detected in cells expressing HMG2/1-CYP1A1, HMG2/1-delta CYP1A1 and CYP1A1, respectively. Replacement of the N-terminal membrane anchor domain of human NADPH-cytochrome P450 oxidoreductase by the HMG2/1 peptide also resulted in a functional fusion enzyme, which was able to interact with HMG2/1-CYP1A1 and the yeast endogenous P450 enzyme lanosterol-14 alpha-demethylase.
The Journal of Clinical Endocrinology & Metabolism, 2005
Noonan syndrome is frequently associated with an unclear disturbance of GH secretion. Half the in... more Noonan syndrome is frequently associated with an unclear disturbance of GH secretion. Half the individuals with Noonan syndrome carry a heterozygous mutation of the nonreceptor-type protein tyrosine phosphatase, Src homology region 2-domain phosphatase-2 (SHP-2), encoded by PTPN11, which has a role in GH receptor signaling. The objective of this study was to compare GH secretion and IGF-I/IGF-binding protein-3 (IGFBP-3) levels of the SHP-2 mutation-positive (mut+ group) vs. mutation-negative individuals (mut- group). All children presenting to us with short stature plus at least three typical anomalies of Noonan syndrome or pulmonic stenosis during the last 5 yr (n = 29; 10 females and 19 males) were recruited. Auxological data, dysmorphic features, and cardiac morphology were documented. Hormone levels were measured by RIA. All coding exons of PTPN11 were sequenced after PCR amplification. A prepubertal subgroup (n = 11) was treated with recombinant human GH (rhGH) to promote growth. Sequencing yielded 11 different PTPN11 missense mutations in 16 of the 29 patients (55% mut+). Pulmonic stenosis (81 vs. 15%; P = 0.0007) and septal defects (63 vs. 15%; P = 0.02) were more frequently found in the mut+ group, whereas minor anomalies, cryptorchidism, and learning disabilities were as frequent in the mut+ group as in the mut- group. The mut+ group was younger at presentation (mean +/- sd, 5.1 +/- 2.7 vs. 10.3 +/- 5.2 yr; P = 0.002), but not significantly shorter [-3.15 +/- 0.92 vs. -3.01 +/- 1.35 height sd score (SDS)]. IGF-I levels (-2.03 +/- 0.69 vs. -1.13 +/- 0.89 SDS; P = 0.005) and IGFBP-3 levels (-0.92 +/- 1.26 vs. 0.40 +/- 1.08 SDS; P = 0.006) were significantly lower in the mut+ group. In contrast, GH levels showed a tendency to be higher in the mut+ group during spontaneous secretion at night and arginine stimulation (P > or = 0.075, not significant). The mean change in height SDS after 1 yr of rhGH therapy (0.043 mg/kg.d) was +0.66 +/- 0.21 in the mut+ group (n = 8), but +1.26 +/- 0.36 in the mut- group (n = 3; P = 0.007). Our data suggest that SHP-2 mutations in Noonan syndrome cause mild GH resistance by a postreceptor signaling defect, which seems to be partially compensated for by elevated GH secretion. This defect may contribute to the short stature phenotype in children with SHP-2 mutations and their relatively poor response to rhGH.
Autosomal dominant isolated growth hormone deficiency type II (IGHD II) is mainly caused by splic... more Autosomal dominant isolated growth hormone deficiency type II (IGHD II) is mainly caused by splice site mutations of the GH-1 gene, leading to deletion of amino acids 32–71 of the human growth hormone (hGH). The severe hGH deficit in IGHD II suggests a dominant negative effect of the partially deleted del(32–71)-hGH on the production, storage or secretion of normal wild-type (wt)-hGH in somatotrophic cells of the pituitary. To shed more light on the cellular and molecular basis of IGHD II, we established and analysed diverse clones of the rat somatotrophic cell line GH4C1 stably expressing either wt-hGH, del(32–71)-hGH, or both proteins concomitantly. The cellular morphology of all transfected GH4C1 cell clones showed moderate differences to untransfected GH4C1 cells. On the molecular level, both cDNA-constructs induced transcription but, under normal culture conditions, only wt-hGH protein was found to be synthesised and secreted in readily detectable amounts. By contrast, only after inhibition of proteasomes did high amounts of del(32–71)-hGH show up. The solubility of del(32–71)-hGH in nondenaturing buffer was poor compared to wt-hGH, hinting at molecular aggregation, and several epitopes recognised by monoclonal hGH antibodies were not present on del(32–71)-hGH, confirming the assumption that del(32–71)-hGH must be severely misfolded. Expression of both proteins in Escherichia coli mirrored the findings from the GH4C1 cell clones in terms of solubility and immunological reactivity. The results of the present study indicate that, in IGHD II, somatotrophs continuously have to remove misfolded del(32–71)-hGH via the proteasomal degradation pathway, suggesting a mechanism that may result in chronic cellular stress.
Human GH protein consists of four -helices and contains two disulfide bridges. Isolated GH defici... more Human GH protein consists of four -helices and contains two disulfide bridges. Isolated GH deficiency type II (IGHD II) is mainly caused by heterozygous splice site mutations of GH-1 leading to the disruption of one disulfide bridge (Cys53-Cys165) and to the loss of amino acids (aa) 32-71, which comprise the complete loop between -helices 1 and 2. The mutant GH pro- tein exerts a dominant negative effect on wild-type (wt) GH secretion by unclear mechanisms. For study of the structure- functionrelationshipofGHmutantsconcerningthedominant negative effect, expression vectors harboring mutated GH cD- NAs were transiently cotransfected with a vector encoding wtGH (pwtGH) into GH4C1 cells. Plasmids encoding -galac- tosidase, luciferase, or IGF-binding protein-2 were cotrans- fected with pwtGH and either of the GH mutants. Compared with the control transfection with pwtGH, GH secretion was mildly decreased by coexpressing wtGH and different GH point mutants with isolated disruption of the disulfide bridge Cys53-Cys165. Similar results were observed with GH mutants deleted in aa 32-46 or 32-52. Deletion of more aa (32-53, 32-63, 32-69,32-71)ascendinglydecreasedGHsecretionandcontent in parallel with the increasing length of the deleted stretch. Aninhibitorydose-dependenteffectofdel32-69GHanddel32- 71GH on the activity/amount of coexpressed -galactosidase, luciferase, and IGF-binding protein-2 was found, whereas mRNA levels were unaffected. Hence, the extent of deletion played the major role in expression of the dominant negative effect. The inhibitory effect of GH mutants on heterologously expressed, non-GH proteins suggests that the dominant neg- ative effect is not limited to GH or to proteins of the regulated secretory pathway, but may depend on expression levels. (Endocrinology 146: 1411-1417, 2005)
The glycerol facilitator is known as the only example of a transport protein that catalyzes facil... more The glycerol facilitator is known as the only example of a transport protein that catalyzes facilitated diffusion across the Escherichia coli inner membrane. Here we show that the gene encoding the facilitator, glpF, is the first gene in an operon with glpK, encoding glycerol kinase, at 88 min of the E. coli chromosome. The operon is transcribed counterclockwise. We cloned the glpF gene, demonstrated that it complemented a chromosomal glycerol transport-minus mutation, and identified the gene product. The GlpF protein appeared in the membrane fraction of plasmid-bearing strains and had an apparent Mr of 25,000.
The ongoing emergence of human infections originating from wildlife highlights the need for bette... more The ongoing emergence of human infections originating from wildlife highlights the need for better knowledge of the microbial community in wildlife species where traditional diagnostic approaches are limited. Here we evaluate the microbial biota in healthy mule deer (Odocoileus hemionus) by analyses of lymph node meta-transcriptomes. cDNA libraries from five individuals and two pools of samples were prepared from retropharyngeal lymph node RNA enriched for polyadenylated RNA and sequenced using Roche-454 Life Sciences technology. Protein-coding and 16S ribosomal RNA (rRNA) sequences were taxonomically profiled using protein and rRNA specific databases. Representatives of all bacterial phyla were detected in the seven libraries based on protein-coding transcripts indicating that viable microbiota were present in lymph nodes. Residents of skin and rumen, and those ubiquitous in mule deer habitat dominated classifiable bacterial species. Based on detection of both rRNA and protein-codi...
Assessment of the microbial diversity residing in arthropod vectors of medical importance is cruc... more Assessment of the microbial diversity residing in arthropod vectors of medical importance is crucial for monitoring endemic infections, for surveillance of newly emerging zoonotic pathogens, and for unraveling the associated bacteria within its host. The tick Ixodes ricinus is recognized as the primary European vector of disease-causing bacteria in humans. Despite I. ricinus being of great public health relevance, its microbial communities remain largely unexplored to date. Here we evaluate the pathogen-load and the microbiome in single adult I. ricinus by using 454- and Illumina-based metagenomic approaches. Genomic DNA-derived sequences were taxonomically profiled using a computational approach based on the BWA algorithm, allowing for the identification of known tick-borne pathogens at the strain level and the putative tick core microbiome. Additionally, we assessed and compared the bacterial taxonomic profile in nymphal and adult I. ricinus pools collected from two distinct geogr...
The Tasmanian devil (Sarcophilus harrisii) is threatened with extinction because of a contagious ... more The Tasmanian devil (Sarcophilus harrisii) is threatened with extinction because of a contagious cancer known as Devil Facial Tumor Disease. The inability to mount an immune response and to reject these tumors might be caused by a lack of genetic diversity within a dwindling population. Here we report a whole-genome analysis of two animals originating from extreme northwest and southeast Tasmania, the maximal geographic spread, together with the genome from a tumor taken from one of them. A 3.3-Gb de novo assembly of the sequence data from two complementary next-generation sequencing platforms was used to identify 1 million polymorphic genomic positions, roughly one-quarter of the number observed between two genetically distant human genomes. Analysis of 14 complete mitochondrial genomes from current and museum specimens, as well as mitochondrial and nuclear SNP markers in 175 animals, suggests that the observed low genetic diversity in today's population preceded the Devil Faci...
The glpFK operon maps near minute 88 on the linkage map of Escherichia coli K-12 with glpF promot... more The glpFK operon maps near minute 88 on the linkage map of Escherichia coli K-12 with glpF promoter proximal. The glpF gene encodes a cytoplasmic membrane protein which facilitates the diffusion of glycerol into the cell. The glpK gene encodes glycerol kinase. In the present work, the nucleotide sequence of the 5'-end of the operon, including the control region, the glpF gene, and part of the glpK gene, was determined. The facilitator was predicted to contain 281 amino acids with a calculated molecular weight of 29,780. It is a highly hydrophobic protein with a minimum of six potential transmembrane alpha helices. The transcription start site for the glpFK operon was located 71 base pairs upstream from the proposed translation start codon for glpF. Preceding the transcription start site were sequences similar to the -10 and -35 consensus sequences for bacterial promoters. Binding sites for the cAMP-cAMP receptor protein (CRP) complex and the glp repressor were identified by DNas...
Overproduction of chimeric proteins containing the HMG2/1 peptide, which comprises the seven tran... more Overproduction of chimeric proteins containing the HMG2/1 peptide, which comprises the seven transmembrane domains of Saccharomyces cerevisiae 3-hydroxy-3-methylglutaryl-CoA reductase isozymes 1 and 2, has previously been observed to induce the proliferation of internal endoplasmic reticulum-like membranes. In order to exploit this amplified membrane surface area for the accommodation of heterologous microsomal proteins, we fused sequences coding for human cytochrome P4501A1 (CYP1A1) to sequences encoding the HMG2/1 peptide and expressed the hybrid genes in yeast. The heterologous hybrid proteins were targeted into strongly proliferated membranes, as shown by electron microscopic and immunofluorescent analysis. Fusion proteins comprising the whole CYP1A1 polypeptide (HMG2/1-CYP1A1) exhibited 7-ethoxyresorufin-O-deethylase activity, whereas fusion proteins lacking the N-terminal 56 amino acids of CYP1A1 (HMG2/1-delta CYP1A1) were inactive and appeared to be unable to incorporate protoheme. Similar amounts of heterologous protein were detected in cells expressing HMG2/1-CYP1A1, HMG2/1-delta CYP1A1 and CYP1A1, respectively. Replacement of the N-terminal membrane anchor domain of human NADPH-cytochrome P450 oxidoreductase by the HMG2/1 peptide also resulted in a functional fusion enzyme, which was able to interact with HMG2/1-CYP1A1 and the yeast endogenous P450 enzyme lanosterol-14 alpha-demethylase.
The Journal of Clinical Endocrinology & Metabolism, 2005
Noonan syndrome is frequently associated with an unclear disturbance of GH secretion. Half the in... more Noonan syndrome is frequently associated with an unclear disturbance of GH secretion. Half the individuals with Noonan syndrome carry a heterozygous mutation of the nonreceptor-type protein tyrosine phosphatase, Src homology region 2-domain phosphatase-2 (SHP-2), encoded by PTPN11, which has a role in GH receptor signaling. The objective of this study was to compare GH secretion and IGF-I/IGF-binding protein-3 (IGFBP-3) levels of the SHP-2 mutation-positive (mut+ group) vs. mutation-negative individuals (mut- group). All children presenting to us with short stature plus at least three typical anomalies of Noonan syndrome or pulmonic stenosis during the last 5 yr (n = 29; 10 females and 19 males) were recruited. Auxological data, dysmorphic features, and cardiac morphology were documented. Hormone levels were measured by RIA. All coding exons of PTPN11 were sequenced after PCR amplification. A prepubertal subgroup (n = 11) was treated with recombinant human GH (rhGH) to promote growth. Sequencing yielded 11 different PTPN11 missense mutations in 16 of the 29 patients (55% mut+). Pulmonic stenosis (81 vs. 15%; P = 0.0007) and septal defects (63 vs. 15%; P = 0.02) were more frequently found in the mut+ group, whereas minor anomalies, cryptorchidism, and learning disabilities were as frequent in the mut+ group as in the mut- group. The mut+ group was younger at presentation (mean +/- sd, 5.1 +/- 2.7 vs. 10.3 +/- 5.2 yr; P = 0.002), but not significantly shorter [-3.15 +/- 0.92 vs. -3.01 +/- 1.35 height sd score (SDS)]. IGF-I levels (-2.03 +/- 0.69 vs. -1.13 +/- 0.89 SDS; P = 0.005) and IGFBP-3 levels (-0.92 +/- 1.26 vs. 0.40 +/- 1.08 SDS; P = 0.006) were significantly lower in the mut+ group. In contrast, GH levels showed a tendency to be higher in the mut+ group during spontaneous secretion at night and arginine stimulation (P > or = 0.075, not significant). The mean change in height SDS after 1 yr of rhGH therapy (0.043 mg/kg.d) was +0.66 +/- 0.21 in the mut+ group (n = 8), but +1.26 +/- 0.36 in the mut- group (n = 3; P = 0.007). Our data suggest that SHP-2 mutations in Noonan syndrome cause mild GH resistance by a postreceptor signaling defect, which seems to be partially compensated for by elevated GH secretion. This defect may contribute to the short stature phenotype in children with SHP-2 mutations and their relatively poor response to rhGH.
Autosomal dominant isolated growth hormone deficiency type II (IGHD II) is mainly caused by splic... more Autosomal dominant isolated growth hormone deficiency type II (IGHD II) is mainly caused by splice site mutations of the GH-1 gene, leading to deletion of amino acids 32–71 of the human growth hormone (hGH). The severe hGH deficit in IGHD II suggests a dominant negative effect of the partially deleted del(32–71)-hGH on the production, storage or secretion of normal wild-type (wt)-hGH in somatotrophic cells of the pituitary. To shed more light on the cellular and molecular basis of IGHD II, we established and analysed diverse clones of the rat somatotrophic cell line GH4C1 stably expressing either wt-hGH, del(32–71)-hGH, or both proteins concomitantly. The cellular morphology of all transfected GH4C1 cell clones showed moderate differences to untransfected GH4C1 cells. On the molecular level, both cDNA-constructs induced transcription but, under normal culture conditions, only wt-hGH protein was found to be synthesised and secreted in readily detectable amounts. By contrast, only after inhibition of proteasomes did high amounts of del(32–71)-hGH show up. The solubility of del(32–71)-hGH in nondenaturing buffer was poor compared to wt-hGH, hinting at molecular aggregation, and several epitopes recognised by monoclonal hGH antibodies were not present on del(32–71)-hGH, confirming the assumption that del(32–71)-hGH must be severely misfolded. Expression of both proteins in Escherichia coli mirrored the findings from the GH4C1 cell clones in terms of solubility and immunological reactivity. The results of the present study indicate that, in IGHD II, somatotrophs continuously have to remove misfolded del(32–71)-hGH via the proteasomal degradation pathway, suggesting a mechanism that may result in chronic cellular stress.
Human GH protein consists of four -helices and contains two disulfide bridges. Isolated GH defici... more Human GH protein consists of four -helices and contains two disulfide bridges. Isolated GH deficiency type II (IGHD II) is mainly caused by heterozygous splice site mutations of GH-1 leading to the disruption of one disulfide bridge (Cys53-Cys165) and to the loss of amino acids (aa) 32-71, which comprise the complete loop between -helices 1 and 2. The mutant GH pro- tein exerts a dominant negative effect on wild-type (wt) GH secretion by unclear mechanisms. For study of the structure- functionrelationshipofGHmutantsconcerningthedominant negative effect, expression vectors harboring mutated GH cD- NAs were transiently cotransfected with a vector encoding wtGH (pwtGH) into GH4C1 cells. Plasmids encoding -galac- tosidase, luciferase, or IGF-binding protein-2 were cotrans- fected with pwtGH and either of the GH mutants. Compared with the control transfection with pwtGH, GH secretion was mildly decreased by coexpressing wtGH and different GH point mutants with isolated disruption of the disulfide bridge Cys53-Cys165. Similar results were observed with GH mutants deleted in aa 32-46 or 32-52. Deletion of more aa (32-53, 32-63, 32-69,32-71)ascendinglydecreasedGHsecretionandcontent in parallel with the increasing length of the deleted stretch. Aninhibitorydose-dependenteffectofdel32-69GHanddel32- 71GH on the activity/amount of coexpressed -galactosidase, luciferase, and IGF-binding protein-2 was found, whereas mRNA levels were unaffected. Hence, the extent of deletion played the major role in expression of the dominant negative effect. The inhibitory effect of GH mutants on heterologously expressed, non-GH proteins suggests that the dominant neg- ative effect is not limited to GH or to proteins of the regulated secretory pathway, but may depend on expression levels. (Endocrinology 146: 1411-1417, 2005)
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Papers by Nicola Wittekindt