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PubMed2006

Reconstruction of a swine SLA-I protein complex and determination of binding nonameric peptides derived from the foot-and-mouth disease virus.

No experimental system to date is available to identify viral T-cell epitopes in swine. In order to reconstruct the system for identification of short antigenic peptides, the swine SLA-2 gene was linked to the beta(2)m gene via (G4S)3, a linker encoding a 15-amino acid glycine-rich sequence (G4S)3, using splicing overlap extension-PCR (SOE-PCR). The maltose binding protein (MBP)-SLA-2-(G4S)3-beta(2)m fusion protein was expressed and purified in a pMAL-p2X/Escherichia coli TB1 system. The purified MBP-SLA-2-(G4S)3-beta(2)m protein was cleaved by factor Xa protease, and further purified by DEAE-Sepharose chromatography. The conformation of the SLA-2-(G4S)3-beta(2)m protein was determined by circular dichroism (CD) spectrum. In addition, the refolded SLA-2-(G4S)3-beta(2)m protein was used to bind three nonameric peptides derived from the foot-and-mouth disease virus (FMDV) O subtype VP1. The SLA-2-(G4S)3-beta(2)m-associated peptides were detected by mass spectrometry. The molecular weights and amino acid sequences of the peptides were confirmed by primary and secondary spectra, respectively. The results indicate that the SLA-2-(G4S)3-beta(2)m was 41.6kDa, and its alpha-helix, beta-sheet, turn, and random coil by CD estimation were 78 aa, 149 aa, 67 aa, and 93 aa, respectively. SLA-2-(G4S)3-beta(2)m protein was able to bind the nonameric peptides derived from the FMDV VP1 region: 26-34 (RRQHTDVSF) and 157-165 (RTLPTSFNY). The experimental system demonstrated that the reconstructed SLA-2-(G4S)3-beta(2)m protein complex can be used to identify nonameric peptides, including T-cell epitopes in swine.

Animals↗
PubMed2005

Molecular characterization of the major outer membrane protein of Haemophilus somnus.

The major outer membrane protein (MOMP) of Haemophilus somnus shows antigenic and molecular mass diversity that forms the basis of a preliminary grouping system for H. somnus strains. In this study, the gene encoding MOMP of H. somnus strain 8025 was cloned in three overlapping fragments by PCR techniques, and then sequenced. The gene consists of a 1164-bp open reading frame encoding a deduced 380-amino acid protein with a 19-amino acid signal sequence, giving a mature protein with a calculated molecular mass of 39,913 Da. Significant homology was found between MOMP and porin protein sequences of bacteria in Pasteurellaceae species. When expressed in Escherichia coli, the protein from the MOMP gene directed by the T7 promoter was identical in size (approximately 40 kDa) to native MOMP and reacted with MOMP-specific antibodies. Comparisons of the MOMP gene sequences from six unrelated strains of H. somnus to that of strain 8025 revealed that the genes of three MOMP type 1 strains were highly conserved with that of strain 8025 in length and sequence. However, two MOMP type 3c strains and one MOMP type 3a strain differed markedly from the MOMP of strain 8025 in their 3'-terminal halves. Their deduced MOMP amino acid sequences differed in sequence (3c, 80.5 and 82.7% identity; 3a, 62.4% identity) and in length (3c, 384 and 376; 3a, 316), indicating that the molecular differences are the basis of antigenicity and molecular mass differences of H. somnus MOMP. In the predicted MOMP secondary structure, the variable sequences primarily mapped to putative surface-exposed loops, and a variable and surface-exposed epitope of MOMP-specific antibody was identified in the seventh-largest loop. These findings are useful for understanding the structural and immunological characteristics of H. somnus.

Amino Acid Sequence↗
PubMed2004

Mechanisms of inactivation of hepatitis A virus in water by chlorine dioxide.

In this study, to elucidate the mechanisms of inactivation of hepatitis A virus (HAV) by chlorine dioxide, cell culture, enzyme-linked immunosorbent assay (ELISA), and long-overlapping RT-PCR were used to detect the infectivity, antigenicity, and entire genome of HAV before and after disinfection. The results revealed the complete inactivation of infectivity after a 10-min exposure to 7.5mg of chlorine dioxide per liter; and the highest level of sensitivity in the 5'non-translated regions (5'NTR) (the sequence from bp 1 to 671), inactivation of which took as much time as the inactivation of infectivity of HAV by chlorine dioxide; the complete destruction of antigenicity after a 10-min exposure to 7.5mg of chlorine dioxide per liter. It is suggested that the inactivation mechanism of HAV by chlorine dioxide was due to the loss of the 5'NTR and/or destruction of the antigenicity, which is not similar to that of chlorine (Appl Environ Microbiol 68: 4951).

Chlorine Compounds↗
PubMed2002

Confirmation of an outbreak of parvovirus B19 in a primary school using IgM ELISA and PCR on thumb prick blood samples.

BACKGROUND: Although parvovirus infections are usually benign and self-limiting, it is important to confirm the diagnosis in a public health setting which might involve pregnant women and in which an outbreak could lead to medical consequences. In these situations, microbiological confirmation by thumb prick is a relatively low-invasive method that is simple to carry out. OBJECTIVE AND STUDY DESIGN: Because relatively small blood volumes are obtained in thumb prick blood samples, we compared the results of two different techniques during an outbreak of erythema infectiosum: the usual serological detection of IgM antibodies (ELISA) versus PCR-based detection of viral DNA. RESULTS: In a school-based outbreak, 39 cases (33 schoolchildren, three parents, three pre-schoolers) were registered over a period of 11 weeks. Sera were obtained from 23 of the school cases and two of the three parent cases. Of all thumb prick serum samples, 65% (15/23) tested positive or borderline positive for parvovirus IgM with ELISA, while 70% (16/23) tested positive or borderline positive with PCR. Although the overlap between the two tests was large (11 samples tested positive or borderline positive in both), a substantial number of samples showed contradictory results (nine samples). CONCLUSION: The overall picture of 37 clinical cases of erythema infectiosum and two adult cases with arthritis, linked to a primary school, fits in well with positive diagnostic results by either technique for parvovirus B19, convincingly demonstrating an outbreak of fifth disease. The considerable number of discrepancies in sample results demonstrates that maximum sensitivity of parvovirus testing would require both tests to be performed.

Antibodies, Viral↗
PubMed2006

[Expression and secretion of human bone morphogenetic protein-7 in Pichia pastoris].

The synonymous codons are used in a highly non-random manner in hosts of widely divergent species, which is termed "codon usage bias". Several reports suggest that codon usage bias sometimes frustrate attempts to express high levels of exogenous genes. In this study, we attempted to express mature peptide of human bone morphogenetic protein-7(hBMP7), with optimized codons in P. pastoris expression system. Three low-usage ARG codons (CGG or CGA) of gene fragment coding the mature peptide of hBMP7 have been successfully converted into P. pastoris-preferred ARG codons (AGA) by overlap extension PCR-based multiple-site-directed mutagenesis for a high level expression of hBMP7 mature peptide. The present results showed that the production level (25.45 mg/L) of codon-optimized hbmp7 had a remarkably improvement of 4.6-fold relative to that (5.5 mg/L) of non-codon-optimized hbmp7. Furthermore, a strain haboring multi-copy of codon-optimized hbmp7 expression cassette was screened, and showed a increased level of expression with 2-fold more potent than the single-copy one. The recombinant hBMP7 mature peptide were produced as a 18 kD monomer proteins, and were easily purified from culture supernatants by using ion-exchange chromatography. Functional assay demonstrated that rhBMP7 could induce ectopic cartilage formation, although its inductive ability was much less active than CHO cell-derived hBMP7.

Animals↗
PubMed2005

Complete sequence and structure of the mitochondrial genome of the human tapeworm, Taenia asiatica (Platyhelminthes; Cestoda).

The complete Taenia asiatica mitochondrial genome was amplified by long extension polymerase chain reaction (long PCR) to yield overlapping fragments that were then completely sequenced. The whole mitochondrial genome was 13 703 bp long and contained 12 protein-encoding, 2 ribosomal RNA (small and large subunits), 22 transfer RNA genes and a short non-coding region. Thus, its gene contents are like those typically found in metazoan animal mitochondrial genomes (apart from the absence of atp8). All the genes were transcribed from the same strand. The 3' end 34 bp region of nad4L overlapped with the 5' end portion of nad4. The tRNA genes were 61-69 bp long, and the secondary structures of 18 tRNAs had typical clover-leaf shapes with paired DHU arms. However, trnC, trnS1, trnS2 and trnR had unpaired DHU arms that were 7-12 bp in length. The tRNAs that transferred serine lacked a DHU arm, as is also observed in a number of parasitic platyhelminths and metazoans. However, the trematode trnRs have paired DHU arms. The T. asiatica mtDNA non-coding region was like that in other cestodes since it was composed of a short non-coding region of 72 nucleotides and a long non-coding region of 176 nucleotides separated by a trnL1/, trnS2/, trnL2/, trnR/, nad5 gene cluster. The sequences of the cox1 genes between T. asiatica and T. saginata differ by 4.6%, while the T. asiatica cob gene differs by 4.1% and 12.9% from the cob genes of T. saginata and T. solium, respectively. In conclusion, the T. asiatica mitocondrial genome should provide a resource for comparative mitochondrial genomics and systematic studies of parasitic cestodes.

Animals↗
PubMed2006

Codon optimization of Candida rugosa lip1 gene for improving expression in Pichia pastoris and biochemical characterization of the purified recombinant LIP1 lipase.

An important industrial enzyme, Candida rugosa lipase (CRL) possesses several different isoforms encoded by the lip gene family (lip1-lip7), in which the recombinant LIP1 is the major form of the CRL multigene family. Previously, 19 of the nonuniversal serine codons (CTG) of the lip1 gene hav been successfully converted into universal serine codons (TCT) by overlap extension PCR-based multiple-site-directed mutagenesis to express an active recombinant LIP1 in the yeast Pichia pastoris. To improve the expression efficiency of recombinant LIP1 in P. pastoris, a regional synthetic gene fragment of lip1 near the 5' end of a transcript has been constructed to match P. pastoris-preferred codon usage for simple scale-up fermentation. The present results show that the production level (152 mg/L) of coLIP1 (codon-optimized LIP1) has an overall improvement of 4.6-fold relative to that (33 mg/L) of non-codon-optimized LIP1 with only half the cultivation time of P. pastoris. This finding demonstrates that the regional codon optimization the lip1 gene fragment at the 5' end can greatly increase the expression level of recombinant LIP1 in the P. pastoris system. More distinct biochemical properties of the purified recombinant LIP1 for further industrial applications are also determined and discussed in detail.

Base Sequence↗
PubMed2004

Sp17 gene expression in myeloma cells is regulated by promoter methylation.

The mechanisms underlying sperm protein 17 (Sp17) gene expression in myeloma cells remained unclear. Using reverse transcription-polymerase chain reaction (RT-PCR), Sp17 transcripts were detected in ARK-B, ARP-1, RPMI-8226 and KMS-11 but not in H929, IM-9, MM1-R and U266 cells. Using a panel of primer pairs in methylation-sensitive PCR to amplify overlapping gene segments, our screening studies showed that the HpaII sites at -359 and -350 are involved in the regulation of Sp17 gene expression. To confirm the differences in methylation status between Sp17-positive and Sp17-negative cell lines, KMS-11 cells (Sp17-positive) and IM-9 cells (Sp17-negative) were subjected to the more accurate method of bisulphite conversion. KMS-11 cells were more hypomethylated at these HpaII sites of exon 1 compared to IM-9 cells, indicating the association of hypomethylated promoter with Sp17 gene expression. In addition, the level of methylation at other CpG sites within the promoter sequence was also higher in IM-9 than KMS-11. Exon 1 was cloned into a reporter vector, pCAT*3 Enhancer. Chloramphenicol acetyl transferase (CAT) activity was restored in cells transfected with the recombinant plasmid, indicating the promoter function of exon 1. Exposure of Sp17-negative cell lines to the hypomethylating agent, 5-azacytidine, resulted in the upregulation of Sp17 gene expression. Our results therefore provide evidence for the regulation of Sp17 gene expression by promoter methylation.

Antigens, Surface↗
PubMed2002

NATH, a novel gene overexpressed in papillary thyroid carcinomas.

In this study a replica cDNA screening (RCS) approach to identify genes differentially expressed in papillary thyroid carcinomas (PTC) was used, as compared to non-neoplastic thyroid tissues. RCS is based on hybridization of radioactively labeled cDNA probes made from the biopsies to replica membranes with 15 000 clones from a PTC cDNA library. Among the genes overexpressed in PTC, and especially in clinically aggressive tumors with histologic evidence of poorly differentiated or undifferentiated areas, a novel gene named NATH was found. NATH has two mRNA species, 4.6 and 5.8 kb, both harboring the same open reading frame encoding a putative protein of 866 amino acids. The NATH protein is homologous to yeast N-acetyltransferase (NAT)1 and to mouse NARG1 (mNAT1) and contains four tetratricopeptide repeat (TPR) domains, suggesting that NATH may be part of a multiprotein complex. Overlapping RT-PCR fragments from several PTC biopsies confirmed the NATH mRNA sequence. Northern blots, semiquantitative RT-PCR experiments, TaqMan real-time RT-PCR experiments, and in situ hybridization verified the overexpression of NATH mRNA localized to tumor cells in PTC biopsies. NATH was expressed at a low level in most human adult tissues, including the normal thyroid gland. Increased NATH expression was seen especially in a Burkitt lymphoma cell line and in adult human testis. Recombinant in vitro expression showed that NATH protein was located mainly in the cytoplasm, and was present as a single protein band of the expected 105 kDa molecular weight. Heterologous expression of NATH in the papillary carcinoma cell line (NPA) and 293 cells did not alter the cellular proliferation rate. The biological function of NATH remains to be elucidated, but the overexpression in classic PTC and especially in poorly differentiated or undifferentiated components may indicate a function in the progression of papillary thyroid carcinomas.

Acetyltransferases↗
PubMed2002

Expression of 5-lipoxygenase in pulmonary artery endothelial cells.

Increased expression of 5-lipoxygenase (5LO) in pulmonary artery endothelial cells (PAECs) has been observed in disease states such as pulmonary hypertension and allergen challenge. To understand the function of endothelial 5LO, we examined the expression of this enzyme in normally cultured human PAECs and its characteristics when overexpressed. A small amount of 5LO message and protein was detected by reverse-transcriptase-mediated PCR (RT-PCR) and Western blotting in PAECs. Sequencing of the RT-PCR products that overlapped the entire coding region of 5LO mRNA indicated that the sequence of PAEC 5LO was identical with that of leucocyte 5LO. Incubation of the PAECs with A23187 and arachidonic acid led to a small production of 5-hydroxyeicosatetraenoic acid (5-HETE) (46-98 pmol/4x10(6) cells) but no leukotrienes. Overexpression of 5LO in PAECs by adenovirus-mediated gene transfer revealed that the enzyme was localized in the nucleus. Incubation of the transduced cells with A23187 (5 microM) caused the production of both 5LO products and downstream leukotrienes. The proportions of the produced leukotriene A(4) (LTA(4)) hydrolates (sum of 6-trans-LTB(4) and 12-epi-6-trans-LTB(4)), LTB(4) and cysteinyl leukotriene were approx. 17:14:10. cGMP production in the 5LO-transduced PAECs was decreased by 33+/-14% on stimulation with A23187. These results show that cultured PAECs express a minimal amount of 5LO, which can generate some 5-HETE, but not leukotrienes. However, increased expression of 5LO in PAECs can lead to the production of all downstream leukotrienes, which could potentially cause endothelial dysfunction in the pulmonary vasculature.

Adenoviridae↗
PubMed2002

Multiple mutagenesis of non-universal serine codons of the Candida rugosa LIP2 gene and biochemical characterization of purified recombinant LIP2 lipase overexpressed in Pichia pastoris.

The 17 non-universal serine codons (CTG) in the Candida rugosa LIP2 gene have been converted into universal serine codons (TCT) by overlap extension PCR-based multiple site-directed mutagenesis. An active recombinant LIP2 lipase was overexpressed in Pichia pastoris and secreted into the culture medium. The recombinant LIP2 showed distinguishing catalytic activities when compared with recombinant LIP4 and commercial C. rugosa lipase. The purified enzyme showed optimum activity at pH 7 and a broad temperature optimum in the range 30-50 degrees C. The enzyme retained 80% of residual activity after being heated at 70 degrees C for 10 min. Recombinant LIP2 demonstrated high esterase activity towards long-chain (C12-C16) p-nitrophenyl esters. Tributyrin was the preferred substrate among all triacylglycerols tested for lipolysis. Among cholesteryl esters, LIP2 showed highest lipolytic activity towards cholesteryl laurate. The esterification of myristic acid with alcohols of various chain lengths showed that the long-chain n-octadecanol (C18) was the preferred substrate. In contrast, the esterification of n-propanol with fatty acids of various chain lengths showed that the short-chain butyric acid was the best substrate. From comparative modelling analysis, it appears that several amino acid substitutions resulting in greater hydrophobicity in the substrate-binding site might play an important role in the substrate specificity of LIP2.

Base Sequence↗
PubMed1996

Structure of the thyrotrophin-releasing hormone receptor in human pituitary adenomas.

BACKGROUND AND OBJECTIVE: TRH acts on specific G-protein coupled receptors sited in cells of the anterior pituitary gland. Pituitary tumours expressing either TSH, PRL or GH may respond to TRH by enhanced, blunted or paradoxical hormone release. Non-functioning pituitary tumours may also show abnormal responses to TRH. Little is understood of the mechanisms regulating inappropriate hormone release in these tumours. Activating or inactivating mutations found in G-protein coupled receptors have been implicated in human pathological conditions. Mutations in the G-protein coupled TRH receptor might be involved in the aetiology of pituitary adenomas resulting in aberrant hormone secretion. We therefore screened samples of pituitary adenomas for the presence of somatic mutations in the TRH receptor gene. PATIENTS: Pituitary adenoma tissue samples were obtained at surgery from 50 patients with pituitary adenoma (17 acromegaly, 15 prolactinoma, 11 TSH-secreting and 7 non-functioning adenoma) along with blood samples to provide lymphocyte DNA as control sequence. METHODS: Genomic DNA was extracted from adenoma and lymphocyte samples and the entire coding region of the TRH receptor was amplified using 5 overlapping pairs of PCR primers. The PCR products were analysed for mutations by non-denaturing polyacrylamide gel electrophoresis which reveals single-strand conformational polymorphisms (SSCP) as a mobility shift in product migration. Wild-type and mutant TRH receptor cDNA were similarly analysed to confirm the sensitivity of the method. Additionally, PCR products were ligated into a PCR cloning vector and DNA sequencing carried out to confirm the findings of SSCP analysis. RESULTS: The human TRH receptor retained normal wild-type sequence in the large group of TSH secreting, PRL secreting, GH secreting and non-functioning pituitary adenomas investigated in this study. CONCLUSION: Our observations suggest that the TRH receptor structure is normal in TSH secreting, PRL secreting, GH secreting and non-functioning pituitary adenomas. It is therefore unlikely that the TRH receptor is involved in the pathology associated with the types of pituitary adenomas investigated in this study. It is possible that some other component of the pathway controlling TRH-signalling events may be implicated in pituitary tumorigenesis.

Acromegaly↗
PubMed2000

Structural analysis of the 11beta-hydroxysteroid dehydrogenase type 2 gene in end-stage renal disease.

BACKGROUND: Mutations in the 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) gene cause a rare form of low-renin hypertension leading to end-stage renal disease (ESRD) in some affected subjects. To date, no search for mutations in the HSD11B2 gene was performed in a large population to obtain an estimate its prevalence. METHODS: The HSD11B2 gene was analyzed in 587 subjects, including 260 ESRD patients (either dialysis or transplanted) for mutations in the exons 2 through 5 and corresponding intronic regions by polymerase chain reaction (PCR) using appropriate overlapping primers, gel analysis by single strand conformational polymorphism (SSCP), and sequencing of identified migration variants. RESULTS: The prevalence of single-nucleotide polymorphisms (SNPs) in ESRD patients and controls was 26%. The following genetic variants were found among all subjects investigated: exon 2 T442G (Leu148/Val, N = 70) and C470A (Thr156/Thr, N = 67), exon 3 G534A (Glu178/Glu, N = 69), and exon 5 C1274T (Asp388/Asp, N = 2). Four SNPs were identified in intron 4 only. In the control population, the prevalence of the variants Leu148 and Thr156 was 14% each. Glu178 was 11%, while no variants were found in exon 5. In ESRD patients, the prevalence of the variant Leu148 was 9%, and Thr156 was 8%. Glu178 was 13%, while the Asp388 variant was 0.7%. In patients with a short duration between the time of diagnosis of the renal disease and the onset of ESRD, the prevalence of the Leu148 and Glu178 variants was higher than in subjects with slowly progressing renal disease. The 11betaHSD2 activity of all of these SNPs is predictably unaltered. CONCLUSIONS: There is a high prevalence of SNPs of the HSD11B2 gene, without causing exonic mutations generating a 11betaHSD2 enzyme with altered activity. Based on statistical analyses, the frequency of homozygosity for mutated alleles of the HSD11B2 gene can be derived as <1/250,000 when a Caucasian population is considered.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗
PubMed2004

DNM1DN: a new class of paralogous genomic segments (duplicons) with highly conserved copies on chromosomes Y and 15.

Screening a testis cDNA selection library for Y-linked genes yielded 79 cDNAs. Of these, 9 matched the 3' region of the dynamin 1 gene (DNM1) on chromosome 9q34 with >90% identity. Fluoresence in situ hybridisation and PCR amplification were used to localise a large number of DNM1-like sequences to human chromosomes 15 and Y. PCR amplification of overlapping Y-linked YACs allowed a more accurate mapping of the Y-linked DNM1-like cDNAs to a euchromatic locus in close proximity to heterochromatin at Yq11.23. A search of the genome database identified 64 highly homologous copies of the DNM1 fragment. Most of these copies were localised to chromosomes 15 and Y, but others mapped to chromosomes 5, 8, 10, 12, 19 and 22. These sequences exhibit all the major features of a duplicon and have been designated DNM1DN (DNM1 duplicon). Evolutionary studies using fluorescence in situ hybridisation indicate that transposition of the DNM1DN sequence to chromosome 15 took place earlier in primate evolution than the transposition to the Y chromosome. The translocation to the Y took place at a time following the divergence of a common ancestor from gorilla, approximately 4-7 million years ago.

Animals↗
PubMed2006

Construction and expression of a single chain Fv fragment against pharmacologically active paeoniflorin in Escherichia coli, and its potential use in an enzyme-linked immunosorbent assay.

A recombinant single chain variable-fragment (scFv) antibody against paeoniflorin (PF) was produced using the hybridoma cell line C31B9. Variable regions of heavy (V (H)) and light (V (L)) chain antibody genes were directly cloned from cDNA resources of hybridoma C31B9 and assembled using splicing by overlap extension (SOE)-PCR using a (Gly (4)Ser) (3) linker DNA. The constructed scFv genes were cloned into pET28a vectors for the generation of recombinant proteins in Escherichia coli. Most of the recombinant proteins were expressed in inclusion bodies. The yield of refolded and purified scFv was 1.89 mg per 100 mL of cell culture. The recombinant scFv displayed cross-reactivity as its mother monoclonal antibody (MAb) C31B9. Therefore, the newly expressed scFv protein was applied to quantitative ELISA to determine the total paeoniflorin (PF) and albiflorin (Alb) concentrations in peony root samples. Using PF as a standard compound, the full linear range of the assay was extended from 0.78 to 25 microg/mL. The results obtained by ELISA employing both the recombinant scFv and the original MAbC31B9 showed a reasonably good agreement with each other.

Antibodies, Monoclonal↗
PubMed1993

Purification, characterization, and cDNA cloning of an NADPH-cytochrome P450 reductase from mung bean.

We report here the isolation and deduced amino acid sequence of the flavoprotein, NADPH-cytochrome P450 (cytochrome c) reductase (EC 1.6.2.4), associated with the microsomal fraction of etiolated mung bean seedlings (Vigna radiata var. Berken). An 1150-fold purification of the plant reductase was achieved, and SDS/PAGE showed a predominant protein band with an apparent molecular mass of approximately 82 kDa. The purified plant NADPH-P450 reductase gave a positive reaction as a glycoprotein, exhibited a typical flavoprotein visible absorbance spectrum, and contained almost equimolar quantities of FAD and FMN per mole of enzyme. Specific antibodies revealed the presence of unique epitopes distinguishing the plant and mammalian flavoproteins as demonstrated by Western blot analyses and inhibition studies. Peptide fragments from the purified plant NADPH-P450 reductase were sequenced, and degenerate primers were used in PCR amplification reactions. Overlapping cDNA clones were sequenced, and the deduced amino acid sequence of the mung bean NADPH-P450 reductase was compared with equivalent enzymes from mammalian species. Although common flavin and NADPH-binding sites are recognizable, there is only approximately 38% amino acid sequence identity. Surprisingly, the purified mung bean NADPH-P450 reductase can substitute for purified rat NADPH-P450 reductase in the reconstitution of the mammalian P450-catalyzed 17 alpha-hydroxylation of pregnenolone or progesterone.

Amino Acid Sequence↗
PubMed1994

Canine X chromosome-linked hereditary nephritis: a genetic model for human X-linked hereditary nephritis resulting from a single base mutation in the gene encoding the alpha 5 chain of collagen type IV.

Many families with X-chromosome linked hereditary nephritis (HN) have mutations in the gene on the X chromosome that codes for the alpha 5 chain of collagen type IV. Canine X-linked HN is an animal model for human X-linked HN. To study the alpha 5(IV) gene in this model, we used the nucleotide sequence published for the human alpha 5(IV) cDNA to construct sets of primers covering approximately 95% of the complete cDNA. cDNA from both affected and normal dog kidneys was amplified by PCR in nine overlapping regions. The nucleotide sequence encoding the noncollagenous domain NC1 hybridized to the human X chromosome and was 93% identical at the DNA level and 97% identical at the protein level to the human alpha 5(IV) NC1 domain, confirming that the canine alpha 5(IV) cDNA had been amplified. Sequence analysis of the alpha 5(IV) cDNA detected a single nucleotide substitution, G-->T, in affected dogs, changing a codon for a conserved glycine residue (GGA) to a stop codon (TGA). When genomic DNA was amplified, the same abnormality was found in exon 35. Using the canine NC1 domain cDNA as a probe for Northern analysis, two transcripts of approximately 8.6 kb and approximately 6.7 kb were identified in both normal and affected male dog kidney RNA. However, the abundance of both transcripts was decreased by a factor of approximately 10 in the affected dog. These results establish at the molecular level that canine X-linked HN is a model for human X-linked HN. This model provides an opportunity to determine the efficacy of new therapies and to investigate the role of the alpha 5(IV) chain in type IV collagen assembly.

Amino Acid Sequence↗
PubMed2003

Formation of disulfide bonds and homodimers of the major cat allergen Fel d 1 equivalent to the natural allergen by expression in Escherichia coli.

Dander from the domestic cat (Felis domesticus) is one of the most common causes of IgE-mediated allergy. Attempts to produce tetrameric folded major allergen Fel d 1 by recombinant methods with structural features similar to the natural allergen have been only partially successful. In this study, a recombinant folded Fel d 1 with molecular and biological properties similar to the natural counterpart was produced. A synthetic gene coding for direct fusion of the Fel d 1 chain 2 N-terminally to chain 1 was constructed by overlapping oligonucleotides in PCR. Escherichia coli expression resulted in a non-covalently associated homodimer with an apparent molecular mass of 30 kDa defined by size exclusion chromatography. Furthermore, each 19,177-Da subunit displayed a disulfide pattern identical to that found in the natural Fel d 1, i.e. Cys3(1) Cys73(2), Cys44(1)-Cys48(2), Cys70(1)-Cys7(2), as determined by electrospray mass spectrometry after tryptic digestion. Circular dichroism analysis showed identical folds of natural and recombinant Fel d 1. Furthermore, recombinant Fel d l reacted specifically with serum IgE, inducing expression of CD203c on basophils and lymphoproliferative responses in cat-allergic patients. The results show that the overall fold and immunological properties of the recombinant Fel d 1 are very similar to those of natural Fel d 1. Moreover, the recombinant Fel d 1 construct provides a tool for defining the three-dimensional structure of Fel d 1 and represents a reagent for diagnosis and allergen-specific immunotherapy of cat allergy.

Allergens↗