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J W Martens

Publications and source records attributed to J W Martens.

15 recordsLinked to original sources
PubMed2001

NF-1C, Sp1, and Sp3 are essential for transcription of the human gene for P450c17 (steroid 17alpha-hydroxylase/17,20 lyase) in human adrenal NCI-H295A cells.

Cytochrome P450c17 catalyzes steroid 17alpha-hydroxylase and 17,20 lyase activities, which are required for the biosynthesis of cortisol and sex steroids. Human P450c17 is expressed in a cAMP-responsive, cell-specific, developmentally programmed fashion, but little is known about its transcriptional regulation. Expression of deletion mutants of up to 2,500 bp of human 5'-flanking DNA in human adrenal NCI-H295A cells indicated that most regulatory activity was confined to the first 227 bp. Deoxyribonuclease I footprinting of the proximal promoter identified the TATA box, an steroidogenic factor-1 site, and three previously uncharacterized sites at -107/85, at -178/-152, and at -220/-185. EMSAs and methylation interference assays suggested that the -107/-85 site and the -178/-152 site bind members of the NF-1 (nuclear factor-1) family of transcription factors. An NF-1 consensus sequence generated similar DNA/protein complexes, and antibodies against NF-1C2/CTF2 supershifted the complexes formed by the -107/-85 site, the -178/-152 site, and the NF-1 consensus site. Western blots of nuclear extracts from NCI-H295A cells probed with this NF-1 antiserum identified two NF-1 isoforms between 50 and 55 kDa. The presence of NF-1C2 (CTF2) and CTF5 in NCI-H295A cells was demonstrated by RT-PCR and sequencing. Mutation of both the -107/-85 and the -178/-152 NF-1 sites reduced basal transcription by half. Supershift assays showed that the ubiquitous proteins Sp1 and Sp3 both bind to the -227/-184 region, and that mutation of their binding sites reduced transcription by 75%. Mutation of the Sp1/Sp3 site plus the two NF-1 sites eliminated almost all detectable transcription. Thus, Sp1 and Sp3 binding to the -227/-184 site and NF-1C proteins binding to the -107/-85 and the -178/-152 sites are crucial for adrenal transcription of the human gene for P450c17.

Adrenal Gland Neoplasms↗
PubMed2000

Enzymatic activities of P450c17 stably expressed in fibroblasts from patients with the polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder affecting approximately 5-10% of women of reproductive age. The clinical features of PCOS include oligo/anovulation, hyperandrogenemia, and hyperinsulinemia. Because P450c17 is the single enzyme catalyzing both 17alpha-hydroxylase and 17,20-lyase activities in the ovary and adrenal, some have suggested that defects in P450c17 may cause the hyperandrogenism of PCOS. Previous studies have shown that serine hyperphosphorylation of P450c17 increases the enzyme's 17,20-lyase activity, thereby favoring androgen production, and that serine phosphorylation of the insulin receptor beta-chain (IR-beta) inhibits IR-beta tyrosine phosphorylation, causing insulin resistance in vitro. We previously suggested that a gain of function mutation in a single serine kinase might cause the hyperandrogenism and insulin resistance observed in PCOS patients by excessive phosphorylation of both P450c17 and IR-beta. To test this hypothesis, we obtained fibroblasts from nine previously studied patients: three controls, three PCOS patients with normal levels of IR-beta serine phosphorylation, and three PCOS patients with increased levels of IR-beta serine phosphorylation. Initial studies showed that such skin fibroblasts could not be transfected effectively by calcium phosphate, diethylaminoethyl-dextran, lipofection or adenovirus procedures. Therefore, we employed a retroviral infection system to stably express human P450c17 in the primary cultures of fibroblast cells from the PCOS patients and controls and measured the resulting 17alpha-hydroxylase and 17,20-lyase activity. The cells were analyzed in a blinded fashion until the study was complete. The 17alpha-hydroxylase and 17,20-lyase activities in each cell line correlated well with the amount of P450c17 protein expressed, but there was no correlation between either enzymatic activity (or their ratio) with the clinical phenotype of the cells' donors even when results were corrected for the number of P450c17 complementary DNA inserts per cell line. Overnight incubation with 1 micromol/L insulin also did not affect enzymatic activity. Thus, we were unable to find evidence for the hypothesis that in PCOS a single abnormal kinase hyperphosphorylates both IR-beta, causing insulin resistance, and P450c17, causing hyperandrogenism. However, because fibroblasts do not normally express either P450c17 or the accessory proteins needed for its optimal activity, these results cannot exclude a role for serine phosphorylation in the hyperandrogenism and insulin resistance of PCOS.

Adolescent↗
PubMed1999

A limited repertoire of mutations of the luteinizing hormone (LH) receptor gene in familial and sporadic patients with male LH-independent precocious puberty.

Herein, we report mutation analysis of the LH receptor gene in 17 males with LH-independent precocious puberty, of which 8 were familial and 9 had a negative family history. A total of 7 different mutations (all previously reported) were detected in 12 patients. Among 10 European familial male-limited precocious puberty (FMPP) patients who had a LH receptor gene mutation, none had the Asp578Gly mutation, which is responsible for the vast majority of cases in the U.S. The restricted number of activating mutations of the LH receptor observed in this and other studies of FMPP strongly suggests that an activating phenotype is associated with very specific sites in the receptor protein. Clinical follow-up of the 5 patients who did not have LH receptor mutations shows that such cases most likely do not have true FMPP. LH receptor mutation analysis provides a sensitive tool for distinguishing true FMPP from other causes of early-onset LH-independent puberty in males.

Amino Acid Sequence↗
PubMed1998

Activating and inactivating mutations in LH receptors.

The glycoprotein hormones luteinizing hormone (LH) and human chorionic gonadotropin (hCG), both of which act through the LH receptor, play an important role in the regulation of both male and female gonadal function. Testicular androgen production by the Leydig cells is almost entirely dependent on LH/hCG action, while in the ovary LH triggers both oestrogen and progestin secretion and ovulation of the Graaffian follicles. In this review we discuss the various mutations that have been found in the LH receptor gene in the human and the lessons that can be learned from the phenotypes of the carriers of these mutations.

Female↗
PubMed1998

Distinct gene arrangement in the Buzura suppressaria single-nucleocapsid nucleopolyhedrovirus genome.

The genome organization of the Buzura suppressaria single-nucleocapsid nucleopolyhedrovirus (BusuNPV) was largely elucidated and compared to those of other baculoviruses. A detailed physical map was constructed for the restriction enzymes BamHI, BglI, BglII, EcoRI, HindIII, KpnI, PstI, XbaI and XhoI. The 120.9 kbp viral genome was cloned as restriction fragments into a plasmid library from which about 43.5 kbp of dispersed sequence information was generated. Fifty-two putative open reading frames homologous to those of other baculoviruses were identified and their location in the genome of BusuNPV was determined. Although the gene content of BusuNPV is similar to that of Autographa californica multiple-nucleocapsid nucleopolyhedrovirus, Bombyx mori nucleopolyhedrovirus and Orgyia pseudotsugata multiple-nucleocapsid nucleopolyhedrovirus, the gene order is, however, significantly different from that observed in the other viruses, which have a high degree of collinearity. A new approach (GeneParityPlot) was developed to represent the differences in gene order among baculoviruses when limited sequence information is available and to take advantage of the high degree of gene conservation. The data obtained show that BusuNPV is a distinct baculovirus species and the analyses suggest that gene distribution along baculovirus genomes may be used as a phylogenetic marker.

Animals↗
PubMed1998

A homozygous mutation in the luteinizing hormone receptor causes partial Leydig cell hypoplasia: correlation between receptor activity and phenotype.

Leydig cell hypoplasia (LCH) is characterized by a decreased response of the Leydig cells to LH. As a result, patients with this syndrome display aberrant male development ranging from complete pseudohermaphroditism to males with micropenis but with otherwise normal sex characteristics. We have evaluated three brothers with a mild form of LCH. Analysis of their LH receptor (LHR) gene revealed a homozygous missense mutation resulting in a substitution of a lysine residue for a isoleucine residue at position 625 of the receptor. In vitro analysis of this mutant LHR, LHR(I625K), in HEK293 cells indicated that the signaling efficiency was significantly impaired, which explains the partial phenotype. We have compared this mutant LHR to two other mutant LHRs, LHR(A593P) and LHR(S616Y), identified in a complete and partial LCH patient, respectively. Although the ligand-binding affinity for all three mutant receptors was normal, the hormonal response of LHR(A593P) was completely absent and that of LHR(S616Y) and LHR(I625K) was severely impaired. Low cell surface expression explained the reduced response of LHR(S616Y), while for LHR(I625K) this diminished response was due to a combination of low cell surface expression and decreased coupling efficiency. For LHR(A593P), the absence of a reduced response resulted from both poor cell surface expression and a complete deficiency in coupling. Our experiments further show a clear correlation between the severity of the clinical phenotype of patients and overall receptor signal capacity, which is a combination of cell surface expression and coupling efficiency.

Adult↗
PubMed1997

Characterization of the ecdysteroid UDP-glucosyltransferase gene of a single nucleocapsid nucleopolyhedrovirus of Buzura suppressaria.

A putative ecdysteroid UDP-glucosyltransferase (egt) gene was identified in the single nucleocapsid nucleopolyhedrovirus of Buzura suppressaria (BusuNPV). This is the first egt gene to be characterized in a SNPV, suggesting that egt genes are prevalent in nucleopolyhedroviruses and possibly in all baculoviruses. The open reading frame (ORF) of the gene is 1539 nucleotides (nt) long, encoding a putative protein (EGT) of 513 amino acids (aa) with a M of 58922. The 5' noncoding region contains three possible TATA boxes. A polyadenylation signal, AATAAA, was found downstream of the translation stop codon. A putative signal peptide of 16 residues was present at the N-terminus of the EGT. The BusuNPV egt gene has a high degree of nucleotide and amino acid sequence homology to multiple nucleocapsid (M) NPV egt genes, the highest being to the Spodoptera exigua MNPV egt. A phylogenetic tree of eleven known EGTs was constructed using maximum parsimony analysis.

Amino Acid Sequence↗
PubMed1997

Inhibin interferes with activin signaling at the level of the activin receptor complex in Chinese hamster ovary cells.

To gain more insight in the mechanism of action of inhibin, we studied the effect of inhibin on activin signaling in Chinese hamster ovary cells. Inhibin specifically counteracted activin-induced expression of a plasminogen activator inhibitor 1 promoter element (3TP) and of the junB gene, but was ineffective when the responses were induced by transforming growth factor-beta. This indicates that inhibin acts only on the activin-specific part of these signaling cascades. Using a constitutively active activin type IB receptor we determined whether inhibin acted at the level of the activin-receptor complex or downstream of it. The mutant activin receptor stimulated the expression of the 3TP promoter in the absence of activin. This stimulation was insensitive to inhibin, indicating that inhibin acts exclusively at or upstream of this activin type I receptor. In addition, competition studies using labeled activin showed that inhibin displaced activin from the activin type II receptors, especially from the activin type IIB receptor, but not from the type I receptors. In conclusion, these data show that in Chinese hamster ovary cells inhibin acts directly at the activin receptor complex, most likely through displacement of activin from the activin type II receptor.

Activin Receptors↗
PubMed1995

Mapping and characterization of the entomocidal domain of the Bacillus thuringiensis CryIA(b) protoxin.

The amino acid sequences necessary for entomocidal activity of the CryIA(b) protoxin of Bacillus thuringiensis were determined. Introduction of stop codons behind codons Arg601, Phe604 or Ala607 showed that amino acid residues C-terminal to Ala607 are not required for insecticidal activity and that activation by midgut proteases takes place distal to Ala607. The two shortest polypeptides, deleted for part of the highly conserved beta-strand, were prone to proteolytic degradation, explaining their lack of toxicity. Apparently, this beta-strand is essential for folding of the molecule into a stable conformation. Proteolytic activation at the N-terminus was investigated by removing the first 28 codons, resulting in a translation product extending from amino acid 29 to 607. This protein appeared to be toxic not only to susceptible insect larvae such as Manduca sexta and Heliothis virescens, but also to Escherichia coli cells. An additional mutant, encoding only amino acid residues 29-429, encompassing the complete putative pore forming domain, but lacking a large part of the receptor-binding domain, was similarly toxic to E. coli cells. This suggests a role for the N-terminal 28 amino acids in rendering the toxin inactive in Bacillus thuringiensis, and indicates that the cytolytic potential of the pore forming domain is only realized after proteolytic removal of these residues by proteases in the insect gut. In line with this hypothesis are results obtained with a mutant protein in which Arg28 at the cleavage site was replaced by Asp. This substitution prevented the protein from being cleaved by trypsin in vitro, and reduced its toxicity to M. sexta larvae.

Animals↗
PubMed1995

Passage of Autographa californica nuclear polyhedrosis virus through the midgut epithelium of Spodoptera exigua larvae.

A special recombinant of Autographa californica multicapsid nuclear polyhedrosis virus (AcNPV) was designed to study the early histopathological events of baculovirus infection in Spodoptera exigua larvae. This recombinant contained a Drosophila melanogaster heat shock 70 promoter driving an Escherichia coli beta-galactosidase (Lac-Z) reporter gene to monitor the presence of early viral gene expression and a second reporter gene, the E. coli beta-glucuronidase (GUS) gene, under control of the very late AcNPV p10 promoter to monitor viral replication. In S. exigua larvae, permissive Spodoptera spp. cultured cells, and nonpermissive D. melanogaster cultured cells early viral gene expression was indicated by the appearance of Lac-Z as early as 3 hr p.i. Late viral gene expression was indicated by the appearance of GUS and occurred only in the permissive cultured cells and larvae. Early and late viral gene expression could be detected simultaneously using differential enzyme histochemistry. Analysis of infected S. exigua larvae revealed that midgut columnar cells and, at a low frequency, midgut regenerative cells were the primary sites of infection. Parental nucleocapsids were apparently transported through columnar cells to underlaying regenerative cells before virus replication and progeny production. Infection of tissues beside the midgut epithelium was not detected prior to viral replication within the midgut, suggesting that infection of the midgut is an important prelude to systemic infection.

Animals↗
PubMed1995

Characterization of baculovirus insecticides expressing tailored Bacillus thuringiensis CryIA(b) crystal proteins.

Full-length, truncated, and mature forms of the CryIA(b) insecticidal crystal protein gene of Bacillus thuringiensis were engineered into the p10 locus of Autographa californica nuclear polyhdrosis virus (AcNPV). A signal sequence of Heliothis virescens juvenile hormone esterase was introduced at the N-terminus of these constructs to induce secretion. All recombinants, except those containing the mature toxin, produced high levels of CryIA(b) ICPs in insect cells. Thirty percent of the intracellular protoxin was N-glycosylated, suggesting that the protoxin was translocated across the ER membrane. Secretion into the medium, however, was limited. The production of the mature toxin was poor as a result of its cytotoxicity to insect cells. In a bioassay against second instar Spodoptera exigua larvae, using a recombinant expressing the Androctonus australis scorpion toxin gene in the same p10 locus as a positive control, the median survival time of AcNPV recombinants expressing the various B. thuringiensis CryIA(b) ICP constructs was not significant different from that of wild-type AcNPV. This suggests that production and/or secretion of B. thuringiensis (pro)toxins by AcNPV p10 recombinant viruses does not increase insecticidal activity since (i) the protoxins produced are inactive and not likely to be activated in vivo; (ii) secretion of the B. thuringiensis protoxins is poor; and (iii) production of the mature toxins results in cytotoxicity.

Animals↗
PubMed1990

Insecticidal activity of a bacterial crystal protein expressed by a recombinant baculovirus in insect cells.

Baculoviruses are insect pathogens with a relatively slow speed of action, and this has limited their use as control agents of insect pests. Introduction into baculoviruses of genes which code for proteins interfering specifically with insect metabolism or metamorphosis, such as toxins, hormones, and enzymes, may enhance the pathogenicity of these viruses. The complete insecticidal crystal protein gene cryIA(b) of Bacillus thuringiensis subsp. aizawai 7.21 was engineered into the nuclear polyhedrosis virus of Autographa californica (AcNPV) in place of the polyhedrin gene. In infected Spodoptera frugiperda cells, the cryIA(b) gene was expressed at a high level without interference with AcNPV production. The crystal protein was found in the cytoplasm of S. frugiperda cells, mainly as large crystals with an ultrastructure similar to that of B. thuringiensis crystals. Infected-cell extracts inhibited feeding of the large cabbage white Pieris brassicae. The toxicity of the crystal protein expressed by AcNPV recombinants was comparable with that of the crystal protein expressed by a corresponding Escherichia coli recombinant.

Journal Article↗