Growth response of adult germ cells to rat androgen-binding protein and human sex hormone-binding globulin

Androgen-binding protein (ABP) is produced by Sertoli cells depending on the development and the stage of the spermatogenic cycle. Germ cell proliferation is at its peak when ABP is at its peak and secreted towards the testicular basal compartment containing spermatogonia and premeiotic spermatocytes. Rat isolated adult germ cell DNA synthesis was studied in vitro in the presence of ABP with and without steroids and in the presence of pure or recombinant sex steroid hormone-binding globulin (SHBG) using thymidine incorporation. Results are: SHBG is able to promote DNA synthesis in the absence of cofactors. Testosterone reacted negatively to the stimulatory effect of SHBG. We conclude that ABP, the physiological steroid-binding protein, should be considered as a paracrine regulator of spermatogenic DNA synthesis in the adult rat.     

Identification of a rat testis-specific gene encoding a potential rat outer dense fibre protein

Histamine H2-receptor antagonistic properties of the anti-ulcer agent T-593, (+/-)-(E)-1-[2-hydroxy-2-(4-hydroxyphenyl)ethyl] -3-[2[[[5-(methylamino)methyl-2-furyl]methyl]thio] ethyl] -2-(methylsulfonyl)guanidine, were investigated on [14C]aminopyrine accumulation in isolated canine gastric mucosal cells and compared with those of ranitidine and famotidine. The potency of T-593-inhibition of [14C]aminopyrine accumulation stimulated by 10(-4) M histamine, with an IC50 value of 1.85 x 10(-6) M, was approximately 5 times greater than that of ranitidine, but half that of famotidine. T-593 did not affect [14C]aminopyrine accumulation stimulated by carbachol or dibutyryl-cAMP. T-593 depressed the maximal response of the histamine concentration-response curve with a dose-related displacement to the right, indicating that the nature of the H2-receptor antagonism of T-593 was insurmountable and included non-competitive inhibition. The inhibitory efficacy of T-593 was time-dependent and was retained after the cells were washed. The inhibitory potency of (-)-S-T-593, one of the enantiomers, on the [14C]aminopyrine accumulation stimulated by histamine was approximately twice that of racemic T-593 and it also behaved as an insurmountable H2-receptor antagonist. However, the potency of (+)-R-T-593 was markedly weak. These results suggest that T-593 has H2-receptor antagonism that is insurmountable and this agent slowly associates and dissociates with the receptor in isolated canine gastric mucosal cells and that the specific substance causing H2-receptor antagonism is (-)-S-T-593.     

Identification of a structural gene encoding a metallothionein-like domain that includes a putative regulator protein for Streptomyces protease gene expression

An open reading frame (termed ORF-PR) encoding a metallothionein-like domain-including protein was found upstream of a previously identified Streptomyces chymotrypsin-type protease gene (sam-P20). Promoter and terminator activities of ORF-PR were detected using the promoterless Streptomyces tyrosinase gene as a reporter gene and expression of ORF-PR was supposed to occur before that of sam-P20 gene. Frameshift mutation analysis showed that the ORF-PR product might act as a repressive regulator of the sam-P20 gene.     

Expression of a gene encoding a ribosomal p40 protein and identification of an active promoter site

The promoter of a gene encoding a ribosome-associated protein of 40 kDa from Arabidopsis thaliana (A-p40) was sequenced and the expression of the gene studied. A-p40 was expressed in the same organs and with the same variations as the eukaryotic elongation factor 1 alpha (eEF1A), another gene coding for a protein involved in translation Arabidopsis plants transformed with a beta-glucuronidase (GUS) gene driven by the A-p40 promoter confirm that A-p40 is expressed in actively dividing and growing cells. eEF1A promoter-GUS fusions have the same pattern of expression. Comparison of cis-acting elements from A-p40 and eEF1A revealed some common elements. A-p40 promoter deletions and transient gene expression in transfected Arabidopsis protopasts allowed the identification of trap40, a cis-acting element regulating gene expression. Gel retardation experiments indicate that eEF1A and A-p40 are regulated by different cis-acting elements. The role of such elements is discussed.     

Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis

Triacylglycerols are quantitatively the most important storage form of energy for eukaryotic cells. Acyl CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the terminal and only committed step in triacylglycerol synthesis, by using diacylglycerol and fatty acyl CoA as substrates. DGAT plays a fundamental role in the metabolism of cellular diacylglycerol and is important in higher eukaryotes for physiologic processes involving triacylglycerol metabolism such as intestinal fat absorption, lipoprotein assembly, adipose tissue formation, and lactation. DGAT is an integral membrane protein that has never been purified to homogeneity, nor has its gene been cloned. We identified an expressed sequence tag clone that shared regions of similarity with acyl CoA:cholesterol acyltransferase, an enzyme that also uses fatty acyl CoA as a substrate. Expression of a mouse cDNA for this expressed sequence tag in insect cells resulted in high levels of DGAT activity in cell membranes. No other acyltransferase activity was detected when a variety of substrates, including cholesterol, were used as acyl acceptors. The gene was expressed in all tissues examined; during differentiation of NIH 3T3-L1 cells into adipocytes, its expression increased markedly in parallel with increases in DGAT activity. The identification of this cDNA encoding a DGAT will greatly facilitate studies of cellular glycerolipid metabolism and its regulation.     

Identification of evolutionarily invariant sequences in the protein C gene promoter

To identify a marker associated with poor outcome in severe malaria that requires no technology, the relationship between the presence of pallor and mortality was reviewed retrospectively in 291 Zambian children with cerebral malaria. The mean (S.D.) haemoglobin concentration among the 222 children assessed as having pallor on admission was significantly lower than that among the 69 children not considered to have pallor [6.0 (1.9) v. 9.2 (1.6) g/dl; P < 0.0005]. Thirty-nine (17.6%) of the children presenting with pallor died, compared with only five (7.2%) of those without pallor (P = 0.036). The adjusted odds of death in children with pallor on admission was 2.8 times higher than that in children without pallor (95% confidence interval = 1.03-7.7; P = 0.044). The clinical observation of pallor may therefore identify children with low haemoglobin concentrations and a high risk of mortality. Whether mothers and village health workers can be taught to recognize pallor in a child with malaria and then to seek early medical attention will need to be determined in further studies.     

Identification and purification of a spinach chloroplast DNA-binding protein that interacts specifically with the plastid psaA-psaB-rps14 promoter region

OBJECTIVE: To catalog a series of rare lesions of the posterior fossa that appeared with unusual initial retrocochlear symptoms and signs and to make the reader more aware of these unusual lesions with a view to improving initial assessment and treatment planning. STUDY DESIGN: The study was a retrospective case review of seven patients. SETTING: Multidisciplinary team evaluation in a tertiary hospital referral center. PATIENTS: Patients with unusual lesions of the cerebellopontine angle and posterior fossa with initial retrocochlear symptoms and signs were included. INTERVENTIONS: Diagnostic and therapeutic. MAIN OUTCOME MEASURES: Hearing preservation and balance function. RESULTS: The rare lesions presented include two aneurysms of the anterior inferior cerebellar artery, one giant basilar artery aneurysm, and one each of the following neoplasms: endodermal cyst, choroid plexus papilloma, cavernous angioma, and ependymoma. CONCLUSIONS: A close working relationship among the otolaryngologist, neurotologist, neurosurgeon, and neuroradiologist is necessary to accurately evaluate these unusual cerebellopontine angle lesions and effect the best treatment outcome.     

Identification of a polar region in transmembrane domain 6 that regulates the function of the G protein-coupled alpha-factor receptor

The alpha-factor pheromone receptor (Ste2p) of the yeast Saccharomyces cerevisiae belongs to the family of G protein-coupled receptors that contain seven transmembrane domains (TMDs). Because polar residues can influence receptor structure by forming intramolecular contacts between TMDs, we tested the role of the five polar amino acids in TMD6 of the alpha-factor receptor by mutating these residues to nonpolar leucine. Interestingly, a subset of these mutants showed increased affinity for ligand and constitutive receptor activity. The mutation of the most polar residue, Q253L, resulted in 25-fold increased affinity and a 5-fold-higher basal level of signaling that was equal to about 19% of the alpha-factor induced maximum signal. Mutation of the adjacent residue, S254L, caused weaker constitutive activity and a 5-fold increase in affinity. Comparison of nine different mutations affecting Ser254 showed that an S254F mutation caused higher constitutive activity, suggesting that a large hydrophobic amino acid residue at position 254 alters transmembrane helix packing. Thus, these studies indicate that Gln253 and Ser254 are likely to be involved in intramolecular interactions with other TMDs. Furthermore, Gln253 and Ser254 fall on one side of the transmembrane helix that is on the opposite side from residues that do not cause constitutive activity when mutated. These results suggest that Gln253 and Ser254 face inward toward the other TMDs and thus provide the first experimental evidence to suggest the orientation of a TMD in this receptor. Consistent with this, we identified two residues in TMD7 (Ser288 and Ser292) that are potential contact residues for Gln253 because mutations affecting these residues also cause constitutive activity. Altogether, these results identify a new domain of the alpha-factor receptor that regulates its ability to enter the activated conformation.     

Identification of ArgBP1, an Arg protein tyrosine kinase binding protein that is the human homologue of a CNS-specific Xenopus gene

Cardiac fibroblasts constitute greater than 90% of the non-myocyte cells in the heart. Previously, it was established that cardiac fibroblasts are predisposed to transformation into a phenotype with muscle-specific features and that transforming growth factor-beta 1 (TGF-beta 1) is a specific inducer of this event. In this study the hypothesis that TGF-beta 1-induced phenotypic modulation of cardiac fibroblasts is associated with their altered proliferative capacity is tested. Therefore the effects of TGF-beta 1 on DNA synthesis in cardiac fibroblasts under normal conditions of cell culture and in response to a potent mitogen, basic fibroblasts growth factor (bFGF) were determined. The results showed that TGF-beta 1 at 15 ng/ml (a concentration that induces fibroblast "transformation") had a regulatory effect on proliferative capacity of cardiac fibroblasts which varied as the function of cell density in culture. In subconfluent and confluent cultures, pre-treatment of cardiac fibroblasts with TGF-beta 1 for 24 h resulted in a dramatic shift in the bFGF-induced stimulation of DNA synthesis. TGF-beta 1-induced inhibition of DNA synthesis in cardiac fibroblasts coincided with their phenotypic modulation as evidenced by the expression of sarcomeric actin mRNA and morphological changes. Cross-linking studies with [125I]-labeled TGF-beta 1 showed the presence of conventional types I, II and III TGF-beta 1 receptor complexes on cardiac fibroblasts and their binding to TGF-beta 1 under the experimental conditions. In summary, these data indicate that the proliferative capacity of cardiac fibroblasts is controlled by TGF-beta 1. They further suggest that the TGF-beta 1-induced phenotypic modulation of cardiac fibroblasts may be extended to include their altered proliferative capacity.     

Effect of preparations of messenger RNA from the spleens of immunized rats on antibody synthesis in rat lymphosarcoma

Messenger RNA was isolated from spleen polysomes of rats immunized with sheep red cells, two methods being employed: a sorption of mRNA on on membrane filters, and poly(U)-cellulose affinity chromatography. The rat transplantable lymphosarcoma cells treated with this RNA started hemolysine synthesis which persisted throughout many cell generations in transplantation of the tumor. The probability is discussed of m-RNA not only functioning as a template for antibody synthesis but also deblocating appropriate genes in recipient cells.