Mapping of mouse gamma crystallin genes on chromosome 1

Restriction fragments analysis of DNA from mouse-hamster somatic-cell hybrid clones revealed that a mouse gamma crystallin cDNA hybridized to genomic sequences located on mouse chromosome 1. Identification of restriction fragment length polymorphisms (RFLPs) in the gamma crystallin sequences of inbred strains of mice permitted the further localization of the gamma crystallin genes (Cryg) to the proximal region of chromosome 1 closely linked to the loci encoding isocitrate dehydrogenase (Idh-1), a low molecular weight (LM) crystallin protein polymorphism (Len-1), and fibronectin (Fn-1). A single recombinant was observed betweenLen-1 and an RFLP in the gamma crystallin gene family, consistent with the hypothesis thatLen-1 is one of the several structural loci encoding gamma crystallin genes.Len-1 is probably located on the centromeric end of theCryg gene family. Linkage ofIdh-1, Cryg, andFn-1 in mice extends the syntenic relationship of those loci to the human, bovine, and rodent genomes and may define a chromosomal region that is generally conserved among mammals. The map position ofCryg, near the eye lens obsolescence (Elo) locus, was confirmed by the discovery that the restriction fragment patterns of gamma crystallin sequences differed between strain C3H/HeJ and the congenic anophthalmic mutant strain, C3H.Elo. Therefore, the gamma crystallin genes were contransferred with the mutantElo gene in the derivation of C3H.Elo. The results establish that LEN-1 is a marker for the gamma crystallin gene family, position the gamma crystallin gene family relative to other markers on mouse chromosome 1, and provide additional evidence that theElo mutation is encoded at a locus closely linked to the gamma crystallin gene cluster. This study found no evidence of recombination hot spots within the gamma crystallin gene cluster.     

Isolation and characterization of a variant somatostatin-14 and two related somatostatins of 34 and 37 residues from lamprey (Petromyzon marinus)

Three major forms of somatostatin were isolated from pancreas of the lamprey (Petromyzon marinus). One of the two major forms is a 14-residue somatostatin (SS-14) having the sequence AGCKNFFWKTFSSC. The homologous substitution Ser for Thr in position 12 is the first example of SS-14 from vertebrate preprosomatostatin gene I having a divergent sequence. The longest form is 37 residues in length (SS-37) and has the sequence ALRAAAVAGSPQQLLPLGQRERKAGCKNFFWKTFSSC. A 34-residue form (SS-34) identical in sequence but truncated at a single Arg residue at position 3 of SS-37 was also isolated. The yields of the three forms were SS-37 (0.43 nmol/g), SS-34 (134 nmol/g), and SS-14 (51.5 nmol/g). The identification of this nested series of somatostatins suggests that prosomatostatin processing in lamprey more closely resembles that observed for procholecystokinin than that of mammalian or other piscine prosomatostatins. Somatostatin-producing cells in the lamprey pancreas were identified by immunostaining using antiserum against SS-34 and anti-serum against mammalian SS-14.     

Genetic Analysis of a Mouse t Complex Locus That Is Homologous to a Kidney Cdna Clone

A mouse kidney cDNA clone, pMK174, identifies restriction fragment length polymorphisms (RFLPs) that map to two unlinked loci. One, designated D17Rp17, has been mapped near quaking, (qk), on chromosome 17 using three sets of recombinant inbred (RI) strains. A study of several t haplotypes resulted in the identification of t-specific alleles of D17Rp17 that map to the proximal half of the t complex. Neither t-specific nor wild-type D17Rp17 alleles are present in chromosomes carrying either the T Orleans (TtOrl) or the T hairpin tail (Thp) deletions. Comparison with other molecular markers indicates that pMK174 identifies a new proximal t complex locus, Rp17. The second locus identified by pMK174, termed D4Rp18, is tentatively assigned to chromosome 4 by mouse-Chinese hamster somatic cell hybrid analysis.     

Regulation of bile acid synthesis via direct effects on the microsomal membrane

Rats treated with ethinylestradiol (5 mg kg-1 day-1 for 5 days) secrete de novo synthesized bile acids at a markedly reduced rate (-57%). Administration of the nonionic detergent Triton WR-1339 to estradiol-treated rats rapidly restored the rate of secretion of de novo synthesized bile acids to control levels. In contrast, when Triton was administered to control rats, the secretion rate of bile acids was unaffected. The reduction in bile acid synthesis displayed by estradiol-treated rats was similar to the 50% decrease in the activity of hepatic microsomal 7 alpha-hydroxylase. The activity of 7 alpha-hydroxylase was also restored to control levels by the administration of Triton to estradiol-treated rats. We examined the possibility that estradiol acts directly on the hepatic microsomes. Adding increasing amounts of estradiol to microsomes obtained from control rats resulted in decreasing activities of 7 alpha-hydroxylase. The inhibition by estradiol of 7 alpha-hydroxylase obtained in vitro occurred with amounts of estradiol that were found to accumulate in the liver via in vivo treatment. Double-reciprocal analysis showed that at and below 50 micrograms of estradiol/0.5 mg of protein uncompetitive inhibition was displayed. Additional experiments showed that adding Triton to microsomes obtained from estradiol-treated rats increased the activity of 7 alpha-hydroxylase to control levels. In contrast, Triton did not increase the activity of 7 alpha-hydroxylase when it was added to control microsomes. These data show for the first time that the estrogenic steroid estradiol acts directly on the microsomes and inhibits both the activity of 7 alpha-hydroxylase and the rate of bile acid synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of in vitro growth phase on the pathogenesis of Salmonella typhimurium in mice

The growth phase of a bacterial (Salmonella typhimurium) culture was shown to have pronounced effects on the pathogenic properties of the harvested bacteria. Salmonellae obtained from a culture in primary (exponential) growth phase (PP) were more readily cleared from the blood and more readily killed by phagocytes than were salmonellae obtained from a more slowly growing secondary growth phase (SP) culture. PP salmonellae were observed to cause death of mice sooner than SP salmonellae. This appeared to be because the more rapid growth of PP, as compared to SP, salmonellae continued in the liver and spleen for several hours following intravenous injection, and more than compensated for their high in vivo death rate. As a result, within 4 h there were approximately 10-fold more live salmonellae in the spleens and livers of mice that had received PP, as compared to SP, salmonellae. This 10-fold difference was maintained until the death of the mice, indicating that after the first 4 h post-inoculation, the net in vivo growth of the salmonellae was the same regardless of their growth phase in the inoculating culture. This transition between PP and SP salmonellae occurred long before a dense stationary phase culture was obtained. Salmonellae grown in minimal media exhibited the biological properties of SP salmonellae and never entered as rapid a growth phase as did salmonellae in complete media.     

Recombinant human interleukin 1 alpha: purification and biological characterization

Interleukin 1 (IL 1) is a polypeptide hormone produced by activated macrophages that affects many different cell types involved in immune and inflammatory responses. The cloning and expression of a murine IL 1 cDNA in Escherichia coli encoding a polypeptide precursor of 270 amino acids has been reported, and expression of the carboxy-terminal 156 amino acids of this precursor in E. coli yields biologically active IL 1. By using the murine IL 1 cDNA as a probe, we have isolated its human homolog from cDNA generated to lipopolysaccharide-stimulated human leukocyte mRNA. Nucleotide sequence analysis of this cDNA predicts a protein of analysis of this cDNA predicts a protein of 271 amino acids (termed IL 1 alpha) which shows congruent to 61% homology to its murine counterpart but only 27% homology to a recently characterized human IL 1 precursor (IL 1 beta). We have expressed the carboxy-terminal 154 amino acids of IL 1 alpha in E. coli, purified this protein to homogeneity, and have compared it with pure recombinant murine IL 1 in several different IL 1 assays based on murine and human cells. Recombinant IL 1 is capable of stimulating T cell and fibroblast proliferation and inducing fibroblast collagenase and prostaglandin production, thus proving that a single molecule has many of the activities previously ascribed to only partially purified IL 1 preparations. Our results indicate that there exists a family of at least two human IL 1 genes (alpha and beta) whose dissimilar protein products have similar biological activities.     

An extra disulfide bridge in the constant domain of Rana catesbeiana immunoglobulin light chains

The immunoglobulins of the bullfrog Rana catesbeiana are unusual in that, in all classes, the light chains are not disulfide bonded to heavy chains or to other light chains. Moreover, the light chains contain six, rather than the usual five, residues of half-cystine. As none of these half-cystines is in the sulfhydryl form or is alkylated after mild reduction, we suggested that the light chains probably contain three intrachain disulfide bridges. We have now carried out experiments to confirm the existence of an extra intrachain disulfide bridge in Rana catesbeiana light chains and to determine its location. Disulfide bridge assignments were based on 1) isolation and sequence analysis of S-(carboxymethyl)cysteine-containing peptides and 2) isolation, from unreduced light chains, of peptides containing a disulfide bridge. Half-cystine residues were found at positions 134 and 194, and these were shown to be joined in the conserved intradomain disulfide bridge. In addition, we found that a residue of half-cystine, located at the third position from the carboxy-terminus, forms a disulfide bridge with a half-cystine at position 119, near the amino-terminus of the domain, the latter residue replacing a proline that has been found at this position in all other light chains. An intrachain disulfide bridge has not been found at this location in any other light chain.     

Studies on Muscarinic Binding Sites in Human Brain Identified with [3H]Pirenzepine

Pirenzepine, a potent antimuscarinic agent with apparent selectivity for a subtype (M1) of muscarinic receptors, was used in tritiated form to characterize its binding to human brain tissue. Specific [3H]pirenzepine binding showed rapid association and dissociation. From kinetic and competitive binding experiments, its KD was 5.5 nM and 9 nM, respectively. Regional distribution of [3H]pirenzepine binding determined in parallel with [3H]quinuclidinyl benzilate binding, a nonselective muscarinic antagonist, indicated a significant correlation for the maximum number of binding sites for the two radioligands in 13 brain regions, with the highest amount of binding for each in the putamen and the least in the cerebellum. Binding for [3H]pirenzepine averaged 57% of that for [3H]quinuclidinyl benzilate, with a range of 20% (cerebellum) to 77% (frontal cortex). Most antidepressants and neuroleptics tested had affinities for [3H]pirenzepine binding sites that were not significantly different from their previously reported values obtained with the use of [3H]quinuclidinyl benzilate.     

ATP-coupled transport of vesicular stomatitis virus G protein between the endoplasmic reticulum and the Golgi

The temperature and ATP dependence of transport of the vesicular stomatitis virus strain ts045 G protein from the endoplasmic reticulum (ER) to an early Golgi compartment containing mannosidase I was studied in the mutant Chinese hamster ovary cell clone 15B. Appearance of G protein containing the Man5GlcNAc2 oligosaccharide species occurred after a shift to the permissive temperature with a lag period of 5 min and without detectable formation of the intermediate Man7GlcNAc2 and Man6GlcNAc2 species. Two biochemically distinct transport steps were detected during transport from the ER to the Golgi. An initial step is temperature sensitive, thermoreversible, and requires a high threshold of cellular ATP for maximal rate of transport (80% of the normal cellular ATP pool). Export from the ER is inhibited at 65% of the normal cellular ATP pool. Prolonged incubation at reduced levels of cellular ATP or at the restrictive temperature resulted in the accumulation of G protein in either the Man8GlcNAc2 species or the Man7GlcNAc2 and Man6GlcNAc2 species, respectively. Reversal of the temperature-sensitive block is ATP coupled. A second step is insensitive to incubation at the restrictive temperature and proceeds efficiently when the cellular ATP pool is reduced to 20% of the control. G protein accumulates at this intermediate step during prolonged incubation at 15 degrees C. The data suggest a functional division of processes required for transport of protein between the ER and Golgi compartments. The two steps may reflect the export (budding) and delivery (fusion) of proteins through vesicular trafficking between the ER and Golgi.     

Ontogeny of cells containing insulin, glucagon, pancreatic polypeptide hormone and somatostatin in the bovine pancreas

Antibodies to insulin, glucagon, pancreatic polypeptide hormone (PP) and somatostatin were used in the immunofluorescence histochemical procedure to study the ontogeny of pancreatic endocrine cells containing the four hormones in the bovine fetus of approximately 100 days gestation to term. Pancreatic sections from the bovine neonate and adult were also examined for the cellular distribution of the four hormones. Immunoreactive cells staining for insulin, glucagon, PP and somatostatin were present in the pancreas of all fetuses studied. Each endocrine cell type displayed a characteristic distribution within the developing pancreas and in the neonate and adult. The presence of the four islet hormones relatively early in bovine fetal life suggests that they may be important in intra- and extra-islet metabolism in the fetus.