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J M Kramer

Publications and source records attributed to J M Kramer.

At least 55 records · Page 3Linked to original sources
PubMed1995

Recombinant ob protein reduces feeding and body weight in the ob/ob mouse.

To determine whether the product of the recently cloned ob gene functions as an adipose-related satiety factor, recombinant murine ob protein was administered intraperitoneally to ob/ob mice. Monomeric ob protein given as single morning injections to groups of three animals at seven doses ranging from 5 to 100 micrograms reduced 24-h chow consumption in a dose-dependent manner from values of 81 +/- 6.8% of control (10-micrograms dose, P = 0.04) to 29 +/- 7.7% of control (100-micrograms dose, P < 0.0001). Daily injections of 80 micrograms of ob protein into six ob/ob mice for 2 wk led to an 11 +/- 1.6% decrease in body weight (P = 0.0009) and suppressed feeding to 26 +/- 4.9% of baseline (P < 0.0001), with significant reduction of serum insulin and glucose levels. The effect of recombinant ob protein on feeding was not augmented by cofactors secreted by adipose tissue, nor did exposure of adipose tissue to ob protein affect intracellular ob mRNA levels. Posttranslational modification of ob protein was not required for activity; however, addition of a hexahistidine tag to the amino terminus of the mature ob protein resulted in prolonged suppression of feeding after injection into ob/ob mice. These results demonstrate a direct effect of the ob protein to suppress feeding in the ob/ob mouse and suggest that this molecule plays a critical role in regulating total body fat content.

Adipose Tissue↗
PubMed1994

Human thrombopoietin: gene structure, cDNA sequence, expression, and chromosomal localization.

Thrombopoietin (TPO), a lineage-specific cytokine affecting the proliferation and maturation of megakaryocytes from committed progenitor cells, is believed to be the major physiological regulator of circulating platelet levels. Recently we have isolated a cDNA encoding a ligand for the murine c-mpl protooncogene and shown it to be TPO. By employing a murine cDNA probe, we have isolated a gene encoding human TPO from a human genomic library. The TPO locus spans over 6 kb and has a structure similar to that of the erythropoietin gene (EPO). Southern blot analysis of human genomic DNA reveals a hybridization pattern consistent with a single gene locus. The locus was mapped by in situ hybridization of metaphase chromosome preparations to chromosome 3q26-27, a site where a number of chromosomal abnormalities associated with thrombocythemia in cases of acute myeloid leukemia have been mapped. A human TPO cDNA was isolated by PCR from kidney mRNA. The cDNA encodes a protein with 80% identity to previously described murine TPO and is capable of initiating a proliferative signal to murine interleukin 3-dependent BaF3 cells expressing the murine or human TPO receptor.

Amino Acid Sequence↗
PubMed1994

Mutations in the alpha 2(IV) basement membrane collagen gene of Caenorhabditis elegans produce phenotypes of differing severities.

Type IV collagen forms a network that provides the major structural support of basement membranes. We have determined the nucleotide alterations and phenotypes of 17 mutant alleles of the Caenorhabditis elegans alpha 2(IV) collagen gene let-2. All 17 mutations are within the triple helical (Gly-X-Y) repeat domain of the molecule. Fifteen of the mutations are replacements of Gly-X-Y repeat glycines with aspartate, glutamate or arginine, and they cause a wide range of phenotypes. The mildest alleles are nearly wild-type at 15 and 20 degrees C but embryonic lethal at 25 degrees C, while the most severe allele is embryonic lethal at all three temperatures. Mutations resulting in severe phenotypes are generally located in areas of lower calculated thermal stability of the type IV collagen molecule. An alanine to threonine substitution at position X of a Gly-X-Y triplet immediately following an interruption results in a severe phenotype. This mutation is unusual because substitutions at positions X or Y have not generally been found to cause strong phenotypes in C. elegans or human collagens. An intron splice acceptor mutation causes a strict embryonic lethal phenotype, but does not completely abolish gene function. Pairs of independent mutations affect each of three glycines, indicating a non-random distribution of mutations in the molecule. It is suggested that this clustering results because many glycine substitutions may cause dominant lethal or sterile phenotypes.

Alleles↗
PubMed1994

Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo.

The major regulator of circulating platelet levels is believed to be a cytokine termed thrombopoietin. It is thought to be a lineage-specific cytokine affecting the proliferation and maturation of committed cells resulting in the production of megakaryocytes and platelets. Despite considerable efforts by a number of laboratories, the unequivocal identification of thrombopoietin has proven elusive. Here we report the functional cloning of a murine complementary DNA encoding a ligand for the receptor encoded by the c-mpl proto-oncogene (c-Mpl). The encoded polypeptide has a predicted molecular mass of 35,000 (M(r) 35K). The protein has a novel two-domain structure with an amino-terminal domain homologous with erythropoietin and a carboxy-terminal domain rich in serine, threonine and proline residues and containing seven potential N-linked glycosylation sites. Intraperitoneal injections of mice with recombinant protein increase circulating platelet levels by greater than fourfold after 7 days. These results along with those presented in the accompanying report strongly suggest that the ligand for c-Mpl is thrombopoietin.

Amino Acid Sequence↗
PubMed1994

The human glucagon receptor encoding gene: structure, cDNA sequence and chromosomal localization.

Characterization of the human glucagon-receptor-encoding gene (GGR) should provide a greater understanding of blood glucose regulation and may reveal a genetic basis for the pathogenesis of diabetes. A cDNA encoding a complete functional human glucagon receptor (GGR) was isolated from a liver cDNA library by a combination of polymerase chain reaction and colony hybridization. The cDNA encodes a receptor protein with 80% identity to rat GGR that binds [125I]glucagon and transduces a signal leading to increases in the concentration of intracellular cyclic adenosine 3',5'-monophosphate. Southern blot analysis of human DNA reveals a hybridization pattern consistent with a single GGR locus. In situ hybridization to metaphase chromosome preparations maps the GGR locus to chromosome 17q25. Analysis of the genomic sequence shows that the coding region spans over 5.5 kb and is interrupted by 12 introns.

Amino Acid Sequence↗
PubMed1994

Structures and functions of collagens in Caenorhabditis elegans.

Two types of collagens have been identified in Caenorhabditis elegans corresponding to two types of extracellular matrix, the cuticle and basement membranes. Cuticle collagens are encoded by a developmentally regulated family of approximately 100 genes. Mutations in cuticle collagens can produce animals that are longer or shorter than normal and/or that are helically twisted. Mutations in different collagens can cause different morphological abnormalities, as can different mutations in the same collagen. Genetic interactions between collagen genes have been described and may identify collagens that interact to form the cuticle. Two basement membrane (type IV) collagen genes have been identified in C. elegans. They encode proteins similar in structure to vertebrate type IV collagen. One of the genes produces two alternatively spliced forms, one predominantly expressed in embryos and the other in larvae and adults, suggesting that embryonic basement membranes may have unique properties. Most mutations in the type IV genes cause embryonic lethality, indicating that normal basement membranes are required for embryogenesis. Temperature-sensitive mutations have been used to show that type IV collagen function is also required for larval development and adult fertility.

Amino Acid Sequence↗
PubMed1994

The C. elegans sqt-1 and rol-6 collagen genes are coordinately expressed during development, but not at all stages that display mutant phenotypes.

Mutations in the sqt-1 and rol-6 cuticle collagen genes of Caenorhabditis elegans can cause dramatic alterations in organismal morphology. Genetic interactions and similarities in sequence and mutant phenotypes suggest that the sqt-1 and rol-6 collagen chains may physically interact. We show here that the sqt-1 and rol-6 genes are coordinately expressed during formation of the L2, L3, L4, adult, and L2d stage cuticles. Quantitative analyses indicate that the ratio of the steady-state levels of the sqt-1 and rol-6 mRNAs is approximately 2:1 at each of these stages, consistent with the possibility that they form a single heterotrimeric collagen. Surprisingly, the temporal expression patterns of sqt-1 and rol-6 mRNAs are not completely correlated with the times of appearance of their mutant phenotypes. Both sqt-1 and rol-6 mutant phenotypes appear in L2, L3, L4, adult, and L2d stage animals, and their mRNAs are easily detectable during synthesis of each of these cuticles. However, both sqt-1 and rol-6 mutant animals also display abnormal phenotypes at the dauer stage, but no transcripts from either gene are detected during synthesis of the dauer cuticle. We propose that dauer animals display a mutant phenotype in the absence of mutant collagen because they maintain the abnormal morphology that was generated in the preceding L2d stage.

Animals↗
PubMed1994

Contamination of hospital linen by Bacillus cereus.

An investigation into two cases of post-operative Bacillus cereus meningitis revealed that hospital linen laundered by a batch continuous washing machine was heavily contaminated by B. cereus spores. The washing machine, detergents, other chemical additives and the water supply were eliminated as the source of contamination. It was found that the linen introduced into the washing machine had a high B. cereus spore content and that this was still present after the wash process. The spores were not killed by either the heat disinfection stage of the wash or the addition of chemical disinfectants and were not removed by the dilution in the process. The multiplication of B. cereus was thought to have occurred on used, damp linen stored in plastic bags, particularly when ambient temperatures were high. An increase in the water flow through the washing machine was the only measure associated with a decrease in B. cereus on laundered linen.

Bacillus cereus↗
PubMed1994

In vitro mutagenesis of Caenorhabditis elegans cuticle collagens identifies a potential subtilisin-like protease cleavage site and demonstrates that carboxyl domain disulfide bonding is required for normal function but not assembly.

The importance of conserved amino acids in the amino and carboxyl non-Gly-X-Y domains of Caenorhabditis elegans cuticle collagens was examined by analyzing site-directed mutations of the sqt-1 and rol-6 collagen genes in transgenic animals. Altered collagen genes on transgenic arrays were shown to produce appropriate phenotypes by injecting in vivo cloned mutant alleles. Equivalent alterations in sqt-1 and rol-6 generally produced the same phenotypes, indicating that conserved amino acids in these two collagens have similar functions. Serine substitutions for either of two conserved carboxyl domain cysteines produced LRol phenotypes. Substitution for both cysteines in sqt-1 also resulted in an LRol phenotype, demonstrating that disulfide bonding is important for normal function but not required for assembly. Arg-1 or Arg-4 to Cys mutations in homology block A (HBA; consensus, 1-RXRRQ-5; in the amino non-Gly-X-Y domain) caused RRol phenotypes, while the same alteration at Arg-3 had no effect, indicating that Arg-3 is functionally different from Arg-1 and Arg-4. Substitutions of Arg-4 with Ser, Leu, or Glu also produced the RRol phenotype, while Lys substitutions for Arg-1 or Arg-4 did not generate any abnormal phenotypes. His substitutions for Arg-1 or Arg-4 caused somewhat less severe RRol phenotypes. Therefore, strong positively charged residues, Arg or Lys, are required at positions 1 and 4 for normal function. The conserved pattern of arginines in HBA matches the cleavage sites of the subtilisin-like endoproteinases. HBA may be a cleavage site for a subtilisin-like protease, and cleavage may be important for cuticle collagen processing.

Alleles↗
PubMed1993

Identification, sequence and expression patterns of the Caenorhabditis elegans col-36 and col-40 collagen-encoding genes.

The collagen (Col)-encoding gene family in the nematode, Caenorhabditis elegans, consists of 50-150 members. We have undertaken studies of these genes as part of the analysis of the assembly of the cuticle, the nematode's exoskeleton. We present here the complete nucleotide and deduced amino acid sequences of the col-36 and col-40 genes, both located on chromosome II and encoding cuticle Col. Both Col possess the structural properties found in the type of Col that form the cuticle, such as short Gly-Xaa-Yaa interruptions and Cys clusters at conserved sites. On the basis of identical patterns of conserved cysteines, col-36 and col-40 belong to the col-6 cuticle Col family. Semi-quantitative analysis using reverse transcription-PCR demonstrates that the col-36 transcript is present in L1 larvae and at the L1-L2 and L2d-dauer molts. The col-40 transcript is present in L1 larvae and at the L2d-dauer molt. Different members of the col-6 family are structurally related, but have different developmental expression patterns.

Amino Acid Sequence↗
PubMed1993

Analysis of enterotoxin production by Bacillus cereus from dairy products, food poisoning incidents and non-gastrointestinal infections.

Of 85 strains of Bacillus cereus isolated in Norway from dairy products, 59% were found to be enterotoxigenic, and 15% were psychrotrophic. Six of the isolates (7%) were identified as potential psychrotrophic food-poisoning strains as they were both enterotoxigenic and exhibited good growth at 6 degrees C. Enterotoxin production was detected using the Western immunoblot technique, and a commercially available reversed passive latex agglutination (RPLA) assay (Unipath BCET-RPLA TD950). Both methods gave essentially the same results. In a separate study, the Western immunoblot and RPLA assays were used in a conjunction with the in vivo vascular permeability reaction (VPR) assay to determine enterotoxin production among 25 isolates of Bacillus cereus referred to the PHLS Food Hygiene Laboratory from incidents of diarrhoeal- and emetic-syndrome food poisoning and non-gastrointestinal infections. Eighty-four percent of these isolates were found to be enterotoxigenic by the Western immunoblot and the RPLA assays, and these results were in good agreement with those obtained by the VPR assay. In both studies, the BCET-RPLA kit proved to be a simple and reliable means for determining enterotoxin production by strains of Bacillus cereus.

Animals↗
PubMed1993

Genetic identification, sequence, and alternative splicing of the Caenorhabditis elegans alpha 2(IV) collagen gene.

The nematode Caenorhabditis elegans has two type IV collagen genes homologous to the mammalian alpha 1(IV) and alpha 2(IV) collagen genes. We demonstrate by transgenic rescue of mutant animals that the genetic locus encoding the C. elegans alpha 2(IV) collagen gene is let-2 on the X chromosome. The most severe effect of mutations in let-2 is temperature-sensitive embryonic lethality. The embryonic lethal phenotype is similar to that seen in animals with mutations in the alpha 1(IV) collagen gene, emb-9. The sequence of the entire C. elegans alpha 2(IV) collagen gene is presented. Comparisons with mammalian type IV collagen sequences show high amino acid sequence conservation in the C-terminal NCl domain and of crosslinking residues (Cys and Lys) in the N-terminal 7S domain. RT-PCR analysis shows that transcripts of the C. elegans alpha 2(IV) collagen gene are alternatively spliced. Transcripts contain one of two mutually exclusive exons, exon 9 or 10. These exons encode very similar products, differing primarily in the sequence of a 9-10 amino acid Gly-X-Y interruption. The expression of these alternatively spliced alpha 2(IV) collagen transcripts is developmentally regulated. In embryos over 90% of the alpha 2(IV) collagen mRNA contains exon 9, while larval and adult RNAs contain 80-90% exon 10. This shift in expression of alternative alpha 2(IV) collagen transcripts suggests that C. elegans embryos may require a different form of alpha 2(IV) collagen than do larvae and adults.

Alternative Splicing↗
PubMed1993

Molecular and genetic analyses of the Caenorhabditis elegans dpy-2 and dpy-10 collagen genes: a variety of molecular alterations affect organismal morphology.

We have identified and cloned the Caenorhabditis elegans dpy-2 and dpy-10 genes and determined that they encode collagens. Genetic data suggested that these genes are important in morphogenesis and possibly other developmental events. These data include the morphologic phenotypes exhibited by mutants, unusual genetic interactions with the sqt-1 collagen gene, and suppression of mutations in the glp-1 and mup-1 genes. The proximity of the dpy-2 and dpy-10 genes (3.5 kilobase) and the structural similarity of their encoded proteins (41% amino acid identity) indicate that dpy-2 and dpy-10 are the result of a gene duplication event. The genes do not, however, appear to be functionally redundant, because a dpy-10 null mutant is not rescued by the dpy-2 gene. In addition, full complementation between dpy-2 and dpy-10 can be demonstrated with all recessive alleles tested in trans. Sequence analysis of several mutant alleles of each gene was performed to determine the nature of the molecular defects that can cause the morphologic phenotypes. Glycine substitutions within the Gly-X-Y portion of the collagens can result in dumpy (Dpy), dumpy, left roller (DLRol), or temperature-sensitive DLRol phenotypes. dpy-10(cn64), a dominant temperature-sensitive DLRol allele, creates an Arg-to-Cys substitution in the amino non-Gly-X-Y portion of the protein. Three dpy-10 alleles contain Tc1 insertions in the coding region of the gene. dpy-10(cg36) (DRLol) creates a nonsense codon near the end of the Gly-X-Y region. The nature of this mutation, combined with genetic data, indicates that DLRol is the null phenotype of dpy-10. The Dpy phenotype results from reduced function of the dpy-10 collagen gene. Our results indicate that a variety of molecular defects in these collagens can result in severe morphologic changes in C. elegans.

Alleles↗
PubMed1993

Analysis of mutations in the sqt-1 and rol-6 collagen genes of Caenorhabditis elegans.

Different mutations in the sqt-1 and rol-6 collagen genes of Caenorhabditis elegans can cause diverse changes in body morphology and display different genetic attributes. We have determined the nucleotide alterations in 15 mutant alleles of these genes. Three mutations in sqt-1 and one in rol-6 that cause dominant right-handed helical twisting (RRol) of animals are arginine to cysteine replacements. These mutations are all within a short conserved sequence, on the amino terminal side of the Gly-X-Y repeats, that is found in all C. elegans cuticle collagens. A recessive RRol mutation of rol-6 is a replacement of one of the same conserved arginines by histidine. In contrast, three sqt-1 mutations that cause recessive left-handed helical twisting (LRol) are replacements of a conserved carboxy-terminal cysteine residue with either tyrosine or serine. These results suggest that disulfide bonding is important in collagen organization and that a deficit or surplus of disulfides may cause cuticle alterations of opposite handedness. In contrast to other collagens, glycine replacement mutations in the Gly-X-Y repeats of sqt-1 cause very mild phenotypes. Nonsense mutations of both sqt-1 and rol-6 cause nearly, but not totally, wild-type phenotypes. A nonsense mutation in sqt-1 suppresses the phenotype of rol-6 RRol mutations, suggesting that rol-6 collagen function is dependent on the presence of sqt-1 collagen. Mutations of sqt-1 are not suppressed by a rol-6 nonsense mutation, however, indicating that sqt-1 collagen can function independently of rol-6.

Alleles↗
PubMed1993

Splicing in Caenorhabditis elegans does not require an AG at the 3' splice acceptor site.

The dinucleotide AG, found at the 3' end of virtually all eukaryotic pre-mRNA introns, is thought to be essential for splicing. Reduction-of-function mutations in two Caenorhabditis elegans genes, the receptor tyrosine kinase gene let-23 and the collagen gene dpy-10, both alter the AG at the end of a short (ca. 50-nucleotide) intron to AA. The in vivo effects of these mutations were studied by sequencing polymerase chain reaction-amplified reverse-transcribed RNA isolated from the two mutants. As expected, we find transcripts that splice to a cryptic AG, skip an exon, and retain an unspliced intron. However, we also find significant levels of splicing at the mutated 3' splice site (AA) and at nearby non-AG dinucleotides. Our results indicate that for short C. elegans introns an AG is not required for splicing at either the correct 3' splice site or incorrect sites. Analysis of a splice site mutant involving a longer, 316-nucleotide C. elegans intron indicates that an AG is also not required there for splicing. We hypothesize that elements besides the invariant AG, e.g., an A-U-rich region, a UUUC motif, and/or a potential branch point sequence, are directing the selection of the 3' splice site and that in wild-type genes these elements cooperate so that proper splicing occurs.

Animals↗
PubMed1992

Bacillus cereus meningitis in two neurosurgical patients: an investigation into the source of the organism.

Two patients developed Bacillus cereus meningitis following neurosurgery. During the subsequent investigation into the source of the organism, linen was discovered to be heavily contaminated with B. cereus. No other prolific source of the organism was found. It seems probable that lint from contaminated fabric was the vehicle of transmission of the organism during extended surgery. Linen should be considered as a possible source of B. cereus infection.

Adult↗