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Biomedical subjects

R C Shuster

Publications and source records attributed to R C Shuster.

At least 19 recordsLinked to original sources
PubMed1992

Activation of insulin receptor signaling by a single amino acid substitution in the transmembrane domain.

The insulin receptor is a ligand-activated tyrosine kinase composed of two alpha and two beta subunits. A single transmembrane domain composed of 23 hydrophobic residues is contained in each beta subunit. We examined the role of the transmembrane domain in regulating insulin receptor signaling by inserting a negatively charged amino acid (Asp) for Val938 (V938D). Chinese hamster ovary (CHO) cells were stably transfected with a plasmid containing both the neomycin-resistance gene and either the wild-type or the mutant (V938D) insulin receptor cDNA. Insulin binding increased similarly in CHO cells stably transfected with the wild-type and the V938D-mutant insulin receptor cDNA. Insulin stimulated glucose transport and cell growth in cells expressing the normal insulin receptor. By contrast, in the absence of insulin, glucose transport and cell growth in CHO-V938D cells were as high as in insulin-stimulated control cells and no longer responsive to insulin stimulation. Phosphorylation of the beta subunit of the insulin receptor was also increased in CHO-V938D cells not exposed to insulin. These results support an essential role of the transmembrane domain of the insulin receptor in the transduction of insulin signaling.

Animals↗
PubMed1990

Insulin-receptor autophosphorylation and kinase activity are constitutively increased in fibroblasts cultured from a patient with heritable insulin-resistance.

Mutations in the insulin receptor gene have been described in families with the inherited insulin-resistant syndrome leprechaunism. At a cellular level, these mutations result in decreased insulin binding and impaired insulin stimulation of receptor autophosphorylation and sugar transport. By contrast, we previously found that fibroblasts cultured from leprechaun patient Atl had constitutively increased sugar transport, even though insulin binding was markedly reduced. Here we report that these fibroblasts have basal insulin-receptor autophosphorylation and kinase activity constitutively increased above insulin-stimulated control cells.

Cells, Cultured↗
PubMed1990

Human fibroblasts express the insulin-responsive glucose transporter (GLUT4).

The transport of glucose across the plasma membrane of nonepithelial cells is mediated by a family of facilitative glucose transporters. One glucose transporter is insulin-responsive (GLUT4) and is found in muscle, heart, and fat cells, differentiated cells which are difficult to maintain and study in culture. Cultured dermal fibroblasts, on the other hand, also are insulin-responsive, can be easily maintained in culture, and retain the genetic complement of the donor. In this paper, we evaluate RNA isolated from cultured human fibroblasts for the expression of four different glucose transporters. Northern blot analysis indicated that human fibroblasts expressed the erythrocyte (GLUT1), the fetal skeletal muscle (GLUT3), and the insulin-responsive (GLUT4) glucose transporters, but not the liver glucose transporter (GLUT2). To confirm the presence of GLUT4 mRNA, cDNA was synthesized from human fibroblast RNA, amplified using primers specific for GLUT4 by the polymerase chain reaction, and sequenced. The sequence was identical to that of GLUT4 cDNA. These data indicate that cultured human fibroblasts express at least three genetically distinct facilitative glucose transporters.

Base Sequence↗
PubMed1989

Increased glucose transport by human fibroblasts with a heritable defect in insulin binding.

Insulin and IGF-I binding and their regulation of hexose transport were evaluated in skin fibroblasts cultured from a family (Atl) whose proband had leprechaunism, hypoglycemia, and severe insulin resistance. High affinity insulin binding to proband Atl cells was absent, and partially, but equally, impaired in fibroblasts from his related parents. IGF-I binding to his cultured fibroblasts was within the normal range. Cells from proband Atl had insulin receptor mRNAs similar to control fibroblasts. 3-O-Methyl-D-glucose (OMG) transport by proband Atl was threefold higher than in control fibroblasts (37.7 v 7.6-11 nmol/mL/s) and was insulin-insensitive. Proband Atl fibroblasts had a threefold increase in the Vmax for OMG entry and a concomitant increase in the number of D-glucose-inhibitable cytochalasin B binding sites on their plasma membrane. Similar levels of glucose transporter mRNA were observed in control and proband Atl fibroblasts. These results suggest that fibroblasts from patient Atl have a genetically transmitted mutation in the alpha subunit of their insulin receptor. In the homozygous affected proband, this mutation impairs insulin binding and causes elevated, insulin-insensitive glucose transport. The dysfunction resulting from this mutation is similar to that introduced in Chinese hamster ovary cells by transfection with a truncated alpha subunit.

3-O-Methylglucose↗
PubMed1988

Mitochondrial DNA in anucleate human blood cells.

Homogeneous populations of human blood platelets or erythrocytes were lysed in alkaline EDTA, bound to nitrocellulose and hybridized to a radioactive mtDNA probe. By comparison to standards of known mtDNA concentration, we determined that platelets contained 4 mtDNA molecules per cell. Rhodamine 123 staining revealed an average of 4 mitochondria per platelet indicating that each mitochondrion contains a single mtDNA molecule. No detectable mtDNA was found in erythrocyte lysates. Using the same procedure, we found that in nucleated cells, mitochondria contained multiple mtDNAs per mitochondrion.

Blood Platelets↗
PubMed1984

Evidence that the cro repressor inhibits expression of the bacteriophage lambda P gene at high multiplicities of infection.

The activity of the lambda P gene product at various multiplicities of infection (m.o.i.) was examined in CI- conditions using an assay which measures the disappearance of the rapidly-sedimenting closed-circular (c.c.) form of phage DNA. When cells were infected with lambda CI857 at multiplicities of 5 phage/cell or less, between 65%-75% c.c. DNA was lost during incubation. If the multiplicity of infection was increased to 10 phage/cell or greater, a marked inhibition in the cleavage of c.c. DNA was observed. When bacteria were infected with either lambda CI857CII2002 or lambda CI857cro27 at low m.o.i., the usual 65%-75% decrease in the percentage of c.c. phage DNA occurred during incubation. In contrast, no losses in c.c. DNA were noted after infection with lambda CI857cro27susP3. At high m.o.i., the cleavage of c.c. DNA was inhibited after infection with lambda CI857CII2002, but not after infection with lambda CI857cro27. It is concluded that at high m.o.i. in CI- infections, the expression of gene P is unaffected by the CII gene product, but is inhibited by the increased intracellular levels of cro protein.

Bacteriophage lambda↗
PubMed1980

On the role of the dnaB protein of Escherichia coli in the replication of lambda bacteriophage DNA.

The interaction between the dnaB protein of E. coli and the gene P product of lambda bacteriophage was investigated by measuring the cleavage of closed circular phage DNA after infection of two temperature sensitive dnaB mutants, JG28 and To534 groP- B. Cleavage of superhelical DNA from a lambda pi B mutant phage was observed after infection of either strain whereas superhelical DNA from a wild type phage was only cleaved after infection of JG28. When DNA synthesis in infected cells was blocked by incubation at the nonpermissive temperature, no inhibition of superhelical phage DNA cleavage was observed. It is concluded that in conditions where the dnaB protein has lost the capacity to function in nucleotide polymerization, it is capable of interacting with the lambda replication gene products to introduce a break in the phage DNA.

Bacterial Proteins↗
PubMed1976

Studies on the synthesis of casein messenger RNA during pregnancy in the rabbit.

The quantity of casein mRNA in the rabbit mammary gland was assayed during the course of pregnancy, by translation of casein mRNA in a reticulocyte lysate and by hybridization to DNA complementary to purified casein mRNA. Both methods indicated that the major increases in the concentration of casein mRNA in both polysomal and total cellular RNA occurred between the 18th and 25th day of pregnancy. The change in casein mRNA concentration during this period coincided with a sharp rise in casein synthesis in mammary gland explants suggesting that the levels of casein mRNA determine the rate of casein synthesis in the mammary gland. The sensitivity of the hybridization assay made it possible to detect the presence of casein mRNA in virgin animals and during the very early stages of pregnancy. At day 5, casein mRNA was found associated with polysomes indication that there was probably some casein synthesis at this early stage of gestation. These results suggest that the hormones controlling lactogenesis in the rabbit may function by augmenting the rate of casein mRNA synthesis rather that initiating transcription of previously inactive genes.

Animals↗
PubMed1973

Influence of thymine starvation on the integrity of episomal and chromosomal deoxyribonucleic acids in Escherichia coli CR34 (lambda ind-).

The effect of thymine deprivation on the integrity of phage lambda, sex factor, and chromosomal deoxyribonucleic acid (DNA) in Escherichia coli CR34 (lambda ind(-)) was examined by sedimenting cell lysates through alkaline sucrose gradients. Both sex factor and chromosomal DNAs showed evidence of being degraded during the starvation period. In contrast, no loss of closed circular lambda DNA was observed.

Carbon Radioisotopes↗
PubMed1968

Evidence for a new endonuclease synthesized by lambda bacteriophage.

Infection of nonlysogenic Escherichia coli CR34(S) (Thy(-)) with bacteriophage lambda C(I)857 resulted in the formation of twisted circular double-stranded phage deoxyribonucleic acid (DNA; species I). When such infected bacteria were incubated in the absence of thymine, there was a significant decrease in the amount of species I DNA after 60 min of incubation. A similar loss of species I lambda DNA during incubation in a thymine-deficient medium was also observed after infection of the endonuclease I-deficient strain, E. coli 1100(S) (Thy(-)). This destruction of twisted, circular lambda DNA in thymine-deprived cells did not occur in the presence of chloramphenicol nor in lysogenic E. coli CR34 carrying a noninducible lambda prophage. It is therefore concluded that the endonuclease which attacks this circular configuration of lambda DNA is newly synthesized after infection and is directed by the phage chromosome.

Centrifugation, Zonal↗
PubMed1967

Fate of thymine-containing dimers in the deoxyribonucleic acid of ultravioletirradiated Bacillus subtilis.

The fate of ultraviolet-induced, thymine-containing dimers in the deoxyribonucleic acid (DNA) of Bacillus subtilis was investigated in both the wild type (UV(R)) and an ultraviolet light-sensitive (UV(S)) mutant. During incubation in the dark, dimers were excised from the DNA of the UV(R)B. subtilis, but remained in the DNA of the UV(S) mutant. About 40% of the excised dimers recovered in the wild type were in the acid-soluble fraction; the remainder were in the incubation medium. A UV(S) mutant of Escherichia coli K-12, shown previously to be defective in dimer excision, was irradiated with ultraviolet light and incubated under visible light for 3 hr. About 65% of thymine-containing photoproducts were removed from the DNA. These photoproducts were not recovered in the acid-soluble fraction. In comparison, the UV(S) mutant of B. subtilis lost only 13% of such photoproducts from DNA when exposed to light under the same conditions.

Bacillus subtilis↗