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Biomedical subjects

I A Bernstein

Publications and source records attributed to I A Bernstein.

At least 55 records · Page 3Linked to original sources
PubMed1980

Effect of microtubule-disrupting drugs on protein and RNA synthesis in Physarum polycephalum amoebae.

The effects of the microtubule-disrupting drugs, colchicine, vinblastine, podophyllotoxin, griseofulvin, and lumicolchicine (10(-5) M), on protein and RNA synthesis were studied in Physarum polycephalum amoebae in culture. All, except lumicolchicine, were found to simultaneously reduce the rate of protein synthesis and stimulate RNA synthesis. These results parallel the effects seen in cells exposed to heat shock. Treatment of cells with a microfilament-disrupting drug, cytochalasin B (10 micrograms/ml in ethanol), resulted in a reduced rate of protein synthesis after 2 h compared to a similar effect by vinblastine in 5--15 min. A morphological abnormality, microtubule paracrystals, were seen associated with centrioles in vinblastine-treated cells in which protein synthesis had been reduced by 50%. Vinblastine and podophyllotoxin were shown to interfere with the recovery of protein synthesis after inhibition by low or elevated temperatures. The possible role of microtubules in regulating the translational response of a cell to an external environmental stimulus is discussed.

Centrioles↗
PubMed1980

Differential lectin binding to cellular membranes in the epidermis of the newborn rat.

Three fluorescein-labeled lectins have been shown to exhibit specificity for the surface of cells in different layers of the epidermis in the newborn rat. An isolectin from seeds of Bandeiraea simplicifolia with specificity for alpha-D-galactosyl end groups labeled the basal and lower spinous cells; a lectin from Ulex europaeus exhibiting specificity for alpha-L-fucosyl units outlines the surface of spinous cells, and a second lectin from B. simplicifolia, with specificity for N-acetyl-D-glucosamine, labels the cornified cells. Appropriate blocking experiments have confirmed the specific nature of the binding.

Animals↗
PubMed1980

Molecular markers of differentiation in the epidermis of the newborn rat.

Regulation in epidermal differentiation can best be studied if molecular mechanism can be associated with structural and functional changes. Such recognized associations include the cessation of mitosis through inhibition of DNA replication by a G1-inhibitor present in the suprabasal cells, the biosynthesis of a tonofilament-protein as an early event in keratinization, the biosynthesis of HRP0 (histidine-rich protein) and its polymerization to HRPI during the formation of keratohyalin, the conversion of HRPI to HRPII coincident with the loss of the nucleus from the granular cell, and the aggregation of the stratum corneum basic protein and keratin filaments to form fibers in the cornified cell. To this list can now be added changes in specificity for lectin-binding to the cell surface as the keratinocyte progresses toward the cutaneous surface. This report presents data on a) the conversion of HRPI to HRPII and b) the differential lectin-binding in the epidermis of the newborn rat. HRPI (Mol. Wgt. greater than or equal to 10(6)) and HRPII (Mol. Wgt. 6 X 10(4)) have similar unique amino acid compositions and exhibit extensive-but not complete-homology in primary structures as determined by peptide mapping after exposure to trypsin. When labeled by exposure in vivo to radioactivity histidine, about 75 of the labeled histidine from both HRPI and HRPII appeared in one peptide fraction in the map, HRPI appears to have on histidine-containing fragment which is not present in HRPII. This peptide appears to contain phosphate and to account for the organically-bound phosphate which was found in HRPI but not defected in HRPII. Changes which occur in the lectin-binding specificity of the cell during differentiation may result from either movement or chemical change in carbohydrates at the cell surface. Immunofluorescent studies have shown that an isolectin from Bandieraea simplicifolia with specificity for alpha-D-galactose binds to the surface of basal and lower spinous cells, a lectin from Ulex europaeus with specificity for alpha-L-focus labels spinous cells, and a second lectin from B. simplicifolia with specificity for N-acetyl-D-glucosamine labels cornified cells. The relationship fo these alterations in the carbohydrates of the cell surface in intracellular structural and/or functional changes in unknown.

Amino Acid Sequence↗
PubMed1978

Turnover of metallothioneins in rat liver.

Two electrophoretically distinguishable metallothioneins were isolated from the livers of Cd2+-treated rats and had thiol group/metal ratios of 3:1, a total metal content, in each of these proteins, of 3.6 atoms of Cd2+ + 2.4 atoms of Zn2+/molecule and 4.2 atoms of Cd2+ + 2.8 atoms of Zn2+/molecule and respective apoprotein mol.wts. of 5844 and 6251. Studies with 1 h pulse labels of [3H]cysteine, given after a single injection of ZnCl2 or CdCl2, showed that these metals stimulated radioactive isotope incorporation into the metallothioneins over the control value by 10- and 15-fold respectively. This stimulation was maximal at 4 h after a single CdCl2 injection and decreased to control values by 16 h, suggesting that either a translational event is responding to free intracellular Cd2+ or a short-lived mRNA is being produced or stabilized in response to the metal treatment. In rats chronically exposed to CdCl2, the metallothioneins increased to 0.2% of the liver wet weight from a control value of 2--4 mumol/kg of liver, with a maximum rate of accumulation of 2--3 mumol/h per kg of liver. The turnover of these proteins in control animals was 0.3--0.6 mumoles/h per kg of liver, measured by the rate of disappearance of 203Hg2+, which binds irreversibly to the metallothioneins. Pretreatment with CdCl2 completely stopped the rapid 203Hg turnover observed in untreated animals. Unlike CdCl2, treatment with ZnCl2 increased the concentration of metallothioneins to a new steady-state pool, 11 mumole/kg of liver, after 10 h. The increase in the zinc-thionein pool by exposure to ZnCl2 in vivo was determined to be primarily due to a stimulation of metallothionein biosynthesis.

Amino Acids↗
PubMed1978

Single-strand breakage in DNA of Escherichia coli exposed to Cd2+.

When a growing culture of Escherichia coli was exposed to 3 X 10(-6) M Cd2+, 85 to 95% of the cells lost their ability to form colonies on agar plates. Loss of viability was accompanied by considerable single-strand breakage in the DNA, with no detectable increase in double-strand breaks. A direct correlation appeared to exist between the number of single-strand breaks and the concentrations of Cd2+ to which the cells were exposed. Exposure of DNA in vitro to a Cd2+ concentration of 3 X 10(-6) M or higher, followed by sedimentation in alkaline sucrose gradients, demonstrated no single-strand breaks. Cadmium-exposed cells recovered viability when incubated in Cd2+-free liquid medium containing 10 mM hydroxyurea. During the early period of recovery, there was a lag in the incorporation of labeled thymidine, but cellular DNA, at least in part, appeared to be repaired.

Cadmium↗
PubMed1977

Tonofilament protein from newborn rat epidermis. Isolation, localization, and biosynthesis of marker of epidermal differentiation.

A novel extraction procedure, previously used on the cell walls of dermatophytes, has been applied to the epidermis of newborn rat. A leucine-rich fraction was isolated which contained over 60% of the total epidermal radioactivity from [3H]leucine in 15 to 20% of the total protein. This fraction was further purified by chromatography in DEAE-cellulose and Sephadex G-200. The protein with the highest specific activity from [3H]leucine was isolated and gave a single band in sodium dodecyl sulfate polyacrylamide gels of molecular weight = 58,000. Antibody to this protein gave a single precipitin band by immunodiffusion and immunoelectrophoresis in agar with the purified protein. This antibody ultrastructurally immunolocalized specifically over tonofilaments in all layers of the epidermis, but showed no reaction in the dermis. The synthesis of this protein in vitro was inhibited by puromycin but not by actinomycin D, suggesting ribosomal synthesis involving a relatively long lived messenger.

Amino Acids↗
PubMed1977

Separation of epidermal layers of the newborn rat.

A method is presented for the separation of epidermal strata by the successive elimination of either the basal or basal and spinous cells with 0.24 M NH4Cl at pH 9.5. Histologic evidence suggests that the residual epidermal strata obtained after incubation of the skin with NH4Cl are reproducible; hence, this technique circumvents loss of granular layer histidine-rich protein inherent with trypsin separation and provides an effective procedure for biochemical analysis of arginine-rich and lysine-rich proteins in the various differentiating epidermal cells.

Ammonium Chloride↗
PubMed1976

Molecular aspects of control in epidermal differentiation.

The mammalian epidermis is organized into layers of structurally different cells--the basal, spinous, granular and cornified layers--which represent steps in the differentiative process that terminates in cornification and desquamation. Investigation of the molecular mechanisms that control this ordered sequence of events provides clues to the etiology of certain epidermal pathologies. DNA synthesis and mitosis are normally restricted to the basal layer. Several substances have been implicated in the mitotic control of epidermal cells, the loss of mitotic activity being the first major step in normal keratinization. Investigations performed in this laboratory indicate that isolated differentiated nuclei can replicate their DNA which they are inhibited from doing in situ. Addition of a high speed supernate from homogenized differentiated cells inhibited this synthetic activity in vitro suggesting the existence of a cytoplasmic inhibitor of DNA synthesis. It is not known whether mitotic inhibition in differentiated epidermal cells is a function of the inhibition of DNA replication. Contrary to previous assumptions, recent experimental evidence clearly indicates that, unlike DNA synthesis, RNA synthesis occurs in differentiated cells. Correlated with this synthetic activity is the observation that a protein rich in histidine is specifically formed in the granular cells. This protein appears to be a component of the keratohyalin granules which fill the cells of the granular layer. Investigations were conducted in this laboratory to determine whether control of the synthesis of this protein occurs at the level of translation or transcription. Translation, in vitro, of mRNA obtained from isolated populations of each epidermal cell type suggested that control of protein synthesis in the differentiating epidermis is transcriptional, i.e. only in the granular cell is there an mRNA for the histidine-rich protein. Transcription, in vitro, of chormatin isolated from the separated cell populations produced RNA with a ratio of cytidine to uracil consistent with the predicted mRNA for this protein thus providing additional support for the hypothesis that epidermal differentiation is controlled at the level of 'gene-readout'.

Animals↗
PubMed1976

Synthesis of DNA in isolated nuclei from differentiated mammalin epidermal cells.

Epidermal spinous and granular cells from the newborn rat neither replicate their nuclear DNA nor proliferate in vitro under conditions which support both processes in basal cells. However, as shown by autoradiography, (3H)thymidine and (14C)bromodeoxyuridine do label nuclei removed from spinous cells but not from granular cells. CsCl density gradient centrifugation of DNA obtained from early differentiated nuclei which had been exposed in vitro to (14C)bromodeoxyuridine, indicated that a considerable level of the tracer was present in the nucleic acid and suggested that replication of the genome had occurred. Therefore, spinous cells appear to retain the capability of reproducing nuclear DNA. Since differentiated cells appear to have the "diploid" level of DNA, these observations point to the replication of DNA as a possible locus of the mitotic inhibition which is coincident with epidermal differentiation.

Animals↗
PubMed1975

Intracellular localization of fructose 1,6-bisphosphate aldolase.

Submission of a rat liver homogenate made in 250 mM sucrose-1 mM EDTA to centrifugation between 9,500 times g for 10 min and 105,000 times g for 60 min results in the sedimentation of 60 to 70% of the total cellular fructose 1,6-bisphosphate aldolase (EC 4.1.2.13). Under these conditions only about one-quarter of the total triose phosphate dehydrogenase and phosphoglycerate kinase appears in the microsomal fraction. Ultrastructural immunologic localization techniques have demonstrated that the aldolase is associated with the endoplasmic reticulum, in situ. The binding of this enzyme to the membrane is sensitive to changes in pH with an optimum at 6.0, and to increasing concentrations of NaCl and fructose 1,6-bisphosphate, being about 100-fold more sensitive to the ester than to the inorganic salt.

Animals↗