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Biomedical subjects

J Kruh

Publications and source records attributed to J Kruh.

At least 19 recordsLinked to original sources
PubMed1993

Characterization, purification and cDNA cloning of a rat perchloric-acid-soluble 23-kDa protein present only in liver and kidney.

A novel protein was extracted with 5% perchloric acid from rat liver and kidney. It is absent from other rat organs. Its apparent molecular mass is 23 kDa as determined by HPLC gel filtration. A single band, corresponding to 10 kDa, was observed after SDS/PAGE, suggesting that the protein consists of two subunits with similar molecular masses. This protein can neither be phosphorylated by ATP, nor acetylated. The sequence of the cDNA encoding this protein was determined. Southern-blot analysis showed that the corresponding gene spanned at least 10 kb and contained at least five introns. Zoo-blot analysis at medium stringency strongly suggests that the gene has been conserved during evolution. The amino-acid sequence of this protein with a highly conserved region is similar to that of a heat-shock protein.

Amino Acid Sequence↗
PubMed1992

[Molecular and cellular action of butyrate].

Butyrate has a dramatic effect on transformed cells in culture. This effect disappears as soon as butyrate is removed from the medium. The other short chain fatty acids are much less effective. Butyrate produces an arrest of cell proliferation at the early G1 phase of the cell cycle. The effect is very general and may be used for cell growth synchronization. This compound increases the expression of the c-fos oncogene and inhibits the expression of c-myc in all phases of the cell cycle. Butyrate modulates the expression of several genes. In general it induces the expression of markers of cell differentiation. Many studies have been devoted to hemoglobin synthesis which is induced in erythroleukemia cells. In general it induces the synthesis of embryonic and of fetal hemoglobin, and delays and even suppresses the switch to adult hemoglobin, which could be useful for the treatment of sickle cell anemia and beta thalassemia. This effect of butyrate seems to require specific DNA regulatory sequences. Butyrate induces the synthesis of alkaline phosphatase, placental and intestinal isozymes, especially in cells where these syntheses are ectopic. It has the same effect on peptidic hormone syntheses and also on receptors of thyroid hormone and insulin. It stimulates their synthesis in cells which are poor in receptor and inhibits the synthesis in cells which have high amounts of these receptors. The use of antibiotics and of the run on method strongly suggest that butyrate acts at the transcriptional level. Butyrate inhibits the induction of proteins, including enzymes, by steroid hormones as has been shown for the induction of tyrosine aminotransferase by glucocorticoids, of ovalbumin and transferrin by estradiol in chick oviduct. Butyrate strongly alters cell morphology, usually it produces an enlargement of the cells with formation of protrusions. In HTC cells alteration of nucleoli and of the nuclear shape are observed. All these alterations are reversible and the cells recover the normal morphology upon removal of butyrate. These alterations result at least partly from modifications of the cytoskeleton: induction of vimentin and cytokeratin, formation of microfilaments, of microtubules and of actin fibers. The external matrix is also modified, as are the cell surface glycoproteins, and gangliosides. Most of these alterations are consistent with the loss of transformation characteristics of the cell. The mechanism of action of butyrate has been studied by many authors. It has been well established that butyrate induces an hyperacetylation of histones by inhibiting histone deacetylases, which is consistent with its stimulatory effect on gene expression.4+ and would require transacting proteins. The use of butyrate in therapeutics would require the synthesis of new molecules including butyrate but more active and metabolized at a slower rate. Several such molecules have been synthesized: monobutyrate 3 (or 6) monoacetate glucose, pivalyloxymethyl-butyrate. The use of such molecules in human therapeutics has been suggested, especially in hematology (sickle cell anemia, beta thalassemia) and in cancerology.

Animals↗
PubMed1991

Stimulation of methotrexate resistance and dihydrofolate reductase gene amplification by c-myc.

We have hypothesized that the c-myc oncogene might promote DNA amplification. Resistance to methotrexate (MTX), a widely used cancer chemotherapeutic agent, often results from amplification of the gene coding for the target enzyme, dihydrofolate reductase (DHFR). We report here that gratuitously induced expression of c-myc in rat fibroblasts grown in the presence of MTX greatly increases the number of colonies resistant to the drug. This effect is not related to an alteration of cell growth, and it can also be observed to a lesser extent when c-myc is induced prior to selection in MTX. The DHFR gene is amplified in nearly half of the colonies cultured under selection conditions. Given the likely role of the c-myc product in DNA replication, these results strongly suggest that expression of c-myc plays a role in methotrexate resistance by promoting DNA amplification.

Animals↗
PubMed1990

Sodium butyrate inhibits c-myc and stimulates c-fos expression in all the steps of the cell-cycle in hepatoma tissue cultured cells.

Sodium butyrate decreases the c-myc mRNA and increases the c-fos transcript level in HTC cells. This effect is independent of the cell-cycle phase. Actinomycin D suppresses the effect on c-fos. Cycloheximide increases both mRNA levels. Sodium butyrate suppresses the effect on c-myc and potentializes the effect on c-fos mRNAs. This suggests that sodium butyrate acts at the transcriptional level and that its effect does not result from the arrest of the cells at the G1 phase.

Animals↗
PubMed1989

Detection of minority point mutations by modified PCR technique: a new approach for a sensitive diagnosis of tumor-progression markers.

The detection of point mutations correlated with diseases, in enzymatically amplified DNA sequences (Polymerase Chain Reaction), is currently performed by digestion of PCR products when an existing restriction site disappears at least in one allele of the amplified mutated sequence or by allele specific radiolabeled probes in all other cases. These methods are the most sensitive but they cannot detect a mutation if it is present in less than 5% of the studied cells. We describe here a method based on the introduction of an artificial restriction site, using a modified primer during the PCR, which creates a RFLP indicative of the studied mutation. This RFLP is detected by a radiolabeled oligonucleotide probe which is not related to the mutation. Our approach multiplies the sensitivity by a factor of 1000 and it is practical for use in screening purposes and the detection, after treatment, of the residual disease in human malignancies. Using this method we detected 20% more mutations at codon 12 in the Ki ras oncogene in DNA from colorectal cancers that were undetectable with all the previous methods.

Base Sequence↗
PubMed1989

DNA sequences homologous to mitochondrial genes in nuclei from normal rat tissues and from rat hepatoma cells.

Using specific probes we show that sequences homologous to NADH dehydrogenase Subunit 6, and Cytochrome oxidase Subunits I, II, and III mitochondrial genes are present in nuclear DNA from various tissues. These mitochondrial-like sequences are also present in rat hepatoma nuclear DNA but with an abnormal organization and a higher copy number than in normal hepatocytes.

Animals↗
PubMed1989

RNAs containing mitochondrial ND6 and COI sequences present an abnormal structure in chemically induced rat hepatomas.

We have constructed a cDNA library prepared from an hepatoma cell line (HTC cells) and isolated a clone, pHT 13, which corresponds to mRNAs present at a much higher level in rat hepatomas than in normal hepatocytes. The sequence of the pHT 13 insert has been previously published (Nucleic Acids Res. 1988, 16,10935). This clone contains mitochondrial DNA sequences with an abnormal organization, since it includes part of the NADH dehydrogenase subunit 6 (ND6) and of the cytochrome oxidase subunit I (COI) genes separated by 230 bases instead of 9 kb in mitochondrial genome from normal hepatocytes. In this work we show (1) that RNAs homologous to this clone are present in hepatoma cells but not in normal hepatocytes, (2) that a 3 kb fragment of tumor mitochondrial DNA contains both the ND6 and the COI sequences. The abnormal structure of the DNA is confirmed by Southern blot analysis which shows that distinct types of mitochondrial DNAs are present in hepatoma cells.

Animals↗
PubMed1989

Increased level of the mitochondrial ND5 transcript in chemically induced rat hepatomas.

We have constructed a cDNA library from a hepatoma cell line (HTC cells) and isolated the clones corresponding to mRNAs present at a much higher level in hepatomas than in normal hepatocytes. The characterization of one of these clones is described in this paper. This clone is homologous to part of the mitochondrial ND5 gene (a subunit of NADH-ubiquinone oxidoreductase). The level of this mRNA was found increased in HTC cells and in hepatocytes from diethylnitrosamine-treated rats long before the development of tumors and strongly increased in carcinoma nodules as compared to hepatocytes from nontreated rats. Southern blot analysis showed a mitochondrial DNA heterogeneity in hepatomas with an alteration of the structure of part of the molecules.

Amino Acid Sequence↗
PubMed1988

Increased expression of the N-myc gene during normal and neoplastic rat liver growth.

The activation of N-myc and c-myc genes has been implicated in the genesis of a variety of cancers. In order to determine whether this activation is related to the process of cell growth, we examined N-myc and c-myc expression in rat hepatocytes when growth was stimulated by partial hepatectomy and in rat liver during neoplastic growth induced by diethylnitrosamine. The levels of N-myc and c-myc mRNAs, which are very low in hepatocytes from normal rats, were increased at least 20-fold within 3 h after partial hepatectomy and decreased rapidly by 6 h. A second transient peak of c-myc and N-myc mRNAs occurred around 9 and 48 h, respectively. N-myc and c-myc expressions were also induced in carcinoma nodules by diethylnitrosamine. A high level of N-myc transcript was observed in hepatocytes as early as 1 month after the carcinogen administration, whereas c-myc transcript was detected at a high level only several months later in carcinoma nodules. Our results suggest that the transient expression of N-myc and c-myc oncogenes during the prereplicative stage of liver regeneration may be associated with the entry of hepatocytes into the cell cycle, and that N-myc expression is not limited to tumors of neural characteristics as has been previously shown but is also observed in cancer from epithelial origin.

Animals↗
PubMed1987

The effects of phorbol ester and Ca ionophore on c-fos and c-myc expression and on DNA synthesis in human lymphocytes are not directly related.

Phorbol ester activates human lymphocyte proliferation as measured by [3H]thymidine incorporation and blast transformation. Phorbol ester and calcium ionophore A 23187 induce c-fos and c-myc expression in these cells as shown by the measure of the specific mRNA levels and of the c-fos nuclear protein amount. The combination of these two agents has a synergistic effect on c-fos but not on c-myc expression. Lymphocyte stimulation by phorbol ester for a short time induces c-fos and c-myc expression, but has no effect on [3H]thymidine incorporation. This result indicates that the induction of c-fos and c-myc expression is not sufficient to commit these cells to DNA synthesis.

Adult↗
PubMed1987

Characterization of an arginine-specific protein kinase tightly bound to rat liver DNA.

A new protein kinase has been characterized among the proteins tightly bound to rat liver DNA and released by DNase I and RNase A treatment. This enzyme was separated by gel filtration from this released material. Its apparent molecular mass was found to be 34 kDa and it is made of a single unit. The main characteristic of this protein kinase is that it is arginine-specific. Isolation of phosphoarginine required the use of proteolytic enzymes at alkaline pH since the phosphate bond is highly acid-labile. This protein kinase is able to autophosphorylate and to phosphorylate a single chromosomal protein of 11 kDa also tightly bound to DNA. It uses ATP and dATP as phosphate donors and is cAMP-independent. Its optimal activity requires Mn2+ ions. Vanadate, spermine and heparin have no effect on its activity.

Animals↗
PubMed1987

c-myc oncogene expression inhibits the initiation of myogenic differentiation.

The role of c-myc oncogene expression in myogenic differentiation has been established by transfecting rat myoblasts of the L6 cell line with plasmid pMT-myc, in which the c-myc coding sequences were under the control of the metallothionein I promoter. We observed that the constitutive expression of the exogenous c-myc gene inhibits muscular differentiation. A diminution of the endogenous c-myc gene expression occurs within the first 24 h after the transfer of the cells to a differentiating medium. This early decrease of c-myc expression is required for cell differentiation to occur. We have also observed that exogenous myc gene expression has no effect on endogenous myc expression.

Animals↗
PubMed1987

Expression of c-myc is under dietary control in rat liver.

Expression of c-myc has often been related to the control of growth and differentiation of a variety of cell types. However, in some cases, such a relation has not been found. The rate of cell division is very low in liver, but c-myc expression is yet easily detected. We show here that a short-time physiological fasting results in a dramatic decrease of c-myc expression in rat liver. This effect does not seem to be dependent on glucagon, since administration of glucagon leads to an increase in c-myc mRNA. This is to our knowledge, the first evidence of a physiological variation of proto-oncogene expression linked to food intake, and we suggest that this variation could play a role in liver cell growth control.

Animals↗
PubMed1986

Rat liver nuclear protein kinases NI and NII. Purification, subunit composition, substrate specificity, possible levels of regulation.

Rat liver nuclear protein kinases NI and NII have been purified to homogeneity by an improved method. This method includes a casein-phosvitin-Sepharose column step, which separates the enzymes from the other chromosomal non-histone proteins, and a gel filtration at high ionic strength in the presence of a high concentration of protease inhibitors to separate the two enzymes from each other. NI has an apparent molecular mass of approximately 50 kDa and is composed of a single subunit. NII has an apparent molecular mass of 133 kDa and is composed of two subunits of identical molecular mass. The V and the Km of the two enzymes were determined for several substrates. Both enzymes phosphorylate chromosomal non-histone proteins with partly different specificities as shown by two-dimensional electrophoreses. When incubated in the absence of protease inhibitors, the enzymes were degraded into discrete polypeptides. Autophosphorylation of a polypeptide derived from NII was observed after incubation of the enzyme with ATP. This phosphorylation stimulated the enzyme activity. Several chromosomal proteins coeluted with NII from the casein-phosvitin-Sepharose column. They remained associated with the enzyme in sucrose gradients, during gel filtration performed at physiological ionic strength, and are dissociated at high ionic strength. These proteins were highly phosphorylated when the protein-NII complex was incubated with ATP.

Amino Acids↗
PubMed1986

Separation of nuclear cAMP independent protein kinases NI and NII from their chromosomal protein substrates and enzyme inhibitors by the use of a casein-phosvitin-Sepharose column.

A casein-phosvitin-Sepharose chromatography column allows separation of nuclear protein kinases from their chromosomal phosphoprotein substrates and from at least some protein kinase inhibitors in a single step. The additional step of passing the eluted material through a partially hydrolyzed, dephosphorylated casein-Sepharose column separates the two protein kinases, NI and NII, from each other.

Animals↗
PubMed1986

Isolation and characterization of complementary DNA clones for genes overexpressed in chemically induced rat hepatomas.

In order to characterize the genes overexpressed in an hepatoma cell line, the HTC cells, and in diethylnitrosamine induced solid hepatomas, we constructed a complementary DNA library from HTC cells and performed differential screening with probes from HTC cells, from malignant nodules obtained 70 weeks after the carcinogen treatment, and from hepatocytes from normal rat liver. Eight clones corresponding to messenger RNAs (mRNAs) much more expressed in hepatomas than in hepatocytes from normal liver were isolated. Three, clones pHT 71, pHT 13, and pHT 26, were further analyzed by the study of their corresponding transcripts in hepatocytes from regenerating liver and in the hepatocytes from the nontumorous parts of the liver. Clone pHT 71 corresponds to a single 2.3-kilobase mRNA which is present in high levels in carcinoma nodules in hepatoma cell lines, in the nontumorous parts of the liver, and in hepatocytes isolated from regenerating liver 30 h after partial hepatectomy. Clone pHT 13 hybridizes with three distinct transcripts 3.8, 2.6, and 1.6 kilobases long. High levels of the 3.8- and 1.6-kilobase mRNAs are present in carcinoma nodules, in hepatoma cell lines, and in the nontumorous parts of the liver. However, the levels of these RNAs are similar in hepatocytes from regenerating liver and in hepatocytes obtained from normal rat liver. Clone pHT 26 corresponds to a 0.6-kilobase mRNA which exists at a high level only in cancer nodules and in hepatoma cell lines. We were unable to observe any cross-hybridization between these clones and the oncogenes which have been found to be expressed in hepatomas (c-fos, c-Ha-ras, c-Ki-ras, N-ras, and c-myc). The mRNAs corresponding to the three clones have not been detected in various tissues from normal adult rats. Our study shows that a high level of these mRNAs might be associated with rat liver carcinogenesis.

Animals↗