Cellular & molecular biology research最新文献

MyoD-mediated activation of skeletal muscle genes, which is dependent upon the consensus E-box sequence, involves, at least in one group of muscle genes, another transcription factor, the myocyte enhancer factor-2 (MEF-2). Since the cardiac myosin light chain-2 (MLC-2) gene promoter lacks the functional E-box but contains the activator MEF-2 site, we tested the effect of ectopic expression of MyoD on cardiac MLC-2 promoter function. Here, we demonstrate that either transient or stable expression of MyoD in otherwise nonpermissive C3H10T1/2 fibroblast cells can promote the expression of MLC-2/CAT. Deletion and site-specific mutation analysis demonstrate that the MEF-2 site (Element B) in the MLC-2 promoter is the target of activation by MyoD. Gel mobility shift assay using nuclear extracts from the normal and MyoD-transfected fibroblast cells did not show a difference in the major MEF-2 binding complexes, except for one complex of fast-moving mobility, which suggested that new MEF-2-like regulatory proteins are induced by MyoD.

In monkey kidney cells (TC-7), microinjected with UV-irradiated (103-362 J/m2) SV40 DNA, the expression of viral antigens decreases in a UV-dose-dependent manner and the viral genes are not repaired constitutively. When the viral DNA is microinjected 4 h after UV-irradiation (40 J/m2) of host cells, the expression of viral antigens is restored in all cells. The time course of restoration of viral gene expression function shows that in UV-irradiated cells the repair is induced rapidly and fully within 2 h and the induced state is maintained for 24 h. Intact viral DNA molecules, microinjected during the period of induction of cellular UV repair, are expressed less efficiently than UV-irradiated viral genomes.

The distribution of the enzyme synthesizing nitric oxide (NO) has been characterized in several mammalian enteric nervous systems. Two methods, immunohistochemical staining, employing anti-nitric oxide synthase antibodies, and histochemical localization of NADPH-diaphorase (NADPH-D), have given the same results. On the other hand, few studies have investigated nitric oxide synthase (NOS) in the gastrointestinal mucosa. Our study demonstrated the presence and distribution of the enzyme, NADPH-D, throughout all layers of the neonatal piglet intestinal tract. In the neonatal piglet, NADPH-D activity was found in nerve fibers parallel to the circular and to the longitudinal muscles and in the ganglion cells of Auerbach's plexus. However, the majority of NADPH-D activity was localized to the mucosa. Furthermore, the most intense activity in the mucosa was observed in villous epithelial cells. Other mucosal cells which were NADPH-D positive included the glandular epithelium and crypt cells. In addition, glandular epithelium in the deeper submucosa had very strong NADPH-D activity. Our results support the hypothesis that locally produced NO mediates physiological functions in the intestinal mucosa and submucosa.

We have identified a new member of the helix-loop-helix (H-L-H) and leucine zipper gene families via a reverse transcriptase-polymerase chain reaction based strategy. This new gene, CHUK (conserved helix-loop-helix ubiquitous kinase), may represent the founding member of a new class of interacting chimeric proteins. The nucleotide sequence of a near full-length murine CHUK cDNA clone revealed an encoded polypeptide specifying: a carboxyl-terminal H-L-H domain, an amino terminal serine-threonine kinase catalytic domain, and a leucine zipper-like amphipathic alpha-helix juxtaposed in between the H-L-H and kinase domains. CHUK is highly conserved in evolution and ubiquitously expressed in diverse types of established cell lines, whereas it is differentially expressed in normal murine tissues. The structural features of the CHUK polypeptide suggest that its putative kinase activity may be targetted to H-L-H and/or leucine zipper transcription factors. Alternatively, the dual amphipathic a helices may serve to control its intrinsic kinase activity by interactions with other cellular factors. CHUK may provide new insights into the regulated transmission of cytoplasmic signals to specific nuclear factors manifesting rapid alterations in patterns of cellular gene expression.

The subcellular distribution of soluble and membrane-bound Arg-beta-naphthylamide-hydrolyzing activities was studied in the left and right rat brain during development and aging. During development, the soluble activity was heterogeneous, whereas adult animals showed the highest activity in the synaptosomal fraction. However, except in fetuses, membrane-bound activity was greatest in the microsomal fraction. Except in microsomal and myelin fractions, soluble and membrane-bound activities showed a decrease in 1-wk-old rats compared with fetuses and a subsequent increase to adult levels in 1-mo-old rats. This profile differed in the microsomal fraction, which increased steadily throughout development. In the synaptosomal fraction, both activities were lower in 24-mo-old rats than in 5-mo-old animals. No differences between the hemispheres were observed in soluble or membrane-bound fractions at any age tested.

The standard laboratory diet administered to sand rat (Psammomys obesus) induces the following physiological and immunological changes: hyperglycemia and hypercholesterolemia involving mainly the free fraction of cholesterol, with an elevation of high-density-lipoprotein levels and a decrease in B and T splenic lymphocyte proliferation in the presence of different mitogens PHA-P, Con A and LPS. These results demonstrate the important modification that could be induced in sand rat by the standard laboratory diet as compared with natural diet, and thus the sand rat (P. obesus) appears to be an interesting model for studies on experimental diabetes mellitus.

A rapid and simple spin column assay has been used to study interactions of BiP with substance P (SP) and ATP. At 4 degrees C, the binding of SP to BiP requires ATP and a stable SP-BiP.ATP complex is formed. Nonhydrolyzable ATP analogues or ADP cannot replace ATP. Although ATP converts BiP dimers to monomers, the requirement for ATP for SP binding is not solely due to BiP dissociation, because purified BiP monomers also require ATP for peptide binding. At 37 degrees C, there is rapid binding of SP to BiP even in the absence of ATP and, in fact, ATP at concentrations above 5 microM causes release of SP from BiP. At this higher temperature, there is also rapid hydrolysis of ATP bound to BiP. These results extend our previous results (Brot et al., 1994) that indicated the formation, at low ATP concentrations, of a labile SP.BiP.ATP complex that, after ATP hydrolysis, resulted in a stable SP.BiP.ADP complex.

We cloned and sequenced cDNAs encoding two lysosomal membrane glycoproteins, lgp-A and lgp-B, from Chinese hamster ovary cells. The deduced amino acid sequences of these proteins are similar to those of the other known members of this conserved family (also known as "LAMP" proteins). We used the cDNAs to generate stable lines of hamster lgp-expressing mouse NIH-3T3 cells, rat NRK cells, and monkey CV-1 cells. We also generated hybridomas that secrete antibodies specific for hamster lgp-A and lgp-B, enabling us to distinguish foreign from endogenous lgps in a wider variety of transfected cell lines. One line of mouse NIH-3T3 cells that expresses hamster lgp-B was studied in detail. Whereas most of the hamster lgp-B appeared to be transported to lysosomes in these cells, butyrate-induced overexpression resulted in the accumulation of a significant proportion of the total on the plasma membrane. In addition, overexpression of this foreign lgp-B also resulted in the appearance of the endogenous mouse lgp-A and lgp-B on the plasma membrane. Characterization of this accumulation suggested that it resulted from competition for one or more limited components in the transport pathway(s) to lysosomes. Endocytosis from the plasma membrane appeared to be one step that was saturable.

Recessive mutant gene c for "'cardiac nonfunction" in the mexican axolotl, Ambystoma mexicanum, results in a failure of affected embryos to develop contracting hearts. Mutant embryos survive approximately 4 weeks after fertilization, but eventually die from a lack of circulation. Morphological studies show that mutant hearts lack organized sarcomeric myofibrils. This abnormality can be corrected by co-culturing early mutant hearts with normal anterior endoderm/mesoderm tissues, by culturing them in a medium "conditioned" by this normal tissue, or by RNA isolated from normal endoderm/mesoderm. Additionally, RNA isolated from normal anterior endoderm/mesoderm conditioned medium corrects the mutant hearts in a dose-dependent manner. A cDNA library is constructed using this RNA. On the basis of sequence analyses on this cDNA library, it was estimated that 56% of the total RNA present in the conditioned medium is rRNA, while 44% is nonribosomal RNA. One of the nonribosomal RNAs that showed no significant homology with other known sequences in the Genebank was examined further. An RT-PCR analysis showed that this RNA (designated "N1") is expressed in juvenile skeletal muscle, brain, and heart in significant amounts, less in the lung and not at all in the liver tissue. Affinity-purified polyclonal antipeptide antibodies were produced against the most antigenic portion of the polypeptide which was deduced from this RNA. Western blot analyses of adult heart homogenates, using these antibodies, showed a specific doublet staining at 67 kDa and 65 kDa. These doublets were purified and analyzed for their amino acid composition which showed that both bands most likely belong to the same protein. The N1-protein was further investigated to determine its localization in normal isolated hearts at embryonic stages 35, 38, and 41 and on cross-sections through the heart regions of whole normal embryos at stages 16, 33-34, 37-38, and 41-42 using immunohistochemical techniques and confocal microscopy. In addition, mutant embryos at stage 37-38 were studied for the presence and distribution of the N1-protein on cross-sections through their heart regions. The N1-protein staining was significantly reduced in mutant hearts when compared to normal.

Nucleoside diphosphate kinase (E.C. 2.7.4.6.) is a broad substrate-specific enzyme that catalyzes the phosphorylation of nucleoside diphosphates to the corresponding triphosphates in nucleic acid biosynthesis. In this report, we investigate its spatial and temporal distributions in yeast to understand how the enzyme exerts its gene function(s). Our results show that the enzyme is predominantly cytoplasmic. A substantial amount of enzyme activity (40-50%) may be associated with the cell membrane. Less than 1% of total activity was detected in the nuclear fraction. Approximately 3% was found in the mitochondrial fraction. When yeast cultures were synchronized, we found that Saccharomyces cerevisiae nucleoside diphosphate kinase did not show cell cycle periodicity, as Schizosaccharomyces pombe enzyme did. To explore its link with DNA synthesis, we investigated its relationship with the Cdc8p (dTMP kinase). We demonstrated a physical interaction between these proteins in vitro, as evidenced that the GST:Cdc8p protein affinity column could retain a subpopulation of nucleoside diphosphate kinase activity from yeast crude extract. Furthermore, when GST:Cdc8p protein was expressed in yeast, the protein could bind to the glutathione-agarose, along with nucleoside diphosphate kinase, suggesting that there is an interaction between GST:Cdc8p and nucleoside diphosphate kinase in vivo. Our results provide evidence for at least a two-enzyme complex that may well facilitate nucleotide channeling in the cell.