Desensitization of muscarinic receptors.

When Chinese hamster ovary cells transfected with the gene for M(3)-muscarinic receptors were stimulated with carbachol continuously for 30 min, the response at the end of the stimulation period was about 20% of the early response (2-3 min after the start of the stimulation). Long-term treatment of the cells with phorbol ester abolished the response completely while desensitization was significantly reduced upon pre-treatment of the cells with GF109203X, antisense oligonucleotide against the alpha-isoform of protein kinase C and wortmannin. We conclude that in the Chinese hamster ovary expression system, desensitization of M(3)-muscarinic receptors is dependent on a fast feedback loop including the alpha-isoform of protein kinase C.

The anti-proliferative effect of calcitriol on HL-60 cells is neutralized by uraemic biological fluids.

Background: It has been demonstrated that uraemic serum/ultrafiltrate inhibits cell-mediated immune response in vitro, and that it suppresses calcitriol synthesis and its biological actions.

Methods: In the present in vitro study, the effect of calcitriol, uraemic ultrafiltrate (UUF) and a combination of both on the human promyelocytic leukaemia cell line, HL-60, was studied by evaluating bromodeoxyuridine (BrdU) incorporation into the DNA, luminol-amplified chemiluminescence (CL) production, expression of CD14, and levels of vitamin D receptor mRNA (VDR mRNA) and CD14 mRNA.

Results: The ability of calcitriol to block cell proliferation (37.

Effect of maturation and aging on beta-adrenergic signal transduction in rat kidney and liver.

The characteristics of the beta-adrenergic signal transduction system were analyzed in kidney and liver membrane preparations from neonatal (2-3 days), mature (2 months), and old (2 years) rats. When comparing kidneys from adult to neonatal rats, we found a higher beta-receptor density and a higher percentage of beta(1)-receptor subtype, lower immunoreactive G(salpha)-protein, a lower ratio between the high and low molecular weight splice variant of G(salpha), lower immunoreactive G(ialpha)-protein, and lower basal adenylate cyclase activity. When comparing livers from adult to neonatal rats, we found lower beta-receptor density and basal adenylate cyclase activity.

G-proteins in kidneys of spontaneously hypertensive rats.

G(s alpha)-, total G(i alpha)- and G(q/11alpha)-protein concentrations were investigated by quantitative immunoblotting in membranes of total kidney, renal cortex and medulla as well as in cortical tubules and glomeruli of Spontaneously Hypertensive Rats (SHR) and normotensive Wistar Kyoto rats (WKY), aged 5 weeks, 3 or 8 months. We found that total kidney of 5 week old SHR possess less G(s alpha)-, G(i alpha)- and G(q/11alpha)-proteins than controls. For G(s alpha)-proteins, differences found in total kidney were mirrored both in cortex (tubules and glomeruli) and in medulla.

The receptor encoded by the human C-MET oncogene is expressed in testicular tissue and on human spermatozoa.

Because of its distinctive ability to act as a mitogen, a mitogen and a morphogen, hepatocyte growth factor/scatter factor (HGF/SF) has all the characteristics of a molecule able to function in regulatory networks of motility, such as the spermatogenic epithelium, and this through binding of its receptor p190MET (C-MET). In this study we report the expression of C-MET in the human seminiferous epithelium and on spermatozoa from men being treated for infertility and sperm donors. The presence of C-MET was demonstrated by immunochemistry on the cell membrane of spermatogonia, spermatocytes, spermatids and on spermatozoa, whereas Sertoli cells and Leydig cells did not show expression.

Study of the influence of plasmids on the arbitrary primer polymerase chain reaction fingerprint of Escherichia coli strains.

To study the effect of plasmids on the arbitrary primer-polymerase chain reaction fingerprint of bacterial strains, the Escherichia coli strains DH5, Top10, and W3110 were transformed with plasmids of different sizes: respectively, pUC19, pCEP and two clinically important plasmids carrying resistance to several antibiotics. Total DNA, i.e.

A monoclonal antibody directed against a human cell membrane antigen prevents cell substrate adhesion and tumor invasion.

It was the aim of this study to design mouse monoclonal antibodies (MAbs) that can inhibit the invasion of breast cancer cells in the host tissue. Therefore, MAbs were raised against epitopes on the extracellular domain of SK-BR-3 human breast cancer cells, and biological assays were performed to test the capability of the MAbs to inhibit cell substrate adhesion. MAb 14C5 bound an extracellular plasma membrane antigen of SK-BR-3 and MCF-7 human breast cancer cells and inhibited the cell substrate adhesion of these cells in vitro.

Specificity of satellite activation by tobacco necrosis virus correlates with nucleic acid hybridization pattern between helper virus isolates.

Tobacco necrosis virus (TNV) comprises over 20 different isolates which are usually classified on the basis of serological cross-reactivity of their virus particles or specific activation of satellite virus strains (STNV-1, -2, and -C). We have studied the relationships between five TNV isolates, TNV-A, -G, -CN, -D, and -AC36 which exhibit considerable differences in symptom formation on Phaseolus vulgaris. It is shown that, like TNV-A, TNV-G and -CN support the multiplication of STNV-1 and -2.

Subgenomic RNAs mediate expression of cistrons located internally on the genomic RNA of tobacco necrosis virus strain A.

Upon infection of tobacco protoplasts, the genomic RNA of tobacco necrosis virus strain A (TNV-A) accumulates linearly in time. The accumulation patterns of the two subgenomic RNAs resemble those of endogenous mRNAs in that the peak levels are reached after several hours. The accumulation of the 1.

Structural similarities between the RNAs of two satellites of tobacco necrosis virus.

The complete nucleotide sequence of satellite tobacco necrosis virus 2 (STNV-2) RNA has been determined. It has the same organization as the previously studied STNV-1 RNA. The 5' untranslated regions (about 30 nt) are nearly identical, while the coat protein coding regions (about 600 nt) have 55% nucleotide sequence similarity.

Genome structure of tobacco necrosis virus strain A.

An almost complete sequence of the RNA genome of tobacco necrosis virus (TNV) strain A has been determined. The genome organization is very similar to that of carnation mottle virus (CarMV) and turnip crinkle virus (TCV). The 5'-proximal open reading frame (ORF) encodes a 23-kDa protein and read-through of its amber codon into the second ORF is presumably used for the translation of a 82-kDa protein.

Expression in plants of the cloned satellite tobacco necrosis virus genome and of derived insertion mutants.

Mechanical inoculation of cowpea leaves with cloned full-size copies of satellite tobacco necrosis virus in the presence of tobacco necrosis (helper) virus resulted in the appearance of infectious virus particles. This could be clearly demonstrated by the detection of structural changes in the progeny viral RNA after inoculation with helper virus and hybrid plasmids containing an STNV-DNA copy with small (14-mer) oligodeoxynucleotides inserted at different sites in either the coding or the non-coding region. Furthermore, although orientation and position of the viral cDNA in the different plasmids did not seem to be of major importance for the ensuing infection process, the presence of adjacent complementary homopolymeric regions (dG/dC) at both ends of the STNV nucleotide sequence was necessary.

Controlled synthesis of the coat protein of satellite tobacco necrosis virus in Escherichia coli.

Chimeric plasmids were constructed such that the cloned complete satellite tobacco necrosis virus (STNV) RNA information came under transcriptional control of the leftward promoter (PL) of bacteriophage lambda. The promoter is fully repressed at low temperatures (28 degrees) by the thermolabile repressor product of the lambdacI857 gene, present in the bacterium on a deficient prophage or as part of another plasmid. Synthesis of the STNV coat protein in Escherichia coli could be initiated by heat induction (42 degrees).

The sequence specificity of endonucleases CauI and CauII isolated from chloroflexus aurantiacus.

The type II restriction enzymes CauI and CauII, isolated from Chloroflexus aurantiacus, recognize and cleave (at the position indicated by an arrow) the sequences G decreases G A/T CC and CC decreases G/C GG, respectively. These conclusions are supported by the results from restriction site mapping, sequence analysis by partial chemical degradation, end-group analysis after lambda exonuclease treatment and computer-assisted comparison of DNA sequence data.

Specific binding of eukaryotic initiation factor 2 to satellite tobacco necrosis virus RNA at a 5'-terminal sequence comprising the ribosome binding site.

The mRNA-binding property of eukaryotic initiation factor 2 (eIF-2) was examined by studying its interaction with satellite tobacco necrosis virus (STNV) RNA carrying a (32)P-labeled 5' end. The RNA molecules bound by limiting amounts of eIF-2 were isolated and digested with pancreatic and T1 RNases. Digestion patterns showed that the labeled STNV RNA preparation offered to eIF-2 was heterogeneous, containing more than 30 different 5' ends; by contrast, the RNA selected by eIF-2 possessed predominantly one 5' end, pApGpUp.

3'-Terminal nucleotide sequence (N = 41) of satellite tobacco necrosis virus RNA.

To convert satellite tobacco necrosis virus RNA into a template for complementary DNA (cDNA) synthesis, a poly(A) tail was added to its 3'-end by means of Escherichia coli ATP:RNA adenyltransferase. This product could then be copied by avian myeloblastosis virus RNA-dependent DNA polymerase in the presence of p(dT)(10) as primer. Under appropriate conditions, cDNA was synthesized discontinuously, and limited transcripts were obtained when one or more deoxynucleoside triphosphates were omitted from the reaction mixture.

Sequence of 129 nucleotides at the 3'-terminus of encephalomyocarditis virus RNA.

The sequence of 129 nucleotides next to the poly(A) tail of encephalomyocarditis virus RNA has been determined by rapid gel sequencing of cDNA synthesized with DNA polymerase I or reverse transcriptase and a phasing primer, [5'-32P]p(dT)8dC. The sequence is in accord with (a) the pyrimidine tracts which were mapped in blocks along the cDNA, (B) the sequences of seven characteristic T1 RNase oligonucleotides in the RNA transcribed from the cDNA with RNA polymerase, and (c) a limited amount of sequence deduced by partial spleen phosphodiesterase digestion and depurination of endonuclease IV oligonucleotides. The 3' end shows little secondary structure on its own.

The 3'-Terminal nucleotide sequence of encephalomyocarditis virus RNA.

Poly(A)-containing encephalomyocarditis virus RNA functions as an excellent template for cDNA synthesis in vitro with an RNA-dependent DNA polymerase in the presence of an oligothymidylate primer. Under appropriate conditions, discrete transcripts of increasing chain length were obtained, suitable for sequence analysis. A limited cDNA fragment of 36 nucleotides, primer (dT)10 included, was synthesized when dGTP was omitted from the reaction mixture and its primary structure was elucidated using direct DNA-sequencing methods.