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.

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. Very few differences could be detected when comparing mature to old kidneys or livers. Stimulated adenosine 3',5'-cyclic monophosphate (cAMP) synthesis was tissue- and age-dependent. In liver, G-protein- and beta-receptor-stimulated cAMP synthesis mirrored basal adenylate cyclase activity and was highest in liver from neonatal animals. In contrast, cAMP synthesis was significantly more stimulated in kidneys from mature animals than from neonatal and senescent rats. We conclude that: (i) the stoichiometry of the components within the beta-receptor/G-protein/adenylate cyclase complex is not fixed but is both tissue- and age-dependent; (ii) adenylate cyclase enzyme activity is possibly but not necessarily the rate-limiting step in the beta-receptor-mediated synthesis of cAMP; and (iii) there is in vivo evidence for a preferential co-expression of the large splice variant of the G(s)-protein and beta(2)-receptor subtype. It is speculated that this could have important physiological consequences for the development of the kidney.

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. Decreased G(i alpha)-concentrations were accompanied by lower tubular but higher glomerular levels, while medullar levels were also increased. Decreased G(q/11alpha)-concentrations were reflected in decreased glomerular and medullary concentrations. Kidneys of 3 month old SHR and WKY possessed similar concentrations of all G(alpha)-species. In 8 month old SHR similar G(i alpha)-, but decreased G(s alpha)-and G(q/11alpha)-concentrations were observed. The G(s alpha)-decrease was reflected in cortex and medulla, the G(q/11alpha)-decrease in the medulla. We conclude that the main strain-related differences in G(alpha)-concentrations are seen in prehypertensive SHR.

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. Comparison of C-MET expression on spermatozoa of the 90% Percoll layer of subfertile patients and donors revealed clearly two distinct groups (unpaired t-test, P < 0.001), whereas comparison of C-MET expression on spermatozoa in the 47% Percoll layer was not significantly different between patients and donors. In addition, there was a significant inverse correlation between sperm concentration and the C-MET expression of spermatozoa in the 90% Percoll layer (r = -0.80, 95% confidence interval, -0.92 to -0.55; P < 0.0001), but not with the C-MET expression of spermatozoa in the 47% Percoll layer. In conclusion, the presence of C-MET was demonstrated in the seminiferous epithelium and on mature and immature spermatozoa, indicating a role for this growth factor receptor in the differentiation and/or migration that occurs during human spermatogenesis.

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. both chromosomal and plasmid DNA, was prepared from transformed cells by boiling the cell suspensions and by phenol-chloroform extraction; chromosomal DNA was prepared by the same methods from the non-transformed, plasmid-free strains; plasmid DNA of pUC19 was purchased; plasmid DNA of pCEP was purified from the transformed strains by caesium chloride density gradient centrifugation. Arbitrarily primed polymerase chain reaction was carried out for all of these preparations. Amplification carried out independently with three different primers resulted in similar patterns for the chromosomal preparations whether or not plasmid was present. Amplification of plasmid DNA gave different patterns, characterized by fragments larger than those obtained when total or chromosomal DNA were used as the target. These data illustrate that the plasmids studied here do not influence the chromosomal arbitrarily primed PCR fingerprint, although plasmids alone are amplified in the absence of chromosomal DNA. Experiments comparing different relative concentrations of plasmid and chromosomal DNA indicate that under natural conditions the amount of chromosomal DNA per cell is sufficient to inhibit observable amplification of the plasmid(s) present.

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. The MAb delayed the adhesion of MCF-7 and SK-BR-3 cells on precultured embryonic heart fragments (PHFS). It inhibited the destruction of the PHF by MCF-7 cells and the invasion of the PHF by SK-BR-3 cells. The MAb reacted with an epitope on the cell membrane of in situ and invasive ductal carcinomas of the breast in immunohistochemistry. Poorly differentiated, highly invasive ductal carcinomas show extensive staining of long plasma membrane extensions. Normal multilayered epithelia, normal connective tissue, and tumors derived from these tissues as well as normal breast tissue were negative. From both cell lines a protein complex consisting of two subunits with molecular weight of 50 and 90 kd, respectively, was immunoprecipitated. It is concluded that the 14C5 antigen plays a role in cell substrate adhesion and subsequently also in invasion of breast cancer cells. The 14C5 MAb was able to inhibit cell substrate adhesion and invasion in vitro of breast cancer cells.

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. The ability to activate STNV-1 and -2 is not completely correlated with the virulence of the TNV isolates on Phaseolus as TNV-CN infects Phaseolus very inefficiently. The RNAs of all STNV-1 and -2 supporting TNV isolates were detectable by Northern blot analysis using RNA probes derived from TNV-A, whereas the RNAs of the STNV-C activating isolates (TNV-D and -AC36) were only detected with a TNV-D-derived RNA probe. This indicates that the classification of the TNV isolates on the basis of satellite activation is representative of the evolutionary relationships between the isolates.

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 1.3-kb subgenomic RNA is delayed by 1 h compared with that of the 1.6-kb subgenomic RNA, which illustrates the important role of the subgenomic RNAs in the regulation of TNV-A gene expression. The locations of the 5' nucleotides of the subgenomic RNAs reveal that the 5'-proximal cistrons of the 1.6- and 1.3-kb RNAs encode an 8-kDa protein from open reading frame (ORF) 3 and the coat protein from ORF 5, respectively. In a wheat germ translation system, a synthetic transcript resembling the 1.6-kb RNA expresses both ORFs 3 and 4. Moreover, the synthesis of the 6-kDa protein from ORF 4 depends on the translation efficiency of ORF 3, suggesting that in vivo, ORFs 3 and 4 are both expressed from the 1.6-kb RNA. The major in vitro translation product of TNV-A genomic RNA is the coat protein. We show that the region upstream of the coat protein promotes internal initiation of translation in vitro. However, this region is functionally inactive in vivo, suggesting that TNV-A genomic RNA is not important for coat protein synthesis in plants.

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. The 620-nt-long trailer sequences, with 64% nucleotide sequence conservation, can fold into a phylogenetically conserved secondary structure consisting of three pseudoknots followed by a long-range interaction-born hairpin structure. The significance of these elements is discussed in view of the particular properties (stability, translational competitiveness, and replication) that characterize these RNAs.

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. The third large ORF encodes the 30-kDa coat protein. Two small ORFs are located upstream and one immediately downstream of this coat protein cistron. Extensive sequence similarity was found between the TNV 82-kDa protein and the putative polymerases of TCV, CarMV, cucumber necrosis virus (CNV), maize chlorotic mottle virus (MCMV), red clover necrotic mosaic virus (RCNMV), and barley yellow dwarf virus (BYDV). The TNV coat protein is very similar to southern bean mosaic virus (SBMV) capsid protein. Of the predicted small proteins only a 7.9-kDa protein shows some sequence similarity with a corresponding protein of MCMV, CarMV, and TCV. The others are unique to TNV. Except for the first four nucleotides at the 5' end no homology was found with the RNA of STNV (satellite of TNV).

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. Progeny STNV was obtained which carried oligonucleotide insertions at various positions of the genome.

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). These in vivo results confirm that the 5'-untranslated region of STNV RNA, preceding the initiating AUG, provides adequate genetic information for efficient translation in the bacterial system. However, fast degradation of the bacterially synthesized STNV coat protein was observed. The use of a protease-deficient strain reduced this breakdown considerably.

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..., the 5'-terminal sequence of intact STNV RNA. Binding analysis of individual 5'-terminal fragments generated from isolated, intact, STNV RNA by partial digestion with T1 RNase showed that eIF-2 does not bind detectably to the 32-nucleotide fragment ending with the initiation codon AUG or to shorter ones, but it does bind the 44-nucleotide fragment that contains the ribosome binding site. In addition to the structural features localized at the 5' end of STNV RNA, eIF-2 appears to recognize a conformation found only in larger molecules, because intact RNA and large 5-'-terminal fragments are bound preferentially over smaller ones. However, binding of short 5'-terminal STNV RNA fragments to eIF-2 is specific, as judged by competition with STNV and ribosomal RNA. Finally, binding of eIF-2 to intact STNV RNA leads to a conformational change in the RNA that greatly facilitates cleavage by T1 and P1 RNases at sites in the vicinity of the initiation region. These results show that eIF-2 interacts specifically with the 5'-terminal region of STNV RNA that contains the ribosome binding site and causes local unfolding of the RNA structure.

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. Ten nonsense codons block all three reading frames such that at least 26 nucleotides do not code for protein. The possible function of a homology A-A-U-A-A-A with other polyadenylated RNAs is discussed.