Glucagon desensitization of adenylate cyclase and stimulation of inositol phospholipid metabolism does not involve the inhibitory guanine nucleotide regulatory protein Gi, which is inactivated upon challenge of hepatocytes with glucagon.

Brief exposure of hepatocytes to glucagon, angiotensin or the protein kinase C activator TPA (12-O-tetradecanoylphorbol 13-acetate) caused the inactivation of the inhibitory guanine nucleotide regulatory protein Gi. Glucagon-mediated desensitization of glucagon-stimulated adenylate cyclase activity was seen in hepatocytes from both normal rats and those made diabetic with streptozotocin, where Gi is not functionally expressed. Normal glucagon desensitization was seen in hepatocytes from young animals, 6 weeks of age, which had amounts of Gi in their hepatocyte membranes which were some 45% of that seen in mature animals (3.4 pmol/mg of plasma-membrane protein). Streptozotocin-induced diabetes in young animals abolished the appearance of functional Gi in hepatocyte plasma membranes. Pertussis-toxin treatment of hepatocytes from both normal mature animals and those made diabetic, with streptozotocin, blocked the ability of glucagon or angiotensin or TPA to elicit desensitization of adenylate cyclase. The isolated B (binding)-subunit of pertussis toxin was ineffective in blocking desensitization. Neither induction of diabetes nor treatment of hepatocytes with pertussis toxin inhibited the ability of glucagon and angiotensin to stimulate the production of inositol phosphates in intact hepatocytes. Thus (i) Gi does not appear to play a role in the molecular mechanism of glucagon desensitization in hepatocytes, (ii) the G-protein concerned with receptor-stimulated inositol phospholipid metabolism in hepatocytes appears not to be a substrate for the action of pertussis toxin, (iii) in intact hepatocytes, treatment with glucagon and/or angiotensin can elicit the inactivation of the inhibitory G-protein Gi, and (iv) pertussis toxin blocks desensitization by a process which does not involve Gi.     

Localization of antigens PwA33 and La on lampbrush chromosomes and on nucleoplasmic structures in the oocyte of the urodele Pleurodeles waltl: Light and electron microscopic immunocytochemical studies

Monoclonal antibodies A33/22 and La11G7 have been used to study the distribution of the corre-sponding antigens, PwA33 and La, on the lampbrush chromosome loops and nucleoplasmic structures of P. waltl oocytes, using immunofluorescence, confocal laser scanning microscopy and immunogold labeling. The results obtained with these antibodies have been compared with those obtained with the Sm-antigen-specific monoclonal antibody Y12. All these monoclonal antibodies (mAbs) labeled the matrices of the majority of normal loops along their whole length. Nucleoplasmic RNP granules showed a strong staining with the mAbs La11G7 and Y12 throughout their mass, but with the mAb A33/22, they showed only a weak peripheral labeling in the form of patches on their surface. This patchy labeling was confirmed by confocal laser scanning microscopy. Electron microscopy revealed that this patchy labeling might be due to a hitherto undescribed type of submicroscopic granular structure, around 100 nm in either dimension, formed by 10-nm particles. Such granules were observed either attached to the RNP granules or free in the nucleoplasm, but rarely in relation with the normal loop matrices. These 100-nm granules may have a role in the movement of proteins and snRNPs inside the oocyte nuclei for storage, recycling, and/or degradation. Our results also suggest that all the microscopically visible free RNP granules of the nucleoplasm of P. waltl oocytes correspond to B snurposomes. The granules forming the B (globular) loops showed a labeling pattern similar to that of B snurposomes; their possible relationship is discussed.     

Homologies between members of the germin gene family in hexaploid wheat and similarities between these wheat germins and certain Physarum spherulins

By screening approximately 10(6) plaques in a wheat DNA library with a "full-length" germin cDNA probe, two genomic clones were detected. When digested with EcoRI, one clone yielded a 2.8-kilobase pair fragment (gf-2.8) and the other yielded a 3.8-kilobase pair fragment (gf-3.8). By nucleotide sequencing, each of gf-2.8 and gf-3.8 was found to encode a complete sequence for germin and germin mRNA, and to contain appreciable amounts of 5'- and 3'-flanking sequences. The "cap" site in gf-2.8 was determined by primer extension and the corresponding site in gf-3.8 was deduced by analogy. The mRNA coding sequences in gf-2.8 and gf-3.8 are intronless and 87% homologous with one another. The 5'-flanking regions in gf-2.8 and gf-3.8 contain recognizable sites of what are probably cis-acting elements but there is otherwise little if any significant similarity between them. In addition to putative TATA and CAAT boxes in the 5'-flanking regions of gf-2.8 and gf-3.8, there are AT-rich inverted-repeats, GC boxes, long purine-rich sequences, two 19-base pair direct-repeat sequences in gf-2.8, and a remarkably long (200-base pair) inverted-repeat sequence (approximately 90% homology) in gf-3.8. An 8% difference between the mature-protein coding regions in gf-2.8 and gf-3.8 is reflected by a corresponding 7% difference between the corresponding 201-residue proteins. Most significantly, the same 8% difference between the mature-protein coding regions in gf-2.8 and gf-3.8 is allied with no change whatever in a central part (61-151) of the encoded polypeptide sequences. It seems likely that this central, strongly conserved core in the germins is of first importance in the biochemical involvements of the proteins. When an equivalence is assumed between like amino acids, the gf-2.8 and gf-3.8 germins show significant (approximately 44%) similarity to spherulins 1a and 1b of Physarum polycephalum, a similarity that increases to approximately 50% in the conserved core of germin. Near the middle (87-96) of the conserved core in the germins is a rare PH(I/T)HPRATEI decapeptide sequence which is shared by spherulins (1a and 1b) and germins (gf-2.8 and gf-3.8). These similarities are discussed in the context of evidence which can be interpreted to suggest that the biochemistry of germins and spherulins is involved with cellular, perhaps cell-wall responses to desiccation, hydration, and osmotic stress.(ABSTRACT TRUNCATED AT 400 WORDS)     

Replacement of the folC gene, encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli, with genes mutagenized in vitro.

The folylpolyglutamate synthetase-dihydrofolate synthetase gene (folC) in Escherichia coli was deleted from the bacterial chromosome and replaced by a selectable Kmr marker. The deletion strain required a complementing gene expressing folylpolyglutamate synthetase encoded on a plasmid for viability, indicating that folC is an essential gene in E. coli. The complementing folC gene was cloned into the vector pPM103 (pSC101, temperature sensitive for replication), which segregated spontaneously at 42 degrees C in the absence of selection. This complementing plasmid was replaced in the folC deletion strain by compatible pUC plasmids containing folC genes with mutations generated in vitro, producing strains which express only mutant folylpolyglutamate synthetase. Mutant folC genes expressing insufficient enzyme activity could not complement the chromosomal deletion, resulting in retention of the pPM103 plasmid. Some mutant genes expressing low levels of enzyme activity replaced the complementing plasmid, but the strains produced were auxotrophic for products of folate-dependent pathways. The folylpolyglutamate synthetase gene from Lactobacillus casei, which may lack dihydrofolate synthetase activity, replaced the complementing plasmid, but the strain was auxotrophic for all folate end products.     

Plasminogen activator and collagenase production by cultured capillary endothelial cells

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4α-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells.     

Tunable luminescence of a synthesized furophenanthraquinone derivative: interactions with different solvents

The synthesis is described of a luminescent furophenanthraquinone derivative, 9‐methoxyphenanthro[4,3‐b]furan‐4,5‐dione (MPFD). The biological importance of tetracyclic furophenanthraquinones was considered and the tunable luminescence of MPFD in different solvents was studied to explore the nature of the specific interactions between MPFD and solvents. Observation of dual emission bands and identical nature of the fluorescence excitation spectra of MPFD monitored at the emission wavelength in polar solvents indicated the formation of two different types of species in the excited state, probably due to proton transfer from the solvent to MPFD. Luminescence intensity due to anionic species was found to be increased and the corresponding peak was red shifted with increase in the proton‐donating ability of the solvents, acting as an acid with respect to MPFD. Availability of more acidic protons in the solvent facilitated this phenomenon occurring in the excited state. MPFD also interacted with halogen‐containing solvents by forming electron donor–acceptor charge transfer (CT) complexes. This CT complex formation was dependent on the number of chlorine atoms; the position of the corresponding luminescence band varied with the polarity of the solvent. Extent of the CT increased with increase in the number of chlorine atoms in the dichloro, trichloro and tetrachloro solvents, whereas the luminescence peak due to the CT complex was found to be blue shifted with decrease in solvent polarity. Interaction of the synthesized bioactive MPFD with different solvents deserves biological importance as proton transfer and CT play pivotal roles in biology.     

Kinetic Enrichment of 34S during Proteobacterial Thiosulfate Oxidation and the Conserved Role of SoxB in S-S Bond Breaking

During chemolithoautotrophic thiosulfate oxidation, the phylogenetically diverged proteobacteria Paracoccus pantotrophus, Tetrathiobacter kashmirensis, and Thiomicrospira crunogena rendered steady enrichment of ³⁴S in the end product sulfate, with overall fractionation ranging between −4.6‰ and +5.8‰. The fractionation kinetics of T. crunogena was essentially similar to that of P. pantotrophus, albeit the former had a slightly higher magnitude and rate of ³⁴S enrichment. In the case of T. kashmirensis, the only significant departure of its fractionation curve from that of P. pantotrophus was observed during the first 36 h of thiosulfate-dependent growth, in the course of which tetrathionate intermediate formation is completed and sulfate production starts. The almost-identical ³⁴S enrichment rates observed during the peak sulfate-producing stage of all three processes indicated the potential involvement of identical S-S bond-breaking enzymes. Concurrent proteomic analyses detected the hydrolase SoxB (which is known to cleave terminal sulfone groups from SoxYZ-bound cysteine S-thiosulfonates, as well as cysteine S-sulfonates, in P. pantotrophus) in the actively sulfate-producing cells of all three species. The inducible expression of soxB during tetrathionate oxidation, as well as the second leg of thiosulfate oxidation, by T. kashmirensis is significant because the current Sox pathway does not accommodate tetrathionate as one of its substrates. Notably, however, no other Sox protein except SoxB could be detected upon matrix-assisted laser desorption ionization mass spectrometry analysis of all such T. kashmirensis proteins as appeared to be thiosulfate inducible in 2-dimensional gel electrophoresis. Instead, several other redox proteins were found to be at least 2-fold overexpressed during thiosulfate- or tetrathionate-dependent growth, thereby indicating that there is more to tetrathionate oxidation than SoxB alone.     

Cervical Cytology Specimen Stability in Surepath Preservative and Analytical Sensitivity for HPV Testing with the cobas and Hybrid Capture 2 Tests

None of the commercial HPV tests are U.S. FDA-approved for testing of cervical cytology specimens in SurePath preservative. Still, ~30% of HPV testing is performed on specimens in this formalin-containing preservative. Formalin-induced DNA fragmentation and cross-linking may interfere with HPV detection. We evaluated analytical sensitivity and specimen stability of the cobas 4800 HPV (Roche) and Hybrid Capture 2 HPV (HC2, Qiagen) tests with residual cervical cytology samples in SurePath preservative available within 1 week of collection. Cobas testing was performed with and without heating samples at 120°C for 20 min diluted 1:1 in an alkaline environment (pretreatment) to revert DNA crosslinking. Stability was tested after 2 weeks of storage at ambient temperature followed by ≤10 weeks at 4°C. Analytical sensitivity and positivity rates (HC2, 18%; cobas pretreated, 46%; cobas untreated, 47%) were greater for cobas than HC2 (n = 682). After 6 weeks of storage, mean HC2 ratios were lower (mean 0.9, SD 6.3) but high variability limited statistical power to detect trends. Cobas threshold cycles (Ct’s) increased in untreated (mean 2.1) but not pretreated samples (mean 0.3; n = 110; p≤0.0001). Overall, cobas had greater analytical sensitivity for samples in SurePath preservative. Although pretreatment introduced a manual sample transfer step and 30 min of incubation times, it improved stability without negatively affecting analytical sensitivity. While awaiting results of large trials to evaluate the clinical performance of cobas, the addition of the pretreatment step may improve the detection of HPV, especially after prolonged sample storage.     

Tethering of the platelet-derived growth factor beta receptor to G-protein-coupled receptors. A novel platform for integrative signaling by these receptor classes in mammalian cells

Here we provide evidence to show that the platelet-derived growth factor beta receptor is tethered to endogenous G-protein-coupled receptor(s) in human embryonic kidney 293 cells. The tethered receptor complex provides a platform on which receptor tyrosine kinase and G-protein-coupled receptor signals can be integrated to produce more efficient stimulation of the p42/p44 mitogen-activated protein kinase pathway. This was based on several lines of evidence. First, we have shown that pertussis toxin (which uncouples G-protein-coupled receptors from inhibitory G-proteins) reduced the platelet-derived growth factor stimulation of p42/p44 mitogen-activated protein kinase. Second, transfection of cells with inhibitory G-protein alpha subunit increased the activation of p42/p44 mitogen-activated protein kinase by platelet-derived growth factor. Third, platelet-derived growth factor stimulated the tyrosine phosphorylation of the inhibitory G-protein alpha subunit, which was blocked by the platelet-derived growth factor kinase inhibitor, tyrphostin AG 1296. We have also shown that the platelet-derived growth factor beta receptor forms a tethered complex with Myc-tagged endothelial differentiation gene 1 (a G-protein-coupled receptor whose agonist is sphingosine 1-phosphate) in cells co-transfected with these receptors. This facilitates platelet-derived growth factor-stimulated tyrosine phosphorylation of the inhibitory G-protein alpha subunit and increases p42/p44 mitogen-activated protein kinase activation. In addition, we found that G-protein-coupled receptor kinase 2 and beta-arrestin I can associate with the platelet-derived growth factor beta receptor. These proteins play an important role in regulating endocytosis of G-protein-coupled receptor signal complexes, which is required for activation of p42/p44 mitogen-activated protein kinase. Thus, platelet-derived growth factor beta receptor signaling may be initiated by G-protein-coupled receptor kinase 2/beta-arrestin I that has been recruited to the platelet-derived growth factor beta receptor by its tethering to a G-protein-coupled receptor(s). These results provide a model that may account for the co-mitogenic effect of certain G-protein-coupled receptor agonists with platelet-derived growth factor on DNA synthesis.