A protein induced by NGF in PC12 cells is stored in secretory vesicles and released through the regulated pathway.

We have previously described the isolation of a cDNA clone corresponding to an mRNA rapidly induced to high levels in PC12 cells by treatment with NGF. We report here the complete amino acid sequence of the protein (named VGF8a) as deduced by nucleotide sequencing of overlapping cDNA clones. VGF8a is particularly rich in proline residues and has a conspicuous number of short stretches of basic amino acid residues which may represent potential targets for proteolytic cleavage. Antibodies directed against recombinant VGF8a-beta-galactosidase fusion proteins were used for immunofluorescent staining of the protein in PC12 cells as well as for its localization, by Western blot analysis, in subfractions of cell homogenates. We demonstrate that in PC12 cells, VGF8a protein is stored in secretory vesicles and is released in response to a variety of stimuli that are known to induce the regulated secretion of neurotransmitters.     

Influence of platelet-activating factor on leukotriene D4-induced contractions of the guinea pig parenchymal strip

Platelet-activating factor (PAF) and sulphidopeptide leukotrienes, such as leukotriene D4 (LTD4), are potent constrictors that are probably released simultaneously in a variety of inflammatory respiratory events. The purpose of the present study was to determine whether LTD4-induced contractions of guinea pig parenchymal lung strips (GPPS) are modified in the presence of PAF. The contractile responses of isolated GPPS to cumulative doses of LTD4, acetylcholine, histamine, and potassium chloride in the presence of PAF (0.1 nM, 0.1 microM) were compared with parallel controls. There was no significant alteration of the response to acetylcholine and potassium chloride and the PAF-induced inhibition of the response to histamine, although significant, was not concentration dependent. In contrast, PAF in a concentration range from 0.1 nM to 1.0 microM caused a marked, concentration-dependent reduction of LTD4-induced contractions. Pretreatment with the PAF receptor antagonist, BN52021, prevented the attenuation of LTD4-induced contraction by PAF. The attenuation of LTD4-induced contraction by PAF was also prevented by pretreatment with indomethacin or with the thromboxane synthase inhibitor U63,557A, but not by pretreatment with the lipoxygenase inhibitors BW755c or nordihydroguaiaretic acid. Thus inhibition of LTD4-induced GPPS contraction by PAF is receptor dependent and probably secondary to thromboxane generation. The respiratory smooth muscle response to leukotrienes may be modified significantly by concomitant PAF release.     

Suppression of protein kinase C and the stimulation of glucocorticoid receptor synthesis by dexamethasone in human fibroblasts derived from tumor tissue

Exposure of fibroblasts derived from keloid tissues, desmoid and dermal tissue from individuals with Gardner's syndrome (GS) to dexamethasone resulted in the suppression of protein kinase C (PKC) activity and [3H]thymidine incorporation into DNA, and a 20-fold induction of glutamine synthetase activity. Treatment of GS and keloid fibroblasts with 0.1 microM dexamethasone for 36 h increased glucocorticoid receptor (GR) synthesis, as determined by [35S]methionine labeling and immunoprecipitation with a monoclonal antibody to the human GR. The suppression of PKC activity by dexamethasone was shown to result from a loss of protein mass as determined by immunoblotting using an antibody to PKC type III. In contrast to these results, exposure of fibroblasts isolated from normal tissues to dexamethasone did not result in the suppression PKC and [3H]thymidine incorporation, there was only a sixfold induction of glutamine synthetase, and a decrease of GR synthesis. As no primary receptor binding defect could be detected, the altered response of tumor cells to steroid-occupied receptor indicates a partial post-receptor binding defect in GS and keloid cells.     

Yeast myosin heavy chain mutant: maintenance of the cell type specific budding pattern and the normal deposition of chitin and cell wall components requires an intact myosin heavy chain gene

Recent studies with myosin heavy chain mutants in the slime mold Dictyostelium discoideum and the yeast Saccharomyces cerevisiae indicate that the myosin heavy chain gene is not essential for cell survival under laboratory growth conditions. However, cells lacking a normal myosin heavy chain gene demonstrate substantial alterations in growth and cell division. In this study, we report that a disruption mutant in the rod portion of the yeast myosin heavy chain gene, MYO1, produces abnormal chitin distribution and cell wall organization at the mother-bud neck in a high proportion of dividing cells. It is suggested that this phenotype is the cause of the cell division defect and the osmotic sensitivity of yeast MYO1 mutants. In the absence of a normal MYO1 polypeptide, yeast cells alter their cell type specific budding pattern. It is concluded that an intact myosin heavy chain gene is required to maintain the cell type specific budding pattern and the correct localization and deposition of chitin and cell wall components during cell growth and division.     

Ras-mediated cell cycle arrest is altered by nuclear oncogenes to induce Schwann cell transformation.

The cellular responses to ras and nuclear oncogenes were investigated in purified populations of rat Schwann cells. v-Ha-ras and SV40 large T cooperate to transform Schwann cells, inducing growth in soft agar and allowing proliferation in the absence of added mitogens. Expression of large T alone reduces their growth factor requirements but is insufficient to induce full transformation. In contrast, expression of v-Ha-ras leads to proliferation arrest in Schwann cells expressing a temperature-sensitive mutant of large T at the restrictive temperature. Cells arrest in either the G1 or G2/M phases of the cell cycle, and can re-enter cell division at the permissive temperature even after prolonged periods at the restrictive conditions. Oncogenic ras proteins also inhibit DNA synthesis when microinjected into Schwann cells. Adenovirus E1a and c-myc oncogenes behave similarly to SV40 large T. They cooperate with Ha-ras oncogenes to transform Schwann cells, and prevent ras-induced growth arrest. Thus nuclear oncogenes fundamentally alter the response of Schwann cells to a ras oncogene from cell cycle arrest to transformation.     

Diffusion gradient plates for herbicide toxicity tests on micro-algae and cyanobacteria

Single dimension agar gel diffusion gradient plates were used to test the tolerance of several micro-algae and cyanobacteria to five herbicides. Growth of most of the test organisms was prevented by terbutryn and diuron at concentrations between 1·1 and 2·8 μg/ml (minimum inhibitory concentration, MIC). Chlorpropham was intermediate in toxicity whilst monuron and atrazine generally gave higher MIC values (up to 17·1 μg/ml). The cyanobacteria were mostly more tolerant than the algae, but susceptibility to the herbicides was variable even between strains of an individual algal species. The gradient plate technique offers the advantages of simplicity and low cost in toxicity screening studies with herbicides or other biologically active chemicals and xenobiotics.     

Photooxidative changes of lysozyme with 337.1 nm laser radiation

Summary Initial photoinduced oxidative changes in the protein lysozyme were studied using the 337.1 nm radiation from a pulsed nitrogen laser without exogenous sensitizing compounds. Irradiation of lysozyme and tryptophan in aerated solution results in the temperature and solvent dependent loss of tryptophan absorption and fluorescence, and the appearance of fluorescent daughter products, primarily N-formyl-kynurenine and kynurenine. Exposures that resulted in 15% loss of tryptophan fluorescence produced no measurable loss in enzymatic activity. Fluorescence quenching experiments on irradiated lysozyme at low conversion percentage suggest that an exposed residue (Trp-62) is favored as an initial target of attack.     

Two mRNAs that differ by two nontemplated nucleotides encode the amino coterminal proteins P and V of the paramyxovirus SV5

The "P" gene of the paramyxovirus SV5 encodes two known proteins, P (Mr approximately equal to 44,000) and V (Mr approximately equal to 24,000). The complete nucleotide sequence of the "P" gene has been obtained and is found to contain two open reading frames, neither of which is large enough to encode the P protein. We have shown that the P and V proteins are translated from two mRNAs that differ by the presence of two nontemplated G residues in the P mRNA. These two additional nucleotides convert the two open reading frames to one of 392 amino acids. The P and V proteins are amino coterminal and have 164 amino acids in common. The unique C terminus of V consists of a cysteine-rich region that resembles a cysteine-rich metal binding domain. An open reading frame that contains this cysteine-rich region exists in all other paramyxovirus "P" gene sequences examined, which suggests that it may have important biological significance.     

Ultrastructural features of the apical complex, pellicle, and membranes investing the gamonts of Haemogregarina magna (Apicomplexa: Adeleina)

Mature gamonts of Haemogregarina magna lie within a type of parasitophorous vacuole (Pv) apparently unique to the haemogregarines. The cytoplasm of infected erythrocytes was separated from the parasite by two Pv membranes. An additional membrane, coated on both sides with electron-dense material, closely invested the gamonts. The apical complex of the gamonts includes a conoid, two preconoidal rings, and an elaborate polar ring complex. The latter consisted of the polar ring and approximately 78 posteriorly directed, radially arranged, "tine-like" structures which fuse as they merge anteriorly into the polar ring. Freeze fracture replicas demonstrated that the pellicle of gamonts of H. magna was structurally similar to that of other apicomplexans. The closely apposed inner membranes of the pellicle formed plates which were arranged into strips along the long axis of the gamont. Calculations indicated that 13 such strips are found around the circumference of the gamonts with about six subpellicular microtubules associated with the inner surface of each strip. Gamonts of H. magna share many structural similarities with the kinetes, ookinetes, and sporokinetes of other apicomplexans. We propose that the conoid and polar ring complex are fundamental features of all apicomplexan "kinetes."