Mitogen-Activated Protein Kinase-Dependent and Protein Kinase C-Dependent Pathways Link the m1 Muscarinic Receptor to β-Amyloid Precursor Protein Secretion

Abstract: Full and functionally selective M1 muscarinic agonists (carbachol and AF102B, respectively) activate secretion of the soluble form of amyloid precursor protein (APPs) in PC12 cells expressing the m1 muscarinic receptor (PC12M1 cells). This activation is further augmented by neurotrophins such as nerve growth factor and basic fibroblast growth factor. Muscarinic stimulation activates two transduction pathways that lead to APPs secretion: protein kinase C (PKC)-dependent and mitogen-activated protein kinase (MAPK)-dependent pathways. These pathways operate in parallel and converge with transduction pathways of neurotrophins, resulting in enhancement of APPs secretion when both muscarinic agonist and neurotrophins stimulate PC12M1 cells. These conclusions are supported by the following findings: (a) Only partial blockade of APPs secretion is observed when PKC, p21^ras, or MAPK is fully inhibited by their respective specific inhibitors, GF109203X, S-trans,trans -farnesylthiosalicylic acid, and PD98059. (b) K252a, which blocks PKC and phorbol 12-myristate 13-acetate-induced APPs secretion, enhances both muscarinic-stimulated MAPK activation and APPs secretion. (c) Activation of MAPK in PC12M1 cells by muscarinic agonists is Ras-dependent but PKC-independent and is enhanced synergistically by neurotrophins. These results suggest that muscarinic stimulation of APPs secretion is mediated by at least two independent pathways that converge and enhance the signal for APPs secretion at the convergence point.     

A simplified radioimmunoassay for physiologically active angiotensin peptides [(1-8) octa- and (2-8) heptapeptides]

The availability of a sensitive and highly specific rabbit antiserum and the development of a peptide-extraction method employing glass beads permitted the evolution of a rapid reliable radioimmunoassay that measures the sum of the concentration of angiotensin II and its active metabolite, angiotensin III. At a dilution of 1:32,000 the antiserum is capable of measuring 1 fmol (1 pg) of angiotensin II. Cross reactivities of this antiserum, taking angiotensin II as 1.0, are: angiotensin III, 0.75; angiotensin-(3-8) hexapeptide, 0.11; angiotensin I, 0.006; angiotensin-(1-14) tetradecapeptide, 0.0001. The recovery of angiotensin II added to hormone-free plasma was 73 +/- 2% [mean +/- standard deviation (SD), n = 20]. When 0.9 ml of plasma was extracted, the minimal concentration of angiotensin II and III that could be quantified was 4 fmol/ml. When larger volumes of plasma were extracted, sensitivity was enhanced. Plasma blanks were zero. Intra-assay variability was 7.6% SD and interassay variability was 11.7% SD. Angiotensin II and III concentration in venous plasma of normal volunteers on an ad libitum diet was 15 +/- 8 fmol/ml (mean +/- SD, range less than 4 to 35 fmol/ml). The plasma of a patient with primary aldosteronism had an unmeasurable value (less than 4 fmol/ml). Posture, converting enzyme inhibition, and renal artery stenosis resulted in expected changes of angiotensin concentration.     

The interactions between indomethacin and cytotoxic drugs in mice bearing B-16 melanomas

We have compared the effects of indomethacin alone (100 microgram/mouse/day) with those of indomethacin plus adriamycin, 5-FU, nitrogen mustard, thioTEPA, and vincristine on B-16 tumor cell proliferation in vivo. As we have previously described, after four days of treatment with indomethacin, subcutaneous tumors were slightly smaller and lighter in weight, but contained more melanoma cells. Addition of indomethacin to cytotoxic regimens resulted in either no change or a decrease in the effectiveness of the chemotherapy. In previous studies we demonstrated that treatment of tumor-bearing mice with a long-acting synthetic analogue of PGE2 (di-M-PGE2) stimulated the synthesis of endogenous prostaglandins. In order to evaluate if these endogenously synthesized prostaglandins were responsible for the inhibition of B-16 growth in vivo, mice were treated with di-M-PGE2 or di-M-PGE2 plus indomethacin. Addition of indomethacin did not alter the tumor inhibitory effects of di-M-PGE2.     

Bacillus anthracis Diverges from Related Clades of the Bacillus cereus Group in 16S-23S Ribosomal DNA Intergenic Transcribed Spacers Containing tRNA Genes

Mung bean nuclease treatment of 16S-23S ribosomal DNA intergenic transcribed spacers (ITS) amplified from several strains of the six species of the Bacillus cereus group showed that B. anthracis Davis TE702 and B. mycoides G2 have other intermediate fragments in addition to the 220- and 550-bp homoduplex fragments typical of the B. cereus group. Long and intermediate homoduplex ITS fragments from strains Davis TE702 and G2 and from another 19 strains of the six species were sequenced. Two main types of ITS were found, either with two tRNA genes (tRNA^Ile and tRNA^Ala) or without any at all. Strain Davis TE702 harbors an additional ITS with a single tRNA gene, a hybrid between the tRNA^Ile and tRNA^Ala genes, suggesting that a recombination event rather than a deletion generated the single tDNA-containing ITS. Strain G2 showed an additional ITS of intermediate length with no tDNA and no similarity to other known sequences. Neighbor-joining analysis of tDNA-containing long ITS indicated that B. cereus and B. thuringiensis represent a single clade. Three signature sequences discriminated B. anthracis from B. cereus and B. thuringiensis, indicating that the anthrax agent started evolving separately from the related clades of the B. cereus group. B. mycoides and B. weienstephanensis were very closely related, while B. pseudomycoides appeared the most distant species.     

Qualitative differences in nuclear proteins correlate with neuronal terminal differentiation

1. Protein composition of neuronal nuclei was studied at two stages of brain maturation, i.e., before (embryonic day 16; E16) and after (postnatal day 10; P10) shortening of the nucleosomal repeat length. Glial nuclei were analyzed in parallel as a control. 2. Total nuclear or HCl- and 5% perchloric acid (PCA)-soluble proteins were analyzed by different electrophoretic techniques. 3. Our results show an increase in the concentration of histone H1 zero with differentiation, although the H1 class undergoes an overall decrease. 4. The chromatin of mature neurons is also enriched in the ubiquinated form of histone H2A (A24), while the high-mobility group (HMG) proteins 1 and 2 seem to decrease slightly relative to core histones. 5. Both quantitative and qualitative differences in the abundance of nonhistone proteins relative to histones accompany neuronal terminal differentiation.     

Bacterial expression and isolation of Petunia hybrida 5-enol-pyruvylshikimate-3-phosphate synthase

5-enol-Pyruvylshikimate-3-phosphate synthase (EPSP synthase, EPSPS), an in vivo enzyme target of the herbicide glyphosate (N-phosphonomethyl glycine), was purified from a Petunia hybrida suspension culture line, MP4-G, by a small-scale high-performance chromatographic purification procedure. The cDNA encoding the mature petunia EPSPS (lacking the chloroplast transit sequence) was cloned into a plasmid, pMON342, for expression in Escherichia coli. This clone complemented the EPSPS deficiency of an E. coli aroA- mutant, and the plant enzyme constituted approximately 1% of the total extractable protein. Large-scale purification of the enzyme from E. coli cells resulted in a highly active protein which was homogeneous as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino terminal sequencing. Antibodies raised against the purified enzyme also reacted with the E. coli EPSPS in Western analyses. The availability of large quantities of the plant enzyme will significantly facilitate mechanistic investigations as well as a comparative study with EPSPS from bacteria and fungi.     

Uptake of [14C]GABA by rat brain slices; the effect of Ca2+

The [14C]GABA uptake by slices (0.3 mm thick) of Wistar rat brain cortex was studied for its dependence on the GABA concentration in the medium, time of incubation and the presence of Ca2+. This process is characterized by the absence of saturation; the uptake by slices increases sharply when the concentration of exogenous [14C]GABA reaches 200 microM. Bicucullin (10(-4) M), an antagonist of GABA, inhibits the accumulation of GABA in the concentration of 0.2 microM by 60%, that evidences for a considerable contribution of the receptor binding to this process. The [14C]GABA uptake when Ca2+ is absent in the incubation medium and when its concentration is 10(-3) M is practically the same and comparatively low concentrations of Ca2+ (10(-6)-10(-4] decrease the GABA uptake.     

Immunological unresponsiveness of self-recognizing B lymphocytes to the PBA property of a soluble T cell factor.

Spleen cells cultured in the presence of Con A became activated to polyclonal anti-body synthesis. This effect was found to be mediated by a polyclonal factor released by the activated T cells. Such a factor, contrary to any other polyclonal B cell activator, so far tested, failed to induce resting B cells to synthetize antibodies capable of lysing autologous albumin coupled SRBC. The Unresponsiveness was found to be specific since Con A activated spleen cells were capable of lysing heterologous albumin and HGG coupled SRBC. Moreover, neither active suppressor cells, nor soluble suppressor factors seem to be involved in the lack of response to autologous antigens. It is concluded that the clones of B cells carrying the polyclonal receptor for the T factor and Ig receptors for self are eliminated or functionally inactivated. The implication of these findings for the mechanism of self-non-self discrimination are discussed in relation to the mechanism of immunocyte triggering.     

Myosin-like microfilaments in the human normal and pathological testis.

The immunofluorescent staining of the human normal testis shows that both peritubular and tubular cells bind antimyosin-like antibodies. The same cells are provided with a large amount of microfilaments within the cytoplasm. The thin microfilaments observed by electron microscopy in various cells of the human testis likely correspond to the sites of immunofluorescent staining. In pathological specimens there can be observed a decrease in peritubular smooth muscle cells associated with a decrease in cytoplasmic microfilaments and in the immunofluorescent staining of some cells.