Agrin induces phosphorylation of the nicotinic acetylcholine receptor.

Agrin causes acetylcholine receptors (AChRs) on chick myotubes in culture to aggregate, forming specializations that resemble the postsynaptic apparatus at the vertebrate skeletal neuromuscular junction. Here we report that treating chick myotubes with agrin caused an increase in phosphorylation of the AChR beta, gamma, and delta subunits. H-7, a potent inhibitor of several protein serine kinases, blocked agrin-induced phosphorylation of the gamma and delta subunits, but did not prevent either agrin-induced AChR aggregation or phosphorylation of the beta subunit. Experiments with anti-phosphotyrosine antibodies demonstrated that agrin caused an increase in tyrosine phosphorylation of the beta subunit that began within 30 min of adding agrin to the myotube cultures, reached a plateau by 3 hr, and was blocked by treatments known to block agrin-induced AChR aggregation. Anti-phosphotyrosine antibodies labeled agrin-induced specializations as they do the postsynaptic apparatus. These results suggest that agrin-induced tyrosine phosphorylation of the beta subunit may play a role in regulating AChR distribution.     

PATMAT: a searching and extraction program for sequence, pattern and block queries and databases

A program has been developed that provides molecular biologistswith multiple tools for searching databases, yet uses a verysimple interface. PATMATcan use protein or (translated) DNAsequences, patterns or blocks of aligned proteins as queriesof databases consisting of amino acid or nucleotide sequences,patterns or blocks. The ability to search databases of blocksby ‘on-the-fly’ conversion to scoring matrices providesa new tool for detection and evaluation of distant relationships.PATMAT uses a pull-down, menu-driven interface to carry outits multiple searching, extraction and viewing functions. Eachquery or database type is recognized, reported, and the appropriatesearch carried out, with matches and alignments reported inwindows as they occur. Any of the high scoring matches can beexported to a file, viewed and recalled as a query using onlya few keystrokes or mouse selections. Searches of multiple databasefiles are carried out by user selection within a window. PATMATruns under DOS; the searching engine also runs under UNIX.     

Biological characterization of Trypanosoma cruzi zymodemes: in vitro differentiation of epimastigotes and infectivity of culture metacyclic trypomastigotes to mice

Thirty-one Trypanosoma cruzi isolates from Chile, Peru, and Bolivia were studied in their capacity to differentiate in vitro from epimastigotes to metacyclic trypomastigotes on TAU-3AAG medium. Zymodeme 1 parasites displayed the best level of differentiation, which ranges from 60 to 90% depending on the isolate. Zymodeme 2 parasites exhibited highly heterogenous differentiation rates. This differentiation method permits the obtention of large amounts of metacyclic trypomastigotes from zymodeme 1 parasites. Metacyclic trypomastigotes obtained in vitro were infective to nude Balb/c hybrid mice. Zymodeme 1 parasites produced high parasitemias in this murine model; in contrast, zymodeme 2 parasites displayed lower parasitemias. Of a total of 27 T. cruzi isolates, 20 proved to be infective to mice, 12 gave enough parasites for further studies, and 8 of these were used for biological characterization. Results are compared with the infective clone Dm28 and Tulahuén strains maintained since 1954 in mice.     

Purification and characterization of PLC-beta m, a muscarinic cholinergic regulated phospholipase C from rabbit brain membrane

Two isozymes of phosphoinositide-specific phospholipase C were isolated and purified from salt-washed rabbit brain membranes. The membranes were extensively washed with isotonic, hypertonic and hypotonic buffers prior to solubilization with sodium cholate. Two isozymes (PLC-IV and PLC-beta m) were purified by a combination of DEAE-Sephacel, AH-Sepharose, heparin-Sepharose, AcA-34 gel filtration and mono-Q FPLC chromatographies. The major activity (PLC-beta m) was purified to homogeneity and had an estimated molecular weight of 155,000 on sodium-dodecyl sulfate-polyacrylamide gels (SDS-PAGE). This isozyme was immunologically identified as PLC-beta, an isozyme previously characterized in bovine brain cytosol and 2 M KCl membrane extracts. A second isozyme, PLC-IV, was immunologically distinct from PLC-beta and PLC-gamma and was purified to a stage where three protein bands (Mr 66,000, 61,000 and 54,000) on SDS-PAGE correlated with enzyme activity. The catalytic properties of the isozymes were studied and found to be very similar. The specific activities for PIP2 were greater than those obtained when PI was used. Both PLC-IV and PLC-beta m were Ca2(+)-dependent; near maximal stimulation for PI and PIP2 hydrolysis was observed at 0.5 microM free Ca2+. Sodium pyrophosphate and sodium fluoride stimulated phospholipase C activity of both isozymes. Polyclonal antibodies raised against PLC-beta m were able to inhibit carbachol and GTP gamma S stimulated phospholipase C activity in 2 M KCl washed rabbit cortical membranes. This suggests that in rabbit brain muscarinic cholinergic stimulation regulates PLC-beta m.     

Amerindian mitochondrial DNAs have rare Asian mutations at high frequencies, suggesting they derived from four primary maternal lineages.

The mitochondrial DNA (mtDNA) sequence variation of the South American Ticuna, the Central American Maya, and the North American Pima was analyzed by restriction-endonuclease digestion and oligonucleotide hybridization. The analysis revealed that Amerindian populations have high frequencies of mtDNAs containing the rare Asian RFLP HincII morph 6, a rare HaeIII site gain, and a unique AluI site gain. In addition, the Asian-specific deletion between the cytochrome c oxidase subunit II (COII) and tRNA(Lys) genes was also prevalent in both the Pima and the Maya. These data suggest that Amerindian mtDNAs derived from at least four primary maternal lineages, that new tribal-specific variants accumulated as these mtDNAs became distributed throughout the Americas, and that some genetic variation may have been lost when the progenitors of the Ticuna separated from the North and Central American populations.     

Genes Responsible for Size Reduction of Marine Vibrios during Starvation Are Located on the Chromosome

In a survey of 21 marine Vibrio spp., all responded to nutrient deprivation by undergoing a reduction in size (dwarfing). However, only 43% of these strains possessed one or more plasmids, suggesting that the genes responsible for dwarfing were located on the chromosome rather than on the plasmids. This conclusion was confirmed by the observation that fragmentation and subsequent size reduction occurred in three strains from which the plasmids had been removed by curing. The cured strains lost certain characteristics, such as resistance to some heavy metals and antibiotics, that were restored when the plasmids were reintroduced by either transformation or electroporation.     

Complete moles have paternal chromosomes but maternal mitochondrial DNA

Summary Complete hydatidiform moles contain only paternal chromosomes. To learn more of their origin, we used restriction endonuclease site polymorphisms found in the parental mitochondrial DNAs to demonstrate that moles contain exclusively maternal mitochondrial DNA. Thus, moles must arise from the fusion of one or two sperm with a mature but anucleate ovum.     

Purified interferon as protection against rhinovirus infection.

In a double-blind placebo-controlled study a preparation of human leucocyte interferon purified by affinity chromatography using a monoclonal antibody and applied by repeated nasal sprays reduced the incidence and severity of colds in volunteers challenged with human rhinovirus 9. Although interferon itself caused some symptoms, these were minor compared with the clinical colds. Interferon activity was still detectable in nasal washings as long as 26 hours after the last dose in about half the volunteers on active treatment.