C4B3 allotype with a novel Ch phenotype

The fourth component of complement (C4) has two classes of protein, C4A and C4B, both of which have many allelic forms. The serological determinants Rodgers (Rg1, Rg2) and Chido (Ch1, Ch2, Ch3) are generally associated with C4A and C4B, respectively. The C4B3 allotype has been detected in a single Canadian family that expresses a novel Ch phenotype, Ch:–1, 2, –3. There was no information for the Rg determinants, as the C4A ^* 2B ^* 3 haplotype would normally express Rg on the C4A protein. Other C4B3 allotypes in informative families have different Ch phenotypes, and the relationships of these within extended major histocompatibility complex haplotypes are discussed in this paper.     

Involvement of a chondroitin sulfate proteoglycan in the avoidance of chick epidermis by dorsal root ganglia fibers: a study using beta-D-xyloside

In 7-day chick embryo dorsal root ganglia and epidermis cocultures, nerve fibers avoid the epidermis. Previous studies have indicated that glycoproteic factors, secreted by epidermis, could be involved in this phenomenon. Treatment of epidermis by beta-D-xyloside, a specific proteoglycan synthesis inhibitor, abolishes the avoidance reaction. The same result is obtained when anti-chondroitin sulfate antibodies are added to the culture medium. Using HPLC and 35SO4 labeling combined with chondroitinase and hyaluronidase treatment, it has been demonstrated that chondroitin sulfate is present in the epidermal conditioned medium. This suggests that a chondroitin sulfate proteoglycan secreted by the epidermis is implicated in the neurite avoidance reaction and that epidermis could therefore control its own "noninnervation". In vivo, inhibitory influences by local extracellular components may control the guidance of growth cones during nerve pattern formation.     

Identification of G proteins mediating fungal elicitor-induced dephosphorylation of host plasma membrane H+ -ATPase

Tomato cells (with the Cf-5 resistance gene) were treated withelicitor preparations containing the avr5 gene product fromtwo Cf-5 incompatible races of the fungal pathogen Cladosporiumfulvum (race 2.3 and race 4), or with elicitor preparationscontaining no avr5 gene product from two Cf-5 compatible races(race 5 and race 2.4.5.9 [EC] .11). Elicitor preparations from race2.3 or race 4 caused dephosphorylation of host plasma membraneH⁺ -ATPase in isolated plasma membranes, while the preparationsfrom race 5 or race 2.4.5.9 [EC] .11 did not. GTP()S, AlF₄^–and cholera toxin (CTX) each induced similar dephosphorylationin the absence of active elicitors. The elicitor-induced dephosphorylationof the H⁺ -ATPase was blocked by preincubation of membraneswith an antibody raised against a stimulatory G protein -subunit(anti-G_s This antibody cross-reacted with a 42 kDa polypeptidefrom tomato plasma membranes. A 42 kDa polypeptide was alsoADP-ribosylated by CTX. When plasma membranes were treated withelicitor preparations from race 4 and separated on non-dissociatingPAGE, two proteins were detected on Western blots with the antibodyraised against the -subunit, suggesting the dissociation ofthe trimeric complex. No dissociation of the complex was detectedwith antibodies raised against either the - or ß-subunitswhen the plasma membranes were treated with elicitor preparationsfrom race 5. The results provide evidence for the activationof a stimulatory subtype of trimeric G proteins in the stimulationof elicitor-induced host defences to fungal pathogens. Key words: G protein, dephosphorylation, H⁺ -ATPase, fungal elicitor, tomato     

Stimulation of (Na+K+)-ATPase of rat liver plasma membrane by amino acids

The effect of twelve l-amino acids on the activity of liver plasma membrane (Na⁺K⁺)-ATPase has been tested. Histidine and arginine significantly enhanced the activity. The activtion by histidine showed saturation kinetics with an apparent K_a of about 8 mM, and was evident over a wide range of Na⁺ concentrations. The same amino acid did not significantly affect the Mg²⁺-dependent ATPase activity.     

Subcellular and submitochondrial localization of phospholipid-synthesizing enzymes in Saccharomyces cerevisiae.

Using highly enriched membrane preparations from lactate-grown Saccharomyces cerevisiae cells, the subcellular and submitochondrial location of eight enzymes involved in the biosynthesis of phospholipids was determined. Phosphatidylserine decarboxylase and phosphatidylglycerolphosphate synthase were localized exclusively in the inner mitochondrial membrane, while phosphatidylethanolamine methyltransferase activity was confined to microsomal fractions. The other five enzymes tested in this study were common both to the outer mitochondrial membrane and to microsomes. The transmembrane orientation of the mitochondrial enzymes was investigated by protease digestion of intact mitochondria and of outside-out sealed vesicles of the outer mitochondrial membrane. Glycerolphosphate acyltransferase, phosphatidylinositol synthase, and phosphatidylserine synthase were exposed at the cytosolic surface of the outer mitochondrial membrane. Cholinephosphotransferase was apparently located at the inner aspect or within the outer mitochondrial membrane. Phosphatidate cytidylyltransferase was localized in the endoplasmic reticulum, on the cytoplasmic side of the outer mitochondrial membrane, and in the inner mitochondrial membrane. Inner membrane activity of this enzyme constituted 80% of total mitochondrial activity; inactivation by trypsin digestion was observed only after preincubation of membranes with detergent (0.1% Triton X-100). Total activity of those enzymes that are common to mitochondria and the endoplasmic reticulum was about equally distributed between the two organelles. Data concerning susceptibility to various inhibitors, heat sensitivity, and the pH optima indicate that there is a close similarity of the mitochondrial and microsomal enzymes that catalyze the same reaction.     

Affinities of Histamine H1-Antagonists in Guinea Pig Brain: Similarity of Values Determined from [3H]Mepyramine Binding and from Inhibition of a Functional Response

Affinity constants for five antagonists at histamine H1-receptors in guinea pig brain have been determined from inhibition of the potentiation by histamine of the adenosine-induced accumulation of cyclic AMP in cerebral cortical slices. This action of histamine appeared to be mediated solely through H1-receptors. The affinity constants obtained were similar to those determined on peripheral H1-receptors and from inhibition of high-affinity [3H]mepyramine binding. This provides strong evidence that at least some of the [3H]mepyramine binding sites in guinea pig brain can be identified with functional H1-receptors.     

Cloning, restriction digestion and DNA labeling of large DNA fragments (greater than or equal to 1 kb) in the presence of remelted SeaPlaque GTG agarose gels.

Large DNA fragments (greater than or equal to 1 kb), separated in low melting temperature SeaPlaque GTG agarose gels, can be enzymatically processed directly in the presence of this agarose (in-gel). Time saving protocols are discussed for in-gel processing of large DNA fragments in the presence of remelted SeaPlaque GTG agarose, including cloning into pUC18, nick translation, random priming and restriction digestion. These in-gel molecular biology techniques are as efficient as those using DNA recovered from agarose. The effects of UV irradiation, Mg2+ concentration and agarose concentration on selected in-gel protocols are also discussed.     

Development of sleep during monotonous stimulation as related to individual differences

This study was designed to examine whether the sleep-promoting effect of monotonous stimulation depends on individual differences in strength of the nervous system, as was suggested by Pavlov. Sixty male subjects were divided into three groups, depending on their score on the "strength of excitation" scale of the Strelau Temperament Inventory. Within each group, subjects were randomly assigned to be exposed to either a) a sequence of tones or b) "no tones" (i.e., a quiet room). Dependent variables were latencies to Sleep Stage 1 (SOL 1) and Sleep Stage 2 (SOL 2). The main effects of stimulation and strength of the nervous system were not statistically significant. However, there was a significant interaction between stimulation and strength for both dependent variables. "Weak" subjects tended to fall asleep more rapidly during monotonous stimulation, whereas the reverse was true of "strong" subjects. The results suggest that individual differences might play an important role in the development of sleep during monotonous stimulation.     

Purification and properties of the cyclic 3',5'-AMP-binding protein from the muscle of the Nematode Ascaris suum

The cyclic 3',5'-AMP-binding protein was isolated from the muscle of Ascaris suum and purified to apparent homogeneity. It migrated as a protein with a relative Mr 54,000 on electrophoresis under denaturing conditions. On gel filtration columns it was eluted at a volume corresponding to a protein of Mr greater than 200,000 under conditions which kept the cyclic 3',5'-AMP-binding property intact. The purified catalytic subunit of protein kinase from Ascaris and the C subunit of cyclic 3',5'-AMP-dependent protein kinase from bovine heart were inhibited by the cyclic 3',5'-AMP-binding protein. Gel filtration studies indicated the formation of a stable protein complex between the protein kinase and the cyclic 3',5'-AMP-binding protein from Ascaris.     

Nucleotide sequence analysis of the purEK operon encoding 5''-phosphoribosyl-5-aminoimidazole carboxylase of Escherichia coli K-12.

5'-Phosphoribosyl-5-aminoimidazole (AIR) carboxylase (EC 4.1.1.21) catalyzes step 6, the carboxylation of AIR to 5'-phosphoribosyl-5-aminoimidazole-4-carboxylic acid, in the de novo biosynthesis of purine nucleotides. As deduced from the DNA sequence of restriction fragments encoding AIR carboxylase and supported by maxicell analyses, AIR carboxylase was found to be composed of two nonidentical subunits. In agreement with established complementation data, the catalytic subunit (deduced Mr, 17,782) was encoded by the purE gene, while the CO2-binding subunit (deduced Mr, 39,385) was encoded by the purK gene. These two genes formed an operon in which the termination codon of the purE gene overlapped the initiation codon of the purK gene. The 5' end of the purEK mRNA was determined by mung bean nuclease mapping and was located 41 nucleotides upstream of the proposed initiation codon. The purEK operon is regulated by the purR gene product, and a purR regulatory-protein-binding site related to the sequences found in other pur loci was identified in the purEK operon control region.