Differential phosphorylation of band 3 and glycophorin in intact and extracted erythrocyte membranes

This report presents an analysis of the phosphorylation of human and rabbit erythrocyte membrane proteins which migrate in NaDodSO₄-polyacrylamide gels in the area of the Coomassie Blue-stained proteins generally known as band 3. The phosphorylation of these proteins is of interest as band 3 has been implicated in transport processes. This study shows that there are at least three distinct phosphoproteins associated with the band 3 region of human erythrocyte membranes. These are band 2.9, the major band 3, and PAS-1. The phosphorylation of these proteins is differentially catalyzed by solubilized membrane and cytoplasmic cyclic AMP-dependent and -independent erythrocyte protein kinases. Band 2.9 is present and phosphorylated in unfractionated human and rabbit erythrocyte ghosts but not in NaI- or dimethylmaleic anhydride (DMMA)-extracted membranes. These latter membrane preparations are enriched in band 3 and in sialoglycoproteins. The NaI-extracted ghosts contain residual protein kinase activity which can catalyze the autophosphorylation of band 3 whereas the DMMA-extracted ghosts are usually devoid of any kinase activity. However, both NaI- and DMMA-extracted ghosts, as well as Triton X-100 extracts of the DMMA-extracted ghosts, can be phosphorylated by various erythrocyte protein kinases. The kinases which preferentially phosphorylate the major band 3 protein are inactive towards PAS-1 while the kinases active towards PAS-1 are less active towards band 3. The band 3 protein in the DMMA-extracted ghosts can be cross-linked with the Cu²⁺ -σ-phenanthroline complex. The cross-linking of band 3 does not affect its capacity to serve as a phosphoryl acceptor nor does phosphorylation affect the capacity of band 3 to form cross-links. In addition to band 2.9, the major band 3 and PAS-1, another minor protein component appears to be present in the band 3 region in human erythrocyte membranes. This protein is specifically phosphorylated by the cyclic AMP-dependent protein kinases isolated from the cytoplasm of rabbit erythrocytes. The rabbit erythrocyte membranes lack PAS-1 and the cyclic AMP-dependent protein kinase substrate.     

Studies of the mechanisms for the induction of in vivo tumor immunity. VI. Induction of specific and nonspecific cell-mediated immunity in tumor-bearing hosts and its correlation with transplantation tumor immunity

Studies were performed to determine the development of cell-mediated cytotoxic response at tumor site in C57BL/6 mice bearing progressively growing FBL-3 ascites leukemia. The effectors isolated from tumor ascites are found to be highly cytotoxic for leukemic target cells. The levels of cytotoxicity obtained with effectors isolated from tumor site are generally higher than those obtained with immune mice. This cytotoxicity is both specific and nonspecific. The specific cytotoxicity against tumor-associated antigen is mainly mediated by T cells and the nonspecific cytotoxicity against unrelated tumor cells is mediated largely by macrophages. The T-cell-enriched preparation did not give significant natural killer activity. When testing the ability of these effectors to produce in vivo immunity against the challenge of FBL-3, it was found that only T cells could confer the transplantation-type immunity, but the immunity was transient. The macrophage-enriched preparation isolated from tumor ascites failed to give in vivo protection. These findings indicate that in FBL-3 system, mice with progressively growing tumors are able to develop immune response against tumor cells. However, this immunity is probably interfered with by a suppressor factor(s) or suppressor cells which restrict their activity to eliminate the tumor cells effectively.     

Human interferon omega (omega) binds to the alpha/beta receptor.

It was proposed that human interferon omega (omega) binds to the interferon alpha/beta receptor but not to the interferon gamma receptor. However, since no studies were performed to provide direct evidence for this hypothesis, we carried out cross-linking experiments and saturation binding assays between a 32P-labeled human interferon-alpha (Hu-IFN-alpha) and unlabeled Hu-IFN-alpha A, -beta, -gamma, and -omega. These assays demonstrated that Hu-IFN-alpha A, -beta, and -omega, but not Hu-IFN-gamma, were able to block binding of 32P-labeled Hu-IFN-alpha A to human cells. These results indicate that Hu-IFN-omega binds to the alpha/beta receptor.     

Comparative mapping of a gorilla-derived alpha satellite DNA clone on great ape and human chromosomes

We have isolated an alpha satellite DNA clone, pG3.9, from gorilla DNA. Fluorescence in situ hybridization on banded chromosomes under high stringency conditions revealed that pG3.9 identifies homologous sequences at the centromeric region of ten gorilla chromosomes, and, with few exceptions, also recognizes the homologous chromosomes in human. A pG3.9-like alphoid DNA is present on a larger number of orangutan chromosomes, but, in contrast, is present on only tow chromosomes in the chimpanzee. These results show that the chromosomal subsets of related alpha satellite DNA sequences may undergo different patterns of evolution.by J.B. Rattner     

Prevalence, biotypes, plasmid profile and antimicrobial resistance of Campylobacter isolated from wild and domestic animals from northeast Portugal.

The incidence of Campylobacter jejuni and Campylobacter coli in wild and producing animals has been studied to evaluate their importance as potential reservoirs of campylobacter infection. These organisms were isolated from: 59 chicken (60.2%), 65 swine (59.1%), 31 black rats (57.4%), 61 sparrows (45.5%), 21 ducks (40.5%), 32 cows (19.5%) and 27 sheep (15.3%). Biotypes, plasmid and resistance profiles were studied in order to characterize the isolates. Biotypes I and II of C. jejuni were predominant in all reservoirs except swine, where C. coli I was more frequent. Plasmid prevalence was higher in strains isolated from swine (53.8%) and rats (45.5%). The size of the plasmids ranged from 1.3 to 82 MDa. A 2.3 MDa plasmid was the most frequent, detected in all the reservoirs except ducks. Antimicrobial susceptibility testing revealed that 5.5% of the strains were resistant to ampicillin, 5.5% to tetracycline, 12.6% to erythromycin and 23.5% to streptomycin. Resistance to erythromycin (26.2%) and to streptomycin (58.4%) was particularly high in isolates from swine. Tetracycline resistance was encoded by a 33 or a 41 MDa plasmid and transferred by conjugation.     

Human ETS1 oncoprotein. Purification, isoforms, -SH modification, and DNA sequence-specific binding.

The human ETS1 proto-oncogene proteins have been isolated from the T-cell leukemia line, CEM, by immunoaffinity chromatography and their identity confirmed by NH2-terminal amino acid sequencing. Incubation of CEM cells with N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) indicates that ETS proteins can be modified in their cellular context and that pretreatment of the cells with N-ethylmaleimide (NEM) protects ETS1 proteins from TLCK modification. These data show that ETS1 proteins can exist in at least two different states, -SH-available and -SH-protected. Renatured human ETS1 has DNA sequence-specific binding to the PEA3 (CAGGAAGT) motif. The ETS1.PEA3 complex can be observed by electrophoretic mobility shift assays (EMSA). Purified ETS1 retards a band which is exactly the same size as a complex that is retarded from nuclear extracts prepared from CEM cells. Reduced ETS1 is required to form the ETS1.PEA3 complex, however; modification of the ETS1 -SH groups by either NEM or by TLCk does not inhibit formation of the complex. The ETS1.PEA3 complex formed with TLCK-modified ETS1 has a slower mobility than the complex formed with unmodified ETS1. Zone sedimentation analysis of purified ETS1 indicates that it is the monomer of ETS1 which binds to the PEA3 oligonucleotide.     

Muscarinic Acetylcholine Receptor Enhances Phosphatidylcholine Hydrolysis via Phospholipase D in Bovine Chromaffin Cells in Culture

Although it is well-established that inositol-containing lipids serve as precursors of intracellular second messenger molecules in chromaffin cells, we describe some findings that show the formation of diacylglycerol from phosphatidylcholine in response to agonist-mediated stimulation. Stimulation of chromaffin cells by acetylcholine produced a high turnover of phosphatidylcholine, as suggested by the release of [3H]choline derived from [3H]-phosphatidylcholine in experiments performed with [3H]choline chloride-prelabeled cells. An enhanced breakdown of phosphatidylcholine was also inferred from the finding of an increased formation of [3H]diacylglycerol in chromaffin cells prelabeled with [3H]glycerol. The diacylglycerol mass that accumulated after stimulation showed a distinct temporal course and seemed to exceed the mass that has been reported to be derived from phosphatidylinositol. In keeping with the purported origin from phosphatidylcholine, diacylglycerol showed a high content in [3H]oleate molecular species. Phospholipase D activity measurements and experiments performed in the presence of propranolol (an inhibitor of phosphatidic acid:phosphohydrolase) suggested that phosphatidylcholine is hydrolyzed by a phospholipase D activity, producing phosphatidic acid, which is subsequently degraded to diacylglycerol, rather than by a phospholipase C. Incubation of chromaffin cells in the presence of atropine before addition of acetylcholine showed complete inhibition of the increased formation of [3H]-diacylglycerol, whereas d-tubocurarine failed to do so. Taken together, these results suggest that acetylcholine activates phosphatidylcholine breakdown and diacylglycerol formation in chromaffin cells via a muscarinic-type receptor.     

Amino acids are general growth inhibitors of Nicotiana silvestris in tissue culture

The growth of Nicotiana silvestris in suspension culture is inhibited by all of the common protein amino acids at the millimolar level, except for L-glutamine. A defined experimental system for growth/inhibition studies has been established, and growth studies were carried out with cells that had been maintained in the exponential growth phase for at least 10 generations (EE cells). The following results were obtained after particularly detailed studies with aromatic amino acids. The onset of inhibition was preceded by a duration of normal growth rate which varied within a range of 12 to 48 h. The degree of inhibition was directly proportional to amino acid concentration and inversely related to the initial cell density of the inoculum. A slowed, but still exponential rate of growth persisted during an early phase of inhibition. Under sufficiently severe conditions, this was followed by progressive diminution of growth rate and eventual lysis. The most drastic inhibitory effects caused by aromatic amino acids were in the order: phenylalanine, tryptophan and tyrosine. When EE cells cultivated under conditions of growth inhibition were diluted into fresh medium, immediate resumption of growth at the uninhibited rate occurred and persisted. On the other hand, when growth-inhibited EE cells were diluted into medium containing the same concentration of amino acid used in the first round of growth, an initial burst of uninhibited growth lasting about 24 h was followed by a drastic, progressively declining growth rate which deteriorated to cell death and lysis. When cells in stationary phase were used as an inoculum, as is done in typical growth characterizations with suspension cultures, the sensitivity to inhibition during the subsequent exponential growth phase was several-fold greater than was the case with EE cells. Hypotheses that growth inhibition might be caused by ammonia toxicity, keto-acid toxicity, or by inhibition of nitrate utilization were ruled out. Observations that provide new insight are: (i)growth-inhibited cells undergo drastic plasmolysis, (ii) L-glutamine is an effective antagonist of amino-acid inhibitors, and (iii) growth-inhibited cells exhibit a transient restoration of normal growth rate upon dilution into fresh growth medium. These results implicate a linkage of amino acids with osmotic regulation and nitrogen metabolism.     

Strains of Colletotrichum coffeanum Noack Causing Coffee Berry Disease in Angola and Malawi with Characteristics Different to the Kenya Strain

Strains of Colletotrichum isolated from berries of Coffea arabica from Angola and Malawi were identified as C. coffeanum causing coffee berry disease (CBD). The isolates were highly aggressive to berries of the cultivar ‘Catimor’ and produced high numbers of acervuli and extremely large masses of pink conidia when cultivated in the dark. These strains are apparently different in aggressiveness and morphology to those described in the past from Kenya.