Biochemical Characterization of Annexins I and II Isolated from Pig Nervous Tissue

Five proteins having molecular masses of 90, 67, 37, 36, and 32 kDa (p90, p67, p37, p36, and p32, respectively) were identified in the particulate fractions of pig brain cortex and pig spinal cord prepared in the presence of 0.2 mM Ca2+ and further purified using a protocol previously described for the purification of calpactins. Proteins p90, p37, and p36 are related to annexins I and II. Annexin II, represented by p90, is found as an heterotetramer, composed of two heavy chains of 36 kDa and two light chains of 11 kDa, and as a monomer of 36 kDa. Protein p37, which differs immunologically from p36, is a monomer and could be related to annexin I. All three proteins are Ca(2+)-dependent phospholipid- and F-actin-binding proteins; they are phosphorylated on a serine and on a tyrosine residue by protein kinases associated with synaptic plasma membranes. Purified p36 monomer and p36 heterotetramer proteins bind to actin at millimolar Ca2+ concentrations. The stoichiometry of p36 binding to F-actin at saturation is 1:2, corresponding to one tetramer or monomer of calpactin for two actin monomers (KD, 3 x 10(-6) M). Synaptic plasma membranes supplemented with the monomeric or tetrameric forms of p36 phosphorylate the proteins on a serine residue. The monomer is phosphorylated on a serine residue by a Ca(2+)-independent protein kinase, whereas the heterotetramer is phosphorylated on a serine residue and a tyrosine residue by Ca(2+)-dependent protein kinases. Antibodies to brain p37 and p36 together with antibodies to lymphocytes lipocortins 1 and 2 were used to follow the distribution of these proteins in nervous tissues. Polypeptides of 37, 34, and 36 kDa cross-react with these antibodies. Anti-p37 and antilipocortin 1 cross-react on the same 37- and 34-kDa polypeptides; anti-p36 and antilipocortin 2 cross-react only on the 36-kDa polypeptides.     

H-Y antigen in X,i(Xq) gonadal dysgenesis: Evidence of X-linked genes in testicular differentiation

Summary Three years ago, we detected H-Y antigen in the white blood cells of a phenotypic female with several of the stigmata of Turner's syndrome, and the mosaic karyotype: 45,X/46,X,i(Xq). We surmised at the time that the isochromosome, i(Xq), may have contained occult Y-chromosome-derived material. We have now confirmed the presence of H-Y in this patient and we have obtained evidence for the presence of H-Y in four of five other similar patients, all of whom are notable for carrying at least a single cell line with the karyotype 46,X,i(Xq). Although we cannot categorically exclude the presence of Y-chromosomal genes in the cells of these patients, there is no cytogenetic evidence of structural rearrangement involving the Y in any of the cases. Expression of H-Y antigen in association with i(Xq) thus implies that H-Y structural genes are X-situated, or alternatively that they are autosomal and X-regulated. It would follow that the H-Y⁺ cellular phenotype per se is not a valid marker for the Y-chromosome, and that H-Y genes that have been mapped to the pericentric region of the Y may be regulatory.     

H-Y antigen in 46,XY gonadal dysgenesis

Summary Presence of H-Y antigen has been correlated with testicular differentiation, and absence of H-Y with failure of testicular differentiation, in a variety of mammalian species. To determine more precisely the relationship between expression of H-Y antigen and development of the testis, we studied the cells of phenotypic females with the 46,XY male karyotype. Blood leukocytes were typed H-Y⁺ in five XY females with gonadal dysgenesis, although in other studies blood leukocytes from XY females with gonadal dysgenesis were typed H-Y^-. Thus mere presence of H-Y antigen is not sufficient to guarantee normal differentiation of the testis. In the present paper we review evidence for an additional factor in gonadal organogenesis, the H-Y antigen receptor. We infer that testicular development requires engagement of H-Y and its receptor. It follows that XY gonadal dysgenesis is the consequence of functional absence of the H-Y testis inducer as in the following conditions: failure of synthesis of H-Y or failure of specific binding of H-Y.     

Assembly of the Mitochondrial Membrane System: Nuclear Suppression of a Cytochrome b Mutation in Yeast Mitochondrial DNA

In a previous study, a mitochondrial mutant expressing a specific enzymatic deficiency in co-enzyme QH₂-cytochrome c reductase was described (Tzagoloff, Foury and Akai 1976). Analysis of the mitochondrially translated proteins revealed the absence in the mutant of the mitochondrial product corresponding to cytochrome b and the presence of a new low molecular weight product. The premature chain-termination mutant was used to obtain suppressor mutants with wild-type properties. One such revertant strain was analyzed genetically and biochemically. The revertant was determined to have a second mutation in a nuclear gene that is capable of partially suppressing the original mitochondrial cytochrome b mutation. Genetic data indicate that the nuclear mutation is recessive and is probably in a gene coding for a protein involved in the mitochondrial translation machinery.     

Adaptation to different growth temperatures modifies some mammalian cell survival responses

Chinese hamster (HA-1) cells that have been grown at 37 °C since explant several years ago can adapt themselves to grow at temperatures ranging from 32 to 41 °C. This growth adaptation is accompanied by major phenotypic changes in, for exampie, the cellular responses to 43 and 45 °C heat challenges and to ethanol challenges (0–10% in concentration). Cells grown at 39.5 °C are seen to acquire substantial heat resistance when compared with cells grown at 37 °C; resistance is even more pronounced if the growth temperature is at 41 °C. On the other hand, cells grown at 32 °C become more sensitive to heat than controls. Our results also indicate an increased resistance to ethanol of the 41 °C grown cells. By contrast the cells' X-ray survival response is affected only minimally. The changes seen are phenotypic; upon being returned to 37 °C, HA-1 cells within 34 h regain their ‘normal’ heat responses.     

Inhibition of receptor-mediated uptake of a lysosomal enzyme into fibroblasts by chloroquine, procaine and ammonia

Cultured human skin fibroblasts take up α- -iduronidase by receptor-mediated pinocytosis. Certain lysosomotropic amines such as chloroquine, ammonia and procaine inhibit this process, without affecting the fluid endocytosis of dextran. In contrast to the competitive inhibition by mannose 6-phosphate, the inhibition by amines is non-competitive and is therefore presumed not to affect binding of the enzyme to receptors. The dose response curves are very steep, and equations that best fit the data use a power of inhibitor concentration (i² for procaine, i⁴ for chloroquine), indicating interaction of several amine molecules at the inhibitory site(s). The inhibition is reversed by removal of the amine from the medium and does not result from accelerated efflux of endocytosed enzyme. We suggest that the amines interfere with delivery of receptor-bound enzyme to lysosomes.     

Localization of the nucleolar organizer by computer-aided analysis of a variant no. 21 in a human isolate

Summary A variant chromosome no. 21 consisting of two stalks and two satellites in tandem was detected during a survey of a human isolate. The variant segregated in three generations of a large kindred. One male had the variant no. 21, a metacentric Y, and a 47, XXY complement; however, no other evidence of chromosomal nondisjunction was found. Computer-aided analysis of sequentially stained variant no. 21 chromosomes indicated that silver-stained material corresponded to the proximal stalk region (as defined defined by Giemsa). These data support the hypothesis that human nucleolar organizers are localized to the stalks of acrocentric chromosomes.     

Differential gene expression in whitefly Bemisia tabaci-infested tomato (Solanum lycopersicum) plants at progressing developmental stages of the insect's life cycle

A suppression-subtractive-hybridization (SSH) strategy was used to identify genes whose expression was modified in response to virus-free whitefly Bemisia tabaci ( Bt , biotype A) infestation in tomato ( Solanum lycopersicum ) plants. Thus, forward and reverse SSH gene libraries were generated at four points in the whitefly's life cycle, namely at (1) 2 days (adult feeding and oviposition: phase I); (2) 7 days (mobile crawler stage: phase II); (3) 12 days (second to third instar nymphal transition: phase III) and (4) 18 days (fourth instar nymphal stage: phase IV). The 169 genes with altered expression (up and downregulated) that were identified in the eight generated SSH libraries, together with 75 additional genes that were selected on the basis of their involvement in resistance responses against phytofagous insects and pathogens, were printed on a Nexterion^® Slide MPX 16 to monitor their pattern of expression at the above phases. The results indicated that Bt infestation in tomato led to distinctive phase-specific expression/repression patterns of several genes associated predominantly with photosynthesis, senescence, secondary metabolism and (a)biotic stress. Most of the gene expression modifications were detected in phase III, coinciding with intense larval feeding, whereas fewer changes were detected in phases I and IV. These results complement previously reported gene expression profiles in Bt -infested tomato and Arabidopisis, and support and expand the opinion that Bt infestation leads to the downregulation of specific defense responses in addition to those controlled by jasmonic acid.