Ca2+ and Mg2+-binding and a putative calmodulin type Ca2+-binding site in Synechococcus Photosystem II

Thylakoids and Photosystem II particles prepared from the cyanobacterium Synechococcus PCC 7942 washed with a HEPES/glycerol buffer exhibited low rates of light-induced oxygen evolution. Addition of either Ca²⁺ or Mg²⁺ to both thylakoids and Photosystem II particles increased oxygen evolution independently, maximal rates being obtained by addition of both ions. If either preparation was washed with NaCl, light induced O₂ evolution was completely inhibited, but re-activated in the same manner by Ca²⁺ and Mg²⁺ but to a lower level. In the presence of Mg²⁺, the reactivation of O₂ evolution by Ca²⁺ allowed sigmoid kinetics, implying co-operative binding. The results are interpreted as indicating that not only Ca²⁺, but also Mg²⁺, is essential for light-induced oxygen evolution in thylakoids and Photosystem II particles from Synechococcus PC 7942. The significance of the reactivation kinetics is discussed. Reactivation by Ca²⁺ was inhibited by antibodies to mammalian calmodulin, indicating that the binding site in Photosystem II may be analogous to that of this protein.Abbreviation HEPES n-2-Hydroxyethylpiperazine--2-ethane sulphonic acid     

Rapid authentication of animal cell lines using pyrolysis mass spectrometry and auto-associative artificial neural networks

Summary Pyrolysis mass spectrometry (PyMS) was used to produce biochemical fingerprints from replicate frozen cell cultures of mouse macrophage hybridoma 2C11-12, human leukaemia K562, baby hamster kidney BHK 21/C13, and mouse tumour BW-O, and a fresh culture of Chinese hamster ovary CHO cells. The dimensionality of these data was reduced by the unsupervised feature extraction pattern recognition technique of auto-associative neural networks. The clusters observed were compared with the groups obtained from the more conventional statistical approaches of hierarchical cluster analysis. It was observed that frozen and fresh cell line cultures gave very different pyrolysis mass spectra. When only the frozen animal cells were analysed by PyMS, auto-associative artificial neural networks (ANNs) were employed to discriminate between them successfully. Furthermore, very similar classifications were observed when the same spectral data were analysed using hierarchical cluster analysis. We demonstrate that this approach can detect the contamination of cell lines with low numbers of bacteria and fungi; this approach could plausibly be extended for the rapid detection of mycoplasma infection in animal cell lines. The major advantages that PyMS offers over more conventional methods used to type cell lines and to screen for microbial infection, such as DNA fingerprinting, are its speed, sensitivity and the ability to analyse hundreds of samples per day. We conclude that the combination of PyMS and ANNs can provide a rapid and accurate discriminatory technique for the authentication of animal cell line cultures.     

A Quick Embedding Method for Light Microscopy and Image Analysis of Cotton Fibers

A quick embedding method using UV polymerization of methacrylate plastic has been devised for embedding fibers encased in a polyvinyl chloride tube. The resulting embedments are suitable for light microscopy and image analysis.     

Familial Site-specific Ovarian Cancer Is Linked to BRCA1 on 17q12-21

In a study of nine families with “site-specific” ovarian cancer (criterion: three or more cases of epithelial ovarian cancer and no cases of breast cancer diagnosed at age <50 years) we have obtained evidence of linkage to the breast-ovarian cancer susceptibility gene, BRCA1 on 17q12-21. If the risk of cancer in these families is assumed to be restricted to the ovary, the best estimate of the proportion of families linked to BRCA1 is .78 (95% confidence interval .32–1.0). If predisposition to both breast and ovarian cancer is assumed, the proportion linked is 1.0 (95% confidence interval .46–1.0). The linkage of familial site-specific ovarian cancer to BRCA1 indicates the possibility of predictive testing in such families; however, this is only appropriate in families where the evidence for linkage to BRCA1 is conclusive.     

Fine mapping of the Autosomal Dominant Split Hand/Split Foot Locus on Chromosome 7, Band q21.3-q22.1

Split hand/split foot (SHFD) is a human developmental defect characterized by missing digits, fusion of remaining digits, and a deep median cleft in the hands and feet. Cytogenetic studies of deletions and translocations associated with this disorder have indicated that an autosomal dominant split hand/split foot locus (gene SHFD1) maps to 7q21-q22. To characterize the SHFD1 locus, somatic cell hybrid lines were constructed from cytogenetically abnormal individuals with SHFD. Molecular analysis resulted in the localization of 93 DNA markers to one of 10 intervals surrounding the SHFD1 locus. The translocation breakpoints in four SHFD patients were encompassed by the smallest region of overlap among the SHFD-associated deletions. The order of DNA markers in the SHFD1 critical region has been defined as PON–D7S812–SHFD1–D7S811–ASNS. One DNA marker, D7S811, detected altered restriction enzyme fragments in three patients with translocations when examined by pulsed-field gel electro-phoresis (PFGE). These data map SHFD1, a gene that is crucial for human limb differentiation, to a small interval in the q21.3-q22.1 region of human chromosome 7.     

Inactivation of the Porphyromonas gingivalis fimA gene blocks periodontal damage in gnotobiotic rats.

Fimbrial production by Porphyromonas gingivalis was inactivated by insertion-duplication mutagenesis, using the cloned gene for the P. gingivalis major fimbrial subunit protein, fimA. by several criteria, this insertion mutation rendered P. gingivalis unable to produce fimbrilin or an intact fimbrial structure. A nonfimbriated mutant, DPG3, hemagglutinated sheep erythrocytes normally and was unimpaired in the ability to coaggregate with Streptococcus gordonii G9B. The cell surface hydrophobicity of DPG3 was also unaffected by the loss of fimbriae. However, DPG3 was significantly less able to bind to saliva-coated hydroxyapatite than wild-type P. gingivalis 381. This suggested that P. gingivalis fimbriae are important for adherence of the organism to saliva-coated oral surfaces. Further, DPG3 was significantly less able to cause periodontal bone loss in a gnotobiotic rat model of periodontal disease. These observations are consistent with other data suggesting that P. gingivalis fimbriae play an important role in the pathogenesis of human periodontal disease.     

4-Hydroxybenzoate-coenzyme A ligase from Rhodopseudomonas palustris: purification, gene sequence, and role in anaerobic degradation.

Anaerobic metabolism of most aromatic acids is initiated by coenzyme A thioester formation. Rhodopseudomonas palustris grows well under anaerobic, phototrophic conditions with many aromatic acids, including benzoate and 4-hydroxybenzoate, as a carbon source. A coenzyme A ligase that reacts with 4-hydroxybenzoate was purified from 4-hydroxybenzoate-grown cells of R. palustris. This enzyme required MgATP, reduced coenzyme A, and 4-hydroxybenzoate, benzoate, or cyclohex-1,4-dienecarboxylate for optimal activity but also used phosphopantetheine, cyclohex-2,5-dienecarboxylate, and 4-fluorobenzoate at lower rates. The 4-hydroxybenzoate-coenzyme A ligase differed in molecular characteristics from a previously described benzoate-coenzyme A ligase from R. palustris, and the two ligases did not cross-react immunologically. The gene encoding the 4-hydroxybenzoate enzyme was cloned and sequenced. The deduced gene product showed about 20% amino acid identity with bacterial coenzyme A ligases involved in aerobic degradation of aromatic acids. An R. palustris mutant carrying a disrupted 4-hydroxybenzoate-coenzyme A ligase gene was unable to grow with 4-hydroxybenzoate under anaerobic conditions, indicating that the enzyme is essential for anaerobic degradation of this compound.     

Effect of polyols on the post-thawing motility of pellet-frozen ram spermatozoa

The cryoprotective effects of polyols in the absence and presence of glycerol in Tris-glucose-egg yolk based diluents on the post-thawing motility and acrosome integrity of pellet-frozen ram spermatozoa were examined. Incorporation of adonitol or xylitol (low molecular weight polyols; LMWPs) in diluents improved motility of spermatozoa in the absence of glycerol with maximum motility at 0.3 M (26.9 vs 13.3% mean motile spermatozoa; P < 0.001). Five concentrations of adonitol (0, 150, 300, 450, 600 mM) were examined in diluents containing 5 concentrations of glycerol (0, 1.5, 3.0, 4.5, 6.0% v/v). There was an additive effect of incorporation of 1.5% v/v glycerol with up to 450 mM adonitol (P < 0.001), but at higher levels of glycerol the incorporation of adonitol was detrimental to motility. The acrosome integrity of spermatozoa in diluents containing 0, 150 and 300 mM adonitol was superior to those containing 450 and 600 mM adonitol (46.1 vs 35.1% mean intact acrosomes; P < 0.05). Among the high molecular weight polyols (HMWPs) examined, better recovery of spermatozoa was obtained in diluents containing sorbitol than mannitol or inositol (P < 0.001). Sorbitol or mannitol (300 mM) improved the motility of spermatozoa in diluents without glycerol (P < 0.001), but the incorporation of HMWPs was detrimental in diluents containing glycerol. All five polyols were examined in isotonic diluents containing 360:0, 300:55, 240:110, 180:165, 120:220mM (Tris:polyol; 360 mosmol) and 6.0% v/v glycerol. There was a linear decrease in motility and acrosome integrity of spermatozoa with increasing polyol concentration in the diluent (P < 0.001) except for inositol, which was not detrimental. We conclude that the polyols examined have a cryoprotective effect on pellet-frozen ram spermatozoa except for inositol. However, in our study, no combination of polyols and glycerol was superior in terms of post-thawing motility and acrosome integrity of spermatozoa to 6.0% v/v glycerol alone in Tris-glucose-egg yolk diluents.