Putative candidate genes for blood pressure control in the SHR.BN-RNO8 congenic substrains

The SHR-Lx congenic strain carrying a differential segment of chromosome 8 of BN and PD origin was recently shown to exhibit a significant decrease in blood pressure as compared to the SHR strain. There were two positional candidate genes for blood pressure control mapped to the differential segment: the rat kidney epithelial potassium channel gene (Kcnj1) and brain dopamine receptor 2 gene (Drd2). Bot these genes were separated into SHR.BN-RNO8 congenic substrains. In this communication, we are presenting the assignment of two further putative candidate genes, which might be involved in blood pressure control to the BN/PD differential segment of the SHR-Lx congenic strain. These are: the gene coding for smooth muscle cell specific protein 22 (Sm22) defined by the D8Mcw1 marker and neuronal nicotinic acetylcholine receptor gene cluster, defined by the D8Bord1 marker. Moreover, the glutamate receptor gene Grik4 which also maps to the differential segment of the SHR-Lx should be taken into account. The genetic separation of all these putative candidate genes of blood pressure control is being performed by recombinations and subsequent selection using (SHR×SHR-Lx) intercross population.     

Techniques for the detection of pathogenic Cryptococcus species in wood decay substrata and the evaluation of viability in stored samples

In this study, we evaluated several techniques for the detection of the yeast form of Cryptococcus in decaying wood and measured the viability of these fungi in environmental samples stored in the laboratory. Samples were collected from a tree known to be positive for Cryptococcus and were each inoculated on 10 Niger seed agar (NSA) plates. The conventional technique (CT) yielded a greater number of positive samples and indicated a higher fungal density [in colony forming units per gram of wood (CFU.g^-1) ] compared to the humid swab technique (ST). However, the difference in positive and false negative results between the CT-ST was not significant. The threshold of detection for the CT was 0.05.10³ CFU.g^-1, while the threshold for the ST was greater than 0.1.10³ CFU^-1. No colonies were recovered using the dry swab technique. We also determined the viability of Cryptococcus in wood samples stored for 45 days at 25ºC using the CT and ST and found that samples not only continued to yield a positive response, but also exhibited an increase in CFU.g^-1, suggesting that Cryptococcus is able to grow in stored environmental samples. The ST.1, in which samples collected with swabs were immediately plated on NSA medium, was more efficient and less laborious than either the CT or ST and required approximately 10 min to perform; however, additional studies are needed to validate this technique.     

Alga consumption of four dominant planktonic crustaceans in Lake Balaton (Hungary)

Between 1981–83 the gut contents ofDaphnia galeata, D. cucullata, Eudiaptomus gracilis, andCyclops vicinus were examined with light and scanning electron microscope to obtain information on the feeding of these species in Lake Balaton. The twoDaphnia species feed mainly on abioseston, and it is assumed that their primary nutrient source was organic matter adsorbed onto the surfaces of the abioseston granules plus bacteria and detritus.E. gracilis feeds on algae, showing a preference for green algae and diatoms.C. vicinus is also a prodigious consumer of algae in Lake Balaton, utilizing the whole size spectrum of phytoplankton. Concerning the trophic relationships between phytoplankton and zooplankton in Lake Balaton, that between diatoms and bothE. gracilis andC. vicinus is the most conspicouos. Convincing evidence for an extensive utilization of blue-green algae was not found. Though there is no firm evidence yet, it is likely that theDaphnia are dependent on organic matter adsorbed on the abioseston.     

Charge distribution on the S layer of Bacillus stearothermophilus NRS 1536/3c and importance of charged groups for morphogenesis and function.

The distribution and functional significance of charged groups on the outer and inner faces of the S layer from Bacillus stearothermophilus NRS 1536/3c was investigated. Chemical modification of the exposed amino or carboxyl groups was performed on whole cells, isolated S layers self-assembled in vitro, and cell wall fragments (S layer attached to the peptidoglycan-containing sacculus). Without chemical modification, S layer self-assembly products could be labeled with polycationic ferritin, while S layers on whole cells could not. Following treatment with glutaraldehyde, whole cells were uniformly labeled with polycationic ferritin. Whole cells treated with glutaraldehyde and glycine methyl ester in the presence of carbodiimide did not bind polycationic ferritin significantly above background. Treatment of cell wall fragments with amino-specific, homobifunctional cross-linkers or with carbodiimide alone rendered the S layer protein nonextractable with sodium dodecyl sulfate. After amidation of the accessible carboxyl groups, the modified, guanidine hydrochloride-extractable S layer protomers did not self-assemble into regularly structured lattices. N-Amidination with ethylacetimidate did not interfere with the self-assembly of the isolated protomers. N-Acetylation resulted in a considerable destabilization of the S layer lattice, as seen by the release of a large amount of modified protomers during the reaction. N-Succinylation led to a complete disintegration of the protein lattice. These results indicated that only the inner face of the S layer carried a net negative charge. On both faces, free amino and carboxyl groups of adjacent protomers were arranged in proximity so as to contribute by electrostatic interactions to the cohesion of the protomers in the two-dimensional array. The native charge of the protomers was required for both the in vitro self-assembly of the isolated subunits and the maintenance of the structural integrity of the S layer lattice. Among other functions, the biological significance of the S layers may be in masking the electronegative charge of the cell wall proper.     

Use of regularly structured bacterial cell envelope layers as matrix for the immobilization of macromolecules

Summary Carboxyl groups present on the outer face of the hexagonally ordered S-layer lattices from Bacillus stearothermophilus PV72 and Clostridium thermohydrosulfuricum L111-69 were activated with carbodiimide. The reaction of the activated carboxyl groups with free amino groups of low molecular weight nucleophiles was controlled by labelling with polycationized ferritin, a net positively charged topographical marker for electron microscopy, which densely binds to S-layers possessing free carboxyl groups. Carbodiimide-activated carboxyl groups were also allowed to react with amino groups of ferritin (MW 440 000) and invertase (MW 270 000). Covalent attachment of ferritin was examined by electron microscopy. Using invertase, approximately 1 mg enzyme was bound per mg S-layer protein indicating a high packing density of invertase molecules on the outer face of the S-layer lattice. The immobilized invertase retained 70% of its original activity.     

Ha-ras-1 alleles in Norwegian lung cancer patients

Summary We have examined DNA restriction fragment length polymorphisms (RFLP) of the Ha-ras-1 gene in DNA from 118 lung cancer patients and 123 unaffected controls. When DNA samples were digested with MspI/ HpaII restriction endonucleases. Southern blot analysis demonstrated 4 common, 4 intermediate and 7 different rare alleles in the combined population after hybridization to the pGDa1 probe. Six of the rare alleles were unique for the lung cancer group and 1 rare allele for the control group. The frequency of rare alleles in lung cancer patients (10/236) was significantly different (P<0.01) from the control group (1/246). The lung cancer group also had a significantly lower frequency of the common 2.57 kb fragment than the controls (P<0.02). The results thus indicate that Ha-ras genotyping may be of value in lung cancer risk assessment.     

Glycosphingolipids of the globo-series are associated with the monocytic lineage of human myeloid cells

Neutral glycosphingolipids (neutral GSLs) of the human myeloid leukemia cell lines ML-2, ML-3, HL-60 and THP-1-0 were metabolically labeled with [3H]galactose and [3H]glucosamine, and analyzed by high-performance liquid chromatography. They were compared with unlabeled neutral GSLs from purified human granulocytes and monocytes. Neutral GSLs were identified by retention times and the structures were further confirmed by degradation with specific exoglycosidases. Two neutral GSLs of the globoseries, globotetraosylceramide and globotriaosylceramide were found in monocytes and the monoblastic leukemia line THP-1-0. The leukemia-derived cell-lines, ML-3 and HL-60, representing successively earlier stages of myeloid differentiation, contained respectively less neutral GSLs of the globoseries and an increasing proportion of (neo)lacto neutral GSLs. Granulocytes and the cell line ML-2 contained almost exclusively neutral GSLs of the (neo)lacto series.