Characterisation of yeast phosphoglycerate kinase modified by mutagenesis at residue 21.

Site-directed mutagenesis has been used to produce mutant forms of yeast phosphoglycerate kinase in which the conserved active-site residue, Arg21, has been replaced by a methionine or a lysine. Kinetic results obtained using these mutant enzymes show that their Km for both 3-phospho-D-glycerate and ATP are significantly different from those recorded for the wild-type enzyme. The Vmax for the lysine mutant is reduced by a factor of two from that of the wild-type enzyme whereas the Vmax for the methionine mutant is reduced more than sevenfold. A very clean electron-density-difference map shows little, if any, evidence of a structural change associated with the C-terminal domain, although resonances in the NMR spectra associated with the ATP-binding site (C-terminal domain) are also affected by the mutation as one might expect from the kinetic results. The NMR data show that binding at both the 3-phospho-D-glycerate and the non-productive ATP-binding site (associated with the N-terminal domain) are affected in the mutant in a way which is different to that associated with the wild-type enzyme. These results, taken together with the X-ray and kinetic data, indicate that the non-productive ATP-binding site and the activating anion-binding site are both associated with the basic patch region of yeast phosphoglycerate kinase.     

The change of behaviour of two strains of Leishmania after cultivation in a defined medium

Attempts have been made to characterize two strains of Leishmania that became infective to golden hamsters only after they had been maintained for several years in a chemically defined culture medium. Observations were made on the growth rates of promastigotes in vitro, course of infection in hamsters, morphology of amastigotes, and electrophoretic mobility patterns of eight isoenzymes. Information was obtained about the buoyant densities of n-DNA and k-DNA, and one strain was tested against monoclonal antibodies. The identity of both strains remains obscure.     

Seasonal occurrence of Campylobacter spp. in surface waters and their correlation with standard indicator bacteria

Campylobacter jejuni, Campylobacter coli, and a Campylobacter-like organism were isolated from a number of natural water sources in central Washington, including ponds, lakes, and small mountain streams at elevations ranging from 1,460 to 5,400 feet (ca. 445 to 1,646 m) above sea level. At the two sites where extensive sampling was done, the bacteria were recovered throughout the year. Generally, the recovery rates were highest in the fall and winter months and lowest during the spring and summer months. Campylobacter density did not show significant correlation with microbiological (plate counts of fecal and total coliforms, fecal streptococci, and heterotrophic bacteria) or physical (water temperature, pH, and conductivity) parameters.     

The coupling of biogeochemical cycles of nutrients

For any element which is incorporated into biomass, the biogeochemical cycle of that element in a given ecosystem will be coupled to that of any other element similarly incorporated. The mutual interaction of two such cycles is examined using a simple model in which each cycle is constrained into four compartments. In each cycle the assimilation rate (primary productivity) is related in a non-linear fashion to the two nutrients and to biomass. The interactions are represented by combining a hyperbolic dependence for each nutrient (involving a "Michaelis constant") with a logistic equation governing the dependence of rate on biomass (involving a "carrying capacity"). The response of the model to perturbation (e.g. mobilization of an abiotic reserve) is strongly governed by the values assigned to these constants. The coupled cycles can exhibit positive feed-back with anomalous responses of the steady state and time-dependent solutions may exhibit complex oscillatory behaviour. Both the steady-state sensitivity and the kinetic behaviour of such coupled systems are simplified if the range of atomic ratios permitted by the assimilation process is restricted. It will therefore be of importance to determine under what conditions the assimilation rates for different elements are governed by mass-action effects (Liebig's Law) or by stoichiometric constraints (Redfield ratios).     

Development at Cold-Hardening Temperatures : The Structure and Composition of Purified Rye Light Harvesting Complex II

Light harvesting complex II (LHCII) was purified from cold-hardened (RH) and nonhardened winter rye (RNH) (Secale cereale L. cv Puma) employing a modified procedure of JJ Burke, CL Ditto, CJ Arntzen (Arch Biochem Biophys 187: 252-263). Triton X-100 solubilization of thylakoid membranes followed by three successive precipitations with 100 mm KCl and 10 mm MgCl₂ resulted in yields of up to 25% on a chlorophyll (Chl) basis and a purity of 90 to 95%, based on polypeptide analysis within 4 hours. Polypeptide and pigment analyses, 77 K fluorescence emission and room temperature absorption spectra indicate the LHCII obtained by this modified method is comparable to LHCII obtained by other published methods. Comparison of purified RH and RNH LHCII indicated no significant differences with respect to polypeptide, amino acid, Chl, and carotenoid compositions as well as no differences in lipid content. However, RH LHCII differed from RNH LHCII specifically with respect to the fatty acid composition of phosphatidyldiacylglycerol only. RH LHCII exhibited a 54% lower trans-Δ³-hexadecenoic acid level associated with PG and a 60% lower oligomeric LHCII:monomeric LHCII (LHCII₁:LHCII₃) than RNH LHCII. Both RH and RNH LHCII exhibited a 5-fold enrichment in PG specifically. Complete removal of PG by enzymic hydrolysis resulted in a significant reduction in the oligomeric content of both RH and RNH LHCII such that LHCII₁:LHCII₃ of RH and RNH LHCII preparations were the same. This confirms that this specific compositional change accounts for the structural differences between RH and RNH LCHII observed in situ and in vitro.     

Replication of cauliflower mosaic virus DNA in leaves and suspension culture protoplasts of cotton

Cauliflower mosaic virus (CaMV) replicated in protoplasts and in inoculated leaves of the non-host, cotton (Gossypium hirsutum, L.). Protoplasts prepared from suspension-cultured cotton cells were infected by incubation with liposome-encapsulated CaMV virions. During a 1-week culture period the amount of CaMV nucleic acid as detected by nucleic acid hybridization in the protoplasts increased significantly regardless of whether or not the protoplasts contained vacuoles. In leaves inoculated with CaMV virions or CaMV DNA, viral DNA sequences were found by leaf skeleton hybridization to be located in small circular areas. DNA extracted from ultracentrifugal pellets of homogenates of inoculated leaves contained circular, gapped CaMV DNA only when inocula contained CaMV virions, CaMV DNA, or partial nested dimer CaMV plasmid DNA. When plants had been heavily watered, the CaMV DNA recovered contained degraded CaMV DNA. The results suggest that the host range limitation for CaMV is not due to an inability to replicate or spread locally in inoculated leaves.     

The sequence of rat leukosialin (W3/13 antigen) reveals a molecule with O-linked glycosylation of one third of its extracellular amino acids.

Leukosialin is one of the major glycoproteins of thymocytes and T lymphocytes and is notable for a very high content of O-linked carbohydrate structures. The full protein sequence for rat leukosialin as translated from cDNA clones is now reported. The molecule contains 371 amino acids with 224 residues outside the cell, one transmembrane sequence and 124 cytoplasmic residues. Data from the peptide sequence and carbohydrate composition suggest that one in three of the extracellular amino acids may be O-glycosylated with no N-linked glycosylation sites. The cDNA sequence contained a CpG rich region in the 3' coding sequence and a large 3' non-coding region which included tandem repeats of the sequence GGAT.     

A histocytochemical study of the macrophages present in tissue responses to adult Onchocerca volvulus

Summary Immunocytochemical and histochemical properties of macrophages present in the subcutaneous chronic inflammatory responses surrounding adultOnchocerca volvulus (nodules) in human tissues were examined. Macrophages with strong non-specific esterase (NSE) and acid phosphatase (AcPase) activities but weak adenosine triphosphatase (ATPase) activity and HLA-DR expression (NSE⁺⁺⁺, AcPase⁺⁺⁺, ATPase^–/+, HLA-DR^–/+) were present in the centre of nodules. Many of the cells adhering to the surface of worms were NSE⁺⁺⁺, AcPase⁺⁺⁺, ATPase^–, HLA-DR⁺⁺⁺. The inner zone of the fibrous capsule of nodules contained macrophages with the profile NSE⁺⁺⁺, AcPase^–, ATPase^–/+, HLA-DR^–/+. A fourth type, NSE⁺⁺⁺, AcPase^–/+, ATPase^–/+, HLA-DR⁺⁺⁺, was located in the outer zone of the capsule, frequently within perivascular accumulations of macrophages, lymphocytes and plasma cells. Active fibroblasts were identified at the inner edge of the fibrous capsule by alkaline phosphatase staining. A feature of all nodules examined was the presence of lipid-filled macrophages, demonstrated by Oil Red O stain; these cells were usually situated in zones adjacent to the centre of nodules, and were of the NSE⁺⁺, AcPase⁺⁺, ATPase^–/+, HLA-DR^–/+ type. Lipid accumulation was not found to be related to the clinical status of the patients studied. The origin and functional significance of this lipid is unknown.     

Recessive Lethal Mutations and the Maintenance of Duplication-Bearing Strains of Dictyostelium discoideum

Recessive lethal mutations have been isolated and used to maintain n + 1 aneuploid strains of Dictyostelium discoideum carrying a duplication of part or all of linkage group VII. The recessive lethal mutations, relA351 and relB352, arose spontaneously in diploids; no mutagenic treatment was used in the isolation of these mutations. The probable gene order on linkage group VII is: centromere, relB couA, bsgB, cobA, relA. Maintenance of aneuploids disomic for linkage group VII was made possible by complementation of a rel mutation on each linkage group VII homologue by the corresponding wild-type allele on the other linkage group VII homologue. The duplication-bearing disomic strains were slow-growing and produced faster-growing sectors on the colony edge. Haploid sectors probably arise by a combination of mitotic recombination and subsequent loss of one homologue, diploid sectors may be formed by chromosome doubling to 2n + 2, followed by chromosome loss to return to 2n, and aneuploid sectors may arise by deletion or new mutation.