Roles of structural domains in the morphology and surface anchoring of the tetragonal paracrystalline array of Aeromonas hydrophila. Biochemical characterization of the major structural domain.

The tetragonally arranged S-layer of Aeromonas hydrophila contains two morphological domains. The mature S-layer protein of A. hydrophila has a subunit molecular weight of 52,000, and has been reported to contain two structural domains. Here a mutant has been isolated which produces an S-layer of subunit molecular weight 38,650 as determined by sedimentation analysis. This truncated S-protein was exported via the periplasm to the cell surface, but could not self-assemble into a tetragonal array or be anchored to the cell surface. Instead the truncated protein formed cup-like structures which were purified and characterized biochemically. Automated Edman degradation showed that the truncated protein comprised the amino-terminal structural domain of the S-protein. This domain had an increased hydrophobic amino acid content relative to the wild-type protein, and contained approximately 42% beta-sheet, 10% alpha-helix, and 19% beta-turn. Differences in alpha-helix and beta-turn contents between the wild-type and truncated proteins were observed when the effects of pH and SDS were examined, indicating that the carboxy terminus influences the effects of environmental change on the conformation of the S-protein. This lesser carboxy-terminal array also appears to be required for both correct array morphology, and array anchoring, while the greater amino-terminal domain appears to comprise the major morphological core of the surface array.     

Multiple shoot production from seedling explants of slash pine (Pinus elliottii,Engelm.)

Hypocotylary explants obtained from 30- to 40-day-old slash pine (Pinus elliottii, Engelm.) seedlings treated with 6-benzylaminopurine produced multiple buds that eventually elongated into axillary shoots. The explants were pulse treated (45-s dip) with 6-benzylaminopurine (22.2, 111, 222 M) plus a control and cultured on three different basal media containing activated charcoal (0.5% w/v). Hormonal concentration and basal medium were compared for the number and size of axillary shoots induced after 12 and 29 days. The greatest number of axillary shoots was produced by explants that were pulse treated with 111 M 6-benzylaminopurine and cultured on Gresshoff and Doy medium. The axillary shoots were fewer in number per explant than shoots previously reported resulting from hormonally induced advantitious buds of slash pine, but the axillary shoots developed more rapidly.Abbreviations BA 6-benzylaminopurine - DMSO dimethyl sulfoxide - PAR photosynthetically active radiation     

Isolation and characterization of a human cDNA clone encoding a novel DNA topoisomerase II homologue from HeLa cells

We have isolated and sequenced 3 human DNA topoisomerase II (topo II) partial cDNA clones from a HeLa carcinoma cell cDNA library. Two clones were identical to an internal fragment of HeLa topo II cDNA. The third clone, CAA5, had a different and novel sequence which shared significant nucleotide (62%) and predicted peptide (70%) homologies with a region of the HeLa topo II cDNA. Our results suggest that HeLa cells express at least two homologous forms of DNA topoisomerase II. The new HeLa topo II homologue is discussed in relation to topo II isoenzymes recently described in a Burkitt lymphoma and other cell lines.     

Identification of conditioned medium proteins from human cancer cells adapted to serum-free conditions

Recent studies have shown that human cancer cell lines can be adapted to grow in serum-free, unsupplemented RPMI-1640 (RO) medium. We have developed similar techniques to rapidly identify proteins of interest in serum-free conditioned medium (CM) of human lung cancer cell lines. Classic and variant small cell lung cancer (SCLC) lines were adapted to growth in RO medium. CM from each line was concentrated and fractionated on an anion-exchange column of a fast protein liquid chromatography system. Concentrates of each fraction were loaded onto lanes of minigels of an automated electrophoresis system. Analysis of the chromatograms reveals peaks seen only in CM of the classic SCLC lines. Electrophoretic analysis of the fractions containing these peaks reveal protein bands distinguishing between the subtypes of human SCLC. One protein was purified to homogeneity with subsequent reversed-phase chromatography and identified by protein microsequencing as histone H2B. These automated techniques have general use in the rapid identification of CM proteins associated with the differentiation or progression of the many types of neoplastic cells which can be adapted to growth in RO medium.     

Diversity among strains causing bacterial kidney disease in salmonid fish

A study was made of the biochemical, cultural, morphological, physiological and serological characters of 25 Gram-positive bacterial isolates of bacterial kidney disease in salmonid fish. Two distinct homogenous phena and seven single-member clusters were defined as a result of overall similarity based on analyses with the Jaccard coefficient. One phenon was equated withCorynebacterium pyogenes, but the second represents a novel taxon.     

Pathways for alanine transport in intestinal basal lateral membrane vesicles

Summary Membrane vesicles obtained from the basal lateral membranes of the rat intestinal epithelium were used to study the pathways for neutral amino acid transport.In the absence of sodium there was a stereospecific uptake ofl-alanine which exhibited saturation kinetics (K _m 0.73mm andV _max 5.3 nmol/mg min at 22°C). The activation energy for this process was 8.1 kcal/mole between 5 and 25°C. Preloading the vesicles with alanine increased the unidirectional influx of alanine into the vesicle. Competition experiments indicated that the affinity of the sodium-independent transport system was glutamine > threonine > alanine > phenylalanine > valine > methionine > glycine > histidine > proline, N-MeAIB. These are the characteristics of the classical L transport system.External sodium increased the rate of the stereospecificl-alanine uptake. The Na-dependent flux had aK _m of 0.04mm and aV _max of 0.26 nmol/mg min at 22°, and an activation energy of 9.1 kcal/mole between 5 and 25°C. Competition experiments suggest the existence of three separate pathways for alanine transport in the presence of sodium. A major pathway is shared by all other amino acids tested (i.e., threonine, glutamine, methionine, phenylalanine, valine, proline and N-MeAIB). This resembles the classical A system. A second pathway is unavailable to either phenylalanine or N-MeAIB; this is reminiscent of the classical ASC system; and the third is a novel pathway which is shared by N-MeAIB but not phenylalanine.The sodium-independent and the sodium-dependent transport ofl-alanine was blocked by PCMBS and significantly inhibited by DTP and NEM. It is concluded that the sodium-independent system (the L-like system) accounts for the efflux of neutral amino acids from the epithelium to the blood during the absorption of amino acids from the gut, and that the sodium-dependent transport processes may play an important role in the supply of amino acids to the epithelium in the absence of amino acids from the gut lumen.