Stationary phase-associated protein expression in Mycobacterium tuberculosis: function of the mycobacterial alpha-crystallin homolog.

The majority of active tuberculosis cases arise as a result of reactivation of latent organisms which are quiescent within the host. The ability of mycobacteria to survive extended periods without active replication is a complex process whose details await elucidation. We used two-dimensional gel electrophoresis to examine both steady-state protein composition and time-dependent protein synthetic profiles in aging cultures of virulent Mycobacterium tuberculosis. At least seven proteins were maximally synthesized 1 to 2 weeks following the end of log-phase growth. One of these proteins accumulated to become a predominant stationary-phase protein. N-terminal amino acid sequencing and immunoreactivity identified this protein as the 16-kDa alpha-crystallin-like small heat shock protein. The gene for this protein was shown to be limited to the slowly growing M. tuberculosis complex of organisms as assessed by Southern blotting. Overexpression of this protein in wild-type M. tuberculosis resulted in a slower decline in viability following the end of log-phase growth. Accumulation of this protein was observed in log-phase cultures following a shift to oxygen-limiting conditions but not by other external stimuli. The protein was purified to homogeneity from overexpressing M. smegmatis in two steps and shown to have a significant ability to suppress the thermal denaturation of alcohol dehydrogenase. Collectively, these results suggest that the mycobacterial alpha-crystallin protein may play a role in enhancing long-term protein stability and therefore long-term survival of M. tuberculosis.     

Variable effects of air-drying on leaching losses from tree leaf litter

Leaching of soluble substances may be an important first step in leaf litter decomposition in small streams, but recent research has suggested that large leaching losses (up to 30% of initial mass in 48 h) may be an artifact created by using air-dried leaves in decomposition experiments. In laboratory experiments, we compared 3 d leaching losses from freshly fallen and air-dried senescent leaves of 27 tree species from different regions across Canada. Air-dried leaves from all species leached measurable amounts of original mass (3.6–32.8% dry mass), but leaching losses from fresh leaves (0–35%) were detectable in all but two species. Air-drying increased leaching losses in many species, but in others it reduced leaching losses or had no measurable effect. Results for leaves of the same species collected in different regions or in different years were generally similar, but species within the same genus often behaved very differently. Neither moisture content (fresh or air-dried), leaf thickness, nor cuticle thickness proved of any value as predictors of leaching losses or the effect of air-drying. The propensity of autumn-fallen leaves to leach, whether fresh or air-dried, appears to be a property of the individual tree species.     

SSCP analysis of the tyrosine kinase domain of the insulin receptor gene: Polymorphisms detected in South African black and white subjects

The frequency of DNA polymorphisms in the tyrosine kinase domain (exons 17–21) of the insulin receptor gene was assessed in 30 black and 30 white South Africans, using single-stranded conformation polymorphism and direct sequencing analysis. A comparison of the frequencies of the normal versus the combined polymorphic alleles, found only in exon 17, showed a significant difference between black and white groups (P = 0.037). Received: 25 May 1995 / Revised: 1 September 1995     

Colonization dynamics of river benthos in response to local changes in bed characteristics

1. The effects of small-scale variation in the nature of a river bed upon the colonization dynamics of benthic invertebrates were explored through a series of microhabitat implants placed in a small river in southern Ontario. The implants presented variations in the levels of four microhabitat features: substrate particle size, current regime, added food and light. The sixteen possible combinations of these variables were replicated five times, for a total of eighty implants.
2. After 14 days of exposure, some of the controlled variables had been modified and a number of other variables (sand, fine (FPOM) and coarse (CPOM) particulate organic matter) had been added by the river.
3. Benthic diversity differed among the implants, with sand and CPOM accounting for 15.5% of the variation. Benthic density was more uniform and was weakly related only to added food. Application of the CANOCO program to the data set suggested that, in general, the environmental variables manipulated in this study were not the key factors influencing colonization, although they may have been important in non-linear combination.
4. A number of important positive associations were evident among colonizing species, some of which are thought to reflect those of predator and prey.
5. Control of colonization appears to be through multiple factors (abiotic, biotic, autochthonous, allochthonous) that differ in nature according to species, and which underline the complexity of selection responses of individual taxa to local variations in bed structure. Researchers must acknowledge this multifactorial organization and dynamic nature if workable models of lotic community structure and development are to be forthcoming.     

Electron paramagnetic resonance characterization of tyrosine radical, M+, in site-directed mutants of photosystem II(t).

Photosystem II contains two well-characterized tyrosine radicals, D(.) and Z(.). Z is an electron carrier between the primary chlorophyll donor and the manganese catalytic site and is essential for enzymatic function. On the other hand, D forms a stable radical with no known role in oxygen evolution. D(.) and Z(.) give rise to similar, but not identical, room temperature electron paramagnetic resonance (EPR) signals, which can be distinguished by their decay kinetics. A third room temperature EPR signal has also been observed in site-directed mutants in which a nonredox active amino acid is substituted at the D or Z site. This four-line EPR signal has been shown to have a tyrosine origin by isotopic labeling (Boerner and Barry, 1994, J. Biol. Chem. 269:134-137), but such an EPR signal has never before been observed from a tyrosyl radical. The radical giving rise to this third unique signal has been named M+. Here we provide kinetic evidence that this signal arises from a third redox active tyrosine, distinct from tyrosine D and Z, in the photosystem II reaction center. Isotopic labeling and EPR spectroscopy provide evidence that M is a covalently modified tyrosine.     

Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NSO cell cultures

Investigations of biological effects of prolonged elevation of growth hormone in animals such as mice and rats require large amounts of mouse and rat growth hormone (GH) materials. As an alternative to scarce and expensive pituitary derived materials, both mouse and rat GH were expressed in NSO murine myeloma cells transfected with a vector containing the glutamine synthetase (GS) gene and two copies of mouse or rat GH cDNA. For optimal expression, the mouse GH vector also contained sequences for targeting integration by homologous recombination. Fed-batch culture processes for such clones were developed using a serum-free, glutamine-free medium and scaled up to 250 L production scale reactors. Concentrated solutions of proteins, amino acids and glucose were fed periodically to extend cell growth and culture lifetime, which led to an increase in the maximum viable cell concentration to 3.5×10⁹ cells/L and an up to 10 fold increase in final mouse and rat rGH titers in comparison with batch cultures. For successful scale up, similar culture environmental conditions were maintained at different scales, and specific issues in large scale reactors such as balancing oxygen supply and carbon dioxide removal, were addressed. Very similar cell growth and protein productivity were obtained in the fed-batch cultures at different scales and in different production runs. The final mouse and rat rGH titers were approximately 580 and 240 mg/L, respectively. During fed-batch cultures, the cell growth stage transition was accompanied by a change in cellular metabolism. The specific glucose consumption rate decreased significantly after the transition from the growth to stationary stage, while lactate was produced in the exponential growth stage and became consumed in the stationary stage. This was roughly coincident with the beginning of ammonia and glutamate accumulation at the entry of cells into the stationary stage as the result of a reduced glutamine consumption and periodic nutrient additions.     

Analysis of stimulatory and inhibitory amino acids for development of hamster one-cell embryos in vitro

Hamster embryo development to the blastocyst stage in vitro can be modulated by amino acids. This series of experiments employed both empirically and statistically designed approaches to elucidate which of 20 amino acids inhibit or stimulate development and to devise a complement of amino acids that best supports in vitro development of hamster 1-cell embryos. Development and/or mean cell number were significantly inhibited by the presence of leucine, tyrosine, valine, isoleucine, phenylalanine, arginine, methionine, or cysteine (at 0.5 mM) and isoleucine, phenylalanine, or tryptophan (at 0.05 mM). Three amino acids—glutamine, taurine, and glycine—were stimulatory and in combination improved development; the culture medium containing these amino acids was designated Hamster Embryo Culture Medium-5. Moreover, addition of another eight amino acids—asparagine, aspartic acid, serine, glutamic acid, histidine, lysine, proline and cysteine (medium designated HECM-6)—had a significant stimulatory effect on development over previously formulated culture media for hamster embryos. These results demonstrated that amino acids, alone and in combination, can markedly stimulate or inhibit hamster embryo development in vitro up to the blastocyst stage. Embryo transfer experiments showed that HECM-5 and ?6 (chemically defined, protein-free culture media) supported normal preimplantation embryo development in vitro. This study also indicates that empirically designed embryo culture media formulations can be as effective as those obtained by application of statistical methodologies. © 1995 wiley-Liss, Inc.