Cellulase production by a thermophilic clostridium species

Strain M7, a thermophilic, anaerobic, terminally sporing bacterium (0.6 by 4.0 μm) was isolated from manure. It degraded filter paper in 1 to 2 days at 60 C in a minimal cellulose medium but was stimulated by yeast extract. It fermented a wide variety of sugars but produced cellulase only in cellulose or carboxymethyl-cellulose media. Cellulase synthesis not only was probably repressed by 0.4% glucose and 0.3% cellobiose, but also cellulase activity appeared to be inhibited by these sugars at these concentrations. Both C₁ cellulase (degrades native cellulose) and C_x cellulase (β-1,4-glucanase) activities in strain M7 cultures were assayed by measuring the liberation of reducing sugars with dinitrosalicylic acid. Both activities had optima at pH 6.5 and 67 C. One milliliter of a 48-h culture of strain M7 hydrolyzed 0.044-meq of glucose per min from cotton fibers. The cellulase(s) from strain M7 was extracellular, produced during exponential growth, but was not free in the growth medium until approximately 30% of the cellulose was hydrolyzed. Glucose and cellobiose were the major soluble products liberated from cellulose by the cellulase. ZnCl₂ precipitation appeared initially to be a good method for the concentration of cellulase activity, but subsequent purification was not successful. Isoelectric focusing indicated the presence of four C_x cellulases (pI 4.5, 6.3, 6.8, and 8.7). The rapid production and high activity of cellulases from this organism strongly support the basic premise that increased hydrolysis of native cellulose is possible at elevated temperature.     

The role of lysine-41 of ribonuclease A in the interaction with RNase inhibitor from human placenta

3-N-Carboxymethyl-His-12 and 1-N-carboxymethyl-His-119-RNase A bind to the naturally occurring RNase inhibitor, isolated from human placenta, 1.3 and 3.6 times, respectively, more strongly than does native RNase A. Near-ultraviolet circular dichroism measurements indicate that the conformational change which occurs upon carboxymethylation of either of the active site histidine residues appears different from that which the protein undergoes upon binding of substrate of a substrate analogue. Specific carboxymethylation of Lys-41 of RNase A decreased the strength of the interaction between the enzyme and the RNase inhibitor to about 12% of the initial value. The near-UV CD spectra of Cm-Lys-41-RNase A and of acetimidyl-RNase A (9.3 lysines modified) and carbamylated RNase A (3.0 lysines modified), which also have weaker interactions with RNase inhibitor of 25% and 10%, respectively, show a negative [theta]MRW identical to that of native RNase A at 275 nm but are altered in the positive [theta]MRW at 240 nm. The CD measurements suggest that one or more tyrosine residues of RNase A may be involved in the interaction with inhibitor. The effects of pH and salt concentration suggest that a major part of the protein-protein interaction is probably through nonpolar forces. The strengths of interactions between the inhibitor and pancreatic RNases from several species were very similar. Since Tyr-92 is the only tyrosine residue retained in all of the species studied, this residue may have a key role in the nonpolar interaction. The data presented herein suggest that the interaction between RNase A and the inhibitor involves the positively charged epsilon-NH2 group of Lys-41 of RNase A. This interaction could result in the inactivation of the enzyme.     

Effects of (R)-deoxycoformycin (pentostatin) on intrauterine nucleoside catabolism and embryo viability in the pregnant mouse.

The viability of early mouse embryos is acutely sensitive to (R)-deoxycoformycin (pentostatin), a tight-binding inhibitor of adenosine deaminase (ADA). Previous studies have shown that a single 5-mg/kg dose on day 7 (plug = day 0) of gestation fully inhibits uteroplacental ADA activity within 0.5 h; causes massive cell death in the neural plate and primary mesenchyme by 6 h, major craniofacial anomalies by day 10, and resorption by day 12 (Knudsen et al., '89; Airhart et al., '91). The present study has examined further the developmental toxicity and early effects of this inhibitor on ADA metabolism. (R)-Deoxycoformycin was administered to pregnant CD-1 (ICR) mice as a single intraperitoneal dose of 0.5-10 mg/kg total body weight on days 6-11 of gestation. The major adverse effect, early resorption, was dose dependent and specific to day 7-8 exposure. Treatment with 5 mg/kg on day 7 resulted in 85% resorptions, 15% malformations, and a 24% reduction in mean fetal weight, whereas the same dose of (S)-deoxycoformycin had no effect. Levels of adenosine and 2'-deoxyadenosine, which are the endogenous substrates of ADA, were monitored in the embryo/decidual unit (E/D) by reversed-phase high-performance liquid chromatography (RP-HPLC). In response to the inhibitor, both nucleosides increased transiently in the antimesometrial compartment (antimesometrial decidua + embryo). Peak levels (Cmax) of adenosine and 2'-deoxyadenosine were dose dependent over the range tested (0.05-10 mg/kg). Exposure to 5 mg/kg on day 7 raised adenosine levels within 0.5 h to 42-fold over the basal level of 0.06 nmol/mg protein. There was an even stronger effect on 2'-deoxyadenosine levels, which were elevated 674-fold over the detection limit of 0.0005 nmol/mg protein. Direct exposure to the inhibitor in serum-free E/D culture produced similar results: 50 microM (R)-deoxycoformycin within 1 h raised adenosine levels 26-fold and 2'-deoxyadenosine levels 410-fold. In vivo studies also showed a general correlation between embryolethality and the length of adenine nucleoside pool expansion, apparent for exposure on day 7, 8, or 9 but not on day 6, suggesting that the embryo becomes sensitive to adenosine or 2'-deoxyadenosine once the neural plate has formed.     

VEGETATIVE REPRODUCTION AND SEXUAL LIFE CYCLE OF THE TOXIC DINOFLAGELLATE GYMNODINIUM CATENATUM FROM TASMANIA,AUSTRALIA1

The toxic, chain-forming dinoflagellate Gymnodinium catenatum Graham was cultured from vegetative cells and benthic resting cysts isolated from estuarine waters in Tasmania, Australia. Rapidly dividing, log phase cultures formed long chains of up to 64 cells whereas stationary phase cultures were composed primarily of single cells (23-41 pm long, 27-36 pm wide). Vegetative growth (mean doubling time 3-4 days) was optimal at temperatures from 14.5-20° C, salinities of 23-34% and light irradiances of 50-300 μE·m^?2·s^?1. The sexual life cycle of G. catenatum was easily induced in a nutrient-deficient medium, provided compatible opposite mating types were combined (heterothallism). Gamete fusion produced a large (59-73 μm long, 50-59 μm wide) biconical, posteriorly biflagellate planozygote (double longitudinal flagellum) which after several days lost one longitudinal flagellum and gradually became subspherical in shape. This older planozygote stage persisted for up to two weeks before encysting into a round, brown resting cyst (42-52 μm diam; hypnozygote) with microreticulate surface ornamentation. Resting cysts germinated after a dormancy period as short as two weeks under our culture conditions, resulting in a single, posteriorly biflagellate germling cell (planomeiocyte). This divided to form a chain of two cells, which subsequently re-established a vegetative population. Implications for the bloom dynamics of this toxic dinoflagellate, a causative organism of paralytic shellfish poisoning, are discussed.     

Rapoport's rule: time for an epitaph?

The christening of the decline in the geographic extent of species from high to low latitudes as Rapoport's rule was a bold step. Allowing for a variety of potentially significant complications to the interpretation of empirical studies, evidence that this is indeed a general pattern is, at the very least, equivocal. The present taxonomically and regionally biased set of studies lend support to the recent suggestion that the pattern is a local phenomenon being expressed primarily in the Palaearctic and Nearctic above latitudes of 40-50°N. Five hypotheses have been proposed to explain the generation of latitudinal declines in range size where they do occur, with the past heavy emphasis on a climatic variability mechanism being eroded. Evidence is accruing in support of more than one such mechanism. Whatever the generality of the `rule', it has undoubtedly served to stimulate a consideration of the role of spatial variation in range sizes in several areas of research in ecology and evolution.     

Short repetitive sequences in green algal mitochondrial genomes: potential roles in mitochondrial genome evolution

Current data on green algal mitochondrial genomes suggest an unexpected dichotomy within the group with respect to genome structure, organization, and sequence affiliations. The present study suggests that there is a correlation between this dichotomy on one hand and the differences in the abundance, base composition, and distribution of short repetitive sequences we observed among green algal mitochondrial genomes on the other. It is conceivable that the accumulation of GC- rich short repeated sequences in the Chlamydomonas-like but not Prototheca-like mitochondrial genomes might have triggered evolutionary events responsible for the distinct series of evolutionary changes undergone by the two green algal mitochondrial lineages. The similarity in base composition, nucleotide sequence, abundance, and mode of organization we observed between the short repetitive sequences present in Chlamydomonas-like mitochondrial genomes on one hand and fungal and vertebrate homologs on the other might extend to some of the roles that the short repetitive sequences have been shown to have in the latter. Potential involvements we propose for the short repetitive sequences in the evolution of Chlamydomonas-like mitochondrial genomes include fragmentation and scrambling of the ribosomal-RNA-coding regions, extensive gene rearrangements, coding-region deletions, surrogate origins of replication, and chromosomal linearization.      

Aspergillus terreus and its toxic metabolites as a food contaminant in some Egyptian Bakery products and grains

A. terreus isolates isolated from some bakery products, corn and rice were found to be able to produce territrems. 90% of theA. terreus isolated from bakery products were able to produce territrem A, with a mean of 0.09 ppm, while 80% ofA. terreus isolates produce territrem B with a mean of 0.24 ppm. On the other hand 31.8% of the isolates ofA. terreus from corn were able to produce territrem A with a mean of 0.44 ppm. ConcerningA. terreus isolates from rice, 66.7% were found to produce territrem A, with a mean of 5.28 ppm, and 77.8% of the isolates produced territrem B with a mean of 1.79 ppm.     

Uncapping and deregulation of telomeres lead to detrimental cellular consequences in yeast

Telomeres are the protein-nucleic acid structures at the ends of eukaryote chromosomes. Tandem repeats of telomeric DNA are templated by the RNA component (TER1) of the ribonucleoprotein telomerase. These repeats are bound by telomere binding proteins, which are thought to interact with other factors to create a higher-order cap complex that stabilizes the chromosome end. In the budding yeast Kluyveromyces lactis, the incorporation of certain mutant DNA sequences into telomeres leads to uncapping of telomeres, manifested by dramatic telomere elongation and increased length heterogeneity (telomere deregulation). Here we show that telomere deregulation leads to enlarged, misshapen "monster" cells with increased DNA content and apparent defects in cell division. However, such deregulated telomeres became stabilized at their elongated lengths upon addition of only a few functionally wild-type telomeric repeats to their ends, after which the frequency of monster cells decreased to wild-type levels. These results provide evidence for the importance of the most terminal repeats at the telomere in maintaining the cap complex essential for normal telomere function. Analysis of uncapped and capped telomeres also show that it is the deregulation resulting from telomere uncapping, rather than excessive telomere length per se, that is associated with DNA aberrations and morphological defects.     

Comparative study of selenium requirements of three phytoplankton species: Gymnodinium catenatum, Alexandrium minutum (Dinophyta) and Chaetoceros cf. tenuissimus (Bacillariophyta)

This study investigated the selenium (SE) requirements of three phytoplankton species which commonly bloom in southern Australian estuaries. The present study showed that the toxic dinoflagellate Gymnodinium catenatum Graham had an obligate requirement for Se (IV) in culture. After two transfers (4 weeks = 7 generations) in Se-deficient seawater medium, this phytoplankton species exhibited a decline in growth rate (25%) and biomass yield (90%), while complete cessation of cell division occurred under prolonged (8 weeks = 12 generations) Se starvation. Addition of 10^-9-10^-7 M H2SeO3 to nutrient-enriched seawater medium resulted in increased G.catenatum growth and biomass yields in direct proportion to the Se concentrations offered. In contrast to G.catenatum, Se limitation was observed in the dinoflagellate Alexandrium minutum Halim after four transfers (5 weeks = 20 generations) in Se-deficient medium. Exponential growth rates of A.minutum decreased slightly (5-10%) when Se was not supplied, but biomass yields decreased as much as 80-90%. The diatom Chaetoceros cf. <It>tenuissimus Meunier showed no evidence of Se limitation even after eight transfers (8 weeks; >60 generations) in Se-deficient medium. Variations in growth rates and biomass yields between transfers provide valuable information about the relative potential for Se limitation in the three species studied. In addition, differences in Se requirement between these bloom-forming phytoplankton species suggest that this micronutrient may play a role in structuring phytoplankton communities in southern Australian waters.