Lactate as substrate for glycogen resynthesis after exercise

Muscle glycogen levels in the perfused rat hemicorpus preparation were reduced two-thirds by electrical stimulation plus exposure to epinephrine (10(-7) M) for 30 min. During the contraction period muscle lactate concentrations increased from a control level of 3.6 +/- 0.6 to a final value of 24.1 +/- 1.6 mumol/g muscle. To determine whether the lactate that had accumulated in muscle during contraction could be used to resynthesize glycogen, glycogen levels were determined after 1-3 h of recovery from the contraction period during which time the perfusion medium (flow-through system) contained low (1.3 mmol/l) or high (10.5 or 18 mmol/l) lactate concentrations but no glucose. With the low perfusate lactate concentration, muscle lactate levels declined to 7.2 +/- 0.8 mumol/g muscle by 3 h after the contraction period and muscle glycogen levels did not increase (1.28 +/- 0.07 at 3 h vs. 1.35 +/- 0.09 mg glucosyl U/g at end of exercise). Lactate disappearance from muscle was accounted for entirely by output into the venous effluent. With the high perfusate lactate concentrations, muscle lactate levels remained high (13.7 +/- 1.7 and 19.3 +/- 2.0 mumol/g) and glycogen levels increased by 1.11 and 0.86 mg glucosyl U/g, respectively, after 1 h of recovery from exercise. No more glycogen was synthesized when the recovery period was extended. Therefore, it appears that limited resynthesis of glycogen from lactate can occur after the contraction period but only when arterial lactate concentrations are high; otherwise the lactate that builds up in muscle during contraction will diffuse into the bloodstream.     

Seasonal nitrogen and carbon concentrations in white,brown and woody fine roots of sugar maple (Acer saccharum Marsh)

Small diameter (<1.0-mm) Acer saccharum Marsh roots were separated into white, brown and woody development state classes and analyzed for total N and C concentrations in April, July and October of 1988. White roots had greater concentrations of N and C than either brown or woody roots at each sampling date, and the N concentration of brown roots was consistently greater than that of woody roots. There were no temporal changes in N concentrations in any of the roots. C was slightly elevated in mid-summer in all three classes of roots. The data suggest the possible existence of an N translocation mechanism in ageing and developing fine roots. More research should be undertaken to establish the mechanisms of N loss in developing fine roots.     

Applications of minirhizotrons to understand root function in forests and other natural ecosystems

Minirhizotrons have proved useful to understand the dynamics and function of fine roots. However, they have been used comparatively infrequently in forests and other natural plant communities. Several factors have contributed to this situation, including anomalous root distributions along the minirhizotron surface and the difficulty of extracting data from minirhizotron images. Technical and methodological advances have ameliorated some of these difficulties, and minirhizotrons have considerable potential to address some questions of long standing interest. These questions include more fully understanding the role of roots in carbon and nutrient cycling, rates of root decomposition, responses to resource availability and the functional significance of interactions between plant roots and soil organisms. Maximizing the potential for minirhizotrons to help us better understand the functional importance of fine roots in natural plant communities depends upon using them to answer only those questions appropriate to both their inherent strengths and limitations.     

Resistance to moloney murine sarcoma virus-induced tumorigenesis in NK-deficient beige mice

C57BL/6J-bg^J$\text{bg}\text{bg}$ mice are reported to be less susceptible to tumor induction by threshold doses of Moloney murine sarcoma virus than their +/bg littermates, and there are no significant differences between $\text{bg}\text{bg}$ and +/bg mice in which tumors were induced with respect to tumor latency, size, and regression rate. The difference in tumor frequency cannot be accounted for by M-MSV boosting of activity in $\text{bg}\text{bg}$ mice or by depression of activity in +/bg animals.     

Use of a Plasmid DNA Probe To Monitor Populations of Bacillus pumilus Inoculant Strains in Hay

We are evaluating naturally occurring isolates of Bacillus pumilus for use as microbial hay preservatives. Seven isolates of B. pumilus from hay contained a 42-kb cryptic plasmid (pMGD296). We wished to determine whether pMGD296 could be used as a molecular marker to follow populations of these isolates in hay over time. Southern blots and colony blots of 69 isolates of B. pumilus and other Bacillus spp. were probed with ³²P-labeled pMGD296. Twenty-nine probe-positive isolates were identified; of these, 28 contained a plasmid with a restriction profile identical to that of pMGD296. One isolate from untreated hay contained a 40-kb plasmid (pMGD150) that was homologous to pMGD296 but had a different restriction fragment pattern. Regions of homology between the two plasmids were identified by Southern blotting, and a 1.9-kb HindIII-PstI fragment of pMGD296 lacking strong homology to pMGD150 was cloned in pUC18. The cloned fragment hybridized only with isolates containing pMGD296 and was used to estimate populations of these isolates in treated and untreated hay.     

Construction and use of a computerized DNA fingerprint database for lactic acid bacteria from silage

Efficient selection of new silage inoculant strains from a collection of over 10,000 isolates of lactic acid bacteria (LAB) requires excellent strain discrimination. Toward that end, we constructed a GelCompar II database of DNA fingerprint patterns of ethidium bromide-stained EcoRI fragments of total LAB DNA separated by conventional agarose gel electrophoresis. We found that the total DNA patterns were strain-specific; 56/60 American Type Culture Collection strains of 33 species of LAB could be distinguished. Enterococcus faecium strains ATCC19434 and ATCC35667 had identical total DNA patterns and RiboPrints. Lactobacillus rhamnosus strains ATCC7469 and ATCC27773 also had identical total DNA patterns, but different RiboPrints. EcoRI RiboPrint patterns could distinguish only about 9/23 Lactobacillus plantarum strains and about 6/10 Lactobacillus buchneri strains, whereas all 33 strains could be distinguished by EcoRI total DNA patterns. Despite gel-to-gel variation, new DNA patterns can be readily grouped with existing patterns using GelCompar II. The database contains large homogenous clusters of L. plantarum, E. faecium, L. buchneri, Lactobacillus brevis and Pediococcus species that can be used for tentative taxonomic assignment. We routinely use the DNA fingerprint database to identify and characterize new strains, eliminate duplicate isolates and for quality control of inoculant product strains. The GelCompar II database has been in continuous use for 7 years and contains more than 3600 patterns representing approximately 700 unique patterns from over 300 gels and is the largest computerized DNA fingerprint database for LAB yet reported.     

Cloning and disruption of the b-isopropylmalate dehydrogenase gene (LEU2 ) of Pichia stipitis with URA3 and recovery of the double auxotroph

Transformation of Pichia stipitis is required to advance genetic studies and development of xylose metabolism in this yeast. To this end, we used P. stipitis URA3 (PsURA3) to disrupt P. stipitis LEU2 in a P. stipitis ura3 mutant. A highly fermentative P. stipitis mutant (FPL-DX26) was selected for resistance to 5′-fluoroorotic acid to obtain P. stipitis FPL-UC7 (ura3-3). A URA3:lacZ“pop-out” cassette was constructed containing PsURA3 flanked by direct repeats from segments of the lacZ reading frame. The P. stipitis LEU2 gene (PsLEU2) was cloned from a P. stipitis CBS 6054 genomic library through homology to Saccharomyces cerevisiae LEU2, and a disruption cassette was constructed by replacing the PsLEU2 reading sequence with the PsURA3:lacZ cassette. FPL-UC7 (ura3-3) was transformed with the disruption cassette, and a site-specific integrant was identified by selecting for the Leu⁻ Ura⁺ phenotype. The ura3 marker was recovered from this strain by plating cells onto 5′-fluoroorotate and screening for spontaneous URA3 deletion mutants. Excision of the flanked PsURA3 gene resulted in the Leu⁻Ura⁻ phenotype. The double auxotrophs are stable and can be transformed at a high frequency by PsLEU2 or PsURA3 carried on autonomous-replication-sequence-based plasmids. Received: 17 June 1997 / Received revision: 10 September 1997 / Accepted: 14 October 1997     

Glycine mineralization in situ closely correlates with soil carbon availability across six North American forest ecosystems

Free amino acids (FAA) constitute a significant fraction of dissolved organic nitrogen (N) in forest soils and play an important role in the N cycle of these ecosystems. However, comparatively little attention has been given to their role as labile carbon (C) substrates that might influence the metabolic status of resident microbial populations. We hypothesized that the residence time of simple C substrates, such as FAA, are mechanistically linked to the turnover of endogenous soil C pools. We tested this hypothesis across a latitudinal gradient of forested ecosystems that differ sharply with regard to climate, overstory taxon, and edaphic properties. Using a combined laboratory and field approach, we compared the turnover of isotopically labeled glycine in situ to the turnover of mineralizable soil C (C_min) at each site. The turnover of glycine was rapid (residence times <2 h) regardless of soil type. However, across all ecosystems glycine turnover rates were strongly correlated with indices of soil organic matter quality. For example, C:N ratios for the upper soil horizons explained ~80% of the variability observed in glycine turnover, and there was a strong positive correlation between in situ glycine-C turnover and C_min measured in the laboratory. The turnover of glycine in situ was better explained by changes in soil C availability than cross-ecosystem variation in soil temperature or concentrations of dissolved inorganic N and FAA-N. This suggests the consumption of these low-molecular-weight substrates by soil microorganisms may be governed as much by the overall decomposability of soil C as by N limitation to microbial growth.     

Ephemeral bio-engineers or reef-building polychaetes: how stable are aggregations of the tube worm Lanice conchilega (Pallas, 1766)?

Dense aggregations of tube-worms can stabilize sediments and generate oases for benthic communities that are different and often more diverse and abundant than those of the surroundings. If these features are to qualify as biogenic reefs under nature-conservation legislation such as the EC Habitats Directive, a level of stability and longevity is desirable aside from physical and biological attributes. Lanice conchilega (Pallas, 1766) is widely distributed around the European coast and aggregations of this tube-dwelling polychaete are known to have a positive effect on the biodiversity of associated species in inter- and sub-tidal areas. This increases the value of L. conchilega-rich habitats for higher trophic levels such as birds and fish. However, L. conchilega is currently not recognized as a reef builder primarily due to uncertainty about the stability of their aggregations. We carried out three studies on different spatial and temporal scales to explore a number of properties relating to stability: (1) Individual aggregations of L. conchilega of ～1 m(2) were monitored for up to 1 year, (2) records of L. conchilega from a 258-ha area over a 35-year period were analyzed, (3) the recovery of a population of L. conchilega subjected to disturbances by cultivation of Manila clams (Ruditapes philippinarum) was followed over 3 years. The studies provided evidence about the longevity of L. conchilega aggregations, their resistance to disturbance, their resilience in recovering from negative impact and their large-scale persistence. The results showed that populations of L. conchilega were prone to considerable fluctuation and the stability of aggregations depended on environmental factors and on recruitment. The tube-worms proved to be susceptible to disturbance by cultivation of Manila clams but demonstrated the potential to recover from that impact. The long-term monitoring of a large L. conchilega population in the Bay of Mont Saint Michel (France) indicated that aggregations can persist over many decades with a constant, densely populated core area and an expanding and contracting more thinly populated fringe zone. The stability of aggregations of L. conchilega and the structures they form do not unequivocally fit the currently accepted definition of a reef. However, given L. conchilega's accepted reef-like potential to influence diversity and abundance in benthic communities, we suggest clarifying and expanding the definition of reefs so that species with records of significant persistence in particular areas and which otherwise meet expectations of reefs are included within the definition.