High-performance liquid chromatographic assay of lactic, pyruvic and acetic acids and lactic acid stereoisomers in calf feces, rumen fluid and urine

To facilitate clinical investigation of metabolic acidosis, a high-performance liquid chromatographic method was adapted and validated for the chiral separation of D-(-) and L-(+)-lactic acid in calf feces, rumen fluid and urine. A non-chiral method was also adapted and validated for the separation of pyruvic, acetic and DL-(+/-)-lactic acids in calf feces and DL-(+/-)-lactic and pyruvic acids in rumen fluid. Separation and quantification were achieved using a reversed phase sulphonated polystyrenedivinylbenzene analytical column for pyruvic, acetic and racemic lactic acids and by a 3 microm octadecylsilane (ODS) packed analytical column coated with N,N-dioctyl-L-alanine as the chiral selector for the separation of lactic acid enantiomers with Cu(II)-containing eluents by stereoselective ligand exchange chromatography. Endogenous analytes were present in validation samples over a range of concentrations (0.2-14.8 mmol/l). For the stereoselective assay, mean intra-day accuracy ranged from 90.6 to 108.4% and intra-day precision from 0.3 to 13.8%. For the non-stereoselective assay, mean intra-day accuracy ranged from 90.4 to 108.8% and intra-day precision from 1.5 to 11.1%. The limit of quantitation was 1.0 mmol/l for D- and L-lactic acid, 0.06125 mmol/l for pyruvic acid, 1.0 mmol/l for DL-lactic acid and 1 mmol/l for acetic acid. These assays can be used to study the role of the gastrointestinal tract and kidney in metabolic acidosis.     

Coliform dynamics and the implications for source tracking

In many parts of the world, coliform counts in recreational waters are unacceptably high. In an attempt to rectify this problem, programmes are under way to develop methods that will allow the sources of the faecal contamination thought to be responsible for these elevated counts to be identified. The success of these efforts depends on the validity of several assumptions that underlie many of the proposed methods. One of the critical assumptions is that the clonal composition of the coliform species being monitored in a water body reflects the clonal composition of the species in the host populations responsible for the faecal inputs into that water body. To determine the extent to which among-strain variation in a coliform species might invalidate this assumption, a series of simple mathematical models was proposed and analysed. The first series of models assumed that all cells of species were identical. The question posed was - is the density of a coliform species in a body of water linearly related to the rate at which cells of the species enter the water body via faecal production? The results of these models suggest that, over a wide range of conditions, cell densities in the water body are linearly related to the rate at which cells enter the water body as a result of faecal contamination. This outcome occurs whether or not cells are capable of division in the external environment. When the rate of cell division depends on the concentration of available nutrients then, when nutrient input rates are 'high' and rates of faecal contamination are 'low', this linear relationship does not hold. The second series of models assumed that the coliform species consists of different strains and that these strains differ in their performance in the external environment. The results of these multistrain models show that the relative abundance of strains in the external environment is unlikely to reflect their relative abundance in the faecal inputs to the environment. Consequently, statements such as - domestic animals are responsible for 30% and wildlife for 70% of the faecal inputs to a water body - may well be meaningless.     

Top-down impact of bacterivorous nematodes on the bacterial community structure: a microcosm study

The influence of bacterivorous nematodes (Diplolaimelloides meyli, Diplolaimelloides oschei, Diplolaimella dievengatensis, Panagrolaimus paetzoldi) on the development of a bacterial community growing on decaying cordgrass detritus was studied in laboratory microcosm experiments. Cordgrass leaves were incubated on a sediment surface with a natural bacterial mixture containing bacteria from sediment, cordgrass detritus and habitat water. The four nematode species were applied separately to the microcosms; controls remained without nematodes. Samples were taken seven times over a 65-day period. The bacterial community structure was analysed by means of DGGE of the 16S rRNA genes. Multi Dimensional Scaling showed grouping of the samples per treatment. Analysis of Similarities indicated that the differences between treatments were significantly larger than differences within treatments. Our results suggest that nematodes can have a significant structuring top-down influence on the 'pool' of bacteria growing on the detritus, even at low densities. Dissimilarities were similar between all treatments. Differences in bacterial community composition within the treatments with monhysterids (D. meyli, D. oschei and D. dievengatensis) can be explained by species-specific food preferences. Panagrolaimus paetzoldi on the other hand feeds unselectively, and thus affects the bacterial community differently. A top-down effect of the nematodes on the diversity of the bacterial community was only evident under high grazing pressure, i.e. in the presence of P. paetzoldi.     

Production of tailor-made fructans in sugar beet by expression of onion fructosyltransferase genes

The consumption of fructans as a low caloric food ingredient or dietary fibre is rapidly increasing due to health benefits. Presently, the most important fructan source is chicory, but these fructans have a simple linear structure and are prone to degradation. Additional sources of high-quality tailor-made fructans would provide novel opportunities for their use as food ingredients. Sugar beet is a highly productive crop that does not normally synthesize fructans. We have introduced specific onion fructosyltransferases into sugar beet. This resulted in an efficient conversion of sucrose into complex, onion-type fructans, without the loss of storage carbohydrate content.     

Blood oxygen transport in common map turtles during simulated hibernation

We assessed the effects of cold and submergence on blood oxygen transport in common map turtles (Graptemys geographica). Winter animals were acclimated for 6-7 wk to one of three conditions at 3 degrees C: air breathing (AB-3 degrees C), normoxic submergence (NS-3 degrees C), and hypoxic (PO2=49 Torr) submergence (HS-3 degrees C). NS-3 degrees C turtles exhibited a respiratory alkalosis (pH 8.07; PCO2=7.9 Torr; [lactate]=2.2 mM) relative to AB-3 degrees C animals (pH 7.89; PCO2=13.4 Torr; [lactate]=1.1 mM). HS-3 degrees C animals experienced a profound metabolic acidosis (pH 7.30; PCO2=7.9 Torr; [lactate]=81 mM). NS-3 degrees C turtles exhibited an increased blood O2 capacity; however, isoelectric focusing revealed no seasonal changes in the isohemoglobin (isoHb) profile. Blood O2 affinity was significantly increased by cold acclimation; half-saturation pressures (P50's) for air-breathing turtles at 3 degrees and 22 degrees C were 6.5 and 18.8 Torr, respectively. P50's for winter animals submerged in normoxic and hypoxic water were 5.2 and 6.5 Torr, respectively. CO2 Bohr slopes (Delta logP50/Delta pH) were -0.15, -0.16, and -0.07 for AB-3 degrees C, NS-3 degrees C, and HS-3 degrees C turtles, respectively; the corresponding value for AB-22 degrees C was -0.37. The O2 equilibrium curve (O2EC) shape was similar for AB-3 degrees C and NS-3 degrees C turtles; Hill plot n coefficients ranged from 1.8 to 2.0. The O2EC shape for HS-3 degrees C turtles was anomalous, exhibiting high O2 affinity below P50 and a right-shifted segment above half-saturation. We suggest that increases in Hb-O2 affinity and O2 capacity enhance extrapulmonary O2 uptake by turtles overwintering in normoxic water. The anomalous O2EC shape and reduced CO2 Bohr effect of HS-3 degrees C turtles may also promote some aerobic metabolism in hypoxic water.     

The evolution of endothermy and its diversity in mammals and birds

Many elements of mammalian and avian thermoregulatory mechanisms are present in reptiles, and the changes involved in the transition to endothermy are more quantitative than qualitative. Drawing on our experience with reptiles and echidnas, we comment on that transition and on current theories about how it occurred. The theories divide into two categories, depending on whether selection pressures operated directly or indirectly on mechanisms producing heat. Both categories of theories focus on explaining the evolution of homeothermic endothermy but ignore heterothermy. However, noting that hibernation and torpor are almost certainly plesiomorphic (=ancestral, primitive), and that heterothermy is very common among endotherms, we propose that homeothermic endothermy evolved via heterothermy, with the earliest protoendotherms being facultatively endothermic and retaining their ectothermic capacity for "constitutional eurythermy." Thus, unlike current models for the evolution of endothermy that assume that hibernation and torpor are specialisations arising from homeothermic ancestry, and therefore irrelevant, we consider that they are central. We note the sophistication of thermoregulatory behavior and control in reptiles, including precise control over conductance, and argue that brooding endothermy seen in some otherwise ectothermic Boidae suggests an incipient capacity for facultative endothermy in reptiles. We suggest that the earliest insulation in protoendotherms may have been internal, arising from redistribution of the fat bodies that are typical of reptiles. We note that short-beaked echidnas provide a useful living model of what an (advanced) protoendotherm may have been like. Echidnas have the advantages of endothermy, including the capacity for homeothermic endothermy during incubation, but are very relaxed in their thermoregulatory precision and minimise energetic costs by using ectothermy facultatively when entering short- or long-term torpor. They also have a substantial layer of internal dorsal insulation. We favor theories about the evolution of endothermy that invoke direct selection for the benefits conferred by warmth, such as expanding daily activity into the night, higher capacities for sustained activity, higher digestion rates, climatic range expansion, and, not unrelated, control over incubation temperature and the benefits for parental care. We present an indicative, stepwise schema in which observed patterns of body temperature are a consequence of selection pressures, the underlying mechanisms, and energy optimization, and in which homeothermy results when it is energetically desirable rather than as the logical endpoint.     

The greatest step in vertebrate history: a paleobiological review of the fish-tetrapod transition

Recent discoveries of previously unknown fossil forms have dramatically transformed understanding of many aspects of the fish-tetrapod transition. Newer paleobiological approaches have also contributed to changed views of which animals were involved and when, where, and how the transition occurred. This review summarizes major advances made and reevaluates alternative interpretations of important parts of the evidence. We begin with general issues and concepts, including limitations of the Paleozoic fossil record. We summarize important features of paleoclimates, paleoenvironments, paleobiogeography, and taphonomy. We then review the history of Devonian tetrapods and their closest stem group ancestors within the sarcopterygian fishes. It is now widely accepted that the first tetrapods arose from advanced tetrapodomorph stock (the elpistostegalids) in the Late Devonian, probably in Euramerica. However, truly terrestrial forms did not emerge until much later, in geographically far-flung regions, in the Lower Carboniferous. The complete transition occurred over about 25 million years; definitive emergences onto land took place during the most recent 5 million years. The sequence of character acquisition during the transition can be seen as a five-step process involving: (1) higher osteichthyan (tetrapodomorph) diversification in the Middle Devonian (beginning about 380 million years ago [mya]), (2) the emergence of "prototetrapods" (e.g., Elginerpeton) in the Frasnian stage (about 372 mya), (3) the appearance of aquatic tetrapods (e.g., Acanthostega) sometime in the early to mid-Famennian (about 360 mya), (4) the appearance of "eutetrapods" (e.g., Tulerpeton) at the very end of the Devonian period (about 358 mya), and (5) the first truly terrestrial tetrapods (e.g., Pederpes) in the Lower Carboniferous (about 340 mya). We discuss each of these steps with respect to inferred functional utility of acquired character sets. Dissociated heterochrony is seen as the most likely process for the evolutionarily rapid morphological transformations required. Developmental biological processes, including paedomorphosis, played important roles. We conclude with a discussion of phylogenetic interpretations of the evidence.     

Qualification of analytical instruments for use in the pharmaceutical industry: A scientific approach

The purpose of the use of analytical instruments is to generate reliable data. Instrument qualification helps fulfill this purpose. No authoritative guide exists that considers the risk of instrument failure and combines that risk with users' scientific knowledge and ability to use the instrument to deliver reliable and consistent data. In the absence of such a guide, the qualification of analytical instruments has become a subjective and often fruitless document-generating exercise. Taking its cue from the new FDA initiative, “Pharmaceutical GMP's for the 21st Century,” an efficient, science- and risk-based process for AIQ was discussed at a workshop on analytical instrument qualification. This report represents the distillate of deliberations on the complicated issues associated with the various stages of analytical instrument qualification. It emphasizes AIQ's place in the overall process of obtaining quality reliable data from analytical instruments and offers an efficient process for its performance, one that focuses on scientific value rather than on producing documents. Implementing such a process should remove ambiguous interpretations by various groups.     

Postnatal glucocorticoids induce alpha-ENaC formation and regulate glucocorticoid receptors in the preterm rabbit lung

At birth, lung fluid clearance is coupled to Na+ transport through epithelial Na+ channels (ENaC) in the distal lung epithelium. We evaluated the effect of postnatal glucocorticoids (GC) on lung alpha-ENaC expression in preterm 29-day gestational age (GA) fetal rabbits. Postnatal treatment of 29-day GA fetuses with 0.5 mg/kg of dexamethasone (Dex) iv resulted in a 2- and 22-fold increase in lung alpha-ENaC mRNA expression compared with saline-treated fetuses after 8 and 16 h, respectively. Lung alpha-ENaC protein levels in Dex-treated fetuses were also elevated compared with saline-treated counterparts. The extravascular lung water (EVLW)/dry lung tissue weight ratios of 29-day GA fetuses treated with either saline or Dex decreased over 24 h compared with that observed at birth; however, at 24 h, the EVLW/dry lung tissue weight ratios of saline- and Dex-treated fetuses were similar. Dex-induced alpha-ENaC mRNA and protein levels were attenuated by glucocorticoid receptor (GCR) antagonist RU-486 in fetal distal lung epithelial cells isolated from 29-day GA fetuses, indicating that GC-dependent augmentation of lung alpha-ENaC requires the presence of functional GCR. Lung GCR mRNA expression and protein levels were elevated in 29-day GA fetuses compared with fetuses at earlier GA. Exposure of 29-day GA fetuses to Dex for 16 h caused a 2.1-fold increase in lung GCR mRNA expression, but GCR protein levels were decreased in Dex-treated fetuses after 24 h. We conclude that postnatal treatment of preterm 29-day GA fetal rabbits with GC results in an elevation of lung alpha-ENaC accompanied by an autoregulation of pulmonary GCR.