Novel Rumen Bacterial Diversity in Two Geographically Separated Sub-Species of Reindeer

Svalbard reindeer (Rangifer tarandus platyrhynchus) live under austere nutritional conditions on the high-arctic archipelago of Svalbard, while semi-domesticated Norwegian reindeer (R. tarandus tarandus) migrate between lush coastal summer pastures and inland winter pastures with lichens on mainland Norway. Svalbard reindeer are known to have high rumen concentrations of cellulolytic bacteria, ranging from 15% of the viable population in summer to 35% in winter, compared to only 2.5% in Norwegian reindeer. Their rumen bacterial diversity was investigated through comparative analyses of 16S rRNA gene sequences (∼1.5 kb in length) generated from clone libraries (n = 121) and bacterial isolates (n = 51). LIBSHUFF comparisons of the composition of the two 16S rRNA libraries from Norwegian reindeer showed a significant effect of artificial feeding compared to natural pasture, but failed to yield significant differences between libraries from Norwegian reindeer and Svalbard reindeer. The combined sequences from reindeer were not significantly different from those reported in wild Thompson’s gazelle in Kenya but did differ from those reported in domestic cattle in Japan. A total of 90 distinct operational taxonomic units (OTUs) were identified by employing a criterion of 97% similarity, while the Chao1 index estimated the reindeer bacterial rumen population richness at 698 OTUs. The majority of the clone library sequences (92.5%) represented novel strains with <97% identity to any known sequence in the public database, most of them affiliated with the bacterial phylum Firmicutes (low G+C Gram-positives) related to the order Clostridiales (76.7%), while Gram-negative bacteria in the Bacteriodales (Prevotella–Bacteroides group) contributed to 22.5%. Also, six of the isolates were putatively novel strains, possibly representing new species in the Clostridium subphylum (cluster XIVa), Actinomyces and Butyrivibrio.     

Bacterial Diversity and Distribution in the Holocene Sediments of a Northern Temperate Lake

Sediments contain an abundance of microorganisms. However, the diversity and distribution of microorganisms associated with sediments are poorly understood, particularly in lacustrine environments. We used banding patterns from denaturing gradient gel electrophoresis (DGGE) and 16S rDNA sequences to assess the structure of bacterial communities in the Holocene sediments of a meromictic lake in Minnesota. Cluster analysis of the DGGE banding patterns indicates that the early- and middle-Holocene samples group separately from the late-Holocene samples. About 79% of the recovered bacterial sequences cluster with the α-, β-, δ-, ɛ-, and γ- Proteobacteriaceae and Firmicutes. The remaining ∼21% lack cultured representatives. The taxonomic lineages of bacteria differ statistically among the early-, middle-, and late-Holocene samples, although the difference is smallest between early- and middle-Holocene samples. Early- and middle-Holocene samples are dominated by ɛ-Proteobacteriaceae, and late-Holocene samples are dominated by sequences from uncultured subphyla. We only recovered δ-Proteobacteriaceae in late-Holocene sediments and α- and γ- Proteobacteriaceae in late- and middle-Holocene sediments. Diversity estimates derived from early-, middle-, and late-Holocene clone libraries indicate that the youngest (late-Holocene) samples had significantly greater bacterial diversity than the oldest (early-Holocene) samples, and the middle-Holocene samples contained intermediate levels of diversity. The observed patterns of diversity may be caused by increased bacterial niche-partitioning in younger sediments that contain a greater abundance of labile organic matter than older sediments. D. M. Nelson and S. Ohene-Adjei contributed equally to this work     

Role of RNA structure and susceptibility to RNase E in regulation of a cold shock mRNA, cspA mRNA

Degradation of the cspA mRNA in vivo is very rapid at temperatures greater than 30 degrees C and is moderately dependent on RNase E. Investigations in vitro show that degradosomes prepared from normal or cold-shocked cultures cleave the cspA mRNA preferentially at a single site in vitro between two stem-loops approximately 24 residues 3' to the termination codon and approximately 31 residues from the 3' end. The site of cleavage is independent of the temperature and largely independent of the phosphorylation status of the 5' end of cspA mRNA. A 5' stem-loop, potential occlusion of the initiation and termination codons, temperature-dependent translational efficiency, and the position of the RNase E cleavage site can explain the differential stability of the cspA mRNA.     

Effect of insertional mutations in the pueA and pueB genes encoding two polyurethanases in Pseudomonas chlororaphis contained within a gene cluster

AIMS: To better understand the role of PueA and PueB from Pseudomonas chlororaphis in polyurethane degradation, the present study was conducted to create insertional mutants in their respective genes. METHODS AND RESULTS: Growth kinetic studies showed that the pueA knockout mutant had a greater effect than the pueB knockout mutant. The pueA mutant had an 80% decrease in cell density from that of the wild type, while the pueB mutant had an 18% decrease in cell density. Polyurethane utilization followed Michaelis-Menten kinetics. The pueA and pueB mutants exhibited a 17% and 10% decrease respectively in growth rate using polyurethane when compared with the wild type. CONCLUSIONS: In this present study, pueA and pueB, are shown to be part of an ABC transporter gene cluster that consists of seven open reading frames. Mutational analysis results suggest that PueA may play a more major role in polyurethane degradation than PueB based on cell density and growth rates. SIGNIFICANCE AND IMPACT OF THE STUDY: The results from this study provide a starting point for the eventual enhancement and bioremediation of polyurethane waste. Understanding the role of polyurethane-degrading enzymes is useful for the creation of strains for this purpose.     

Potentiation of electrical and chemical synaptic transmission mediated by endocannabinoids

Endocannabinoids are well established as inhibitors of chemical synaptic transmission via presynaptic activation of the cannabinoid type 1 receptor (CB1R). Contrasting this notion, we show that dendritic release of endocannabinoids mediates potentiation of synaptic transmission at mixed (electrical and chemical) synaptic contacts on the goldfish Mauthner cell. Remarkably, the observed enhancement was not restricted to the glutamatergic component of the synaptic response but also included a parallel increase in electrical transmission. This effect involved the activation of CB1 receptors and was indirectly mediated via the release of dopamine from nearby varicosities, which in turn led to potentiation of the synaptic response via a cAMP-dependent protein kinase-mediated postsynaptic mechanism. Thus, endocannabinoid release can potentiate synaptic transmission, and its functional roles include the regulation of gap junction-mediated electrical synapses. Similar interactions between endocannabinoid and dopaminergic systems may be widespread and potentially relevant for the motor and rewarding effects of cannabis derivatives.     

Behavioral phenotypes of Disc1 missense mutations in mice

To support the role of DISC1 in human psychiatric disorders, we identified and analyzed two independently derived ENU-induced mutations in Exon 2 of mouse Disc1. Mice with mutation Q31L showed depressive-like behavior with deficits in the forced swim test and other measures that were reversed by the antidepressant bupropion, but not by rolipram, a phosphodiesterase-4 (PDE4) inhibitor. In contrast, L100P mutant mice exhibited schizophrenic-like behavior, with profound deficits in prepulse inhibition and latent inhibition that were reversed by antipsychotic treatment. Both mutant DISC1 proteins exhibited reduced binding to the known DISC1 binding partner PDE4B. Q31L mutants had lower PDE4B activity, consistent with their resistance to rolipram, suggesting decreased PDE4 activity as a contributory factor in depression. This study demonstrates that Disc1 missense mutations in mice give rise to phenotypes related to depression and schizophrenia, thus supporting the role of DISC1 in major mental illness.     

Altering the Divalent Metal Ion Preference of RNase E

RNase E is a major intracellular endoribonuclease in many bacteria and participates in most aspects of RNA processing and degradation. RNase E requires a divalent metal ion for its activity. We show that only Mg²⁺ and Mn²⁺ will support significant rates of activity in vitro against natural RNAs, with Mn²⁺ being preferred. Both Mg²⁺ and Mn²⁺ also support cleavage of an oligonucleotide substrate with similar kinetic parameters for both ions. Salts of Ni²⁺ and Zn²⁺ permitted low levels of activity, while Ca²⁺, Co³⁺, Cu²⁺, and Fe²⁺ did not. A mutation to one of the residues known to chelate Mg²⁺, D346C, led to almost complete loss of activity dependent on Mg²⁺; however, the activity of the mutant enzyme was fully restored by the presence of Mn²⁺ with kinetic parameters fully equivalent to those of wild-type enzyme. A similar mutation to the other chelating residue, D303C, resulted in nearly full loss of activity regardless of metal ion. The properties of RNase E D346C enabled a test of the ionic requirements of RNase E in vivo. Plasmid shuffling experiments showed that both rneD303C (i.e., the rne gene encoding a D-to-C change at position 303) and rneD346C were inviable whether or not the selection medium was supplied with MnSO₄, implying that RNase E relies on Mg²⁺ exclusively in vivo.     

Effect of branched-chain fatty acid on lipid dynamics in mice lacking liver fatty acid binding protein gene

Although a role for liver fatty acid protein (L-FABP) in the metabolism of branched-chain fatty acids has been suggested based on data obtained with cultured cells, the physiological significance of this observation remains to be demonstrated. To address this issue, the lipid phenotype and metabolism of phytanic acid, a branched-chain fatty acid, were determined in L-FABP gene-ablated mice fed a diet with and without 1% phytol (a metabolic precursor to phytanic acid). In response to dietary phytol, L-FABP gene ablation exhibited a gender-dependent lipid phenotype. Livers of phytol-fed female L-FABP–/– mice had significantly more fatty lipid droplets than male L-FABP–/– mice, whereas in phytol-fed wild-type L-FABP+/+ mice differences between males and females were not significant. Thus L-FABP gene ablation exacerbated the accumulation of lipid droplets in phytol-fed female, but not male, mice. These results were reflected in the lipid profile, where hepatic levels of triacylglycerides in phytol-fed female L-FABP–/– mice were significantly higher than in male L-FABP–/– mice. Furthermore, livers of phytol-fed female L-FABP–/– mice exhibited more necrosis than their male counterparts, consistent with the accumulation of higher levels of phytol metabolites (phytanic acid, pristanic acid) in liver and serum, in addition to increased hepatic levels of sterol carrier protein (SCP)-x, the only known peroxisomal enzyme specifically required for branched-chain fatty acid oxidation. In summary, L-FABP gene ablation exerted a significant role, especially in female mice, in branched-chain fatty acid metabolism. These effects were only partially compensated by concomitant upregulation of SCP-x in response to L-FABP gene ablation and dietary phytol. gene targeting; phytanic acid     

Chronic kidney disease and automatic reporting of estimated glomerular filtration rate: a position statement

The systematic staging of chronic kidney disease (CKD) by glomerular filtration measurement and proteinuria has allowed the development of rational and appropriate management plans. One of the barriers to early detection of CKD is the lack of a precise, reliable and consistent measure of kidney function.The most common measure of kidney function is currently serum creatinine concentration. It varies with age, sex, muscle mass and diet, and interlaboratory variation between measurements is as high as 20%.The reference interval for serum creatinine concentration includes up to 25% of people (particularly thin, elderly women) who have an estimated glomerular filtration rate (eGFR) that is significantly reduced (< 60 mL/min/1.73 m). The recent publication of a validated formula (MDRD) to estimate GFR from age, sex, race and serum creatinine concentration, without any requirement for measures of body mass, allows pathology laboratories to "automatically" generate eGFR from data already acquired. Automatic laboratory reporting of eGFR calculated from serum creatinine measurements would help to identify asymptomatic kidney dysfunction at an earlier stage. eGFR correlates well with complications of CKD and an increased risk of adverse outcomes such as cardiovascular morbidity and mortality. We recommend that pathology laboratories automatically report eGFR each time a serum creatinine test is ordered in adults. As the accuracy of eGFR is suboptimal in patients with normal or near-normal renal function, we recommend that calculated eGFRs above 60 mL/min/1.73 m be reported by laboratories as "> 60 mL/min/1.73 m", rather than as a precise figure.     

Impact of nutrition on oocyte quality: cumulative effects of body composition and diet leading to hyperinsulinemia in cattle

The present study sought to assess the combined effects of body composition and diet (level of feeding) on the postfertilization developmental potential of oocytes recovered from heifers using ultrasound-guided transvaginal follicular aspiration and to relate oocyte quality to the metabolic status of these animals. By collecting oocytes on repeated occasions spanning several weeks, it was possible to assess the cumulative effects of changes in nutritional status on oocyte quality over this period. Twenty-four heifers of low and moderate body condition were placed on one of two levels of feeding (equivalent to once or twice the maintenance requirements of these animals). Oocytes were recovered at two defined time points within each of three successive estrous cycles and were matured, fertilized, and cultured to the blastocyst stage in vitro. The results show that the effect of feeding level on oocyte quality is dependent on the body condition of the animal, with the high level of feeding being beneficial to oocytes from animals of low body condition but detrimental to oocytes from animals of moderately high body condition. Furthermore, the effects of high levels of feeding on oocyte quality were cumulative, with blastocyst yields for relatively fat heifers on twice the maintenance requirement deteriorating with time relative to yields for relatively thin heifers on the same level of feeding. Finally, a significant proportion of the moderately fat animals on the high level of feeding were hyperinsulinemic, and we show, to our knowledge for the first time in ruminants, that this condition is associated with impaired oocyte quality.