Calculations of apparent ruminal synthesis and intestinal absorption of biotin in dairy cows as influenced by the extraction method

Biotin is present in nature either free or as biocytin, which is only degraded under the action of a specific enzyme: biotinidase. This enzyme is not included in analytical assays generally used. A method for sample preparation using biotinidase was developed in our laboratory before analysis by ELISA. Three cows equipped with duodenal and ileal cannulae were used to compare the effects of methods of sample preparation on calculations of apparent ruminal synthesis and intestinal absorption of biotin. There was no apparent ruminal synthesis of biotin, no matter whether free or total biotin was measured (p = 0.84). Results also suggested that rumen microbes cannot utilize nor degrade biocytin present in the feed. Estimates of apparent intestinal absorption were influenced by the sample preparation method (p = 0.002). Analysis of free biotin caused an artefact, suggesting intestinal synthesis of this vitamin; whereas determination of total biotin concentrations showed that absorption was taking place in the small intestine.     

How does regulatory Ca2+ regulate the Na+-Ca2+ exchanger?

Spatial and temporal regulation of intracellular Ca2+ concentrations is a fundamental requirement for life. The mammalian cardiac Na+-Ca2+ exchanger serves as the main mechanism for Ca2+ efflux after heart contraction. Exchange activity is highly regulated by intracellular Ca2+, which binds two regulatory domains (CBD1 and CBD2) and triggers the full activity of the exchanger. We solved the X-ray crystallographic structure of CBD2 in the presence and absence of Ca2+. Together with mutational analysis of the Ca2+ binding sites, this study reveals the crucial role of one of the two bound Ca2+ ions and helps propose hypotheses on the mechanism of regulation of the exchanger.     

Quantifying the links between land use and population growth rate in a declining farmland bird

Land use is likely to be a key driver of population dynamics of species inhabiting anthropogenic landscapes, such as farmlands. Understanding the relationships between land use and variation in population growth rates is therefore critical for the management of many farmland species. Using 24 years of data of a declining farmland bird in an integrated population model, we examined how spatiotemporal variation in land use (defined as habitats with “Short” and “Tall” ground vegetation during the breeding season) and habitat‐specific demographic parameters relates to variation in population growth taking into account individual movements between habitats. We also evaluated contributions to population growth using transient life table response experiments which gives information on contribution of past variation of parameters and real‐time elasticities which suggests future scenarios to change growth rates. LTRE analyses revealed a clear contribution of Short habitats to the annual variation in population growth rate that was mostly due to fledgling recruitment, whereas there was no evidence for a contribution of Tall habitats. Only 18% of the variation in population growth was explained by the modeled local demography, the remaining variation being explained by apparent immigration (i.e., the residual variation). We discuss potential biological and methodological reasons for high contributions of apparent immigration in open populations. In line with LTRE analysis, real‐time elasticity analysis revealed that demographic parameters linked to Short habitats had a stronger potential to influence population growth rate than those of Tall habitats. Most particularly, an increase of the proportion of Short sites occupied by Old breeders could have a distinct positive impact on population growth. High‐quality Short habitats such as grazed pastures have been declining in southern Sweden. Converting low‐quality to high‐quality habitats could therefore change the present negative population trend of this, and other species with similar habitat requirements.     

The potassium channel MBK1 (Kv1.1) is expressed in the mouse retina

1. The neurons of the retina have electrical properties that are different from those of most of the other neurons of the central nervous system. To identify the voltage-gated ion channels found in the retina, we screened mouse retinal cDNA libraries with oligonucleotide probes homologous to the mammalian K+ channel MBK1 (Kv1.1) and ligated two partial clones to produce a full-length clone with no significant differences from MBK1. 2. Expression of MBK1 mRNA was determined by RNAse protection. MBK1 mRNA was detected in retinal RNA and was also detected in brain, liver, and heart RNAs. 3. We transcribed the full-length clone, injected it into oocytes of Xenopus laevis, and measured the membrane currents 2 to 6 days later. Depolarization from a holding voltage of -90mV induced a slowly activated outward current with a peak value as large as 20 microA. The current inactivated very slowly with a single exponential time course [mean time constant, 6.5 +/- 0.4 sec (SEM) for activation voltage of -10mV]. 4. The outward current was reduced to half-maximal by 0.42 mM tetraethylammonium, 1.1 mM 4-aminopyridine, and 3.2 mM Ba2+ but was not significantly attenuated by Co2+ (1 mM). 5. The reversal potential (measured with tail currents) changed by 53mV per decade change of [K+] from 1 to 77 mM. 6. The voltage for half-maximal activation of the conductance was -26.6mV (+/- 1.7mV), and the voltage required for an e-fold increase in conductance was 6.9mV (+/- 0.5mV). 7. Thus, the mRNA for MBK1 found in the mouse retina causes the expression of a voltage-dependent K+ current which has properties suitable for may retinal neurons.     

Thyroid hormones stimulate renal expression of CFTR.

CFTR is a multifunctional protein of the ATP binding cassette family that may contribute to overall electrolyte homeostasis by acting as a chloride channel in the kidney. In renal tissues CFTR does not exists only in its full-length form, but also as a kidney-specific, truncated splice variant, TNR-CFTR. In this study we show that both forms of CFTR are regulated by thyroid hormones in rat renal tissue. Four groups of male rats were used: control, hypothyroid, hypothyroid with T(4) treatment and hyperthyroid rats. The hypothyroid rats showed a decrease of both CFTR and TNR-CFTR mRNAs (44%, and 49%, respectively, n=5; p<0.05) and proteins (30% and 37%, respectively, n=5, p<0.05) expressions, compared to control group. In hyperthyroid rats, a significant increase in both CFTR and TRN-CFTR mRNAs expressions (43% and 95%, n=5; p<0.05) and proteins (250% and 38%, respectively, n=5, p<0.05) was observed when compared to control group. Treatment of immortalized rat proximal tubule cells (IRPTC) with T(3) (10(-7)M) produced also an increase of CFTR mRNA expression (95%, n=5, p<0.05). Analysis of the promoter region of CFTR transfected to IRPTC showed that T(3) (10(-7) M) stimulates the CFTR promoter (38%, n=4, p<0.05).     

RIN2 Deficiency Results in Macrocephaly, Alopecia, Cutis Laxa, and Scoliosis: MACS Syndrome

Inherited disorders of elastic tissue represent a complex and heterogeneous group of diseases, characterized often by sagging skin and occasionally by life-threatening visceral complications. In the present study, we report on an autosomal-recessive disorder that we have termed MACS syndrome (macrocephaly, alopecia, cutis laxa, and scoliosis). The disorder was mapped to chromosome 20p11.21-p11.23, and a homozygous frameshift mutation in RIN2 was found to segregate with the disease phenotype in a large consanguineous kindred. The mutation identified results in decreased expression of RIN2, a ubiquitously expressed protein that interacts with Rab5 and is involved in the regulation of endocytic trafficking. RIN2 deficiency was found to be associated with paucity of dermal microfibrils and deficiency of fibulin-5, which may underlie the abnormal skin phenotype displayed by the patients.     

The fully-active and structurally-stable form of the mitochondrial ATP synthase of Polytomella sp. is dimeric

Mitochondrial F₁F_O-ATP synthase of chlorophycean algae is a stable dimeric complex of 1,600 kDa. It lacks the classic subunits that constitute the peripheral stator-stalk and the orthodox polypeptides involved in the dimerization of the complex. Instead, it contains nine polypeptides of unknown evolutionary origin named ASA1 to ASA9. The isolated enzyme exhibited a very low ATPase activity (0.03 Units/mg), that increased upon heat treatment, due to the release of the F₁ sector. Oligomycin was found to stabilize the dimeric structure of the enzyme, providing partial resistance to heat dissociation. Incubation in the presence of low concentrations of several non-ionic detergents increased the oligomycin-sensitive ATPase activity up to 7.0–9.0 Units/mg. Incubation with 3% (w/v) taurodeoxycholate monomerized the enzyme. The monomeric form of the enzyme exhibited diminished activity in the presence of detergents and diminished oligomycin sensitivity. Cross-linking experiments carried out with the dimeric and monomeric forms of the ATP synthase suggested the participation of the ASA6 subunit in the dimerization of the enzyme. The dimeric enzyme was more resistant to heat treatment, high hydrostatic pressures, and protease digestion than the monomeric enzyme, which was readily disrupted by these treatments. We conclude that the fully-active algal mitochondrial ATP synthase is a stable catalytically active dimer; the monomeric form is less active and less stable. Monomer-monomer interactions could be mediated by the membrane-bound subunits ASA6 and ASA9, and may be further stabilized by other polypeptides such as ASA1 and ASA5. Alexa Villavicencio-Queijeiro and Miriam Vázquez-Acevedo have contributed equally to this work.     

PRIMA-1 cytotoxicity correlates with nucleolar localization and degradation of mutant p53 in breast cancer cells

PRIMA-1 has been identified as a compound that restores the transactivation function to mutant p53 and induces apoptosis in cells expressing mutant p53. Studies on subcellular distribution of the mutant p53 protein upon treatment with PRIMA-1^Met, a methylated form of PRIMA-1, have suggested that redistribution of mutant p53 to nucleoli may play a role in PRIMA-1 induced apoptosis. Here, we specifically investigated the influence of PRIMA-1 on cellular localization of mutated p53-R280K endogenously expressed in tumour cells. By using immunofluorescence staining, we found a strong nucleolar redistribution of mutant p53 following PRIMA-1 treatment. This subcellular localization was associated to p53 degradation via ubiquitylation. When cells were treated with adriamycin, neither nucleolar redistribution nor mutant p53 down modulation and degradation were observed. Interestingly, cells where p53-R280K was silenced were more sensitive to PRIMA-1 than the parental ones. These results indicate that in some cellular context, the cell sensitivity to PRIMA-1 could depend on the abolition of a gain-of-function activity of the mutated p53, through a protein degradation pathway specifically induced by this compound.     

A quantitative and statistically robust method for the determination of xylem conduit spatial distribution

? Premise of the study: Because of their limited length, xylem conduits need to connect to each other to maintain water transport from roots to leaves. Conduit spatial distribution in a cross section plays an important role in aiding this connectivity. While indices of conduit spatial distribution already exist, they are not well defined statistically. ? Methods: We used point pattern analysis to derive new spatial indices. One hundred and five cross-sectional images from different species were transformed into binary images. The resulting point patterns, based on the locations of the conduit centers-of-area, were analyzed to determine whether they departed from randomness. Conduit distribution was then modeled using a spatially explicit stochastic model. ? Key results: The presence of conduit randomness, uniformity, or aggregation depended on the spatial scale of the analysis. The large majority of the images showed patterns significantly different from randomness at least at one spatial scale. A strong phylogenetic signal was detected in the spatial variables. ? Conclusions: Conduit spatial arrangement has been largely conserved during evolution, especially at small spatial scales. Species in which conduits were aggregated in clusters had a lower conduit density compared to those with uniform distribution. Statistically sound spatial indices must be employed as an aid in the characterization of distributional patterns across species and in models of xylem water transport. Point pattern analysis is a very useful tool in identifying spatial patterns.