Prolonged infusion of amino acids increases leucine oxidation in fetal sheep

Maternal high-protein supplements designed to increase birth weight have not been successful. We recently showed that maternal amino acid infusion into pregnant sheep resulted in competitive inhibition of amino acid transport across the placenta and did not increase fetal protein accretion rates. To bypass placental transport, singleton fetal sheep were intravenously infused with an amino acid mixture (AA, n = 8) or saline [control (Con), n = 10] for ～12 days during late gestation. Fetal leucine oxidation rate increased in the AA group (3.1 ± 0.5 vs. 1.4 ± 0.6 μmol·min(-1)·kg(-1), P < 0.05). Fetal protein accretion (2.6 ± 0.5 and 2.2 ± 0.6 μmol·min(-1)·kg(-1) in AA and Con, respectively), synthesis (6.2 ± 0.8 and 7.0 ± 0.9 μmol·min(-1)·kg(-1) in AA and Con, respectively), and degradation (3.6 ± 0.6 and 4.5 ± 1.0 μmol·min(-1)·kg(-1) in AA and Con, respectively) rates were similar between groups. Net fetal glucose uptake decreased in the AA group (2.8 ± 0.4 vs. 3.9 ± 0.1 mg·kg(-1)·min(-1), P < 0.05). The glucose-O(2) quotient also decreased over time in the AA group (P < 0.05). Fetal insulin and IGF-I concentrations did not change. Fetal glucagon increased in the AA group (119 ± 24 vs. 59 ± 9 pg/ml, P < 0.05), and norepinephrine (NE) also tended to increase in the AA group (785 ± 181 vs. 419 ± 76 pg/ml, P = 0.06). Net fetal glucose uptake rates were inversely proportional to fetal glucagon (r(2) = 0.38, P < 0.05), cortisol (r(2) = 0.31, P < 0.05), and NE (r(2) = 0.59, P < 0.05) concentrations. Expressions of components in the mammalian target of rapamycin signaling pathway in fetal skeletal muscle were similar between groups. In summary, prolonged infusion of amino acids directly into normally growing fetal sheep increased leucine oxidation. Amino acid-stimulated increases in fetal glucagon, cortisol, and NE may contribute to a shift in substrate oxidation by the fetus from glucose to amino acids.     

Sequence-dependent sorting of recycling proteins by actin-stabilized endosomal microdomains

The functional consequences of signaling receptor endocytosis are determined by the endosomal sorting of receptors between degradation and recycling pathways. How receptors recycle efficiently, in a sequence-dependent manner that is distinct from bulk membrane recycling, is not known. Here, in live cells, we visualize the sorting of a prototypical sequence-dependent recycling receptor, the beta-2 adrenergic receptor, from bulk recycling proteins and the degrading delta-opioid receptor. Our results reveal a remarkable diversity in recycling routes at the level of individual endosomes, and indicate that sequence-dependent recycling is an active process mediated by distinct endosomal subdomains distinct from those mediating bulk recycling. We identify a specialized subset of tubular microdomains on endosomes, stabilized by a highly localized but dynamic actin machinery, that mediate this sorting, and provide evidence that these actin-stabilized domains provide the physical basis for a two-step kinetic and affinity-based model for protein sorting into the sequence-dependent recycling pathway.     

Characterization of a novel mutation in the cardiac ryanodine receptor that results in catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmogenic disease that manifests as syncope or sudden death during high adrenergic tone in the absence of structural heart defects. It is primarily caused by mutations in the cardiac ryanodine receptor (RyR2). The mechanism by which these mutations cause arrhythmia remains controversial, with discrepant findings related to the role of the RyR2 binding protein FKBP12.6. The purpose of this study was to characterize a novel RyR2 mutation identified in a kindred with clinically diagnosed CPVT.Single-strand conformational polymorphism analysis and direct DNA sequencing were used to screen the RyR2 gene for mutations. Site-directed mutagenesis was employed to introduce the mutation into the mouse RyR2 cDNA. The impact of the mutation on the interaction between RyR2 and a 12.6 kDa FK506 binding protein (FKBP12.6) was determined by immunoprecipitation and immunoblotting and its effect on RyR2 function was characterized by single cell Ca²⁺ imaging and [³H]ryanodine binding.A novel CPVT mutation, E189D, was identified. The E189D mutation does not alter the affinity of the channel for FKBP12.6, but it increases the propensity for store-overload-induced Ca²⁺ release (SOICR). Furthermore, the E189D mutation enhances the basal channel activity of RyR2 and its sensitivity to activation by caffeine.The E189D RyR2 mutation is causative for CPVT and functionally increases the propensity for SOICR without altering the affinity for FKBP12.6. These observations strengthen the notion that enhanced SOICR, but not altered FKBP12.6 binding, is a common mechanism by which RyR2 mutations cause arrhythmias.Key words: arrhythmia, calcium, death sudden, genetics, ion channels     

Conditioning shelter dogs to sit

Human contact in the shelter may lessen effects of change in environment and smooth transition into a home. Training can increase a dog's interaction with people in a shelter environment. Experiments were conducted to determine how rapidly shelter dogs learn to sit, if the dogs can retain sitting behavior over time, and if sitting transfers to novel locations and people. Two experiments trained shelter dogs (n = 21) to sit when a stranger approached over a 10-trial session. Food and a verbal cue or a clicker reinforced the sit. The experiments measured latency to sit for each trial. Latency to sit decreased significantly over trials. Another experiment included reinforcement given to dogs (n = 20) on a noncontingent basis or for sitting. Five days of the experiment (condition training) were in the same room with the same experimenter. The last 4 days (testing) varied by both experimenter and location (familiar or strange). Results indicate that short training sessions are effective for teaching shelter dogs to sit, that dogs can retain sitting behavior over 2 days, and that training transfers to novel people and situations.     

An association between vasomotion and oxygen extraction

Vasomotion is defined as a spontaneous local oscillation in vascular tone whose function is unclear but may have a beneficial effect on tissue oxygenation. Optical reflectance spectroscopy and laser Doppler fluximetry provide unique insights into the possible mechanisms of vasomotion in the cutaneous microcirculation through the simultaneous measurement of changes in concentration of oxyhemoglobin ([HbO(2)]), deoxyhemoglobin ([Hb]), and mean blood saturation (S(mb)O(2)) along with blood volume and flux. The effect of vasomotion at frequencies <0.02 Hz attributed to endothelial activity was studied in the dorsal forearm skin of 24 healthy males. Fourier analysis identified periodic fluctuations in S(mb)O(2) in 19 out of 24 subjects, predominantly where skin temperatures were >29.3°C (X(2) = 6.19, P < 0.02). A consistent minimum threshold in S(mb)O(2) (mean: 39.4%, range: 24.0-50.6%) was seen to precede a sudden transient surge in flux, inducing a fast rise in S(mb)O(2). The integral increase in flux correlated with the integral increase in [HbO(2)] (Pearson's correlation r(2) = 0.50, P < 0.001) and with little change in blood volume suggests vasodilation upstream, responding to a low S(mb)O(2) downstream. This transient surge in flux was followed by a sustained period where blood volume and flux remained relatively constant and a steady decrease in [HbO(2)] and equal and opposite increase in [Hb] was considered to provide a measure of oxygen extraction. A measure of this oxygen extraction has been approximated by the mean half-life of the decay in S(mb)O(2) during this period. A comparison of the mean half-life in the 8 normal subjects [body mass index (BMI) <26.0 kg/m(2)] of 12.2 s and the 11 obese subjects (BMI >29.5 kg/m(2)) of 18.8 s was statistically significant (Mann Whitney, P < 0.004). The S(mb)O(2) fluctuated spontaneously in this saw tooth manner by an average of 9.0% (range 4.0-16.2%) from mean S(mb)O(2) values ranging from 30 to 52%. These observations support the hypothesis that red blood cells may act as sensors of local tissue hypoxia, through the oxygenation status of the hemoglobin, and initiate improved local perfusion to the tissue through hypoxic vasodilation.     

Mitochondrial protein patterns correlating with impaired insulin secretion from INS-1E cells exposed to elevated glucose concentrations

Extended hyperglycaemia leads to impaired glucose-stimulated insulin secretion (GSIS) and eventually beta-cell apoptosis in individuals with type 2 diabetes mellitus. In an attempt to dissect mechanisms behind the detrimental effects of glucose, we focused on measuring changes in expression patterns of mitochondrial proteins. Impaired GSIS was observed from INS-1E cells cultured for 5 days at 20 or 27 mM glucose compared to cells cultured at 5.5 or 11 mM glucose. After culture, mitochondria were isolated from the INS-1E cells by differential centrifugation. Proteins of the mitochondrial fraction were bound to a strong anionic surface (SAX2) protein array and mass spectra generated by SELDI-TOF-MS. Analysis of the spectra revealed proteins with expression levels that correlated with the glucose concentration of the culture medium. Indeed, such differentially expressed proteins created patterns of protein changes, which correlated with impairment of GSIS. In conclusion, the study reveals the first glucose-induced differentially expressed patterns of beta-cell mitochondrial proteins obtained by SELDI-TOF-MS.     

Diversity of Basidiomycetes in Michigan Agricultural Soils

We analyzed the communities of soil basidiomycetes in agroecosystems that differ in tillage history at the Kellogg Biological Station Long-Term Ecological Research site near Battle Creek, Michigan. The approach combined soil DNA extraction through a bead-beating method modified to increase recovery of fungal DNA, PCR amplification with basidiomycete-specific primers, cloning and restriction fragment length polymorphism screening of mixed PCR products, and sequencing of unique clones. Much greater diversity was detected than was anticipated in this habitat on the basis of culture-based methods or surveys of fruiting bodies. With “species” defined as organisms yielding PCR products with ≥99% identity in the 5′ 650 bases of the nuclear large-subunit ribosomal DNA, 241 “species” were detected among 409 unique basidiomycete sequences recovered. Almost all major clades of basidiomycetes from basidiomycetous yeasts and other heterobasidiomycetes through polypores and euagarics (gilled mushrooms and relatives) were represented, with a majority from the latter clade. Only 24 of 241 “species” had 99% or greater sequence similarity to named reference sequences in GenBank, and several clades with multiple “species” could not be identified at the genus level by phylogenetic comparisons with named sequences. The total estimated “species” richness for this 11.2-ha site was 367 “species” of basidiomycetes. Since >99% of the study area has not been sampled, the accuracy of our diversity estimate is uncertain. Replication in time and space is required to detect additional diversity and the underlying community structure.     

Addressing dimensions of “The Great Moral Wrong”: How inequity in music education is polarizing the academic potential of Australian students

The intrinsic and extrinsic benefits of music education incorporate academic, social, emotional, and behavioral spheres. To deny students access to music education essentially locks them out of such benefits, limiting their potential. Music education programs have declined significantly in Australia in the last few decades and access to such programs serves as a reminder of the widening gap of inequality in the Australian school system. The reasons for the decline in music education, both at a preservice and inservice level, and the political landscape which has influenced and undermined the concept of a level playing field in public education are interrelated. Through extensive data analysis, a picture of how and why music education serves as an example of wider issues of access and equity in Australian schools will be demonstrated.     

Evidence for the secondary sexual development of the anal fin in female kokanee salmon Oncorhynchus nerka

This study examines whether the anal fin undergoes secondary sexual development similar to other reproductive traits in salmonids. This hypothesis was tested by comparing the anal‐fin size of female kokanee salmon Oncorhynchus nerka that were in the early and late stages of sexual development. Females in an advanced stage of maturation had significantly larger anal fins relative to females in an early state of maturation (+4–7%), indicating that the anal fin undergoes secondary sexual development. The magnitude of this secondary growth was comparable with snout length (+9–10%), which is known to undergo secondary sexual development in female salmonids. When morphological trait dimensions were compared between the sexes, the anal fin was the only morphological trait found to have a female‐biased sexual size dimorphism. This is the first study to show that the anal fin of female salmonids undergoes secondary sexual development.     

Monitoring the prevention of amyloid fibril formation by alpha-crystallin. Temperature dependence and the nature of the aggregating species

The molecular chaperone, alpha-crystallin, has the ability to prevent the fibrillar aggregation of proteins implicated in human diseases, for example, amyloid beta peptide and alpha-synuclein. In this study, we examine, in detail, two aspects of alpha-crystallin's fibril-suppressing ability: (a) its temperature dependence, and (b) the nature of the aggregating species with which it interacts. First, the efficiency of alpha-crystallin to suppress fibril formation in kappa-casein and alpha-synuclein increases with temperature, despite their rate of fibrillation also increasing in the absence of alpha-crystallin. This is consistent with an increased chaperone ability of alpha-crystallin at higher temperatures to protect target proteins from amorphous aggregation [GB Reddy, KP Das, JM Petrash & WK Surewicz (2000) J Biol Chem275, 4565-4570]. Second, dual polarization interferometry was used to monitor real-time alpha-synuclein aggregation in the presence and absence of alphaB-crystallin. In contrast to more common methods for monitoring the time-dependent formation of amyloid fibrils (e.g. the binding of dyes like thioflavin T), dual polarization interferometry data did not reveal any initial lag phase, generally attributed to the formation of prefibrillar aggregates. It was shown that alphaB-crystallin interrupted alpha-synuclein aggregation at its earliest stages, most likely by binding to partially folded monomers and thereby preventing their aggregation into fibrillar structures.