The preferential mobilisation of C20 and C22 polyunsaturated fatty acids from the adipose tissue of the chick embryo: potential implications regarding the provision of essential fatty acids for neural development

The effects of 10 day clenbuterol administration on cardiac and skeletal muscle capillarities were studied, particularly in terms of the distribution of arteriolar and venular capillaries and their capillary density, in young (10-week-old) and middle-aged (37-week-old) male Wistar rats. Rats of the treated groups were fed a diet containing 2 mg kg-1 clenbuterol hydrochloride. In both young and middle aged rats, clenbuterol treatment increased the body wt and the weights of the heart and hindlimb muscles. The mean fibre cross-sectional area was significantly increased after the treatment in the left ventricle, soleus, plantaris and both deep and superficial portions of gastrocnemius (P < 0.01). In the left ventricle, the total capillary density and the density of venular capillaries were decreased after the treatment in both young (9 and 13%, respectively) and middle-aged rats (10 and 11%, respectively). A decrease in total capillary density was also observed in all skeletal muscles examined. In both young and middle-aged rats, the capillary-to-fibre (C:F) ratio and the proportion of each capillary did not change after the treatment in both the left ventricle and skeletal muscles. Clenbuterol significantly decreased the activity of succinate dehydrogenase in all skeletal muscles examined (P < 0.01). These results suggest that clenbuterol increased the diffusion distance for oxygen in the left ventricle and skeletal muscles. These changes may reduce the oxygen supply to tissues and increase muscle fatigability.     

Vascular rejection post heart transplantation is associated with positive flow cytometric cross-matching

28 isolates of canine parvovirus type-2 (CPV-2) were obtained from dogs with hemorrhagic gastroenteritis in Italy. The antigenic structure of CPV-2 isolates was characterized, using four discriminating monoclonal antibodies. In addition, four vaccinal strains were examined. Similar to reports from Australia and the United Kingdom, a much higher prevalence of CPV-2a (25/28 isolates) was observed than the other variant type, CPV-2b (3/28 isolates). DNA fragments (2.2 kbp) of representative strains of CPV-2, CPV-2a and CPV-2b were amplified by the polymerase chain reaction (PCR) and the products were digested by the restriction enzymes (RE) RsaI, HpaII, HindIII and PvuII. The RvaI enzyme allows the differentiation of CPV-2 from CPV-2a and CPV-2b.     

Genetic control of differential baseline breathing pattern

The purpose of the present study was to determine the genetic control of baseline breathing pattern by examining the mode of inheritance between two inbred murine strains with differential breathing characteristics. Specifically, the rapid, shallow phenotype of the C57BL/6J (B6) strain is consistently distinct from the slow, deep phenotype of the C3H/HeJ (C3) strain. The response distributions of segregant and nonsegregant progeny were compared with the two progenitor strains to determine the mode of inheritance for each ventilatory characteristic. The BXH recombinant inbred (RI) strains derived from the B6 and C3 progenitors were examined to establish strain distribution patterns for each ventilatory trait. To establish the mode of inheritance, baseline breathing frequency (f), tidal volume, and inspiratory time (TI) were measured five times in each of 178 mature male animals from the two progenitor strains and their progeny by using whole body plethysmography. With respect to f and TI, the two progenitor strains were consistently distinct, and segregation analyses of the inheritance pattern suggest that the most parsimonious genetic model for response distributions of f and TI is a two-loci model. In similar experiments conducted on 82 mature male animals from 12 BXH RI strains, each parental phenotype was represented by one or more of the RI strains. Intermediate phenotypes emerged to confirm the likelihood that parental strain differences in f and TI were determined by more than one locus. Taken together, these studies suggest that the phenotypic difference in baseline respiratory timing between male B6 and C3 mice is best explained by a genetic model that considers at least two loci as major determinants.     

Neurobiology and sexual orientation: current relationships

Despite great progress in the neurosciences, our understanding of the determinants of sexual orientation is incomplete. The authors review for the clinician/neuropsychiatrist studies pertaining to the formation of sexual orientation in the following areas: hormone effects on sexual behavior (animal and human); the complicated relationship between gender identity, gender role, and sexual orientation in humans; cross-cultural studies of homosexuality; behavioral observations in pseudohermaphrodites and offspring of mothers treated with hormones during pregnancy; brain studies of homosexual and heterosexual individuals; and genetic studies. The authors conclude that human sexual orientation is complex and diversely experienced and that a biopsychosocial model best fits the current state of knowledge in the field.     

Structure of tetrameric human phenylalanine hydroxylase and its implications for phenylketonuria

Phenylalanine hydroxylase (PheOH) catalyzes the conversion of L-phenylalanine to L-tyrosine, the rate-limiting step in the oxidative degradation of phenylalanine. Mutations in the human PheOH gene cause phenylketonuria, a common autosomal recessive metabolic disorder that in untreated patients often results in varying degrees of mental retardation. We have determined the crystal structure of human PheOH (residues 118-452). The enzyme crystallizes as a tetramer with each monomer consisting of a catalytic and a tetramerization domain. The tetramerization domain is characterized by the presence of a domain swapping arm that interacts with the other monomers forming an antiparallel coiled-coil. The structure is the first report of a tetrameric PheOH and displays an overall architecture similar to that of the functionally related tyrosine hydroxylase. In contrast to the tyrosine hydroxylase tetramer structure, a very pronounced asymmetry is observed in the phenylalanine hydroxylase, caused by the occurrence of two alternate conformations in the hinge region that leads to the coiled-coil helix. Examination of the mutations causing PKU shows that some of the most frequent mutations are located at the interface of the catalytic and tetramerization domains. Their effects on the structural and cellular stability of the enzyme are discussed.     

Performance and tissue zinc and metallothionein accumulation in chicks fed a high dietary level of zinc

Four experiments were conducted to identify several factors that might improve the accuracy and reproducibility of Zn bioavailability assays for chicks. Response of tissue Zn and metallothionein (MT) concentrations to various elevated levels and soluble sources of dietary Zn were measured, as well as the effect of delaying high Zn administration until 7 d posthatching to alleviate the detrimental effect of Zn sulfate on feed intake to 3 wk of age. Bone Zn increased (P < 0.01) in all experiments in response to increasing dietary Zn concentrations. Liver and pancreas MT were affected (P < 0.01) by a source by age interaction and variability that made this criterion unsuitable for bioavailability assays. Lastly, 1-d-old chicks were used to study the effect of delaying feeding of a high-Zn diet up to 7 d of age. The basal diet was fed continuously for 21 d as a control. A diet containing 1,000 ppm Zn was either fed continuously from Day 1, or started on Day 3, 5, or 7. Chicks given high Zn on Day 3, 5, or 7 decreased (P < 0.01) feed intake within 24 h of feeding. Delayed feeding of high dietary Zn might help to alleviate decreased feed intake observed in previous studies. Delaying the onset of high Zn feeding by several days may help alleviate feed intake problems observed with Zn sulfate. Use of either Zn gluconate or Zn acetate as a standard in assays or use of MT synthesis as a bioavailability criterion will probably not be useful to improve accuracy of the estimates.     

Superior vena cava syndrome after heart transplantation: percutaneous treatment of a complication of bicaval anastomoses

The aims of this study were to establish a working rabbit heart model of regional myocardial ischaemia in which electrophysiologic parameters and arrhythmogenesis could be correlated and to explore the mechanisms underlying the antiarrhythmic activity of lignocaine. Monophasic action-potential duration (MAPD90), effective refractory period (ERP), and conduction delay were measured at three ventricular sites in isolated hearts paced at 3.3 Hz. The hearts were treated before and throughout 30 min of ischaemia and 15 min of reperfusion with a vehicle or 20 microM lignocaine. In both groups, ischaemia produced a similar shortening in MAPD90. Lignocaine decreased ERP shortening during ischaemia from -56+/-4 to -32+/-6 ms. An ischaemia-induced increase in conduction delay was greater in the lignocaine than the control group (49+/-7 vs. 11+/-2 ms). Ischaemia-induced dispersion of repolarisation was reduced by lignocaine from 66+/-4 to 32+/-7 ms, and dispersion of refractoriness was decreased from 57+/-6 to 16+/-3 ms. Lignocaine decreased inducibility of ventricular fibrillation (VF) during ischaemia from 86 to 25%. We conclude that, in this model, the antiarrhythmic activity of lignocaine during regional ischaemia is associated with an increase in ischaemia-induced conduction delay and reduced dispersion of repolarisation and refractoriness.     

Glucosamine-induced insulin resistance in 3T3-L1 adipocytes is caused by depletion of intracellular ATP

Glucosamine, which enters the hexosamine pathway downstream of the rate-limiting step, has been routinely used to mimic the insulin resistance caused by high glucose and insulin. We investigated the effect of glucosamine on insulin-stimulated glucose transport in 3T3-L1 adipocytes. The Delta-insulin (insulin-stimulated minus basal) value for 2-deoxyglucose uptake was dramatically inhibited with increasing concentrations of glucosamine with an ED50 of 1.95 mM. Subcellular fractionation experiments demonstrated that reduction in insulin-stimulated 2-deoxyglucose uptake by glucosamine was due to an inhibition of translocation of both Glut 1 and Glut 4 from the low density microsomes (LDM) to the plasma membrane. Analysis of the insulin signaling cascade revealed that glucosamine impaired insulin receptor autophosphorylation, insulin receptor substrate (IRS-1) phosphorylation, IRS-1-associated PI 3-kinase activity in the LDM, and AKT-1 activation by insulin. Measurement of intracellular ATP demonstrated that the effects of glucosamine were highly correlated with its ability to reduce ATP levels. Reduction of intracellular ATP using azide inhibited Glut 1 and Glut 4 translocation from the LDM to the plasma membrane, insulin receptor autophosphorylation, and IRS-1 tyrosine phosphorylation. Additionally, both the reduction in intracellular ATP and the effects on insulin action caused by glucosamine could be prevented by the addition of inosine, which served as an alternative energy source in the medium. We conclude that direct administration of glucosamine can rapidly lower cellular ATP levels and affect insulin action in fat cells by mechanisms independent of increased intracellular UDP-N-acetylhexosamines and that increased metabolism of glucose via the hexosamine pathway may not represent the mechanism of glucose toxicity in fat cells.     

Sanger DNA-sequencing reactions performed in a solid-phase nanoreactor directly coupled to capillary gel electrophoresis

A miniaturized, solid-phase nanoreactor was developed to prepare Sanger DNA-sequencing ladders which was directly interfaced to a capillary gel electrophoresis system. A biotinylated fragment of the rat brain actin gene (1 kbp) was amplified by PCR and attached to the interior wall of an (aminoalkyl)silane-derivatized fused-silica capillary tube via a biotin/streptavidin/biotin linkage. Coverage of the capillary wall with the biotinylated DNA averaged 77 +/- 10%. Stability of the anchored template under pressure (33 nL/s) and electroosmotic flows (11.3 nL/s) were favorable, requiring rinsing for > 150 h to reduce the surface coverage by only 50%. In addition, the immobilized template was stable toward temperatures required for preparing sequencing ladders, even under cycling conditions. Standard Sanger dideoxynucleotide termination performed in a large-volume (approximately 8 microL) solid-phase reactor using the thermally stable polymerase enzymes Taq and Vent and the polymerases T7 and Bst with off-line slab gel electrophoresis and autoradiographic detection indicated that acceptable fragment generation was achieved only in the case of the thermally stable polymerases. Banding was not apparent for T7 and Bst since all reagents were inserted into the column in a single plug at the beginning of the reaction. A small volume reactor (volume approximately 62 nL) was then used to perform DNA polymerase reactions and was coupled directly to a capillary gel column for separation. The capillary reactor was placed inside a thermocycler to control the temperature during chain extension and was directly connected to the gel column via zero dead volume fused-silica connectors. The complementary DNA fragments generated (C-track only) in the reactor were denatured using heat and directly injected onto the gel-filled capillary for size separation with detection accomplished using near-IR laser-induced fluorescence. Extension and single-base separation resolution of the C-track, which was directly injected onto the gel column, was estimated to be > 450 bases from the primer annealing site with plate numbers ranging from 1 x 10(6) to 2 x 10(6)/m.