The mechanism of excitation-contraction coupling in phenylephrine-stimulated human saphenous vein

The human saphenous vein (HSV) is the most widely used graft in coronary artery revascularization procedures and is susceptible to spasm perioperatively. The aim of this study is to elucidate the mechanism(s) of agonist-induced excitation-contraction coupling in this vessel. Isometric contraction experiments were combined with in situ smooth muscle intracellular Ca(2+) concentration ([Ca(2+)](i)) imaging by confocal microscopy of intact undistended HSV segments during activation with phenylephrine (PE; 50 microM). Stimulation with PE produced a sustained contraction. Preincubation with 5 microM nifedipine, a blocker of the L-type voltage-operated Ca(2+) channel, or 50 microM SKF-96365, a blocker of both the voltage- and receptor-operated channels, reduced force generation by 25-30%. Ca(2+) imaging revealed that PE elicited only a transient rise in [Ca(2+)](i), suggesting that Ca(2+) plays only a minor role. However, a requirement for basal Ca(2+) levels was demonstrated when PE contractions could not be maintained in Ca(2+)-free medium. In light of the transient Ca(2+) response, it appears that signals other than Ca(2+) must maintain the tonic contraction elicited by PE, such as those that sensitize the myofilaments to Ca(2+). Application of HA-1077 (a Rho kinase inhibitor) at the peak of the contraction completely abolished the plateau phase of the response, whereas application of genistein (a tyrosine kinase inhibitor) reduced this phase by approximately 50%. The foregoing results suggest that, whereas the transient Ca(2+) signal can contribute to the development of force, maintenance of the plateau phase of the PE contraction in the HSV is the result of myofilament Ca(2+) sensitization by Rho kinase and tyrosine phosphorylation. The elucidation of the mechanisms of excitation-contraction coupling in the HSV may be useful for the development of therapeutic strategies for the alleviation of vein graft spasm.     

Cloning and characterization of the Burkholderia vietnamiensis norM gene encoding a multi-drug efflux protein

Polymyxin B-sensitive mutants in Burkholderia vietnamiensis (Burkholderia cepacia genomovar V) were generated with a mini-Tn5 encoding tetracycline resistance. One of the transposon mutants had an insertion in the norM gene encoding a multi-drug efflux protein. Expression of B. vietnamiensis norM in an Escherichia coli acrAB deletion mutant complemented its norfloxacin hypersensitivity, indicating that the protein functions in drug efflux. However, no effect on antibiotic sensitivity other than sensitivity to polymyxin B was observed in the B. vietnamiensis norM mutant. We demonstrate that increased polymyxin sensitivity in B. vietnamiensis was associated with the presence of tetracycline in the growth medium, a phenotype that was partially suppressed by expression of the norM gene.     

Treating mites in a bat colony: a case study

Since many bats used in research are wild-caught, dealing with mite infestation can present a unique challenge to the animal care staff. The authors tested the efficacy of fipronil spray for eliminating mites from a bat colony.     

Chondroitin sulfate disrupts axon pathfinding in the optic tract and alters growth cone dynamics

Little is known about the cues that guide retinal axons across the diencephalon en route to their midbrain target, the optic tectum. Here we show that chondroitin sulfate proteoglycans are differentially expressed within the diencephalon at a time when retinal axons are growing within the optic tract. Using exposed brain preparations, we show that the addition of exogenous chondroitin sulfate results in retinal pathfinding errors. Retinal axons disperse widely from their normal trajectory within the optic tract and extend aberrantly into inappropriate regions of the forebrain. Time-lapse analysis of retinal growth cone dynamics in vivo shows that addition of exogenous chondroitin sulfate causes intermittent stalling and increases growth cone complexity. These results suggest that chondroitin sulfate may modulate the guidance of retinal axons as they grow through the diencephalon towards the optic tectum.     

Myosin VIIa,harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle

Deaf-blindness in three distinct genetic forms of Usher type I syndrome (USH1) is caused by defects in myosin VIIa, harmonin and cadherin 23. Despite being critical for hearing, the functions of these proteins in the inner ear remain elusive. Here we show that harmonin, a PDZ domain-containing protein, and cadherin 23 are both present in the growing stereocilia and that they bind to each other. Moreover, we demonstrate that harmonin b is an F-actin-bundling protein, which is thus likely to anchor cadherin 23 to the stereocilia microfilaments, thereby identifying a novel anchorage mode of the cadherins to the actin cytoskeleton. Moreover, harmonin b interacts directly with myosin VIIa, and is absent from the disorganized hair bundles of myosin VIIa mutant mice, suggesting that myosin VIIa conveys harmonin b along the actin core of the developing stereocilia. We propose that the shaping of the hair bundle relies on a functional unit composed of myosin VIIa, harmonin b and cadherin 23 that is essential to ensure the cohesion of the stereocilia.     

Chronic neuropeptide Y infusion during lactation suppresses pup growth and reduces the length of lactational infertility in rats

In lactating rats, food restriction potentiates the already high levels of hypothalamic neuropeptide Y (NPY). To investigate the role that high levels of NPY might play in the prolongation of lactational infertility that typically accompanies a food restricted lactation we investigated the effects of chronic central infusions of NPY in ad libitum-fed lactating females. First, we compared the effects of intracerebroventricular (icv) infusion of NPY from Days 12-19 postpartum at a dose of 14.4 microg/day with a similar treatment in nonlactating females. In subsequent experiments we examined the effects of NPY infusions into the lateral ventricle at doses of 6 or 20 mug/day or unilaterally into the medial preoptic area at a dose of 1 microg/day from either Days 12-19 or 7-21 postpartum. Effects on food intake; female body weight; and, where appropriate, litter weight and length of lactational diestrus were compared between NPY and vehicle-treated females. As expected NPY infusion produced a robust increase in body weight and food intake in nonlactating females that was accompanied by a suppression of cyclicity. By contrast NPY treatment in lactating rats resulted in a marked decrease in litter growth and an earlier termination of lactational diestrus.     

The X-ray crystal structure of human gamma S-crystallin C-terminal domain.

gammaS-crystallin is a major human lens protein found in the outer region of the eye lens, where the refractive index is low. Because crystallins are not renewed they acquire post-translational modifications that may perturb stability and solubility. In common with other members of the betagamma-crystallin superfamily, gammaS-crystallin comprises two similar beta-sheet domains. The crystal structure of the C-terminal domain of human gammaS-crystallin has been solved at 2.4 A resolution. The structure shows that in the in vitro expressed protein, the buried cysteines remain reduced. The backbone conformation of the "tyrosine corner" differs from that of other betagamma-crystallins because of deviation from the consensus sequence. The two C-terminal domains in the asymmetric unit are organized about a slightly distorted 2-fold axis to form a dimer with similar geometry to full-length two-domain family members. Two glutamines found in lattice contacts may be important for short range interactions in the lens. An asparagine known to be deamidated in human cataract is located in a highly ordered structural region.     

NK cell activity during human cytomegalovirus infection is dominated by US2-11-mediated HLA class I down-regulation

A highly attractive approach to investigate the influence and hierarchical organization of viral proteins on cellular immune responses is to employ mutant viruses carrying deletions of various virus-encoded, immune-modulating genes. Here, we introduce a novel set of deletion mutants of the human CMV (HCMV) lacking the UL40 region either alone or on the background of a deletion mutant devoid of the entire US2-11 region. Deletion of UL40 had no significant effect on lysis of infected cells by NK cells, indicating that the expected enhancement of HLA-E expression by specific peptides derived from HCMV-encoded gpUL40 leader sequences was insufficient to confer target cell protection. Moreover, the kinetics of MHC class I down-regulation by US2-11 genes observed at early and late phases postinfection with wild-type virus correlated with increased susceptibility to NK lysis. Thus, the influence of HCMV genes on NK reactivity follows a hierarchy dominated by the US2-11 region, which encodes all viral genes capable of down-modulating expression of classical and non-classical MHC class I molecules. The insights gained from studies of such virus mutants may impact on future therapeutic strategies and vaccine development and incorporate NK cells in the line of defense mechanisms against HCMV infection.     

A novel class II-binding motif selects peptides that mediate organ-specific autoimmune disease in SWXJ,SJL/J,and SWR/J mice

Idiopathic dilated cardiomyopathy (DCM) is responsible for approximately 25% of all cases of congestive heart failure. We have recently shown that immunization of autoimmune-susceptible SWXJ mice with whole cardiac myosin leads to T cell-mediated experimental autoimmune myocarditis (EAMC) and DCM. We have now identified two disease-inducing peptides from cardiac alpha-myosin heavy chain (CAMHC). Our approach involved the use of a novel MHC class II-binding motif contained in several peptides known to be immunogenic in SWXJ (H-2(q,s)) mice or in the parental SJL/J (H-2(s)) or SWR/J (H-2(q)) mouse strains. Two of four CAMHC peptides containing the -KXXS- peptide motif were found to be immunogenic. Immunization of SWXJ or parental SJL/J and SWR/J mice with CAMHC peptides palpha406-425 or palpha1631-1650 resulted in EAMC and DCM, characterized by inflammation, fibrosis, and decompensated right-sided ventricular dilatation. Despite mediating high incidences of severe disease, both peptides were found to be cryptic determinants, thereby providing further evidence for the importance and perhaps predominance of self crypticity in autoimmunity. Both peptides showed dual parental I-A(q) and I-A(s) restriction and mediated passive transfer of disease with activated CD4(+) T cells. An intact motif was necessary for antigenicity because loss of activity occurred in peptides containing nonconservative substitutions at the motif's terminal lysine and serine residues. Our studies provide a new model for EAMC and DCM in strains of mice widely used in autoimmune studies. Moreover, the -KXXS- motif may be particularly useful in implicating previously overlooked proteins as autoimmune targets and in facilitating the development of new organ-specific autoimmune mouse models for human diseases.