Ablation of PLB exacerbates ischemic injury to a lesser extent in female than male mice: protective role of NO

Recent studies suggest a role for phospholamban phosphorylation during ischemia and reperfusion. The role of phospholamban in ischemia was studied by subjecting hearts from male and female wild-type (MWT/FWT) and phospholamban-knockout (MKO/FKO) mice to 20 min of ischemia-40 min of reperfusion while (31)P NMR spectra were acquired. ATP and pH values fell lower during ischemia, and postischemic contractility was less, in MKO and FKO versus WT hearts. After shorter ischemia (15 min), recoveries of contraction, ATP, and pH were greater in FKO than MKO hearts. To examine the role of nitric oxide (NO) synthases (NOS) in the protection in FKO versus MKO hearts, we utilized 1 microM l-NAME, a NOS inhibitor, or 100 microM S-nitroso-N-acetylpenicillamine (SNAP), an NO donor. Recoveries of function, ATP, and pH were less in l-NAME-treated FKO than untreated FKO hearts and greater in SNAP-treated MKO than untreated MKO hearts. In conclusion, phospholamban ablation increased ischemic injury in both males and females; however, female hearts were less susceptible than male hearts. Protection in females was decreased by a NOS inhibitor and mimicked in males by an NO donor, implying that protection was NOS mediated.     

Collection and culture techniques for gelatinous zooplankton

Gelatinous zooplankton are the least understood of all planktonic animal groups. This is partly due to their fragility, which typically precludes the capture of intact specimens with nets or trawls. Specialized tools and techniques have been developed that allow researchers and aquarists to collect intact gelatinous animals at sea and to maintain many of these alive in the laboratory. This paper summarizes the scientific literature on the capture, collection, and culture of gelatinous zooplankton and incorporates many unpublished methods developed at the Monterey Bay Aquarium in the past 15 years.     

Ca(2+) loading and adrenergic stimulation reveal male/female differences in susceptibility to ischemia-reperfusion injury

To compare ischemia-reperfusion injury in males versus females under hypercontractile conditions, perfused hearts from 129J mice pretreated with 3 mmol/l Ca(2+) or 10(-8) mol/l isoproterenol +/- 10(-6) mol/l N(omega)-nitro-L-arginine methyl ester (L-NAME) were subjected to 20 min of ischemia and 40 min of reperfusion while (31)P NMR spectra were acquired. Basal contractility increased equivalently in female versus male hearts with isoproterenol- or Ca(2+) treatment. Injury was equivalent in untreated male versus female hearts but was greater in isoproterenol or Ca(2+)-treated male than female hearts, as indicated by lower postischemic contractile function, ATP, and PCr. Endothelial nitric oxide (NO) synthase (eNOS) expression was higher in female than male hearts, neuronal NOS (nNOS) did not differ, and inducible NOS (iNOS) was undetectable. Ischemic NO production was higher in female than male hearts, and L-NAME increased injury in female isoproterenol-treated hearts. In summary, isoproterenol or high Ca(2+) pretreatment increased ischemia-reperfusion injury in males more than females. eNOS expression and NO production were higher in female than male hearts, and L-NAME blocked female protection. Females were therefore protected from the detrimental effects of adrenergic stimulation and Ca(2+) loading via a NOS-mediated mechanism.     

Uniformity,ideality, and hydrogen bonds in transmembrane alpha-helices

Protein environments substantially influence the balance of molecular interactions that generate structural stability. Transmembrane helices exist in the relatively uniform low dielectric interstices of the lipid bilayer, largely devoid of water and with a very hydrophobic distribution of amino acid residues. Here, through an analysis of bacteriorhodopsin crystal structures and the transmembrane helix structure from M2 protein of influenza A, some helices are shown to be exceptionally uniform in hydrogen bond geometry, peptide plane tilt angle, and backbone torsion angles. Evidence from both the x-ray crystal structures and solid-state NMR structure suggests that the intramolecular backbone hydrogen bonds are shorter than their counterparts in water-soluble proteins. Moreover, the geometry is consistent with a dominance of electrostatic versus covalent contributions to these bonds. A comparison of structure as a function of resolution shows that as the structures become better characterized the helices become much more uniform, suggesting that there is a possibility that many more uniform helices will be observed, even among the moderate resolution membrane protein structures that are currently in the Protein Data Bank that do not show such features.     

Protein profiled features patterned via confocal microscopy

Protein patterns were printed using conventional microlithographic materials in a bilayer arrangement and unconventional exposure tools. The bilayer resist stack consisted of a lower poly(tert-butyl methacrylate) layer and an upper diazonaphtoquinone/novolak layer. The protein features were printed in either 'contact printing', or 'step and repeat' mode. The latter printing mode can be managed in a flow-cell consisting of a standard microscope slide and cover slip, spaced apart by about 20 microm, as follows: (i) the exposure step is carried out in the cell using focused 488 nm beam of a confocal laser scanning microscope; (ii) the development step is performed by flowing the photoresist developer through the cell; (iii) the selective deposition of the protein (FITC-labelled avidin) is achieved via the flow of the protein solution through the cell until a desired contrast has been reached; (iv) the control of the process is assured using on-line monitoring of the photo-activated red fluorescence of the developing resist layer, and of the green fluorescence of the FITC-protein patterns, respectively. The protein printing technique uses equipment routinely available in biological laboratory. The 'step and repeat' patterning yields high and controllable resolution. The process can be applied in the fabrication of medical microanalysis devices.     

Genetic analysis of the relationship between activation loop phosphorylation and cyclin binding in the activation of the Saccharomyces cerevisiae Cdc28p cyclin-dependent kinase

We showed recently that a screen for mutant CDC28 with improved binding to a defective Cln2p G1 cyclin yielded a spectrum of mutations similar to those yielded by a screen for intragenic suppressors of the requirement for activation loop phosphorylation (T169E suppressors). Recombination among these mutations yielded CDC28 mutants that bypassed the G1 cyclin requirement. Here we analyze further the interrelationship between T169E suppression, interaction with defective cyclin, and G1 cyclin bypass. DNA shuffling of mutations from the various screens and recombination onto a T169E-encoding 3' end yielded CDC28 mutants with strong T169E suppression. Some of the strongest T169E suppressors could suppress the defective Cln2p G1 cyclin even while retaining T169E. The strong T169E suppressors did not exhibit bypass of the G1 cyclin requirement but did so when T169E was reverted to T. These results suggested that for these mutants, activation loop phosphorylation and cyclin binding might be alternative means of activation rather than independent requirements for activation (as with wild type). These results suggest mechanistic overlap between the conformational shift induced by cyclin binding and that induced by activation loop phosphorylation. This conclusion was supported by analysis of suppressors of a mutation in the Cdk phosphothreonine-binding pocket created by cyclin binding.