Mammalian Rad9 plays a role in telomere stability, S- and G2-phase-specific cell survival, and homologous recombinational repair

The protein products of several rad checkpoint genes of Schizosaccharomyces pombe (rad1+, rad3+, rad9+, rad17+, rad26+, and hus1+) play crucial roles in sensing changes in DNA structure, and several function in the maintenance of telomeres. When the mammalian homologue of S. pombe Rad9 was inactivated, increases in chromosome end-to-end associations and frequency of telomere loss were observed. This telomere instability correlated with enhanced S- and G2-phase-specific cell killing, delayed kinetics of gamma-H2AX focus appearance and disappearance, and reduced chromosomal repair after ionizing radiation (IR) exposure, suggesting that Rad9 plays a role in cell cycle phase-specific DNA damage repair. Furthermore, mammalian Rad9 interacted with Rad51, and inactivation of mammalian Rad9 also resulted in decreased homologous recombinational (HR) repair, which occurs predominantly in the S and G2 phases of the cell cycle. Together, these findings provide evidence of roles for mammalian Rad9 in telomere stability and HR repair as a mechanism for promoting cell survival after IR exposure.     

Neurochemical evidence for two types of presynaptic alpha2-adrenoceptors

Neurochemical and pharmacological evidence has been obtained that noradrenergic varicosities (in mouse and rat vas deferens) and cholinergic varicosities (in the Auerbach's plexus) contain heterogenous alpha₂-adrenoceptors through which the release of [³H]noradrenaline and [³H]acetylcholine can be modulated. The quantitative data also support the hypothesis that different noradrenaline and xylazine sensitive alpha₂-adrenoceptors are present prejunctionally in the vas deferens and Auerbach's plexus preparations. Prazosin, although it has a presynaptic inhibitory effect on alpha₂-adrenoceptors of noradrenergic axon terminals, has no effect on cholinergic axon terminals. These data suggest that there are two different types of alpha₂-adrenoceptors at the presynaptic axon terminals.Special Issue Dedicated to Dr. Abel Lajtha     

Diverse post-translational modifications of the pannexin family of channel-forming proteins

The pannexin family of channel-forming proteins is composed of 3 distinct but related members called Panx1, Panx2, and Panx3. Pannexins have been implicated in many physiological processes as well as pathological conditions, primarily through their function as ATP release channels. However, it is currently unclear if all pannexins are subject to similar or different post-translational modifications as most studies have focused primarily on Panx1. Using in vitro biochemical assays performed on ectopically expressed pannexins in HEK-293T cells, we confirmed that all 3 pannexins are N-glycosylated to different degrees, but they are not modified by sialylation or O-linked glycosylation in a manner that changes their apparent molecular weight. Using cell-free caspase assays, we also discovered that similar to Panx1, the C-terminus of Panx2 is a substrate for caspase cleavage. Panx3, on the other hand, is not subject to caspase digestion but an in vitro biotin switch assay revealed that it was S-nitrosylated by nitric oxide donors. Taken together, our findings uncover novel and diverse pannexin post-translational modifications suggesting that they may be differentially regulated for distinct or overlapping cellular and physiological functions.     

Ubiquitin-protein conjugates accumulate in the lysosomal system of fibroblasts treated with cysteine proteinase inhibitors.

Mouse fibroblasts (3T3-L1 cells) accumulate detergent- and salt-insoluble aggregates of proteins conjugated to ubiquitin when incubated in the presence of inhibitors of lysosomal cysteine cathepsins, including E-64. These ubiquitin-protein conjugates co-fractionate with lysosomes on density gradients and are found in multivesicular dense bodies which by electron microscopy appear to be engaged in microautophagy. Both E-64 and ammonium chloride increase the intracellular concentration of free ubiquitin, but only E-64 leads to the formation of insoluble lysosomal ubiquitin-protein conjugates. The results are discussed in relation to the possible intracellular roles of ubiquitin conjugation.     

Transformation of Nε-CBZ-l-lysine to CBZ-l-oxylysine using l-amino acid oxidase from Providencia alcalifaciens and l-2-hydroxy-isocaproate dehydrogenase from Lactobacillus confusus

Summary Biotransformations were developed to oxidize N-carbobenzoxy(CBZ)-l-lysine and to reduce the product keto acid to l-CBZ-oxylysine. Lysyl oxidase (l-lysine: O₂ oxidoreductase, EC 1.4.3.14) from Trichoderma viride was relatively specific for l-lysine and had very low activity with N-substituted derivatives. l-Amino acid oxidase (l-amino acid: O₂ oxidoreductase [deaminating], EC 1.4.3.2) from Crotalus adamanteus venom had low activity with l-lysine but high activity with N-formyl-, t-butyoxycarbonyl(BOC)-, acetyl-, trifluoroacetyl-, or CBZ-l-lysine. l-2-Hydroxyisocaproate dehydrogenase (EC 1.1.1.-) from Lactobacillus confusus catalyzed the reduction by NADH of the keto acids from N-acetyl-, trifluoroacetyl-, formyl- and CBZ-l-lysine but was inactive with the products from oxidation of l-lysine, l-lysine methyl ester, l-lysine ethyl ester or N-t-BOC-l-lysine. Providencia alcalifaciens (SC9036, ATCC 13159) was a good microbial substitute for the snake venom oxidase and also provided catalase (H₂O₂:H₂O₂ oxidoreductase EC 1.11.1.6). N-CBZ-l-Lysine was converted to CBZ-l-oxylysine in 95% yield with 98.5% optical purity by oxidation using P. alcalifaciens cells followed by reduction of the keto acid using l-2-hydroxyisocaproate dehydrogenase. NADH was regenerated using formate dehydrogenase (formate: NAD oxidoreductase, EC 1.2.1.2) from Candida boidinii. The Providencia oxidase was localized in the particulate fraction and catalase activity was predominantly in the soluble fraction of sonicated cells. The pH optima and kinetic constants were determined for the reactions. Correspondence to: R. L. Hanson     

Image averaging of flexible fibrous macromolecules: the clathrin triskelion has an elastic proximal segment.

We have developed computational techniques that allow image averaging to be applied to electron micrographs of filamentous molecules that exhibit tight and variable curvature. These techniques, which involve straightening by cubic-spline interpolation, image classification, and statistical analysis of the molecules' curvature properties, have been applied to purified brain clathrin. This trimeric filamentous protein polymerizes, both in vivo and in vitro, into a wide range of polyhedral structures. Contrasted by low-angle rotary shadowing, dissociated clathrin molecules appear as distinctive three-legged structures, called "triskelions" (E. Ungewickell and D. Branton (1981) Nature 289, 420). We find triskelion legs to vary from 35 to 62 nm in total length, according to an approximately bell-shaped distribution (mu = 51.6 nm). Peaks in averaged curvature profiles mark hinges or sites of enhanced flexibility. Such profiles, calculated for each length class, show that triskelion legs are flexible over their entire lengths. However, three curvature peaks are observed in every case: their locations define a proximal segment of systematically increasing length (14.0-19.0 nm), a mid-segment of fixed length (approximately 12 nm), and a rather variable end-segment (11.6-19.5 nm), terminating in a hinge just before the globular terminal domain (approximately 7.3 nm diameter). Thus, two major factors contribute to the overall variability in leg length: (1) stretching of the proximal segment and (2) stretching of the end-segment and/or scrolling of the terminal domain. The observed elasticity of the proximal segment may reflect phosphorylation of the clathrin light chains.     

Effects of aortic irregularities on blood flow

Anatomic aortic anomalies are seen in many medical conditions and are known to cause disturbances in blood flow. Turner syndrome (TS) is a genetic disorder occurring only in females where cardiovascular anomalies, particularly of the aorta, are frequently encountered. In this study, numerical simulations are applied to investigate the flow characteristics in four TS patient- related aortic arches (a normal geometry, dilatation, coarctation and elongation of the transverse aorta). The Quemada viscosity model was applied to account for the non-Newtonian behavior of blood. The blood is treated as a mixture consisting of water and red blood cells (RBC) where the RBCs are modeled as a convected scalar. The results show clear geometry effects where the flow structures and RBC distribution are significantly different between the aortas. Transitional flow is observed as a jet is formed due to a constriction in the descending aorta for the coarctation case. RBC dilution is found to vary between the aortas, influencing the WSS. Moreover, the local variations in RBC volume fraction may induce large viscosity variations, stressing the importance of accounting for the non-Newtonian effects.