The bovine seminal plasma protein PDC-109 extracts phosphorylcholine-containing lipids from the outer membrane leaflet

The bovine seminal plasma protein PDC-109 modulates the maturation of bull sperm cells by removing lipids, mainly phosphatidylcholine and cholesterol, from their cellular membrane. Here, we have characterized the process of extraction of endogenous phospholipids and of their respective analogues. By measuring the PDC-109-mediated release of fluorescent phospholipid analogues from lipid vesicles and from biological membranes (human erythrocytes, bovine epididymal sperm cells), we showed that PDC-109 extracts phospholipids with a phosphorylcholine headgroup mainly from the outer leaflet of these membranes. The ability of PDC-109 to extract endogenous phospholipids from epididymal sperm cells was followed by mass spectrometry, which allowed us to characterize the fatty acid pattern of the released lipids. From these cells, PDC-109 extracted phosphatidylcholine and sphingomyelin that contained an enrichment of mono- and di-unsaturated fatty acids as well as short-chain and lyso-phosphatidylcholine species. Based on the results, a model explaining the phospholipid specificity of PDC-109-mediated lipid release is presented. Astrid Tannert and Anke Kurz have contributed equally to this work. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Homozygous mutation in SPATA16 is associated with male infertility in human globozoospermia

Globozoospermia is a rare (incidence <0.1% in male infertile patients) form of teratozoospermia, mainly characterized by round-headed spermatozoa that lack an acrosome. It originates from a disturbed spermiogenesis, which is expected to be induced by a genetic factor. Several family cases and recessive mouse models with the same phenotype support this expectation. In this study, we present a consanguineous family with three affected brothers, in whom we have identified a homozygous mutation in the spermatogenesis-specific gene SPATA16. This is the first example of a nonsyndromic male infertility condition in humans caused by an autosomal gene defect, and it could also mean that the identification of other partners like SPATA16 could elucidate acrosome formation.     

Biochemical characterization of isocitrate dehydrogenase from Methylococcus capsulatus reveals a unique NAD+-dependent homotetrameric enzyme

The gene encoding isocitrate dehydrogenase (IDH) of Methylococcus capsulatus (McIDH) was cloned and overexpressed in Escherichia coli. The purified enzyme was NAD⁺-dependent with a thermal optimum for activity at 55–60°C and an apparent midpoint melting temperature (T _m) of 70°C. Analytical ultracentrifugation (AUC) revealed a homotetrameric state, and McIDH thus represents the first homotetrameric NAD⁺-dependent IDH that has been characterized. Based on a structural alignment of McIDH and homotetrameric homoisocitrate dehydrogenase (HDH) from Thermus thermophilus (TtHDH), we identified the clasp-like domain of McIDH as a likely site for tetramerization. McIDH showed moreover, higher sequence identity (48%) to TtHDH than to previously characterized IDHs. Putative NAD⁺-IDHs with high sequence identity (48–57%) to McIDH were however identified in a variety of bacteria showing that NAD⁺-dependent IDHs are indeed widespread within the domain, Bacteria. Phylogenetic analysis including these new sequences revealed a close relationship with eukaryal allosterically regulated NAD⁺-IDH and the subfamily III of IDH was redefined to include bacterial NAD⁺- and NADP⁺-dependent IDHs. This apparent relationship suggests that the mitochondrial genes encoding NAD⁺-IDH are derived from the McIDH-like IDHs.     

Rac is a dominant regulator of cadherin-directed actin assembly that is activated by adhesive ligation independently of Tiam1

Classic cadherins function as adhesion-activated cell signaling receptors. On adhesive ligation, cadherins induce signaling cascades leading to actin cytoskeletal reorganization that is imperative for cadherin function. In particular, cadherin ligation activates actin assembly by the actin-related protein (Arp)2/3 complex, a process that critically affects the ability of cells to form and extend cadherin-based contacts. However, the signaling pathway(s) that activate Arp2/3 downstream of cadherin adhesion remain poorly understood. In this report we focused on the Rho family GTPases Rac and Cdc42, which can signal to Arp2/3. We found that homophilic engagement of E-cadherin simultaneously activates both Rac1 and Cdc42. However, by comparing the impact of dominant-negative Rac1 and Cdc42 mutants, we show that Rac1 is the dominant regulator of cadherin-directed actin assembly and homophilic contact formation. To pursue upstream elements of the Rac1 signaling pathway, we focused on the potential contribution of Tiam1 to cadherin-activated Rac signaling. We found that Tiam1 or the closely-related Tiam2/STEF1 was recruited to cell-cell contacts in an E-cadherin-dependent fashion. Moreover, a dominant-negative Tiam1 mutant perturbed cell spreading on cadherin-coated substrata. However, disruption of Tiam1 activity with dominant-negative mutants or RNA interference did not affect the ability of E-cadherin ligation to activate Rac1. We conclude that Rac1 critically influences cadherin-directed actin assembly as part of a signaling pathway independent of Tiam1. actin cytoskeleton; Cdc42; E-cadherin     

Co-product allocation in life cycle assessments of seafood production systems: Review of problems and strategies

Background, Aim and Scope  

As Life Cycle Assessment is being increasingly applied to study fisheries and aquaculture systems, the LCA methodology must be adapted to address the unique aspects of these systems. The focus of this methodological paper is the specific allocation problems faced in studying seafood production systems and how they have been addressed to date.     

Evaluation of demineralised,freeze-dried,irradiated bone allografts in the treatment of osseous defects in the oral cavity

Demineralised, freeze-dried bone allografts (DFDBA) have been used extensively by dentists in the treatment of periodontal and periapical osseous defects resulting from inflammatory diseases. Their use in India however, is limited by the availability of quality allografts and the high cost of imported alternatives. A study was conducted to assess the osteogenic potential of DFDBA prepared for the first time in India by the Tata Memorial Hospital (TMH) Tissue Bank. The DFDBA was used in the treatment of osseous defects after removal of periapical lesions associated with devitalised teeth in 10 healthy patients. At the 6-month recall visit all the patients showed a remarkable decrease in the grades of mobility, and 9 out of the 10 patients showed radiographic evidence of complete healing of the osseous defects with evidence of normal bony trabaeculae. These findings indicate that the indigenously prepared DFDBA is a cost effective, biocompatible material with osteogenic potential that can be used effectively in treating osseous defects of periapical lesions associated with non vital teeth.     

Recombinant production of <Emphasis Type="Italic">Streptococcus equisimilis</Emphasis> streptokinase by <Emphasis Type="Italic">Streptomyces lividans</Emphasis>

Background  

Streptokinase (SK) is a potent plasminogen activator with widespread clinical use as a thrombolytic agent. It is naturally secreted by several strains of beta-haemolytic streptococci. The low yields obtained in SK production, lack of developed gene transfer methodology and the pathogenesis of its natural host have been the principal reasons to search for a recombinant source for this important therapeutic protein. We report here the expression and secretion of SK by the Gram-positive bacterium Streptomyces lividans. The structural gene encoding SK was fused to the Streptomyces venezuelae CBS762.70 subtilisin inhibitor (vsi) signal sequence or to the Streptomyces lividans xylanase C (xlnC) signal sequence. The native Vsi protein is translocated via the Sec pathway while the native XlnC protein uses the twin-arginine translocation (Tat) pathway.