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The isolation and biochemical characterization of the human sperm tail fibrous sheath (FS) is described for the first time. Initially, the solubilization properties of the FS were assessed immunocytochemically using GDA-J/F3 and RT97 monoclonal antibodies (MoAbs) and morphologically by electron microscopy. Following extensive investigations to optimize the conditions for the FS isolation, a simple method was developed which involved sequential extraction of the flageller components with Triton-dithiothreitol (DTT) and urea-DTT. The procedure was monitored by phase contrast microscopy and the purity of the FS preparations was confirmed by electron microscopy. SDS-PAGE of the isolated FS revealed seven major protein bands with mol. wt of 97, 76, 62, 55, 33, 28 and 25 kDa. In Western blotting, the reaction of RT97 MoAb with supernatants from the various extraction steps and the isolated FS indicated that its target antigen (AJ-p97) was an integral FS product and that disulphide bonding was probably involved in its stabilization. The reactivity of normal and aprotruded sperm tails with GDA-J/F3 and RT97 MoAbs was not affected by Triton while the GDA-J/F3 staining of the cytoplasmic matrix of other abnormal spermatids was abolished, thus suggesting variation in the biochemical properties of GDA-J/F3 in normal and abnormal germ cells. These and other data indicate that the FS could be a modified form of intermediate filament. [ABSTRACT FROM AUTHOR]

Aims: Stable genetic background makes individuals from the Mediterranean island of Sardinia ideal to define the predictive power of islet-related autoantibodies (IRAs): glutamic acid decarboxylase antibodies (GADA), tyrosine phosphatase-like antibodies (IA-2A), islet cell antibodies (ICA) to identify T1DM progressors. The aims of the present study were: (1) determination of IRAs reference limits in healthy non-diabetic Sardinian schoolchildren (SSc). (2) Predictive power evaluation of IRAs as single or combined determination to identify islet to identify T1DM progressors., Methods: Between 1986 and 1994, 8448 SSc were tested for IRAs. All were followed up for 10 years. The predictive power of single or combination of IRAs was determined as hazard ratio (HR), sensitivity, specificity, area under the ROC curve, negative and positive predictive value (NPV, PPV)., Results: All 43 progressors to T1DM, but three showed at least one autoantibody positivity. HR for any single-autoantibody positivity was 55.3 times greater when compared to SSc negative for all IRAs. Any single autoantibody performed at least 64.9 % sensitivity with PPV always lower than 16 %. The best performing combination was ICA, plus IA-2A (showing 52.6 % sensitivity, 99.8 % specificity, 0.76 area under the ROC curve, 51.3 % PPV and 99.8 % NPV., Conclusions: Determination of IRAs reference limits in healthy SSc by standard statistical methods is crucial to establish the power of IRAs as progression markers to T1DM. Our data offer a solid rationale for future testing of ICA and IA-2A as routine laboratory markers to identify individuals at high risk of T1DM in the general population.

CoA synthase mediates the last two steps in the sequence of enzymatic reactions, leading to CoA biosynthesis. We have recently identified cDNA for CoA synthase and demonstrated that it encodes a bifunctional enzyme possessing 4'-phosphopantetheine adenylyltransferase and dephospho-CoA kinase activities. Molecular cloning of CoA synthase provided us with necessary tools to study subcellular localization and the regulation of this bifunctional enzyme. Transient expression studies and confocal microscopy allowed us to demonstrate that full-length CoA synthase is associated with the mitochondria, whereas the removal of the N-terminal region relocates the enzyme to the cytosol. In addition, we showed that the N-terminal sequence of CoA synthase (amino acids 1-29) exhibits a hydrophobic profile and targets green fluorescent protein exclusively to mitochondria. Further analysis, involving subcellular fractionation and limited proteolysis, indicated that CoA synthase is localized on the mitochondrial outer membrane. Moreover, we demonstrate for the first time that phosphatidylcholine and phosphatidylethanolamine, which are the main components of the mitochondrial outer membrane, are potent activators of both enzymatic activities of CoA synthase in vitro. Taken together, these data provide the evidence that the final stages of CoA biosynthesis take place on mitochondria and the activity of CoA synthase is regulated by phospholipids.

Improved molecular understanding of the pathogenesis of type 2 diabetes is essential if current therapeutic and preventative options are to be extended. To identify diabetes-susceptibility genes, we have completed a primary (418-marker, 9-cM) autosomal-genome scan of 743 sib pairs (573 pedigrees) with type 2 diabetes who are from the Diabetes UK Warren 2 repository. Nonparametric linkage analysis of the entire data set identified seven regions showing evidence for linkage, with allele-sharing LOD scores > or =1.18 (P< or =.01). The strongest evidence was seen on chromosomes 8p21-22 (near D8S258 [LOD score 2.55]) and 10q23.3 (near D10S1765 [LOD score 1.99]), both coinciding with regions identified in previous scans in European subjects. This was also true of two lesser regions identified, on chromosomes 5q13 (D5S647 [LOD score 1.22] and 5q32 (D5S436 [LOD score 1.22]). Loci on 7p15.3 (LOD score 1.31) and 8q24.2 (LOD score 1.41) are novel. The final region showing evidence for linkage, on chromosome 1q24-25 (near D1S218 [LOD score 1.50]), colocalizes with evidence for linkage to diabetes found in Utah, French, and Pima families and in the GK rat. After dense-map genotyping (mean marker spacing 4.4 cM), evidence for linkage to this region increased to a LOD score of 1.98. Conditional analyses revealed nominally significant interactions between this locus and the regions on chromosomes 10q23.3 (P=.01) and 5q32 (P=.02). These data, derived from one of the largest genome scans undertaken in this condition, confirm that individual susceptibility-gene effects for type 2 diabetes are likely to be modest in size. Taken with genome scans in other populations, they provide both replication of previous evidence indicating the presence of a diabetes-susceptibility locus on chromosome 1q24-25 and support for the existence of additional loci on chromosomes 5, 8, and 10. These data should accelerate positional cloning efforts in these regions of interest.

A perinuclear theca protein of the human sperm subacrosome was detected using LH43 monoclonal antibody which was originally raised against human keratinocytes. Using indirect immunofluorescence, the antibody stained the acrosomal zone (AZ) of dried ejaculated spermatozoa but did not react with viable cells, thus suggesting that the antigen was intracellular. This was confirmed by immunogold electron microscopy which also revealed the ultrastructural localisation of the antigen to the subacrosomal fibrils. Throughout spermatogenesis the antigen was only detected on the AZ of developed testicular spermatozoa and its expression was continued during their epididymal passage. Biochemically, the protein was insoluble in Triton, and dithiothreitol (DTT) was required for its solubilisation. In Western blotting of sperm and keratinocyte lysates, the antibody detected similar 90-kDa protein doublets (AJ-p90). These biochemical features exclude the identity of AJ-p90 with various cyto- and karyo-skeletal antigens, including the intermediate filaments and microfilaments. AJ-p90 therefore represents a novel product of the subacrosomal perinuclear theca. The significance of these data is discussed together with the importance of the antibody for probing the perinuclear theca in normal and abnormal germ cells, including multinucleated spermatids which also showed reactivity with the antibody.