Reproductive biology of the starry smooth-hound shark Mustelus asterias: geographic variation and implications for sustainable exploitation.

Examination of the reproductive biology of Mustelus asterias in the north-east Atlantic Ocean highlighted apparent geographical variation in maturity, fecundity and ovarian cycle between Atlantic and Mediterranean populations. The stretch total length (L(ST) ) and age at 50% maturity for Atlantic males and females were estimated at 78 cm L(ST) and 4-5 years and 87 cm L(ST) and 6 years, respectively. Size at maturity of females was considerably smaller than in Mediterranean specimens (96 cm L(ST) ). Ovarian fecundity ranged from eight to 27 oocytes and uterine fecundity from six to 18 embryos. The gestation period was c. 12 months, followed by a resting period of c. 12 months, resulting in a biennial cycle. Females stored sperm in the oviducal gland and, unlike Mediterranean specimens, no uterine compartments were observed in Atlantic specimens. This study reveals the existence of strong, possibly adaptive, divergence in life-history traits in an elasmobranch, whose northern populations may be more susceptible to overexploitation than previously believed.

Monitoring of intra-operative nociception: skin conductance and surgical stress index versus stress hormone plasma levels.

'Surgical Stress Index' and the 'Number of Fluctuations in Skin Conductance.s⁻¹, use different methods to analyse sympathetic tone and so provide an estimate of peri-operative analgesia. The aim of our study was to investigate the relationship between these methods and stress hormone plasma levels. In 20 patients scheduled for elective surgery, values of the two methods, mean arterial blood pressure, heart rate and blood samples (to measure plasma levels of adrenaline, noradrenaline, adrenocorticotrophic hormone and cortisol) were obtained at five time points. Changes in Surgical Stress Index and the Number of Fluctuations in Skin Conductance.s⁻¹ only partially reflected changes in plasma noradrenaline levels. Surgical Stress Index, heart rate and blood pressure, but not the 'Number of Fluctuations in Skin Conductance.s⁻¹ changed in response to changes in depth of analgesia by showing significant differences between before and after a bolus of fentanyl. However, the overall predictive ability of both methods was poor.

Prostatic secretory protein (PSP94) expression in human female reproductive tissues, breast and in endometrial cancer cell lines.

PSP94 (beta microseminoprotein, beta MSP) is one of the three major proteins secreted by the normal human prostate gland. Using reverse transcriptase polymerase chain reaction (RT-PCR) and Southern blotting, PSP94 transcripts were shown in human endometrium, myometrium, ovary, breast, placenta and in the human endometrial cancer cell lines KLE and AN3 CA. Primers used in these studies were specific for human prostate PSP94, and were derived from its flanking non-coding regions. The results were confirmed by sequence analysis of two independently derived clones from normal human breast tissues and the other two from KLE cells respectively. The sequences were identical with the coding sequence of human prostate PSP94 cDNA. Using RNA from the endometrial tissues, two different transcripts of approximately 487 bp, equivalent to prostate PSP94 and approximately 381 bp, corresponding to prostate PSP57, its alternately spliced form, were amplified by RT-PCR. Human ovary, breast, placenta and endometrial cancer cell lines (KLE, AN3 CA), however, showed only the full length, approximately 487 bp, PSP94 transcript. We further demonstrated by in situ hybridization that PSP94 mRNA is expressed specifically in the glandular epithelial cells, and not in the stroma of both the human endometrial and breast tissues. Further, using image analysis of in situ hybridization data, the levels of PSP94 mRNA in the cycling endometrial tissues and in breast confirmed the differential levels of expression in the cycling endometrium (P<0.005). This study distinctly demonstrated significant expression of PSP94 mRNA in human uterine, breast and other female reproductive tissues as well in the endometrial cancer cell lines, suggesting that it may have a role in these tissues as a local autocrine paracrine factor.

Recombinant vaccinia viruses and gene gun vectors expressing the large subunit of Schistosoma mansoni calpain used in a murine immunization-challenge model.

Schistosomiasis is a parasitic disease affecting over 200 million people every year in tropical regions of the world. Drug treatment and other existing control measures are costly and have failed to eliminate the incidence of infection, morbidity and mortality due to Schistosoma mansoni infection. Vaccination of susceptible individuals using recombinant vaccines encoding key S. mansoni antigens may be the most effective and least expensive means of controlling schistosomiasis. A candidate vaccine antigen is p80, the large subunit of the S. mansoni protease, calpain. In our vaccine studies, we have employed both the wild-type p80 and a mutant p80 (mut p80) in which an active site amino acid was genetically altered to create a less proteolytically-active enzyme. Two vaccine delivery approaches were implemented using p80 or mut p80 as vaccine antigen: recombinant vaccinia virus (RVV) inoculation and DNA immunization via the Accell gene gun (GG) delivery system. RVV's expressing p80 and mut p80 were generated and tested for recombinant protein expression in vitro. These RVV's were tested for protective capacity in mouse challenge studies. Neither subcutaneous nor intranasal vaccinations with RVV-p80 or RVV-mut p80 were capable of significantly reducing the mean worm burdens of vaccinated mice. A GG-RVV combination immunization regime using WRG-vectors encoding p80 and mut p80 for GG priming and the RVV's for boosting prior to S. mansoni challenge infection was performed and no significant protection was obtained over two repeated studies. However, duplicate challenge studies involving GG immunization of mice with WRG-vectors encoding p80 or mut p80 revealed that 3 inoculations of mice with WRG-full5' mut p80 (containing the full 5' untranslated region of mut p80) provided 60% protection which was statistically significant (p < 0.05). These preliminary in vivo studies demonstrate the potential for further study of the protection afforded by gene gun-delivered WRG-full5' mut p80 into subsequently-challenged mice. Such studies may pave the way to effective vaccination of humans using WRG DNA vectors expressing a schistosomal calcium-activated neutral protease.

Detection of PSP94 and its specific binding sites in the prostate adenocarcinoma cell line LNCaP.

PURPOSE: To evaluate the expression of prostate secretory protein of 94 amino acids (PSP94) and PSP94 binding proteins in the LNCaP cell line. MATERIALS AND METHODS: The reverse-transcription polymerase chain reaction (RT-PCR) and Southern blot hybridization were employed to assay the expression of PSP94. Immunoprecipitation with specific polyclonal antibodies was used to detect PSP94 secreted by the LNCaP cells. The binding proteins were assayed by equilibrium binding assays. RESULTS: PSP94 was expressed and secreted in the LNCaP cells. As well as, LNCaP cells expressed surface membrane proteins capable of binding PSP94 in a specific and saturable manner. Exposure of LNCaP cells to exogenous PSP94 resulted in the up-regulation of PSP94 binding sites, indicating functional interactions for PSP94 and its receptor in this cell line. CONCLUSION: The expression of PSP94 and its receptors may be partially regulated by an autocrine pathway in the LNCaP cell line.

Identification of binding proteins for PSP94 in human prostate adenocarcinoma cell lines LNCaP and PC-3.

BACKGROUND: Prostatic secretory protein of 94 amino acids (PSP94) is one of the predominant proteins found in human seminal fluid. Limited information is available regarding a physiological function for PSP94. An important step in the elucidation of this function is the determination of the mechanism of interaction of PSP94 with potential cellular targets. METHODS: Equilibrium binding assay was employed to demonstrate specific binding of biotinylated-PSP94 to the LNCaP and PC-3 cell lines. Binding proteins were partially purified by PSP94 affinity-chromatography from LNCaP, PC-3 cells, and prostate tissues. RESULTS: Binding of biotinylated-PSP94 to LNCaP and PC-3 cells was saturable and time and temperature dependent. The binding could be specifically competitively inhibited by unlabelled PSP94. Two types of PSP94 binding sites with distinct affinity (Kd) and density (Bmax) were determined by Scatchard analysis for each of the two cell lines. For the LNCaP cells, these values were Kd 1 = 0.75 nM and Bmax1 = 300 fmol/mg protein and Kd 2 = 4.5 nM, Bmax2 = 780 fmol/mg protein, respectively. Similar affinity and density results were obtained for PC-3 cells: Kd 1 = 0.83 nM, Bmax1 = 250 fmol/mg protein, and Kd 2 = 5.0 nM, Bmax2 = 700 fmol/mg. The binding of biotinylated-PSP94 to the LNCaP cells was competitively inhibited by the partially purified proteins. Analysis of these proteins SDS-PAGE showed three main bands and the molecular weights of these three bands were approximately 180, 100 and 60 kD, respectively. CONCLUSIONS: The data showed the presence of specific binding proteins to the PSP94 in LNCaP, PC-3 cells, and prostate tissue.

Kinesin light chain in a eubacterium.

A eubacterial homolog of a kinesin light chain gene has been isolated and characterized from the cyanobacterium Plectonema boryanum. Although the eubacterial and eukaryotic kinesin light chains are highly similar in amino acid sequence, the eubacterial sequence differs in several distinguishing structural features, including the absence of a putative PEST domain and the presence of additional highly conserved imperfect tandem repeats. Two soluble kinesin light chain antigens have been identified from whole-cell lysates by immunoblot analysis. Attempts to identify a canonical kinesin heavy-chain gene or protein were unsuccessful, suggesting that a kinesin heavy chain may be absent or unnecessary for kinesin light-chain function in this eubacterium. Our findings establish that certain basal elements of eukaryotic cellular transport appear to be resident in eubacteria. We discuss the possibility that the eukaryotic kinesin light chain was acquired by lateral gene transfer.

A new scalable purification procedure for prostatic secretory protein (PSP94) from human seminal plasma.

A simple three-step procedure for the purification of native prostate secretory protein (PSP94) from human seminal plasma is described. The steps are ammonium sulfate fractionation followed by a Macro-Prep S support (cation) flowthrough process and the final Macro-Prep high Q support (anion exchange) chromatography using two step-gradient elution. The benefits of this procedure lie in its simplicity, speed, and relatively inexpensive materials, thus providing advantages over the previously reported schemes. The purity of the product as judged by single band on SDS-polyacrylaminde gel electrophoresis was equivalent to that attained by analytical HPLC anion exchange procedure. Yields were in the range of 18-25 mg highly pure PSP94 per 50 ml of seminal plasma. The desalted, lyophilized, purified PSP94 sample was characterized by SDS-PAGE, Western blot, and N-terminal sequencing. All parameters tested confirm its identity. Preliminary data show that this procedure is suitable for a large-scale production. The direct quantitation of PSP94 by SDS-PAGE and densitometric image analysis at various purification steps for evaluating the recovery of PSP94 is described. The results obtained show that this is an efficient strategy for preparation of highly purified native PSP94.

Characterization of a small variable surface glycoprotein from Trypanosoma vivax.

Several biochemical properties of a variant surface glycoprotein (VSG) from the parasite Trypanosoma (Duttonella) vivax have been determined. ILDat 2.1 VSG is approximately 40 kDa in size making this the smallest trypanosome VSG described to date. The glycolipid anchor of ILDat 2.1 VSG is resistant to treatment with T. brucei-derived phospholipase C and data based on lectin affinity chromatography, incorporation of radiolabelled sugar and treatment with endoglycosidase H suggest that the T. vivax VSG bears little carbohydrate. cDNA to ILDat 2.1 VSG mRNA has been cloned and the encoded protein sequence includes the N-terminal amino acid peptide sequence derived from native VSG. The molecular weight of the VSG predicted from the translated cDNA sequence is similar to that of the native molecule and in support of the biochemical data it is devoid of sites for N-linked glycosylation. Examination of the deduced ILDat 2.1 VSG protein sequence reveals that it is most similar to T. congolense VSGs in the distribution of Cys residues and like the former it does not contain any of the defined VSG C-terminal domain types. However, unlike T. congolense VSGs it does not readily fit into the currently described VSG N-terminal domain types. Our studies suggest that ILDat 2.1 VSG is distinct from any of the previously characterized VSGs.

Trypanosoma brucei: analysis of cytoplasmic Ca2+ during differentiation of bloodstream stages in vitro.

The highly regulated intracellular concentration of calcium (Ca2+) is a well-described regulator of diverse cellular events, including cell cycle control. In the present study we have addressed the regulation of cytosolic Ca2+ in differentiation events in the life cycle of the protozoan parasite Trypanosoma brucei. Bloodstream form (BSF) trypanosomes include the mitotically active long slender forms (LS) which differentiate to two nondividing stages--intermediate (INT) which transform into short stumpy (SS) forms. An axenic in vitro culture system was used to cultivate LS to a density greater than 1.0 x 10(6) cells/ml/day. Populations of the intermediate BSF (INT) and SS were derived from cultured LS by treatment with difluoromethyl ornithine (DFMO, 100 microM) for 2 and 4 days, respectively. A semiquantitative reverse transcriptase-coupled polymerase chain reaction protocol (SQ-RT-PCR) was developed to objectively distinguish the three BSF by monitoring the relative levels of stage-specific mRNAs--cytochrome oxidase II (COXII), variant surface glycoprotein, and procyclin during the differentiation of LS to SS, showing an increase in COXII and procyclin mRNA expression during this process of differentiation. Basal cytosolic Ca2+ levels [Ca2+]i of populations of LS, INT, and SS were studied using Indo-1 dual emission fluorometry. [Ca2+]i was maximal in dividing LS cells and was shown to decrease coincidentally with early events in the process of differentiation to INT and SS. Thapsigargin (1 microM), reported to cause the release of Ca2+ from the endoplasmic reticulum, elevated [Ca2+]i by about 30-60 nM in all BSF; however, the total thapsigargin-releasable stores decreased in parallel with the decrease in basal [Ca2+]i. Control treatments verified that elevations in [Ca2+]i in response to thapsigargin were intracellular in origin. These results may reflect the cessation of cytosolic Ca2+ transients involved in the regulation of mitosis as the parasite exits from the cell cycle and differentiates from rapidly dividing LS to the nondividing SS.

Alternative splicing of PSP94 (prostatic secretory protein of 94 amino acids) mRNA in prostate tissue.

While performing reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of total mRNA from prostate cancer specimens, two forms of PSP94 cDNA were detected. RT-PCR products were analysed by Southern blotting and probing with exon-specific oligonucleotides. In the short form of PSP94 mRNA, designated as PSP57, exon III was found to be deleted. The two mRNA forms were confirmed by cloning and sequencing of the RT-PCR products and were found to result from alternative splicing. The alternatively spliced form, PSP57, was characterized by sequence analysis. PSP94 and PSP57 possess identical exons I and II, including identical secretion signal peptide and the 5' untranslated sequences. PSP57 has a frame-shifted exon IV and encodes a putative 57 amino acid protein with a novel, highly basic C-terminus of 41 amino acids. PSP57 mRNA was detected in other urogenital tissues (kidney, bladder) and in most tumor cell lines tested, but was not detectable in other tissues such as breast and lung. In prostate tumor cell lines, PSP57 mRNA was aberrantly spliced and localized in the nuclear fraction of the cell. Our results suggest the possible existence of a novel PSP protein that originates from alternative splicing of PSP94 mRNA in urogenital tissues.

Characterization of a C3 receptor on the envelope of Schistosoma mansoni.

The surface of the syncytial epithelium of the human parasite, Schistosoma mansoni, consists of an apical plasma membrane (APM) and an overlying envelope (En). The rapid turnover of these membranes is an adaptation to parasitism and is influenced by ambient signals emanating from the host's immune system. The third component of complement (C3) has been shown to stimulate the synthesis of the En via a C3-binding protein (C3bp) and a Ca(2+)-dependent signal transduction mechanism. Using ELISA the C3bp was found to be restricted to the En. In addition, cross-linking of iodinated C3 to En proteins with the homobifunctional noncleavable disuccinimidyl suberate reagent yielded a receptor-C3 complex in excess of 250 kDa, and SDS-PAGE analysis of solubilized En proteins that were radiolabeled and chromatographed on C3-Sepharose revealed a 130-kDa protein that specifically bound to the C3 beads. In further experiments, using a photoactivatable radiolabeled cross-linker, the Denny-Jaffe reagent, C3 transferred the radiolabel to a 130-kDa En protein. Metabolic labeling experiments have demonstrated that this C3bp is synthesized by the parasite and, more importantly, antibodies raised against the C3bp blocked En synthesis in vivo. Also, the surface localization of the C3bp was demonstrated using immunolabeling electron microscopy. The data presented herein strongly suggest that the 130-kDa schistosome En protein is a C3bp responsible for renewal of the En in response to C3 binding.

Characterization of Ca(2+)-dependent neutral protease (calpain) from human blood flukes, Schistosoma mansoni.

Calcium-dependent, neutral cysteine-proteases (calpain) were purified from human blood flukes, Schistosoma mansoni. The electrophoretic mobilities, Western blot analyses and high specificity to peptide inhibitors confirmed the presence of both calpain I and II in the purified preparation. The schistosome calpains were localized in the surface syncytial epithelium and underlying musculature. Using peptide inhibitors, calpain was shown to function as a mediator of the surface membrane synthetic process. Since there was also no immunological cross-reactivity between vertebrate and schistosome calpains using antibodies affinity-purified from native and recombinant schistosome calpains, this protease may be usefully investigated as forming the basis of a molecular vaccine against schistosomiasis.

An integral membrane glycoprotein associated with an endocytic compartment of Trypanosoma vivax: identification and partial characterization.

A 65-kD glycoprotein (gp65) of Trypanosoma (Duttonella) vivax was identified using a murine monoclonal antibody (mAb 4E1) that had been raised against formalin-fixed, in vitro-propagated, uncoated forms. Intracellular localization studies utilizing the mAb in immunofluorescence on fixed, permeabilized T. vivax bloodstream forms and immunoelectron microscopy on thin sections of Lowicryl K4M-embedded cells revealed labeling of vesicles and tubules in the posterior portion of the parasite. Some mAb-labeled vesicles contained endocytosed 10 nm BSA-gold after incubation of the parasites with the marker for 5-30 min at 37 degrees C, and the greatest degree of colocalization was observed after 5 min. Double labeling experiments using the mAb and a polyclonal anti-variant surface glycoprotein (VSG) antibody to simultaneously localize both gp65 and VSG demonstrated that there was little overlap in the distribution of these antigens. Thus, gp65 is associated with tubules and vesicles that are involved in endocytosis but which appear to be distinct from VSG processing pathways within the cell. Using the mAb for immunoblot analyses, gp65 was shown to be enriched in a fraction of solubilized membrane proteins eluted from either immobilized Con A or Ricinus communis agglutinin and was found to possess carbohydrate linkages cleaved by both endoglycosidase H and O-glycosidase, suggesting the presence of N- and O-linked glycans. Protease protection and crosslinking experiments suggest that gp65 is a transmembrane protein with trypsin cleavage and NH2-crosslinking sites on the lumenal face of the vesicles.

Schistosoma mansoni: characterization and identification of calcium-binding proteins associated with the apical plasma membrane and envelope.

Calcium-binding proteins (CaBPs) of Schistosoma mansoni were purified by hydrophobic affinity chromatography. Metabolically labeled CaBPs were characterized using SDS-polyacrylamide gel electrophoresis followed by fluorography. A number of CaBPs were detected in total tissue extracts, apical plasma membrane, and soluble fractions of the apical bilayer complex, ranging from 15 to 205 kDa in their molecular masses. No CaBPs were discerned in the envelope of the apical bilayer complex. Two CaBPs were positively identified as calmodulin and gelsolin via immunoblot analyses. The possible role of CaBPs in surface signal transduction mechanisms has also been briefly discussed.

Localization of a 24-kilodalton glycoprotein in adult Schistosoma mansoni using immunofluorescence and immunoelectron microscopy.

Studies on schistosome protective immune responses have focused mainly on antigens of the parasite's syncytial surface. One of the characterized schistosome antigens, a 24-kDa glycoprotein, has been considered important in mechanisms of immune evasion by the parasites. In the present study, using affinity-purified antibodies to the 24-kDa protein for immunofluorescence and immunoelectron microscopy, we demonstrated an association of the 24-kDa antigen with the discoid bodies (the major syncytial inclusion bodies; DBs) and the surface membrane complex (most likely the apical plasma membrane) of adult Schistosoma mansoni. This is consistent with previous observations that the 24-kDa antigen appeared to be localized to the syncytial membrane and DB fractions. The present results also support the suggestion that the DBs are the precursor organelles of the apical plasma membrane.

Isolation of a highly enriched plasma membrane fraction of Trypanosoma brucei by free-flow electrophoresis.

A procedure is described whereby a highly enriched plasma membrane fraction was isolated from Trypanosoma brucei by the technique of preparative free-flow electrophoresis. The purity of the plasma membrane fraction was monitored by electron microscopy and by marker enzymology, and is compared to those obtained by previous methods. Proteins associated with plasma membrane fraction were analyzed by SDS-PAGE and phase separated in Triton X-114.

Mapping of segmental antigenic determinants on structurally related variant surface glycoproteins of Trypanosoma brucei.

To study common and variant specific antigenic determinants on variant surface glycoproteins from Trypanosoma brucei, we have selected four serologically cross-reacting variant populations. Monoclonal antibodies were raised against the purified variant surface glycoproteins from each variant trypanosome population. Six monoclonal antibodies bind to segmental epitopes and one binds to a topographically assembled epitope. Amino acid compositions of these variant surface glycoproteins reveal striking conservation of certain residues including cysteine and charged amino acids. We also find that all seven monoclonal antibodies used in this study bind to protein determinants not exposed on the surface of the living trypanosome. Only one monoclonal antibody exhibits homologous specificity, while the remainder display cross-reactivity for three or all four variant surface glycoproteins. In addition, polyacrylamide gel electrophoresis peptide mapping and Western blots probed with each monoclonal antibody reveal significant peptide homologies. Furthermore, two pairs of monoclonal antibodies recognize two epitopes that are possibly immunodominant. The significance of these findings is discussed in terms of the structural similarities and differences among variant surface glycoproteins.

Physical studies of Trypanosoma brucei variant surface glycoproteins and their antigenic determinants.

Secondary structure determinations have been carried out on two antigenically related variant surface glycoproteins (VSG's) from Trypanosoma brucei, WaTat 1.1 and WaTat 1.12. The two molecules, which bear highly homologous amino-terminal sequences, showed subtle differences in their circular dichroism (CD). Computer analysis revealed that the contribution of alpha helix to the secondary structure of the VSG's was 49% for WaTat 1.1 and 52% for WaTat 1.12. Unfolding studies using guanidine hydrochloride suggested that the WaTat 1.12 VSG was slightly more resistant than WaTat 1.1 VSG to the effect of this reagent. The membrane form of WaTat 1.1 VSG purified by reverse-phase high-performance liquid chromatography gave CD and fluorescence spectra indicative of a partially unfolded or denatured molecule. It was also shown that the antigenic differences between the VSG's were due to surface-oriented topographically assembled epitopes which were highly sensitive to structural perturbations. Monoclonal antibodies specific for these epitopes bound to four discreet determinants on WaTat 1.1, one of which was absent from WaTat 1.12.