A new isolation procedure and further characterisation of the lectin from the red marine alga Ptilota serrata

Ptilota serrata has been shown previously to contain a lectin (PSL) which is non-specific for human blood groups. We report here a new isolation procedure for PSL using a single step affinity chromatography technique on cross-linked guar gum and further characterisation studies. PSL was inhibited by galactose and its derivatives. The carbohydrates o-nitrophenyl-N-acetyl-α-D-galactoside, p-nitrophenyl-N-acetyl-β-D-galactoside and lactose were strong inhibitors. The glycoproteins porcine stomach mucin, asialo bovine mucin and asialofetuin were also inhibitory. The Mr of PSL, determined by gel filtration, was 55,470. SDS-PAGE revealed one single protein band with Mr of 18,390, indicating the native protein to be a trimer of apparently identical subunits. PSL was shown to contain large amounts of acidic and hydroxyl amino acids but low in basic amino acids. This revised version was published online in August 2006 with corrections to the Cover Date.     

Host Range and Interference Studies of Three Classes of Pig Endogenous Retrovirus

Recent interest in the use of porcine organs, tissues, and cells for xenotransplantation to humans has highlighted the need to characterize the properties of pig endogenous retroviruses (PERVs). Analysis of a variety of pig cells allowed us to isolate and identify three classes of infectious type C endogenous retrovirus (PERV-A, PERV-B, and PERV-C) which have distinct env genes but have highly homologous sequences in the rest of the genome. To study the properties of these env genes, expression plasmids for the three env genes were constructed and used to generate retrovirus vectors bearing corresponding Env proteins. Host range analyses by the vector transduction assay showed that PERV-A and PERV-B Envs have wider host ranges, including several human cell lines, compared with PERV-C Env, which infected only two pig cell lines and one human cell line. All PERVs could infect pig cells, indicating that the PERVs have a potential to replicate in pig transplants in immunosuppressed patients. Receptors for PERV-A and PERV-B were present on cells of some other species, including mink, rat, mouse, and dog, suggesting that such species may provide useful model systems to study infection and pathogenicity of PERV. In contrast, no vector transduction was observed on nonhuman primate cell lines, casting doubt on the utility of nonhuman primates as models for PERV zoonosis. Interference studies showed that the three PERV strains use receptors distinct from each other and from a number of other type C mammalian retroviruses.Pig-to-human xenotransplantation has the potential to alleviate the shortage of allogeneic organs for transplantation (1, 25). In addition, it may also allow the development of novel therapies by providing unlimited supplies of cells and tissues (9, 11, 13, 18). Recently, substantial progress has been made in overcoming immunological barriers to cross-species transplantation (25, 27). At the same time, however, serious concerns that zoonotic infections might occur as a result of xenotransplantation have been expressed (1, 6, 30). Our report that an established pig cell line produces a porcine endogenous retrovirus (PERV) that can infect human cells fueled these concerns (23). Subsequently, the isolation of human tropic PERV from stimulated miniswine peripheral blood lymphocytes (38) has shown that normal pig cells can also produce potentially hazardous virus. PERVs may be difficult to eliminate from donor animals because multiple copies of PERV genomes are present in normal pig genomes (2, 16, 23). PERV infection may have serious impact on the health of not only transplant recipients but also the human population at large, if spread of an undetected infectious agent into the community were to take place (3, 31). To assess the risk posed by the PERVs for pig-to-human transplantation, a greater understanding of the properties of the PERVs is required.Sequence analyses indicate that the infectious PERVs are closely related to one another in their gag and pol genes, with maximum amino acid divergence of around 5% (2, 16a, 23). The PERVs are members of the mammalian type C retrovirus genus showing closest homology to the gibbon ape leukemia virus (GALV) pol gene, with about 70% amino acid identity, and 60 to 70% identity to murine leukemia viruses (MLV). However, three distinct env genes have now been identified in PERV clones. Two of these env genes, PERV-A and PERV-B, were cloned from human 293 cells infected with PK15 virus (16). The third distinct class of PERV env gene, here designated PERV-C, was reported as a part of a full-length PERV genome isolated from miniature swine lymphocytes (PERV-MSL) and from a swine lymphoma (PERV-Tsukuba-1) (2, 32). The three types show marked differences in the VRA, VRB, and PRO regions of SU surface glycoprotein (2, 16). Differences in these regions determine the host range specificity of the different classes of MLV (4, 5). These observations suggest that the PERVs belong to three distinct classes with different host range specificities. To test this idea, the functions of the three types of PERV env gene were examined and correlated to production, infection, and replication of PERVs in cell culture. Recombinant retrovirus vectors bearing PERV Env proteins were developed and their host ranges, cell tropism, and interference with each other as well as with other type C retroviruses were examined. The results of these experiments are the subject of this report.     

Variable Substitution Rates of the 18 Domain Sequences in Artemia Hemoglobin

The Artemia hemoglobin is a dimer comprising two nine-domain covalent polymers in quaternary association. Each polymer is encoded by a gene representing nine successive globin domains which have different sequences and are presumed to have been copied originally from a single-domain gene. Two different polymers exist as the result of a complete duplication of the nine-domain gene, allowing the formation of either homodimers or the heterodimer. The total population size of 18 domains comprising nine corresponding pairs, coupled with the probability that they reflect several hundred million years of evolution in the same lineage, provides a unique model in which the process of gene multiplication can be analyzed. The outcome has important implications for the reliability of local molecular clocks. The two polymers differ from each other at 11.7% of amino acid sites; however when corresponding individual domains are compared between polymers, amino acid substitution fluctuates by a factor of 2.7-fold from lowest to highest. This variation is not obvious at the DNA level: Domain pair identity values fluctuate by 1.3-fold. Identity values are, however, uncorrected for multiple substitutions, and both silent and nonsilent changes are pooled. Therefore, to determine the variability in relative substitution rates at the DNA level, we have used the method of Li (1993, J Mol Evol 36:96–99) to determine estimates of nonsynonymous (K _A ) and synonymous (K _S ) substitutions per site for the nine pairs of domains. As expected, the overall level of silent substitutions (K _S of 56.9%) far exceeded nonsilent substitutions (K _A of 6.7%); however, for corresponding domain pairs, K _A fluctuates by 2.3-fold and K _S by 1.7-fold. The large discrepancies reflected in the expressed protein have accrued within a single lineage and the implication is that divergence dates of different genera based on amino acid sequences, even with well-studied proteins of reasonable size, can be wrong by a factor well in excess of 2. Received: 4 June 1997 / Accepted: 17 December 1997     

Use of pH 9.5 Tris-HCI Buffer Containing 5% Urea for Antigen Retrieval Immunohistochemistry

Successful antigen retrieval (AR) immunohistochemistry is dependent on the temperature, heating time, and pH value of the AR solutions. There is no single standardized AR solution, however, that is suitable for all antibodies “routinely” used in surgical pathology for immunostaining archival tissue sections. We tested a variety of AR solutions varying in pH value, chemical composition, and molarity. Based upon preliminary results, we compared three AR solutions: 0.1 M Tris-HCI buffer, pH 9.5, containing 5% urea, 0.1 M Tris-HCI buffer pH 9.5 without urea, and citrate buffer, pH 6.0. Each AR solution was tested with a panel of 34 antibodies using microwave heating for antigen retrieval. The heating conditions were standardized at 10 min and an automated stainer was used to standardize the immunostaining method. The Tris-HC1 containing urea was superior to pH 6.0 citrate buffer for 22 antibodies. In 12 cases, Tris-HC1 with urea was also superior to Tris-HC1 alone. In 12 cases, the intensity was similar for all three retrieval solutions. The staining obtained with Tris-HC1 with urea was equal to or better than with pH 6.0 citrate buffer in all cases. The Tris-HC1 with urea solution is satisfactory for AR of most antibodies employed in routine surgical pathology.     

Isolation of microtubule-associated proteins from carrot cytoskeletons: a 120 kDa map decorates all four microtubule arrays and the nucleus

A method for biochemically isolating microtubule-associated proteins (MAPs) from the detergent-extracted cytoskeletons of carrot suspension cells has been devised. The advantage of cytoskeletons is that filamentous proteins are enriched and separated from vacuolar contents. Depolymerization of cytoskeletal microtubules with calcium at 4°C releases MAPs which are then isolated by association with taxol stabilized neurotubules. Stripped from microtubules (MTs) by salt, then dialysed, the resulting fraction contains a limited number of high molecular weight proteins. Turbidimetric assays demonstrate that this MAP fraction stimulates polymerization of tubulin at concentrations at which it does not self-assemble. By adding it to rhodamine-conjugated tubulin, the fraction can be seen to form radiating arrays of long filaments, unlike MTs induced by taxol. In the electron microscope, these arrays are seen to be composed of mainly single microtubules. Blot-affinity purified antibodies confirm that two of the proteins decorate cellular microtubules and fulfil the criteria for MAPs. Antibodies to an antigenically related triplet of proteins about 60–68 kDa (MAP 65) stain interphase, preprophase band, spindle and phragmoplast microtubules. Antibodies to the 120 kDa MAP also stain all of the MT arrays but labelling of the cortical MTs is more punctate and, unlike anti-MAP 65, the nuclear periphery is also stained. Both the anti-65 kDa and the anti-120 kDa antibodies stain cortical MTs in detergent-extracted, substrate-attached plasma membrane disks ('footprints'). Since the 120 kDa protein is detected at two surfaces (nucleus and plasma membrane) known to support MT growth in plants, it is hypothesized that it may function there in the attachment or nucleation of MTs.     

Physiological and nutritional determinants of protease secretion by Clostridium sporogenes: characterization of six extracellular proteases

Summary Proteases were the principal secretory proteins of Clostridium sporogenes and were optimally produced after active growth at 37° C. Glucose, ammonia and peptides repressed protease production. Protease formation was maximal in cultures grown at pH 6.5, but proteolytic activity exhibited a pH optimum of 7.0–8.0. Protease activity in culture filtrates was stimulated by divalent metal ions (Ca²⁺, Mn²⁺ and Co²⁺) and was strongly inhibited by ethylene diaminetetraacetate (EDTA) and thimerosal. Non-denaturing polyacrylamide gel electrophoresis and HPLC gel filtration demonstrated the presence of six major proteases of low molecular mass (approx. 15–35 kDa). The enzymes were partially purified from non-denaturing gels. Each hydrolysed azocoll and azocasein, but differed in their activity against a range of native collagen substrates. All six enzymes degraded human placental collagen (Type IV) but only one had a broad substrate specificity, being able to hydrolyse the more recalcitrant collagens (Types I, II and III). Experiments with individual proteases showed that their activities were strongly inhibited (40–85%) by 5 mM EDTA, indicating that they were metalloproteases. The enzymes exhibited different susceptibilities to inhibition by either 3 mM phenylmethylsulphonyl-fluoride (PMSF), 5 mM thimerosal, or 10 mM cysteine, which respectively inhibit serine, thiol and metalloproteases.     

The effects of orthovanadate,vanadyl and peroxides of vanadate on glucose metabolism in skeletal muscle preparations in vitro

The insulin-like effects of various vanadium compounds (orthovanadate, vanadyl and peroxides of vanadate) on rates of glucose oxidation, lactate formation and glycogen synthesis were measured in isolated incubated epitrochlearis (mainly type 11 fibres) and soleus (mainly type I fibres) muscle preparations. There was a small stimulation of the rate of glucose utilisation in soleus muscle preparations in vitro by orthovanadate (1 mM). Orthovanadate or vanadyl, at 1 mM, had little effect on the rates of lactate formation or glycogen synthesis in isolated incubated epitrochlearis muscle preparations. In contrast, peroxides of vanadate (peroxovanadates, at 1 mM) significantly stimulated glucose utilisation in both soleus and epitrochlearis muscle preparations in vitro. The stimulation of the rate of glycogen synthesis was associated with an increase in the percentage of glycogen synthase in the I (or a) form. Peroxovanadates were administered in the drinking water to rats made insulin deficient by streptozotocin treatment. There was no decrease in the elevated level of blood glucose over an 8 day administration period. (Mol Cell Biochem 109: 157–162, 1992)     

In situ hybridization studies using a molecular probe that maps to Xq27–Xq28

Summary The locus DXS98, which is recognized by the sequence p4D-8, is closely linked to the FRAXA locus. In this study we present data that confirm the existing mapping data, sublocalizing this sequence to the Xq27 region immediately proximal to the fragile site at Xq27.3.     

Molecular characterisation of two alpha-1-antitrypsin deficiency variants: proteinase inhibitor (Pi) NullNewport (Gly115→Ser) and (Pi) Z Wrexham (Ser−19→Leu)

Summary Two single point mutations in the alpha-1-antitrypsin gene, resulting in AAT deficiency, have been characterised in heterozygotes by DNA amplification and direct sequencing. The mutations result in amino acid substitutions, Gly¹¹⁵Ser and Ser^–19Leu, in the leader sequence, respectively, and have been designated Pi Null_Newport and Pi Z Wrexham. In the two families studied the mutations occur on chromosomes which also carry the common mutation causing Z deficiency. Individuals with such a deficiency are, therefore, compound heterozygotes. It is not known if these particular mutations would only cause a mild form of AAT deficiency in the absence of the Z mutation as they do not appear to cause predictable folding abnormalities. They do, however, result in severe deficiency when the Z mutation occurs in the same gene.