Schistosomes express two forms of cathepsin D.

We report here a cDNA and its deduced amino acid sequence encoding a cathepsin D-like, aspartic protease expressed by adult stages of the human blood fluke Schistosoma mansoni. The cDNA encodes a short signal peptide, a pro-enzyme peptide of 37 amino acid residues, and a mature enzyme of 377 residues which has strong homology with mammalian cathepsins D. This aspartic protease, although 84% identical in amino acids of the mature enzyme region to the previously reported cathepsin D from the Asian schistosome S. japonicum, differs remarkably from the S. japonicum enzyme in having a carboxyl terminal extension of 43 amino acid residues. These cathepsins D of schistosomes may play pivotal roles in the degradation of hemoglobin obtained by the parasites from ingested host erythrocytes.

Proteolytic degradation of host hemoglobin by schistosomes.

Schistosomes acquire amino acids for growth, development, and reproduction by catabolizing hemoglobin obtained from ingested host erythrocytes. While the biochemical pathway(s) involved has not been determined definitively, a number of proteases including schistosome legumain and cathepsin L-, D-, B- and C-like enzymes have been ascribed roles in the degradation of hemoglobin to diffusible peptides. Transcripts encoding these schistosome proteases, which appear to be expressed in the gastrodermis and cecum of the schistosome, have been reported. Because these enzymes are candidate targets at which to direct novel anti-schistosomal therapies, the comparative biochemistry of these and their counterpart mammalian proteases is now the focus of research in a number of laboratories. This paper reviews reports dating from 40 years ago to the present on how schistosomes digest host-derived hemoglobin, and interprets apparent anomalies in some earlier compared to later reports, the latter having benefited from the availability of PCR and gene cloning technologies. More specifically, the review concentrates on five proteolytic enzymes, and their associated genes, which have been ascribed key roles in the pathway of hemoglobin degradation.

Salt fluoridation and dental caries in Jamaica.

PURPOSE: In 1987, Jamaica initiated a comprehensive island-wide salt fluoridation program. A survey was conducted in 1995 to monitor the impact of salt fluoridation among children in Jamaica. METHODS: Dental examinations of 1,120 children aged 6-8, 12, and 15 years were conducted according to World Health Organization criteria to assess dental caries, fluorosis, the presence of and need for dental sealants, and Community Periodontal Treatment Needs (CPI). RESULTS: Age specific DMFT means observed in 1995 were 0.2 at age 7, 0.4 at age 8, 1.1 at age 12 and 3.0 at age 15. The mean DMFT scores in children 6, 12 and 15 years of age were dramatically lower than the corresponding scores of 1.7, 6.7 and 9.6 obtained at the baseline examination in 1984 for children of the same age groups, respectively (baseline data for 7- and 8-year-olds were not collected). The mean percentage of sound permanent teeth for all age groups was 90% in 1995. The percentage of children caries-free at baseline was 27.6% for 6 years, 2.8% for 12 years and 0.3% for 15 years of age. In 1995, the percentage of caries-free children (permanent teeth) was 61%. In 1984, 23 children were scored as having very mild or mild fluorosis. In 1995, five children were scored in the same categories of fluorosis, using Dean's criteria; thus, fluorosis remained at negligible levels in 1995. CONCLUSIONS: The oral health survey conducted in Jamaica in 1995 indicated a significant decline in dental caries compared with findings in 1984. The major change in Jamaica during the interval was the introduction of salt fluoridation in 1987. Dental fluorosis was low in the 1995 survey.

Measuring utilization and impact of home care services: a systems model approach for cost effectiveness.

The relevance of home care research to policy questions is discussed as framework for study on "effects" (precursors and sequelae) of home care. This study used a large, multi-service agency's longitudinal (8-year) case records (N = 2436) to examine a system model for relationships among entry characteristics, utilization of services, and need for services upon discharge from home care. Deducing case-mix from utilization patterns, pay plan at entry was identified as best of the available predictors of both duration and intensity (using multivariate analysis). Duration and intensity, dual contributors to "total visits," were found to vary inversely and were predicted by different entering pay plans. While 1/3 of all cases were discharged to informal or self care, that was the most prevalent exit status of the clients (49%) who entered directly from hospital care. The methods used in disaggregating and analyzing these retrospectively-coded case records suggest that home services research: 1. distinguish type, intensity, and duration as components of "total visits" which combine to account for costs of care; 2. find concomitants of functional level (such as pay plan) which are accessible for designating case mix for purpose of projecting service use; 3. measure effectiveness in terms relevant to stated objectives of the long term care system, which need to acknowledge mortality and to separate service needs at entry room those at exist from the series of formal and informal providers on a continuum of care.

Nonuniform contraction in the isolated cat papillary muscle.

Microspheres infused into the coronary microcirculation were used as markers to define segments within isolated cat papillary muscles. Video recording and analysis provided measurements of the variations of segment lengths as the muscles contracted at lengths of 76-100% Lmax. In all muscles, segments in the center region were found to shorten during muscle isometric contraction while those in the end regions lengthened. Central shortening was typically 10-15%. In the passive state, segment lengths varied directly with muscle length over a broad range characterized by low force. Segments in the center region, however, displayed an abrupt transition to high stiffness at a certain length while end regions continued to stretch. Force-length relationships obtained for the presumably healthy center segment are significantly different from those obtained for the whole muscle. These results suggest that there may be major difficulties with the interpretation of mechanical measurements on papillary muscles unless contractile inhomogeneity is eliminated or taken into account.

A method for the isolation of schistosome eggs and miracidia free of contaminating host tissues.

A novel method for the isolation of schistosome eggs and miracidia from livers of mice infected with Schistosoma japonicum or S. mansoni is described. The method employed collagenase B to degrade the interstitial matrix of mouse liver tissue, after which the schistosome eggs were separated from the liver cells by 2 single-step density centrifugations through Percoll. Using this procedure sufficient quantities of miracidia were obtained to generate a cDNA library. Southern blot analysis demonstrated that miracidia isolated by this method were free from contaminating host DNA.

Characterisation of two distinct HKT1-like potassium transporters from Eucalyptus camaldulensis.

Potassium is an essential macronutrient in higher plants. It plays an important physiological role in stoma movements, osmoregulation, enzyme activation and cell expansion. The demand for potassium can be substantial, especially when the plant concerned is a Eucalyptus tree in excess of 50 m tall. We have isolated two cDNAs, EcHKT1 and EcHKT2, from Eucalyptus camaldulensis (river red gum) which are expressed in leaves, stems and roots. These encode potassium transporter polypeptides with homology to the wheat K+-Na+ symporter, HKT1. EcHKT1 and EcHKT2 both complemented the K+-limited growth of an Escherichia coli K+-uptake-deficient triple mutant. EcHKT1 and EcHKT2 also mediated Na+ and K+ uptake when expressed in Xenopus oocytes. A comparison of the EcHKT1 and EcHKT2 sequences and their transport properties indicated that these cDNAs represent two K+ transporters with distinct functional characteristics. The functional and structural conservation between these two E. camaldulensis genes and the wheat HKT1 suggests that they play an important, albeit elusive, physiological role.

Characterization and cloning of the cathepsin L proteinases of Schistosoma japonicum.

Adult Schistosoma japonicum parasites synthesize and secrete both cathepsin L and cathepsin B cysteine proteinases. The specific activities of cathepsin L were many-fold higher than that of cathepsin B. The cDNAs encoding two distinct cathepsin L proteinases, here termed cathepsin L1 and L2, were isolated. The deduced amino acid sequences of the mature cathepsin L1 and L2 were approximately 41% identical, and moreover, S. japonicum cathepsin L2 showed more similarity with human cathepsin L than with schistosome cathepsin L1. Schistosome cathepsin L proteinases may be involved in the digestion of hemoglobin obtained from host erythrocytes. However, since we detected their presence in schistosome eggs, the release of these enzymes by eggs trapped in the liver and other organs may be associated with the granulomatous responses which characterize the pathology of human schistosomiasis.

Generation, identification, and evaluation of expressed sequence tags from different developmental stages of the Asian blood fluke Schistosoma japonicum.

Here we report 658 expressed sequence tags (ESTs) generated from the 5'-termini of clones randomly selected from directional cDNA libraries constructed from mRNAs from three developmental stages of Schistosoma japonicum. Putative identifications were assigned to 46. 2% of the ESTs; 6.4% were previously known from S. japonicum, 5.6% were previously known from S. mansoni, 34.2% were known from other organisms, and the remaining 53.8% may represent S. japonicum-specific genes. These 658 ESTs appeared to be derived from 457 unique genes, which together represent 2 to 3% of the 15,000 to 20,000 genes predicted to occur in the schistosome genome.

Recombinant expression and localization of Schistosoma mansoni cathepsin L1 support its role in the degradation of host hemoglobin.

Cysteine proteinases expressed by schistosomes appear to play key roles in the digestion of host hemoglobin, the principal source of amino acid nutrients utilized by these parasites. We have shown previously that the predominant cysteine proteinase activity in soluble extracts and excretory/secretory (ES) products of adults of Schistosoma mansoni and S. japonicum is cathepsin L-like in its substrate specificity. However, biochemical analysis of the cathepsin L activity in extracts and ES products of schistosomes has been complicated by the presence of at least two distinct forms of schistosome cathepsin L, termed SmCL1 and SmCL2. We now report the purification and enzyme characteristics of active, recombinant SmCL1 which was obtained by transforming Saccharomyces cerevisiae with an expression plasmid encoding the preproenzyme of SmCL1. Recombinant SmCL1 was secreted by the transformed yeast into the culture media from which it was purified by gel filtration and ion-exchange chromatography. The purified enzyme exhibited substrate specificity against synthetic peptidyl substrates (e.g., Boc-Val-Leu-Lys-NHMec and Z-Phe-Arg-NHMec; kcat/Km = 17.25 and 6.24 mM-1 s-1, respectively) and against gelatin and hemoglobin, characteristic of cathepsin L. Immunoblot analysis using antiserum raised against recombinant SmCL1 demonstrated that native SmCL1 of 33 kDa was present in ES products and soluble extracts of S. mansoni. Using this antiserum and thin tissue sections, we localized the native SmCL1 to the gastrodermis and to the tegument of adult schistosomes. Recombinant SmCL1 was capable of degrading human hemoglobin at pH 4.0 to 4.5 but not higher, suggesting that denaturation of hemoglobin by low pH, as found in the cecum of the adult schistosome, may be necessary for its catalysis by cathepsin L and other gut-associated proteinases. Together, these results support a role for SmCL1 in the degradation of host hemoglobin within the gut of the schistosome.

Cathepsin C from Schistosoma japonicum--cDNA encoding the preproenzyme and its phylogenetic relationships.

A cDNA encoding preprocathepsin C was isolated from adults of the asian blood fluke Schistosoma japonicum. The deduced amino acid sequence of S. japonicum cathepsin C comprised 458 amino acid residues; 22 NH2-terminal residues corresponding to the signal peptide, 199 residues corresponding to the propeptide and 237 COOH-terminal residues corresponding to the mature enzyme region. The amino acid sequence of this preprocathepsin showed 43% and 50% identity to that of human and rat, respectively. The preproenzyme shared only 59% identity with the sequence for a cathepsin C reported from Schistosoma mansoni, differing from it in active-site residues and in its potential N-glycosylation sites. Northern-blot analysis showed that S. japonicum cathepsin C was expressed in greater quantities in female than in male parasites. Phylogenetic analysis utilizing the mature enzyme sequences of S. japonicum and other cathepsin Cs demonstrated that cathepsin Cs and cathepsin Bs shared a common ancestry. The unusually long prosegment observed in cathepsin C from S. japonicum and from other species was compared to that of cathepsin Bs and cathepsin Ls. The extension contained two blocks of residues which were highly conserved among cathepsin Cs. The COOH terminus of the prosegment exhibited a composite of features present in the prosegments of cathepsin Ls and cathepsin Bs. Most significantly, given the common ancestry of cathepsin B and cathepsin C, the prosegment of cathepsin C included ERFNIN-like motifs and other residues more characteristic of non-cathepsin-B-like members of the papain superfamily such as cathepsin L.