Incorporation of log odds of positive lymph nodes into the AJCC TNM classification improves prediction of survival in oral cancer.

OBJECTIVES: To assess the prognostic performance of a new N classification that incorporates the log odds of positive lymph nodes (LODDS) into the routinely used pathological N classification for oral squamous cell carcinoma (OSCC) patients. DESIGN: Retrospective cohort study utilising LODDS into pN category was performed, and the AJCC TNM stage and T-New N-M stage were compared with respect to 5-year disease-specific survival (DSS) rates. The discriminability was evaluated from the linear trend chi-square test, Akaike information criterion (AIC) and Harrell's c-statistic. SETTING: Medical centrer in Taiwan. PARTICIPANTS: A total of 463 patients received primary surgery and neck dissection between 2004 and 2013 for OSCC. MAIN OUTCOME MEASURES: The discriminability for 5-year DSS rates. RESULTS: The median follow-up period was 54 months, the mean patient age was 54 ± 11 years and 428 patients (92.4%) were male. The patients with higher LODDS had worse 5-year DSS rates. Incorporation of LODDS into the prognostic model based on the seventh edition of the TNM classification significantly improved discriminative performance for 5-year DSS with a lower AIC (1883 versus 1897), and higher prediction accuracy (Harrell's c-statistic: 0.768 versus 0.764). CONCLUSIONS: By utilising a merger of the LODDS and pN classifications to create a new N classification has better discriminatory and predictive ability than pathological TNM staging and could help identify high-risk patients for intense adjuvant therapy.

Experimental study of fat grafting under negative pressure for wounds with exposed bone.

BACKGROUND: The combination of fat grafting and negative pressure (VAC) therapy represents a synergistic interaction of all essential components for wound healing. The aim of this experimental study was to determine whether it could promote healing of wounds with exposed bone. METHODS: Full-thickness wounds with denuded bone in Sprague-Dawley rats were treated with either polyurethane foam dressing, fat grafting alone, polyurethane foam dressing with VAC, or polyurethane foam dressing with VAC combined with a single, or two administrations of fat graft. Wound healing kinetics, tissue growth, cell proliferation (Ki-67) and angiogenesis (platelet endothelial cell adhesion molecule 1 and α-smooth muscle actin) were investigated. Messenger RNA levels related to angiogenesis (vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF)), profibrosis (platelet-derived growth factor A and transforming growth factor ß), adipocyte expression (fatty acid-binding protein (FABP) 4 and peroxisome proliferator activated receptor γ), and extracellular matrix remodelling (collagen I) were measured in wound tissues. RESULTS: Wounds treated by VAC combined with fat grafting were characterized by cell proliferation, neoangiogenesis and maturation of functional blood vessels; they showed accelerated granulation tissue growth over the denuded bone compared with VAC- or foam dressing-treated wounds. Fat grafting alone over denuded bone resulted in complete necrosis. Expression of angiogenesis markers (VEGF and b-FGF) and adipocyte expression factors (FABP-4) was upregulated in wounds treated with VAC combined with fat grafting. CONCLUSION: Fat grafting with VAC therapy may represent a simple but effective clinical solution for a number of complex tissue defects, and warrants testing in clinical models. SURGICAL RELEVANCE: The combination of fat grafting and vacuum therapy represents a synergistic interaction of regenerative cells, hospitable wound matrix and stimulating micromechanical forces. It could accelerate complex wound healing through cell proliferation, neoangiogenesis and maturation of functional blood vessels. The efficacy of a multimodal wound healing approach is established in this experimental model; it could easily be translated into clinical trials of treatment for difficult wounds.

Thermal response of a dental tissue induced by femtosecond laser pulses.

This paper reports a theoretical and experimental study for thermal transport in a thin slice of human tooth induced by a 120 fs, 800 nm pulse laser at a repetition rate of 1 kHz. The surface reflectivity of enamel and the convection heat transfer coefficient were determined using an inverse heat transfer analysis. Instead of a fully three-dimensional modeling, two simplified two-dimensional (2D) planar and axisymmetric heat conduction models were proposed to simulate the temperature fields. The temperature responses obtained from the 2D planar and axisymmetric model agree well with the experimental measurements. On the other hand, the one-dimensional (1D) result significantly differs from the 2D axisymmetric one, suggesting that care should be taken when a 1D thermal model is considered for estimating temperature response.

Leishmania donovani. Hamster macrophage interactions in vitro: cell entry, intracellular survival, and multiplication of amastigotes.

An in vitro system was developed for studying host-parasite cellular interactions in visceral leishmaniasis with amastigotes isolated from infected spleens of hamsters and their peritoneal macrophages maintained by an improved method. The culture system supports the growth of Leishmania donovani amastigotes with different parasite/macrophage ratios for up to 2 wk, yielding results more consistent and reproducible than previously possible. Results indicated that the "forms" of the amastigotes (with or without adherent host membranes) and the "state" of the macrophages (with or without stimulation in vivo by thioglycollate or in vitro by aging) had no effect on the growth rate of the parasites, which, however, seems to vary with the macrophage subpopulations. An electron microscope study suggests that amastigotes are ingested through phagocytosis by the macrophages and become lodged in loose phagosomes. Additional evidence with quantitative data is presented to support the earlier findings that phagosome-lysosome fusion occurs after the interiorization of the parasites and that they not only survive but multiply in these vacuoles. During the postinfection periods, reorientation of amastigotes in vacuolar space results in the appearance of three types of parasitophorous vacuoles (parasites in loose vacuoles, in tight-fitting vacuoles or abutting in part against the inner lining of vacuoles). The last category may be the predominant type giving rise to the variations observed. Exogenously introduced dense marker accumulated in these parasitophorous vacuoles of the macrophages infected for several days indicating a continuous accessibility of amastigotes to the ambient mestruum via phagosome-lysosome vacuolar system of the host cells. This finding may have significant implications in parasite nutrition, host immunity, and chemotherapy of leishmaniasis.

Genomic Signature of Mismatch Repair Deficiency in Areca Nut-Related Oral Cancer.

Areca nut (AN) chewing contributes to an increase of oral squamous cell carcinoma (OSCC) cases in South and Southeast Asia; however, genomic events underlying the carcinogenesis process of AN-related OSCC remain unclear. Here, we comprehensively describe the genomic and transcriptome alterations of 113 Chinese OSCC patients (89 AN related and 24 AN negative) by whole-exome sequencing and RNA sequencing, and we compared the genomic differences between AN-related and AN-negative samples by integrating sequencing data of 325 OSCC patients from The Cancer Genome Atlas database and 50 from a published Taiwanese study. We identified 11 significantly mutated genes for OSCC, including 4 novel ones (ATG2A, WEE1, DST, and TSC2), of which WEE1 and ATG2A mutated with significantly higher rates in AN-related samples (P = 0.04 and P = 0.003, respectively). Mutational signature analysis revealed that AN-related OSCCs were specially characterized by the genomic signature of mismatch repair deficiency (dMMR), which could also predict the prognosis status of AN-related OSCC. In addition, an elevated PD-L1 expression was also observed in both AN-related patients (P = 3.71 × 10-11) and those with a high dMMR level (P = 1.99 × 10-4). Further differential expression analysis and in vitro experiments confirmed the role of dMMR in the development of OSCC induced by AN exposure. Taken together, this study first revealed the molecular profiles and highlighted the role of dMMR in AN-related OSCC among the Chinese population and identified that AN-related OSCC may represent a potential cohort for effective anti-PD-1/L1 immunotherapy.

MicroRNA deregulation and pathway alterations in nasopharyngeal carcinoma.

MicroRNAs (miRNAs) are a family of small non-coding RNA molecules of about 20-23 nucleotides in length, which negatively regulate protein-coding genes at post-transcriptional level. Using a stem-loop real-time-PCR method, we quantified the expression levels of 270 human miRNAs in 13 nasopharyngeal carcinoma (NPC) samples and 9 adjacent normal tissues, and identified 35 miRNAs whose expression levels were significantly altered in NPC samples. Several known oncogenic miRNAs, including miR-17-92 cluster and miR-155, are among the miRNAs upregulated in NPC. Tumour suppressive miRNAs, including miR-34 family, miR-143, and miR-145, are significantly downregulated in NPC. To explore the roles of these dysregulated miRNAs in the pathogenesis of NPC, a computational analysis was performed to predict the pathways collectively targeted by the 22 significantly downregulated miRNAs. Several biological pathways that are well characterised in cancer are significantly targeted by the downregulated miRNAs. These pathways include TGF-Wnt pathways, G1-S cell cycle progression, VEGF signalling pathway, apoptosis and survival pathways, and IP3 signalling pathways. Expression levels of several predicted target genes in G1-S progression and VEGF signalling pathways were elevated in NPC tissues and showed inverse correlation with the down-modulated miRNAs. These results indicate that these downregulated miRNAs coordinately regulate several oncogenic pathways in NPC.

Isolation of intracellular symbiotes by immune lysis of flagellate protozoa and characterization of their DNA.

A new method dependent on immune lysis is described for the isolation of intracellular symbiotes from two species of flagellate protozoa Blastocrithidia culicis and Crithidia oncopelti. The symbiote-containing flagellates are exposed to complement and antisera prepared in rabbits against symbiote-free organisms. The immune lysis seems to weaken the plasma membranes of the flagellates so that subsequent application of gentle shearing force liberates the intracellular entities in an undamaged condition. The symbiotes are then separated from other cellular components by DNAse digestion and differential centrifugation. The average recovery of symbiotes isolated by this method is 20%. Light and electron microscopy establishes the structural integrity and numerical abundance of isolated symbiotes in the final fractions. Integrity of symbiotes is further indicated by the high activity of a marker enzyme, uroporphyrinogen I synthetase. The DNA's of symbiote-containing and symbiote-free flagellates, and of isolated symbiotes were purified and compared after isopycnic centrifugation. The comparison establishes the presence of DNA's in symbiotes of both species. The guanine-cytosine (G-C) content of symbiote DNA differs from that of host DNA's in C. oncopelti, but resembles that of kinetoplast DNA in B. culicis. The latter observation was further shown by heat denaturation study. Renaturation kinetics indicate that the genome complexity of symbiote DNA in B. culicis is similar to that of bacteria.

A glycosylphosphatidylinositol (GPI)-negative phenotype produced in Leishmania major by GPI phospholipase C from Trypanosoma brucei: topography of two GPI pathways.

The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide), are glycosylphosphatidylinositol (GPI) anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPI-PLC) in L. major in order to examine the topography of the protein-GPI and polysaccharide-GPI pathways. In L. major cells expressing GPI-PLC, cell-associated gp63 could not be detected in immunoblots. Pulse-chase analysis revealed that gp63 was secreted into the culture medium with a half-time of 5.5 h. Secreted gp63 lacked anti-cross reacting determinant epitopes, and was not metabolically labeled with [3H]ethanolamine, indicating that it never received a GPI anchor. Further, the quantity of putative protein-GPI intermediates decreased approximately 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. However, GPI-PLC cleaved polysaccharide-GPI intermediates (glycoinositol phospholipids) in vitro. Thus, reactions specific to the polysaccharide-GPI pathway are compartmentalized in vivo within the endoplasmic reticulum, thereby sequestering polysaccharide-GPI intermediates from GPI-PLC cleavage. On the contrary, protein-GPI synthesis at least up to production of Man(1 alpha 6)Man(1 alpha 4)GlcN-(1 alpha 6)-myo-inositol-1-phospholipid is cytosolic. To our knowledge this represents the first use of a catabolic enzyme in vivo to elucidate the topography of biosynthetic pathways. GPI-PLC causes a protein-GPI-negative phenotype in L. major, even when genes for GPI biosynthesis are functional. This phenotype is remarkably similar to that of some GPI mutants of mammalian cells: implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma are discussed.

Human cutaneous lieshmania in a mouse macrophage line: propagation and isolation of intracellular parasites.

A mouse macrophage line, J774G8, supports continuous and prolific intracellular growth of Leishmania mexicana amazonensis, the etiological agent of a South American cutaneous leishmaniasis. The intracellular parasites from these infected cultures can be isolated with high recovery rate and purity by simple Percoll gradient centrifugation.

Nutritional significance of symbiotic bacteria in two species of hemoflagellates.

Symbiote-free strains of Blastocrithidia culicis and Crithidia oncopelti, obtained by chloramphenicol treatment, were compared nutritionally with normal, symbiote-containing strains. The symbiotic bacteria spare the flagellates requirements for exogenous hemin and for other nutritional factors present in liver extract.

Multiplication of a human parasite (Leishmania donovani) in phagolysosomes of hamster macrophages in vitro.

Leishmania donovani, the etiological agent of human visceral leishmaniasis, was grown in hamster peritoneal macrophages in vitro. By electron microscopy, using a lysosomal marker, these parasitic protozoa were seen to multiply within host cell phagolysosomes. The survival mechanism of this intracellular parasite is based apparently upon resistance to macrophage lysosomal enzymic digestion.

The 63-kilobase circular amplicon of tunicamycin-resistant Leishmania amazonensis contains a functional N-acetylglucosamine-1-phosphate transferase gene that can be used as a dominant selectable marker in transfection.

Tunicamycin (TM)-resistant Leishmania amazonensis has been found previously to contain amplified chromosomal DNA, existing exclusively as extrachromosomal circles of 63 kb. Fragments of this DNA cloned into plasmids were functionally analyzed by transfection of wild-type cells. A clone with a 15-kb fragment of the 63-kb circle was initially found to confer TM resistance. A library of the 15-kb fragment was then prepared and used in toto to transfect wild-type cells. The transfectants that emerged after selection were found to contain a plasmid with an insert of 4.6 kb. Evidence from deletion experiments suggests that this is the minimal transfection-effective fragment. Sequencing of the 4.6-kb DNA revealed 1.4-kb homolog of N-acetylglucosamine-1-phosphate transferase genes. The L. amazonensis gene is similar to those from two other sources in their deduced peptide sequence by 65 to 70% and in hydropathic characteristics. The L. amazonensis gene is amplified by more than 128-fold over the wild type and overproduces a major transcript of 2.4 kb in all transfectants. The endogenous copy of this gene was amplified by polymerase chain reaction from the wild type and cloned into pX-NEO, a Leishmania expression vector. Amplification of this plasmid in the transfectants by selection with G418 simultaneously made them resistant to TM. Evidence provided thus indicates that the 1.4-kb DNA is an N-acetylglucosamine-1-phosphate transferase gene whose amplification is responsible for TM resistance in Leishmania variants and transfectants.

Correlation between liver cirrhosis and risk of death from oral cancer: Taiwan cohort study.

BACKGROUND: A nationwide population-based cohort was used to examine the severity of liver cirrhosis and risk of mortality from oral cancer. METHODS: The cohort consisted of 3583 patients with oral cancer treated by surgery between 2008 and 2011 in Taiwan. They were grouped on the basis of normal liver function (n = 3471), cirrhosis without decompensation (n = 72) and cirrhosis with decompensation (n = 40). The primary endpoint was mortality. Hazard ratios of death were also determined. RESULTS: The mortality rates in the respective groups were 14.8 per cent, 20.8 per cent and 37.5 per cent at one year (p < 0.001). The adjusted hazard ratios of death at one year for each group compared to the normal group were 2.01 (p = 0.021) for cirrhotic patients without decompensation, 4.84 (p < 0.001) for those with decompensation and 2.65 (p < 0.001) for those receiving chemotherapy. CONCLUSION: Liver cirrhosis can be used to predict one-year mortality in oral cancer patients. Chemotherapy should be used with caution and underlying co-morbidities should be managed in cirrhotic patients to reduce mortality risk.

Cloning and sequencing a putative pyrophosphate-dependent phosphofructokinase gene from Entamoeba histolytica.

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) gene from Entamoeba histolytica was cloned from its genomic library and sequenced. The open reading frame has 1149 bp and codes for a protein of 41.5 kDa. The deduced amino acid sequence of E. histolytica PPi-PFK has 25 to 28% identity to the PPi-PFKs from Propionibacterium freudenreichii, Naegleria fowleri and potato. The amino acid residues known to contribute to the active site of PPi-PFK from P. freudenreichii are conserved.

Stage-independent splicing of transcripts two heterogeneous neighboring genes in Leishmania amazonensis.

Gene expression in trypanosomatid protozoa is largely regulated posttranscriptionally, e.g., 5' splice leader addition and 3' polyadenylation of mRNAs. We examined these events in Leishmania by mapping the splice sites of the transcripts from two different, but closely linked single-copy genes 2.3 kb apart. The coding regions of the approx. 1 kb upstream gene (P36) and the approx. 1.4 kb downstream gene (NAGT) produce approx. 2 and 3 kb mRNAs, respectively. Both genes were overexpressed in cells that were transfected with this bicistronic unit (> or = 7.5 kb), taking advantage of the NAGT as a selectable marker for tunicamycin-resistance. The transcripts from both genes were spliced constitutively at both ends, irrespective of their episomal or chromosomal expression in both leishmanial stages. Primer extension of the 5' UTRs and S1 nuclease protection of the 3' UTRs initially identified the major splice sites, corresponding to the genomic sequence at -205 bp and + approx. 900 bp of P36, and -1012 bp and + approx. 600 bp of NAGT. These splice sites, consistent with the size of the major transcripts, are among those mapped precisely by sequencing RT-PCR amplified 5' and 3' UTRs. The additional sites mapped by the latter are minor alternatives, especially abundant for transcripts of the downstream NAGT. All these minor splice sites are closer than the major splice sites to the coding region, indicating that the most distant splice sites are preferentially used. This preference creates a 387 bp 'gap' with polypyrimidine tracts in the intergenic region consistent with the model coupling splice leader addition with polyadenylation in pre-mRNA processing. The stage-independence of these events suggests that the 7.5 kb dicistronic unit is suitable for constructing Leishmania-specific constitutive expression vectors.

Leishmania donovani-macrophage binding mediated by surface glycoproteins/antigens: characterization in vitro by a radioisotopic assay.

A radioisotopic assay was developed to quantitate the binding of Leishmania donovani promastigotes to hamster peritoneal macrophages in vitro. The binding was temperature dependent and required no serum factors. Binding was reduced by preloading macrophages with zymosan granules or unlabeled promastigotes, but not with latex leads or opsonized erythrocytes. Binding was reduced by 10 mM EGTA that was reversible by the addition at an equimolar concentration of calcium, but not magnesium ions. Sialic acid, D-glucose, D-mannose and their derivatives reduced the binding, whereas L-fucose, D-galactose and their related sugars did not. Pretreatment of promastigotes with neuraminidase, alpha-mannosidase, alpha-N-acetylglucosaminidase or beta-glucosidase reduced their binding to macrophages. Prior trypsinization of either macrophages or promastigotes also substantially reduced the binding. At 4 degrees C, prior opsonization of promastigotes with subagglutination titers of antiserum doubled the level of binding but in combination with Protein A reduced it to 50% of its normal binding level. Prior opsonization of macrophages decreased their binding to promastigotes significantly at 4 or 37 degrees C. The results indicate that binding of Leishmania donovani promastigotes to hamster peritoneal macrophages is a ligand-receptor interaction involving their antigenic surface membrane proteins. The binding ligands of the parasites appear to have at least sialol, glucosyl, mannosyl and N-acetylglucosaminyl terminal residues as binding determinants. Thus, receptor-mediated endocytosis, defined in a broader sense, appears to be the mechanism by which leishmanias gain entry into macrophages.

N-glycosylation as a biochemical basis for virulence in Leishmania mexicana amazonensis.

Promastigotes of Leishmania mexicana amazonensis grown in vitro under different conditions showed variable degrees of virulence, as determined quantitatively by the size of the lesions and the number of intracellular parasites produced in mice and in cultured macrophages, respectively. Promastigotes newly transformed from amastigotes gave the highest degree of virulence, which decreased progressively with periods of their continuous in vitro cultivation. This loss of virulence was prevented by making virulent wildtype promastigotes resistant to tunicamycin, an inhibitor of N-acetylglucosamine-1-phosphate transferase in the dolichol pathway of protein glycosylation. In the wildtype cells, the progressive loss of virulence during a period of two years was marked by a gradual decrease in the activity of the glycosyltransferase, incorporation of 2-D-[3H]mannose and the expression of a surface glycoprotein (gp63). Virulent and avirulent wildtype cells differed in these activities by 3-4 fold. In contrast, during equivalent periods of in vitro cultivation, tunicamycin-resistant cells were found to consistently maintain the biochemical phenotypes of the virulent wildtype, except for a disproportional elevation of the glycosyltransferase activity. Thus, only a portion of the over-produced enzyme is relevant to leishmanial virulence in the drug-resistant variants. The bulk of its activity in these cells serves to overcome the inhibitory effect of tunicamycin responsible for their resistance to this drug, as shown previously. A role of N-glycosylation in leishmanial virulence is now indicated by studying cells of this phenotype selected by two independent methods. It is inferred that leishmanial virulence may be regulated by the level of the glycosyltransferases for N-glycosylation of proteins, e.g. gp63 in relation to their expression as virulent determinants.

Heme requirement and acquisition by extracellular and intracellular stages of Leishmania mexicana amazonensis.

The inability to synthesize heme, a well known metabolic defect of trypanosomatid protozoa, accounts for their growth requirement for heme compounds in vitro. We now extend this finding to a pathogen Leishmania mexicana amazonensis, especially the intracellular replicative stage of amastigotes in the macrophage. We measured the level of heme and its biosynthetic enzymes, aminolevulinate dehydratase and porphobilinogen deaminase in the parasites and in infected and non-infected macrophages of J774G8 line. Succinylacetone was used to inhibit heme biosynthesis. Leishmanias transform and grow only in medium containing either heme (usually supplied as hemin) or protoporphyrin IX (the latter is leishmanicidal at high concentrations). We detected 1.2, 8.5 and 25 pmol mg-1 protein of heme in amastigotes, promastigotes and macrophages, respectively. The activities of porphobilinogen deaminase and aminolevulinate dehydratase in macrophages were 70 and 2400 pmol h-1 mg-1 protein, respectively. Leishmania-infected macrophages gave the same results and leishmanias had negligible activities of these enzymes. Succinylacetone at 10(-9)-10(-3) M had no effect on leishmanias, but dose-dependently inhibited the activity of aminolevulinate dehydratase to a negligible level and lowered that of porphobilinogen deaminase in macrophages, resulting in a maximum of 66% reduction in intracellular heme. Amastigotes grew equally well in succinylacetone-treated and control untreated macrophages. The results suggest that L. m. amazonensis, incapable of heme biosynthesis, acquires heme exogenously from the culture medium, i.e., fetal bovine serum, independent of the heme synthesized by the macrophages.

Identification by extrachromosomal amplification and overexpression of a zeta-crystallin/NADPH-oxidoreductase homologue constitutively expressed in Leishmania spp.

A gene which overexpresses a 36-kDa protein (p36) in tunicamycin-resistant Leishmania was mapped by transfection and overexpression to the upstream region of the drug maker in the extrachromosomal amplicon. Complete sequencing of this region revealed a single open reading frame of about 1 kb. Authenticity of the cloned gene is verified by immunologic specificity of its recombinant products and sequence identity with a p36 peptide. The gene shares an overall sequence similarity of about 50% with members of the eukaryote alcohol dehydrogenase family at the amino acid level, including essentially all 13 evolutionarily conserved residues and a nucleotide-binding domain. The binding ligands for both structurally and catalytically important zinc atoms are absent, similar to the zeta-crystallin/NADPH:quinone oxidoreductase gene. Consistent with hydrophilicity of its primary sequence and the presence of a nucleotide binding site, p36 is a soluble molecule non-sedimentable at 105,000 x g and binds Blue Sepharose, elutable only with NADPH. The p36 gene is expressed constitutively in both stages of the wild-type and is conserved among all Leishmania species examined, suggestive of its functional significance different from evolutionarily related homologues.

Acid protease activity of a major surface membrane glycoprotein (gp63) from Leishmania mexicana promastigotes.

A unique protease with activity optimal at pH 4.0 and trailing toward the alkaline pH spectrum was detected with intact glutaraldehyde-fixed promastigotes of Leishmania mexicana amazonensis, indicating surface localisation of the enzyme. That this surface protease may be a virulence factor is suggested by its apparent roles in multiple steps during leishmanial infections of macrophages. Indeed, its specific activity was 2-2.5 fold higher on virulent cells than on avirulent cells. Several lines of evidence indicate that this acid protease activity is expressed by the major surface glycoprotein (gp63) of L. m. amazonensis. Monoclonal antibody affinity purified gp63 degraded serum albumin, hemoglobin, complement C3, immunoglobulin G and purified rat liver lysosomal proteins in their native forms. The specific activity is about 20-fold higher at pH 4.0 than at pH 7.5 and is about four-fold higher at the body temperature of the mammalian host (37 degrees C) than at that of the insect host (27 degrees C). The protease activity is sodium dodecyl sulphate-sensitive. Among various protease inhibitors tested, only heavy metal ions (1 mM), 1,10-orthophenanthroline (1 mM) and bestatin (100 ng ml-1) significantly inhibited gp63 acid protease activity by up to 80%. N-linked oligosaccharides of gp63 appear to be important for the stability of this molecule, possibly by preventing its autodegradation. Purified gp63 effected limited proteolysis of human complement C3 molecules at the physiological serum pH of 7.5 in a manner, which supports the idea of its participation in complement-receptor mediated endocytosis of promastigotes by macrophages.