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Topical lidocaine has been recently marketed as a new treatment for post-herpetic neuralgia. The aim of our study was to characterize the absorption profile of and systemic exposure to lidocaine from patch and gel formulations in normal volunteers, patients with post-herpetic neuralgia, and patients with acute herpes zoster. The bioavailability of lidocaine from the patch formulation averaged 3%, and was similar after single and repeated doses. Systemic exposure to lidocaine and monoethylglycinexylidide (MEGX), the primary active metabolite of lidocaine, after application of lidocaine gel or patches was minimal in normal volunteers, patients with post-herpetic neuralgia, and patients with acute herpes zoster. Considering the benefit versus risk of topical lidocaine, systemic absorption and toxicity of lidocaine seems not to be a significant risk., (Copyright 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association)

We report a case of an infant presenting with the rare association of tetralogy of Fallot, hypoplasia of the pulmonary arteries, and stenotic bicuspid aortic valve. Surgical correction, performed at 16 months of age, included aortic valvular commissurotomy, opening the right ventricular outflow tract (transannular patch), and ventricular septal defect closure. The postoperative course was favorable, and the child was discharged from the hospital. Three months after the procedure, the patient is in excellent condition, without cardiac medication.

Ulcerative colitis and colonic Crohn's disease (together known as inflammatory bowel disease or IBD) are both associated with increased risk for colorectal cancer. Although it is customary to emphasize differences in the biology of IBD-associated and sporadic colon cancer, we believe these are far outweighed by the similarities. These similarities suggest that they might have similar pathogenic mechanisms. Because the normal colon is arguably in a continual state of low-grade inflammation in response to its microbial flora, it is reasonable to speculate that both IBD-associated and sporadic colon cancer might be the consequence of bacteria-induced inflammation.

Ulcerative colitis and Crohn's disease (together known as Inflammatory Bowel Disease or IBD) are both associated with increased risk for colorectal cancer. Although it is conventional to emphasise differences between IBD-associated and sporadic colon cancer, such as a lower rate of Adenomatosis Polyposis Coli mutations and earlier p53 mutations, IBD-associated cancer has a similar dysplasia-cancer sequence to sporadic colon cancer, similar frequencies of major chromosomal abnormalities and of microsatellite instability and similar glycosylation changes. This suggests that IBD-associated colon cancer and sporadic colon cancer might have similar pathogenic mechanisms. Because the normal colon is arguably in a continual state of low-grade inflammation in response to its microbial flora, it is reasonable to suggest that both IBD-associated and sporadic colon cancer may be the consequence of bacteria-induced inflammation. We have speculated that the glycosylation changes might result in recruitment to the mucosa of bacterial and dietary lectins that might otherwise pass harmlessly though the gut lumen. These could then lead to increased inflammation and/or proliferation and thence to ulceration or cancer. The glycosylation changes include increased expression of onco-fetal carbohydrates, such as the galactose-terminated Thomsen-Friedenreich antigen (Gal beta1,3GalNAc alpha-), increased sialylation of terminal structures and reduced sulphation. These changes cannot readily be explained by alterations in glycosyltransferase activity but similar changes can be induced in vitro by alkalinisation of the Golgi lumen. Consequences of these changes may be relevant not only for cell-surface glycoconjugates but also for intracellular glycoconjugates.

Recent molecular characterizations of microbial communities from deep-sea hydrothermal sites indicate the predominance of bacteria belonging to the epsilon subdivision of Proteobacteria (epsilon Proteobacteria). Here, we report the first enrichments and characterizations of four epsilon Proteobacteria that are directly associated with Alvinella pompejana, a deep sea hydrothermal vent polychete, or with hydrothermal vent chimney samples. These novel bacteria were moderately thermophilic sulfur-reducing heterotrophs growing on formate as the energy and carbon source. In addition, two of them (Am-H and Ex-18.2) could grow on sulfur lithoautrotrophically using hydrogen as the electron donor. Optimal growth temperatures of the bacteria ranged from 41 to 45 degrees C. Phylogenetic analysis of the small-subunit ribosomal gene of the two heterotrophic bacteria demonstrated 95% similarity to Sulfurospirillum arcachonense, an epsilon Proteobacteria isolated from an oxidized marine surface sediment. The autotrophic bacteria grouped within a deeply branching clade of the epsilon Proteobacteria, to date composed only of uncultured bacteria detected in a sample from a hydrothermal vent along the mid-Atlantic ridge. A molecular survey of various hydrothermal vent environments demonstrated the presence of two of these bacteria (Am-N and Am-H) in more than one geographic location and habitat. These results suggest that certain epsilon Proteobacteria likely fill important niches in the environmental habitats of deep-sea hydrothermal vents, where they contribute to overall carbon and sulfur cycling at moderate thermophilic temperatures.

Goblet cell depletion occurs in various forms of colitis, but its mechanism is unknown. We have investigated two linked hypotheses: (i) that bacterial peptides, such as formyl-methionyl-leucyl-phenylalanine (fMLP), interact with epithelial cells inducing the release of chemokines, including interleukin-8 (IL-8), which in turn leads to the recruitment of neutrophils which release mucin secretagogues; (ii) that fMLP acts directly on epithelial cells to cause mucus secretion. Studies were performed to measure the effects of fMLP on the synthesis and secretion of IL-8 and mucus by the goblet cell differentiated colon cancer cell lines HT29-MTX (methotrexate-conditioned HT29 colonic adenocarcinoma cell line) and LS174T, and to assess the effects of neutrophil-derived secretagogues on goblet cell secretion in these cell lines. fMLP (0.1 microM) increased the secretion of IL-8 by 105% (P<0.0001) in HT29-MTX cells and by 401% (P<0.0001) in LS174T cells. fMLP also increased the synthesis and secretion of mucins by these cell lines, with maximal effects of 65% above control values for synthesis (P<0.01) and 73% for secretion (P<0.01). A dose-related increase (up to 67%; P<0.01) in mucin secretion was demonstrated in HT29-MTX cells in response to incubation with supernatant from activated neutrophils. This effect was largely (83%; P<0.02) inhibited by ICI 200,355, a specific inhibitor of neutrophil elastase. In conclusion, the bacterial peptide fMLP and neutrophil elastase are both potent mucus secretagogues for colon epithelial cells. fMLP also elicits release of the potent neutrophil chemoattractant IL-8 from colon epithelial cells. These findings support the hypothesis that the mucosal inflammation and mucus depletion seen in ulcerative colitis could result from interaction between bacterial peptides and the mucosa.

Increased mucosal expression of TF, the Thomsen-Friedenreich oncofetal blood group antigen (galactose beta1-3 N-acetylgalactosamine alpha-) occurs in colon cancer and colitis. This allows binding of TF-specific lectins, such as peanut agglutinin (PNA), which is mitogenic to the colorectal epithelium. To identify the cell surface TF-expressing glycoprotein(s), HT29 and Caco2 colon cancer cells were surface-labeled with Na[(125)I] and subjected to PNA-agarose affinity purification and electrophoresis. Proteins, approximately 110-180 kDa, present in HT29 but not Caco2 were identified by Western blotting as high molecular weight splice variants of CD44 (CD44v). Selective removal of TF antigen by Streptococcus pneumoniae endo-alpha-N-acetylgalactosaminidase substantially reduced PNA binding to CD44v. Immunoprecipitated CD44v from HT29 cell extracts also expressed sialyl-Tn (sialyl 2-6 N-acetylgalactosaminealpha-). Incubation of PNA 15 microg/ml with HT29 cells caused no additional proliferative effect in the presence of anti-CD44v6 mAb. In colon cancer tissue extracts (N = 3) PNA bound to CD44v but not to standard CD44. These data show that CD44v is a major PNA-binding glycoprotein in colon cancer cells. Because CD44 high molecular weight splice variants are present in colon cancer and inflammatory bowel disease tissue but are absent from normal mucosa, these results may also explain the increased PNA reactivity in colon cancer and inflammatory bowel disease. The coexpression of oncofetal carbohydrate antigens TF and sialyl-Tn on CD44 splice variants provides a link between cancer-associated changes in glycosylation and CD44 splicing, both of which correlate with increased metastatic potential.

Mucins in ulcerative colitis and colon cancer share common properties of reduced sulfation and increased oncofetal carbohydrate antigen expression. It has previously been shown that there is no simple correlation between these changes and the activity of the relevant glycosyl-, sialyl-, and sulfo-transferases. We examined mucin sulfation and expression of oncofetal Thomsen-Friedenreich (TF) antigen (galactosyl beta1-3N-acetylgalactosamine alpha-) in the goblet cell-differentiated human colon cancer cell line LS174T following treatment with bafilomycin A(1, )which raises intra-Golgi pH, or monensin, which disrupts medial-trans Golgi transport. Cells were dual-labeled with sodium [(35)S]-sulfate and D-[6-(3)H(N)]-glucosamine hydrochloride, or labeled with L-[U-(14)C]-threonine alone. Mucin was purified using Sepharose CL-4B gel filtration. Mucin sulfo-Lewis(a) and TF antigen expression were assessed using the F2 anti-sulfo-Lewis(a) monoclonal antibody and peanut agglutinin binding respectively. Bafilomycin (0.01 microM; 48 h) reduced total mucin sulfation, expressed relative to incorporation of glucosamine, to 0.50 +/- 0.04 d.p.m. [(35)S]-sulfate per d.p.m. [(3)H]-glucosamine compared to control, 0.84 +/- 0.05 (p < 0.001, n = 16). This was accompanied by 50.3 +/- 8.0% increased expression of TF antigen (p < 0.01) and 50.1 +/- 5.5% decreased expression of sulfo-Lewis(a) (p < 0.01). The reduced sulfate:glucosamine ratio was largely due to increased incorporation of glucosamine into newly synthesized mucin rather than reduction in total sulfate incorporation. In contrast, monensin only reduced total mucin glycosylation at concentrations > 0.1 microM and had no significant effect on mucin sulfation or TF expression. Intra-Golgi alkalinization affects mucin glycosylation, resulting in decreased mucin sulfation and increased expression of TF antigen, changes that mimic those seen in cancerous and premalignant human colonic epithelium.

A highly integrated, morphologically diverse bacterial community is associated with the dorsal surface of Alvinella pompejana, a polychaetous annelid that inhabits active high-temperature deep-sea hydrothermal vent sites along the East Pacific Rise (EPR). Analysis of a previously prepared bacterial 16S ribosomal DNA (rDNA) library identified a spirochete most closely related to an endosymbiont of the oligochete Olavius loisae. This spirochete phylotype (spirochete A) comprised only 2.2% of the 16S rDNA clone library but appeared to be much more dominant when the same sample was analyzed by denaturing gradient gel electrophoresis (DGGE) and the terminal restriction fragment length polymorphism procedure (12 to 18%). PCR amplification of the community with spirochete-specific primers used in conjunction with DGGE analysis identified two spirochete phylotypes. The first spirochete was identical to spirochete A but was present in only one A. pompejana specimen. The second spirochete (spirochete B) was 84.5% similar to spirochete A and, more interestingly, was present in the epibiont communities of all of the A. pompejana specimens sampled throughout the geographic range of the worm (13 degrees N to 32 degrees S along the EPR). The sequence variation of the spirochete B phylotype was less than 3% for the range of A. pompejana specimens tested, suggesting that a single spirochete species was present in the A. pompejana epibiotic community. Additional analysis of the environments surrounding the worm revealed that spirochetes are a ubiquitous component of high-temperature vents and may play an important role in this unique ecosystem.

Recently, we described a novel simian immunodeficiency virus (SIVlhoest) from a wild-caught L'Hoest monkey (Cercopithecus lhoesti) from a North American zoo. To investigate whether L'Hoest monkeys are the natural host for these viruses, we have screened blood samples from 14 wild animals from the Democratic Republic of Congo. Eight (57%) were found to be seropositive for SIV. Nearly full-length genome sequences were obtained for SIV isolates from three of these monkeys and compared to the original isolate and to other SIVs. The four samples of SIVlhoest formed a distinct cluster in phylogenetic trees. Two of these isolates differed on average at only about 5% of nucleotides, suggesting that they were epidemiologically linked; otherwise, the SIVlhoest isolates differed on average by 18%. Both the level of diversity and the pattern of its variation along the genome were very similar to those seen among isolates of SIVagm from vervet monkeys, pointing to similarities in the nature of, and constraints on, SIV evolution in these two species. Discordant phylogenetic relationships among the SIVlhoest isolates for different genomic regions indicated that mosaic viruses have been generated by recombination, implying that individual monkeys have been coinfected by more than one strain of SIV. Taken together, these observations provide strong evidence that L'Hoest monkeys constitute a natural reservoir for SIV.

The human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) appear to have originated by cross-species transmission of simian immunodeficiency virus (SIV) from asymptomatically infected African primates. Few of the SIVs characterized to date efficiently infect human primary lymphocytes. Interesting, two of the three identified to infect such cultures (SIVsm and SIVcpz) have appeared in human populations as genetically related HIVs. In the present study, we characterized a novel SIV isolate from an East African monkey of the Cercopithecus genus, the l'hoest monkey (C. l'hoesti), which we designated SIVlhoest. This SIV isolate efficiently infected both human and macaque lymphocytes and resulted in a persistent infection of macaques, characterized by high primary virus load and a progressive decline in circulating CD4 lymphocytes, consistent with progression to AIDS. Phylogenetic analyses showed that SIVlhoest is genetically distinct from other previously characterized primate lentiviruses but clusters in the same major lineage as SIV from mandrills (SIVmnd), a West African primate species. Given the geographic distance between the ranges of l'hoest monkeys and mandrills, this may indicate that SIVmnd arose through cross-species transmission from close relatives of l'hoest monkeys that are sympatric with mandrills. These observations lend support to the hypothesis that the primate lentiviruses originated and coevolved within monkeys of the Cercopithecus genus. Regarded in this light, lentivirus infections of primates not belonging to the Cercopithecus genus may have resulted from cross-species transmission in the not-too-distant past.

1. One hypothesis for the link between inflammatory bowel disease and primary sclerosing cholangitis is that neutrophil activators, such as bacterial chemotactic peptides or neutrophil granule products themselves, pass from the inflamed colon to the liver via an enterohepatic circulation. However, there are no data on biliary concentrations of neutrophil granule products in patients with active and inactive inflammatory bowel disease.2. Gall bladder bile was obtained at laparotomy from 42 patients with ulcerative colitis and 21 patients with Crohn's disease. Biliary lactoferrin and myeloperoxidase concentrations were quantified by ELISA.3. In active ulcerative colitis, the mean lactoferrin concentration in gall bladder bile of 2.8+/-0.40 mg/l was higher than that seen after colectomy (1.2+/-0.11 mg/l; P<0.0001) or in patients with pouchitis (1.8+/-0.34 mg/l; P=0.06). In active Crohn's colitis, the mean lactoferrin concentration was 3.7+/-0.9 mg/l, compared with 1.1+/-0. 24 mg/l in the post-colectomy group (P<0.05) and 3.1+/-0.71 mg/l in those with active ileitis or ileocolitis. In contrast, biliary myeloperoxidase concentrations were low and comparable in all groups, with a mean concentration in the 42 patients with ulcerative colitis of 11.2+/-1.9 microgram/l.4. In contrast to myeloperoxidase, biliary lactoferrin concentrations are increased in active ulcerative colitis and Crohn's disease, and fall with colectomy and with disease remission. These findings indirectly support the hypothesis that bacterial chemotactic peptides (which induce selective degranulation of neutrophil secondary granules), and/or lactoferrin itself, undergo an enterohepatic circulation.

The genomes of simian immunodeficiency viruses isolated from African green monkeys (SIVagm) contain a single accessory gene homolog of human immunodeficiency virus type 1 (HIV-1) vpr. This genomic organization differs from that of SIVsm-SIVmac-HIV-2 group viruses, which contain two gene homologs, designated vpr and vpx, which in combination appear to share the functions of HIV-1 vpr. The in vitro role of the SIVagm homolog was evaluated with molecularly cloned, pathogenic SIVagm9063-2. These studies revealed that this gene shares properties of HIV-1 vpr, such as nuclear and virion localization. In addition, SIVagm mutants with inactivating mutations of vpr are unable to replicate in nondividing cells, such as macaque monocyte-derived macrophages, but replicate to almost wild-type levels in a susceptible human T-cell line. The transport of virus preintegration complexes into the nucleus in primary macrophages, as measured by the production of unintegrated circular viral DNA, is less efficient for the mutant viruses than it is for the wild-type virus. SIVagm mutants also replicate inefficiently in primary macaque peripheral blood mononuclear cells, with a propensity for substitutions that remove the inserted inactivating stop codon. These data, in conjunction with recent findings that the Vpr protein is capable of inducing G2 arrest, are consistent with designation of this SIVagm accessory gene as vpr to reflect its shared functions and properties with HIV-1 vpr.

Background: All 5-aminosalicylic acid (5-ASA) preparations are potentially nephrotoxic, but there has been concern that newer delivery systems may increase this risk, either because of altered absorption or altered metabolism. Previous studies of 5-ASA absorption and excretion have usually either been performed in healthy controls or have only examined short-term therapy. 5-ASA and N-acetyl-5-ASA have therefore been measured in blood samples, and N-acetyl-5-ASA in urine samples, from patients with ulcerative colitis on long-term maintenance with different 5-ASA preparations and compared with sensitive markers of renal damage., Methods: Patients receiving mesalazine (Asacol) (n = 13), sulphasalazine (n = 12) or olsalazine (Dipentum) (n = 8), all at doses within the recommended range were studied. Six-hour and trough serum concentrations of 5-ASA and N-acetyl-5-ASA and 24-h urinary excretion of N-acetyl-5-ASA were measured by high-performance liquid chromatography., Results: Absorption of 5-ASA, assessed as 24-h excretion of N-acetyl-5-ASA expressed as molar % of ingested dose, was greater in patients receiving mesalazine, 23.25 +/- 10.65% (mean +/- s.d.; n = 13), than those receiving sulphasalazine (11.16 +/- 10.52%, n = 12; P = 0.003) or olsalazine (9.70 +/- 3.89%, n = 8; P < 0.002). The ratio of 5-ASA: N-acetyl-5-ASA in the serum 6 h after dose was also greater with mesalazine (1.02 +/- 0.44, mean +/- s.d.) than sulphasalazine (0.54 +/- 0.44, P < 0.02) or olsalazine (0.38 +/- 0.44, P < 0.005). Urinary markers of tubular damage were increased in four of 33 patients, but showed no correlation with concentration of 5-ASA or N-acetyl-5-ASA in serum and N-acetyl-5-ASA in urine, nor with lifetime dose or average daily dose of 5-ASA., Conclusions: In patients with ulcerative colitis receiving maintenance 5-ASA therapy there was greater absorption and less acetylation of 5-ASA from mesalazine (Asacol) compared with sulphasalazine or olsalazine, but no evidence from this study that this resulted in increased nephrotoxicity.

Although ovine lentiviruses have been described in the United States since the early part of this century, North American strains of sheep lentiviruses remain relatively uncharacterized at the molecular level. The LTR of a North American ovine lentivirus, OLV-CU1, was found to be closely related at the molecular and functional levels to visna virus, the Icelandic ovine lentivirus. Sequence analysis of the LTR revealed high identity to other ovine and caprine lentiviruses in key regulatory elements of the upstream promoter region (-25 to -115). However, the R region of the LTR was much less homologous. Transcriptional control of OLV-CU1 in transient transcriptional assays required a conserved putative AP-4 region and possibly an AP-1 like element in the upstream promoter region for moderate to high levels of transcription, much like visna virus. In contrast to visna virus, the downstream region beyond the transcriptional start site was required for virus-specific transactivation.

1. We speculated that corticosteroids might cause beneficial stimulation of mucus synthesis, since this is a known action of carbenoxolone, itself a corticosteroid, and has also been proposed as a possible mechanism for the protective effect of smoking on ulcerative colitis. We have therefore compared the effects of corticosteroids including carbenoxolone, and nicotine on mucin synthesis, assessed by incorporation of N-[3H]acetylglucosamine into mucin by colonic epithelial biopsies in culture. 2. In histologically normal biopsies from the left colon, hydrocortisone and prednisolone caused a very marked concentration-dependent increase in mucin synthesis, with maximal effect (580 and 300% of control values respectively) at 6 mumol/l [P < 0.001, n = 35 biopsies (seven patients)] and 1.5 mumol/l [P < 0.001, n = 35 (seven patients)] respectively. The maximal effect of hydrocortisone was significantly greater than that of prednisolone (P < 0.05). Carbenoxolone, 0.17 mmol/l, also increased mucin synthesis in the left colon by 242% [P < 0.05, n = 15 (three patients)]. In contrast, these corticosteroids caused only a small, non-significant increase in mucin synthesis in the histologically normal right colon; fludrocortisone, 2 and 20 mumol/l, and aldosterone, 0.1-10 mumol/l, had no effect. Nicotine significantly increased mucin synthesis (180-220% of control values) between 62.5 nmol/l and 6.25 mumol/l (P < 0.05 at all concentrations) in both the right and left colon. In biopsies from the relatively uninvolved right colon of patients with ulcerative colitis, corticosteroids and nicotine caused relatively smaller increases in mucin synthesis. 3. The marked stimulation of mucin synthesis by corticosteroids suggests that this may account, at least in part, for their therapeutic effect in ulcerative colitis.

Studies of primate lentivirus phylogeny over the past decade have established a minimum of five related, but genetically distinct, groups of simian immunodeficiency virus (SIV), each originating from a different African primate species. The hypothesis that HIV-2 (and SIVmac) arose by cross-species transmission from sooty mangabeys (Cercocebus atys has been strengthened by a more detailed characterization of the SIVsm/SIVmac/HIV-2 group of viruses. SIV from all four subspecies of African green monkeys (SIVagm) have been characterized with an apparent chimeric genome structure of SIVagm from West African green monkeys. Although these naturally infected primates remain healthy, cross-species transmission to other primate species may result in immunodeficiency, as caused by SIVsm infection of macaque monkeys (Macaca sp.) and recently, SIVagm infection of pig-tailed macaques (M. nemestrina). Studies of variation within infected individuals have been facilitated by adaptation of the techniques of heteroduplex analysis and single-stranded conformational polymorphism of PCR generated fragments.

Previous studies have shown the presence in faeces of sulphatases, sialidases, glycosidases, and proteases relevant to mucus degradation, but the relative role of these enzymes in the degradation of colonic mucus has been unclear. A total mucinase assay using 14C threonine biologically labelled human colonic mucin as substrate was therefore developed in this study. Faecal mucinase activity of a pooled normal faecal filtrate was capable of removing 80% of the 14C threonine label from mucin within eight hours incubation, but 20% remained intact despite prolonged incubation. The pH profile of mucinase activity is broad (pH 4.5-9.5) suggesting contribution from multiple enzymes. Mucinase activity was reduced by preincubation with 100 micrograms/ml chymostatin (82.8%), 0.5 mg/ml EDTA (91.6%), and 4 g/l bismuth subsalicylate (72.0%). All 55 faecal samples studied contained detectable mucinase activity, measured as dpm release/micrograms protein/hour, which was greater in samples from patients with ulcerative colitis (n = 17, median 52.7, interquartile range 32.9-66.9), than controls (n = 26, 34.4, 26.8-40.4, p < 0.02) or patients with Crohn's disease (n = 12, 35.5, 17.5-55.7, p < 0.05). There was, however, no significant difference in faecal mucinase activity between inactive and active ulcerative colitis. These results suggest that faecal mucinase activity is one factor contributing to the thin mucus layer in ulcerative colitis and represents a potential target for drug treatment.

Avian gastrin is a gastric acid secretagogue, but is structurally more closely related to mammalian cholecystokinin, which is functionally distinct from gastrin. This apparently anomalous structure/activity relationship raises important issues for understanding the evolution of regulatory peptides and the mechanisms that control their expression. To clarify the possible mechanisms, we have determined the sequence of a 6.5-kb BamHI genomic DNA fragment that includes the entire avian gastrin transcriptional unit. The complete cDNA sequence, determined by anchored PCR, encoded a precursor of 105 amino acids. Conserved primary amino acid structures were limited to a decapeptide determining biological activity, and essential sites for post-translational processing. Significantly, however, the genomic regulatory regions, and introns, were unlike those of any previously reported gastrin/cholecystokinin gene. The avian gastrin gene contained no recognizable TATA-box motif, a feature unique to this gene family, but had a G+C-rich region immediately upstream of the transcription initiation site, and a Z-DNA purine-pyrimidine repeat sequence. Moreover, physiological regulation of the avian gastrin gene differed markedly from that observed in mammals, in that the important local paracrine downregulation by antral somatostatin was absent; instead, evidence for a hormonal regulation was demonstrated. The data indicate that genomic regulatory elements within regulatory peptide families such as the gastrin/cholecystokinin family, and mechanisms of physiological control, are not conserved during evolution, even though biologically important amino acid sequence information is retained.

Increased binding of the lectin peanut agglutinin is a common feature in epithelial malignancy and hyperplasia. This may have considerable functional importance in the intestine by allowing interaction between the epithelium and mitogenic lectins of dietary or microbial origin. Peanut agglutinin binds the disaccharide Thomsen-Friedenreich (TF, T or core 1) blood group antigen, Gal beta (1-3) GalNAc alpha-, but is not totally specific for this site. Consequently, there has been controversy about the presence of this structure in colon cancer; studies with anti-TF monoclonal antibodies have failed to detect it. We have examined the presence of TF antigen in colonic mucus glycoprotein (mucin) using endo-alpha-N-acetylgalactosaminidase (O-Glycanase), which specifically catalyzes the hydrolysis of TF antigen from glycoconjugates. Samples of adenocarcinoma, inflammatory bowel disease (ulcerative colitis), and normal mucin were treated with O-glycanase, the liberated disaccharide was separated from the glycoprotein and analyzed using dual CarboPac PA-100 column high performance anion-exchange chromatography coupled with pulsed amperometric detection. O-Glycanase treatment released increased amounts of TF antigen from both colonic adenocarcinoma (8.0 +/- 3.9 ng/micrograms protein, n = 11; P < 0.0001 ANOVA) and ulcerative colitis mucin (3.3 +/- 0.3 ng/micrograms protein, n = 5; P = 0.04) compared with mucin samples from histologically normal mucosa distant from carcinoma (1.5 +/- 1.1 ng/micrograms protein, n = 9). However, after mild acid treatment to remove sialic acids and fucose, releasable TF antigen was increased in all nine of these histologically normal mucin samples (5.5 +/- 2.6 ng/micrograms protein, P < 0.0002). We conclude that TF antigen is an oncofetal antigen which is expressed in colon cancer, but is concealed by further glycosylation (sialylation and/or fucosylation) in the normal colonic mucosa.

Sheep lung dipeptidase was released from a plasma membrane preparation by digestion with a phosphatidylinositol-specific phospholipase C. The dipeptidase was purified to homogeneity using affinity chromatography followed by high performance liquid chromatography. The NH2-terminal sequence was determined and employed to prepare a probe to clone the c-DNA of the enzyme. The primary structure of sheep lung dipeptidase, deduced from the c-DNA exhibited a high homology to kidney dipeptidases cloned from other animal species. Northern analysis detected the m-RNA of the dipeptidase in lung, kidney, and intestinal tissues of the sheep.

The extent of virus genetic variation within tissues and peripheral blood mononuclear cells (PBMC) from two simian immunodeficiency virus (SIV)-infected macaques was analyzed. The products of PCR amplification of two regions, region 1 (SIV V1 region) and region 2 (region corresponding to the human immunodeficiency virus V3 cysteine loop and part of the C3 region immediately downstream), of the SIV envelope were examined for single-stranded conformation polymorphism followed by sequence analysis of selected clones. The V1 region of the SIV envelope of viruses present within lymphoid tissues displayed extensive heterogeneity, while viral populations within the PBMC and brain appeared to be less variable. Region 2 heterogeneity in both animals was generally confined to three residues in a tissue-specific manner. In addition, virus from the brains of both animals appeared to be distinct compared with viruses present in other tissues and PBMC of the same animal, both in the pattern of PCR-single-stranded conformation polymorphism SCP and in the sequence of region 2. These studies revealed that the tissues of SIV-infected macaques were a reservoir for viral variants distinct from those seen in PBMC.

Preparations for Joint Commission on Accreditation of Healthcare Organizations (JCAHO) surveys are rarely described in the literature. This article is intended to provide much-needed information on survey preparation processes, not only to Department of Veterans Affairs medical centers, but also to all hospitals undergoing surveys of biopsychosocial delivery of care. Beginning in 1995, JCAHO will apply biopsychosocial reviews to all assessments of patients. Facilities whose preparations include those detailed in this article will be better prepared for such reviews.

The mechanisms of action of gastrin and gastrin releasing peptide (GRP) in stimulating gastric acid secretion were examined in the anaesthetized turkey. Gastrin and GRP produced dose-dependent increases in acid secretion that were inhibited by the gastrin/CCK-B antagonist CI988. The antagonist did not affect the acid secretory responses to histamine or carbachol. A GRP antagonist (M216140) inhibited the acid response to GRP, but not gastrin. The results suggest that in birds, GRP stimulates acid secretion by release of gastrin, which acts in turn on classical gastrin/CCK-B receptors in the proventriculus.

Neutral reduced oligosaccharides are in general not sufficiently retained to achieve adequate separation and reproducible chromatography using high-pH anion-exchange chromatography. We describe a method to increase the retention using two columns in series. This method has been applied to the separation of oligosaccharides purified from human meconium glycoproteins, obtained as their alditols after alkaline-borohydride release of oligosaccharides. The neutral oligosaccharide alditols were significantly retained upon two CarboPac PA-100 columns, connected in series, and eluted in 80 mM sodium hydroxide between 4 and 10 min. Three sialylated alditols studied were substantially retained and could be eluted in a gradient of 0-500 mM sodium acetate-80 mM sodium hydroxide between 10 and 45 min. The elution patterns were based on their size, charge and linkage, such that oligosaccharide alditol isomers could be separated.

High performance gel filtration on monodisperse cross-linked agarose (Superose 6) has been assessed as a system for purification of mucus glycoproteins. Comparison with the conventional two-step purification of mucus glycoprotein by Sepharose CL4B gel filtration followed by caesium chloride density gradient centrifugation shows that purification by high performance gel filtration is at least as thorough, yielding mucin that is free from non-mucin glycoproteins as defined by buoyant density, mobility on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and absence of Concanavalin A binding (mannose-containing) material. This technique allows mucus glycoprotein to be purified from lyophilized crude mucin in 120 min compared with approximately 72 h using the conventional techniques. This makes the comparative study of mucus glycoprotein changes in disease states much more feasible.

A lentivirus has been isolated from a Finnish ewe with ovine progressive pneumonia in a closed upstate New York flock. We demonstrated that the virus, designated ovine lentivirus strain CU1 (OLV-CU1), is biologically, biochemically and molecularly related to, but distinct from, previously described sheep and goat lentiviruses. Nine of 32 ewes (from the affected flock) with precipitating antibodies for ovine lentivirus also produced antibodies that were able to neutralize the infectivity of OLV-CU1. The virus replicated in cultured sheep fibroblasts and caused the formation of large multi-nucleated cells. OLV-CU1-specific RNA transcripts found in infected cells and virion antigenic proteins were similar to those of other small ruminant lentiviruses. However, the virus was distinguished from other isolates at the DNA level by nucleic acid hybridization, restriction endonuclease mapping and partial sequencing of the virus genome.

Worldwide, traffic deaths are 500,000 per year and growing. Four nations, China, India, Russia, and the United States of America, have reached levels of 45,000-50,000 traffic deaths per year, although the nature of the problem is quite different in fully motorized countries from what it is in motorizing countries. In motorizing countries the death rates per 10,000 motor vehicles are much higher than in motorized countries, and also the traffic stream is much more varied. Research for the reduction of traffic injuries is funded at a proportionately much lower level than that for heart disease, cancer, or AIDS. Part of the reason for this is the failure of governments to view and to treat the problem of traffic injuries as a public health problem, but rather to view it solely as a transport problem or as a police matter. A case is made for global mechanisms for the transfer of traffic injury reducing technology from fully motorized nations to motorizing nations.

Chickens treated with the H+/K+ ATPase inhibitor omeprazole, to inhibit gastric acid secretion, failed to gain weight and showed decreased food intake compared with controls. The gastrin antagonist PD134308 reversed the action of omeprazole on food intake. Since exogenous gastrin decreased food intake, and since omeprazole increased plasma gastrin concentrations, the results suggest that elevated plasma gastrin in chicken exerts a satiety effect.

Statewide crash data bases from nine states were subjected to time series analyses to detect changes in injuries associated with onset of seat belt laws in the respective states. In each of 18 analyses involving drivers covered by the law observed casualties were below the number forecast on the basis of prior experience and assuming that no law had been enacted. In the case of others, not covered by the law, observed injuries were equally often above or below forecast. Relative to covered drivers not only were the numbers below forecast, but in 12 of the 18 instances there was a statistically significant indication of an abrupt decrease the month the law began.

This study investigated the effects of a peer teaching procedure, combined with student letter-writing activities, on the acquisition and generalization of capitalization skills. Three students, aged 9 years, obtained instruction from peer partners that included (a) an introduction and review of capitalization rules, (b) feedback on each participant's previous capitalization work, and (c) guided and independent practice on sentences that required capitalization. All three students demonstrated acquisition of the capitalization rules after participating in the teaching sessions with one or two peer partners. In addition, there were increases in capitalization accuracy in participants' letters to peers who did not serve as teaching partners, thus demonstrating a measure of across-peer (stimulus) generalization. Mixed results were obtained on a response generalization task (sentence writing). Finally, sentencing-writing activities also showed that two of the peer partners substantially improved their use of capitalization skills as a result of teaching the target students.

The North Carolina Seat Belt Law required an evaluation of the effectiveness of the act with a report of the findings to the Legislature three years after the law went into effect. This paper addresses changes in statewide belt usage and in occupant injury associated with that law. Observational data collected bimonthly from a probability sample of 72 sites stratified by geographic region, rural/urban location, road type, and time of day show that belt use rose from a baseline rate of 25% to a warning ticket phase rate of 45%. Belt use then reached 78% upon enforcement and is now nearly 64%. Time series analysis showed that statistically significant reductions in percentages of moderate and serious injuries occurred at the beginning of both the warning ticket and the enforcement phases. Forecasts of injuries and deaths were also developed from the time series models and were compared with observed totals. Warning tickets brought about a modest 5.4% reduction in serious injuries; fatalities among occupants covered by the law showed no change. In contrast, the subsequent enforcement phase saw a reduction of 11.6% in fatalities and 14.6% in serious or worse injuries. This represents an estimated annual savings of 131 lives and over 2,300 serious injuries in North Carolina during the 18 months following onset of enforcement.

The isolation of bombesin-related peptides in chicken proventriculus was monitored by radioimmunoassay using a C-terminal specific bombesin antibody. Two peptides were identified, one corresponded to the 27-residue, chicken gastrin-releasing peptide (GRP-27) previously identified; the other corresponded to its C-terminal hexapeptide. Chicken GRP-27 stimulated pancreatic and gastric acid secretion in anaesthetized turkeys, but the hexapeptide was inactive. No evidence could be found to suggest that the hexapeptide was an artifact of degradation generated during extraction or isolation. It is proposed that the hexapeptide is produced either by chymotryptic-like cleavage of GRP-27 or by trypsin-like cleavage followed by two cycles of dipeptidylaminopeptidase cleavage. This type of biosynthetic processing may be more common than formerly supposed.

Sheep lung dipeptidase was released from a lung membrane preparation by digestion with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis. The total enzyme activity released into the supernatant was 4- to 5-fold greater than that measured in the intact membrane prior to solubilization. The release of the peptidase from the membrane by this treatment is typical of proteins anchored to the lipid bilayer by a covalent attachment of phosphatidylinositol via a C-terminal glycolipid extension. The solubilized lung peptidase was further purified by ammonium sulfate fractionation followed by affinity chromatography and high-pressure liquid chromatography. A linear relationship between log molecular weight and elution volume for proteins of known molecular weight was established using a Toya Soda TSK 3000 high-pressure liquid chromatography column, and the molecular weight of the lung dipeptidase was estimated at 105,000. The peptidase activity against glycyldehydrophenylalanine of the purified enzyme co-chromatographed in high-pressure liquid chromatography with the activity that converted leukotriene D4 to leukotriene E4. In kinetic studies using leukotriene D4 as substrate, the relationship between the rate of hydrolysis and enzyme concentration was shown to be linear over the range 20 ng to 98 ng enzyme. Values of Km and Vmax for the dipeptidase using leukotriene D4 as substrate were 43 +/- 6 microM and 11,200 +/- 400 nmol/min per mg, respectively. Inhibition of the conversion of leukotriene D4 to leukotriene E4 was observed with a series of inhibitory agents. Cilastatin, bestatin and chloracetyldehydrophenylalanine were all effective at the micromolar level with cilastatin proving to be the most effective inhibitor. Dithiothreitol was effective within the millimolar range.

Purified human renal dipeptidase was shown to exhibit no detectable activity against substrates that are characteristic for other known mammalian peptidases. The enzymic activities that were assayed were: aminopeptidase A, aminopeptidase B, aminopeptidase M, aminopeptidase P, and tripeptidase. A quantitative assay for renal dipeptidase was developed which measures the rate of release of glycine from glycylpeptides by pre-column derivatization of the amino acid with phenylisothiocyanate followed by high-performance liquid chromatography. The ratio of Vmax/Km for a series of dipeptides was used as an index of the enzyme's preference for substrates. According to the data obtained, the enzyme prefers that a bulky, hydrophobic group of the dipeptide be located at the N-terminal position. This suggests that the substrate-binding site of the enzyme may provide a hydrophobic pocket to accommodate the hydrophobic moiety at the N-terminus of the dipeptide. The unsaturated dipeptide substrate, glycyldehydrophenylalanine, was employed in spectrophotometric assays to provide kinetic analyses of enzymic inhibition. The inhibitory effect of dithiothreitol was immediate, and the kinetic data indicated reversible, competitive inhibition. These results suggest that the inhibitor competes with substrate for a coordination site of zinc within the active site of the enzyme. The reaction of renal dipeptidase with the transition-state peptide analog, bestatin, was time dependent, and velocity measurements were made after the inhibitor had been incubated with the enzyme until constant rates were observed. These steady-state rate measurements, made following preincubation of enzyme with inhibitor, were employed to show that bestatin caused apparent non-competitive inhibition of the enzyme. The inhibitory effect of the beta-lactam inhibitor, cilastatin, upon the oligomeric dipeptidase was shown to be competitive. Graphical analysis of this inhibition indicated that the subunits of the enzyme react independently during enzymic catalysis and that the catalytic event is not influenced by cooperativity between sites on the subunits. The conversion of leukotriene D4 to leukotriene E4 in the presence of human renal dipeptidase was demonstrated by HPLC procedures. This bioconversion reaction was quantitated by derivatizing the glycine produced by cleavage of the cysteinylglycine bond and isolating this derivative as a function of time. The relationship between the purified enzyme concentration and enzyme activity against leukotriene D4 was shown to be linear over the enzyme concentration range of 1 ng through 69 ng in this assay.(ABSTRACT TRUNCATED AT 400 WORDS)

Renal dipeptidase, previously identified as a component of renal microvilli, has been investigated to determine its orientation within this organelle. Digestion of porcine renal microvilli with papain released essentially all of aminopeptidase M, an outer membrane marker enzyme from the microvilli within one hour; whereas less than 10% of renal dipeptidase was released under the same conditions. Antibody to purified renal dipeptidase produced 50% inhibition of the purified enzyme at an antibody/antigen molar ratio of 2:1. Inhibition by the renal dipeptidase-directed antibody was not observed when the enzyme was bound within the microvillar structure. Demembranation of the microvilli with Triton X-100 resulted in a distribution of 68% of renal dipeptidase in the insoluble pellet and 32% in the soluble supernatant. The same detergent treatment released 92% of animopeptidase M into the supernatant. These results indicate that renal dipeptidase is not located at the luminal surface of the microvillus membrane where it would be available for release of papain, inhibition by antibody, or solubilization by detergent. Fractionation of the Triton-insoluble pellet with 2 M NaCl resulted in the release of 64% of the peptidase into a pellicle fraction separated from insoluble pellet and soluble supernatant. Finally extraction of Triton-insoluble pellet with 0.05 mM ATP-0.10 mM MgCl2 X 6 H2O solubilized 57% of the renal dipeptidase.

In contrast to other kinds of phospholipases, phospholipases D that are toxic for humans and animals are not commonly encountered as constituents of venoms or as products of pathogenic microorganisms. Toxic phospholipases D are present, however, in the venom of the brown recluse spider (Loxosceles reclusa) and in supernatants or filtrates of cultures of Corynebacterium pseudotuberculosis. Although the two enzyme toxins are derived from phylogenetically disparate entities, they are similar in molecular weight, charge, substrate specificity, and in several biological activities. They are immunologically distinguishable.

The ability to adapt to a large daily intake of unleavened bread made from wheaten wholemeals of high extraction rate was examined in two young Americans who had not previously consumed fiber, phytate-, and phosphate-rich bread of this type. Adaptation was evaluated by comparing balances of zinc, calcium, magnesium, and phosphorus for a 12 day period after ten days of bread consumption with those during a similar period after 50 days and also by observing changes in blood composition during this period of bread consumption. Significantly negative balances of zinc, magnesium and phosphorus were observed in the initial period. However, all had become positive or nearly so after 50 days. By contrast, calcium balances which were also negative during the initial period remained negative at the end of the study. Plasma calcium and zinc concentrations had declined to low normal or subnormal values at this time. The failure to overcome the disturbing effects of consumption of wholemeal bread upon calcium metabolism occurred despite daily exposure to sunshine for many hours under conditions favorable for Vitamin D biosynthesis.

Kinetic analyses of monoanion inhibition and 15Cl nuclear magnetic resonance at 5.88 MHz were employed to study monoanion interactions with the zinc metalloenzyme, renal dipeptidase. The enzyme-catalyzed hydrolysis of glycyldehydrophenylalanine exhibited competitive inhibition when the reaction rate was determined in the presence of the monovalent anions fluoride, chloride, bromide, iodide, azide, nitrate, or thiocyanate or upon the addition of the divalent anion, sulfate. Competitive inhibition was produced by these anions. One anion was bound per enzyme molecule, and except in the case of fluoride all of the anions appeared to bind at the same site. Cyanide ion produced a much more effective inhibition of renal dipeptidase than the other monoanions, and it was shown that two cyanide ions were bound per enzyme molecule. An investigation of the effect of pH upon monoanion inhibition suggested that the anion inhibitors bind to the group with a pK of approximately 7.8. Complete dissociation of this group (approximately pH 8.4) eliminates the inhibitory effect of anions. The 35Cl line broadening produced by renal dipeptidase in 0.5 M NaCl solutions was 100 times more effective than that produced by equivalent concentrations of aquozinc(II). The line broadening was dependent upon the concentration of the metalloenzyme and independent of the frequency of the exciting radiation. When zinc ion was removed from the metalloenzyme by dialysis or when chloride was titrated from the metalloenzyme by cyanide, line broadening was decreased. Treatment of renal dipeptidase with saturating concentrations of the competitive inhibitor, guanosine triphosphate, in the presence of 0.5 M NaCl also produced a significant decrease in the 35Cl line width. The 35Cl line broadening produced by renal dipeptidase was shown to decrease with increasing pH through the range pH 5.8-10.8. This line-width variation with pH appeared to result from the titration of a site on the metalloprotein with an approximate pK of 7.4. Temperature studies of 35Cl line broadening by the metalloenzyme in the presence of chloride and cyanide inhibitors suggest that the fast exchange process pertains and that the dominant relaxation mechanism is quadrupolar in nature.

Theoretical predictions from a simulation model of insect distributions and dispersal among isolated food plants have been tested with data gathered from 13 Cinnabar moth populations. Agreement with the predictions was good. Egg batch size was equal to or slightly larger than the number which could be supported by the average food plant. Egg batch distribution was more clumped when density was high and when egg batch size was small relative to food plant size. The tendency for larval dispersal was lower in populations in areas where plants were widely spaced. These findings indicate that the Cinnabar moth has the genetic or phenotypic flexibility to adjust egg batch size, egg distribution, and larval dispersal to characteristics of the habitat. A hypothesized model is proposed to describe the interactions between larval dispersal, food plant response to defoliation, and population fluctuations for the Cinnabar moth and its food plant, tansy ragwort.

Heat-extracted antigens from seven species of Bacteroides were used in passive hemagglutination and counterimmunoelectrophoretic tests. Sera from 87 normal persons (group I) and 15 patients with ulcerative colitis (group II) were of low and equal reactivity in passive hemagglutination tests; all positive tests were eliminated by 2-mercaptoethanol reduction of the sera. When these same sera were tested by counterimmunoelectrophoresis with six of the Bacteroides antigens, no significant difference in the percentage of positive reactions was noted. However, using the chi-square test, the seventh antigen, prepared from Bacteroides vulgatus, successfully distinguished the two populations at the 0.025 level. Counterimmunoelectrophoretic tests with the B. vulgatus antigen also provided a means to separate the patients in group II with active disease from those in remission at a P value of 0.01. All the sera from 12 patients with defined Crohn's disease activity indexes reacted with the B. vulgatus antigen in counterimmunoelectrophoretic tests. Reduction and alkylation of patient sera with 2-mercaptoethanol and iodoacetamide removed detectable antibody in 78% of the samples, which suggested a dominant role of immunoglobulin M in the response to Bacteroides antigens.

Isolated renal brush border microvilli vesicles were employed to study the uptake of radiolabel from L-Ala. [3H]Gly and D-Ala.[3H]Gly as well as to determine the presence of dipeptidase activity. Microvilli vesicles were prepared from porcine kidney cortex by differential centrifugation through hypotonic Tris buffer containing Mg2+. The microvilli vesicles transiently accumulated radiolabel from L-Ala. [3H]Gly to higher levels than were initially present in the incubation medium (overshoot phenomenon). This accumulation was dependent on the presence of an inward-directed (extravesicular greater than intravesicular) Na+ gradient and was osmotically sensitive and linear with respect to microvilli protein concentration. Analysis of intravesicular contents revealed that all 3H uptake from L-Ala. [3H]Gly appeared as free glycine. Hydrolysis studies demonstrated the rate of L-Ala.[3H]Gly hydrolysis to free alanine and [3H[glycine by the microvilli to be greatly in excess of their rate of radiolabel uptake from this dipeptide. In addition, the uptake profiles and kinetic constants for vesicular uptake of radiolabel from L-Ala.[3H]Gly and free glycine were demonstrated to be identical when measured by double-labeling techniques in the same experiments. These results indicate that L-Ala.[3H]Gly is hydrolyzed at the external surface of the microvilli with the [3H]glycine released being transported into the vesicles by a Na+ gradient-dependent system identical to that employed for free glycine. Microvilli vesicle uptake of radiolabel from D-Ala.[3H]Gly exhibited no Na+ dependent "over-shoot" effect. D-Ala.[3H]Gly was completely resistant to microvilli-catalyzed hydrolysis. Analysis of the microvilli for renal dipeptidase, an enzyme with hydrolytic activity against a wide range of L-dipeptides, revealed this enzyme to be enriched in the microvilli vesicles to a degree equivalent to that observed for marker enzymes for renal microvilli. Renal dipeptidase catalyzed hydrolysis of L-Ala.Gly but not D-Ala.Gly, as was the case with microvilli-catalyzed hydrolysis of the dipeptides. With its location in the renal brush border microvilli and its hydrolytic action against L-dipeptides, renal dipeptidase my act at the luminal surface of the proximal tubule cell to hydrolyze L-dipeptides present in the glomerular filtrate, with the resultant free amino acids transported across the brush border microvilli by Na+ gradient-dependent processes.

Polyacrylamide gel electrophoresis of Loxosceles reclusa venom demonstrated that only one of seven or eight major (plus three or four minor) protein components caused necrosis in guinea pig skin. Sephadex gel filtration separated the venom into three major peaks, the second peak of which contained the dermonecrotic activity. Hyperimmunization of rabbits with increasing doses of venom from L. reclusa produced potent precipitating antisera, and the rabbits became resistant to lesion development. Ouchterlony-type immunodiffusion and immunoelectrophoretic studies revealed six to seven distinct precipitation lines, one of which stained intensely for esterase activity. Immunohistochemical techniques failed to detect any protease, lipase, catalase, acid phosphatase, alkaline phosphatase, or amylase activity in the venom. The spreading activity of recluse spider venom in guinea pig skin was inhibited as much as 71% by antivenom. Venom preincubated with antivenom was unable to incite lesions in guinea pig skin. Passive immunization of guinea pigs 18 h before an injection of venom conferred venom resistance upon the animals. Local injections of antivenom immediately after intradermal injections of venom markedly reduced the dermal lesion. Heparin reduced the local and systemic effects of venom when preincubated with whole venom or when administered systemically before an intradermal injection of venom. Treatment of whole venom with the chelating agent ethylenediaminetetraacetate did not inhibit its necrotic activity. Transfer studies from a 24-h lesion indicated that the necrotic activity was localized and remained active in tissue for at least 24 h but not for 5 days. No lesions developed when high concentrations of venom were intradermally injected into the skin of sacrificed guinea pigs, indicating that an interaction of body constituents and venom is essential for the development of a lesion.

An antidiuretic hormone-inactivating peptidase located in renal plasma membranes of porcine kidney medulla has been studied. Treatment of antidiuretic hormone (lysine vasopressin) with renal plasma membranes resulted in a progressive loss of biological activity as measured by the rat pressor assay. The reaction of 2,4,6-trinitrobenzenesulfonic acid with released amino groups was employed to follow the peptidase-catalyzed hydrolysis of the hormone. An 83-fold purification of the membrane-bound peptidase was achieved by Lubrol PX solubilization of the membranes followed by DEAE-cellulose, hydroxylopatite, and 8% agarose column chromatography. The molecular weight of the peptidase was 442 000 as determined by 8% agarose gel filtration. An analysis of the antidiuretic hormone hydrolysis products by thin-layer chromatography revealed the presence of trinitrophenyl-glycinamide. The release of glycinamide from the hormone as a function of time was demonstrated. Mg2+ had a slight inhibitory effect and Ca2+ had a strong inhibitory effect on the peptidase activity.

Venom from the poisonous brown recluse spider (Loxosceles reclusa) catalyses the lysis of dithiothreitol-treated human erythrocytes when incubated with serum complement but not in heat inactivated serum, a characteristic of complement system activation via the alternate pathway. This activity of the venom was shown to reside in a Sephadex G-75 fraction of the venom which also contains the dermonecrotoxin. Isoelectric focusing of this fraction identified the complement inactivating molecules(s) in the region near pH=6. Analysis of complement after interaction with venom indicated a loss of haemolytic C3. Immunoelectrophoretic development of venom-complement mixtures with anti C3 proactivator revealed the appearance of the C3 activator. These data indicate that recluse spider venom activates the alternate complement pathway.