The HLA-B 3906 allele imparts a high risk of diabetes only on specific HLA-DR/DQ haplotypes.

AIMS/HYPOTHESIS: We investigated the risk associated with HLA-B*39 alleles in the context of specific HLA-DR/DQ haplotypes. METHODS: We studied a readily available dataset from the Type 1 Diabetes Genetics Consortium that consists of 2,300 affected sibling pair families genotyped for both HLA alleles and 2,837 single nucleotide polymorphisms across the major histocompatibility complex region. RESULTS: The B*3906 allele significantly enhanced the risk of type 1 diabetes when present on specific HLA-DR/DQ haplotypes (DRB1 0801-DQB1 0402: p = 1.6 × 10(-6), OR 25.4; DRB1 0101-DQB1 0501: p = 4.9 × 10(-5), OR 10.3) but did not enhance the risk of DRB1 0401-DQB1 0302 haplotypes. In addition, the B 3901 allele enhanced risk on the DRB1 1601-DQB1 0502 haplotype (p = 3.7 × 10(-3), OR 7.2). CONCLUSIONS/INTERPRETATION: These associations indicate that the B 39 alleles significantly increase risk when present on specific HLA-DR/DQ haplotypes, and HLA-B typing in concert with specific HLA-DR/DQ genotypes should facilitate genetic prediction of type 1 diabetes, particularly in a research setting.

Statistical analysis of Peptide electron transfer dissociation fragmentation mass spectrometry.

It is well established that protein sequence determination may be achieved by mass spectrometric analysis of protonated tryptic peptides subjected to collisional activation. When separated by nanoflow HPLC, a high percentage of peptides from complex mixtures of proteins can usually be identified. Recently, alternative, radical-driven fragmentation approaches of electron capture dissociation and the more common electron transfer dissociation (ETD) have been introduced and made widely available. In order to utilize these techniques in large scale proteomics studies, it is important to characterize the performance of these fragmentation processes on peptides formed by a range of enzymatic cleavages. In this study, we present a statistical analysis of the ion types that are observed from peptides produced by different enzymes and highlight the different characteristics of ETD spectra of doubly charged precursors in comparison to precursors of higher charge states.

Plasma clearance of neostigmine and pyridostigmine in the dog.

1 The pharmacokinetics of neostigmine and pyridostigmine was studied in conscious dogs by the use of a cross-over design. 2 Both neostigmine and pyridostigmine were cleared from plasma in a biexponential manner. 3 The apparent volume of distribution of pyridostigmine was invariably greater than that of neostigmine, and its fast disposition half-life was approximately three times longer. 4 The whole body clearance and the urinary elimination of pyridostigmine was approximately twice that of neostigmine. 5 The slow disposition half-life of pyridostigmine was approximately three times longer than that of neostigmine, suggesting that the longer duration of action of pyridostigmine is related to the differential clearance of the two quarternary amines from plasma.

Pharmacological manipulation of adynamic ileus: controlled randomized double-blind study of ceruletide on intestinal motor activity after elective abdominal surgery.

In a double-blind placebo-controlled trial of patients undergoing elective abdominal surgery (n = 91), a single intravenous infusion of ceruletide (2.5 ng kg-1 min-1 for 1 hour) resulted in audible bowel sounds in 42/47 patients as opposed to 30/44 receiving placebo (P less than 0.025). Excessive bowel sounds were noted in 16 patients in the ceruletide group and four receiving placebo (P less than 0.01). Significantly more patients (P less than 0.01) in the ceruletide group (22/45 versus 9/44) passed flatus per rectum between the second and third post-operative day. Ceruletide infusion was accompanied by a significant increase in the incidence of nausea and vomiting (P less than 0.005, P less than 0.0025) but these side effects were short-lived. These results indicate that ceruletide is likely to be a useful therapeutic agent for acute intestinal adynamic motility disorders.

Proteinase-like peptidase activities and oestrogen receptor levels in breast cancer tissue.

The relationship between proteinase-like peptidase activities and oestrogen receptor levels and status in breast cancer tissue homogenates from 61 patients with breast cancer has been evaluated. With Spearman's rank-order correlation analysis, significant positive correlations were observed between receptor levels and the activities of cathepsin-(B + L)-like, cathepsin-H-like, trypsin-like, plasminogen-activator-like and elastase-like peptidases. In addition, the activities of all but the latter enzyme were significantly higher in patients with receptor-rich tumours than in receptor-poor tumours, and this may have implications for future treatment regimens for patients with oestrogen-receptor-rich tumours. The findings reported are consistent with the suggestion that in breast cancer there may be an association between steroid receptors and proteolytic enzymes such that the release of these enzymes may be under hormonal control.

Breast carcinoma cellularity and its relation to oestrogen receptor content.

The cellularity of 104 primary breast carcinomata was determined by semiautomated image analysis to allow the relation between cellularity and oestrogen receptor content values to be assessed. The oestrogen receptor content of tumours detected by a steroid binding assay showed no correlation with cellularity, although a possible weak negative correlation was observed between tumour cellularity and oestrogen receptor content detected by enzyme immunoassay. It is concluded that there is no single direct correlation between tumour cellularity and oestrogen receptor content.

Effect of repeated intraperitoneal chemotherapy with epirubicin on the hepatic transport of bile acids and their enterohepatic circulation.

The effect of repeated intraperitoneal perfusion with epirubicin on the clearance of 14C-taurocholate by the liver and the enterohepatic circulation of the synthetic bile acid 75-SeHCAT were investigated using a rat model. After six treatments there was no significant difference in the transport rate constants (plasma to liver, liver to bile and liver to plasma) between and within the saline and epirubicin groups. Similarly, there were no differences detected between the groups for the parameters derived from these transport rate constants. Thus the initial plasma clearance after six perfusions was 39 +/- 9, and 36 +/- 11 ml/min/kg in the epirubicin and saline groups respectively. The excretory efficiency of the liver at this stage was identical: 67 +/- 10% in the epirubicin treated animals and 67 +/- 6% in the saline controls. A deterioration in the SeHCAT retention was observed after repeated intraperitoneal perfusion in both groups. This was significant only in the cytotoxic group, between the first and sixth epirubicin perfusion: 59 +/- 9% vs 48 +/- 9% at 24 h (P = 0.03), 31 +/- 8% vs 22 +/- 5% at 48 h (P = 0.019) and was not cumulative beyond this stage. These findings indicate that repeated intraperitoneal perfusion chemotherapy with epirubicin does not impair bile acid clearance by the hepatocyte. The decrease in the retention of SeHCAT is unlikely to be the result of epirubicin-induced ileal mucosal damage since the reduction was not cumulative beyond 48 h of administration of the compound.

Inhibition of proteinase-like peptidase activities in serum and tissue from breast cancer patients.

The inhibitory profiles of several proteinase-like peptidases active on synthetic peptide (MCA) substrates, present in sera and 100,000g supernatants of malignant tissue from patients with breast cancer, have been studied using a series of known inhibitors including epoxysuccinyl peptides (E-64, Ep-475), Z-Phe-Phe-diazomethane, PMSF, iodoacetamide, 1-10-O-phenanthroline, leupeptin, aprotinin, elastatinal and alpha 2-macroglobulin. While in general the inhibition profiles confirmed reported substrate specificities some anomalies were observed. In particular, the serum activities on two cathepsin B substrates were unaffected by specific cysteine proteinase inhibitors and in breast tissue only 20-37% of activity towards these two substrates was apparently due to the presence of endopeptidases. However, the potent inhibition of other proteinase-like activities by the epoxysuccinyl peptides and leupeptin, or similar inhibitors, may be useful agents in the study of methods of combating tumour spread.

Comparability of hamster with human faecal unconjugated bile acids in a model of colorectal cancer.

The relationship between experimental colorectal carcinogenesis and bile acids has usually been investigated in the rat, a species with a markedly different bile acid profile from man. In this study, we show that the hamster faecal bile acid profile is similar to that in man. Rectal cancer was induced in hamsters using twice weekly instillations of N-methyl-N-nitro-N-nitrosoguanidine (MNNG) for 4 weeks at doses of 1-8 mg kg-1. The medcian (range) faecal bile acid concentrations of tumour-bearing hamsters (0.52, 0.46-0.84 mumoles g-1 faeces) was reduced compared to controls (1.08, 0.95-1.65, mumoles g-1) and non-tumour bearing MNNG treated hamsters (1.18, 0.64-1.42 mumoles g-1), largely due to a decrease in cholic acid derivatives (all p less than 0.05) at least). This model may be more suitable for studying the relationship between colorectal cancer and bile acids.

Effects of dietary N-3 and saturated fats on growth rates of the human colonic cancer cell lines SW-620 and LS 174T in vivo in relation to tissue and plasma lipids.

Ninety nude mice were inoculated subcutaneously with 1 x 10(7) cells of the human colonic cancer cell lines, SW-620 and LS174T. Tumour growth was assessed weekly for three weeks whilst the animals were receiving one of three diets: control (4.6% fat), coconut (20% fat, saturated fatty acids) and Maxepa (20% fat; n-3 fatty acids). At the end of the study SW-620 tumour weights (mean +/- SD, gm) were: control = 0.38 +/- 0.22, coconut = 0.43 +/- 0.31, Maxepa 0.20 +/- 0.16; the LS174T tumour weights were control = 1.33 +/- 1.27, coconut = 0.47 +/- 0.74, Maxepa = 0.38 +/- 0.56 (p less than 0.001, analysis of covariance). The Maxepa diet produced significant retardation in tumour growth (p less than 0.001). This was associated with reduced levels of linoleic acid and arachidonic acid in adipose tissue and tumour lipids with incorporation of n-3 fatty acids (all p less than 0.01 at least, analysis of variance). Moreover, the Maxepa diet produced significant reductions of plasma cholesterol, phospholipids and triglycerides (all p less than 0.01).

Changes in intragastric bile acid composition in patients receiving cimetidine postoperatively.

Enterogastric reflux has been implicated as a possible etiologic mechanism in gastritis both after partial gastrectomy and in those with an intact pylorus. We studied the effects of cimetidine on bile acid concentration and composition by high-performance liquid chromatography. The gastric aspirates collected for this study came from 27 prospectively randomized patients receiving intravenous cimetidine (200 mg every 6 hours) and 25 patients given a placebo. Total bile acid concentration of aspirates was determined spectrophotometrically. Marked differences were noted in conjugated bile acids. Glycochenodeoxycholic acid, a toxic dihydroxy bile acid, was decreased after cimetidine compared with results from the placebo. The ratio of less toxic trihydroxylated to more toxic dihydroxylated bile acids was significantly increased. Enterogastric reflux itself seemed unaltered by cimetidine; likewise, the concentration of total bile acids in the cimetidine group was similar to that among patients receiving placebo. These changes in bile salt composition with cimetidine may help explain its salutary effects in gastritis, over and above its ability to reduce gastric hydrogen ion secretion.

Fluorogenic compound hydrolysis as a measure of toxicity-induced cytoplasmic viscosity and pH changes.

If a fluorogenic compound, such as fluorescein diacetate, is added to a water solution containing living cells it becomes hydrolyzed by intracellular esterases into a fluorochrome whose fluorescence can be used to monitor the cytoplasmic pH and the cytoplasmic viscosity of the cells. In this paper we have used this technique to measure the effects of different concentrations of Co2+ and Cd2+ ions on the cytoplasmic pH and the cytoplasmic viscosity of a single cell culture. Our results indicate that the observed decrease in the efficiency of the intracellular hydrolyzation of fluorogenic substances in the presence of different concentrations of heavy metals could be caused by both a decrease in the cytoplasmic pH and an increase in the cytoplasmic viscosity. A decrease in cytoplasmic pH would decrease the effectiveness of the intracellular enzymes, whereas an increase in cytoplasmic viscosity would decrease diffusion which would also reduce the effectiveness of the reaction. The dependence of the reciprocal of the cytoplasmic viscosity on the concentration of these metals correlates well with published results on their toxicity.

Detection and quantification of protein adduction by electrophilic fatty acids: mitochondrial generation of fatty acid nitroalkene derivatives.

Nitroalkene fatty acid derivatives manifest a strong electrophilic nature, are clinically detectable, and induce multiple transcriptionally regulated anti-inflammatory responses. At present, the characterization and quantification of endogenous electrophilic lipids are compromised by their Michael addition with protein and small-molecule nucleophilic targets. Herein, we report a trans-nitroalkylation reaction of nitro-fatty acids with beta-mercaptoethanol (BME) and apply this reaction to the unbiased identification and quantification of reaction with nucleophilic targets. Trans-nitroalkylation yields are maximal at pH 7 to 8 and occur with physiological concentrations of target nucleophiles. This reaction is also amenable to sensitive mass spectrometry-based quantification of electrophilic fatty acid-protein adducts upon electrophoretic resolution of proteins. In-gel trans-nitroalkylation reactions also permit the identification of protein targets without the bias and lack of sensitivity of current proteomic approaches. Using this approach, it was observed that fatty acid nitroalkenes are rapidly metabolized in vivo by a nitroalkene reductase activity and mitochondrial beta-oxidation, yielding a variety of electrophilic and nonelectrophilic products that could be structurally characterized upon BME-based trans-nitroalkylation reaction. This strategy was applied to the detection and quantification of fatty acid nitration in mitochondria in response to oxidative inflammatory conditions induced by myocardial ischemia-reoxygenation.

Formation and separation by reversed-phase high-performance liquid chromatography of fluorescent and UV-absorbing bile salt derivatives.

A method is described for the production of bile salt derivatives via the substituent hydroxyl groups on the steroid nucleus that permits derivatization of taurine and glycine conjugates. The method involves the oxidation of the hydroxyl groups at C-3 alpha or C-7 alpha on the nucleus by the action of hydroxysteroid dehydrogenases. The keto bile salts produced are isolated and reacted with 2,4-dinitrophenylhydrazine or Dns-hydrazine to yield ultraviolet absorbing or fluorescent derivatives, respectively. The bile salt hydrazones were separated using a reversed-phase C18 radial compression cartridge with a methanol-phosphate buffer gradient elution. Although the 3-keto derivatives of chenodeoxycholic and deoxycholic acids were not resolved, the 7-keto Dns-hydrazone of chenodeoxycholic acid was separated from 3-keto derivative of deoxycholic acid.

Effect of portacaval transposition on hepatic function in dogs with obstruction to the hepatic venous outflow.

Complete hepatic venous outflow obstruction (COB) in dogs resulted in impairment of hepatic function and cellular injury. Thus both the serum albumin concentration and the plasma clearance of indocyanine green (ICG) were reduced, whilst serum glycoproteins, aminotransferases and alkaline phosphatase levels were increased. Partial outflow block (POB)resulted in less severe functional changes although a significant decrease in serum albumin was also observed. The changes in liver function in dogs with portacaval transposition (PCT) and COB were generally similar to those in dogs with COB alone. However, the increases in serum enzyme activities and the decrease in plasma clearance of ICG were greater in the group with PCT. A late improvement in the clearance of ICG in dogs with COB and PCT was probably related to retrograde portal flow. When the hepatic lesion was only moderate (POB) PCT had little effect on either the liver function tests or plasma clearance of ICG, although no improvement in function was observed. It is concluded that PCT has no beneficial effect on dogs with either severe or moderate hepatic lesion and may cause further functional deterioration.

Blood flow during obstruction to the venous effect of portacaval transposition on hepatic outflow from the canine liver.

When portacaval transposition (PCT) was performed in dogs in combination with a complete hepatic venous outflow block (COB) procedure, estimated hepatic blood flow (EHBF) declined steadily. Four weeks after this combined procedure mean flow was only 46% of the value obtained 3 days after operation, but this did not prevent the development of ascites which occurs in dogs with COB alone. Complete reversal of portal flow was demonstrated in the longest surviving dog. When PCT was performed in dogs with established partial outflow block EHBF remained at preoperative levels whether portal hypertension was present or not prior to the second operation. This maintenance of hepatic blood flow was due to continuing venous flow into the portal bed. It would appear that PCT is probably of little value as a treatment for portal hypertension and bleeding oesophageal varices in cases of advanced cirrhosis, but may be of some benefit in a few selected cases where the hepatic lesion is less severe.

Compartmental analysis of the plasma clearance of tetrabromsulphthalein and dibromsulphthalein in the normal dog and in patients with liver disease.

A comparison has been made of the plasma clearances of the anionic dye analogues, tetrabromsulphthalein (BSP) and dibromsulphthalein (dBSP), analysed by means of a two-compartmental model. In the normal conscious dog all three transport rate constants and initial and steady state plasma clearances were greater with dBSP than BSP. On the other hand the equivalent liver volume (ELV) and storage capacity were lower with dBSP. In patients with various hepatic disorders dBSP values were greater than those obtained with BSP apart from rate constant b (liver to plasma). Patients with advanced liver impairment in general exhibited lower hepatic uptake, clearance and storage of both dyes compared to patients with mild forms of liver disease. The ratio of ELV values for BSP and dBSP appeared to be related to overall hepatic function, with ratios greater than 1 in the normal dogs and less than 1 in the patients.

High-performance liquid chromatography of bile acids with a reversed-phase radial compression column.

A reversed-phase radial compression column (Waters Assoc. Radial-Pak C18, 10 micrometers, 8 mm I.D.) has been evaluated for the separation of bile acid standards by high-performance liquid chromatography. With a mobile phase of methanol-water (75:25, v/v) containing 2.5% (v/v) acetic acid and adjusted to pH 5.25 with 10 M sodium hydroxide, a mixture of fifteen bile acids could be resolved, and the ten major conjugated bile acids of human bile were well separated in under 20 min at a flow-rate of 2.0 ml min-1. More rapid separations could be achieved at higher flow-rates. Refractive index detection permitted quantitation of 5 nmoles or less.