Genetic variants of hemophilia B: detection by means of a specific PTC inhibitor.

Hemophilia B can be divided into at least two mutant forms different from the mild, moderate, and severe categories previously described. In about 90% of hemophilia B patients, PTC-inhibitor-neutralizing activity is reduced in proportion to PTC clotting activity. In about 10% of the patients, PTC-inhibitor-neutralizing activity is fully effective, whereas PTC clotting activity is reduced. Extensive pedigree studies indicate that the presence or absence of inhibitor-neutralizing activity is genetically determined. It is suggested that those hemophilia B mutants with decreased inhibitor-neutralizing material produce decreased amounts of PTC-protein. It is further suggested that those with normal levels of inhibitor-neutralizing material produce normal amounts of PTC-protein, which is structurally altered so as to lose procoagulant activity but which retains inhibitor-neutralizing activity. The latter group may be analogous to CRM(+) mutants described in bacteria and Neurospora.

Diethylnitrosamine-induced pancreatic neoplasms in the fish Rivulus ocellatus marmoratus.

Diethylnitrosamine [(DEN) CAS: 55-18-5] caused neoplasms of the exocrine pancreas in rivulus, Rivulus ocellatus marmoratus, a self-fertilizing hermaphroditic fish, after a single exposure to 810, 270, or 130 mg DEN/liter or after a continuous exposure to 50, 17, or 9 mg DEN/liter. Rivulus developed pancreatic adenomas after a single exposure. After continuous exposure, pancreatic adenomas, cystadenomas, and adenocarcinomas developed. Adenocarcinoma developed in fish first exposed as larvae but not in those first exposed as juveniles. Adenocarcinoma of pancreatic or biliary origin invaded the intestinal muscularis layer and mucosa. Dedifferentiation of acinar cells preceded the formation of adenomas, and atypical acinar cells formed parts of some of the ductlike structures in the pancreatic neoplasms.

Papillomas on fish exposed to chlorinated wastewater effluent.

The presence of carcinogenic and mutagenic chemical(s) in the effluent of a wastewater treatment plant was indicated by papillomas developing on caged black bullheads (Ictalurus melas), hepatic enzyme induction in exposed fish, and Ames test mutagenicity of organic extracts of the wastewater. Although virus-like particles have been reported in papillomas of several other fish species, no evidence was obtained for the presence of viruses in the black bullhead papillomas. Mutagenic and carcinogenic chemicals were not identified in the wastewater, but chlorination was implicated as a factor contributing to the induction of the papillomas. The prevalence of papillomas on wild black bullheads exposed to the effluent decreased from 73 to 23% after the amount of residual chlorine (CAS: 7782-50-5) in the effluent leaving the chlorine contact chamber was reduced from 1.3-3.1 mg/liter to 0.25-1.2 mg/liter.

The beta-carotene and retinol efficacy trial (CARET) for chemoprevention of lung cancer in high risk populations: smokers and asbestos-exposed workers.

CARET is a multicenter, two-armed, double-masked randomized chemoprevention trial in Seattle, Portland, San Francisco, Baltimore, Connecticut, and Irvine, to test whether oral administration of beta-carotene (30 mg/day) plus retinyl palmitate (25,000 IU/day) can decrease the incidence of lung cancer in high risk populations, namely, heavy smokers and asbestos-exposed workers. The intervention combines the antioxidant action of beta-carotene and the tumor suppressor mechanism of vitamin A. As of April 30, 1993, CARET had randomized 1,845 participants in the 1985-1988 pilot phase plus 13,260 "efficacy" participants since 1989; of these, 4,000 are asbestos-exposed males and 11,105 are smokers and former smokers (44% female). Accrual is complete everywhere except Irvine, which was the last center added (1991), and the safety profile of the regimen to date has been excellent. With 14,420 smokers, 4,010 asbestos-exposed participants, and 114,100 person-years through February 1998, we expect CARET to be capable of detecting a 23% reduction in lung cancer incidence in the two populations combined and 27, 49, 32, and 35% reductions in the smokers, female smokers, male smokers, and asbestos-exposed subgroups, respectively. CARET is highly complementary to the alpha-tocopherol-beta-carotene study in Finland and the Harvard Physicians Health Study (beta-carotene alone) in the National Cancer Institute portfolio of major cancer chemoprevention trials.

Age-related down regulation of hepatic cytochrome P1-450, P3-450, catalase and CuZn-superoxide dismutase RNA.

The influence of age on liver gene expression was investigated in two strains of H-2 congenic mice. In B10.RIII mice (H-2r), basal P1- and P3-450 RNA levels progressively decreased 65 and 95%, respectively, between 4 and 28 months of age (P less than or equal to 0.05). Polyaromatic hydrocarbon (PAH) induced P1- and P3-450 RNA levels decreased about 50% during this time (P less than or equal to 0.05). In contrast, in C57BL/10 mice (H-2b) little or no change was detected in basal or induced P1- or P3-450 RNA levels. CuZn-superoxide dismutase RNA decreased 80 to 90% between 4 and 9 months of age in B10.RIII mice, while a quantitatively smaller decrease of 50 to 65% was found in C57BL/10 mice (P less than or equal to 0.05). Catalase RNA decreased approximately 80% between 4 and 9 months of age in B10.RIII mice, and a similar decrease was found in C57BL/10 mice. Down regulation of these genes may explain the reduced activities of the cognate hepatic enzymes, and reduced xenobiotic metabolism found in older animals.

Endogenous enzymes cause structural and chemical artifacts in methacrylate- and celloidin-embedded sections of unfixed freeze-dried tissues.

Bovine exocrine pancreas and fish (Rivulus ocellatus marmoratus) liver containing pancreatic acini were cryofixed, freeze-dried, and embedded in methacrylate or double-embedded in celloidin and paraffin. In chemically unfixed sections incubated in aqueous solutions, dissolution of zymogen granules was coincident with loss of tissue structure and antigenicity. Type II-S soybean protease inhibitor at 150 mg/liter during section flotation and in aqueous reagents used for immunohistochemistry prevented these artifacts and allowed the use of more dilute antibody solutions. Loss of glycogen from fish hepatocytes was most rapid in areas adjacent to pancreatic acini. Rapid loss of glycogen was attributed to amylase and was prevented by using poly-L-lysine instead of 3-aminopropyltriethoxysilane slide adhesive and by using alcoholic solutions during PAS staining. Inhibition of endogenous enzymes is an important consideration in the development of histological protocols with freeze-dried tissue sections.

The Women's Health Trial Feasibility Study in Minority Populations: design and baseline descriptions.

The Women's Health Trial: Feasibility Study in Minority Populations (WHT:FSMP), a randomized trial of 2208 women, was conducted to investigate three questions. First, can women from minority and low-socioeconomic-status populations be recruited in numbers sufficient to evaluate a dietary intervention designed to lower fat intake. Second, the efficacy of a low fat, increased fruit/vegetable/ grain product intervention for reducing fat consumption. Third, will participation in the intervention lower plasma cholesterol and estradiol levels relative to the controls. The baseline results showed that an adequate number of minority and low SES women could be recruited to test the study hypotheses. A diverse study population of postmenopausal women consuming a high fat diet was recruited: 28% of participants were Black, 16% were Hispanic, 11% had less than a high school level of education, and 15.5% had household incomes of < $15,000.

Some applications of categorical data analysis to epidemiological studies.

Several examples of categorized data from epidemiological studies are analyzed to illustrate that more informative analysis than tests of independence can be performed by fitting models. All of the analyses fit into a unified conceptual framework that can be performed by weighted least squares. The methods presented show how to calculate point estimate of parameters, asymptotic variances, and asymptotically valid chi 2 tests. The examples presented are analysis of relative risks estimated from several 2 x 2 tables, analysis of selected features of life tables, construction of synthetic life tables from cross-sectional studies, and analysis of dose-response curves.

Pooling 12 nomifensine studies for efficacy generalizability.

Twelve parallel group, randomized, double-blind studies of nomifensine's safety and efficacy in the treatment of depressed patients were combined into three pools according to common protocols. This approach permitted evaluation of 1) efficacy results for studies with moderate-sized pools of patients, 2) the degree to which efficacy was generalizable to depressed patients in the general population, and 3) the conditions under which pooled active vs. active (imipramine vs. nomifensine) studies could be regarded as pivotal in support of efficacy. Results showed that nomifensine's superiority over placebo was generalizable to patients with a wide range of characteristics, including age 60 years or older. An appropriate statistical profile of more pronounced nomifensine responders would include patients with a duration of present episode less than 4 months who are acutely depressed, exhibit more severe symptoms, and have been previously hospitalized or treated with other psychotropic medications. A comprehensive assessment and power analysis of the pooled active vs. active studies provided strong evidence for comparability of nomifensine and imipramine.

Aging alters hepatic expression of insulin receptor and c-jun mRNA in the mouse.

The clear association between species and life span suggests that aging, like development, is genetically orchestrated. To explore this hypothesis, the expression of mRNA for a number of transcription regulatory and signal transduction proteins was investigated during aging of B10.RIII, C57BL/10 and B10.BR mice. mRNA for glucocorticoid receptor, CCAAT and enhancer binding protein, transcription factor Sp1 and RNA polymerase II elongation factor S-II were unchanged between 4 and 24 months of age in these mice. These factors are required for the normal transcription of many genes, perhaps explaining their steady rates of expression throughout life. Insulin-like growth factor I mRNA also remained unchanged. By contrast, mRNA for the insulin receptor and transcription factor c-jun changed significantly during aging. c-Jun mRNA decreased approximately 55% between 4 and 12 months of age and then increased by 24-25 months of age to levels approximately equal to those found in young mice. Insulin receptor mRNA increased approximately 30% by 24-25 months of age in all strains of mice. These results suggest that factors determining the steady state level of these mRNAs are altered in level or activity during aging. Assessing the causes and significance of these changes will require further study. However, our results demonstrate that alterations in the expression of specific regulatory genes occur during aging.

Pathology of diethylnitrosamine toxicity in the fish Rivulus marmoratus.

Rivulus marmoratus were exposed to 0, 10, 21, 45, 95, or 200 mg/liter diethylnitrosamine (DEN) for 6 weeks and examined 12 weeks after the end of exposure. Fatty change, hepatocellular glycogenosis, multiple basophilic foci, enlarged and distorted cells with or without an enlarged nucleus, and hyaline bodies and cytological alterations observed after exposure to DEN. Hemangiomas, cholangiomas, biliary cystadenomas, and glandular, trabecular and anaplastic hepatocellular carcinomas were observed at the 18th week. Only those fish exposed to 95 mg/liter DEN had cavernous hemangiomas and peliosis-like lesions, which could be a preneoplastic lesion preceding cavernous hemangiomas. Adenomatous hyperplasia of thyroid and granulomas were other chronic reactions caused by DEN toxicosis.

Preliminary description of lesions in juvenile largemouth bass injected with largemouth bass virus.

Juvenile largemouth bass Micropterus salmoides were intraperitoneally injected with largemouth bass virus (LMBV), a member of the genus Ranavirus, family Iridoviridae. Moribund fish which had been injected with 10(6.2) tissue culture infectious doses, 50% endpoint (TCID50), were sampled 4 d after injection; other largemouth bass injected with this dose died between 3 and 5 d after injection. Fish injected with 10(2.8) TCID50 of LMBV were also examined after 4 d and had lesions similar to those of fish injected with the high dose. Clinical signs included darker pigmentation, inflammation and necrosis at the site of injection, distended abdomen, corkscrew swimming, and lateral recumbency. Internally, fish had focally pale livers, bright red spleens and reddened intestinal ceca. Histologically acute fibrinous peritonitis affected the surface of all organs in the peritoneal cavity, but deeper portions of organs appeared normal. There was also necrosis of the gastrointestinal mucosa. Except for the injection site, lesions were confined to the peritoneal cavity.

Detection of Yersinia ruckeri in rainbow trout blood by use of the polymerase chain reaction.

We evaluated a polymerase chain reaction (PCR) method for detecting Yersinia ruckeri, the bacterial pathogen causing enteric redmouth disease (ERM), in blood of rainbow trout Oncorhynchus mykiss. Identification of the PCR product was confirmed by Southern blot hybridization with a 32P-labeled oligonucleotide probe matching a sequence within the small subunit ribosomal RNA gene of Y. ruckeri. Following a 1 h immersion of rainbow trout in water with 4.5 x 10(6) colony-forming units of Y. ruckeri l(-1), the PCR was positive for all blood samples from 1 h (first sample) to 5 d and was negative from 9 to 30 d (last sample). Fish in this experiment did not show signs of disease, probably because they had been vaccinated against Y. ruckeri. To test this method with naturally infected fish, 42 rainbow trout from hatcheries were examined. Four of these fish had clinical signs of ERM and were infected with Y. ruckeri based on bacteriological culture. The PCR method detected Y. ruckeri in blood, intestine, liver, and trunk kidney from the 4 fish with ERM and from 5 additional rainbow trout that were bacteriologically negative for Y. ruckeri. Three of 5 rainbow trout from streams receiving effluent from hatcheries were positive for Y. ruckeri when tested with PCR, although there was no growth of Y. ruckeri on culture plates inoculated with the same samples. Samples were successfully stored for 1 wk in lysis buffer at 25 degrees C. This study demonstrated that a non-lethal blood sample can be used with PCR to detect Y. ruckeri.

Partial nucleotide sequence of the porcine corticosteroid-binding globulin (CBG) cDNA and specification of CBG expression sites in postnatal pigs.

The purpose of this study was to develop a porcine CBG cDNA probe in order to examine the porcine CBG mRNA expression in major tissues from the postnatal pig. The reverse transcriptase-polymerase chain reaction (RT-PCR) was conducted to develop the porcine CBG cDNA probe using total RNA extracted from liver of 40-day-old pig. The RT-PCR product was subcloned into the pGEM vector (Promega, Madison, WI) and subjected to restriction enzyme treatments and DNA sequencing. Northern blot analysis was conducted using total RNA extracted from samples (approximately 200 mg) of liver, lung, kidney, and whole adrenal tissue that were collected from pigs on day 3 (n = 2) or day 40 (n = 2) postpartum. A 500 bp partial porcine CBG cDNA encoded 166 amino acids and had 83, 78, and 77% homology to a 494-nucleotide sequence of CBG from sheep, human, and rabbit, respectively. The deduced peptide sequence of the partial porcine CBG showed 77, 62, 60, and 51% homology to sheep, human, rabbit, and rat CBG sequences, respectively. An approximately 1.53 kb CBG mRNA was detected only in the liver tissue. In conclusion, the development of a partial CBG cDNA for swine makes it possible to study the ontogeny and the regulation of CBG synthesis at the molecular level and, based on tissues examined in this study, the liver appears to be the primary source of CBG biosynthesis in the postnatal pig.

Effect of tumor necrosis factor-alpha on progesterone production by granulosa cells in laying hens of different genetic lines.

In vitro progesterone production by granulosa cells in the presence or absence of human recombinant tumor necrosis factor-alpha (hrTNF-alpha) was measured at 10, 20, and 30 wk of egg production in White Leghorn hens selected for high (HA)- or low-antibody (LA) response to sheep red blood cell challenge. Isolated granulosa cells from the three largest preovulatory follicles (F1-F3) were incubated with 5 or 250 ng/ml hrTNF-alpha, and progesterone production was determined by the use of a validated radioimmunoassay. F1, F2 and F3 granulosa cells from HA hens produced more (P < or = 0.05) progesterone (140.8, 107.2, and 49.7 ng/ml) than LA hens (109.4, 78.9, and 26.9 ng/ml). The treatment of granulosa cells with hrTNF-alpha consistently inhibited (P < or = 0.05) progesterone secretion by all follicles among HA and LA hens, but not always at both doses. Generally, 5 ng/ml hrTNF-alpha was the maximum inhibitory dose. In the laying hen, a decrease in steroid production in response to cytokines may upset the steroid balance created by follicular hierarchy and inhibit or delay ovulation.

Gill lesions and death of bluegill in an acid mine drainage mixing zone.

The toxicity of an acid mine drainage (AMD) mixing zone was investigated by placing bluegill (Lepomis macrochirus) at the confluence of a stream contaminated by AMD and a stream having neutral pH. A mixing channel receiving water from both streams was assembled in the field, during July and October 1996, to determine the toxicity of freshly mixed and aged water (2.9-7.5 min). The AMD stream had elevated concentrations of Al and Fe, which precipitated upon mixing, and of Mn, which did not precipitate in the mixing zone. Fish exposed to freshly mixed water had higher mortality than fish exposed to water after aging. Precipitating Al, but not Fe, accumulated on the gills of bluegill, and accumulation was more rapid early during the mixing process than after aging. Fish exposed for 3.5 h to freshly mixed water had hypertrophy and hyperplasia of gill filament and lamellar epithelial cells. Similar lesions were observed after 6.0 h in fish exposed to water aged after mixing. Results demonstrated that Al was the predominant metal accumulating on the gills of fish in this AMD mixing zone, and that mixing zones can be more toxic than AMD streams in equilibrium.

Granulomatous inflammation and monstrous giant cells in response to intraperitoneal hormone implants in channel catfish (Ictalurus punctatus).

Plastic implants (2.7 mm maximum dimension) of an ethyl vinyl acetate copolymer (EVAc) matrix, containing inulin, bovine serum albumin (BSA) and luteinizing hormone releasing hormone (LHRH), were covered with impervious EVAc and then surgically placed into the peritoneal cavity of 1-year-old channel catfish, Ictalurus punctatus. In fish kept in cold water (13 degrees C), 10 per cent of the implants per month were encapsulated by granulation tissue. In fish kept in warm water (27 degrees C), 20 per cent of the implants per month were encapsulated, with a total of 86 per cent encapsulated at 5 months. In addition to fibroblasts and capillaries, the granulation tissue included macrophages, neutrophils, lymphocytes, plasma cells, multinucleated giant cells and a matrix of collagen fibres. The density of the fibrous capsule increased with time. In a separate investigation, it was found that the thickness of the capsule was directly proportional to the degree of exposure of the EVAc matrix to the fish (exposure influenced by the rate of dissolution of the capsule content). Monstrous giant cells with up to 600 nuclei per 5 microns thick section were seen in capsules around implants. On intraperitoneally implanted cover glasses, whole giant cells contained up to 6000 nuclei and were interconnected by cytoplasmic bridges. Signs of neoplasia, implant expulsion or massive adhesions were not seen.

Follicular development and superovulation response in cows administered multiple FSH injections early in the estrous cycle.

To determine whether follicular development, superovulation and embryo production were affected by the absence or presence of a dominant follicle, cows were administered injections of FSH twice daily in the early (Days 2 to 6, estrus=Day 0) or middle stage (beginning on Day 10 or 11) of the estrous cycle. Treatment with FSH early in the cycle stimulated follicular development in 83 to 100% of all cows from 4 groups evaluated at different times after PGF2alpha treatment on Days 6 and 7. However, the proportion of cows with >2 ovulations varied from 31 to 62.5%, indicating that induction of follicular development may occur in the absence of superovulation. When compared with cows treated in the middle of the cycle, no differences were observed in the proportion of cows with >2 ovulations (31 vs 20%), ovulation rate. (26.0+/-6.3 vs 49.6+/-25.8), production of ova/embryos (13.3+/-3.2 vs 14.4+/-3.4), or the number of transferable embryos (8.0+/-3.6 vs 5.4+/-1.5; early vs middle, respectively). The proportion of the total number of embryos collected that were suitable for transfer was greater (P<0.01) in cows treated early in the cycle (60%) than at midcycle (37.5%). The diameter of the largest follicle observed by ultra-sound prior to initiation of FSH treatment in the early stage of the cycle (10.0+/-2.0 mm) was smaller (P<0.05) than at midcycle (16.8+/-1.3 mm). These results demonstrate that superinduction of follicular development is highly consistent after FSH treatment at Days 2 to 6 of the cycle and that superovulation and embryo production are not less variable than when FSH is administered during the middle of the cycle. However, superovulation in the early stage of the cycle may increase the proportion of embryos suitable for transfer.

Uptake of channel catfish virus from water by channel catfish and bluegills.

Radiolabeled channel catfish virus entered the gills of juvenile channel catfish (Ictalurus punctatus) and was then concentrated in the gut and the liver over 48 hours. Diminution of radioactivity was not detected in these tissues over the course of the experiment. Bluegills (Lepomis macrochirus) were capable of clearing the virus during a period of 48 hours. [3H]Thymidine alone had a different distribution in the channel catfish than did labeled virus.

Molecular cloning of fragments of the channel catfish virus (Herpesviridae) genome and expression of the encoded mRNA during infection.

Eleven EcoRI DNA fragments from the genome of an isolate of channel catfish virus (CCV) were cloned into the bacterial vector pUC19. The cloned DNA fragments ranged in size from approximately 200 base pairs to greater than 5,400 base pairs and accounted for about 13.5% of the 130,000-base pair CCV genome. Nine of these CCV DNA fragments encoded sequences that were expressed during late CCV infection. Channel catfish (total length, 4 cm) injected with CCV expressed CCV mRNA at detectable amounts in greater than or equal to 1 tissues. Uninjected control fish failed to express CCV-specific mRNA or expressed CCV-specific mRNA at lower amounts because of the presence of endogenous CCV. Tissue samples from clinically normal channel catfish fingerlings from 2 other farms as well as from adult brood stock also expressed CCV-specific mRNA. The results suggest that CCV can persist in a dormant or transcriptionally active state without causing clinical disease.