Molecular ecology of a facultative swine waste lagoon.

AIMS: To investigate the microbial ecology of three facultative swine waste lagoons. METHODS AND RESULTS: Phylogenetic analysis of sequences in a 16S rRNA gene clone library and fluorescence in situ hybridization (FISH) analyses were used to assess bacterial diversity in a swine waste lagoon. FISH analysis and Gram-staining were used to compare the microbial communities of all three swine waste lagoons. Six operational taxonomic units were in high relative abundance and corresponded to the following phylotypes; Thiolamprovum, Verrucomicrobia, Acholeplasma, Turicibacter, Clostridium and Bacteroides. PCR was employed to detect the genes apsA and dsrAB which encode for enzymes specifically associated with dissimilatory sulfate-reduction within sulfate-reducing bacteria (SRB). Amplification of these genes confirmed their presence within the lagoons. CONCLUSIONS: All lagoons were dominated by purple sulfur bacteria, affiliated to Thiolamprovum pedioforme. The molecular identification of fermentative bacteria and SRB indicate the following metabolic processes within such facultative ponds: sulfur-cycling, fermentation, inter-species hydrogen transfer and carbon cycling. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides the first molecular evidence for the existence of a sulfur cycle which is linked to phototrophic sulfide oxidation by purple bacteria and organotrophic sulfate-reduction by SRB.

Predictive, pre-natal and diagnostic genetic testing for Huntington's disease: the experience in Canada from 1987 to 2000.

Predictive and pre-natal testing for Huntington's Disease (HD) has been available since 1987. Initially this was offered by linkage analysis, which was surpassed by the advent of the direct mutation test for HD in 1993. Direct mutation analysis provided an accurate test that not only enhanced predictive and pre-natal testing, but also permitted the diagnostic testing of symptomatic individuals. The objective of this study was to investigate the uptake, utilization, and outcome of predictive, pre-natal and diagnostic testing in Canada from 1987 to April 1, 2000. A retrospective design was used; all Canadian medical genetics centres and their affiliated laboratories offering genetic testing for HD were invited to participate. A total of 15 of 22 centres (68.2%), currently offering or ever having offered genetic testing for HD, responded, providing data on test results, demographics, and clinical history. A total of 1061 predictive tests, 15 pre-natal tests, and 626 diagnostic tests were performed. The uptake for predictive testing was approximately 18% of the estimated at-risk Canadian population, ranging from 12.5% in the Maritimes to 20.7% in British Columbia. There appears to have been a decline in the rate of testing in recent years. Of the predictive tests, 45.0% of individuals were found to have an increased risk, and a preponderance of females (60.2%) sought testing. A greater proportion of those at < or = 25% risk sought predictive testing once direct CAG mutation analysis had become available (10.9% after mutation analysis vs 4.7% before mutation analysis, p = 0.0077). Very few pre-natal tests were requested. Of the 15 pre-natal tests, 12 had an increased risk, resulting in termination of pregnancy in all but one. Diagnostic testing identified 68.5% of individuals to be positive by mutation analysis, while 31.5% of those with HD-like symptoms were not found to have the HD mutation. The positive diagnostic tests included 24.5% of individuals with no known prior family history of HD.

The relationship between steroidogenic factor 1 and DAX-1 expression and in vitro gonadotropin secretion in human pituitary adenomas.

The orphan nuclear receptors, steroidogenic factor 1 (SF-1) and DAX-1, are involved in gonadotroph differentiation, and SF-1 has been shown to activate the LH-beta and glycoprotein hormone alpha-subunit (alpha GSU) gene promoters. Pituitary adenomas from 34 patients [13 somatotroph tumors, 4 prolactinomas, and 17 clinically nonfunctioning pituitary adenomas (NFPAs)] were enzymatically dispersed and cultured in vitro for 48 h. Tissue culture medium was collected and assayed for LH, FSH, and alpha GSU; messenger RNA was extracted from adherent cells, and expression of SF-1 and DAX-1 messenger RNA was determined by RT-PCR and verified by direct DNA sequencing. The presence of DAX-1 protein in tumor tissue was confirmed by immunocytochemistry. DAX-1 was demonstrated in all NFPAs, 7 of 13 somatotroph tumors and 0 of 4 prolactinomas. SF-1 expression occurred in 8 of 16 NFPAs, 4 of 12 somatotroph tumors, and 1 of 4 prolactinomas. LH secretion in vitro was greater in NFPAs that were SF-1 positive (P < 0.05). Neither FSH secretion nor alpha GSU secretion in vitro were significantly related to the expression of SF-1 or DAX-1. SF-1-positive somatotroph tumors immunostained positively for LH-beta and/or FSH-beta and secreted gonadotropins in vitro. SF-1 expression is associated with the in vitro secretion of LH by NFPAs. A proportion of somatotroph tumors also express SF-1 and DAX-1 and secrete gonadotropin hormones in vitro.

Cytogenetic aspects of the Canadian early and mid-trimester amniotic fluid trial (CEMAT).

Cytogenetic results from a large multicentre randomized controlled study of 2108 amniotic fluids obtained at 11+0-12+6 weeks (EA) and 1999 fluids at 15+0-16+6 weeks (MA) were compared. There was no statistically significant difference in the rate of chromosome abnormalities (EA =1.9 per cent; MA=1.7 per cent) or level III mosaicism (EA=0.2 per cent; MA= 0.2 per cent) between the groups. Level I and Level II mosaicism occurred more frequently in MA. Maternal cell contamination was not significantly different between the groups, but maternal cells only were analysed from one bloody EA fluid. The number of repeat amniocenteses because of cytogenetic problems was 2.2 per cent in the EA group compared with only 0.3 per cent in the MA group. On average, culture of EA fluids required one day more than MA fluids. Although both culture success (97.7 per cent) and accuracy (99.8 per cent) were high for patients randomized to the EA group, routine amniocentesis prior to 13 weeks' gestation is not recommended for clinical reasons including an increased risk of fetal loss and talipes equinovarus.

Pain control following elective gastrointestinal surgery: is epidural anesthesia warranted?

Fifty-nine patients undergoing elective major gastrointestinal surgery were entered into a prospective, randomized trial between January 1993 and July 1994 comparing the effectiveness, side effects, and hospital costs of postoperative epidural anesthesia (Group 1, n = 29) and intramuscular narcotic injections (Group 2, n = 30). Epidural catheters were inserted by a team that supervised catheter care and infusion rates in the postoperative period. The nonepidural group received intramuscular injections on a regular basis. Patients filled out visual analog scales to measure levels of pain ( 1 = minimal, 10 = maximal) every eight hours. Patient activity, bowel, and urinary function were recorded by the nursing staff. Control of pain (as measured by the daily average visual analog score) was more effective in Group 1 (P < .001) on postoperative days 1-3 (1.3 vs 3.6 on day 1, 0.7 vs 2.6 on day 2, 0.9 vs 3 on day 3). There was no significant difference in mean values between groups 1 and 2 with respect to first ambulation on the hospital ward, onset of liquid diet, intake of solid food, first spontaneous voiding, first bowel movement, length of hospitalization, or charge of hospitalization ($13,439 +/- 7,452 vs $11,821 +/- 6,630). We conclude that epidural anesthesia significantly lessens incisional pain following major elective lower gastrointestinal surgery when compared to analgesic injections alone. However, while not statistically significant, the overall charge was increased by 14% in the epidural group. This finding should be examined in light of the relatively low pain level in patients receiving narcotic injections alone.

Long-term outcome in women less than 30 years of age with breast cancer.

BACKGROUND AND OBJECTIVES: Young age at diagnosis is associated with poor prognosis in female breast cancer, but few studies report long-term outcome in women less than 30 years of age. We evaluated 30-year survival in this patient population. METHODS: A retrospective analysis was performed of 29 women less than 30 years of age who were diagnosed with breast cancer between the years 1953 and 1983. All but two patients were followed either until death or for a minimum of 30 years. RESULTS: Actuarial 30-year survival was 19% for the entire group and 10% for women with invasive ductal carcinoma. Twenty-two (92%) of 24 deaths were due to metastatic breast cancer, including three deaths occurring after disease-free intervals of more than 12 years. CONCLUSIONS: Breast cancer in our study population of women less than 30 years of age was a highly lethal disease, particularly in patients with invasive ductal carcinoma. The phenomenon of late death after a long disease-free interval is important in the interpretation of data reflecting newer forms of breast cancer treatment.

Linkage of Niemann-Pick disease type D to the same region of human chromosome 18 as Niemann-Pick disease type C.

Niemann-Pick type II disease is a severe disorder characterized by accumulation of tissue cholesterol and sphingomyelin and by progressive degeneration of the nervous system. This disease has two clinically similar subtypes, type C (NPC) and type D (NPD). NPC is clinically variable and has been identified in many ethnic groups. NPD, on the other hand, has been reported only in descendants of an Acadian couple who lived in Nova Scotia in the early 18th century and has a more homogeneous expression resembling that of less severely affected NPC patients. Despite biochemical differences, it has not been established whether NPC and NPD are allelic variants of the same disease. We report here that NPD is tightly linked (recombination fraction .00; maximum LOD score 4.50) to a microsatellite marker, D18S480, from the centromeric region of chromosome 18q. Carstea et al. have reported that the NPC gene maps to this same site; therefore we suggest that NPC and NPD likely result from mutations in the same gene.

Giant diverticula of the colon: a clinical assessment.

Giant colonic diverticulum is a rare complication of diverticular disease. In the English literature, only 81 cases have been described. Twelve patients had complications caused by the giant diverticulum. Seventy patients were treated operatively, and three died. Elective resection of the diverticulum and the adjacent colon with primary anastomosis is the ideal treatment. The significant number of complications caused by the giant diverticulum and the low morbidity and mortality rate associated with surgical treatment reinforce the importance of accurate diagnosis and elective treatment of this disorder.

Minor malformations and physical measurements in autism: data from Nova Scotia.

In the context of an epidemiological study of autism in Nova Scotia, subjects were evaluated for minor physical anomalies and physical measurements. Normal control children, children with autism and their siblings, and children with developmental disabilities and their siblings were compared. Posterior rotation of the external ears was found to be a characteristic related to autism specifically, rather than to developmental disabilities in general. Small feet and normal-to-large hands also were observed in the autism group. Children with autism had a significant reduction in interpupillary distance, but not intercanthic distance or head circumference. In contrast, children with other developmental disabilities were notable for general small stature, which affected the hands, feet, eyes, and head size, as well as height. Abnormal ear configuration was the minor malformation most characteristic of the developmental disability group, and the subset of Down syndrome children had single transverse creases of the palm and epicanthic folds that resulted in significantly increased rates of these anomalies in the developmentally disabled controls. Siblings of the two disabled groups were not significantly different from normal controls on any of the measures that characterized children with autism or other developmental disabilities. The results agree with those of several previous studies, which have suggested that abnormalities of the ears are the general category of minor anomalies most associated with autism. Recent evidence regarding the embryological origin of autism suggests that the ear effects may be an important marker of the initiating events that lead to the disorder.

Detection of a R173W mutation in the porphobilinogen deaminase gene in the Nova Scotian "foreign Protestant" population with acute intermittent porphyria: a founder effect.

OBJECTIVES: Acute intermittent porphyria (AIP) is caused by mutations in the porphobilinogen deaminase (PBGD) gene that disrupt the function of the enzyme. Many mutations that lead to decreased PBGD activity have been described. An Arg to Trp substitution at codon 173 (CGG-->TGG in exon 10) and designated R173W, which leads to a CRIM-negative phenotype, has been reported in Swedish, Finnish, Scottish, and South African kindreds, and in a Nova Scotian proband with fatal AIP. In this work, we investigated the presence of this mutation in a Nova Scotian patient population presenting with AIP. DESIGN AND METHODS: Single-strand conformation polymorphism analysis and DNA sequencing by TA cloning and Sanger's dideoxy chain termination method, were used to confirm the maternal transmission of this mutation to the proband. The mutation also eliminates an Ncil (also Mspl) endonuclease restriction site, which allows for detection of the mutant allele by polymerase chain reaction amplification and restriction enzyme digestion. RESULTS: The family of the Nova Scotian proband and four other AIP kindreds showed the presence of the same mutation. These five families are descendants of German, Swiss, and French immigrants historically known as the "Foreign Protestants," who were recruited to Nova Scotia in the 1750s. CONCLUSION: In all these families, descent from one couple that settled in Nova Scotia in 1751 has been identified by genealogy research, consistent with a founder effect within this population. This is the first identified mutation in PBGD causing AIP that has been linked to a founder effect in descendants of an immigrant population to North America, and which could be traced to such a distant background, similar to the South African variegate porphyria mutation.

Stability and oligomeric equilibria of refolded interleukin-1beta converting enzyme.

We report the preparation and characterization of interleukin-1beta converting enzyme (ICE) refolded from its p20 and p10 protein fragments. Refolded ICE heterodimer (p20p10) was catalytically active but unstable, and in size exclusion chromatography eluted at an apparent molecular mass of 30 kDa. The mechanisms of the observed instability were pH-dependent dissociation at low enzyme concentrations, and autolytic degradation of the p10 subunit at high concentrations. Binding and subsequent removal of a high affinity peptidic inhibitor increased the apparent molecular mass to 43 kDa (by size exclusion chromatography), and significantly increased its stability and specific activity. Chemical cross-linking and SDS-polyacrylamide gel electrophoresis analysis of the 43-kDa size exclusion chromatography conformer revealed a 60-kDa species, which was absent in the 30-kDa conformer, suggesting that inhibitor binding caused formation of a (p20p10)2 homodimer. The observation of a reversible equilibrium between ICE (p20p10) and (p20p10)2 suggests that analogous associations, possibly between ICE and ICE homologs, can occur in vivo, resulting in novel oligomeric protease species.

Primary lymphoproliferative disorders of the breast.

Primary breast lymphoproliferative disorders are rare lesions and include both the malignant lymphomas and the benign pseudolymphomas. We reviewed 4,491 consecutive cases of breast cancer diagnosed and treated between 1973 and 1988. Patients with lymphoma in other sites and those with lymphomas limited to axillary nodes were excluded. RESULTS. Five patients (0.11%) presented with primary lymphoreticular lesions, of which three were primary non-Hodgkin's lymphoma and two were pseudolymphomas. Patients were followed clinically through to the present time or until death occurred. Surgical procedures included incisional or excisional biopsy in four patients and modified radical mastectomy in one. Two patients received chemo-therapy and one received radiotherapy. One patient with pseudolymphoma subsequently developed infiltrating ductal carcinoma of the same breast. Three patients with primary breast non-Hodgkin's lymphoma died within the follow-up period, with a mean survival of 33 months. CONCLUSIONS. We conclude that primary breast lymphoma is a rare and aggressive breast malignancy with a poor prognosis despite different treatment options.

Long-term management of diverticulitis in young patients.

PURPOSE: This study was designed to determine the natural history of documented diverticulitis that resolves after treatment with intravenous antibiotics and bowel rest in patients under the age of 50. METHODS: Records of 40 patients aged 50 or under who were hospitalized with the diagnosis of acute diverticulitis between 1980 and 1984 were reviewed to obtain data regarding how the diagnosis was made. Patients successfully treated with antibiotics were contacted five to nine years after their attack and surveyed via telephone questionnaire about symptoms, recurrent attacks, and surgical interventions. RESULTS: A total of 40 patients were included in the study. Ten patients (25 percent) required surgery during initial admission, and 30 patients were discharged with resolution of their symptoms after treatment with intravenous antibiotics and bowel rest. A five-year to nine-year follow-up was obtained on patients treated medically, one-third of whom underwent operation for diverticulitis during this period, and two-thirds of whom did not require surgery during the follow-up period. All operations were elective with single-stage resections. CONCLUSION: Based on our data, we do not recommend surgery in this population after a single episode of diverticulitis that resolves after treatment with antibiotics.

Antepartum diagnosis of pancreatic carcinoma: a case report.

A pregnant patient found to have primary carcinoma of the pancreas is discussed. The extreme rarity of this condition is emphasized. There are only a handful of cases reported in the world literature, and the diagnoses in these reports were made almost exclusively post partum. We could find only two reported cases in which the diagnosis was made ante partum, those by Gamberdella in 1984 and by Duff and Greene in 1985. The case we present demonstrates the difficult management decisions that arise when the diagnosis of pancreatic carcinoma is made in the midst of gestation.

Endoscopic localization of colon cancers.

In order to determine the accuracy of endoscopic localization of colon cancers, the endoscopic location was compared to the actual location at the time of operation in 320 patients who underwent resection of intraabdominal colon cancer between 1983 and 1988. The endoscopic location was correct in 86% of the cases. There were 44 endoscopic errors, including seven missed cancers. One-third of all endoscopic errors occurred when the tumor was in the cecum. We conclude that endoscopy is an accurate method of localizing colon cancers. However, with the advent of laparoscopic surgery and the loss of the ability to palpate the colon, the 14% of endoscopic errors take on a greater importance and additional means for localizing tumors should be pursued in selected cases.