Improved, computer-generated system for pyocin typing of Pseudomonas aeruginosa.

We applied numerical clustering algorithms to the selection of a new indicator strain set for the pyocin typing of Pseudomonas aeruginosa. The new indicator set is composed of selected indicator strains from the sets described in 1966 by Gillies and Govan (J. Pathol. Bacteriol. 91:339-345) and in 1974 by Jones, Zakanycz, Thomas, and Farmer (Appl. Microbiol. 27:400-406) and is designated the G-F set. This indicator set consists of 14 indicator strains which typed 99.5% of 114 test cultures, has a high degree of discrimination (10 patterns encompass 50% of the test strains), and provides 62.3% reproducibility of the same typing pattern in duplicate tests done on different days. The G-F set of indicator strains provides slightly higher percentages of typable cultures than either of the other two sets, has greater discriminatory capability, and is more reproducible than they are. We recommend that the G-F set of indicator strains be used instead of the two other sets for pyocin typing of P. aeruginosa. We also tested a recently described overlay procedure for pyocin testing of P. aeruginosa and found it to be superior to previous methods in that it is easier to perform, it provides answers in only 24 h instead of 48 h, and it can be used to type mucoid strains (which previous techniques could not readily do). Thus, the application of numerical clustering algorithms and use of a revised typing procedure have produced an improved system for pyocin typing of P. aeruginosa. Similar procedures may be applicable to other typing systems.     

Role of computed tomography in screening for hepatocellular carcinoma in patients with cirrhosis

One hundred patients with cirrhosis underwent abdominal computed tomography (CT) using a delayed contrast technique to determine liver and spleen volume. These scans were reviewed to screen this "at risk" population for hepatocellular carcinoma (HCC). Fifteen of the 100 screened patients had focal abnormalities suspicious for HCC. On biopsy, only 1 patient was shown to have HCC. The other 14 patients had either fatty infiltration or focal regeneration. In the same time interval, a total of 10 patients had histologically proven HCC. All presented with symptoms and died within 4 months of diagnosis. The results show that focal hepatic lesions can be detected by CT but in this population the lesions may not be due to HCC. The incidence of HCC was approximately 1%, probably reflecting a truly low incidence in this population.     

Relationship of oxidative damage to the hepatocarcinogenicity of the peroxisome proliferators di(2-ethylhexyl)phthalate and Wy-14,643

Quantitative comparisons of the time course of biochemical andmorphological changes induced by peroxisome proliferators resultingin low and high incidences of hepatic cancer have not been conductedpreviously under bioassay conditions. [4-Chloro-6-(2,3 xylidino)-2-pyrimidyl-thio]aceticacid (Wy-14,643) at 0.1% in the diet produced a much higherincidence of hepatic cancer in male rats than 1.2% di(2-ethylhexyl)phthalate(DEHP) in the diet. Both diets, however, caused similar degreesof peroxisome proliferation. To investigate this differencein carcinogenicity, H₂O₂-detoxification mechanisms and indicesof oxidative damage were evaluated in male F-344 rats fed 1.2%DEHP or 0.1% Wy-14,643 for up to one year. DEHP or Wy-14,643treatment increased hepatic catalase activity 25% from 8 to365 days. DEHP or Wy-14,643 treatment decreased hepatic glutathioneperoxidase activity by 50% from 8 to 365 days. Glutathione concentrationswere not affected by 151 days of DEHP or Wy-14,643 feeding.The similar effects of DEHP and Wy on H₂O₂ detoxification enzymesand glutathione concentrations suggests that these factors arenot responsible for the widely different carcinogenicities ofWy-14,643 and DEHP. Hepatic vitamin E concentrations were 50%lower in rats receiving Wy-14,643 for 151 days as compared torats fed DEHP or control diets. Lipofuscin, which was containedwithin lysosomes, was increased 3-fold after 39 days of DEHPand remained at this level up to 365 days of treatment. In comparison,lipofuscin was increased 4-fold after 18 days of Wy-14,643 andcontinued to accumulate in a linear manner reaching values 30-foldover controls after 365 days of treatment. DEHP treatment for39–365 days increased the activities of the lysosomalenzymes -fucosidase, ß-galactosidase and N-acetylglucosaminidase50–100%. The same enzyme activities were increased 4-foldafter 39–365 days of Wy-14,643. Lysosomal cathepsin Bactivity was unchanged by DEHP but doubled by 151 and 365 daysof Wy-14,643. Acid phosphatase activity was unchanged by DEHPbut increased by 50% after 151 and 365 days of Wy-14, 643. Inaddition, conjugated dienes were increased (45%) only in ratsreceiving Wy-14,643 for 151 and 365 days. These data show forthe first time that the magnitude and time course of lipofuscindeposition, induction of lysosomal enzymes and conjugated dieneaccumulation, is correlated closely with the degree of carcinogenicity.Wy-14,643-induced decreases in hepatic vitamin E concentrationscould contribute to the observed accumulation of conjugateddienes at later time points. The data suggest that lipofuscinaccumulation is an early biomarker that is quantitatively predictiveof the carcinogenicity of the peroxisome proliferators DEHPand Wy-14,643.     

Clinical and molecular study in congenital muscular dystrophy with partial laminin alpha 2 (LAMA2) deficiency

Complete laminin alpha2 (LAMA2) deficiency causes approximately half of congenital muscular dystrophy (CMD) cases. Many loss-of-function mutations have been reported in these severe, neonatal-onset patients, but only single missense mutations have been found in milder CMD with partial laminin alpha2 deficiency. Here, we studied nine patients diagnosed with CMD who showed abnormal white-matter signal at brain MRI and partial deficiency of laminin alpha2 on immunofluorescence of muscle biopsy. We screened the entire 9.5 kb laminin alpha2 mRNA from patient muscle biopsy by direct capillary automated sequencing, single strand conformational polymorphism (SSCP), or denaturing high performance liquid chromatography (DHPLC) of overlapping RT-PCR products followed by direct sequencing of heteroduplexes. We identified laminin alpha2 sequence changes in six of nine CMD patients. Each of the gene changes identified, except one, was novel, including three missense changes and two splice-site mutations. The finding of partial laminin alpha2 deficiency by immunostaining is not specific for laminin alpha2 gene mutation carriers, with only two patients (22%) showing clear causative mutations, and an additional three patients (33%) showing possible mutations. The clinical presentation and disease progression was homogeneous in the laminin alpha2-mutation positive and negative CMD patients.     

Search for intracranial aneurysm susceptibility gene(s) using Finnish families

Background

Cerebrovascular disease is the third leading cause of death in the United States, and about one-fourth of cerebrovascular deaths are attributed to ruptured intracranial aneurysms (IA). Epidemiological evidence suggests that IAs cluster in families, and are therefore probably genetic. Identification of individuals at risk for developing IAs by genetic tests will allow concentration of diagnostic imaging on high-risk individuals. We used model-free linkage analysis based on allele sharing with a two-stage design for a genome-wide scan to identify chromosomal regions that may harbor IA loci.

Methods

We previously estimated sibling relative risk in the Finnish population at between 9 and 16, and proceeded with a genome-wide scan for loci predisposing to IA. In 85 Finnish families with two or more affected members, 48 affected sibling pairs (ASPs) were available for our genetic study. Power calculations indicated that 48 ASPs were adequate to identify chromosomal regions likely to harbor predisposing genes and that a liberal stage I lod score threshold of 0.8 provided a reasonable balance between detection of false positive regions and failure to detect real loci with moderate effect.

Results

Seven chromosomal regions exceeded the stage I lod score threshold of 0.8 and five exceeded 1.0. The most significant region, on chromosome 19q, had a maximum multipoint lod score (MLS) of 2.6.

Conclusions

Our study provides evidence for the locations of genes predisposing to IA. Further studies are necessary to elucidate the genes and their role in the pathophysiology of IA, and to design genetic tests.     

Effect of acute insulin administration on biliary lipid secretion by the diabetic rat.

The insulin-deficient state induces profound changes in bile formation. The present work was to test the effect of acute insulin administration on lipid secretion into bile in diabetic rats. Diabetes was induced by streptozotocin injection (6 mg/100 g body-weight, i.p., 6 days before the experiments). Bile formation was stimulated by taurocholate infusion (0.5 mumol/min/100 g body-weight). Intravenous administration of insulin (bolus: 100 mU/100 g body-weight, plus infusion: 5 mU/min/100 g body-weight) induced choleresis accompanied by a slight and transient enhancement in bile acid output which was similar to that found in lecithin and cholesterol output in the control group. However, insulin induced a rapid and significant (P less than 0.05) reduction in biliary lipid output in the diabetic rats. These results suggest that insulin may play an important role in mechanisms other than synthesis involved in the supply of biliary lipids towards the canaliculi.     

Cap formation in a B-lymphocyte cell line is inhibited by pertussis toxin and phorbol ester.

We have examined concanavalin A (Con A)-induced cap formation in a B-lymphocyte derived cell line, LAZ-559. Treatment with pertussis toxin (PT) or phorbol-12-myristate-13-acetate (PMA) prior to exposure of the cells to Con A abolished the capping reaction. The possible role of calcium mobilization was tested using cells pre-loaded with the fluorescent dye Quin2. Both PT and PMA caused inhibition of calcium mobilization at concentrations similar to those observed for the inhibition of capping. The possible identity of the substrate for pertussis toxin was examined by carrying out ADP-ribosylation of the isolated plasma membranes using [alpha-32P]NAD and pertussis toxin. Several bands were observed at molecular weights of 109,000, 43,000, 34,000 and 22,000. Comparative labelling with cholera toxin revealed a separate band at 42,000. The bands at 43,000 and 34,000 are PT specific. Of these, the 43,000 band comigrated with the PT substrate that has been shown to regulate capping in human neutrophils (Lad et al., 1985a, 1986b). PMA-induced phosphorylation was examined in 32P-loaded cells, and multiple bands were observed to be labelled in a dose-dependent manner, at least two of which were very similar in mobility to the PT substrate. Our results suggest that regulation of calcium mobilization and the control of capping via a PMA-sensitive, GTP-binding protein are probably general phenomena observable in multiple cell systems.