Frequent loss of heterozygosity in early non-small cell lung cancers at chromosome 9p21 proximal to the CDKN2a gene.

Deletions involving the chromosome 9p21 region have been reported as frequent events in non-small cell lung cancer (NSCLC). To investigate potential tumor-suppressor gene (TSG) loci within the 9p21 region, which also harbors the candidate TSG locus CDKN2a, we studied 32 cases of primary NSCLC for loss of heterozygosity (LOH). Tumor and paired normal lung cells were microdissected from lung tissue imprints and all samples screened using PCR-LOH analysis with 15 9p markers. In addition, 3 NSCLC cell lines and their matched normal lung and tumor DNA were similarly analyzed. LOH at the marker D9S259, which is proximal to the CDKN2a locus, was found most frequently (52%), while LOH at D9S942, the marker closest (5 kb) to the CDKN2a gene, was seen in only 17%. Homozygous loss of markers close to CDKN2a was, however, detected in 2 of the 3 cell lines and one accompanying tumor sample. We propose that a TSG in the region of deletion proximal to the CDKN2a gene within 9p21 may play a significant role in the pathogenesis and progression of NSCLC.

Homozygous and hemizygous deletions of 9p centromeric to the interferon genes in lung cancer.

Cytogenetic analysis has indicated that deletion of chromosome 9p occurs in a significant number of non-small cell lung and mesothelioma tumors. Using paired oligonucleotide primers, we have undertaken an extensive analysis of 9p markers to determine homozygous and heterozygous loss of marker sequences. Homozygous loss of D9S169 and D9S171, both of which map centromeric to the IFN gene cluster, were noted in three cell lines (27%) and hemizygous deletions of one or both of these loci was found in a further six cell lines (54%). These data suggest the presence of a potential tumor suppressor gene for lung cancer in proximity to D9S169 and D9S171 at 9p21.

A soluble recombinant fusion protein of the transmembrane envelope protein of equine infectious anaemia virus for ELISA.

The use of the bacterial expression vector, pGex, to produce an abundant, soluble fusion protein of gp45 from equine infectious anaemia virus is described. Purification of the recombinant protein was achieved by one step affinity chromatography on immobilized glutathione using competitive elution so no harsh conditions were required. This provides a readily available antigen that is defined, plentiful and cheap. Yields of 3.5 mg of purified soluble protein/litre of bacterial culture were obtained. This antigen was found to be suitable for ELISA. Background reactivity to either the glutathione-S-transferase (GST) fusion partner by immune sera or the EIA-GST fusion protein by normal sera were negligible.

Breastfeeding success with preterm quadruplets.

Although the incidence of triplet and quadruplet birth has increased in the United States, few research-based guidelines are available for assisting mothers of these multiple births with breastfeeding. The purpose of this case study is to report a successful breastfeeding experience of a mother with preterm quadruplets. The quadruplets were born by cesarean delivery at 34 weeks' gestation and weighed from 1,820 g to 2,240 g. In-hospital breastfeeding experiences were managed by the authors, according to research-based guidelines for breastfeeding preterm neonates and infants. During the first month after discharge of the four newborns, the mother breastfed 12-34 times daily. Mean daily weight gains for the quadruplets during this time varied from 30 g to 54 g, indicative of adequate maternal milk supply. Nurses in maternity and neonatal specialties can apply the findings from this study to similar cases of mothers who want to breastfeed multiple neonates or infants.

Comparison of diagnostic procedures for porcine leptospirosis.

Kidneys and matched serum samples were obtained from 368 pigs slaughtered at three Victorian abattoirs, and originating from 42 farms. Macroscopic lesions (white spots) were observed on 102 of the kidneys. Serum samples were tested by the microscopic agglutination test (MAT) and by an IgM enzyme immunoassay (EIA). Kidneys were cultured for leptospires, examined histologically after Warthin-Starry silver staining and after immunogold silver staining (IGSS), and tested for leptospiral DNA by DNA hybridization. Forty-four infected pigs were identified by culture or immunogold silver staining of kidneys or by high MAT titres (greater than or equal to 1024). Infection was demonstrated in 7.5% of visibly normal kidneys, in 23.5% of kidneys with white spots, and in 48% of kidneys with large white spots, of 1 cm diameter or greater. The apparent (maximum) sensitivities of diagnostic procedures for detecting infection were as follows: MAT (at a titre of either 64 or 1024) 95%; IgM EIA 82%; culture 61%; presence of white spots 55%; IGSS 52%; presence of large white spots 30%; Warthin-Starry silver staining 20%. IGSS, Warthin-Starry staining and DNA hybridization all appeared to be highly specific. Of 22 kidney sections identified as positive by IGSS, 13 showed intact leptospires, and these kidneys were all culture-positive. Nine others showed leptospiral antigen in the kidney tubules but no intact leptospires. Only five of these kidneys were culture-positive.

Leptospira interrogans serovar hardjo is not a major cause of bovine abortion in Victoria.

The aim of this study was to determine whether evidence could be obtained of foetal infection with Leptospira interrogans serovar hardjo in aborted foetuses collected from dairy farms. Material from 197 abortions occurring over a wide area of Victoria was collected over 3 years. None of 195 foetal kidney cultures or 7 cultures from membranes was positive for leptospiral organisms. Immunogold silver staining for leptospires was performed on sections of kidneys, lungs or heart from 156 foetuses, with negative results. Evidence of transient leptospiral infection in 11 of 123 foetuses was obtained by foetal heart blood serology. Two isolates of L. interrogans serovar hardjo were obtained from the urine of milking cows. These strains were examined by restriction endonuclease analysis and both were shown to be of the genotype Hardjobovis, as have been all Australian isolates studied so far. It appears that foetal infection with serovar hardjo is not associated with any substantial proportion of bovine abortions in Victoria, in contrast to the situation in Northern Ireland. The apparent absence from Victoria of the pathogenic genotype Hardjoprajitno is a possible explanation.

Microbial Ecology of the Gut in Laboratory Stocks of the Migratory Grasshopper, Melanoplus sanguinipes (Fab.) (Orthoptera: Acrididae).

Mean pH values in pooled samples of foregut, midgut, and hindgut from adult Melanoplus sanguinipes, which had been raised in the laboratory on barley shoots and wheat bran, were 5.15, 6.39, and 5.98, respectively. Homogenates of midgut/hindgut sections and frass (feces) yielded colony counts of bacteria by the spread plate method of 5.7 to 5.9 and 5.3 to 5.5 log(10) colonies per mg, respectively; there were no significant differences (P > 0.05) between counts obtained on several media or on media incubated aerobically or anaerobically. There was no evidence of significant populations of protozoa, fungi, or obligately anaerobic bacteria associated with the gut. A total of 168 pure strains of bacteria isolated from the gut sections were characterized and assigned to 11 taxonomic groups, including Enterococcus spp., Serratia liquefaciens, Pseudomonas spp., and Enterobacter spp. Numbers of Enterococcus spp. in the gut were 2 to 3 orders of magnitude higher than those of the other genera. Strains representing only four of the groups were recovered from bran fed to the grasshoppers; the barley shoots, which were raised in sterile soil, appeared virtually sterile. Examination of the gut wall by scanning electron microscopy revealed the presence of epimural bacteria in the foregut and hindgut but not in the midgut. The distribution of epimural cocci and bacilli differed with the gut section examined. Numerous spherical to ovoid structures up to 10 mum in diameter, which were not identified, were associated with the microvillous surface of the midgut epithelium. Acetate was present in gut, hemolymph, and frass, and it was shown that representative isolates of Enterococcus spp. and Enterobacter agglomerans produced acetate when incubated in an aqueous suspension of bran. The egestion time of solid digesta, as measured with methylene blue-stained barley shoots, was 3.0 to 5.7 h. The results show that M. sanguinipes supported extensive indigenous populations of luminal and epimural bacteria in the gut which were composed predominantly of facultatively anaerobic species; the relatively short egestion time, indicating rapid passage of digesta through the gut, was consistent with the microscopic appearance of digesta residues in frass and could account, at least in part, for the absence of a significant population of obligately anaerobic bacteria from the gut.

Isolation and presumptive identification of adherent epithelial bacteria ("epimural" bacteria) from the ovine rumen wall.

One hundred sixty-one strains of adherent bacteria were isolated under anaerobic conditions from four sites on the rumen epithelial surface of sheep fed hay or a hay-grain ration. Before isolation of bacteria, rumen tissue was washed six times in an anaerobic dilution solution, and viable bacteria suspended in the washings were counted. Calculation indicated that unattached bacteria would have been removed from the tissue by this procedure, but a slow and progressive release of attached bacteria also occurred. Nevertheless, a wide range of characteristic morphological types remained associated with the epithelium as demonstrated by scanning electron microscopy. Most of these types were represented among the isolates. Characterization and presumptive identification of the isolates showed that 95.0% belonged to previously described genera of functionally significant rumen bacteria, including Butyrivibrio sp. (31.1%), Bacteroides sp. (22.4%), Selenomonas ruminantium (9.9%), Succinivibrio dextrinosolvens (8.7%), Streptococcus bovis (8.1%), Propionibacterium sp. (4.3%), Treponema sp. (3.1%), and Eubacterium sp., Lachnospira multiparus, and Ruminococcus flavefaciens (2.5% each). Eight isolates (5.0%) were not identified. L. multiparus was recovered only from hay-fed animals; all other genera were obtained from animals fed either ration. All S. bovis strains and two strains each of Bacteroides sp. and Butyrivibrio sp. were aerotolerant; all other strains were strictly anaerobic. Bacteria representing the gram-positive, facultatively anaerobic flora associated with rumen wall tissue (R. J. Wallace, K.-J. Cheng, D. Dinsdale, and E. R. Ørskov, Nature (London) 279:424-426, 1979) were therefore not recovered by the techniques used; instead a different fraction of the adherent population was isolated. The term "epimural" is proposed to describe the flora associated with the rumen epithelium.