Integrated genomic map from uropathogenic Escherichia coli J96.

Escherichia coli J96 is a uropathogen having both broad similarities to and striking differences from nonpathogenic, laboratory E. coli K-12. Strain J96 contains three large (>100-kb) unique genomic segments integrated on the chromosome; two are recognized as pathogenicity islands containing urovirulence genes. Additionally, the strain possesses a fourth smaller accessory segment of 28 kb and two deletions relative to strain K-12. We report an integrated physical and genetic map of the 5,120-kb J96 genome. The chromosome contains 26 NotI, 13 BlnI, and 7 I-CeuI macrorestriction sites. Macrorestriction mapping was rapidly accomplished by a novel transposon-based procedure: analysis of modified minitransposon insertions served to align the overlapping macrorestriction fragments generated by three different enzymes (each sharing a common cleavage site within the insert), thus integrating the three different digestion patterns and ordering the fragments. The resulting map, generated from a total of 54 mini-Tn10 insertions, was supplemented with auxanography and Southern analysis to indicate the positions of insertionally disrupted aminosynthetic genes and cloned virulence genes, respectively. Thus, it contains not only physical, macrorestriction landmarks but also the loci for eight housekeeping genes shared with strain K-12 and eight acknowledged urovirulence genes; the latter confirmed clustering of virulence genes at the large unique accessory chromosomal segments. The 115-kb J96 plasmid was resolved by pulsed-field gel electrophoresis in NotI digests. However, because the plasmid lacks restriction sites for the enzymes BlnI and I-CeuI, it was visualized in BlnI and I-CeuI digests only of derivatives carrying plasmid inserts artificially introducing these sites. Owing to an I-SceI site on the transposon, the plasmid could also be visualized and sized from plasmid insertion mutants after digestion with this enzyme. The insertional strains generated in construction of the integrated genomic map provide useful physical and genetic markers for further characterization of the J96 genome.

Type-specific contributions to chromosome size differences in Escherichia coli.

The Escherichia coli genome varies in size from 4.5 to 5.5 Mb. It is unclear whether this variation may be distributed finely throughout the genome or is concentrated at just a few chromosomal loci or on plasmids. Further, the functional correlates of size variation in different genome copies are largely unexplored. We carried out comparative macrorestriction mapping using rare-restriction-site alleles (made with the Tn10dRCP2 family of elements, containing the NotI, BlnI, I-CeuI, and ultra-rare-cutting I-SceI sites) among the chromosomes of laboratory E. coli K-12, newborn-sepsis-associated E. coli RS218, and uropathogenic E. coli J96. These comparisons showed just a few large accessory chromosomal segments accounting for nearly all strain-to-strain size differences. Of 10 sepsis-associated and urovirulence genes, previously isolated from the two pathogens by scoring for function, all were colocalized exclusively with one or more of the accessory chromosomal segments. The accessory chromosomal segments detected in the pathogenic strains from physical, macrorestriction comparisons may be a source of new virulence genes, not yet isolated by function.

A national profile of nursing centers. Arenas for advanced practice.

Escalating costs of health care and decreasing accessibility to care are growing dilemmas in America. Primary care is being delivered in nurse-managed clinics, or nursing centers, by nurses qualified for advanced nursing practice. The purpose of this study was to build a profile of the structure and function of nursing centers, noting major differences between those that are academic-based and those that are not. Questionnaires were developed and mailed to 234 subjects/agencies in every major region of the United States. Responses were tabulated and percentages were calculated to make comparisons and build a profile of nursing centers, based on the following factors identified in the literature and by nurse experts: (a) funding and fees, (b) organizational structure, (c) time elements, (d) demographics, (e) services, (f) qualifications and categories of staff, (g) functions of staff (h) research endeavors, (i) major barriers, and (j) major sources of support.

Elevation of lymphocyte CD45 protein tyrosine phosphatase activity during mitosis.

CD45 is an abundant cell-surface protein tyrosine phosphatase (PTPase) of hematopoietic cells that mediates specific tyrosine dephosphorylation in lymphocyte Ag activation. The hypothesis that CD45 PTPase activity varies during progression through the cell cycle was examined in this study. Expression of CD45 PTPase activity was determined in pre-B lymphoma (70Z/3.12) and cytolytic T lymphocyte (CTLL-2) cell lines after fractionation by counterflow centrifugal elutriation into cell cycle-stage-enriched subpopulations. For both cell lines, CD45 PTPase activity was elevated 2- to 10-fold in late G2 + M fractions compared with the PTPase activity of asynchronous cells. FACS and SDS-PAGE analyses indicated that there was only a minor increase in CD45 protein expression during progression through the cell cycle, suggesting that increased CD45 PTPase activity was a result of increased enzymatic activity. Cyclin B expression in 70Z/3.12 cells was evaluated in elutriated subpopulations and intact cyclin B was present in all cell cycle phases from G1 to late G2 + M, disappearing in mitosis and just before those fractions containing maximum CD45 activity. This observation indicated that the peak PTPase activity of CD45 occurred in late mitosis or in cytokinesis. The elevation of CD45 PTPase activity in mitosis supports the hypothesis that CD45 has a role in phosphorylation events occurring during cell division.

Are routine chest radiographs prior to angiography of any value?

Chest radiographs are frequently requested prior to diagnostic angiography, though there is no published evidence of their clinical utility. This study was undertaken to evaluate their contribution to patient management. The routine chest radiographs obtained prior to peripheral and coronary angiography in 240 patients were prospectively reviewed for abnormalities likely to affect management. Two hundred and twenty (91.7%) examinations were performed, of which 164 were obtained within 24 h of angiography. Previous radiographs were available in 154 patients (64.2%). One hundred and sixteen radiographs were normal. There were 117 abnormalities on the radiographs of 104 patients, mainly cardiac enlargement and heart failure. No angiogram was postponed or cancelled because of abnormalities detected on a routine radiograph, although radiographic findings led to a change in the volume of contrast medium injected into dilated aortic roots in 10 patients undergoing cardiac catheterization. Pre-angiography radiographs had no effect on the practice of peripheral angiography. In only one patient were further investigations and therapy instigated because of findings, but even in this case these findings were present on previous studies. We conclude that routine pre-angiography chest radiographs are not necessary in the absence of specific clinical indications.

Purification to apparent homogeneity by immunoaffinity chromatography and partial characterization of the GM3 ganglioside-forming enzyme, CMP-sialic acid:lactosylceramide alpha 2,3-sialyltransferase (SAT-1), from rat liver Golgi.

CMP-sialic acid:lactosylceramide alpha 2,3-sialyltransferase (SAT-1) has been purified approximately 40,000-fold to apparent homogeneity from rat liver Golgi. The enzyme was solubilized from Golgi vesicles in 5% lauryldimethylamine oxide and "partially" purified by affinity chromatography twice on CMP-hexanolamine and once on lactosylceramide aldehyde-Sepharose 4B. Final purification was achieved by immunoaffinity chromatography on M12GC7-Gel 10. The M12GC7 monoclonal antibody specifically inhibits and immunoprecipitates SAT-1 activity. Identification of the protein, with an apparent molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of about 60,000 daltons, was confirmed by Western blot and immunodetection with M12GC7. SAT-1 specifically catalyzes the transfer of N-acetylneuraminic acid (NeuAc, sialic acid) to lactosylceramide (Gal beta 1-4Glc beta 1-O-ceramide), forming GM3 ganglioside. Studies on substrate specificity indicate that the preferred acceptors have the general structure saccharide beta 1-O-ceramide, a disaccharide being preferred to a monosaccharide. SAT-1 is a glycoprotein. The carbohydrate moieties are detected with specific lectins. Deglycosylation of SAT-1 with N-glycanase results in an increase in a 43,000-dalton band. The two-dimensional electrophoretogram of SAT-1 indicates a pI range of 5.7-6.2 for the 60,000-dalton protein.

Special considerations in the purification of the GM3 ganglioside-forming enzyme, CMP-sialic acid:lactosylceramide alpha 2-3 sialyltransferase (SAT-1): effects of protease inhibitors on rat hepatic SAT-1 activity.

Co-purification of an endogenous proteolytic activity has been proposed as the cause for the size heterogeneity of sialyltransferases. Reported herein are results on the effects of various protease inhibitors, sulfhydryl-reducing agents and antimicrobial agents on SAT-1 activity. Addition of protease inhibitors to immunoaffinity-purified rat liver SAT-1 dramatically affects its activity. All protease inhibitors examined, with the exception of PMSF, inhibited the purified enzyme. The most inhibitory were the cysteine (thiol) protease inhibitors. This effect is less spectacular when the effect of these inhibitors was studied on SAT-1 activity in Golgi-enriched microsomes, although the inhibition was greatest by the cysteine protease inhibitors. One dramatic effect, found in both cases, was the apparent activation of SAT-1 activity in the presence of beta-mercaptoethanol.

Special considerations in the purification of the GM3 ganglioside forming enzyme, CMP-sialic acid:lactosylceramide alpha 2-3 sialyltransferase (SAT-1): solubilization of SAT-1 with lauryldimethylamine oxide.

Lauryldimethylamine oxide (LDAO) was employed in the purification of the GM3 ganglioside forming enzyme, CMP-sialic acid:lactosylceramide alpha 2-3 sialyltransferase (SAT-1) (4). This detergent has advantages over the typically employed Triton detergents in the solubilization and stabilization of this sialyltransferase. Crude protein fractions solubilized from rat liver Golgi by several such detergents are very similar in composition as determined by two-dimensional gel electrophoresis. However, LDAO appears to activate and stabilize SAT-1 activity. It is possible that SAT-1 activation involves the structurally similar hydrophobic moieties and quaternary amino groups of LDAO and phosphatidylcholine.

Development of Down's syndrome infants with and without heart defects and changes in their caretaking environment.

The purpose of this study is two-fold: (1) to describe developmental and family characteristics of infants with Down's syndrome who were enrolled in an intervention programme; and (2) to examine developmental and caretaking patterns related to maternal age and congenital heart defects. Infants and their home environment were assessed at about 3 1/2 months and at 24 months of age. A sample of normal infants of equivalent developmental age were also tested for comparison. We found that while the infants with Down's syndrome showed a significant decline in their developmental quotient compared to the normals, their home environments resembled those of the normal group, particularly at the pretest. Yet, there were more improvements in the homes of the group of normal infants than in the homes of the infants with Down's syndrome, mainly in maternal responsivity. Older mothers restricted their infants with Down's syndrome more than younger mothers, but otherwise they did not seem to differ from each other. Infants with Down's syndrome without heart defects were provided with a better home environment than were infants with Down's syndrome with a heart defect. These results are discussed in the light of the biological limitations placed on the Down's syndrome infants and difficulties in parental adaptation to the diagnosis of Down's syndrome.

Psychiatric consultation-liaison in the acute physical disabilities setting.

This paper provides an overview of psychiatric consultation-liaison services in a general hospital setting. The patient most frequently referred for consultation-liaison services is the individual experiencing depression secondary to physical illness. The dimensions of occupational therapy intervention with this population is featured, including assessment, intervention strategies, and documentation guidelines useful in the treatment planning.

Automated differential leucocyte counting by the Geometric Data Hematrak system: eighteen months experience in a private pathology laboratory.

The Geometric Data Hematrak is a computerized electronic pattern recognition system designed to scan Wright's stained blood films. The instrument consists of a microscopy and data processing computer which classifies leucocytes by a digital image processing system into various cell types. Additional features include facilities for automatic appraisal of red cell morphology and estimation of platelet numbers. We report our experience with the routine use of a Hematrak Model 360 over an 18 mth period, during which time approximately 60 000 blood films have been processed by this system.