Construction and characterization of three region-specific microdissection libraries for human chromosome 18

Three region-specific libraries for the entire human chromosome 18 were constructed using microdissection and MboI linker-adaptor microcloning techniques. The libraries included 18pter-p11.1 (designated 18P library), 18q 11.1-q12.3 (18Q1 library), and 18q21.1-qter (18Q2 library). Samples of the microclones from each library were analyzed in detail. The insert sizes ranged between 50–600 bp, with a mean of 180–220 bp for the three libraries. The libraries contained approximately 40–60% microclones with unique sequence inserts. More than 30 unique sequence microclones from each library were analyzed by Southern blot hybridization to demonstrate that they are human specific and were derived from chromosome 18. The human gemomic HindIII fragments hybridized to each microclone were determined and microclones crosshybridized to rodent species were identified. These region-specific libraries and the unique sequence microclones from the libraries are useful reagents for (1) isolating hughly polymorphic microsatellite markers for refined linkage analysis, (2) identifying corresponding YAC, BAC or other clones with large inserts for contig assembly and high resolution physical mapping, (3) isolating cDNA clones from the dissected region, and (4) convenient sequencing of the microclones to prepare high density markers and sequence-tagged sites (STSs). Such applications have been demonstrated in a series of similarly constructed microdissection libraries from other regions of the human genome.     

Genomic Analysis of a Pathogenicity Island in Uropathogenic Escherichia coli CFT073: Distribution of Homologous Sequences among Isolates from Patients with Pyelonephritis, Cystitis, and CatheterAssociated Bacteriuria and from Fecal Samples

Urinary tract infection is the most frequently diagnosed kidney and urologic disease and Escherichia coli is by far the most common etiologic agent. Uropathogenic strains have been shown to contain blocks of DNA termed pathogenicity islands (PAIs) which contribute to their virulence. We have defined one of these regions of DNA within the chromosome of a highly virulent E. coli strain, CFT073, isolated from the blood and urine of a woman with acute pyelonephritis. The 57,988-bp stretch of DNA has characteristics which define PAIs, including a size greater than 30 kb, the presence of insertion sequences, distinct segmentation of K-12 and J96 origin, GC content (42.9%) different from that of total genomic DNA (50.8%), and the presence of virulence genes (hly and pap). Within this region, we have identified 44 open reading frames; of these 44, 10 are homologous to entries in the complete K-12 genome sequence, 4 are nearly identical to the sequences of E. coli J96 encoding the HlyA hemolysin, 11 encode P fimbriae, and 19 show no homology to J96 or K-12 entries. To determine whether sequences found within the junctions of the PAI of CFT073 were common to other uropathogenic strains of E. coli, 11 probes were isolated along the length of the PAI and were hybridized to dot blots of genomic DNA isolated from clinical isolates (67 from patients with acute pyelonephritis, 38 from patients with cystitis, 49 from patients with catheter-associated bacteriuria, and 27 from fecal samples). These sequences were found significantly more often in strains associated with the clinical syndromes of acute pyelonephritis (79%) and cystitis (82%) than in those associated with catheter-associated bacteriuria (58%) and in fecal strains (22%) (P < 0.001). From these regions, we have identified a putative iron transport system and genes other than hly and pap that may contribute to the virulent phenotype of uropathogenic E. coli strains.     

Actions of some anions on electrical properties and mechanical threshold of frog twitch muscle

1. Using two micro-electrodes in a point-voltage clamp technique, the effects of the lyotropic anions, NO(3) (-), Br(-), I(-), SCN(-), and CH(3)SO(4) (-), and of SO(4) (2-) on the mechanical threshold and electrical properties of frog sartorius muscle were studied.2. In chloride Ringer solution the spike threshold was -59 mV, mechanical threshold -48 mV, and the threshold for delayed rectification of the total current at about 100 msec -52 mV.3. When Cl(-) was replaced by one of the lyotropic anions, the effective resistance determined at -100 mV tended to increase. But, because of the variability of the effective resistance in individual fibres, most lyotropic anions did not cause a statistically significant increase in the effective resistance. Only I(-) and SO(4) (2-) significantly increased the effective resistance.4. Most lyotropic anions had no significant effect on the spike threshold; I(-), at 58 mM, lowered it slightly. Sulphate raised the threshold.5. Tetrodotoxin (0.1 mug/ml.) abolished the spikes, but did not affect the mechanical and delayed rectification thresholds. It was, therefore, used to pre-treat all preparations for determining these thresholds.6. All lyotropic anions lowered the mechanical and the delayed rectification thresholds, the order of effectiveness being approximately SCN(-) > I(-) > NO(3) (-) > CH(3)SO(4) (-) > Br(-). As in Cl(-) Ringer, the two thresholds lay very close together in every case. Sulphate raised slightly both the mechanical and delayed rectification thresholds, again in close parallel.7. This close agreement of the mechanical and delayed rectification thresholds is not caused by movement artifact, because in fibres in which visible contractions were eliminated with hypertonic solutions the delayed rectification thresholds were the same as those in contracting fibres.8. In spite of the close agreement, reasons are given to doubt a direct causal relationship between the mechanical and delayed rectification thresholds.9. Nitrate apparently had little effect on the rate of inactivation of the outward current, or on the relation between steady-state inactivation and membrane potential.     

Enterotoxicity and cytotoxicity of Vibrio parahaemolyticus thermostable direct hemolysin in in vitro systems

Vibrio parahaemolyticus is a marine bacterium known to be a common cause of seafood gastroenteritis worldwide. The thermostable direct hemolysin (TDH) has been proposed to be a major virulence factor of V. parahaemolyticus. TDH causes intestinal fluid secretion as well as cytotoxicity in a variety of cell types. In this study, we investigated the interplay between the hemolysin's enterotoxic and cytotoxic effects by using both human and rat cell monolayers. As revealed by microspectrofluorimetry, the toxin causes a dose-dependent increase in intracellular free calcium in both Caco-2 and IEC-6 cells. This effect was reversible only when low toxin concentrations were tested. The TDH-activated ion influx pathway is not selective for calcium but admits ions such sodium and manganese as well. Furthermore, in the same range of concentration, the hemolysin triggers a calcium-dependent chloride secretion. At high concentrations, TDH induces a dose-dependent but calcium-independent cell death as assessed by functional, biochemical, and morphological assays.     

Current trends and new approaches in the management of diabetes mellitus

Current trends in the management of type 2 diabetes mellitus, based on the 20-year United Kingdom Prospective Diabetic Study, include intensive treatment to control the blood glucose level and blood pressure in order to prevent or delay microvascular and cardiovascular complications. In the new millennium, type 2 diabetes will become epidemic in developing countries. If diabetes were to develop in 10% of the 1.2 billion population of China, the expense of intensive treatment would be immense. Laboratory tests are useful for detecting risk factors before the onset of the disease and convincing the general public to take preventive measures. Glucose tolerance testing is one of these tests. When glucose tolerance is impaired, 25% of beta-cell function is lost. Determining the plasma proinsulin level is another useful evaluation; impaired glucose tolerance accompanied by increased plasma proinsulin level is indicative of an enhanced risk that type 2 diabetes will develop within 5 years. Educating the public about eating a healthy diet and exercising may prevent the development of diabetes and thereby reduce the global prevalence of type 2 diabetes.     

Genetic predetermination of quantitative expression of HLA antigens in platelets and mononuclear leukocytes

In view of the potential functional importance of quantitative expression of HLA antigens, a series of studies were conducted to determine the relative quantities of specific HLA-A and -B antigens expressed in MNLs and platelets of HLA-phenotyped family members and unrelated individuals. An mAb that reacts with a well-defined monomorphic epitope in the α₃ domain of the heavy chains of HLA molecules was developed and used to quantify each HLA-A or -B antigen on western blots of IEF gels. The results of these studies demonstrated that the relative quantities of HLA-A and -B antigens in platelets and MNLs of an individual did not change over time. Further studies showed that the relative quantities of HLA-A and -B antigens for haplotypes shared among the first-degree relatives were always the same and followed Mendelian inheritance. In contrast, the relative quantities of HLA-A and -B antigens for a haplotype shared by unrelated individuals varied significantly. All these findings support the hypothesis that the quantitative expression of HLA antigens is genetically predetermined and may play important roles in determining disease susceptibility and severity. Human Immunology 38, 243–250 (1993)     

Influence of the Length of the Lipooligosaccharide α Chain on Its Sialylation in Neisseria meningitidis

The sialylation of lipooligosaccharide (LOS) in Neisseria meningitidis plays a role in the resistance of the organism to killing by normal human serum. The length of the alpha chain extending out from the heptose I [Hep (I)] moiety of LOS influenced sialylation of N. meningitidis LOS in vitro and in vivo. The alpha chain required a terminal Gal and a trisaccharide or longer oligosaccharide to serve as an acceptor for sialylation. The disaccharide lactose (Galbeta1-4Glc) in the alpha chain of immunotype L8 LOS could not function as an acceptor for the sialyltransferase, probably due to steric hindrance imposed by the neighboring Hep (II) with phosphorylethanolamine and another group attached.     

Genome-wide profiling of oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is a common malignancy, the incidence of which is particularly high in some Asian countries due to the geographically linked areca quid (AQ) chewing habit. In this study, array-based comparative genomic hybridization was used to screen microdissected OSCCs for genome-wide alterations. The highest frequencies of gene gain were detected for TP63, Serpine1, FGF4/FGF3, c-Myc and DMD. The highest frequencies of deletion were detected for Caspase8 and MTAP. Gained genes, classified by hierarchical clustering, were mainly on 17q21-tel; 20q; 11q13; 3q27-29 and the X chromosome. Among these, gains of EGFR at 7p, FGF4/FGF3, CCND1 and EMS1 at 11q13, and AIB1 at 20q were significantly associated with lymph node metastasis. The genomic profiles of FHIT and EXT1 in AQ-associated and non-AQ-associated OSCCs exhibited the most prominent differences. RT-PCR confirmed the significant increase of TP63 and Serpine1 mRNA expression in OSCC relative to non-malignant matched tissue. A significant increase in Serpine1 immunoreactivity was observed from non-malignant matched tissue to OSCC. However, there was no correlation between the frequent genomic loss of Caspase 8 and a significant decrease in Caspase8 expression. These data demonstrate that genomic profiling can be useful in analysing pathogenetic events involved in the genesis or progression of OSCC.     

Neonatal sepsis in the neonatal intensive care unit: characteristics of early versus late onset.

Neonatal sepsis is a major cause of death in newborns despite sophisticated neonatal intensive care. This retrospective study reviewed the clinical characteristics of cases of culture-proven sepsis in a neonatal intensive care unit from January 1992 to December 2001. Patients were divided into those with onset of sepsis in the first 7 days of life (early-onset group) and those with onset after the seventh day of life (late-onset group). A total of 270 cases with 325 episodes of sepsis and 353 isolated pathogens were identified and included in the study. The male-to-female ratio was 1.4. The majority of cases of sepsis occurred in low birth weight (75.9%) and premature babies (76.7%). Late onset occurred in 71.9% of cases. Patients with late onset had a lower mortality rate than those with early onset (11.3% vs 28.9%). Coagulase-negative staphylococci (20.1%) was the most common organism isolated, but infection with Pseudomonas aeruginosa was associated with the highest morality rate (55.0%). Late-onset sepsis was significantly more common in very low birth weight and premature infants. The most frequently encountered pathogens in the early-onset group were group B streptococci (GBS) and Escherichia coli, while in the late-onset group, the organisms were coagulase-negative staphylococci and Enterobacteriaceae, including E. coli, Klebsiella pneumoniae, and Acinetobacter baumannii. GBS infection resulted in the highest mortality when the onset of sepsis was within the first 24 hours of life.