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1.  Evaluating two methods for fingerprinting genomes of Actinobacillus actinomycetemcomitans 
Oral microbiology and immunology  1993;8(6):337-343.
The arbitrary primer polymerase chain reaction (AP-PCR) and Southern blot restriction fragment length polymorphism (RFLP) were used to genotype the periodontal pathogen A. actinomycetemcomitans. Total genomic DNA from 73 strains was extracted by conventional methods. Three random-sequence 10-base oligonucleotide primers were chosen for AP-PCR. The amplified DNA products were separated electrophoretically in a 1% agarose gel containing ethidium bromide and the banding patterns were compared among different strains. For RFLP analysis, DNA was digested with EcoRI, separated on a 0.8% agarose gel and transferred to a nylon membrane. The membrane was probed with a previously characterized 5.2 kilobases (kb) DNA fragment cloned from A. actinomycetemcomitans strain Y4. The probe was labeled with digoxigenin, and hybridized fragments were detected with anti-digoxigenin antibody. AP-PCR produced 4–10 DNA bands in the 0.5–5 kb regions and distinguished 9, 13 or 17 genotypes, depending on the specific primer used. Southern blot RFLP analysis revealed 12 hybridization patterns consisting of 1 or 2 DNA fragments (2–23 kb). The addition of the Southern blot analysis to the AP-PCR analysis gave rise to a total of 30 DNA profiles among the 73 A. actinomycetemcomitans study strains. The results indicate that both AP-PCR and Southern blot analysis are useful in clonal analysis of A. actinomycetemcomitans.
PMCID: PMC3534794  PMID: 7908736
Actinobacillus actinomycetemcomitans; DNA fingerprinting; polymerase chain reaction; restriction fragment length polymorphism; DNA probe; Southern blot
2.  The ionizing radiation-induced replication protein A phosphorylation response differs between ataxia telangiectasia and normal human cells. 
Molecular and Cellular Biology  1993;13(12):7222-7231.
Replication protein A (RPA), the trimeric single-stranded DNA-binding protein complex of eukaryotic cells, is important to DNA replication and repair. Phosphorylation of the p34 subunit of RPA is modulated by the cell cycle, occurring during S and G2 but not during G1. The function of phosphorylated p34 remains unknown. We show that RPA p34 phosphorylation is significantly induced by ionizing radiation. The phosphorylated form, p36, is similar if not identical to the phosphorylated S/G2 form. gamma-Irradiation-induced phosphorylation occurs without new protein synthesis and in cells in G1. Mutation of cdc2-type protein kinase phosphorylation sites in p34 eliminates the ionizing radiation response. The gamma-irradiation-induced phosphorylation of RPA p34 is delayed in cells from ataxia telangiectasia, a human inherited disease conferring DNA repair defects and early-onset tumorigenesis. UV-induced phosphorylation of RPA p34 occurs less rapidly than gamma-irradiation-induced phosphorylation but is kinetically similar between ataxia telangiectasia and normal cells. This is the first time that modification of a repair protein, RPA, has been linked with a DNA damage response and suggests that phosphorylation may play a role in regulating DNA repair pathways.
PMCID: PMC364792  PMID: 8246944
3.  Rate of change of left ventricular ejection fraction during exercise is superior to the peak ejection fraction for predicting functionally significant coronary artery disease. 
British Heart Journal  1993;70(6):507-512.
OBJECTIVE--To detect and characterise rapid temporal changes in the left ventricular response to exercise in patients with ischaemic heart disease and to relate these changes to the functional severity of coronary artery disease. BACKGROUND--The gamma camera does not allow the detection of rapid changes in cardiac function during exercise radionuclide ventriculography, the monitoring of which may improve the assessment of patients with ischaemic heart disease. METHODS--A miniature nuclear probe (Cardioscint) was used to monitor continuously left ventricular function during exercise in 31 patients who had coronary angiography for suspected coronary artery disease. A coronary angiographic jeopardy score was calculated for each patient. RESULTS--The coronary jeopardy score ranged from 0 to 12 (median 4). Ejection fraction fell significantly during exercise from 46% to 34%. Patients were divided into two groups based on the response of their ejection fraction to exercise. In 14 patients (group I), the peak change in ejection fraction coincided with the end of exercise, whereas in the other 17 patients (group II) the peak change in ejection fraction occurred before the end of exercise, resulting in a brief plateau. The peak change in ejection fraction and the time to its occurrence were independent predictors of coronary jeopardy (r = -0.59, p < 0.001 for peak change and r = -0.69, p < 0.001 for time to that change). The rate of change in ejection fraction was the strongest predictor of coronary jeopardy (r = -0.81, p < 0.001). In group I the peak change in ejection fraction was a poor predictor severity of coronary disease (r = -0.28, NS), whereas the time to peak and the rate of change in ejection fraction were good predictors (r = -0.65 and r = -0.73, p < 0.01). In group II the peak, the time to the peak, and the rate of change in ejection fraction were good predictors of coronary jeopardy (r = -0.75, r = -0.61, and r = -0.83, p < 0.01). CONCLUSION--The rate of change of ejection fraction during exercise can be assessed by continuous monitoring of left ventricular function with the nuclear probe, and is the best predictor of functionally significant coronary artery disease.
PMCID: PMC1025380  PMID: 8280514
4.  Mosquitocidal toxins of bacilli and their genetic manipulation for effective biological control of mosquitoes. 
Microbiological Reviews  1993;57(4):838-861.
The identification, cloning, and characterization of protein toxins from various species of bacilli have demonstrated the existence of mosquitocidal toxins with different structures, mechanisms of action, and host ranges. A start has been made in understanding the polypeptide determinants of toxicity and insecticidal activity, and the purification of toxins from recombinant organisms may lead to the elucidation of their X-ray crystal structures and the cloning of brush border membrane receptors. The results of cloning mosquitocidal toxins in heterologous microorganisms show the potential of expanding the range of susceptible mosquito species by combining several toxins of different host specificity in one cell. Toxins have been expressed in new microorganisms with the potential for increasing potency by persisting at the larval feeding zone. The powerful tools of bacterial genetics are being applied to engineer genetically stable, persistent toxin expression and expand the insecticidal host ranges of Bacillus sphaericus and Bacillus thuringiensis strains. These techniques, together with modern formulation technology, should eventually lead to the construction of mosquitocidal microorganisms which are effective enough to have a real impact on mosquito-borne diseases.
PMCID: PMC372941  PMID: 7905597
5.  Choice in local anaesthesia. 
PMCID: PMC504649  PMID: 8110668
6.  Location of IS200 on the genomic cleavage map of Salmonella typhimurium LT2. 
Journal of Bacteriology  1993;175(23):7624-7628.
Locations of six Tn10s, closely linked to each of the six IS200s on the genomic cleavage map of Salmonella typhimurium LT2, were determined by digestion with XbaI and BlnI and separation of the fragments by pulsed-field gel electrophoresis; the locations were then further defined by P22-mediated joint transduction. The orientation of each IS200 with respect to its linked Tn10 was determined by Southern blotting. The locations of IS200-I, IS200-III, and IS200-V were confirmed to be close to sufD, melB, and purC, as previously indicated. IS200-II is jointly transduced with cysG. IS200-IV is near fliA; the linked Tn10 is inserted in fli, making the strain nonmotile. IS200-VI is jointly transduced with aspC but not with aroA. IS200 is transposed to a seventh site in some strains, while remaining in the other six locations described above. These data indicate that genome analysis by pulsed-field gel electrophoresis can locate the positions of Tn10s with accuracy sufficient to predict P22-mediated joint transduction.
PMCID: PMC206918  PMID: 8244931
7.  Signals in chicken beta-globin DNA influence chromatin assembly in vitro. 
Molecular and Cellular Biology  1993;13(12):7596-7603.
We have confirmed the result that chicken beta-globin gene chromatin, which possesses the characteristics of active chromatin in erythroid cells, has shortened internucleosome spacings compared with bulk chromatin or that of the ovalbumin gene, which is inactive. To understand how the short (approximately 180-bp) nucleosome repeat arises specifically on beta-globin DNA, we have studied chromatin assembly of cloned chicken beta-globin DNA in a defined in vitro system. With chicken erythrocyte core histones and linker histone H5 as the only cellular components, a cloned 6.2-kb chicken beta-globin DNA fragment assembled into chromatin possessing a regular 180 +/- 5-bp repeat, very similar to what is observed in erythroid cells. A 2-kb DNA subfragment containing the beta A gene and promoter region, but lacking the downstream intergenic region between the beta A and epsilon genes, failed to generate a regular nucleosome array in vitro, suggesting that the intergenic region facilitates linker histone-induced nucleosome alignment. When the beta A gene was placed on a plasmid that contained a known chromatin-organizing signal, nucleosome alignment with a 180-bp periodicity was restored, whereas nucleosomes on flanking plasmid sequences possessed a 210-bp spacing periodicity. Our results suggest that the shortened 180-bp nucleosome spacing periodicity observed in erythroid cells is encoded in the beta-globin DNA sequence and that nucleosome alignment by linker histones is facilitated by sequences in the beta A-epsilon intergenic region.
PMCID: PMC364831  PMID: 8246976
8.  The glnB region of the Escherichia coli chromosome. 
Journal of Bacteriology  1993;175(22):7441-7449.
We present sequences of the glnB gene of Escherichia coli and of two open reading frames (ORFs) located directly upstream of glnB and transcribed in the same direction. The major transcriptional start sites for glnB are located between ORF-2 and glnB, but some transcription of glnB is initiated at the promoter for ORF-1. The putative amino acid sequence of the ORF-2 product has high homology to that of response regulators which by phosphorylation acquire the ability to activate transcription of sigma 54-dependent promoters. The product of ORF-1 showed no similarity to other known proteins. The product of neither ORF-1 nor ORF-2 is necessary for the ability of PII, the product of glnB, to bring about the repression of glutamine synthetase in response to nitrogen excess. On the other hand, the product of hmpA, a gene located on the other side of glnB and transcribed in the opposite direction, appears to play an auxiliary role in this process.
PMCID: PMC206889  PMID: 8226691
9.  A soluble Bacillus cereus cytochrome P-450cin system catalyzes 1,4-cineole hydroxylations. 
Applied and Environmental Microbiology  1993;59(11):3889-3893.
A cytochrome P-450-dependent monooxygenase system that catalyzes the stereospecific hydroxylation of the monoterpene substrate 1,4-cineole was demonstrated in cell-free preparations of Bacillus cereus UI-1477. 1,4-Cineole hydroxylations were catalyzed by a 100,000 x g (1-h)-centrifuging soluble, hexane-inducible enzyme that activated and incorporated molecular oxygen into hydroxylated products; required NADH; was inhibited by SKF-525A, imidazole, metyrapone, and octylamine; and displayed a 452-nm peak in the carbon monoxide difference absorption spectrum. The constant 7:1 ratio of endo/exo alcohol products formed when 1,4-cineole was hydroxylated by normal cells, hexane-induced cells, and cell extracts suggested that a single enzyme designated cytochrome P-450cin was responsible for both reactions.
PMCID: PMC182545  PMID: 8285692
10.  Identification of a killer cell-specific regulatory element of the mouse perforin gene: an Ets-binding site-homologous motif that interacts with Ets-related proteins. 
Molecular and Cellular Biology  1993;13(11):6690-6701.
The gene encoding the cytolytic protein perforin is selectively expressed by activated killer lymphocytes. To understand the mechanisms underlying the cell-type-specific expression of this gene, we have characterized the regulatory functions and the DNA-protein interactions of the 5'-flanking region of the mouse perforin gene (Pfp). A region extending from residues +62 through -141, which possesses the essential promoter activity, and regions further upstream, which are able to either enhance or suppress gene expression, were identified. The region between residues -411 and -566 was chosen for further characterization, since it contains an enhancer-like activity. We have identified a 32-mer sequence (residues -491 to -522) which appeared to be capable of enhancing gene expression in a killer cell-specific manner. Within this segment, a 9-mer motif (5'-ACAGGAAGT-3', residues -505 to -497; designated NF-P motif), which is highly homologous to the Ets proto-oncoprotein-binding site, was found to interact with two proteins, NF-P1 and NF-P2. NF-P2 appears to be induced by reagents known to up-regulate the perforin message level and is present exclusively in killer cells. Electrophoretic mobility shift assay and UV cross-linking experiments revealed that NF-P1 and NF-P2 may possess common DNA-binding subunits. However, the larger native molecular mass of NF-P1 suggests that NF-P1 contains an additional non-DNA-binding subunit(s). In view of the homology between the NF-P motif and other Ets proto-oncoprotein-binding sites, it is postulated that NF-P1 and NF-P2 belong to the Ets protein family. Results obtained from the binding competition assay, nevertheless, suggest that NF-P1 and NF-P2 are related to but distinct from Ets proteins, e.g., Ets-1, Ets-2, and NF-AT/Elf-1, known to be expressed in T cells.
PMCID: PMC364732  PMID: 8413265
11.  Distinctive effects of the carboxyl-terminal sequence of the insulin-like growth factor I receptor on its signaling functions. 
Journal of Virology  1993;67(11):6835-6840.
We have shown previously that the extracellular sequences of the human insulin receptor (IR) and the insulin-like growth factor I receptor (IGFR) have an inhibitory effect on protein tyrosine kinase (PTK) activity and on the biological functions of their respective Gag-receptor fusion proteins. To study the role of IGFR carboxyl sequence in modulation of the Gag-IGFR PTK and biological activities, five mutants, CM1, CM2, CM3, CM4, and CM5, containing carboxyl deletions of 17, 27, 47, 67, and 88 amino acids (aa), respectively, were constructed from the parental virus UIGFR encoding the Gag-IGFR. Deletion of up to 27 aa had little effect on the cell-transforming and PTK activities of UIGFR. Deletions of 47 aa in CM3 abolished PTK and transforming activities. Surprisingly, a further deletion of 20 aa in CM4 beyond that in CM3 reactivated the kinase and transforming activities. CM5, containing a deletion of 20 aa beyond that in CM4, had only marginal transforming and PTK activities. We conclude that deletion of the carboxyl region of the Gag-IGFR inactivates, instead of activating as in the case with Gag-IR, its transforming activity and the amino acid sequence 1250 to 1310 is essential for PTK and transforming activities. Analysis of the ability of the full-length IGFR and its mutant receptors described above to associate with phosphatidylinositol 3 kinase indicated that the association required PTK activity and tyrosine phosphorylation of the receptors and correlated well with their transforming activities. The carboxyl 88 aa are not essential for the association.
PMCID: PMC238128  PMID: 7692086
12.  A passive transfer model of the organ-specific autoimmune disease, bullous pemphigoid, using antibodies generated against the hemidesmosomal antigen, BP180. 
Journal of Clinical Investigation  1993;92(5):2480-2488.
Subepidermal blistering associated with the human skin diseases bullous pemphigoid and herpes gestationis has been thought to be an IgG autoantibody-mediated process; however, previous attempts to demonstrate the pathogenicity of patient autoantibodies have been unsuccessful. An immunodominant and potentially pathogenic epitope associated with these blistering diseases has recently been mapped to the extracellular domain of a human epidermal antigen, BP180. Patient autoantibodies that react with this well-defined antigenic site failed to crossreact with the murine form of this autoantigen and thus could not be assayed for pathogenicity in a conventional passive transfer mouse model. As an alternative, rabbit polyclonal antibodies were generated against a segment of the murine BP180 protein homologous with the human BP180 autoantibody-reactive site and were passively transferred into neonatal BALB/c mice. The injected animals developed a subepidermal blistering disease that closely mimicked bullous pemphigoid and herpes gestationis at the clinical, histological, and immunological levels. Autoantibodies that recognize the human BP180 ectodomain are therefore likely to play an initiatory role in the pathogenesis of bullous pemphigoid and herpes gestationis.
PMCID: PMC288433  PMID: 7693763
13.  New high-toxicity mosquitocidal strains of Bacillus sphaericus lacking a 100-kilodalton-toxin gene. 
Applied and Environmental Microbiology  1993;59(10):3470-3473.
Five new high-toxicity mosquitocidal strains of Bacillus sphaericus were isolated in Singapore. They all belong to phage group 8 and have binary toxin (51.4- plus 41.9-kDa) genes located on the chromosome but lack a 100-kDa-toxin gene. These strains of B. sphaericus constitute a new subgroup, as only two weakly toxic strains in phage group 8 have previously been described and all the known high-toxicity strains have both binary toxin and 100-kDa-toxin genes.
PMCID: PMC182475  PMID: 7902695
14.  Quantitation of enteroviral RNA by competitive polymerase chain reaction. 
Journal of Clinical Microbiology  1993;31(10):2634-2640.
The polymerase chain reaction (PCR) is a new diagnostic technique for the detection of enteroviral infection; however, it currently provides only qualitative results. The aim of this study was to adapt PCR for the accurate quantitation of enteroviral RNA in clinical specimens. For this purpose, we designed a standard RNA which was homologous to sequences at the 5' end of the coxsackie B3 enterovirus genome but contained a single-base-pair mutation which created a novel internal restriction site. Serial dilutions of this standard template RNA were mixed with a fixed concentration of coxsackie B3 enterovirus RNA. The viral and standard templates were reversed transcribed to cDNA and coamplified by PCR, and a comparison of the radioactive PCR products was made. Since the templates were both present in a single reaction tube and competed for the same primers, the ratio of products remained proportional throughout the amplification process. By this approach, a fourfold-difference in viral titer was clearly distinguishable. Moreover, we were able to accurately quantitate as few as 15 50% tissue culture infectious doses, which reflects common clinical viral titers. This study lays the foundation for quantitation of enteroviral RNA in clinical specimens and establishes a technique that can readily be applied to the diagnosis of enteroviral infection.
PMCID: PMC265955  PMID: 8253959
15.  Pharmacokinetics of [18F]fleroxacin in healthy human subjects studied by using positron emission tomography. 
Antimicrobial Agents and Chemotherapy  1993;37(10):2144-2152.
Positron emission tomography (PET) with [18F]fleroxacin was used to study the pharmacokinetics of fleroxacin, a new broad-spectrum fluoroquinolone, in 12 healthy volunteers (9 men and 3 women). The subjects were infused with a standard therapeutic dose of fleroxacin (400 mg) supplemented with approximately 20 mCi of [18F]fleroxacin. Serial PET images were made and blood samples were collected for 8 h, starting at the initiation of the infusion. The subjects were then treated with unlabeled drug for 3 days (400 mg/day). On the fifth day, infusion of radiolabeled drug, PET imaging, and blood collection were repeated. In most organs, there was rapid accumulation of radiolabeled drug, with stable levels achieved within 1 h after completion of the infusion. Especially high peak concentrations (in micrograms per gram) were achieved in the kidney (> 34), liver (> 25), lung (> 20), myocardium (> 19), and spleen (> 18). Peak concentrations of drug more than two times the MIC for 90% of Enterobacteriaceae strains tested (> 10-fold for most organisms) were achieved in all tissues except the brain and remained above this level for more than 6 to 8 h. The plateau concentrations in tissues (2 to 8 h, in micrograms per gram +/- standard error of the mean) of drug were as follows: brain, 0.83 +/- 0.032; myocardium, 4.53 +/- 0.24; lung, 5.80 +/- 0.48; liver, 7.31 +/- 0.33; spleen, 6.00 +/- 0.47; bowel, 3.53 +/- 0.74; kidney, 8.85 +/- 0.64; bone, 2.87 +/- 0.29; muscle, 4.60 +/- 0.33; prostate, 4.65 +/- 0.48; uterus, 3.87 +/- 0.39; breast, 2.68 +/- 0.11; and blood, 2.35 +/- 0.09. Concentrations of fleroxacin in tissue were similar in males and females, before and after pretreatment with unlabeled drug.
PMCID: PMC192242  PMID: 8257137
16.  recA mutations that reduce the constitutive coprotease activity of the RecA1202(Prtc) protein: possible involvement of interfilament association in proteolytic and recombination activities. 
Journal of Bacteriology  1993;175(20):6518-6529.
Twenty-eight recA mutants, isolated after spontaneous mutagenesis generated by the combined action of RecA1202(Prtc) and UmuDC proteins, were characterized and sequenced. The mutations are intragenic suppressors of the recA1202 allele and were detected by the reduced coprotease activity of the gene product. Twenty distinct mutation sites were found, among which two mutations, recA1620 (V-275-->D) and recA1631 (I-284-->N), were mapped in the C-terminal portion of the interfilament contact region (IFCR) in the RecA crystal. An interaction of this region with the part of the IFCR in which the recA1202 mutation (Q-184-->K) is mapped could occur only intermolecularly. Thus, altered IFCR and the likely resulting change in interfilament association appear to be important aspects of the formation of a constitutively active RecA coprotease. This observation is consistent with the filament-bundle theory (R. M. Story, I. T. Weber, and T. A. Steitz, Nature (London) 335:318-325, 1992). Furthermore, we found that among the 20 suppressor mutations, 3 missense mutations that lead to recombination-defective (Rec-) phenotypes also mapped in the IFCR, suggesting that the IFCR, with its putative function in interfilament association, is required for the recombinase activity of RecA. We propose that RecA-DNA complexes may form bundles analogous to the RecA bundles (lacking DNA) described by Story et al. and that these RecA-DNA bundles play a role in homologous recombination.
PMCID: PMC206762  PMID: 8407828
17.  Integration of simian virus 40 into cellular DNA occurs at or near topoisomerase II cleavage hot spots induced by VM-26 (teniposide). 
Molecular and Cellular Biology  1993;13(10):6190-6200.
Inhibition of DNA topoisomerase II in simian virus 40 (SV40)-infected BSC-1 cells with a topoisomerase II poison, VM-26 (teniposide), resulted in rapid conversion of a population of the SV40 DNA into a high-molecular-weight form. Characterization of this high-molecular-weight form of SV40 DNA suggests that it is linear, double stranded, and a recombinant with SV40 DNA sequences covalently joined to cellular DNA. The majority of the integrants contain fewer than two tandem copies of SV40 DNA. Neither DNA-damaging agents, such as mitomycin and UV, nor the topoisomerase I inhibitor camptothecin induced detectable integration in this system. In addition, the recombination junctions within the SV40 portion of the integrants correlate with VM-26-induced, topoisomerase II cleavage hot spots on SV40 DNA. These results suggest a direct and specific role for topoisomerase II and possibly the enzyme-inhibitor-DNA ternary cleavable complex in integration. The propensity of poisoned topoisomerase II to induce viral integration also suggests a role for topoisomerase II in a pathway of chromosomal DNA rearrangements.
PMCID: PMC364678  PMID: 8413219
18.  Neither CD14 nor serum is absolutely necessary for activation of mononuclear phagocytes by bacterial lipopolysaccharide. 
Infection and Immunity  1993;61(10):4452-4461.
The stimulation of mononuclear phagocytes by lipopolysaccharide (LPS) is facilitated by the binding of complexes of LPS and LPS-binding protein to CD14. Although it is clear that CD14 is involved in LPS-induced signaling, other investigators have hypothesized the existence of additional signaling pathways in macrophages. We sought to determine whether CD14-independent pathways of monocyte activation might exist. Washed human mononuclear cells responded with reduced sensitivity to LPS in the absence of serum. Anti-CD14 monoclonal antibody (MAb) inhibited the response to LPS in serum-free conditions, but this was easily reversed at higher concentrations of LPS. We established a human monocytic cell line, designated SFM (derived from THP-1), in serum-free medium to examine LPS responses under defined conditions. Differentiation of SFM cells with 1,25-dihydroxycholecalciferol promoted the expression of abundant cell surface CD14. Differentiated SFM cells responded to LPS despite the complete absence of serum proteins for > 20 generations of growth. LPS stimulation of differentiated SFM cells was inhibited by anti-CD14 MAbs only when serum was present. In contrast to anti-CD14 MAb, the LPS antagonists lipid IVa and Rhodobacter sphaeroides lipid A inhibited monocyte activation under serum-free conditions, implying that these compounds compete with LPS at a site distinct from CD14. Undifferentiated SFM cells (expressing minimal CD14) still responded to LPS in serum-free conditions, and anti-CD14 MAb had little inhibitory effect. The addition of purified LPS-binding protein or human serum promoted a CD14-dependent pathway of monocyte activation by LPS in these cells. We conclude that monocytes do not absolutely require serum proteins to be stimulated by LPS and that CD14-independent LPS signaling pathways exist which are inhibitable by lipid IVa and R. sphaeroides lipid A.
PMCID: PMC281179  PMID: 7691750
19.  Intravenous glucose suppresses glucose production but not proteolysis in extremely premature newborns. 
Journal of Clinical Investigation  1993;92(4):1752-1758.
To ascertain whether the inability to suppress glucose production and increase glucose utilization in response to glucose infusion is an inherent characteristic of immature individuals, we determined glucose rate of appearance (R(a)) in minimally stressed, clinically stable, extremely premature infants (approximately 26-wk gestation) at two glucose infusion rates (6.2 +/- 0.4 and 9.5 +/- 0.5 mg/kg per min). We also assessed whether an increase in glucose delivery suppresses proteolysis by measuring the R(a) of phenylalanine and leucine. Glucose R(a) (and utilization) increased significantly at the higher glucose infusion rate (7.9 +/- 0.5 vs. 9.8 +/- 0.6 mg/kg per min). Glucose production persisted at the lower glucose infusion rate but was suppressed to nearly zero at the higher rate (1.7 +/- 0.5 vs. 0.3 +/- 0.1 mg/kg per min). Proteolysis was unaffected by the higher glucose infusion rate as reflected by no change in the rates of appearance of either phenylalanine (96 +/- 5 vs. 95 +/- 3 mumol/kg per h) or leucine (285 +/- 20 vs. 283 +/- 14 mumol/kg per h). Thus, clinically stable, extremely premature infants suppress glucose production and increase glucose utilization in response to increased glucose infusion, demonstrating no inherent immaturity of these processes. In contrast, increasing the rate of glucose delivery results in no change in whole body proteolysis in these infants. The regulation of proteolysis in this population remains to be defined.
PMCID: PMC288336  PMID: 8408627
20.  Defective splicing of mRNA from one COL1A1 allele of type I collagen in nondeforming (type I) osteogenesis imperfecta. 
Journal of Clinical Investigation  1993;92(4):1994-2002.
Osteogenesis imperfecta (OI) type I is the mildest form of heritable bone fragility resulting from mutations within the COL1A1 gene. We studied fibroblasts established from a child with OI type I and demonstrated underproduction of alpha 1 (I) collagen chains and alpha 1 (I) mRNA. Indirect RNase protection suggested two species of alpha 1 (I) mRNA, one of which was not collinear with fully spliced alpha 1 (I) mRNA. The noncollinear population was confined to the nuclear compartment of the cell, and contained the entire sequence of intron 26 and a G-->A transition in the first position of the intron donor site. The G-->A transition was also identified in the genomic DNA. The retained intron contained an in-frame stop codon and introduced an out-of-frame insertion within the collagen mRNA producing stop codons downstream of the insertion. These changes probably account for the failure of the mutant RNA to appear in the cytoplasm. Unlike other splice site mutations within collagen mRNA that resulted in exon skipping and a truncated but inframe RNA transcript, this mutation did not result in production of a defective collagen pro alpha 1 (I) chain. Instead, the mild nature of the disease in this case reflects failure to process the defective mRNA and thus the absence of a protein product from the mutant allele.
PMCID: PMC288367  PMID: 8408653
21.  Loss of cell-cell and cell-matrix adhesion molecules in colorectal cancer. 
British Journal of Cancer  1993;68(3):507-514.
Adhesion molecules are thought to play a vital role in the induction and maintenance of tissue differentiation and their loss or down-regulation has been implicated in the neoplastic process. Recent studies have shown that the morphoregulatory activities are a consequence of interactive processes between several cell adhesion molecules rather than the function of a single molecule. Therefore, we have investigated a panel of adhesion molecules including members of the integrin, cadherin and immunoglobin superfamily in colorectal cancer. Twenty-eight consecutive colorectal adenocarcinomas were stained using an avidin-biotin indirect immunoperoxidase technique. Our results showed a consistent loss of the alpha 2 and beta 1 integrin subunits (21/28 = 75% and 22/28 = 78.6% respectively) and a decrease in expression of E-cadherin in 5/5 poorly differentiated adenocarcinomas. Carcinoembryonic antigen expression was preserved but with basolateral accentuation seen in tumours. There was no statistical correlation with Dukes' stage. These results provide further evidence that in colorectal cancer there is a widespread deregulated expression of cell-cell and cell-matrix adhesion molecules. Changes in the expression and function of adhesion molecules which regulate growth and differentiation may play a role in the behaviour of colorectal cancer.
PMCID: PMC1968382  PMID: 8353041
22.  Use of personal measurements for ozone exposure assessment: a pilot study. 
Environmental Health Perspectives  1993;101(4):318-324.
During summer 1991, we collected indoor, outdoor, and personal ozone concentration data as well as time-activity data in State College, Pennsylvania. These concentrations were measured for 23 children and their homes using passive ozone samplers. Outdoor concentrations were also measured at a stationary ambient monitoring site. Results from this pilot study demonstrate that fixed-site ambient measurements may not adequately represent individual exposures. Outdoor ozone concentrations showed substantial spatial variation between rural and residential regions. Ignoring this spatial variation by using fixed-site measurements to estimate personal exposures can result in an error as high as 127%. In addition, evidence from our pilot study indicates that ozone concentrations of a single indoor microenvironment may not represent those of other indoor microenvironments. Personal exposures were significantly correlated with both indoor (r = 0.55) and outdoor (r = 0.41) concentrations measured at home sites. Multiple regression analyses identified indoor ozone concentrations as the most important predictors of personal exposures. However, models based on time-weighted indoor and outdoor concentrations explained only 40% of the variability in personal exposures. When the model included observations for only those participants who spent the majority of their day in or near their homes, an R2 of 0.76 resulted when estimates were regressed on measured personal exposures. It is evident that contributions from diverse indoor and outdoor microenvironments must be considered to estimate personal ozone exposures accurately.
PMCID: PMC1519793  PMID: 8275989
23.  The v-Src SH3 domain binds phosphatidylinositol 3'-kinase. 
Molecular and Cellular Biology  1993;13(9):5225-5232.
Fibroblasts transformed by v-src or by related oncogenes encoding activated tyrosine kinases contain elevated levels of polyphosphoinositides with phosphate at the D-3 position of the inositol ring, as a result of the activation of phosphatidylinositol (PI) 3'-kinase. v-src-transformed cells also contain increased levels of PI 3'-kinase activity immunoprecipitable with anti-phosphotyrosine antibodies; furthermore, PI 3'-kinase can be detected in association with the v-Src tyrosine kinase. To identify regions of v-Src that can interact with PI 3'-kinase, the v-Src SH2 and SH3 domains were expressed in bacteria and incubated with lysates of normal chicken embryo fibroblasts. In vitro, the v-Src SH3 domain, but not the SH2 domain, bound PI 3'-kinase in lysates of uninfected chicken embryo fibroblasts. Substitutions of two highly conserved SH3 residues implicated in ligand binding abolished the ability of the v-Src SH3 domain to associate with PI 3'-kinase. Furthermore, the v-Src SH3 domain bound in vitro to the amino-terminal region of the p85 alpha subunit of PI 3'-kinase. These results suggest that the v-Src SH3 domain may mediate an interaction between the v-Src tyrosine kinase and PI 3'-kinase, by direct binding to p85.
PMCID: PMC360211  PMID: 7689147
24.  The time of administration of 3'-azido-3'-deoxythymidine (AZT) determines its host toxicity with possible relevance to AZT chemotherapy. 
3'-Azido-3'-deoxythymidine (AZT) is the drug most widely used in the treatment of AIDS. Its major drug-related toxicity is bone marrow suppression, which limits the dose of AZT that can be used. It is essential that AZT be phosphorylated for antiviral effect. We have recently demonstrated that thymidine kinase (TK), the initial enzyme in AZT anabolism, follows a circadian pattern in rat bone marrow. The present study was undertaken to determine whether AZT toxicity is related to the time of its administration and whether the variation in toxicity is correlated with the circadian variation in TK activity. Male Sprague-Dawley rats were housed under standardized conditions of light and dark (lights on 0600 to 1800 and lights off 1800 to 0600) for 4 weeks. The animals were randomly divided into seven groups; six groups were administered AZT by intraperitoneal injection at the same dose of 750 mg/kg of body weight at various times (0400, 0800, 1200, 1600, 2000, and 2400), and one group was used as a control. AZT-related toxic effects, including bone marrow toxicity, differed significantly among the treatment groups, depending on the time of AZT administration (by analysis of variance and Cosinor analysis, P < 0.001). The least toxicity was observed in rats receiving AZT at 1600 (10 h after light onset [10 HALO], in late sleep span) and the greatest toxicity was observed in those injected at 0400 (22 HALO, in late activity span). To verify these results, we administered AZT by intraperitoneal injection at an approximately 50% lethal dose (1,500 mg/kg) to two groups of rats, one at 1200 (6 HALO, in the middle of the sleep span) and the other at 2400 (18 HALO, in the middle of the activity span). AZT lethality was significantly higher in rats receiving AZT at 2400 (18 HALO, in the middle of the activity span). Further statistical analysis demonstrated that the variation in AZT toxicity was correlated with the circadian variation in TK activity in bone marrow of the same species (peak activity at 0400 [22 HALO, in late activity span] and trough activity at 1600 [10 HALO, in late sleep span]), suggesting that the circadian variation in TK activity may be the biochemical basis for the observed circadian variation in AZT toxicity. These results may be useful in the design of improved AZT chemotherapeutic regimens.
PMCID: PMC188068  PMID: 8239582
25.  Insertion and deletion mutations in the repA4 region of the IncFII plasmid NR1 cause unstable inheritance. 
Journal of Bacteriology  1993;175(17):5350-5358.
Mutants of IncFII plasmid NR1 that have transposons inserted in the repA4 open reading frame (ORF) are not inherited stably. The repA4 ORF is located immediately downstream from the replication origin (ori). The repA4 coding region contains inverted-repeat sequences that are homologous to the terC inverted repeats located in the replication terminus of the Escherichia coli chromosome. The site of initiation of leading-strand synthesis for replication of NR1 is also located in repA4 near its 3' end. Transposon insertions between ori and the right-hand terC repeat resulted in plasmid instability, whereas transposon insertions farther downstream did not. Derivatives that contained a 35-bp frameshift insertion in the repA4 ORF were all stable, even when the frameshift was located very near the 5' end of the coding region. This finding indicates that repA4 does not specify a protein product that is essential for plasmid stability. Examination of mutants having a nest of deletions with endpoints in or near repA4 indicated that the 3' end of the repA4 coding region and the site of leading-strand initiation could be deleted without appreciable effect on plasmid stability. Deletion of the pemI and pemK genes, located farther downstream from repA4 and reported to affect plasmid stability, also had no detectable effect. In contrast, mutants from which the right-hand terC repeat, or both right- and left-hand repeats, had been deleted were unstable. None of the insertion or deletion mutations in or near repA4 affected plasmid copy number. Alteration of the terC repeats by site-directed mutagenesis had little effect on plasmid stability. Plasmid stability was not affected by a fus mutation known to inactivate the termination function. Therefore, it appears that the overall integrity of the repA4 region is more important for stable maintenance of plasmid NR1 than are any of the individual known features found in this region.
PMCID: PMC206589  PMID: 8396115

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