Reverse genetic analysis of the glutathione metabolic pathway suggests a novel role of PHGPX and URE2 genes in aluminum resistance in Saccharomyces cerevisiae.

We have taken a systematic genetic approach to study the potential role of glutathione metabolism in aluminum (Al) toxicity and resistance, using disruption mutants available in Saccharomyces cerevisiae. Yeast disruption mutants defective in phospholipid hydroperoxide glutathione peroxidases (PHGPX; phgpx1 Delta, phgpx2 Delta, and phgpx3Delta), were tested for their sensitivity to Al. The triple mutant, phgpx1 Delta/2Delta/3Delta, was more sensitive to Al (55% reduction in growth at 300 microM Al) than any single phgpx mutant, indicating that the PHGPX genes may collectively contribute to Al resistance. The hypersensitivity of phgpx3Delta to Al was overcome by complementation with PHGPX3, and all PHGPX genes showed increased expression in response to Al in the wild-type strain (YPH250), with maximum induction of approximately 2.5-fold for PHGPX3. Both phgpx3Delta and phgpx1Delta/2Delta/3Delta mutants were sensitive to oxidative stress (exposure to H(2)O(2) or diamide). Lipid peroxidation was also increased in the phgpx1Delta/2Delta/3Delta mutant compared to the parental strain. Disruption mutants defective in genes for glutathione S-transferases (GSTs) (gtt1Delta and gtt2Delta), glutathione biosynthesis (gsh1Delta and gsh2Delta), glutathione reductase (glr1Delta) and a glutathione transporter (opt1Delta) did not show hypersensitivity to Al relative to the parental strain BY4741. Interestingly, a strain deleted for URE2, a gene which encodes a prion precursor with homology to GSTs, also showed hypersensitivity to Al. The hypersensitivity of the ure2Delta mutant could be overcome by complementation with URE2. Expression of URE2 in the parental strain increased approximately 2-fold in response to exposure to 100 microM Al. Intracellular oxidation levels in the ure2Delta mutant showed a 2-fold (non-stressed) and 3-fold (when exposed-to 2 mM H(2)O(2)) increase compared to BY4741; however, the ure2Delta mutant showed no change in lipid peroxidation compared to the control. The phgpx1Delta/2Delta/3Delta and ure2Delta mutants both showed increased accumulation of Al. These findings suggest the involvement of PHGPX genes and a novel role of URE2 in Al toxicity/resistance in S. cerevisiae.

Physical mapping of barley genes using an ultrasensitive fluorescence in situ hybridization technique.

The primary objective of this study was to elucidate gene organization and to integrate the genetic linkage map for barley (Hordeum vulgare L.) with a physical map using ultrasensitive fluorescence in situ hybridization (FISH) techniques for detecting signals from restriction fragment length polymorphism (RFLP) clones. In the process, a single landmark plasmid, p18S5Shor, was constructed that identified and oriented all seven of the chromosome pairs. Plasmid p18S5Shor was used in all hybridizations. Fourteen cDNA probes selected from the linkage map for barley H. vulgare 'Steptoe' x H. vulgare 'Morex' (Kleinhofs et al. 1993) were mapped using an indirect tyramide signal amplification technique and assigned to a physical location on one or more chromosomes. The haploid barley genome is large and a complete physical map of the genome is not yet available; however, it was possible to integrate the linkage map and the physical locations of these cDNAs. An estimate of the ratio of base pairs to centimorgans was an average of 1.5 Mb/cM in the distal portions of the chromosome arms and 89 Mb/cM near the centromere. Furthermore, while it appears that the current linkage maps are well covered with markers along the length of each arm, the physical map showed that there are large areas of the genome that have yet to be mapped.

Isolated septic arthritis caused by penicillin-resistant Streptococcus pneumoniae.

Streptococcus pneumoniae is a common cause of infection in the pediatric population, as well as an important cause of septic arthritis. The increased prevalence of drug-resistant S pneumoniae in North America has renewed interest in the use of pneumococcal vaccines. We describe the case of a child with isolated acute septic arthritis caused by infection with penicillin-resistant S pneumoniae.

Successful treatment of an infant with Chromobacterium violaceum sepsis.

Chromobacterium violaceum sepsis, a rarely reported phenomenon, has a high mortality rate. We report a unique case of C. violaceum sepsis in an infant. A 4-month-old girl presented to our institution with fever, pustular skin lesions, and distended abdomen, as well as diminished activity and mental status. Radiological investigation revealed brain, lung, and hepatic abscesses. The infant was successfully treated with trimethoprim-sulfamethoxazole and ciprofloxacin.

The relationship between volume status, hydration, and radiographic findings in the diagnosis of community-acquired pneumonia.

BACKGROUND: Many clinicians believe the radiographic expression of community-acquired pneumonia (CAP) is affected by the fluid volume status of the patient. However, there are very few data to support or refute this concept. With this study we began to examine the relationship between admission fluid volume status and the radiographic expression of CAP. METHODS: Using a retrospective chart review, we examined 376 consecutive inpatient encounters with the diagnosis of pneumonia at discharge from a community teaching hospital. Patients were evaluated by age, sex, admission serum sodium, blood urea nitrogen (BUN) level, creatinine, and fluid administered in the first 48 hours of treatment. We classified these patients as either showing radiographic progression (P) or no radiographic progression (NP) by comparison of admission and follow-up radiographs. RESULTS: A total of 125 patient encounters satisfied inclusion criteria for the study. Using the Student t test we noted a statistically significant difference between the P and NP groups for BUN level (P=.02), volume of fluid administered during the first 48 hours (P=.04), and marginally for age (P=.05). The P group had higher BUN levels (mean=34 vs 24), more 48-hour fluid intake (mean=5824 mL vs 4764 mL), and younger age (mean=59 years vs 66 years) than the NP group. Logistic regression poorly predicted which patients would have worsening infiltrate on the second radiograph. CONCLUSIONS: Elevated admission BUN level and higher fluid volume administered in the first 48 hours of admission were associated with worsening radiographic findings of pneumonia after hydration. Prospective studies are needed for confirmation of our results.

Tuberculous enteritis: a case report.

Tuberculous enteritis is an unusual diagnosis in the United States. Because this entity is rare and the symptoms are not specific, the physician must have a high index of suspicion. We report the case of a young man with tuberculous involvement of the gastrointestinal tract who required surgical intervention for small bowel obstruction.

Direct measurement of aluminum uptake and distribution in single cells of Chara corallina.

Quantitative information on the uptake and distribution of Al at the cellular level is required to understand mechanisms of Al toxicity, but direct measurement of uptake across the plasma membrane has remained elusive. We measured rates of Al transport across membranes in single cells of Chara corallina using the rare (26)Al isotope, an emerging technology (accelerator mass spectrometry), and a surgical technique for isolating subcellular compartments. Accumulation of Al in the cell wall dominated total uptake (71-318 microgram m(-2) min(-1)), although transport across the plasma membrane was detectable (71-540 ng m(-2) min(-1)) within 30 min of exposure. Transport across the tonoplast was initially negligible, but accelerated to rates approximating uptake across the plasma membrane. The avacuolate protoplasm showed signs of saturation after 60 min, but continued movement across the plasma membrane was supported by sequestration in the vacuole. Saturation of all compartments was observed after 12 to 24 h. Accumulation of Al in the cell wall reflected variation in [Al(3+)] induced by changes in Al supply or complexing ligands, but was unaffected by pH. In contrast, transport across the plasma membrane peaked at pH 4.3 and increased when [Al(3+)] was reduced by complexing ligands. Cold temperature (4 degrees C) reduced accumulation in the cell wall and protoplasm, whereas 2,4-dinitrophenol and m-chlorocarbonylcyanidephenyl hydrazone increased membrane transport by 12- to 13-fold. Our data suggest that the cell wall is the major site of Al accumulation. Nonetheless, membrane transport occurs within minutes of exposure and is supported by subsequent sequestration in the vacuole. The rapid delivery of Al to the protoplasm suggests that intracellular lesions may be possible.

FISH landmarks for barley chromosomes (Hordeum vulgare L.).

Barley metaphase chromosomes (2n = 14) can be identified by fluorescence in situ hybridization (FISH) and digital imaging microscopy using heterologous 18S rDNA and 5S rDNA probe sequences. When these sequences are used together, FISH landmark signals were seen so that all 7 chromosomes were uniquely identified and unambiguously oriented. The chromosomal location of the landmark signals was determined by FISH to a barley trisomic series using the 18S and 5S probes labeled with different fluorophores. The utility of these FISH landmarks for barley physical mapping was also demonstrated when an Amy-2 cDNA clone and a BAC clone were hybridized with the FISH landmark probes.

Flexor tenosynovitis due to Mycobacterium asiaticum.

Mycobacteria other than tuberculosis infections of the hand are seen with increasing frequency. A case of Mycobacterium asiaticum flexor tenosynovitis is described. Successful management included radical flexor tenosynovectomy and therapy with oral minocycline and clarithromycin.

The risk of peripheral vein phlebitis associated with chlorhexidine-coated catheters: a randomized, double-blind trial.

OBJECTIVE: To evaluate the risk of phlebitis associated with chlorhexidine-coated polyurethane catheters in peripheral veins. DESIGN: A randomized, double-blinded trial comparing chlorhexidine-coated polyurethane catheters with uncoated polyurethane catheters. SETTING: A university hospital. PATIENTS: Adult medicine and surgery patients. INTERVENTIONS: Certified registered nurse anesthetists or an infusion team consisting of nurses and physicians inserted the catheters. Catheter insertion sites were scored twice daily for evidence of phlebitis. At the time catheters were removed, a quantitative blood culture was performed, and catheters were sonicated for quantitative culture. RESULTS: Of 221 evaluable catheters, phlebitis developed in 18 (17%) of 105 coated catheters, compared to 27 (23%) of 116 uncoated catheters (relative risk [RR], 0.74; 95% confidence interval [CI95], 0.43-1.26; P = .32). By survival analysis, chlorhexidine-coated catheters had a lower risk of phlebitis during the first 3 days (P = .06), but not when all catheters were considered in both patient groups (P = .31). In the absence of catheter colonization, the incidence of phlebitis was 21% (16/76) and 24% (20/86) for coated and uncoated catheters, respectively (P = .85), whereas in the presence of catheter colonization, the incidence of phlebitis was 14% (1/7) and 80% (4/5) for coated and uncoated catheters, respectively (RR, 0.18; CI95, 0.03-1.15; P = .07). CONCLUSION: The risk of phlebitis in the presence of catheter colonization was 82% lower for chlorhexidine-coated polyurethane catheters compared to otherwise identical uncoated catheters.

The reliability and validity of the Tekdyne hand dynamometer: Part I.

The purpose of this study was to evaluate the reliability of Tekdyne hand dynamometer in a controlled loading environment using the Instron 1331. Test-retest reliability, the intertool reliability of three different Tekdyne hand dynamometers, and the effects of surface area and the configuration of the forces applied to the Tekdyne hand dynamometer were studied. In addition, intertool reliability between a Jamar dynamometer modified with foam padding and the same Jamar dynamometer without padding was calculated to explore this device as an alternative measurement device. Intratool and intertool reliabilities of the three Tekdyne tools were high (ICC = 0.993-10.998, SEM = 0.106-0.045 psi and ICC = 0.995, SEM = 0.080 psi, respectively) when tested on the Instron. Both the surface area and the configuration of the force applied to the Tekdyne dynamometer appeared to influence the output reading of this device. The measurements obtained on the Tekdyne hand dynamometer correlated well with those obtained with the Jamar hand dynamometer when a controlled load was applied (r = 0.988). The Tekdyne hand dynamometer is a reliable tool in a controlled loading environment; however, further study is needed to determine its validity with respect to the Jamar dynamometer for testing human grip strength.

The reliability and validity of the Tekdyne hand dynamometer: Part II.

The purpose of this study was to evaluate the reliability and validity of the Tekdyne hand dynamometer for measuring grip strength as compared with the Jamar dynamometer. Additionally, it was hypothesized that one to six weeks after carpal tunnel release (CTR) grip strength would appear to be greater when tested on a softer tool than when tested on the Jamar dynamometer. The Tekdyne, the standard Jamar instrument (SJ) and a Jamar dynamometer modified with foam padding (MJ) were compared in 48 subjects without upper-extremity abnormality and 30 subjects following open-palm CTR. The Tekdyne and the Jamar hand dynamometers were well correlated in the presence and in the absence of upper-extremity abnormality (r = 0.975 and r = 0.871, respectively). Tekdyne intrasession reliabilities in both the non-surgical group and the surgical group were high (ICC = 0.954, SEM = 0.290 psi and ICC = 0.958, SEM = 0.219 psi). The Tekdyne intersession reliability of the non-surgical group was high (ICC = 0.971, SEM = 0.22 psi). There was no statistically significant difference between the ratios of the non-operated grip strengths across the three measurement devices, suggesting that the softer device did not promote greater force production by the operated hand.

Regulation of arylsulphatase A and sulphogalactolipid turnover by cortisol in myelinogenic cultures of cells dissociated from embryonic mouse brain.

Myelinogenic cultures of cells dissociated from embryonic mouse brain were used to study the regulation of myelination-associated molecules by cortisol. Cortisol in physiological concentrations (0.03 microM) caused an increased accumulation of myelination-associated sulphogalactolipids. It also stimulated the myelin- and oligodendroglia-specific cyclic nucleotide phosphohydrolase. The increase in sulphogalactolipid content was caused by a cortisol-concentration-dependent inhibition in arylsulphatase A activity and not by an increase in either cerebroside sulphotransferase activity or an increase in availability of adenosine 3'-phosphate 5'-phosphosulphate. Of several steroid hormones tested only the glucocorticoid types brought about these changes. The relationship between net sulphogalactolipid accumulation and arylsulphatase A inhibition induced by cortisol was confirmed by sulphogalactolipid turnover studies. Depending on whether a single-phase or a two-phase decay calculation is used, the turnover of sulphogalactolipid with cortisol present was decreased at 22 days in culture by either 62% or 65% respectively of that without cortisol. This decrease in turnover can be attributed completely to the decrease of arylsulphatase activity by cortisol to 63% of the value for normal cells grown under the same conditions.

Serial enzyme-linked immunosorbent assays for antibody to Candida antigens during induction chemotherapy for acute leukemia.

Two or more sera were collected from 68 adult patients who received a total of 100 courses of induction chemotherapy for acute leukemia. Sera were tested by ELISAs for antibody to candidal mannan and a major cytoplasmic antigen. Twenty episodes of induction chemotherapy were accompanied by invasive candidiasis of skin, esophagus, or deeper organs. Levels of antibody to mannan rose more than 2.75 times normal activity in nine of these episodes and were already above this level in all sera in two episodes. Levels of antibody to mannan exceeded 2.75 times normal in only two of the remaining episodes in which invasive candidiasis was not observed. An eightfold increase in antibody to mannan occurred at some point in five of the 20 episodes complicated by invasive candidiasis but in only one of the remaining 80 episodes. Antibody to the major cytoplasmic antigen was detected infrequently in sera from episodes complicated by invasive candidiasis. Further studies of precise serial measurements of antibody to mannan therefore appear warranted.

Quantitation of antibody to Candida mannan by enzyme-linked immunosorbent assay.

Early clinical diagnosis of invasive candidiasis is difficult. To facilitate rapid diagnosis of Candida infections, we developed an ELISA to quantitate levels of antibody to Candida mannan. The test was standardized by analysis of a nonselected inpatient population to determine a cutoff point defining the upper 5% of such a population as test-positive. Passively acquired sera from patients in intensive care units, patients with neoplastic disease or recent renal allografts, and other patients were analyzed. There was no significant difference between the number of positive tests obtained from patients in whom candidiasis was considered but cultures were negative and from the nonselected inpatient population. Positive tests were obtained from 18.5% of patients with Candida mucocutaneous colonization or infection and 40% and 63.6% of patients with probable and proven invasive candidiasis, respectively. Patients with neoplastic disease had lower test sensitivity than patients in other test categories. These results demonstrate the usefulness of a simple, rapid, standardized test for quantitation of levels of antibody activity to Candida mannan in the serodiagnosis of candidiasis.

Oral neomycin dosage schedules for suppression of ammonia production by bowel flora.

To better define a minimal but optimal dose of oral neomycin to suppress ammonia production by bowel flora, several dosage regimens were examined in normal healthy volunteers. Fecal urease activity was quantitatively determined and was used as an indirect measure of intrinsic ammonia production by bowel flora. Large doses of neomycin were found to exert inhibition of fecal urease for many days. There was considerable variation in enzymatic activity among subjects even after adjustments were made for protein content of the stool. Depending on the dose, there was a 1- to 3-day lag in neomycin effect on stool urease activity and several days of continued effect. The most effective regimen of those studied was a loading dose of 6 g of neomycin given in three divided doses on day 1, followed by 1 g twice daily.

Daily change in pineal N-acetyltransferase activity in a diurnal mammal, the ground squirrel.

Pineal N-acetyltransferase (NAT) activity in the ground squirrel, a diurnal mammal, was found to have a daily fluctuation with peak activity during the dark time. This same daily change is found in nocturnal mammals and diurnal birds. NAT may play an important role in keeping track of light and dark cycles.