Structure of the human argininosuccinate synthetase gene and an improved system for molecular diagnostics in patients with classical and mild citrullinemia

Deficiency of argininosuccinate synthetase (ASS) causes citrullinemia, an autosomal recessive inherited defect of the urea cycle. Most patients described so far have presented with the classical form of the disease. There are also patients with a mild form of citrullinemia in whom the exact molecular basis and clinical relevance are uncertain. Mutations in the human ASS gene have not yet been described in mildly affected or asymptomatic patients with citrullinemia. The genomic sequence of the human ASS gene is not precisely known making mutation analysis difficult. Here, the entire genomic DNA sequence and mutations in the ASS gene of patients with the classical and mild form of the disease are described. The mutations c.1168G-->A (G390R) and IVS13+5 G-->A and the novel mutation c.323G-->T (R108L) have been found to be associated with classical citrullinemia, whereas the novel mutations c.535T-->G (W179R), and c.1085G-->T (G362V) have been detected on alleles of the mildly affected patients. Thus, mutations found in the human ASS gene of asymptomatic children with biochemical abnormalities and in some cases enzymatically proven citrullinemia have allowed us to classify these cases as ASS-deficient patients. The elucidation of the structure of the human ASS gene has made possible the use of intronic primers for molecular analysis of patients with mild disease and the classical form, and provides another option for prenatal diagnostics in affected families with the severe type.     

Fine needle aspiration of the thyroid: can an image processing system improve differentiation?

OBJECTIVE: To differentiate thyroid nodules by means of fine needle aspiration with subsequent computer-aided diagnosis. STUDY DESIGN: Fine needle aspiration biopsies obtained from 137 patients for whom histopathologic diagnosis was available were investigated: 16 hyperplastic nodules (12%), 39 adenomas (28%), 25 follicular thyroid carcinomas (18%), 19 follicular variants of papillary thyroid carcinoma (14%) and 38 papillary thyroid carcinomas (28%). From each stained specimen 100 cell scenes were scanned and analyzed, constituting a total of 62,325 cell images. RESULTS: All the entities described could be well discriminated from each other by automated image analysis methods. Both the diagnosis of tumor type and the differentiation between benign and malignant could be achieved with a sensitivity of .98. CONCLUSION: With only 7-10 calculated cell features (texture line analysis) and classification with decision trees, a tool for high-quality cell image diagnosis is available. Subtypes of cells characterized by the calculated features could be found in all the specimens and could be assigned to the malignancies with high statistical significance. The method increases the relevance of image processing as an additional diagnostic tool for cytologic examination of thyroid nodules.     

Kinetic and spectroscopic studies of Tritrichomonas foetus low-molecular weight phosphotyrosyl phosphatase. Hydrogen bond networks and electrostatic effects

Virtually all of the eukaryotic low-molecular weight protein tyrosine phosphatases (LMW PTPases) studied to date contain a conserved, high-pK(a) histidine residue that is hydrogen bonded to a conserved active site asparagine residue of the phosphate binding loop. However, in the putative enzyme encoded by the genome of the trichomonad parasite Tritrichomonas foetus, this otherwise highly conserved histidine is replaced with a glutamine residue. We have cloned the gene, expressed the enzyme, demonstrated its catalytic activity, and examined the structural and functional roles of the glutamine residue using site-directed mutagenesis, kinetic measurements, and NMR spectroscopy. Titration studies of the two native histidine residues in the T. foetus enzyme as monitored by (1)H NMR revealed that H44 has a pK(a) of 6.4 and H143 has a pK(a) of 5.3. When a histidine residue was introduced in place of the native glutamine at position 67, a pK(a) of 8.2 was measured for this residue. Steady state kinetic methods were employed to study how mutation of the native glutamine to alanine, asparagine, and histidine affected the catalytic activity of the enzyme. Examination of k(cat)/K(m) showed that Q67H exhibits a substrate selectivity comparable to that of the wild-type (WT) enzyme, while Q67N and Q67A show reduced activity. The effect of pH on the reaction rate was examined. Importantly, the pH-rate profile of the WT TPTP enzyme revealed a much more clearly defined acidic limb than that which can be observed for other wild-type LMW PTPases. The pH-rate curve of the Q67H mutant shows a shift to a lower pH optimum relative to that seen for the wild-type enzyme. The Q67N and Q67A mutants showed curves that were shifted to higher pH optima. Although the active site of this enzyme is likely to be similar to that of other LMW PTPases, the hydrogen bonding and electrostatic changes afford new insight into factors affecting the pH dependence and catalysis by this family of enzymes.     

High-performance liquid chromatographic assay of metabolites of thioguanine and mercaptopurine in capillary blood

The main metabolites of the cytotoxic drugs thioguanine (6TG) and mercaptopurine (6MP) can be measured conveniently in red blood cells (RBC). Isolation of RBC, however, is laborious and requires some milliliters of blood. This HPLC assay allows measurements of thiopurine metabolites in very small blood samples obtained from the finger-tip. The metabolites, derivatives of 6TG and methylmercaptopurine (6MeMP), were extracted and hydrolized with perchloric acid to liberate the corresponding base. 6MeMP is completely transformed under these conditions to 4-amino-5-(methylthio)carbonyl imidazole. The chromatographic separation of 6TG and this imidazole was performed in a single run under isocratic conditions within 10 min using a 70 mm column. The quantification limit was 0.5 nmol/ml for 6TG and 3 nmol/ml blood for 6MeMP. The accuracy was 83% for 6TG (CV=3%) over the concentration range of 0.5-20 nmol/ml blood and 102% (CV=4%) for 6MeMP over the range of 3-150 nmol/ml blood. The intra-assay CV ranged from 5.4 to 7.4% for 6TG and from 6.2 to 10.6% for 6MeMP. The inter-assay CV was 7.5 and 9.5% in a pooled blood sample. The levels in RBC in whole blood were nearly coincident with those obtained in separated RBC, isolation of RBC therefore is not necessary for these measurements, if the drugs are given per os in the day before blood sampling. The concentration of 6MeMP nucleotides is more dependent on the given 6MP dose than the concentration of 6TG nucleotides. Intraindividual variations were small at unchanged drug doses, interindividual metabolite concentrations were highly variable.     

Mutual influences of Pseudomonas aeruginosa and Desulfovibrio desulfuricans on their adhesion to stainless steel

The mutual influences of Pseudomonas aeruginosa PAO1 and Desulfovibrio desulfuricans subsp. desulfuricans (ATCC 29577) on their adhesion to stainless steel were investigated in batch and column experiments. It was found that P. aeruginosa promoted the adhesion of D. desulfuricans under conditions of turbulence, but not under quiescent conditions. The enhancement involved the alignment of most D. desulfuricans along P. aeruginosa cells and was attributed to the additional interaction surface area provided by adhered P. aeruginosa to aligning D. desulfuricans cells. A slightly positive effect of preadhered D. desulfuricans on the adhesion of P. aeruginosa was found. Under condition of laminar flow, substantially better adhesion of D. desulfuricans to confluent P. aeruginosa biofilms than to steel was observed. The mutual influences are discussed in terms of more favorable adhesion energies and the influence of changed hydraulic conditions due to the roughness of P. aeruginosa biofilms.     

Anomalous fluorescence enhancement of Cy3 and cy3.5 versus anomalous fluorescence loss of Cy5 and Cy7 upon covalent linking to IgG and noncovalent binding to avidin

This study provides a critical examination of protein labeling with Cy3, Cy5, and other Cy dyes. Two alternate situations were tested. (i) Antibodies were covalently labeled with Cy dye succinimidyl ester at various fluorophore/protein ratios and the fluorescence of the labeled antibodies was compared to that of free Cy dye. (ii) Fluorescent biotin derivatives were synthesized by derivatizing ethylenediamine with one biotin and one Cy3 (or Cy5) residue. The fluorescence properties of these biotin-Cy dye conjugates were examined at all ligand/(strept)avidin ratios (0 相似文献   

Mapping of Drosophila mutations using site-specific male recombination.

Although recombination does not usually occur in the male Drosophila germline, site-specific recombination can be induced at the ends of P elements. This finding suggested that male recombination could be used to map Drosophila mutations. In this article, we describe the general method and its application to the mapping of two EMS-induced female-sterile mutations, grauzone and cortex. Within two months, the grauzone gene was mapped relative to seven different P-element insertion sites, and cortex was mapped relative to 23 different P-elements. The results allowed us to map grauzone to a region of about 50 kb, and cortex distal to the chromosomal region 33E. These experiments demonstrate that P-element-induced site-specific male recombination is an efficient and general method to map Drosophila autosomal mutations.     

Smaller lungs in women affect exercise hyperpnea

We subjected 29 healthy young women (age: 27 ± 1 yr) with a wide range of fitness levels [maximal oxygenuptake (O_{2 max}): 57 ± 6 ml · kg¹ · min¹;35-70ml · kg¹ · min¹]to a progressive treadmill running test. Our subjects had significantly smaller lung volumes and lower maximal expiratory flow rates, irrespective of fitness level, compared with predicted values for age-and height-matched men. The higher maximal workload in highly fit(O_{2 max} > 57 ml · kg¹ · min¹,n = 14) vs. less-fit(O_{2 max} < 56 ml · kg¹ · min¹,n = 15) women caused a higher maximalventilation (E) with increased tidal volume (VT)and breathing frequency (f_b) atcomparable maximal VT/vitalcapacity (VC). More expiratory flow limitation (EFL; 22 ± 4% ofVT) was also observed duringheavy exercise in highly fit vs. less-fit women, causing higherend-expiratory and end-inspiratory lung volumes and greater usage oftheir maximum available ventilatory reserves.HeO₂ (79% He-21%O₂) vs. room air exercise trialswere compared (with screens added to equalize external apparatusresistance). HeO₂ increasedmaximal expiratory flow rates (20-38%) throughout the range ofVC, which significantly reduced EFL during heavy exercise. When EFL wasreduced with HeO₂, VT,f_b, andE (+16 ± 2 l/min) weresignificantly increased during maximal exercise. However, in theabsence of EFL (during room air exercise),HeO₂ had no effect onE. We conclude that smaller lungvolumes and maximal flow rates for women in general, and especiallyhighly fit women, caused increased prevalence of EFL during heavyexercise, a relative hyperinflation, an increased reliance onf_b, and a greater encroachment onthe ventilatory "reserve." Consequently,VT andE are mechanically constrained duringmaximal exercise in many fit women because the demand for highexpiratory flow rates encroaches on the airways' maximum flow-volumeenvelope.

     

Cytometric patterns reveal growth states of Shewanella putrefaciens

Bacterial growth is often difficult to estimate beyond classical cultivation approaches. Low cell numbers, particles or coloured and dense media may disturb reliable growth assessment. Further difficulties appear when cells are attached to surfaces and detachment is incomplete. Therefore, flow cytometry was tested and used for analysis of bacterial growth on the single‐cell level. Shewanella putrefaciens was cultivated as a model organism in planktonic or biofilm culture. Materials of smooth and rough surfaces were used for biofilm cultivation. Both aerobic and anaerobic as well as feast and famine conditions were applied. Visualization of growth was also done using Environmental Scanning and Phase Contrast Microscopy. Bioinformatic tools were applied for data interpretation. Cytometric proliferation patterns based on distributions of DNA contents per cell corresponded distinctly to the various lifestyles, electron acceptors and substrates tested. Therefore, cell cycling profiles of S. putrefaciens were found to mirror growth conditions. The cytometric patterns were consistently detectable with exception of some biofilm types whose resolution remained challenging. Corresponding heat maps proved to be useful for clear visualization of growth behaviour under all tested conditions. Therefore, flow cytometry in combination with bioinformatic tools proved to be powerful means to determine various growth states of S. putrefaciens, even in constrained environments. The approach is universal and will also be applicable for other bacterial species.     

Differential Survival for Men and Women with HIV/AIDS-Related Neurologic Diagnoses

Objectives

Neurologic complications of human immunodeficiency virus (HIV) infection and acquired immune deficiency syndrome (AIDS) frequently lead to disability or death in affected patients. The aim of this study was to determine whether survival patterns differ between men and women with HIV/AIDS-related neurologic disease (neuro-AIDS).

Methods

Retrospective cohort data from a statewide surveillance database for HIV/AIDS were used to characterize survival following an HIV/AIDS-related neurologic diagnosis for men and women with one or more of the following conditions: cryptococcosis, toxoplasmosis, primary central nervous system lymphoma, progressive multifocal leukoencephalopathy, and HIV-associated dementia. A second, non-independent cohort was formed using university-based cases to confirm and extend the findings from the statewide data. Kaplan-Meier analysis was used to compare the survival experiences for men and women in the cohorts. Cox regression was employed to characterize survival while controlling for potential confounders in the study population.

Results

Women (n=27) had significantly poorer outcomes than men (n=198) in the statewide cohort (adjusted hazard ratio=2.31, 95% CI: 1.22 to 4.35), and a similar, non-significant trend was observed among university-based cases (n=17 women, 154 men). Secondary analyses suggested that this difference persisted over the course of the AIDS epidemic and was not attributable to differential antiretroviral therapy responses among men and women.

Conclusions

The survival disadvantage of women compared to men should be confirmed and the mechanisms underlying this disparity elucidated. If this relationship is confirmed, targeted clinical and public health efforts might be directed towards screening, treatment, and support for women affected by neuro-AIDS.