Expression and biochemical characterization of human glucose-6- phosphate dehydrogenase in Escherichia coli: a system to analyze normal and mutant enzymes

We have developed a system to characterize normal and mutated glucose-6- phosphate dehydrogenase (G6PD) enzymes in vitro. Normal or mutant G6PD cDNA was subcloned into a pGEX-3X vector, which allowed production of a functional fusion protein in Escherichia coli. When we compared the recombinant normal enzyme with authentic human G6PD, indistinguishable Km values for glucose-6-phosphate (G6P) and NADP were obtained, and the utilization rates for two substrate analogues (2-deoxy G6P and deamino NADP) also showed no difference between the enzymes. This system was used to assay a biochemically uncharacterized variant, G6PD Taipei (493 A-->G; 165 Asn-->Asp), plus two other known mutations (487 G-->A; 163 Gly-->Ser and 592 C-->T; 198 Arg-->Cys) that are located close to or within the putative G6P binding domain. Our results show that the G6PD activities of these three mutants were greatly reduced. No significant alteration in G6PD kinetics was observed for both 487 and 493 mutations. However, a drastic reduction in the Km for G6P (4-fold decrease) and tremendous increases in utilization rates of 2-deoxy G6P (32-fold increase) and deamino NADP (6-fold increase) were associated with the 592 mutation. This results suggests that arginine 198 in human G6PD, possibly located within the putative G6P binding domain, may play an important role in binding the substrate G6P. In addition, we and others have recently identified that at least nine different types of mutations are responsible for G6PD deficiency in Chinese. In this report, we also present the occurrence rate of each mutation present in the population of Taiwan.     

Excessive bronchoconstriction induced by histamine and effects of volume history in patients with bronchial asthma

Abstract The aim of this study was to examine the inter-relationships between the different effects of deep breaths and histamine provocation on airway function in patients with bronchial asthma. Group 1 consisted of 38 consecutive out-patients with newly diagnosed mild asthma, group 2 consisted of 20 patients with bronchial asthma of varying severity who were studied during clinical remission. We measured bronchial responsiveness (BR) to histamine inhalation as the dose of histamine which provoked a 20% fall in FEV₁ (PD₂₀). The fall in forced vital capacity (FVC) after inhaling the highest dose of histamine during each BR test was calculated and expressed as percentage of the value measured at baseline (δFVC in percentage). We studied the effects of deep breaths on airway caliber in group 2 patients by comparing isovolumic flow rates on partial (P) and maximal (M) forced expiratory flow volumes curves expressed as the M/P ratio. The changes in residual volume (RV) after deep breaths (δRV) were expressed as a percentage of the largest VC measured on the composite M and P curves. The patients in group 1 had significantly higher PD₂₀ and lower δFVC than patients in group 2. There was, however, no significant correlation between PD₂₀ and δFVC measurements in individual patients (r<0.1, P>0.05). The M/P ratio was significantly related to δFVC (r=?0.6, P<0.006). There was also a significant positive relation between the magnitude of increase in residual volume following deep breaths (δRV) and the degree of fall in FVC following histamine inhalation (δFVC) (r= 0.65, P= 0.001). This significant relationship between the degree of airway closure after a deep breath and airway closure after histamine challenge is a new finding. In patients with bronchial asthma, the effects of a deep breath on airway function may be indicative of the tendency for airway closure during BR testing.     

Diagnosis and Management of Schwannomas Originating from the Cervical Vagus Nerve

Introduction

A schwannoma is a benign, slow growing, encapsulated nerve sheath tumour. Presentation of a schwannoma is a diagnostic and management challenge.

Methods

Internet searches of PubMed/MEDLINE^® for all articles listing schwannomas of the vagus nerve in the cervical/neck region (1980–2012) were undertaken to ascertain diagnostic pitfalls. The references of all articles were cross-checked to include all pertinent contributions. Further articles were traced through reference lists.

Results

Schwannomas are solitary, well circumscribed and medial to the carotid sheath. Preoperative diagnoses of schwannomas in the lateral part of the neck can cause confusion with its nerve of origin (ie whether it arises from the vagus nerve or a sympathetic chain). Computed tomography and magnetic resonance imaging reveal valuable information regarding the location and origin of the tumour as well as aiding surgical planning. The diagnosis can be confirmed intraoperatively. Postoperative recovery of neurological function is dependent on the type of surgery. Histopathological studies searching for classical features and immunohistochemical staining for S100 also confirm the diagnosis.

Conclusions

Schwannomas should be considered in the differential diagnoses of unusual masses in the neck. Preoperative imaging elicits valuable information regarding the location and origin of schwannomas and histopathology confirms the diagnosis.     

Asynchronous regulation of splicing events within protein 4.1 pre-mRNA during erythroid differentiation

Protein 4.1 is an 80-kD structural component of the red blood cell (RBC) cytoskeleton. It is critical for the formation of the spectrin/actin/protein 4.1 junctional complex, the integrity of which is important for the horizontal strength and elasticity of RBCs. We and others have previously shown that multiple protein 4.1 mRNA isoforms are generated from a single genomic locus by several alternative mRNA splicing events, leading to the insertion or skipping of discrete internal sequence motifs. The physiologic significance of these motifs: (1) an upstream 17-nucleotide sequence located at the 5' end of exon 2 that contains an in-frame ATG initiation codon, the inclusion of which by use of an alternative splice acceptor site in exon 2 allows the production of a 135-kD high-molecular-weight isoform present in nonerythroid cells; (2) exon 16, which encodes a 21-amino acid (21aa) segment located in the 10-kD "spectrin/actin binding domain" (SAB), the presence of which is required for junctional complex stability in RBCs. Previous studies by our group and others suggested that, among blood cells, this exon was retained only in mature mRNA in the erythroid lineage. Exon 16 is one of a series of three closely linked alternatively spliced exons, generating eight possible mRNA products with unique configurations of the SAB. In this communication, we report studies of the expression of both the translation initiation region and the SAB region during induced erythroid maturation in mouse erythroleukemia (MEL) cells. We have found that only two of eight possible combinatorial patterns of exon splicing at the SAB region are encountered: the isoform lacking all three exons, present in predifferentiated cells, and the isoform containing only exon 16, which increases in amount during erythroid differentiation. The protein isoform containing the 21aa segment encoded by exon 16 efficiently and exclusively incorporates into the membrane, whereas the isoform lacking this 21aa segment remains in the cytoplasm, as well as the membrane. In contrast with exon 16, the erythroid pattern of exon 2 splicing, i.e., skipping of the 17-base sequence at the 5' end, was found to be already established in the uninduced MEL cells, suggesting strongly that this regulated splicing event occurs at an earlier stage of differentiation. Our results demonstrate asynchronous regulation of two key mRNA splicing events during erythroid cell maturation. These findings also show that the splicing of exon 16 alters the intracellular localization of protein 4.1 in MEL cells, and appears to be essential for its targeting to the plasmalemma.     

Transcultural and psychometric validation of the Dispositional Resilience Scale (DRS-15) in Chinese adult women

Purpose

The aim of this study was to report translation and transcultural adaptation of the 15-item Dispositional Resilience Scale in traditional Chinese (C-DRS-15) and evaluate its psychometric properties.

Methods

The DRS is a self-report instrument that measures psychological hardiness. We followed an international standard of cross-cultural translation and validation of patient-reported outcome measures to create the Chinese version. Then, the translated C-DRS-15 was validated on 542 Chinese women from a population-based sample in Hong Kong.

Results

The internal consistency and criterion-related validity were investigated. Exploratory and confirmatory factor analysis revealed that the C-DRS-15 was supported by a modified three-factor structure in our Chinese sample (RMSEA = .06, CFI = .94, TLI = .92, and SRMR = .06). The reliability (Cronbach’s α coefficient = .78) and validity were satisfactory. Total resilience score was negatively correlated with depression (p < .001), with non-depressed women scoring higher on the C-DRS-15.

Conclusions

The C-DRS-15 was demonstrated to be a reliable and valid measurement to assess hardiness in Chinese women.