Efficient expression of the gene for spinach phosphoribulokinase in Pichia pastoris and utilization of the recombinant enzyme to explore the role of regulatory cysteinyl residues by site-directed mutagenesis

Phosphoribulokinase (PRK), unique to photosynthetic organisms, is regulated in higher plants by thioredoxin-mediated thiol-disulfide exchange in a light-dependent manner. Prior attempts to overexpress the higher plant PRK gene in Escherichia coli for structure-function studies have been hampered by sensitivity of the recombinant protein to proteolysis as well as toxic effects of the protein on the host. To overcome these impediments, we have spliced the spinach PRK coding sequence immediately downstream from the AOX1 (alcohol oxidase) promoter of Pichia pastoris, displacing the chromosomal AOX1 gene. The PRK gene is now expressed, in response to methanol, at 4-6% of total soluble protein, without significant in vivo degradation of the recombinant enzyme. This recombinant spinach PRK is purified to homogeneity by successive anion-exchange and dye-affinity chromatography and is shown to be electrophoretically and kinetically indistinguishable from the authentic spinach counterpart. Site-specific replacement of all of PRK's cysteinyl residues (both individually and in combination) demonstrates a modest catalytically facilitative role for Cys-55 (one of the regulatory residues) and the lack of any catalytic role for Cys-16 (the other regulatory residue), Cys-244, or Cys-250. Mutants with seryl substitutions at position 55 display non-hyperbolic kinetics relative to the concentration of ribulose 5-phosphate. Sulfate restores hyperbolic kinetics and enhances kinase activity, presumably reflecting conformational differences between the position 55 mutants and wild-type enzyme. Catalytic competence of the C16S-C55S double mutant proves that mere loss of free sulfhydryl groups by oxidative regulation cannot account entirely for the accompanying total inactivation.     

Colonic diverticulosis in Hong Kong: distribution pattern and clinical significance

This is a retrospective study on the prevalence of diverticulosis in Hong Kong Chinese adults. Eight-hundred and fifty-eight consecutive barium enema examinations over a period of 18 months (January 1995-June 1996) were analysed. Results show that the prevalence of diverticulosis in our community is 25.1% with no significant difference between male and female adults. The prevalence is lower than Western countries but higher than in Asia. Moreover, the peak prevalence is at the 50-79 years age group with lower prevalence in the older age groups. This may be explained by the rapid rise in prevalence in the younger age groups so that the age-related increase in prevalence become obscured. We postulate that this may be due to Western cultural influence in our diet and lifestyle. There is no significant difference in the symptomatology of patients with and without diverticulosis, supporting the idea that diverticulosis alone is usually asymptomatic. There is predominance of right hemicolon involvement in our subjects, in contrast to the left hemicolon predominance in the Caucasian population. Of patients with diverticulosis, 55.3% have only right-sided involvement and 32.6% have bilateral involvement. Only 12.1% of patients with diverticulosis have exclusively left hemicolon involvement. Caecal and ascending colon diverticula are found in 6.4% and 17.6% of all the adults under study, respectively. Clinicians caring for patients from our community should take note of this high frequency of caecal and ascending colon diverticulosis as caecal and ascending colon diverticulitis is notoriously difficult to diagnose clinically.     

Subclavian mycotic aneurysm presenting as mediastinal abscess

A 60-year-old man was admitted to a hospital for evaluation of intermittent fever, dysphagia, hoarseness, and general chest discomfort. Great vessel mycotic aneurysm was suspected when antibiotic trials failed and chest X-ray showed paraaortic mass with pleural effusion mimicking mediastinitis. Although the correct diagnosis of mycotic aneurysm of innominate artery was made thereafter and vigorous treatment was initiated immediately, this patient succumbed to overwhelming sepsis, probably due to a 2-week delay in another hospital. This case is reported to remind readers of the possibility of this unusual location of mycotic aneurysm. A high index of suspicion should be maintained to make an earlier diagnosis and obtain better prognosis. Computed tomography and 3D magnetic resonance angiography also significantly improve the diagnosis when mycotic aneurysm location is unusual and presentation is equivocal.     

Spontaneous thrombosis of a basilar artery traumatic aneurysm in a child

Traumatic aneurysms are rare and occur most commonly in young adults; however, the relative frequency in the pediatric population is high, owing to the low prevalence of congenital saccular aneurysms in children. Traumatic aneurysms typically involve the anterior circulation, and spontaneous thrombosis is uncommon; hence, surgery is usually necessary. We present a case of a posttraumatic aneurysm in a child that occurred after a fall from a large height and that spontaneously thrombosed.     

Aconitine poisoning following the ingestion of Chinese herbal medicines: a report of eight cases

BACKGROUND: Traditional Chinese medicines often contain 'chuanwu' and 'caowu', the roots of certain Aconitum species which are thought to have an anti-inflammatory effect in many conditions. Excessive amounts of these materials, which contain diterpene alkaloids particularly aconitine, can produce toxic effects and occasional fatalities. AIMS: This study was conducted to document the adverse effects related to these herbal medicines which resulted in hospital admission and to determine the outcome in these patients. METHODS: A retrospective survey was conducted of patients admitted to the Prince of Wales Hospital, Hong Kong with suspected adverse effects from Chinese herbal medicines containing chuanwu or caowu over a two year period from 1989 to 1991. RESULTS: Eight patients were identified with features of mild to moderate intoxication including nausea and vomiting, paraesthesiae or numbness in the mouth and extremities, hypotension and ventricular extrasystoles. The management of aconitine poisoning is essentially supportive and in-hospital observation with ECG monitoring should be continued for at least 24 hours because of the risk of cardiovascular collapse and ventricular arrhythmias. The medical profession and general public should be alerted to the potential toxicity of these herbs and their usage should be controlled by legislation in Hong Kong as it is in some other countries.     

An ampD gene in Pseudomonas aeruginosa encodes a negative regulator of AmpC beta-lactamase expression

The ampD and ampE genes of Pseudomonas aeruginosa PAO1 were cloned and characterized. These genes are transcribed in the same orientation and form an operon. The deduced polypeptide of P. aeruginosa ampD exhibited more than 60% similarity to the AmpD proteins of enterobacteria and Haemophilus influenzae. The ampD product transcomplemented Escherichia coli ampD mutants to wild-type beta-lactamase expression.     

Individual differences in trajectory of intellectual development over 45 years of adulthood

The hypothesis that individual differences in adult intellectual development reflect variation in life context and personality was examined in 132 World War II (WWII) veterans. Intelligence data from the Canadian Army M Test were available for 3 occasions, WWII, 1984-1986 (Int85), and 1989-1991 (Int90). Performance declined over the 45 years on 5 subtests but improved on the 6th, Vocabulary. Correlations between WWII and Int90 scores showed considerable stability of individual differences in intelligence over 45 years. Younger age predicted less decline over 45 years on all subtests; a more engaged lifestyle predicted less decline on most subtests, and better health and greater introversion predicted less decline on some subtests. Predictors of change since Int85 were similar to those for the whole period. Implications for theories of adult intellectual development are discussed.     

Usage and adverse effects of Chinese herbal medicines

Oestrogen receptor (ER) expression in breast cancer is regarded as a phenotype that may change during the natural history of the disease or during endocrine therapy. It has been suggested that in up to 70% of tumours that show acquired resistance the mechanism may be changed in ER status from positive to negative. This paper proposes an alternative hypothesis that ER expression in a stable phenotype in breast cancer. The paper reviews the literature on ER expression during the natural history of breast cancer in patients and also presents data on the effect of endocrine therapy on ER expression. If the alternative hypothesis is true it has important implications for treatment from chemoprevention to acquired endocrine resistance in advanced disease. Equally, if the hypothesis is true, attempts to develop laboratory models of endocrine resistance where ER-positive tumours become ER negative need to be re-evaluated.