The role of cysteine residues of spinach ferredoxin-NADP+ reductase As assessed by site-directed mutagenesis

To investigate the functional role of the cysteine residues present in the spinach ferredoxin-NADP+ oxidoreductase, we individually replaced each of the five cysteine residues with serine using site-directed mutagenesis. All of the mutant reductases were correctly assembled in Escherichia coli except for the C42S mutant protein. C114S and C137S mutant enzymes apparently showed structural and kinetic properties very similar to those of the wild-type reductase. However, C272S and C132S mutations yielded enzymes with a decreased catalytic activity in the ferredoxin-dependent reaction (14 and 31% of the wild type, respectively). Whereas the C132S was fully competent in the diaphorase reaction, the C272S mutant flavoprotein showed a 35-fold reduction in catalytic efficiency with respect to the wild-type enzyme (0.4 versus 14.28 microM-1 s-1) due to a substantial decrease of kcat. NADP+ binding by the C272S mutant enzyme was apparently quantitatively the same (Kd = 37 microM) but qualitatively different, as shown by the differential spectrum. Stopped-flow experiments showed that the enzyme-FAD reduction rate was considerably decreased in the C272S mutant reductase, along with a much lower yield of the charge-transfer transient species. It is inferred from these data that the charge transfer (FAD-NADPH) between the reductase and NADPH is required for hydride transfer from the pyridine nucleotide to flavin to occur with a rate compatible with catalysis.     

Stabilised p53 facilitates aneuploid clonal divergence in colorectal cancer

Mutations in the p53 tumour suppressor gene are amongst the most frequent genetic abnormalities acquired in tumours. Recent studies in vitro suggest that mutant p53 destabilises the genome and facilitates development of aneuploidy. Here, in a study of 83 colorectal carcinomas, we demonstrate that alterations in p53 (detected by immunocytochemical stabilisation) precede and apparently facilitate divergence of aneuploid sub-clones. Aneuploidy in these tumours (but not those with normal p53) is predominantly in the subtetraploid range, suggesting that endoreduplication is important in its origin. This association with a specific phase of carcinoma progression is not shared by other commonly acquired genetic abnormalities in these tumours. These observations highlight the critical role of p53 in the regulation of abnormal chromosome replication and afford an explanation for the association between p53 abnormalities, aneuploidy and biological aggression in cancer.     

What decides: high mortality in paediatric acute renal failure?

Forty paediatric cases of A.R.F. (Acute Renal Failure) of various aetiology were included in the study. 60% of patients were less than 4 years of age with male predominance. 80% cases reported to us very late with oligoanuria of more than 24 hours (2-7 days). Diarrhoea, vomiting and fever were other dominant symptoms. Maximum cases were severely anaemic (87.5%) with mean Hb 7.73 +/- 1.9 gm%. 40% cases were of underweight while only one case (2.5%) was of over weight, inspite of volume excess in 40% cases. All 24 cases, who were estimated for serum albumin, found to have marked hypoalbuminemia. Mortality was found to be as high as 65% inspite of effective peritoneal dialysis in all cases. High mortality seems to be due to profound anuria of many days (because of marked delay in reaching the hospital), fever and malnutrition besides other factors as aetiology.     

Rapid relief of anxiety in cancer patients with both alprazolam and placebo

This study evaluated 36 cancer patients who were enrolled in a randomized, double-blind, placebo-controlled trial conducted over a 4-week period to evaluate the efficacy of alprazolam in the treatment of anxiety associated with cancer. Hamilton Anxiety Scale scores declined significantly between baseline and the end of the first week of the study in both treatment groups. There was no significant difference in response between the patients receiving alprazolam and placebo. Similar results were obtained from other instruments. These results suggest that nondrug factors or spontaneous improvement may play a more important role than pharmacotherapy in the treatment of anxiety associated with cancer.     

Activation of PKCalpha downstream from caspases during apoptosis induced by 7-hydroxystaurosporine or the topoisomerase inhibitors, camptothecin and etoposide, in human myeloid leukemia HL60 cells

We previously demonstrated that the anticancer agent and protein kinase C (PKC) inhibitor 7-hydroxystaurosporine (UCN-01) induces apoptosis independently of p53 and protein synthesis in HL60 cells. We now report the associated changes of PKC isoforms. PKCalpha, betaI, betaII, delta, and zeta activities were measured after immunoprecipitation of cytosols from UCN-01-treated HL60 cells. UCN-01 had no effect on PKCzeta and inhibited kinase activity of PKCbetaI, betaII, and delta. PKCalpha activity was initially inhibited at 1 h, and subsequently increased as cells underwent apoptosis 3 h after the beginning of UCN-01 treatment. Camptothecin (CPT) and etoposide (VP-16) also markedly enhanced PKCalpha activity during apoptosis in HL60 cells. However, CPT did not affect PKCbetaI, betaII and zeta, and activated PKCdelta. PKCalpha activation was not due to increased protein levels or proteolytic cleavage but was associated with PKCalpha autophosphorylation in vitro and increased phosphorylation in vivo. We also found that not only PKC delta but also PKC betaI was proteolytically activated in HL60 cells during apoptosis. The PKCalpha activation and hyperphosphorylation were abrogated by N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethylketone (z-VAD-fmk) under conditions that abrogated apoptosis. z-VAD-fmk also prevented PKCdelta and betaI proteolytic activation. Together these findings suggest that caspases regulate PKC activity during apoptosis in HL60 cells. At least two modes of activation were observed: hyperphosphorylation for PKCalpha and proteolytic activation for PKC delta and betaI.     

Minocycline

Periodontal diseases are bacterial infections and antimicrobial agents appear to offer great potential in their treatment and prevention. One such chemotherapeutic agent is minocycline. The aim of this paper is to review the literature on this antibiotic concerning in vitro and in vivo studies, its pharmacokinetics and secondary effects.     

Lumbar lordosis. Effects of sitting and standing

STUDY DESIGN: The effect of sitting versus standing posture on lumbar lordosis was studied retrospectively by radiographic analysis of 109 patients with low back pain. OBJECTIVE: To document changes in segmental and total lumbar lordosis between sitting and standing radiographs. SUMMARY OF BACKGROUND DATA: Preservation of physiologic lumbar lordosis is an important consideration when performing fusion of the lumbar spine. The appropriate degree of lumbar lordosis has not been defined. METHODS: Total and segmental lumbar lordosis from L1 to S1 was assessed by an independent observer using the Cobb angle measurements of the lateral radiographs of the lumbar spine obtained with the patient in the sitting and standing positions. RESULTS: Lumbar lordosis averaged 49 degrees standing and 34 degrees sitting from L1 to S1, 47 degrees standing and 33 degrees sitting from L2 to S1, 31 degrees standing and 22 degrees sitting from L4 to S1, and 18 degrees standing and 15 degrees sitting from L5 to S1. CONCLUSION: Lumbar lordosis while standing was nearly 50% greater on average than sitting lumbar lordosis. The clinical significance of this data may pertain to: 1) the known correlation of increased intradiscal pressure with sitting, which may be caused by this decrease in lordosis; 2) the benefit of a sitting lumbar support that increases lordosis; and 3) the consideration of an appropriate degree of lordosis in fusion of the lumbar spine.     

Tumor necrosis factor-induced E-selectin expression on vascular endothelial cells

OBJECTIVE: To determine the tumor necrosis factor (TNF) receptor type involved in induction of E-selectin expression on vascular endothelial cells. DESIGN: Prospective, in vitro repeated-measures analysis of cellular responses. SETTING: Research laboratory in an academic medical center. SUBJECTS: Cultured human umbilical vein endothelial cells. INTERVENTIONS: Human umbilical vein endothelial cells were incubated with recombinant human TNF (rhTNF) to induce the expression of E-selectin on their surfaces. To block rhTNF from binding to receptors, the cells were incubated with monoclonal antibodies against TNF receptors (anti-CD120a and anti-CD120b). TNF-induced E-selectin expression of the endothelial cells, with and without blocking antibodies, was then determined using indirect immunofluorescence and flow cytometry. MEASUREMENTS AND MAIN RESULTS: Blocking of either CD120a or CD120b receptors individually resulted in inhibition of TNF-induced E-selectin expression on human umbilical vein endothelial cells. When both antibodies were added, the inhibition of TNF-induced E-selectin expression was synergistic. Inhibition of E-selectin expression was dependent on both TNF concentrations and antibody concentrations. CONCLUSIONS: Both CD120a and CD120b receptors are involved in TNF-induced E-selectin expression on human umbilical vein endothelial cells. Blocking of both or one receptor type can reduce or totally inhibit expression of E-selectin on human umbilical vein endothelial cells, but the response is dependent on both TNF and antibody concentrations.     

Recommendations for the prevention of coronary artery disease in Asians: a scientific statement of the International College of Nutrition

PURPOSE: Two types of glass wool were used to remove leukocytes in semen for evaluation of reactive oxygen species production by spermatozoa in oligozoospermic patients with leukocytospermia. METHODS: Semen samples were prepared using fine-structure glass wool (SpermFertil) and coarse-structure glass wool. In each treatment group, native semen was evaluated for sperm concentration, percentage motility, viability, leukocyte concentration, and production of reactive oxygen species. RESULTS: Electron microscopically, SpermFertil showed a higher number of leukocytes attached to the fibers compared to coarse-structure glass wool. Leukocytes in native semen and after glass wool filtration as determined by peroxidase cytochemistry confirmed this observation. Reactive oxygen species decreased from 45.303 counts/10(7) viable cells in native semen to 15.806 counts/10(7) cells in coarse structure wool and 7.465 counts/10(7) cells in Spermfertil, respectively. CONCLUSIONS: Removal of leukocytes from semen of oligozoospermic patients by means of glass wool filtration is a useful method to distinguish production of reactive oxygen species by leukocytes versus sperm cells.