Vitamin D administration to tuberculous children and its value

Our study was done to assess the value of administration of vitamin D to tuberculous children. The study included twenty four newly diagnosed tuberculous children; eleven males and thirteen females. Their age ranged from one and half to thirteen years. Thirteen patients were extra thoracic type of T.B., while only seven were intrathoracic and the rest were mixed. They were randomly divided into two Groups according to the treatment administered: Group A patients were given Rifampicin, Isoniazid and Streptomycin. Group B received in addition vitamin D. After eight weeks therapy, the patients of each group were evaluated regarding clinical, laboratory, and radiological improvement. Vitamin D level is raised after treatment in both Groups A and B, but this rise is not significant. It also showed insignificant difference between the two groups. Vitamin D level showed very high significant decrease in tuberculous children than matched healthy controls (non tuberculous children). Calcium was significantly elevated after treatment in Group A whereas no significant change was detected in Group B. Phosphorous was highly significantly elevated after treatment in Group A, whereas in Group B it is just significantly elevated. Alkaline phosphatase level in both groups A and B were slightly decreased after treatment. However, this decrease was not significant. Clinical improvement was more evident in Group B patients (those taking vitamin D) as compared to Group A patients. The same was noted with X-ray and Sonographic findings. We concluded that vitamin D therapy may be of great value in addition to antituberculous drugs in the treatment of tuberculous children, and its use is highly recommended.     

Metabolism and pharmacokinetics of N1,N11-diethylnorspermine

The pharmacokinetics and metabolism of N1,N11-diethylnorspermine (DENSPM) is described. When administered to dogs as an intravenous bolus, DENSPM was shown to have a plasma half-life of 72.8 +/- 11.8 min, with an early distribution phase half-life of approximately 4 min and an apparent volume of distribution of 0.216 +/- 0.032 liter/kg. The renal clearance half-life was 59.7 +/- 7.6 min, with 48.8 +/- 12.5% of the drug recovered in the urine between 0-4 hr unchanged. In three other experiments, the drug was administered to dogs by constant rate intravenous infusion over periods ranging from 10 min to 2 hr. Analysis of plasma concentration-time data and urinary excretion data yielded pharmacokinetic parameters in general agreement with the intravenous bolus experiments. DENSPM metabolites were identified in both beagle dog and mouse tissues. Tissues were sampled from a single beagle 24 hr posttreatment, and rodent samples were examined at 12, 24, 48, and 96 hr posttreatment. Both the concentration of DENSPM and the metabolic profile were shown to vary in the lung, liver, spleen, and kidney. Although all the tissues examined contained DENSPM and its metabolites, the liver and kidney had the highest level of metabolites that included N1-ethylnorspermine, N1-ethylnorspermidine, N1-ethyl-1,3-diaminopropane, and norspermidine. These data suggest that DENSPM is metabolized by N-deethylation and step-wise removal of aminopropyl equivalents by spermine/spermidine N1-acetyltransferase/polyamine oxidase, a metabolic pathway unique to the polyamines.     

Control of glucan-induced systemic granulomatosis by cyclosporine A

This study has shown that cyclosporine A (CyA), under certain conditions, is a powerful inhibitor of intravascular and extravascular monocyte/macrophage accumulation. Experiments were carried out in Lewis rats in which intravenous injection of particulate glucan calls forth a striking granulomatous response in lung, liver, and spleen and produces a marked stimulation of splenic erythro- and myelopoiesis. In agreement with the results of others, there was also a considerable elevation in monocyte/macrophage chemoattractant levels in the bronchoalveolar lavage fluid, which is held to be a key reaction in the pathogenesis of the histologic lesions. Treatment of the animals with subcutaneous injections of CyA prevented the rise in the chemoattractant activity and suppressed the granulomatous organ infiltration as well as the splenic hemopoiesis. The findings supply new insights into the activities of CyA and would support its clinical use in macrophage-dominated diseases.     

Retrephination keratoplasty for high astigmatism after penetrating keratoplasty

PURPOSE: We report preliminary results of a new procedure for correcting high astigmatism after penetrating keratoplasty. METHODS: The procedure entails full-thickness trephination along the original donor-recipient junction with careful suturing in a combined interrupted and running fashion. Four eyes of four patients with severe astigmatism and myopia after penetrating keratoplasty underwent the procedure. RESULTS: High preoperative cylinder ranging from 4.50 to 16.00 D (mean 9.00 D) was reduced to 0.50 to 3.50 D (mean 1.90 D) at the last examination (between 3 to 6 months). Spherical equivalent myopia ranging from -2.00 to -10.25 D (mean -4.90 D) was essentially unchanged at plano to -9.00 D (-4.70 D) at the last examination. Overall, there was a mean refractive cylinder reduction of 7.10 D (79%). CONCLUSION: Retrephination after penetrating keratoplasty appears to be an acceptable alternative for correcting high astigmatism, and had only a small effect on the level of myopia.     

Biosynthesis of chondroitin sulfate. Purification of glucuronosyl transferase II and use of photoaffinity labeling for characterization of the enzyme as an 80-kDa protein

A photoaffinity analogue, [beta-32P]5-azido-UDP-GlcA, was used to photolabel the enzymes that utilize UDP-GlcA in cartilage microsomes and rat liver microsomes. SDS-polyacrylamide gel electrophoresis analysis of photolabeled cartilage microsomes, which are specialized in chondroitin sulfate synthesis, showed a major radiolabeled band at 80 kDa and other minor radiolabeled bands near 40 and 60 kDa. Rat liver microsomes, which are enriched for enzymes of detoxification by glucuronidation, had a different pattern with multiple major labeled bands near 50-60 and 35 kDa. To determine that the photolabeled 80-kDa protein is the GlcA transferase II, we have purified the enzyme from cartilage microsomes. This membrane-bound enzyme, involved in the transfer of GlcA residues to non-reducing terminal GalNAc residues of the chondroitin polymer, has now been solubilized, stabilized, and then purified greater than 1350-fold by sequential chromatography on Q-Sepharose, heparin-Sepharose, and WGA-agarose. The purified enzyme exhibited a conspicuous silver-stained protein band on SDS-polyacrylamide gel electrophoresis that coincided with the major radiolabeled band of 80 kDa. SDS-polyacrylamide gel analysis of photoaffinity-labeled active fractions from the Q-Sepharose, heparin-Sepharose, and WGA-agarose also indicated only the single radiolabeled band at 80 kDa. Intensity of photolabeling in each of the fractions examined coincided with enzyme activity. The photolabeling of this 80-kDa protein was saturable with the photoprobe and could be inhibited by the addition of UDP-GlcA prior to the addition of the photoprobe. Thus, the photolabeling with [beta-32P]5-azido-UDP-GlcA has identified the GlcA transferase II as an 80-kDa protein. The purified enzyme was capable of transferring good amounts of GlcA residues to chondroitin-derived pentasaccharide with negligible transfer to pentasaccharides derived from hyaluronan or heparan.     

Mechanistic studies of the dual phosphorylation of mitogen-activated protein kinase

Previous work on the responses of mitogen-activated protein (MAP) kinase cascade components in a Xenopus oocyte extract system demonstrated that p42 MAP kinase (MAPK) exhibits a sharp, sigmoidal stimulus/response curve, rather than a more typical hyperbolic curve. One plausible explanation for this behavior requires the assumption that MAP kinase kinase (MAPKK) carries out its dual phosphorylation of p42 MAPK by a distributive mechanism, where MAPKK dissociates from MAPK between the first and second phosphorylations, rather than a processive mechanism, where MAPKK carries out both phosphorylations before dissociating. Here we have investigated the mechanism through which a constitutively active form of human MAPKK-1 (denoted MAPKK-1 R4F or MAPKK-1*) phosphorylates Xenopus p42 MAPK in vitro. We found that the amount of monophosphorylated MAPK formed during the phosphorylation reaction exceeded the amount of MAPKK-1* present, which would not be possible if the phosphorylation occurred exclusively by a processive mechanism. The monophosphorylated MAPK was phosphorylated predominantly on tyrosine, but a small proportion was phosphorylated on threonine, indicating that the first phosphorylation is usually, but not invariably, the tyrosine phosphorylation. We also found that the rate at which pulse-labeled monophosphorylated MAPK became bisphosphorylated depended on the MAPKK-1* concentration, behavior that is predicted by the distributive model but incompatible with the processive model. These findings indicate that MAPKK-1* phosphorylates p42 MAPK by a two-collision, distributive mechanism rather than a single-collision, processive mechanism, and provide a mechanistic basis for understanding how MAP kinase can convert graded inputs into switch-like outputs.     

Human MSH2 binds to trinucleotide repeat DNA structures associated with neurodegenerative diseases

The expansion of trinucleotide repeat sequences is associated with several neurodegenerative diseases. The mechanism of this expansion is unknown but may involve slipped-strand structures where adjacent rather than perfect complementary sequences of a trinucleotide repeat become paired. Here, we have studied the interaction of the human mismatch repair protein MSH2 with slipped-strand structures formed from a triplet repeat sequence in order to address the possible role of MSH2 in trinucleotide expansion. Genomic clones of the myotonic dystrophy locus containing disease-relevant lengths of (CTG)n x (CAG)n triplet repeats were examined. We have constructed two types of slipped-strand structures by annealing complementary strands of DNA containing: (i) equal numbers of trinucleotide repeats (homoduplex slipped structures or S-DNA) or (ii) different numbers of repeats (heteroduplex slipped intermediates or SI-DNA). SI-DNAs having an excess of either CTG or CAG repeats were structurally distinct and could be separated electrophoretically and studied individually. Using a band-shift assay, the MSH2 was shown to bind to both S-DNA and SI-DNA in a structure-specific manner. The affinity of MSH2 increased with the length of the repeat sequence. Furthermore, MSH2 bound preferentially to looped-out CAG repeat sequences, implicating a strand asymmetry in MSH2 recognition. Our results are consistent with the idea that MSH2 may participate in trinucleotide repeat expansion via its role in repair and/or recombination.     

Glutamine extraction by the gut is reduced in depleted [corrected] patients with gastrointestinal cancer

In order to investigate the expression of MUC5AC mucin in normal gastric mucosa and gastric carcinomas, we produced 3 monoclonal antibodies (MAbs) using a MUC5AC synthetic peptide. The immunohistochemical study was performed using one of these MAbs (CLH2) which reacted with the different designs of peptides based on the MUC5AC tandem repeat and with native and deglycosylated mucin extracted from gastric tissues. CLH2 immunoreactivity was restricted to foveolar and mucopeptic neck cells in normal gastric mucosa. No reactivity was observed in type-I intestinal metaplasia. Out of 66 gastric carcinomas, 42 (63.6%) expressed MUC5AC. Most diffuse carcinomas were positive (83.3%), whereas only 59.3% of intestinal and 40.0% of atypical carcinomas expressed MUC5AC (p < 0.05). Gastric carcinomas with mixed pattern showed immunoreactivity in diffuse areas and decreased immunoreactivity in intestinal areas. Every early gastric carcinoma expressed MUC5AC, in contrast to 58.6% of advanced carcinomas (p < 0.05). A trend toward decreased immunoreactivity was observed in deep areas of advanced carcinomas in comparison with the respective superficial areas. Taking together the specific staining of foveolar and mucopeptic neck cells and the absence of immunoreactivity in intestinal metaplasia, we conclude that MUC5AC expression may be used as a marker of gastric differentiation. This assumption is further supported by the finding of MUC5AC immunoreactivity in most diffuse carcinomas, which usually display morphologic and histochemical signs of gastric differentiation. The expression of MUC5AC in early gastric carcinomas, regardless of their histologic type, suggests that all gastric carcinomas retain at least some cells with a gastric phenotype during the first steps of neoplastic development.     

Magnetic resonance microscopy--a new tool for the toxicologic pathologist

Factor VIII and factor V share a repetitive domain structure of A1-A2-B-A3-C1-C2. To define the region(s) within the factor VIII heavy chain that result in inefficient expression of the recombinant protein, we expressed a series of factor VIII/factor V chimeras that contained heterologous sequences from the A1 and/or A2 domains. Substitution of the factor VIII A1 domain dramatically reduced secretion of factor V approximately 500-fold, whereas substitution of the factor VIII A2 domain had minimal effect on secretion. Conversely, substitution of the factor V A1 domain increased secretion of factor VIII approximately 3-fold, whereas substitution of the factor V A2 domain actually reduced secretion approximately 4-fold. Pulse chase experiments confirmed that reduced expression levels were due to decreased secretion rather than instability of secreted protein. Smaller substitutions did not further localize within the A1 domain the regions responsible for inefficient secretion.