Cerebrospinal Fluid Nitrite/Nitrate Levels in Neurologic Diseases

Abstract: Nitric oxide has been proposed to mediate cytotoxic effects in inflammatory diseases. To investigate the possibility that overproduction of nitric oxide might play a role in the neuropathology of inflammatory and noninflammatory neurological diseases, we compared levels of the markers of nitric oxide, nitrite plus nitrate, in the CSF of controls with those in patients with various neurologic diseases, including Huntington's and Alzheimer's disease, amyotrophic lateral sclerosis, and HIV infection. We found that there were no significant increases in the CSF levels of these nitric oxide metabolites, even in patients infected with HIV or in monkeys infected with poliovirus, both of which have significantly elevated levels of the neurotoxin quinolinic acid and the marker of macrophage activation, neopterin. However, CSF quinolinic acid, neopterin, and nitrite/nitrate levels were significantly increased in a small group of patients with bacterial and viral meningitis.     

Two tumor models of curative adoptive chemoimmunotherapy using tumor-infiltrated spleen cells with potent antitumor cytotoxicity stimulated by antigen-sharing tumors

Previously we have established curative protocols for adoptive chemoimmunotherapy (ACIT) of mice bearing different plasmacytomas that are known to bear cross-reacting antigens: (a) the cure of mice bearing an early-stage, nonpalpable MOPC-315 tumor by a very low dose of cyclophosphamide (10 mg/kg) and cultured MOPC-315-tumor-infiltrated (TI) spleen cells (25×10⁶) and (b) the cure of mice bearing a late-stage, relatively drug-resistant, highly metastatic RPC-5 tumor with cyclophosphamide (100 mg/kg) and cultured RPC-5 TI spleen cells (25×10⁶–50×10⁶). In both models, the spleen cells were obtained from mice bearing a late-stage tumor and were cultured for 5 days in the presence of polyethyleneglycol 6000 and autochthonous tumor cells as a source of tumor antigen. Here we show that RPC-5 tumor cells could substitute for MOPC-315 tumor cells in the 5-day culture of MOPC-315 TI spleen cells so that they became curative in ACIT for mice bearing an early-stage MOPC-315 tumor. Similarly, MOPC-315 tumor cells could substitute for RPC-5 tumor cells in the 5-day culture of RPC-5 TI spleen cells so that they became curative in ACIT of mice bearing a late-stage RPC-5 tumor. In addition, RPC-5 TI spleen cells cultured with either MOPC-315 or RPC-5 tumor cells were effective in curing all mice bearing an early-stage MOPC-315 tumor by ACIT. However, MOPC-315 TI spleen cells whether cultured with MOPC-315 or RPC-5 tumor cells, were much less effective than cultured RPC-5 TI spleen cells in curing mice bearing a late-stage RPC-5 tumor by ACIT (although the survival of these mice was extended significantly). Interestingly, whereas RPC-5 TI spleen cells cultured with either MOPC-315 or RPC-5 tumor cells were as effective as MOPC-315 TI spleen cells cultured under the same conditions in lysing MOPC-315 tumor cells in vitro, MOPC-315 TI spleen cells that had been cultured with either MOPC-315 or RPC-5 tumor cells exerted a much weaker in vitro cytotoxic T lymphocyte activity against RPC-5 tumor cells than did RPC-5 TI spleen cells that had been cultured under the same conditions.Work was supported by research grant CA-30088 from the National Cancer Institute and IM-435 from the American Cancer Society. M. B. M. was supported by Career Development Award CA-01350 from the National Cancer InstituteThis work is in partial fulfillment of the requirements for the Ph. D. degree     

Studies on Cartilage: Electron Microscope Observations on Normal Rabbit Ear Cartilage

Normal rabbit ear cartilage studied with the light and electron microscope shows chondrocytes in which large lipide spherules, and an abundance of glycogen, a few small mitochondria, and relatively few elements of the endoplasmic reticulum can be identified. The chondrocytes contain, in addition, a material which stains strongly with acid fuchsin and appears in the electron microscope as a relatively dense felt-work. In electron micrographs, the matrix of normal rabbit ear cartilage consists of two components: a uniformly distributed moderately dense substance which appears as a fine meshwork without any particular pattern extending from cartilage cell border to cartilage cell border; and a three-dimensional anastomotic network of more dense material, which is best described as "felt-like" lying between the cells. The similarity between the felt-like material of the matrix and the elastic fibers described in previous electron microscope observations is discussed.     

EVIDENCE FOR POST-TRANSLATIONAL MODIFICATION OF TRIOSE PHOSPHATE ISOMERASE (TPI) IN ISOËTES (ISOËTACEAE)

As part of an electrophoretic study on Isoëtes, a number of Neotropical and North American species were examined for allozyme variation in TPI. Three of these species—I. storkii, I. flaccida, and I. mexicana—exhibit three distinct zones of TPI activity. The two most anodally migrating zones are comparable to the two zones found in most angiosperms and in several other species of Isoëtes. The single or three-banded phenotypes produced at these loci correspond, respectively, to the homozygous and heterozygous patterns typical of a dimeric enzyme. The most cathodal zone (zone III) differs in producing either single or two-banded phenotypes. Analyses of these three zones indicate a nearly perfect correlation between zones II and III in putative allelic constitution and relative allelic mobility. Explanations involving TPI gene duplications and/or null alleles fail to account for the peculiar banding characteristics and origin of activity zone III. An alternative hypothesis involving a protease duplication and differential post-translational modification is postulated. This hypothesis adequately explains the zone III phenotypes and fixation of the third activity zone in the species examined. Amino acid sequencing is suggested as the most direct test of this hypothesis. The taxonomic distribution of TPI III generally supports a previous, morphologically-based, hypothesis on species relationships in Isoëtes. The presence of this zone is regarded as an independent synapomorphy for a major clade of Neotropical Isoëtes.     

Site-directed mutagenesis of the katG gene of Mycobacterium tuberculosis: effects on catalase–peroxidase activities and isoniazid resistance

Recent studies examining the molecular mechanisms of isoniazid (INH) resistance in Mycobacterium tuberculosis have demonstrated that a significant percentage of drug-resistant strains are mutated in the katG gene which encodes a catalase–peroxidase, and the majority of these alterations are missense mutations which result in the substitution of a single amino acid. In previous reports, residues which may be critical for enzymatic activity and the drug-resistant phenotype have been identified by evaluating INH-resistant clinical isolates and in vitro mutants. In this study, site-directed mutagenesis techniques were utilized to alter the wild-type katG gene from M. tuberculosis at 13 of these codons. The effects of these mutations were determined using complementation assays in katG -defective, INH-resistant strains of Mycobacterium smegmatis and Mycobacterium bovis BCG. This mutational analysis revealed that point mutations in the katG gene at nine of the 13 codons can cause drug resistance, and that enzymatic activity and resistance to INH are inversely related. In addition, mutations in the mycobacterial catalase–peroxidase which reduce catalase activity also decrease peroxidase activity.