Structural characterization of the cyanelle peptidoglycan of Cyanophora paradoxa by 252Cf plasma desorption mass spectrometry and fast atom bombardment/tandem mass spectrometry

A strategy for the structural characterization of the four major NaBH4-reduced peptidoglycan monomers derived from muramidase-digested peptidoglycan from the cyanelles of the flagellate Cyanophora paradoxa Korschikoff is described. Initial molecular weight determination of these glycopeptides was performed by positive and negative ion plasma desorption mass spectrometry. Due to the presence of two pairs of disaccharide tripeptide and disaccharide tetrapeptide monomers differing in mass by 112 units, respectively, an as yet unknown peptidoglycan modification either at the carbohydrate or at the peptide moiety was assumed. beta-Elimination of the disaccharide unit from the unreduced peptidoglycan monomers yielded the corresponding (modified) N1-lactyltripeptides and -tetrapeptides, respectively. These peptides, N-terminally blocked with lactic acid, unambiguously showed the modification to be located on the peptide moiety. By positive ion fast atom bombardment/hybrid tandem mass spectrometry of the reduced peptidoglycan monomers as well as of the corresponding deglycosylated monomers (= N1-lactylpeptides) the modification was determined to be linked to the glutamic acid moiety. Based on combined data from plasma desorption mass spectrometry, tandem mass spectrometry, accurate mass measurement and amino acid analysis of the acid hydrolysate after derivatization with o-phthaldialdehyde by high-performance liquid chromatography we could establish the structure of the modification as N-acetylputrescine. Finally, the confirmation of the linkage of the glutamic acid to diaminopimelic acid via the gamma-COOH was based on the presence of a-type peptide backbone fragment ions in the positive ion plasma desorption mass spectra of the modified N1-lactylpeptides.     

Interleukin-6 cDNA transfected Lewis lung carcinoma cells show unaltered net tumour growth rate but cause weight loss and shortened survival in syngeneic mice

HuIL-6 cDNA, cloned into a neomycin resistant conferring expression vector, BMGNeo, was transfected into Lewis Lung Carcinoma (LLC) cells. LLC cells (5 x 10(6) ml-1) transfected with IL-6 cDNA (LLC-IL6) secreted IL-6 into the culture supernatant at a concentration of 9.9 ng ml-1 within 48 h. When 1,000,000 of untransfected LLC, BMGNeo vector transfected LLC (LLC-Neo) or LLC-IL6 cells were transplanted into C57BL/6 mice subcutaneously, the mean +/- s.d. of survival times of these mice were 33.3 +/- 9.7, 34.3 +/- 7.1 and 17.0 +/- 3.1 days, respectively. The survival time of LLC-IL6 cells transplanted mice was significantly shorter than that of LLC (P < 0.01) or LLC-Neo (P < 0.01) cells transplanted mice without a measurable difference of tumour size. Plasma concentration of IL-6 steadily increased in LLC-IL6 transplanted mice. Body weight and serum albumin were significantly lower in LLC-IL6 transplanted mice than in LLC transplanted mice. Mouse IL-1 alpha and mouse TNF-alpha were not detected in the plasma of LLC-IL6 transplanted mice. These data suggested that secretion of IL-6 from LLC cells was unable to alter net tumour growth rate but rather caused a state similar to cachexia without detectable increase of IL-1 alpha and TNF-alpha in the plasma. This state may be responsible for the shortened survival of LLC-IL6 tumour-bearing mice.     

Elucidation of mitochondrial effects by tetrahydroaminoacridine (tacrine) in rat, dog, monkey and human hepatic parenchymal cells

Tetrahydroaminoacridine (tacrine) causes morphological and functional changes in the endoplasmic reticulum, ribosomes, and mitochondria in the liver of humans and animals. In order to investigate species differences as well as to understand the morphological changes, we examined the effects of tacrine on respiration and electron transport in mitochondria isolated from rat, dog, monkey, and human liver. Tacrine produced significantly decreased respiratory control ratios (RCR) in all species at concentrations ranging from 5 to 25 microg/ml. Human mitochondria were more sensitive to tacrine effects with RCR decreased 24% at 5 microg/ml while other species were unaffected at this concentration. The tacrine effects were characterized by increased hepatic mitochondrial State 4 respiration in rats and decreased State 3 respiration in humans. Mitochondria from aged rats were more sensitive to the effects of tacrine than mitochondria from young animals, with significantly decreased RCR at 10 microg/ml in aged rats while mitochondria from young rats were unaffected at this concentration. Concomitant with the respiratory changes, mitochondrial DNA synthesis was impaired. Since tacrine undergoes extensive biotransformation, we also explored the possibility that metabolites could exert detrimental effects. The ranking order of potency for decreasing RCR caused by monohydroxylated metabolites was: tacrine > 4-OH and 7-OH > 2-OH, 1-OH, and velnacrine with the latter group of metabolites having no effect on mitochondrial respiration at concentrations up to 50 microg/ml. In vivo administration of 20 mg/kg tacrine to rats for up to 20 days caused a paradoxical increase in RCR and P/O on Day 1 and decreased RCR on Days 9 and 20, the later findings being consistent with in vitro data. From these data we propose that tacrine does not necessarily have to be metabolized to exert effects on mitochondria at different sites in the electron transport chain that differ among species. These effects are exacerbated in mitochondria from older animals and humans appear to be more sensitive than the laboratory animals studied.     

Cancer prevention--behavior changes: the short and the long of it

BACKGROUND: Behavioral factors play an important role in the adoption and maintenance of nutritional modifications, delivered either through chemopreventive regimens or through dietary adaptation. A body of research supports a protective role of fruits and vegetables, dietary fiber, and eating a low-fat diet in reducing cancer risk. There is only limited knowledge, however, about the cancer-preventing properties of specific micronutrients, apart from beta-carotene; about optimal levels of intake of differing micronutrients; and about patterns of food intake associated with reduced cancer risk. Thus, it would seem preferable, overall, to be able to recommend cancer prevention through dietary modification rather than through the administration of individual agents. METHODS: Studies of dietary adherence in cancer prevention have yielded varying success rates, but are generally quite promising. We have developed a model that encompasses the range of behavioral, psychological, social, and systemic variables thought to influence adherence to nutritional regimens. RESULTS: The model is being utilized to influence the form and content of nutritional regimens and to promote short-term change which can then be sustained as long-term lifestyle modification. The model can also be applied to evaluate adherence to nutritional or other behavioral modifications and to determine the factors predictive of success.     

Clinical and historical predictors of dental caries on radiographs

Jet injection can be used to introduce genes into the cells of differentiated tissues of living animals and organ cultures. When a solution of plasmid DNA is jet injected into a selected tissue or organ, cells lying in or near the path of the jet injection are transfected with the DNA and the introduced gene(s) are expressed. Since there is minimal morbidity from each jet injection, multiple injections can be performed at the same or nearby sites. Both mRNA and protein expression from transfected genes can be quantitated using standard methods. In addition, the technique is an efficient means of DNA immunization. Methodology for using jet injection to transfer plasmid DNA into the cells of skin, fat, mammary gland, and muscle are described.     

Spatial learning deficits in mice with a targeted glucocorticoid receptor gene disruption

Previous studies in rats using the Morris water maze suggested that the processing of spatial information is modulated by corticosteroid hormones through mineralocorticoid and glucocorticoid receptors in the hippocampus. Mineralocorticoid receptors appear to be involved in the modulation of explorative behaviour, while additional activation of glucocorticoid receptors facilitates the storage of information. In the present study we used the water maze task to examine spatial learning and memory in mice homozygous and heterozygous for a targeted disruption of the glucocorticoid receptor gene. Compared with wild-type controls, homozygous and heterozygous mice were impaired in the processing of spatial but not visual information. Homozygous mutants performed variably during training, without specific platform-directed search strategies. The spatial learning disability was partly compensated for by increased motor activity. The deficits were indicative of a dysfunction of glucocorticoid receptors as well as of mineralocorticoid receptors. Although the heterozygous mice performed similarly to wild-type mice with respect to latency to find the platform, their strategy was more similar to that of the homozygous mice. Glucocorticoid receptor-related long-term spatial memory was impaired. The increased behavioural reactivity of the heterozygous mice in the open field points to a more prominent mineralocorticoid receptor-mediated function. The findings indicate that (i) the glucocorticoid receptor is of critical importance for the control of spatial behavioural functions, and (ii) mineralocorticoid receptor-mediated effects on this behaviour require interaction with functional glucocorticoid receptors. Until the development of site-specific, inducible glucocorticoid receptor mutants, glucocorticoid receptor-knockout mice present the only animal model for the study of corticosteroid-mediated effects in the complete absence of a functional receptor.