Inhibition of ornithine decarboxylase potentiates nitric oxide production in LPS-activated J774 cells

We have examined whether modulation of the polyamine biosynthetic pathway, through inhibition by alpha-difluoromethylornithine (DFMO) of the rate limiting enzyme, ornithine decarboxylase (ODC), modulates NO synthesis in J774 macrophages. DFMO potentiated LPS-stimulated nitrite production in both a concentration- and time-dependent manner, increasing nitrite levels by 48+/-5% at 10 mM. This effect was observed in cells pre-treated with DFMO for 24 h prior to stimulation with LPS. Addition of DFMO 12 h after LPS failed to potentiate LPS-induced nitrite production. Supplementation of the culture medium with horse serum (10%) in place of foetal calf serum (10%) caused no significant change in either LPS-induced nitrite production or in the ability of DFMO (10 mM) to potentiate LPS-induced NO synthesis. Metabolism of L-[3H]arginine to L-[3H]citrulline by partially purified inducible nitric oxide synthase (iNOS) was not significantly altered by either DFMO (1-10 mM) or by putrescine (0.001-1 mM), spermidine (0.001-1 mM) or spermine (0.001-1 mM). iNOS activity was also unaffected by 1 mM EGTA but was markedly attenuated (70+/-0.07%) by L-NMMA (100 microM). Pre-incubation of cells with DFMO (10 mM; 24 h) prior to activation with LPS resulted in enhanced (approximately 2 fold) iNOS protein expression. These results show that DFMO potentiates LPS-induced nitrite production in the murine macrophage cell line J774. Since the only known mechanism of action of DFMO is inhibition of ODC, and thus polyamine biosynthesis, we conclude that expression of iNOS can be critically regulated by endogenous polyamines.     

Growth response of human coronary smooth muscle cells to angiotensin II and influence of angiotensin AT1 receptor blockade

BACKGROUND: Adolescents and young adults are four times more likely to be victims of sexual assault than women in all other age groups. In the vast majority of these cases, the perpetrator is an acquaintance of the victim. Date rape is a subset of acquaintance rape where nonconsensual sex occurs between two people who are in a romantic relationship. METHODS: We conducted a MEDLINE and Current Concepts search for articles relating to date rape and then systematically reviewed all relevant articles. RESULTS: Lifetime prevalence of date or acquaintance rape ranges from 13% to 27% among college-age women and 20% to a high of 68% among adolescents. Demographic characteristics that increase vulnerability to date rape include younger age at first date, early sexual activity, earlier age of menarche, a past history of sexual abuse or prior sexual victimization, and being more accepting of rape myths and violence toward women. Other risk factors include date-specific behaviors such as who initiated, who paid expenses, who drove, date location and activity, as well as the use of alcohol or illicit drugs such as flunitrazepam (Rohypnol). Alcohol use that occurs within the context of the date can lead to: the misinterpretation of friendly cues as sexual invitations, diminished coping responses, and the female's inability to ward off a potential attack. CONCLUSIONS: Longitudinal research designs are needed to further our understanding of sexual violence among adolescents and young adults and the most effective ways to eliminate it. Understanding and comparing research findings would be easier if consensus regarding the definitions of date rape, sexual aggression, and sexual assault was obtained. Finally, primary and secondary date and acquaintance rape prevention programs must be developed and systematically evaluated.     

Enhanced B-1 cell development, but impaired IgG antibody responses in mice deficient in secreted IgM

The role of endogenous natural IgM in promoting the adaptive Ab response was investigated in newly constructed mutant mice in which B cells do not secrete IgM but still express surface IgM and IgD and undergo class switching to express other Ig isotypes. While the mutant mice had relatively normal numbers of conventional B (B-2) cells in all tissues examined, unexpectedly, B-1 cells in the peritoneum and spleen were approximately threefold more abundant. The elevated levels of B-1 cells were already detectable at 4 wk of age and were stably maintained throughout life. The levels of serum IgG2a, IgG3, and IgA were also elevated in the mutant mice at an early age. IgG2a response to a T cell-independent Ag was augmented, whereas IgG Ab responses to suboptimal doses of a T cell-dependent Ag were impaired. The latter defect was associated with fewer splenic germinal centers, impaired Ab affinity maturation, and less Ag trapping on follicular dendritic cells. Together, these findings demonstrate a physiologic role of natural IgM in the feedback regulation of B-1 cell development, the regulation of IgG2a production, and the promotion of efficient B-2 cell Ab responses.     

A C-terminal fragment of Agouti-related protein increases feeding and antagonizes the effect of alpha-melanocyte stimulating hormone in vivo

Agouti-related protein (Agrp) is present in rat and human hypothalamus and is structurally related to agouti protein. Overexpression of either of these proteins results in obesity. However the effect of exogenous Agrp and its in vivo interaction with alpha-melanocyte stimulating hormone (alphaMSH), the likely endogenous melanocortin 3 and 4 receptor (MC3-R and MC4-R) agonist, have not been demonstrated. We report that 1 nmol of Agrp(83-132), a C-terminal fragment of Agrp, when administered intracerebroventricularly (ICV) into rats, increased food intake over a 24-h period (23.0+/-1.4 g saline vs 32.9+/-2.3 g Agrp, p<0.05). The hyperphagia was similar to that seen when 1 nmol of the synthetic MC3-R and MC4-R antagonist SHU9119 was given i.c.v. (19.6+/-1.8 g saline vs 32.5+/-1.7 g SHU9119, p<0.001). Both Agrp(83-132) and SHU9119 blocked the reduction in 1-h food intake of i.c.v. alphaMSH at the beginning of the dark phase. This effect occurred independently of whether the antagonists were administered simultaneously, or nine hours prior, to the alphaMSH. We have also shown Agrp(83-132) is an antagonist at the MC3-R and MC4-R, with similar inhibition of cAMP activation to that previously reported for the full length peptide. In conclusion, Agrp(83-132) administered i.c.v. increases feeding with long lasting effects and is able to inhibit the action of alphaMSH. This interaction may be mediated by the MC3-R and/or MC4-R.     

A RAPD-based comparison of thermophilic bacilli from milk powders

The similarity of strains of thermophilic Geobacillus stearothermophilus (formerly Bacillus stearothermophilus), Anoxybacillus flavithermus (formerly Bacillus flavothermus), Bacillus licheniformis and Bacillus subtilis isolated from separate milk powder production runs from multiple factories was examined using a random amplified polymorphic DNA (RAPD) protocol. As a result of the analysis of the RAPD fingerprints and data relating to general growth and biochemical tests, over 98% of the 1470 isolates examined (grown at 55 degrees C) were assigned to the species G. stearothermophilus, A. flavithermus, B. licheniformis and B. subtilis. The G. stearothermophilus isolates were identified as being nearly identical to G. stearothermophilus (DSMZ 22; equivalent to ATCC 12980), or G. stearothermophilus var. calidolactis (DSMZ 1550). Three groups of isolates were found to be related to A. flavithermus (DSMZ 2641) by partial small ribosomal subunit (16S) sequence comparisons and shown to be interrelated by RAPD analyses with multiple primer sets. The thermophilic isolates of B. licheniformis were positively identified by comparison with type strains of B. licheniformis DSMZ 13 and DSMZ 8785. All of the B. subtilis strains shared bands in their RAPD profiles and were similar to a common B. subtilis type strain (DSMZ 10 and DSMZ 347). Overall, the most common and prevalent group of strains (group A) was demonstrated to be closely related to G. stearothermophilus (DSMZ 22).     

Particle growth by acid-catalyzed heterogeneous reactions of organic carbonyls on preexisting aerosols

Aerosol growth by the heterogeneous reactions of different aliphatic and alpha,beta-unsaturated carbonyls in the presence/absence of acidified seed aerosols was studied in a 2 m long flow reactor (2.5 cm i.d.) and a 0.5-m3 Teflon film bag under darkness. For the flow reactor experiments, 2,4-hexadienal, 5-methyl-3-hexen-2-one, 2-cyclohexenone, 3-methyl-2-cyclopentenone, 3-methyl-2-cyclohexenone, and octanal were studied. The carbonyls were selected based on their reactivity for acid-catalyzed reactions, their proton affinity, and their similarity to the ring-opening products from the atmospheric oxidation of aromatics. To facilitate acid-catalyzed heterogeneous hemiacetal/acetal formation, glycerol was injected along with inorganic seed aerosols into the flow reactor system. Carbonyl heterogeneous reactions were accelerated in the presence of acid catalysts (H2SO4), leading to higher aerosol yields than in their absence. Aldehydes were more reactive than ketones for acid-catalyzed reactions. The conjugated functionality also resulted in higher organic aerosol yieldsthan saturated aliphatic carbonyls because conjugation with the olefinic bond increases the basicity of the carbonyl leading to increased stability of the protonated carbonyl. Aerosol population was measured from a series of sampling ports along the length of the flow reactor using a scanning mobility particle sizer. Fourier transform infrared spectrometry of either an impacted liquid aerosol layer or direct reaction of carbonyls as a thin liquid layer on a zinc selenide FTIR disk was employed to demonstrate the direct transformation of chemical functional groups via the acid-catalyzed reactions. These results strongly indicate that atmospheric multifunctional organic carbonyls, which are created by atmospheric photooxidation reactions, can contribute significantly to secondary organic aerosol formation through acid-catalyzed heterogeneous reactions. Exploratory studies in 25- and 190-m3 outdoor chambers were also implemented to demonstrate the formation of high molecular weight organic structures. The reaction of ozone with alpha-pinene to generate secondary organic aerosols (SOAs) was performed in the presence of background aerosol consisting of a mixture of wood soot and diesel soot. Results strongly suggest that indigenous sulfuric acid associated with the combustion of fossil fuels (e.g., diesel soot) can initiate acid-catalyzed heterogeneous reactions of SOAs on the particle phase.     

Amplitude and phase microscopy for sizing of spherical particles

We describe a numerical vector diffraction model based on Mie theory that describes the imaging of spherical particles by bright-field, confocal, and interferometric microscopes. The model correctly scales the amplitude-scattered field relative to the incident field so that the forward-scattered and incident light can be interfered to correctly model imaging with copolarization transmission microscopes for the first time to our knowledge. The model is used to demonstrate that amplitude and phase imaging with an interferometric microscope allows subwavelength particle sizing. Furthermore, we show that the phase channel allows much smaller particles to be sized than amplitude-only measurements. The model is validated by experimental measurements.     

Hierarchical responses of soil invertebrates (earthworms) to toxic metal stress

The concept of a hierarchical cascade of biological responses to stress occurring across different levels of biological organization is an underlying principle of both theoretical and regulatory ecology/ecotoxicology. This study investigates the reality of this cascade for earthworms exposed to toxic metal stress. Gene expression was the most sensitive endpoint (EC50 = 616 microg Zn g(-1)) followed by the integrity of coelomocyte lysosomal membrane (EC50 = 645 microg Zn g(-1)). This confirms that, in accordance with the cascade concept, suborganism level endpoints respond at lower metal concentrations than higher organization endpoints. The relative sensitivity of the higher organization parameters was not as predicted by the cascade. Organic material removal was more sensitive (EC50 = 997 microg Zn g(-1)) than L. rubellus reproduction (EC50 = 3236 microg Zn g(-1)), L. rubellus population size (EC50 = 5000-11500 microg Zn g(-1)), and earthworm community diversity (EC50 = 1737 microg Zn g(-1)). This can be attributed to (1) the relative insensitivity of L. rubellus to metals and (2) general toxic effects of metals on the earthworm energy budget (and thus feeding). On the basis of these results, it can be concluded that predictive assessments of the consequences of environmental stressors needs to include approaches that respect the relative sensitivities of different taxa, while retrospective appraisals should exploit the sensitivity of low organization level responses.