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Mesenteric lymph contains unidentified proinflammatory mediators that increase in concentration after hemorrhage. In the search for candidate mediators, we examined mesenteric lymph for the presence of proinflammatory substances that are known to be produced in the gut: (a) antimicrobial peptides and antimicrobial proteins produced in the Paneth cells of the intestine (alpha-defensin 4, secretory phospholipase A2 [sPLA2], and Reg 2 protein) and (b) asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NOS. Anesthetized male rats were hemorrhaged to 40 mmHg and maintained at that pressure by intermittent blood withdrawal until the pressure fell to less than 40 mmHg (decompensation) at which point they were resuscitated with three times the shed blood volume of Ringer's lactate solution administered over 1 h. Mesenteric lymph samples were analyzed for ADMA by enzyme-linked immunosorbent assay and for alpha-defensin 4, sPLA2, and Reg2 by Western blotting. Protein concentration in lymph was unchanged by hemorrhage, but alpha-defensin 4 increased significantly (12-fold greater than control) as did ADMA (2-fold greater than control). The sPLA2 could not be detected in lymph, and Reg 2 was unchanged during hemorrhage. During resuscitation, lymph flow tended to increase, but the concentration of ADMA and alpha-defensin 4 by volume did not increase. Reg 2 decreased during resuscitation. The results indicate that ADMA and immunoreactive product to alpha-defensin 4 may contribute to the increase in inflammatory activity of mesenteric lymph during hemorrhage, but they are unlikely to be the mediators responsible for the increase in the concentration of inflammatory mediators in postresuscitation lymph.

Hypoxia is an often seen problem resulting from conditions such as ischemia, hemorrhage, stroke, premature birth, and other cardiovascular difficulties. To find useful remedies that are capable of ameliorating its casualty is an essential effort. Although the underlying mechanisms of the hypoxia-induce injury and cell death are still not fully understood, it has been shown that hypoxia induces nitric oxide (NO) overproduction and inducible nitric oxide synthase (iNOS) overexpression that play important roles in producing injury including increases in polymorphonuclear neutrophils (PMN) infiltration to injured tissues and leukotriene B4 (LTB4) generation. Moreover, it has been evident that transcription factors responsible for iNOS expression are also altered by hypoxia. Hypoxia also increases intracellular Ca2+ concentration, tumor necrosis factor-alpha, lipid peroxidation, prostaglandin E2 production, activity of caspase-3 and -9, and release of cytochrome c from mitochondria, apoptosis inducible factor, and endonuclease G. However, it has been shown that downregulation of iNOS can limit cell injury caused by hypoxia. In our laboratory, we have found that treatment with either iNOS inhibitors or iNOS siRNA inhibits iNOS expression, reduces lipid peroxidation, apoptosome formation, and cellular caspase-3 activity, preserves cellular ATP levels, and increases cell survival. Therefore, iNOS inhibition may be a novel mechanism for protection from hypoxia-induced injury and cell death.

Background: Recently, a technique which departs radically from conventional approaches has been proposed. This novel technique utilizes biological objects such as viruses as nano-templates for the fabrication of nanostructure elements. For example, rod-shaped viruses such as the M13 phage and tobacco mosaic virus have been successfully used as biological templates for the synthesis of semiconductor and metallic nanowires., Results and Discussion: Low wave number (

Hemorrhage in mice results in decreased ATP levels in the jejunum, lung, kidney, heart, and brain but not in liver tissue lysates, albeit at variable levels and time kinetics. The decreased protein expression and activity of pyruvate dehydrogenase (PDH) accounted for the hemorrhage-induced ATP loss. Treatment with geldanamycin (GA; 1 microg/g body wt), a known inducer of heat shock protein (HSP)70, inhibited the hemorrhage-induced ATP loss in the jejunum, lung, heart, kidney, and brain. GA was found to increase PDH protein, preserve PDH enzymatic activity, and inhibit mucosal injury in jejunum tissues. GA-induced HSP70i was found to form complexes with PDH protein. HSP70 gene transfer into intestinal epithelial cells promoted PDH and ATP levels, whereas HSP70 short interfering RNA limited them. We conclude that agents able to increase the expression of HSP70 and PDH may be of value in reducing pathology resulting from hemorrhage-associated ATP loss.

External bioenergy (EBE, energy emitted from a human body) has been shown to increase intracellular calcium concentration ([Ca2+]i, an important factor in signal transduction) and regulate the cellular response to heat stress in cultured human lymphoid Jurkat T cells. In this study, we wanted to elucidate the underlying mechanisms. A bioenergy specialist emitted bioenergy sequentially toward tubes of cultured Jurkat T cells for one 15-minute period in buffers containing different ion compositions or different concentrations of inhibitors. [Ca2+], was measured spectrofluorometrically using the fluorescent probe fura-2. The resting [Ca2+]i in Jurkat T cells was 70 +/- 3 nM (n = 130) in the normal buffer. Removal of external calcium decreased the resting [Ca2+]i to 52 +/- 2 nM (n = 23), indicating that Ca2+ entry from the external source is important for maintaining the basal level of [Ca2+]i. Treatment of Jurkat T cells with EBE for 15 min increased [Ca2+]i by 30 +/- 5% (P < 0.05, Student t-test). The distance between the bioenergy specialist and Jurkat T cells and repetitive treatments of EBE did not attenuate [Ca2+]i responsiveness to EBE. Removal of external Ca2+ or Na+, but not Mg2+, inhibited the EBE-induced increase in [Ca2+]i. Dichlorobenzamil, an inhibitor of Na+/Ca2+ exchangers, also inhibited the EBE-induced increase in [Ca2+]i in a concentration-dependent manner with an IC50 of 0.11 +/- 0.02 nM. When external [K+] was increased from 4.5 mM to 25 mM, EBE decreased [Ca2+]i. The EBE-induced increase was also blocked by verapamil, an L-type voltage-gated Ca2+ channel blocker. These results suggest that the EBE-induced [Ca2+]i increase may serve as an objective means for assessing and validating bioenergy effects and those specialists claiming bioenergy capability. The increase in [Ca2+]i is mediated by activation of Na+/Ca2+ exchangers and opening of L-type voltage-gated Ca2+ channels.

Sodium cyanide (NaCN)-induced chemical hypoxia is known to increase intracellular free calcium concentration and reduce cell survival, but its effect on gene expression has not been studied. In this study, we designed primers to conduct a rapid and reliable assay for the expression of mRNA of inducible nitric oxide synthase (iNOs), tumor suppressor protein p53, Bcl-2, heat shock protein 70 (HSP-70), and beta-actin in human intestinal epithelial T84 cells and Jurkat T cells. NaCN-induced chemical hypoxia increased iNOs and HSP-70 mRNA in both types of cells, whereas p53 and Bcl-2 mRNA were singularly induced in T84 cells and Jurkat T cells, respectively. In both cell types, treatment of hypoxic cells with a reversible selective iNOs inhibitor, Now-nitro-L-arginine (LNNA), blocked iNOs, Bcl-2, and HSP-70 mRNA, but increased p53. The NaCN-induced hypoxia was also found to increase caspase-3 cellular activity in both cell types. Treatment with LNNA alone decreased the basal caspase-3 cellular activity. A prior treatment of LNNA significantly inhibited the NaCN-induced increase in the cellular activity of this apoptotic enzyme. This is the first report to show that NaCN-induced chemical hypoxia alters both stress-related gene expression and caspase-3 cellular activity and can be regulated by the iNOs inhibitor LNNA. Since NaCN has been included in the 'National chemical terrorism threat' list, by the US Department of Defense, our studies provide useful insight in the development of molecular sensors to detect early exposure to this chemical terrorism threat.

It has been shown that expression of heat shock proteins (HSPs) can interfere with the effectiveness of therapeutic cytotoxic drugs. In this study, we investigated the regulation of expression of HSPs in human epidermoid A-431 cells. Two known protein tyrosine kinase inhibitors were studied. Treatment of cells with herbimycin A increased production of inducible HSP 70 kDa (HSP-70i) in a concentration-dependent manner, whereas genistein did not. The increase induced by herbimycin A was observed within 2 h, reached a peak at 6 h, and remained above the basal level 3 days later. Pretreatment with genistein inhibited the herbimycin A-induced increase in HSP-70i. Herbimycin A treatment increased levels of HSP-70i mRNA in cells, suggesting that herbimycin A increases HSP-70i by promoting transcription. Treatment with genistein or genistein combined with herbimycin A did not increase HSP-70i mRNA, suggesting that the inhibitory effect of genistein also occurs at the level of mRNA production. Herbimycin A increased intracellular Ca2+ concentration ([Ca2+]i), but treatment with genistein decreased it. Chelation of [Ca2+]i with BAPTA blocked the herbimycin A-induced increase in HSP-70i mRNA and HSP-70i protein. Herbimycin A induced the phosphorylation of heat shock factor 1 (HSF1), while genistein reduced HSF1 production. The ability of genistein to inhibit the herbimycin A-induced increase in HSP-70i is not associated with genistein's capacity to decrease basal [Ca2+]i, but because it decreases HSFI production. The herbimycin A-induced increase in HSP-70i protected cells from hypoxia injury.

Background: External bioenergy (energy emitted from the body) can influence a variety of biological activities. It has been shown to enhance immunity, promote normal cell proliferation, increase tumor cell death, and accelerate bone fracture recovery. In this study, we investigated whether external bioenergy could alter intracellular calcium concentration ([Ca2+]i, an important factor in signal transduction) and regulate the cellular response to heat stress in cultured human lymphoid Jurkat T cells., Methods: A practitioner emitted bioenergy toward tubes of cultured Jurkat cells for one 15-minute period. [Ca2+]i was measured spectrofluorometrically using the fluorescent probe indo-1. The heat shock protein 72 kd was detected using 35S-methionine prepulse and Western blot analysis., Results: The resting [Ca2+]i in Jurkat T cells was 90+/-3 nM in the presence of external calcium. The removal of external calcium decreased the resting [Ca2+]i to 54+/-2 nM, indicating that Ca2+ entry from the external source is important for maintaining the basal level of [Ca2+]i. In the presence of external Ca2+, treatment of Jurkat T cells with external bioenergy for 15 minutes increased [Ca2+]i by 22+/-3%. [Ca2+]i remained elevated in these cells for 2 hours. Surprisingly, we also observed that [Ca2+]i increased by 11+/-1% if cells were simply placed in the area where external bioenergy had been used. This lingering effect of external bioenergy dissipated within 24 hours. Treatment with external bioenergy reduced cellular responses to heat stress and did not induce the production of heat shock protein 72 kd, which is known to provide cytoprotection., Conclusions: These results suggest that externally applied bioenergy can upregulate [Ca2+]i and downregulate the cellular response to stress. The association between the external bioenergy and increases in [Ca2+]i may be a good index for detecting presence of bioenergy.