O2 sensitivity and H2 production activity of hydrogenases—A review

Hydrogenases are metalloproteins capable of catalyzing the interconversion between molecular hydrogen and protons and electrons. The iron–sulfur clusters within the enzyme enable rapid relay of electrons which are either consumed or generated at the active site. Their unparalleled catalytic efficiency has attracted attention, especially for potential use in H₂ production and/or fuel cell technologies. However, there are limitations to using hydrogenases, especially due to their high O₂ sensitivity. The subclass, called [FeFe] hydrogenases, are particularly more vulnerable to O₂ but proficient in H₂ production. In this review, we provide an overview of mechanistic and protein engineering studies focused on understanding and enhancing O₂ tolerance of the enzyme. The emphasis is on ongoing studies that attempt to overcome O₂ sensitivity of the enzyme while it catalyzes H₂ production in an aerobic environment. We also discuss pioneering attempts to utilize the enzyme in biological H₂ production and other industrial processes, as well as our own perspective on future applications.     

The structure of AcrIE4-F7 reveals a common strategy for dual CRISPR inhibition by targeting PAM recognition sites

Bacteria and archaea use the CRISPR-Cas system to fend off invasions of bacteriophages and foreign plasmids. In response, bacteriophages encode anti-CRISPR (Acr) proteins that potently inhibit host Cas proteins to suppress CRISPR-mediated immunity. AcrIE4-F7, which was isolated from Pseudomonas citronellolis, is a fused form of AcrIE4 and AcrIF7 that inhibits both type I-E and type I-F CRISPR-Cas systems. Here, we determined the structure of AcrIE4-F7 and identified its Cas target proteins. The N-terminal AcrIE4 domain adopts a novel α-helical fold that targets the PAM interaction site of the type I-E Cas8e subunit. The C-terminal AcrIF7 domain exhibits an αβ fold like native AcrIF7, which disables target DNA recognition by the PAM interaction site in the type I-F Cas8f subunit. The two Acr domains are connected by a flexible linker that allows prompt docking onto their cognate Cas8 targets. Conserved negative charges in each Acr domain are required for interaction with their Cas8 targets. Our results illustrate a common mechanism by which AcrIE4-F7 inhibits divergent CRISPR-Cas types.     

The role of Kupffer cell alpha(2)-adrenoceptors in norepinephrine-induced TNF-alpha production

Although previous studies have demonstrated that plasma levels of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) increase during early sepsis, the precise mechanism responsible for its upregulation remains to be elucidated. Since recent studies have shown that the gut is an important source of norepinephrine (NE) release during early sepsis and enterectomy prior to the onset of sepsis attenuates TNF-alpha production, we hypothesized that gut-derived NE plays a major role in upregulating TNF-alpha via the activation of alpha(2)-adrenoceptors on Kupffer cells. To confirm that NE increases TNF-alpha synthesis and release, Kupffer cells were isolated from normal rats and incubated with NE (20 or 50 nM) or another alpha(2)-adrenergic agonist clonidine (50 nM) without addition of Escherichia coli endotoxin. Supernatant levels of TNF-alpha were then measured. In additional animals, intraportal infusion of NE (20 microM) with or without the specific alpha(2)-adrenergic antagonist yohimbine (1 mM) at a rate of 13 microl/min was carried out for 2 h. Plasma and Kupffer cell levels of TNF-alpha were assayed thereafter. Moreover, the effects of NE and yohimbine on TNF-alpha production was further examined using an isolated perfused liver preparation. The results indicate that both NE and clonidine increased TNF-alpha release by approximately 4-7-fold in the isolated cultured Kupffer cells. Similarly, intraportal infusion of NE in vivo or in isolated livers increased TNF-alpha synthesis and release which was inhibited by co-infusion of yohimbine. Furthermore, the increased cellular levels of TNF-alpha in Kupffer cells after in vivo administration of NE was also blocked by yohimbine. These results, taken together, suggest that gut-derived NE upregulates TNF-alpha production in Kupffer cells through an alpha(2)-adrenergic pathway, which appears to be responsible at least in part for the increased levels of circulating TNF-alpha observed during early sepsis as well as other pathophysiologic conditions such as trauma, hemorrhagic shock, or gut ischemia/reperfusion.     

Protein kinase C epsilon suppresses Abeta production and promotes activation of alpha-secretase

Deposition of plaques containing Abeta is considered important in the pathogenesis of Alzheimer's disease. Phorbol esters that activate protein kinase C (PKC) promote alpha-secretase-mediated processing of the beta amyloid precursor protein (APP), which generally reduces formation of Abeta. To determine which PKC isozymes mediate this process, we studied CHO cells that express human APP751. Phorbol 12-myristate, 13-acetate (PMA)-stimulated APP secretion, which was reduced by a general PKC inhibitor bisindoylmaleimide I, but not by G? 6976, which inhibits PKCalpha, beta, gamma, and mu. Since PKCdelta and epsilon were the only other PMA-sensitive isozymes present, we studied cells that express selective peptide inhibitors of these isozymes. Expression of the PKCepsilon inhibitor inhibited PMA-induced APPs secretion and suppression of Abeta production. In contrast, the PKCdelta inhibitor had no effect. These results provide evidence that PKCepsilon decreases Abeta production by promoting alpha-secretase mediated cleavage of APP.     

Immune enhancing effect of a growth hormone secretagogue

Growth hormone (GH) has been known to enhance immune responses, whether directly or through the insulin like growth factor-1, induced by GH. Recently a nonpeptidyl small m.w. compound, a GH secretagogue (GHS), was found to induce the production of GH by the pituitary gland. In this study, we examined the effect of GHS in immunological functions of 5- to 6-wk-old and 16- to 24-month-old mice. In young mice, we observed a significant increase in PBLs, but T and B cell-proliferative responses were not consistently enhanced. The old mice, treated with GHS for 3 wk, did not show increases in peripheral lymphocytes, but they exhibited a statistically significant increase in thymic cellularity and differentiation. When inoculated with a transplantable lymphoma cell line, EL4, the treated old mice showed statistically significant resistance to the initiation of tumors and the subsequent metastases. Generation of CTL to EL4 cells was also enhanced in the treated mice, suggesting that GHS has a considerable immune enhancing effect, particularly in the old mice. We have also found that GHS promoted better thymic engraftment in bone marrow transplant of SCID mice. We found more cycling cells in the spleens of treated mice, suggesting that GHS may exert its immune enhancing effect by promoting cell division in lymphoid cells. These observations ascribe to GHS a novel therapy possible for aging, AIDS, and transplant individuals, whose immune functions are compromised.     

Estradiol replacement in ovariectomized rats increases the hepatic concentration and biliary secretion of alpha-tocopherol and polyunsaturated fatty acids

Previously, we showed that estradiol replacement in ovariectomized rats produced prominent increases in serum and liver alpha-tocopherol (alphaTP). The present study was conducted to examine whether the estrogen-induced increase in the liver concentrations of alphaTP affects its biliary secretion and the fatty acid compositions of hepatic and biliary lipids. Ten ovariectomized rats were assigned to two groups: five rats were implanted subcutaneously with time-release estradiol pellets (OXE; 25 microg/day/rat) and five with placebo (OXP). Twice daily rats were pair-fed a modified AIN-93G diet containing soybean oil. At 5 weeks, bile was collected via a bile cannula hourly for 8 hours during duodenal infusion of a lipid emulsion (565 micromol triolein and 396 micromol Na-taurocholate/24 mL phosphate buffered saline, pH 6.45) at 3.0 mL/hr. During the 8-hour period, no difference was noted in the hourly rate of bile flow (0.95 mL/hr in OXE rats vs. 0.99 mL/hr in OXP rats). The biliary output of alphaTP for 8 hours was higher in OXE rats (51.6 +/- 3.6 nmol) than OXP rats (31.7 +/- 2.9 nmol). Likewise, the liver concentration of alphaTP was higher in OXE rats (81.9 +/- 3.5 nmol/g liver) than in OXP rats (53.3 +/- 7.4 nmol/g liver). The biliary secretion of phospholipids (PL) for 8 hours was significantly (P < 0.05) higher in OXE rats (55.1 +/- 4.9 micromol) than in OXP rats (42.3 +/- 4.7 micromol). Among the PL fatty acids, the outputs of 20:4 and 22:6n-3 were increased most markedly by estradiol replacement. The total outputs of 22:6n-3 for 8 hours in OXE and OXP rats were 2.95 +/- 0.20 micromol and 1.37 +/- 0.23 micromol, respectively. In the liver, the concentrations of PL 22:5n-3 and 22:6n-3 were elevated significantly in OXE rats. The present results suggest that estradiol may protect hepatic PL and membranes against oxidative damage by improving the liver status of alphaTP.     

A postmitotic role for Isl-class LIM homeodomain proteins in the assignment of visceral spinal motor neuron identity

LIM homeobox genes have a prominent role in the regulation of neuronal subtype identity and distinguish motor neuron subclasses in the embryonic spinal cord. We have investigated the role of Isl-class LIM homeodomain proteins in motor neuron diversification using mouse genetic methods. All spinal motor neuron subtypes initially express both Isl1 and Isl2, but Isl2 is rapidly downregulated by visceral motor neurons. Mouse embryos lacking Isl2 function exhibit defects in the migration and axonal projections of thoracic level motor neurons that appear to reflect a cell-autonomous switch from visceral to somatic motor neuron character. Additional genetic mutations that reduce or eliminate both Isl1 and Isl2 activity result in more pronounced defects in visceral motor neuron generation and erode somatic motor neuron character. Thus, an early phase of high Isl expression and activity in newly generated motor neurons permits the diversification of visceral and somatic motor neuron subtypes in the developing spinal cord.     

Residential sunlight exposure is associated with a decreased risk of prostate cancer

The possibility that exposure to sunlight reduces the risk of clinical prostate cancer has been strongly suggested by ecologic data. However, data on prostate cancer risk in relation to sunlight exposure in individuals are sparse. We analyzed data from the First National Health and Nutrition Examination Survey (NHANES I) Epidemiologic Follow-up Study in order to test the hypothesis that residential sunlight exposure reduces the risk of prostate cancer. We identified 153 men with incident prostate cancer from a cohort of 3414 white men who completed the baseline interview and dermatologic examination in 1971-1975 and were followed up to 1992. We used Cox proportional hazards modeling to estimate relative risks (RR) and 95% confidence intervals (CI) for measures of residential sunlight exposure, adjusting for age, family history of prostate cancer, and dietary intake of fat and calcium. Residence in the South at baseline (RR = 0.68, CI = 0.41-1.13), state of longest residence in the South (RR = 0.62, CI = 0.40-0.95), and high solar radiation in the state of birth (RR = 0.49, CI = 0.30-0.79) were associated with significant and substantial reductions in prostate cancer risk. These data support the hypothesis that sunlight exposure reduces the risk of prostate cancer and have important implications for prostate cancer prevention.