The exchange activities of [Fe] hydrogenase (iron–sulfur-cluster-free hydrogenase) from methanogenic archaea in comparison with the exchange activities of [FeFe] and [NiFe] hydrogenases

[Fe] hydrogenase (iron–sulfur-cluster-free hydrogenase) catalyzes the reversible reduction of methenyltetrahydromethanopterin (methenyl-H4MPT+) with H2 to methylene-H4MPT, a reaction involved in methanogenesis from H2 and CO2 in many methanogenic archaea. The enzyme harbors an iron-containing cofactor, in which a low-spin iron is complexed by a pyridone, two CO and a cysteine sulfur. [Fe] hydrogenase is thus similar to [NiFe] and [FeFe] hydrogenases, in which a low-spin iron carbonyl complex, albeit in a dinuclear metal center, is also involved in H2 activation. Like the [NiFe] and [FeFe] hydrogenases, [Fe] hydrogenase catalyzes an active exchange of H2 with protons of water; however, this activity is dependent on the presence of the hydride-accepting methenyl-H4MPT+. In its absence the exchange activity is only 0.01% of that in its presence. The residual activity has been attributed to the presence of traces of methenyl-H4MPT+ in the enzyme preparations, but it could also reflect a weak binding of H2 to the iron in the absence of methenyl-H4MPT+. To test this we reinvestigated the exchange activity with [Fe] hydrogenase reconstituted from apoprotein heterologously produced in Escherichia coli and highly purified iron-containing cofactor and found that in the absence of added methenyl-H4MPT+ the exchange activity was below the detection limit of the tritium method employed (0.1 nmol min−1 mg−1). The finding reiterates that for H2 activation by [Fe] hydrogenase the presence of the hydride-accepting methenyl-H4MPT+ is essentially required. This differentiates [Fe] hydrogenase from [FeFe] and [NiFe] hydrogenases, which actively catalyze H2/H2O exchange in the absence of exogenous electron acceptors

Analytical approaches to photobiological hydrogen production in unicellular green algae

Several species of unicellular green algae, such as the model green microalga Chlamydomonas reinhardtii, can operate under either aerobic photosynthesis or anaerobic metabolism conditions. A particularly interesting metabolic condition is that of “anaerobic oxygenic photosynthesis”, whereby photosynthetically generated oxygen is consumed by the cell’s own respiration, causing anaerobiosis in the culture in the light, and induction of the cellular “hydrogen metabolism” process. The latter entails an alternative photosynthetic electron transport pathway, through the oxygen-sensitive FeFe-hydrogenase, leading to the light-dependent generation of molecular hydrogen in the chloroplast. The FeFe-hydrogenase is coupled to the reducing site of photosystem-I via ferredoxin and is employed as an electron-pressure valve, through which electrons are dissipated, thus permitting a sustained electron transport in the thylakoid membrane of photosynthesis. This hydrogen gas generating process in the cells offers testimony to the unique photosynthetic metabolism that can be found in many species of green microalgae. Moreover, it has attracted interest by the biotechnology and bioenergy sectors, as it promises utilization of green microalgae and the process of photosynthesis in renewable energy production. This article provides an overview of the principles of photobiological hydrogen production in microalgae and addresses in detail the process of induction and analysis of the hydrogen metabolism in the cells. Furthermore, methods are discussed by which the interaction of photosynthesis, respiration, cellular metabolism, and H(2) production in Chlamydomonas can be monitored and regulated

Immunocytology improves prognostic impact of peritoneal tumour cell detection compared to conventional cytology in gastric cancer

AIMS: Studies on the value of peritoneal tumour cell dissemination for prognosis in gastric cancer using various methods to detect tumour cells have produced conflicting conclusions. We studied the incidence and prognostic relevance of microscopic intraperitoneal tumour cell dissemination in gastric cancer, comparing conventional and immunocytological detection. METHODS: Peritoneal wash-outs of 111 consecutive gastric patients without overt peritoneal carcinomatosis, including 75 curatively resected patients, were studied. Sixty patients with benign disorders served as controls. 100 ml of warm NaCl 0.9% was instilled intraoperatively and 20 ml was reaspirated. The specimens were stained peri-operatively with H&E. In the last 47 patients (30 of whom were curatively resected) additional immunostaining with the HEA-125 antibody was performed. The results of cytology were correlated with the TNM categories and with post-operative follow-up. RESULTS: Of the patients, 42.3% and 48.9% were positive when conventional and immunocytological staining were employed, respectively. Conventional cytology was significantly associated with the pT and M categories. Immunocytology was significantly associated with the pT, pN and M caterogies. In four of 30 curatively resected patients (13.3%), the results of conventional and immunocytology were different. Three patients with positive immunocytology but negative conventional cytology died during follow-up (median follow-up 45.3 months), whereas one patient with positive conventional but negative immunocytology is still alive. In an univariate analysis 4 years post-surgery, positive immunocytology was significantly associated with an unfavourable prognosis in patients with curatively resected gastric cancer. While only 28.6% (six of 21) of the patients with negative immunocytology had died, this proportion increased to 77.8% (seven of nine) with positive immunocytology (P=0.018). The mean survival of negative vs positive patients amounted to 1205+/-91 vs 772+/-147 days (P=0.007). In contrast, in conventional cytology we found no significantly different survival time between negative and positive patients. CONCLUSIONS: Immunocytology seems to be superior to conventional cytology and should be preferred