Purification and N-terminal amino acid sequence of fructose-6-phosphate phosphoketolase from Bifidobacterium longum BB536

AIMS: The key enzyme in the fructose-6-phosphate shunt in bifidobacteria, Fructose-6-phosphate phosphoketolase (F6PPK; E.C. 4.1.2.22.), was purified to electrophoretic homogeneity for the first time from Bifidobacterium longum (BB536). METHODS AND RESULTS: A three-step procedure comprising acetone fractionation followed by fast protein liquid chromatography (FPLC) resulted in a 30-fold purification. The purified enzyme had a molecular mass of 300 +/- 5 kDa as determined by gel filtration. It is probably a tetramer containing two different subunits with molecular masses of 93 +/- 1 kDa and 59 +/- 0.5 kDa, as determined by SDS-PAGE. CONCLUSION: The deduced N-terminal amino acid sequences of the two subunits revealed no significant similarity between them and other proteins when compared to the data bases of EMBL and SWISS-PROT, indicating that this could be the first report on N-terminal amino acid sequence of F6PPK. SIGNIFICANCE AND IMPACT OF THE STUDY: The data from this study will be used to design oligonucleotide probe specific for bifidobacteria and to study the gene encoded F6PPK.     

Accelerated endothelial dysfunction in mild prediabetic insulin resistance: the early role of reactive oxygen species

Insulin resistance is well established as an independent risk factor for the development of type 2 diabetes and cardiovascular atherosclerosis. Most studies have examined atherogenesis in models of severe insulin resistance or diabetes. However, by the time of diagnosis, individuals with type 2 diabetes already demonstrate a significant atheroma burden. Furthermore, recent studies suggest that, even in adolescence, insulin resistance is a progressive disorder that increases cardiovascular risk. In the present report, we studied early mechanisms of reduction in the bioavailability of the antiatheroscerotic molecule nitric oxide (NO) in very mild insulin resistance. Mice with haploinsufficiency for the insulin receptor (IRKO) are a model of mild insulin resistance with preserved glycemic control. We previously demonstrated that 2-mo-old (Young) IRKO mice have preserved vasorelaxation responses to ACh. This remained the case at 4 mo of age. However, by 6 mo, despite no significant deterioration in glucose homeostasis (Adult), IRKO mice had marked blunting of ACh-mediated vasorelaxation [IRKO maximum contraction response (E(max)) 66 +/- 5% vs. wild type 87 +/- 4%, P < 0.01]. Despite the endothelial dysfunction demonstrated, aortic endothelial nitric oxide synthase (eNOS) mRNA levels were similar in Adult IRKO and wild-type mice, and, interestingly, aortic eNOS protein levels were increased, suggesting a compensatory upregulation in the IRKO. We then examined the potential role of reactive oxygen species in mediating early endothelial dysfunction. The superoxide dismutase mimetic Mn(III)tetrakis(1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP) restored ACh relaxation responses in the Adult IRKO (E(max) to ACh with MnTMPyP 85 +/- 5%). Dihydroethidium fluorescence of aortas and isolated coronary microvascular endothelial cells confirmed a substantial increase in endothelium-derived reactive oxygen species in IRKO mice. These data demonstrate that mild insulin resistance is a potent substrate for accelerated endothelial dysfunction and support a role for endothelial cell superoxide production as a mechanism underlying the early reduction in NO bioavailability.     

Patterning with clocks and genetic cascades: Segmentation and regionalization of vertebrate versus insect body plans

Oscillatory and sequential processes have been implicated in the spatial patterning of many embryonic tissues. For example, molecular clocks delimit segmental boundaries in vertebrates and insects and mediate lateral root formation in plants, whereas sequential gene activities are involved in the specification of regional identities of insect neuroblasts, vertebrate neural tube, vertebrate limb, and insect and vertebrate body axes. These processes take place in various tissues and organisms, and, hence, raise the question of what common themes and strategies they share. In this article, we review 2 processes that rely on the spatial regulation of periodic and sequential gene activities: segmentation and regionalization of the anterior–posterior (AP) axis of animal body plans. We study these processes in species that belong to 2 different phyla: vertebrates and insects. By contrasting 2 different processes (segmentation and regionalization) in species that belong to 2 distantly related phyla (arthropods and vertebrates), we elucidate the deep logic of patterning by oscillatory and sequential gene activities. Furthermore, in some of these organisms (e.g., the fruit fly Drosophila), a mode of AP patterning has evolved that seems not to overtly rely on oscillations or sequential gene activities, providing an opportunity to study the evolution of pattern formation mechanisms.     

Cairo-East Bank effluent re-use study 3 — Effect of field crop irrigation with secondary treated wastewater on biological and chemical properties of soil and groundwater

Large scale field trials were conducted in fertile soil and desert (virgin) soil to evaluate the effect of irrigation with secondary treated wastewater from two wastewater treatment plants in Cairo on biological and chemical properties of soil and groundwater. Soil samples were taken for physical and chemical analysis after crop harvest. Groundwater monitoring wells were installed in and around the experimental soil sites. Considerable amounts of macronutrients (NPK) were applied to the grown crops through the treated wastewater irrigation: N (19–79%), P (23–181%) and K (85–357%) of the recommended fertilizer rates according to the crop and the experimental site. Soil physical and chemical analysis showed that both soils are widely variable in water holding capacity, organic matter, pH value, CaCO₃, salinity, cation exchange capacity, and soil bulk density in the topsoil (0–30 cm). Groundwater samples which were examined for the presence of pathogenic bacteria (salmonella), faecal coliform bacteria and helminth ova. At the virgin soil site, 10–57% of the samples from each well contained salmonella, whereas at the fertile soil, salmonella was not detected in five wells, and the occurrence in the other four wells was 10–20% of the samples. The numbers of faecal coliforms were similar at both sites, in the range 10²–10³ MPN per 100 mL. Small numbers of parasite ova were also found in the majority of wells, with a greater number occurring at the virgin soil. The groundwater in both sites was similar and of poor quality, and would be unsuitable for potable or irrigation purposes.     

Corneal Confocal Microscopy Detects Small Fibre Neuropathy in Patients with Upper Gastrointestinal Cancer and Nerve Regeneration in Chemotherapy Induced Peripheral Neuropathy

There are multiple neurological complications of cancer and its treatment. This study assessed the utility of the novel non-invasive ophthalmic technique of corneal confocal microscopy in identifying neuropathy in patients with upper gastrointestinal cancer before and after platinum based chemotherapy. In this study, 21 subjects with upper gastrointestinal (oesophageal or gastric) cancer and 21 healthy control subjects underwent assessment of neuropathy using the neuropathy disability score, quantitative sensory testing for vibration perception threshold, warm and cold sensation thresholds, cold and heat induced pain thresholds, nerve conduction studies and corneal confocal microscopy. Patients with gastro-oesophageal cancer had higher heat induced pain (P = 0.04) and warm sensation (P = 0.03) thresholds with a significantly reduced sural sensory (P<0.01) and peroneal motor (P<0.01) nerve conduction velocity, corneal nerve fibre density (CNFD), nerve branch density (CNBD) and nerve fibre length (CNFL) (P<0.0001). Furthermore, CNFD correlated significantly with the time from presentation with symptoms to commencing chemotherapy (r = -0.54, P = 0.02), and CNFL (r = -0.8, P<0.0001) and CNBD (r = 0.63, P = 0.003) were related to the severity of lymph node involvement. After the 3^rd cycle of chemotherapy, there was no change in any measure of neuropathy, except for a significant increase in CNFL (P = 0.003). Corneal confocal microscopy detects a small fibre neuropathy in this cohort of patients with upper gastrointestinal cancer, which was related to disease severity. Furthermore, the increase in CNFL after the chemotherapy may indicate nerve regeneration.