Lysosomal leukocyte beta-D-glucuronidase during enzyme replacement therapy in Fabry disease

OBJECTIVE: Fabry disease results from a deficiency in the activity of alpha-d-galactosidase A and subsequent accumulation of neutral glycosphingolipids in lysosomes. This study investigated whether lysosomal enzymes can indicate biochemical changes in the lysosomal apparatus induced by enzyme replacement therapy (ERT). DESIGN AND METHODS: Eight patients were monitored by clinical and biochemical tests and several lysosomal glycohydrolases were measured in plasma and leucocytes. RESULTS: Before starting ERT, beta-d-glucuronidase in leukocytes was markedly increased. After 1 month of therapy, enzyme levels dropped in all patients. In the patients who regularly followed the therapy, the enzyme levels remained stable for the next 20 months. In one patient who interrupted therapy for 2 months, the enzyme levels rose again. CONCLUSIONS: Lysosomal enzymes can be useful for monitoring biochemical changes in patients with Fabry disease receiving ERT. Though these findings refer to only a small number of patients, the correlation between beta-d-glucuronidase levels and ERT is interesting and might serve as a basis for further studies to define the potential of this enzyme in monitoring the effects of ERT in lysosomal storage disorders.     

Intestinal anisakiasis in Italy: case report

A case of intestinal anisakiasis caused by Anisakis sp. larva type I in a woman from Italy who consumed raw marinated anchovies, is reported. The diagnosis was based on the morphological features characteristic of anisakid larval stages, which were readily recognized in a large granuloma removed after emergency surgical treatment.     

Survivin is regulated by interleukin‐4 in colon cancer stem cells

Colorectal cancer has provided an important model to test the stem cell hypothesis of cancer origin, which implies that cancer arises as a result of genetic aberrations in stem cells leading to deregulation of the proliferation/differentiation balance. We and others have demonstrated that, similarly to other solid tumors, colon carcinogenesis and progression are dictated by highly apoptosis‐resistant stem‐like cells. Our data have suggested that protection from apoptosis is achieved by autocrine production of interleukin‐4 (IL‐4) through up‐regulation of anti‐apoptotic mediators. In this study, we extend our analysis to another apoptosis inhibitor widely expressed in tumors, namely survivin (also known as BIRC‐5, baculoviral IAP repeat‐containing protein 5). We show that this protein, with important roles in cell death counteraction and mitotic progression control, is regulated by the IL‐4 pathway in colon rectal cancer stem cells (CR‐CSC). Hence, the presence of IL‐4 increases survivin levels in our model while cytokine neutralization has opposing effects. Treatment with cytokine neutralizing agent or with leflunomide, Stat6 inhibitor, have similar consequences on survivin localization, increasing its nuclear pool, an observation known to be correlated with a good prognosis in colon cancer patients. These results demonstrate that IL‐4, through activation of the STAT‐6 signaling pathway, is involved in survivin expression levels as well as its localization. These findings shed more light on the molecular mechanisms involved in IL‐4‐mediated chemoresistance. J. Cell. Physiol. 225: 555–561, 2010. © 2010 Wiley‐Liss, Inc.     

Response surface methodology as an approach to determine the optimal activities of xylose reductase and xylitol dehydrogenase enzymes from Candida Mogii

A central composite experimental design leading to a set of 16 experiments with different combinations of pH and temperature was performed to attain the optimal activities of xylose reductase (XR) and xylitol dehydrogenase (XDH) enzymes from Candida mogii cell extract. Under optimized conditions (pH 6.5 and 38°C), the XR and XDH activities were found to be 0.48 U/ml and 0.22 U/ml, respectively, resulting in an XR to XDH ratio of 2.2. Stability, cofactor specificity and kinetic parameters of the enzyme XR were also evaluated. XR activity remained stable for 3 h under 4 and 38°C and for 4 months of storage at −18°C. Studies on cofactor specificity showed that only NADPH-dependent XR was obtained under the cultivation conditions employed. The XR present in C. mogii extracts showed a superior K _m value for xylose when compared with other yeast strains. Besides, this parameter was not modified after enzyme extraction by aqueous two-phase system.     

Synergy of fatty acid and reactive alkenal activation of proton conductance through uncoupling protein 1 in mitochondria

The kinetics of proton transport through mammalian UCP1 (uncoupling protein 1) expressed in yeast mitochondria were measured. There was little or no UCP1 activity in the absence of added palmitate, but significant activity in its presence. The activator 4-HNE (4-hydroxy-2-nonenal) had little effect when added alone, but significantly enhanced proton conductance in the presence of added palmitate. Activation of the proton conductance of UCP1 was synergistic: proton conductance in the presence of both palmitate and 4-HNE was significantly greater than the sum of the individual effects. Mitochondria from control yeast transformed with empty vector showed no such synergy, showing that synergy is a property of UCP1. Activation by the 4-HNE analogue trans-cinnamate showed essentially the same characteristics as activation by 4-HNE. Mitochondria from brown adipose tissue also showed synergistic activation of GDP-sensitive proton conductance by palmitate and 4-HNE. These results show that reactive alkenals activate the proton conductance of UCP1 more strongly when fatty acids are also added, with implications for both mechanistic and physiological models of UCP1 activation.     

The effect of insulin on the uptake and metabolic fate of glucose in isolated perfused hearts of dyslipemic rats

Male Wistar rats chronically (15 weeks) fed a sucrose-rich diet (SRD; 63% w/w) developed hypertriglyceridemia and impaired glucose homeostasis. Hearts from these animals were isolated and perfused using the Langendorff recirculating method. Glucose at levels similar to those found in the animal in vivo was used as the only exogenous substrate. The hearts were perfused for 30 minutes in the presence or absence of insulin (30 mU/mL) in the perfusion medium. In the absence of the hormone, glucose uptake was impaired and the glucose utilization was reduced, with a significant increase of lactate release. Glucose oxidation, which was estimated from the activation state of the enzyme pyruvate dehydrogenase complex (PDHc), was depressed mainly due to both an increase of PDH kinase and a decrease of PDHa (active form of PDHc) activities. Although the addition of insulin in the perfusion medium improved the above parameters, it was unable to normalize them. The present results suggest that at least two different mechanisms might contribute to insulin resistance and to the impaired glucose metabolism in the perfused hearts of the dyslipemic SRD-fed animals: (1) reduced basal and insulin-stimulated glucose uptake and its utilization or (2) increased availability and oxidation of lipids (low PDHa and high PDH kinase activities), which in turn decrease glucose uptake and utilization. Thus, this nutritional experimental model may be useful to study how impaired glucose homeostasis, increases plasma free fatty acid levels and hypertriglyceridemia could contribute to heart tissue malfunction.     

Evaluation of DNA fragmentation in llama (Lama glama) sperm using the sperm chromatin dispersion test

The integrity of sperm chromatin is now viewed as an important factor in male fertility and in early embryonic development. The objectives of this study were: (1) adapt the simple and inexpensive sperm chromatin dispersion (SCD) test to evaluate DNA fragmentation in llama sperm and establish the halo patterns observed in this species, (2) determine an effective and reliable positive control for this technique and (3) evaluate correlation between the SCD test and the toluidine blue (TB) stain. To adapt the SCD test, three different mercaptoethanol (ME) concentrations were assayed (2.5%, 5% and 10% ME). To determine an effective positive control, three treatments (incubation at 100 °C for 30 min, incubation with 0.3 M NaOH for 30 min at room temperature and exposure to UV light for 2h) were assayed. The concentration selected to use in the SCD test was 5% ME, because it produced the largest halo while still conserving the structure of the core. Four DNA dispersion patterns were clearly observed: (I) nuclei with large DNA dispersion halos; (II) nuclei with medium halos; (III) nuclei with very small halos and (IV) nuclei with no halo. All treatments used as positive controls were effective in producing DNA fragmentation. A high correlation (r=0.84, P=0.03) was observed between spermatozoa without halos and TB positive cells. To conclude, SCD patterns in llama sperm have been established as well as a repeatable positive control for the assay. The SCD test and TB stain are simple and inexpensive techniques that can be used to evaluate DNA damage in llama sperm.