Retinol-induced mdr1 and mdr3 modulation in cultured rat Sertoli cells is attenuated by free radical scavengers

The effects of retinol on modulation of mdr genes in Sertoli cells were investigated. The hypothesis that free radical scavengers may attenuate the effect of retinol was also tested. Sertoli cells isolated from 15-day-old Wistar rats were cultured for 48 h and then treated with retinol for 24 h with or without free radical scavengers (1 mM mannitol, 0.1 mM Trolox or superoxide dismutase [200 U/ml]). Expression of mdr1, mdr2 and mdr3 genes was monitored by RT-PCR. Mitochondrial superoxide production was used as an index of ROS production. Expression of mdr1 and mdr3 was inhibited by retinol treatment (7 microM, 24 h), while mdr2 was not detected in response to any of the treatments. We also observed that retinol supplementation (7 microM, 24 h) increased superoxide production. The observed inhibition of mdr genes was attenuated by all co-treatments, suggesting that retinol-induced ROS are required for inhibition of mdr1 and mdr3 expression. The results suggest that retinol may play an important role in the modulation of the mdr gene family in cultured rat Sertoli cells and that these effects appear to be mediated by ROS.     

Improvement in xylitol production from sugarcane bagasse hydrolysate achieved by the use of a repeated-batch immobilized cell system

Candida guilliermondii cells were immobilized in Ca-alginate beads and used for xylitol production from concentrated sugarcane bagasse hydrolysate during five successive fermentation batches, each lasting 48 hours. The bioconversion efficiency of 53.2%, the productivity of 0.50 g/l x h and the final xylitol concentration of 23.8 g/l obtained in the first batch increased to 61.5%, 0.59 g/l x h and 28.4 g/l, respectively, in the other four batches (mean values), with variation coefficients of up to 2.3%.     

Complete conversion of D,L-5-monosubstituted hydantoins with a low velocity of chemical racemization into D-amino acids using whole cells of recombinant Escherichia coli

A reaction system was developed for the production of D-amino acids from D,L-5-monosubstituted hydantoins with a very slow rate of spontaneous racemization. For this purpose the D-hydantoinase and D-carbamoylase from Agrobacterium radiobacter NRRL B11291 were cloned in separate plasmids and expressed in Escherichia coli. The third enzyme, hydantoin racemase, was cloned from Agrobacterium tumefaciens C58. The hydantoin racemase amino acid sequence was significantly similar to those previously described. A reaction system consisting of recombinant Escherichia coli whole cell biocatalysts containing separately expressed D-hydantoinase, D-carbamoylase, and hydantoin recemase showed high substrate specificity and was effective toward both aliphatic and aromatic D,L-5-monosubstituted hydantoins. After optimizing reaction conditions (pH 8 and 50 degrees C), 100% conversion of D,L-5-(2-methylthioethyl)-hydantoin (15 mM) into D-methionine was obtained in 30 min.     

MEG (Model Extender for Gepasi): a program for the modelling of complex, heterogeneous, cellular systems

SUMMARY: We describe a program for the construction of spatially distributed metabolic models, which may then be simulated using the metabolic simulator GEPASI: This is useful for the modelling of heterogeneous systems whether as liquid cultures or as spatially organised systems with specified interconnections.     

A reciprocal translocation between ’Garfi’ almond and ’Nemared’ peach

A map with 51 markers (46 RFLPs and five isozymes) was constructed using an interspecific F₂ population between ’Garfi’ almond (Prunus amygdalus Batsch.) and ’Nemared’ peach [Prunus persica (L.) Batsch.]. This map was developed by selecting markers covering most of the distance of the eight linkage groups from previously constructed Prunus maps. The markers studied in this population mapped to seven linkage groups instead of the eight expected in Prunus. Markers belonging to groups 6 and 8 in previous maps formed a single group in the ’Garfi’×’Nemared’ F₂ and several marker pairs placed in different groups in other maps exhibited tight linkages. The study of pollen fertility and chromosome behavior during meiosis in the F₁ generation allowed us to confirm the hypothesis that a reciprocal translocation exists between ’Garfi’ and ’Nemared’. Based on independent evidence of linkage between markers and pollen fertility data in the F₂ population, we concluded that the breakpoint of the reciprocal translocation was placed between markers AC50 and AG26A in group 6 and between markers AG112A and FG230A in group 8. Received: 28 June 2000 / Accepted: 17 October 2000     

Integrating retroviral cassette extends gene delivery of HSV-1 expression vectors to dividing cells

Retroviral vectors have long been used in a wide variety of gene transfer applications but have certain drawbacks, such as small cargo size, limited tropism, and low titers. HSV expression vectors overcome these disadvantages, but, because they persist in target cells as nonreplicative episomes, they are not retained in all the progeny of dividing cells. Chimeric HSV/AAV products that can mediate transgene integration in human mitotic cells have been constructed, but, to date, genetic modification of dividing cells in animal models using HSV products has not been possible. Here, we report the construction of hybrid HSV/retroviral vectors that exhibit up to 50-fold higher transgene integration efficiency compared to vectors containing only HSV-1 components. Efficient integration of a retroviral transgene cassette encoding pac in human cells required expression of the Moloney murine leukemia virus gag-pol genes, but in murine cells, could also be mediated by endogenous activities, albeit at a lower level. Gene delivery was equally efficient in BHK21, a cell line resistant to retroviral infection, and transgene retention and expression were observed to be stable for least one month in Hs683 human glioma cells. These vectors have wide applications for the genetic modification of many cell types.     

Structural and functional characterization explains loss of dNTPase activity of the cancer-specific R366C/H mutant SAMHD1 proteins

Elevated intracellular levels of dNTPs have been shown to be a biochemical marker of cancer cells. Recently, a series of mutations in the multifunctional dNTP triphosphohydrolase (dNTPase), sterile alpha motif and histidine–aspartate domain–containing protein 1 (SAMHD1), have been reported in various cancers. Here, we investigated the structure and functions of SAMHD1 R366C/H mutants, found in colon cancer and leukemia. Unlike many other cancer-specific mutations, the SAMHD1 R366 mutations do not alter cellular protein levels of the enzyme. However, R366C/H mutant proteins exhibit a loss of dNTPase activity, and their X-ray structures demonstrate the absence of dGTP substrate in their active site, likely because of a loss of interaction with the γ-phosphate of the substrate. The R366C/H mutants failed to reduce intracellular dNTP levels and restrict HIV-1 replication, functions of SAMHD1 that are dependent on the ability of the enzyme to hydrolyze dNTPs. However, these mutants retain dNTPase-independent functions, including mediating dsDNA break repair, interacting with CtIP and cyclin A2, and suppressing innate immune responses. Finally, SAMHD1 degradation in human primary-activated/dividing CD4+ T cells further elevates cellular dNTP levels. This study suggests that the loss of SAMHD1 dNTPase activity induced by R366 mutations can mechanistically contribute to the elevated dNTP levels commonly found in cancer cells.     

Effects of Ocean Acidification on the Ballast of Surface Aggregates Sinking through the Twilight Zone

The dissolution of CaCO₃ is one of the ways ocean acidification can, potentially, greatly affect the ballast of aggregates. A diminution of the ballast could reduce the settling speed of aggregates, resulting in a change in the carbon flux to the deep sea. This would mean lower amounts of more refractory organic matter reaching the ocean floor. This work aimed to determine the effect of ocean acidification on the ballast of sinking surface aggregates. Our hypothesis was that the decrease of pH will increase the dissolution of particulate inorganic carbon ballasting the aggregates, consequently reducing their settling velocity and increasing their residence time in the upper twilight zone. Using a new methodology for simulation of aggregate settling, our results suggest that future pCO₂ conditions can significantly change the ballast composition of sinking aggregates. The change in aggregate composition had an effect on the size distribution of the aggregates, with a shift to smaller aggregates. A change also occurred in the settling velocity of the particles, which would lead to a higher residence time in the water column, where they could be continuously degraded. In the environment, such an effect would result in a reduction of the carbon flux to the deep-sea. This reduction would impact those benthic communities, which rely on the vertical flow of carbon as primary source of energy.     

Mechanisms of Hearing Loss after Blast Injury to the Ear

Given the frequent use of improvised explosive devices (IEDs) around the world, the study of traumatic blast injuries is of increasing interest. The ear is the most common organ affected by blast injury because it is the body’s most sensitive pressure transducer. We fabricated a blast chamber to re-create blast profiles similar to that of IEDs and used it to develop a reproducible mouse model to study blast-induced hearing loss. The tympanic membrane was perforated in all mice after blast exposure and found to heal spontaneously. Micro-computed tomography demonstrated no evidence for middle ear or otic capsule injuries; however, the healed tympanic membrane was thickened. Auditory brainstem response and distortion product otoacoustic emission threshold shifts were found to be correlated with blast intensity. As well, these threshold shifts were larger than those found in control mice that underwent surgical perforation of their tympanic membranes, indicating cochlear trauma. Histological studies one week and three months after the blast demonstrated no disruption or damage to the intra-cochlear membranes. However, there was loss of outer hair cells (OHCs) within the basal turn of the cochlea and decreased spiral ganglion neurons (SGNs) and afferent nerve synapses. Using our mouse model that recapitulates human IED exposure, our results identify that the mechanisms underlying blast-induced hearing loss does not include gross membranous rupture as is commonly believed. Instead, there is both OHC and SGN loss that produce auditory dysfunction.