A 3,387?bp 5′-flanking sequence of the goat alpha-S1-casein gene provides correct tissue-specific expression of human granulocyte colony-stimulating factor (hG-CSF) in the mammary gland of transgenic mice

A new expression vector containing the 1,944 bp 5'-flanking regulatory region together with exon 1 and intron 1 of the goat alpha-S1-casein gene (CSN1S1), the full-sized human granulocyte colony-stimulating factor gene (hGCSF) and the 3'-flanking sequence of the bovine CSN1S1, was created. The vector DNA was used for generation of four mouse transgenic lines. The transgene was integrated into chromosomes 8 and 12 of two founders as 2 and 5 copies, respectively. Tissue-specific secretion of hG-CSF into the milk of transgenic mice was in the range of 19-40 μg/ml. RT-PCR analysis of various tissues of the transgenic mice demonstrated that expression of hGCSF was detected in only the mammary gland in the progeny of all founders. Moreover, cells were shown to be positive for hG-CSF by immunofluorescent analysis in the mammary glands but not in any other tissues. There were no signs of mosaic expression in the mammary gland. Trace amounts of hG-CSF were detected in the serum of females of two transgenic lines during lactation only. However, no transgenic mice showed any changes in hematopoiesis based on the number of granulocytes in blood. Immunoblotting of hG-CSF in the milk of transgenic mice revealed two forms, presumably the glycosylated and non-glycosylated forms. The hematopoietic activity of hG-CSF in the milk of transgenic females is comparable to that of recombinant G-CSF. In general, the data obtained in this study show that the new expression vector is able to provide correct tissue-specific expression of hG-CSF with high biological activity in transgenic mice.     

Porcine Adipose Tissue-Derived Mesenchymal Stem Cells Retain Their Proliferative Characteristics,Senescence, Karyotype and Plasticity after Long-Term Cryopreservation

We and others have provided evidence that adipose tissue-derived mesenchymal stem cells (ASCs) can mitigate rat cardiac functional deterioration after myocardial ischemia, even though the mechanism of action or the relevance of these findings to human conditions remains elusive. In this regard, the porcine model is a key translational step, because it displays heart anatomic-physiological features that are similar to those found in the human heart. Towards this end, we wanted to establish the cultural characteristics of porcine ASCs (pASCs) with or without long-term cryostorage, considering that allogeneic transplantation may also be a future option. Compared to fresh pASCs, thawed cells displayed 90–95% viability and no changes in morphological characteristics or in the expression of surface markers (being pASCs characterized by positive markers CD29⁺; CD90⁺; CD44⁺; CD140b⁺; CD105⁺; and negative markers CD31⁻; CD34⁻; CD45⁻ and SLA-DR⁻; n = 3). Mean population doubling time was also comparable (64.26±15.11 hours to thawed cells vs. 62.74±18.07 hours to fresh cells) and cumulative population doubling increased constantly until Passage 10 (P10) in the entire cell population, with a small and gradual increase in senescence (P5, 3.25%±0.26 vs. 3.47%±0.32 and P10, 9.6%±0.29 vs. 10.67%±1.25, thawed vs. fresh; SA-β-Gal staining). Chromosomal aberrations were not observed. In addition, under both conditions pASCs responded to adipogenic and osteogenic chemical cues in vitro. In conclusion, we have demonstrated the growth characteristics, senescence, and the capacity of pASCs to respond to chemical cues in vitro and have provided evidence that these properties are not influenced by cryostorage in 10% DMSO solution.     

Lysosomal calcium homeostasis defects, not proton pump defects, cause endo-lysosomal dysfunction in PSEN-deficient cells

Presenilin (PSEN) deficiency is accompanied by accumulation of endosomes and autophagosomes, likely caused by impaired endo-lysosomal fusion. Recently, Lee et al. (2010. Cell. doi: http://dx.doi.org/10.1016/j.cell.2010.05.008) attributed this phenomenon to PSEN1 enabling the transport of mature V0a1 subunits of the vacuolar ATPase (V-ATPase) to lysosomes. In their view, PSEN1 mediates the N-glycosylation of V0a1 in the endoplasmic reticulum (ER); consequently, PSEN deficiency prevents V0a1 glycosylation, compromising the delivery of unglycosylated V0a1 to lysosomes, ultimately impairing V-ATPase function and lysosomal acidification. We show here that N-glycosylation is not a prerequisite for proper targeting and function of this V-ATPase subunit both in vitro and in vivo in Drosophila melanogaster. We conclude that endo-lysosomal dysfunction in PSEN(-/-) cells is not a consequence of failed N-glycosylation of V0a1, or compromised lysosomal acidification. Instead, lysosomal calcium storage/release is significantly altered in PSEN(-/-) cells and neurons, thus providing an alternative hypothesis that accounts for the impaired lysosomal fusion capacity and accumulation of endomembranes that accompanies PSEN deficiency.     

Land use and nitrogen export in the Piracicaba River basin, Southeast Brazil

Anthropogenic N inputs and riverine export were determined for a meso-scale river basin in one of the most developed and economically important regions of South America. The Piracicaba River basin is located in southeastern Brazil and drains into a tributary of the Paraná River. The basin supports over 3 million people (about 2% of the population of Brazil) with intensive agricultural and industrial activities. During two years from 1995 to 1997, biweekly samples were collected at 10 stations along the Piracicaba River and its tributaries for analyses of dissolved and particulate N. The average annual flux of dissolved inorganic N and total N increased by a factor of 15 and 20 times, respectively, from the headwaters to the lower reaches of the main channel, whereas discharge increased by only 7 times. On a per area basis, the export of TN varied according to land use and was significantly correlated to the net input of anthropogenic N. Among 10 sub-catchments composing the basin, areas mostly covered by pasture and forest had the lowest export, whereas more agricultural and urban areas had higher export. The amount of N exported from each sub-catchment varied widely, but inputs were consistently higher than fluvial outputs. Losses and retention of N occurred throughout the basin but were especially high in the sub-catchment with a main-stem reservoir, suggesting that aquatic processing plays an important role in controlling riverine N export. Total net anthropogenic input to the Piracicaba River basin was 4,500 (± 900) kg N km^–2 yr^–1 of which about 40% was exported via fluvial outputs.     

PnTx3-6 a spider neurotoxin inhibits K+-evoked increase in [Ca2+](i) and Ca2+-dependent glutamate release in synaptosomes

The present experiments investigated the effect of a neurotoxin purified from the venom of the spider Phoneutria nigriventer. This toxic component, P. nigriventer toxin 3-6 (PnTx3-6), abolished Ca(2+)-dependent glutamate release with an IC(50) of 74.4nM but did not alter Ca(2+)-independent secretion of glutamate when brain cortical synaptosomes were depolarized by KCl (33mM). This effect was most likely due to interference with the entry of calcium through voltage activated calcium channels (VACC), reducing the increase in the intrasynaptosomal free calcium induced by membrane depolarization with an IC(50) of 9.5nM. We compared the alterations induced by PnTx3-6 with the actions of toxins known to block calcium channels coupled to exocytosis. Our results indicate that PnTx3-6 inhibition of glutamate release and intrasynaptosomal calcium involves P/Q type calcium channels and this toxin can be a valuable tool in the investigation of calcium channels.     

Tissue distribution evaluation of stealth pH-sensitive liposomal cisplatin versus free cisplatin in Ehrlich tumor-bearing mice

Multidrug resistance and drug toxicity represent major obstacles to cancer chemotherapy. Drug delivery systems, such as liposomes, offer improved chemical stability of encapsulated drugs, enhanced accumulation in tumors and decreased toxicity. The aim of this study was to evaluate the tissue distribution of stealth pH-sensitive liposomes containing cisplatin (SpHL-CDDP), compared with free cisplatin (CDDP), in solid Ehrlich tumor-bearing mice. After administering a 6 mg/kg single intravenous bolus injection of either free radiolabeled cisplatin or SpHL containing radiolabeled cisplatin, blood and tissues were analyzed for cisplatin content by determining radioactivity using an automatic scintillation apparatus. The area under the CDDP concentration-time curve (AUC) obtained for blood after SpHL-CDDP administration was 2.1 fold larger when compared with free CDDP treatment. The longer circulation of SpHL-CDDP led to a higher tumor AUC, and the determination of the ratio between AUC in each tissue and that in blood (Kp) showed a higher accumulation of CDDP in SpHL-CDDP administrated tumors. The SpHL-CDDP was also significantly uptaken by the liver and spleen. The distribution of SpHL-CDDP in these organs was extensive, revealing a high extravasation of CDDP to the tissues. The SpHL-CDDP kidney uptake was also greater than that of free CDDP; however, the Kp value found was lower. This indicates that the SpHL-CDDP led to a reduction of CDDP retention by renal tissue. Thus, these results indicate that the SpHL-CDDP may indeed be useful in alleviating renal damage induced by CDDP and thus represents a promising delivery system for cancer treatment through CDDP.     

Clonal Transmission of ESBL-Producing <Emphasis Type="Italic">Klebsiella</Emphasis> spp. at a University Hospital in Brazil

The present study was designed to determine the prevalence and extended-spectrum β-lactamase (ESBL) types in clinical isolates of Klebsiella spp. at a university hospital located in the Brazilian southern region (Ribeirão Preto, São Paulo) as well as their antibiotic susceptibility and genetic profiles. This study included 147 non-repeat Klebsiella spp. isolates collected from January to June 2000, of which 23 K. pneumoniae and 8 K. oxytoca were selected as ESBL producers by using the Oxoid combination disk method and Etest ESBL strip. β-lactamases were characterized by IEF, PCR and sequencing assays using primers for ESBL genes. Antibiotic susceptibility was evaluated by MicroScan system. Dissemination of two major clones of ESBL-producing Klebsiella spp. occurred in the hospital. According to the results obtained in this study there was a clonal spread of CTX-M-producing K. oxytoca in five clinics and dissemination of ESBL-producing K. pneumoniae in the nursery and pediatrics wards.     

Antimicrobial effects of a pulsed electromagnetic field: an in vitro polymicrobial periodontal subgingival biofilm model

Abstract

The objective was to test the influence of a pulsed electromagnetic field (PEMF) on bacterial biofilm colonization around implants incorporated with healing abutments. Healing abutments with (test group) and without (control group) active PEMF devices were placed in a multispecies biofilm consisting of 31 different bacterial species. The biofilm composition and total bacterial counts (x10⁵) were analyzed by checkerboard DNA-DNA hybridization. After 96?h, the mean level of 7 out of the 31 bacterial species differed significantly between groups, namely Eubacterium nodatum, Fusobacterium nucleatum ssp. nucleatum, Streptococcus intermedius, Streptococcus anginosus, Streptococcus mutans, Fusobacterium nucleatum ssp. Vicentii and Capnocytophaga ochracea were elevated in the control group (p?<?0.05). The mean total bacterial counts were lower in the Test group vs the control group (p?<?0.05). An electromagnetic healing cap had antimicrobial effects on the bacterial species and can be used to control bacterial colonization around dental implants. Further clinical studies should be conducted to confirm these findings.     

A weak base-generating system suitable for selective manipulation of lysosomal pH

pH varies widely among the different intracellular compartments. The establishment and maintenance of a particular pH appears to be critical for proper organellar function. This has been deduced from experiments where intraorganellar pH was altered by means of weak acids or bases, ionophores or inhibitors of the vacuolar H(+)-ATPase (V-ATPase). These manipulations, however, are not specific and simultaneously alter the pH of multiple compartments. As a result, it is difficult to assign their effect to a defined organelle. To circumvent this limitation, we designed and implemented a procedure to selectively manipulate the pH of a compartment of choice, using lysosomes as a model organelle. The approach is based on the targeted and continuous enzymatic generation of weak electrolyte, which enabled us to overcome the high buffering capacity of the lysosomal lumen, without altering the pH of other compartments. We targeted jack-bean urease to lysosomes and induced the localized generation of ammonia by providing the membrane-permeant substrate, urea. This resulted in a marked, rapid and fully reversible alkalinization that was restricted to the lysosomal lumen, without measurably affecting the pH of endosomes or of the cytosol. The acute alkalinization induced by urease-urea impaired the activity of pH-dependent lysosomal enzymes, including cathepsins C and L, without altering endosomal function. This approach, which can be extended to other organelles, enables the analysis of the role of pH in selected compartments, without the confounding effects of global disturbances in pH or vesicular traffic.