Taurine deficiency in maned wolves (Chrysocyon brachyurus) maintained on two diets manufactured for prevention of cystine urolithiasis

This study assesses the long‐term effects of an experimental diet vs. a commercially available manufactured diet, intended to reduce clinical disease related to cystinuria, on the taurine status of captive maned wolves. For 13 weeks, two pairs of maned wolves were maintained on the commercially available maintenance diet, whereas two individually housed wolves were maintained on the experimental diet. All six wolves, at the beginning and at the end of the diet trial, had severely decreased plasma concentrations of taurine (as compared to the normal canine reference range of 60–120 nmol/ml) (National Research Council [2003] National Academies Press) with average taurine concentrations of 16 nmol/ml at the beginning of the study and 3 nmol/ml at the end of the study. There was no statistically significant difference in the taurine concentrations between animals on the maintenance vs. experimental diets. Both diets were supplemented subsequently with taurine at a concentration of 0.3%. All study animals were eventually switched to the taurine‐supplemented version of the commercially manufactured maintenance diet and subsequent samplings were carried out to monitor plasma taurine concentrations. A final sampling, carried out approximately 5 months after the initiation of taurine supplementation, showed an average taurine concentration within the target canine reference range (90.25 nmol/ml). There are numerous physiologic (e.g., possible unique metabolism and requirements for taurine in this species as compared to other canids) and dietary factors (e.g., effects of the types and concentrations of fiber and protein on nutrient availability, taurine metabolism, and enterohepatic circulation of taurine‐conjugated bile salts; impaired taurine synthesis secondary to low cysteine availability) that could be potential contributors to the development of taurine deficiency in the maned wolves in this study. Taurine supplementation should be considered in maned wolves maintained on diets intended for reduction of cystinuria‐related complications. Zoo Biol 0:1–14, 2005. © 2005 Wiley‐Liss, Inc.     

Effects of acrylamide, latrunculin, and nocodazole on intracellular transport and cytoskeletal organization in melanophores

The effects of acrylamide (ACR), nocodazole, and latrunculin were studied on intracellular transport and cytoskeletal morphology in cultured Xenopus laevis melanophores, cells that are specialized for regulated and bidirectional melanosome transport. We used three different methods; light microscopy, fluorescence microscopy, and spectrophotometry. ACR affected the morphology of both microtubules and actin filaments in addition to inhibiting retrograde transport of melanosomes but leaving dispersion unaffected. Using the microtubule-inhibitor nocodazole and the actin filament-inhibitor latrunculin we found that microtubules and actin filaments are highly dependent on each other, and removing either component dramatically changed the organization of the other. Both ACR and latrunculin induced bundling of microtubules, while nocodazole promoted formation of filaments resembling stress fibers organized from the cell center to the periphery. Removal of actin filaments inhibited dispersion of melanosomes, further concentrated the central pigment mass in aggregated cells, and induced aggregation even in the absence of melatonin. Nocodazole, on the other hand, prevented aggregation and caused melanosomes to cluster and slowly disperse. Dispersion of nocodazole-treated cells was induced upon addition of alpha-melanocyte-stimulating hormone (MSH), showing that dispersion can proceed in the absence of microtubules, but the distribution pattern was altered. It is well established that ACR has neurotoxic effects, and based on the results in the present study we suggest that ACR has several cellular targets of which the minus-end microtubule motor dynein and the melatonin receptor might be involved. When combining morphological observations with qualitative and quantitative measurements of intracellular transport, melanophores provide a valuable model system for toxicological studies.     

Replication protein A directs loading of the DNA damage checkpoint clamp to 5'-DNA junctions

The heterotrimeric checkpoint clamp comprises the Saccharomyces cerevisiae Rad17, Mec3, and Ddc1 subunits (Rad17/3/1, the 9-1-1 complex in humans). This DNA damage response factor is loaded onto DNA by the Rad24-RFC (replication factor C-like complex with Rad24) clamp loader and ATP. Although Rad24-RFC alone does not bind to naked partial double-stranded DNA, coating of the single strand with single-stranded DNA-binding protein RPA (replication protein A) causes binding of Rad24-RFC via interactions with RPA. However, RPA-mediated binding is abrogated when the DNA is coated with RPA containing a rpa1-K45E (rfa1-t11) mutation. These properties allowed us to determine the role of RPA in clamp-loading specificity. The Rad17/3/1 clamp is loaded with comparable efficiency onto naked primer/template DNA with either a 3'-junction or a 5'-junction. Remarkably, when the DNA was coated with RPA, loading of Rad17/3/1 at 3'-junctions was completely inhibited, thereby providing specificity to loading at 5'-junctions. However, Rad17/3/1 loaded at 5'-junctions can slide across double-stranded DNA to nearby 3'-junctions and thereby affect the activity of proteins that act at 3'-termini. These studies show a unique specificity of the checkpoint loader for 5'-junctions of RPA-coated DNA. The implications of this specificity for checkpoint function are discussed.     

Germinal center function in the spleen during simian HIV infection in rhesus monkeys

Infection with HIV-1, SIV, or simian HIV is associated with abnormalities in the number, size, and structure of germinal centers (GCs). To determine whether these histopathologic abnormalities are associated with abnormalities in Ab development, we analyzed nucleotide sequences of Igs from splenic GCs of simian HIV-infected macaques. Virus-specific GCs were identified in frozen splenic tissue sections by inverse immunohistochemistry using rHIV-1 gp120 as a probe. B cells from envelope-specific GCs were isolated from these sections using laser capture microdissection. Their Igs were amplified from cDNA using nested PCR, then cloned and sequenced. Nucleotide sequences were recovered from nine multimember clonal lineages. Within each lineage, sequences had similar V-D-J or V-J junctions but differed by somatic mutations distributed throughout the variable domain. The clones were highly mutated, similar to that previously reported for HIV-1-specific human IgG Abs. The average clone had 37 mutations in the V region, for a frequency of 0.11 mutations/base. The mutational pattern was strikingly nonrandom, with somatic mutations occurring preferentially at RGYW/WRCY hotspots. Transition mutations were favored over transversions, with C-->T and G-->A replacements together accounting for almost one-third of all mutations. Analysis of replacement and silent mutations in the framework and CDRs suggests that the Igs were subjected to affinity selection. These data demonstrate that the process of Ab maturation is not seriously disrupted in GCs during the early stages of immunodeficiency virus infection, and that Env-specific Igs developing in GCs are subject to extensive somatic mutation and profound selection pressures.     

Salmonella typhimurium coordinately regulates FliC location and reduces dendritic cell activation and antigen presentation to CD4+ T cells

During infection, Salmonella transitions from an extracellular-phase (STEX, growth outside host cells) to an intracellular-phase (STIN, growth inside host cells): changes in gene expression mediate survival in the phagosome and modifies LPS and outer membrane protein expression, including altered production of FliC, an Ag recognized by immune CD4+ T cells. Previously, we demonstrated that systemic STIN bacteria repress FliC below the activation threshold of FliC-specific T cells. In this study, we tested the hypothesis that changes in FliC compartmentalization and bacterial responses triggered during the transition from STEX to STIN combine to reduce the ability of APCs to present FliC to CD4+ T cells. Approximately 50% of the Salmonella-specific CD4+ T cells from Salmonella-immune mice were FliC specific and produced IFN-gamma, demonstrating the potent immunogenicity of FliC. FliC expressed by STEX bacteria was efficiently presented by splenic APCs to FliC-specific CD4+ T cells in vitro. However, STIN bacteria, except when lysed, expressed FliC within a protected intracellular compartment and evaded stimulation of FliC-specific T cells. The combination of STIN-mediated responses that reduced FliC bioavailability were overcome by dendritic cells (DCs), which presented intracellular FliC within heat-killed bacteria; however, this ability was abrogated by live bacterial infection. Furthermore, STIN bacteria, unlike STEX, limited DC activation as measured by increased MHC class II, CD86, TNF-alpha, and IL-12 expression. These data indicate that STIN bacteria restrict FliC bioavailability by Ag compartmentalization, and together with STIN bacterial responses, limit DC maturation and cytokine production. Together, these mechanisms may restrain DC-mediated activation of FliC-specific CD4+ T cells.     

Breast cancer resistance protein (BCRP/ABCG2) is expressed by progenitor cells/reactive ductules and hepatocytes and its expression pattern is influenced by disease etiology and species type: possible functional consequences.

Breast cancer resistance protein (BCRP/ABCG2) is an ATP-binding cassette transport protein that is expressed in several organs including the liver. Previous studies have shown that ABC transport proteins play an important pathophysiological role in several liver diseases. However, to date, expression pattern and possible role of BCRP in human liver diseases and animal models have not been studied in detail. Here we investigated the expression pattern of BCRP in normal liver, chronic parenchymal and biliary human liver diseases, and parallel in different rat models of liver diseases. Expression was studied by immunohistochemistry and additionally by RT-PCR analysis in Thy-1-positive rat oval cells. Bile ducts, hepatic progenitor cells, reactive bile ductules, and blood vessel endothelium were immunoreactive for BCRP in normal liver and all types of human liver diseases and in rat models. BCRP was expressed by the canalicular membrane of hepatocytes in normal and diseased human liver, but never in rat liver. Remarkably, there was also expression of BCRP at the basolateral pole of human hepatocytes, and this was most pronounced in chronic biliary diseases. In conclusion, BCRP positivity in the progenitor cells/reactive ductules could contribute to the resistance of these cells to cytotoxic agents and xenotoxins. Basolateral hepatocytic expression in chronic biliary diseases may be an adaptive mechanism to pump bile constituents back into the sinusoidal blood. Strong differences between human and rat liver must be taken into account in future studies with animal models.     

Evaluation of six methods for extraction and purification of viral DNA from urine and serum samples

The sensitivity and reliability of PCR for diagnostic and research purposes require efficient unbiased procedures of extraction and purification of nucleic acids. One of the major limitations of PCR-based tests is the inhibition of the amplification process by substances present in clinical samples. This study used specimens spiked with a known amount of plasmid pBKV (ATCC 33-1) to compare six methods for extraction and purification of viral DNA from urine and serum samples based on recovery efficiency in terms of yield of DNA and percentage of plasmid pBKV recovered, purity of extracted DNA, and percentage of inhibition. The most effective extraction methods were the phenol/chloroform technique and the silica gel extraction procedure for urine and serum samples, respectively. Considering DNA purity, the silica gel extraction procedure and the phenol/chloroform method produced the most satisfactory results in urine and serum samples, respectively. The presence of inhibitors was overcome by all DNA extraction techniques in urine samples, as evidenced by semiquantitative PCR amplification. In serum samples, the lysis method and the proteinase K procedure did not completely overcome the presence of inhibitors.     

In vitro efficacy of Linezolid on clinical strains of Mycobacterium tuberculosis and other mycobacteria

Linezolid, an oxazolidinone that acts by inhibiting protein synthesis, was evaluated in strains of tuberculosis and non-tubercular mycobacteria resistant to one or more drugs isolated in northern Sardinia. The in vitro activity of Linezolid (Pfizer) was assessed on different isolates of Mycobacterium spp. from clinical samples by the Proportional Method. Linezolid demonstrated an excellent activity against the 24 strains of M. tuberculosis and against M. gordonae, M. marinum, M. aurum, M. phlei, and M. avium, with MIC values ranging from 0.5 to 2 microg/ml. Linezolid can be used in combination with the standard antitubercular medications, or as an effective therapeutic alternative in infections caused by M. tuberculosis or by other species of non-tubercular mycobacteria.