An in vitro test for measuring cytotoxicity of mercuric chloride to human kidney epithelial cells by specific enzyme release

Mercuric compound toxicity is well documented in animals and man for practically all organs. The recent development of cell culture techniques appeared as a novel fruitful tool in toxicology, especially in renal toxicology. Heavy metal induced renal cell alterations can be evaluated by membrane permeability damages.The present study evaluates mercuric chloride nephrotoxic effect in human kidney epithelial cells by measuring the release of two specific nephrotoxicity marker enzymes, Gamma Glutamyl Transferase (GGT) and Alkaline Phosphatase (ALP) in the culture medium. Cultured kidney epithelial cells were exposed to different HgCl₂ concentrations (5, 10, 20, and 50 g). Cultures were examined after 6 and 24 hours exposure. A good correlation between mercury dose and toxic effect, and exposure time and toxic effect was found. Enzymes were significantly released into the culture medium for 5 g and 10 g HgCl₂/ml after 6 hours exposure; and after 24 hours exposure, enzymes were released for 5 g/ml only.It appears that the specific tubular enzyme release in the culture medium is a good in vitro test for quantification of specific tubular damage.     

Preparation and biological activity of taxol acetates

Synthesis and biological activity of 2′-acetyltaxol and 7-acetyltaxol are reported. Activity is measured $\text{in}\text{vivo}$ by cytotoxicity toward the macrophage-like cell line J774.2, and $\text{in}\text{vitro}$ by promotion of microtubule assembly in the absence of exogenous GTP. Addition of an acetyl moiety at C-2′ results in loss of $\text{in}\text{vitro}$ activity but not cytotoxicity. The properties of 7-acetyltaxol are similar to those of taxol in its effects on cell replication and on $\text{in}\text{vitro}$ microtubule polymerization. Therefore a free hydroxyl group at C-7 is not required for $\text{in}\text{vitro}$ activity and this position is available for structural modifications.     

Construction of Novel Shuttle Vectors for Use between Moderately Halophilic Bacteria andEscherichia coli

Shuttle vectors useful for the genetic manipulation of several moderately halophilic bacteria have been constructed. These vectors are based on the minimal replicon of pCM1, a cryptic plasmid fromChromohalobacter marismortui,combined with the useful properties of pUC18 plasmid (i.e., small size, high copy number, multiple cloning sites,lacZfragment), as well as with the trimethoprim resistance gene as a selection marker for moderate halophiles. These vectors can be efficiently transferred by RP4-mediated conjugation fromEscherichia colito the moderate halophilesChromohalobacter marismortui, Deleya halophila, Halomonas elongata, Halomonas subglaciescola,andVolcaniella eurihalina.     

Efficient transformation of a Yersinia enterocolitica strain (serotype O:3) by pBR322 DNA

Abstract The Yersinia enterocolitica strain MS201 (serotype O:3) was transformed by pBR322 DNA extracted from an Escherichia coli strain. The pBR322 DNA extracted from an Escherichia coli strain. The pBR322 DNA extracted from the transformed Y. enterocolitica is able to transform plasmid-free MS201 at a significantly higher frequency, suggesting the existence of a restriction-modification system in MS201.     

Assembly of Bacillus subtilis phage phi29. 1. Mutants in the cistrons coding for the structural proteins.

The effect of mutations in the cistrons coding for the phage structural proteins has been studied by analyzing the phage-related structures accumulated after restrictive infection. Infection with susmutants in cistron 8, lacking both the major head and the fiber protein, does not produce any phage-related structure, suggesting a single route for the assembly of phage phi29; infection with ts mutants in this cistron produces isometric particles. Mutants is cistron 9, coding for the tail protein, TP1, produce DNA-free prolate heads with an internal core; these particles are abortive and contain the head proteins HPO, HP1 and HP3, the upper collar protein NP2 and the nonstructural proteins p7, p15 and p16. Mutants in cistron 10, coding for the upper collar protein, NP2, produce DNA-free isometric heads also with an internal core; they contain the head proteins and the nonstructural protein p7, suggesting that this protein forms the internal core. Mutants in cistrons 11 and 12, coding for the lower collar protein, NP3, and the neck appendages, NP1, respectively, give rise to the formation of DNA-containing normal capsids and DNA-free prolate particles, more rounded at the corners than the normal capsids and with an internal core; the DNA-containing 11-particles are formed by the head proteins and the upper collar protein; the DNA-free 11-particles contain, besides these proteins, the nonstructural protein p7 and a small amount of proteins p15 and 16. The DNA-containing 12-particles have all the normal phage structural proteins except the neck appendages, formed by protein NP1; the DNA-free particles are similar to the DNA-free 11-particles. After restricitive infection mutant sus14(1241) has a delayed lysis phenotype and produces a phage burst higher than normal, after artificial lysis. It produces DNA-containing particles, identical to wild-type phage, which have all the normal phage structural proteins, and DNA-free prolate particles, more rounded at the corners than the final phage particles and with an internal core; the last particles contain the same proteins as the DNA-free 11 or 12-particles. These particles could represent a prohead state, ready for DNA encapsulation. None of the DNA-containing particles have the nonstructural proteins p7, p15 or p16, suggesting that these proteins are released from the proheads upon DNA encapsulation.     

Breakthrough pulmonary Aspergillus fumigatus infection with multiple triazole resistance in a Spanish patient with chronic myeloid leukemia

BackgroundAn allogeneic hematopoietic cell transplantation (allo-HCT) patient presented with chronic pulmonary aspergillosis associated to pulmonary graft versus host disease (GVHD) and was treated for a long time with several antifungal agents that were administered as prophylaxis, combination therapies, and maintenance treatment. The patient suffered from a breakthrough invasive pulmonary aspergillosis due to Aspergillus fumigatus after long-term antifungal therapy.Material and methodsSeveral isolates were analyzed. First isolates were susceptible in vitro to all azole agents. However, after prolonged treatment with itraconazole and voriconazole a multiple azole resistant A. fumigatus isolate was cultured from bronchoalveolar lavage (BAL) when the patient was suffering from an invasive infection, and cavitary lesions were observed.ResultsAnalysis of the resistant mechanisms operating in the last strain led us to report the first isolation in Spain of an azole resistant A. fumigatus strain harboring the L98H mutation in combination with the tandem repeat (TR) alteration in CYP51A gene (TR-L98H). Long-term azole therapy may increase the risk of resistance selecting strains exhibiting reduced susceptibility to these compounds. However, since the isolates were genetically different the suggestion that could be made is that the resistance was not induced during the prolonged azole therapy but the patient might simply have acquired this resistant isolate from the environment, selected by the therapy.ConclusionsThese findings suggest that in all long-term treatments with antifungal agents, especially with azoles, repeated sampling and regular susceptibility testing of strains isolated is necessary as resistant isolates could be selected.     

CXCL12-Mediated Murine Neural Progenitor Cell Movement Requires PI3Kβ Activation

The migratory route of neural progenitor/precursor cells (NPC) has a central role in central nervous system development. Although the role of the chemokine CXCL12 in NPC migration has been described, the intracellular signaling cascade involved remains largely unclear. Here we studied the molecular mechanisms that promote murine NPC migration in response to CXCL12, in vitro and ex vivo. Migration was highly dependent on signaling by the CXCL12 receptor, CXCR4. Although the JAK/STAT pathway was activated following CXCL12 stimulation of NPC, JAK activity was not necessary for NPC migration in vitro. Whereas CXCL12 activated the PI3K catalytic subunits p110α and p110β in NPC, only p110β participated in CXCL12-mediated NPC migration. Ex vivo experiments using organotypic slice cultures showed that p110β blockade impaired NPC exit from the medial ganglionic eminence. In vivo experiments using in utero electroporation nonetheless showed that p110β is dispensable for radial migration of pyramidal neurons. We conclude that PI3K p110β is activated in NPC in response to CXCL12, and its activity is necessary for immature interneuron migration to the cerebral cortex.