Chemical synthesis and optical, structural, and surface characterization of InP-In2O3 quantum dots.

InP-In2O3 colloidal quantum dots (QDs) synthesized by a single-step chemical method without injection of hot precursors (one-pot) were investigated. Specifically, the effect of the tris(trimethylsilyl)phosphine, P(TMS)3, precursor concentration on the QDs properties was studied to effectively control the size and shape of the samples with a minimum size dispersion. The effect of the P(TMS)3 precursor concentration on the optical, structural, chemical surface, and electronic properties of InP-In2O3 QDs is discussed. The absorption spectra of InP-In2O3 colloids, obtained by both UV-Vis spectrophotometry and photoacoustic spectroscopy, showed a red-shift in the high-energy regime as the concentration of the P(TMS)3 increased. In addition, these results were used to determine the band-gap energy of the InP-In2O3 nanoparticles, which changed between 2.0 and 2.9 eV. This was confirmed by Photoluminescence spectroscopy, where a broad-band emission displayed from 2.0 to 2.9 eV is associated with the excitonic transition of the InP and In2O3 QDs. In2O3 and InP QDs with diameters ranging approximately from 8 to 10 nm and 6 to 9 nm were respectively found by HR-TEM. The formation of the InP and In2O3 phases was confirmed by X-ray Photoelectron Spectroscopy.

Detection and differentiation of Entamoeba histolytica and Entamoeba dispar in clinical samples through PCR-denaturing gradient gel electrophoresis.

Amebiasis is one of the twenty major causes of disease in Mexico; however, the diagnosis is difficult due to limitations of conventional microscopy-based techniques. In this study, we analyzed stool samples using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) to differentiate between Entamoeba histolytica (pathogenic) and E. dispar (non-pathogenic). The target for the PCR amplification was a small region (228 bp) of the adh112 gene selected to increase the sensitivity of the test. The study involved 62 stool samples that were collected from individuals with complaints of gastrointestinal discomfort. Of the 62 samples, 10 (16.1%) were positive for E. histolytica while 52 (83.9%) were negative. No sample was positive for E. dispar. These results were validated by nested PCR-RFLP (restriction fragment length polymorphism) and suggest that PCR-DGGE is a promising tool to differentiate among Entamoeba infections, contributing to determine the specific treatment for patients infected with E. histolytica, and therefore, avoiding unnecessary treatment of patients infected with the non-pathogenic E. dispar.

Botulinum neurotoxin type A inhibits synaptic vesicle 2 expression in breast cancer cell lines.

AIM: It is known that botulinum neurotoxin type A (BoNTA) improves some kinds of cancer (e.g. prostate) and that synaptic vesicle glycoprotein 2 (SV2) is the molecular target of this neurotoxin. Besides having potential therapeutic value, this glycoprotein has recently been proposed as a molecular marker for several types of cancer. Although the mechanisms of cancer development and the improvement found with botulinum treatment are not well understood, the formation of the botulinum-SV2 complex may influence the presence and distribution of SV2 and the function of vesicles. To date, there are no reports on the possible effect of botulinum on breast cancer of unknown causes, which have a great impact on women's health. Thus we determined the presence of SV2 in three breast cancer cell lines and the alterations found with botulinum application. MATERIALS AND METHODS: With and without adding 10 units of botulinum, SV2 protein expression was determined by optical densitometry in T47D, MDA-MB-231 and MDA-MB-453 cell lines and the distribution of SV2 was observed with immunochemistry (hematoxylin staining). RESULTS: The SV2 protein was abundant in the cancer cells herein tested, and maximally so in T47D. In all three cancer cell lines botulinum diminished SV2 expression, which was found mostly in the cell periphery. CONCLUSION: SV2 could be a molecular marker in breast cancer. Its expression and distribution is regulated by botulinum, suggesting an interesting control mechanism for SV2 expression and a possible alternative therapy. Further studies are needed in this sense.

Synaptic vesicle protein 2 (SV2) isoforms.

New molecular markers of cancer had emerged with novel applications in cancer prevention and therapeutics, including for breast cancer of unknown causes, which has a high impact on the health of women worldwide. The purpose of this research was to determine protein and mRNA expression of synaptic vesicle 2 (SV2) isoforms A, B and C in breast cancer cell lines. Cultured cell lines MDA-MB-231, SKBR3, T47D were lysed and their protein and mRNA expression analyzed by real-time PCR and western blot technique, respectively. SV2A, B proteins were identified in non-tumor (MCF-10A) and tumor cell lines (MDA-MB-231 and T47D) while SV2C only was found in the T47D cell line. Furthermore, the genomic expression was consistent with protein expression for a such cell line, but in MDA-MB-231 there was no SV2B genomic expression, and the SV2C mRNA and protein were not found in the non tumoral cell line. These findings suggest a possible cellular transdifferentiation to neural character in breast cancer, of possible relevance to cancer development, and point to possible use of SV2 as molecular marker and a vehicle for cancer treatment with botulinum toxin.

Entamoeba histolytica: DNA carrier vesicles in nuclei and kinetoplast-like organelles (EhkOs).

Entamoeba histolytica, the protozoan responsible for human amoebiasis, has a complex genome, whose linear chromosomes and DNA circles have so far eluded detailed analysis. We report the detection by transmission electron microscopy of nuclear vesicles (0.05-0.3 microm in diameter) carrying DNA in E. histolytica trophozoites. In late anaphase many of these nuclear vesicles were found to be organized in structures of approximately 2.5 x 1 microm, in association with chromosomes and microtubules. In glutaraldehyde-fixed and detergent-treated trophozoites, nuclear vesicles displayed a non-membranous envelope. Binding of phosphotungstate stain and recognition by serum from patients with systemic lupus erythematosus indicated that these vesicles contain DNA. Similar DNA carrier vesicles were found in the cytoplasm and in the E. histolytica kinetoplast-like organelle (EhkO). By Feulgen staining, we detected DNA carrier vesicles entering or leaving the nuclei, suggesting a structural relationship between the nuclear vesicles and the vesicles present in the EhkOs.

Circular and linear DNA molecules in the Entamoeba histolytica complex molecular karyotype.

Entamoeba histolytica genome was analysed by pulsed field gel electrophoresis under conditions to separate linear chromosomes in the 170-1400 kb range. We identified linear DNA molecules of 227, 366, 631, 850, 1112 and 1361 kb (mean sizes obtained by three different methods) and we estimated their reorientation times and migration velocities at various experimental conditions. DNA shift mobility assays, using ethidium bromide, suggested that bands migrating at 227 and 631 kb contain linear and circular DNA, whereas a band at 436 kb has only circular DNA. We obtained a regression equation relating sizes of supercoiled DNA molecules with their migration velocities during a pulse at constant electric field and temperature. We also developed a computer program (EHPATTERNS) that predicts the migration per pulse and the resolution order of circular and linear E. histolytica DNA at different pulse times and constant driving and frictional forces. The simulation showed that linear DNA molecules frequently co-migrate with circular molecules, but circular molecules change when the pulse time varies. This molecular mixture generates broad bands and difficulties in the interpretation of the molecular karyotype of E. histolytica.

Yeast exoglucanases. Where redundancy implies necessity.

Three exoglucanase genes have been described in Saccharomyces cerevisiae. The bulk of the exoglucanase (EXO) activity is encoded by the EXG1 gene, whose primary gene product is differentially glycosylated during its transit to the cell surface to yield three isoenzymes: EXOI, EXOII and EXOII1/2. EXOII, the major isoenzyme, carries two short oligosaccharides, each one consisting of an inner core with a single branch of the outer chain, attached to both potential glycosylation sites present in the molecule (Asn165 and Asn325). EXOI and EXOII1/2 are minor representatives. The second carries a single short sugar residue attached to Asn165 whereas the former elongate the outer chain of, at least, one oligosaccharide as other cell wall mannoproteins. The protein portion of the EXGI gene product is cleaved in Golgi by the Kex2 protease. A different exoglucanase, encoded by a second gene (EXG2), has been characterized as a heavily glycosylated, membrane bound 200 kDa glycoprotein. Finally, a third exoglucanase, encoded by the SSG gene, is synthesized during sporulation of diploids. Exoglucanases similar to those encoded by the EXG1 gene have been detected in other yeasts and characterized in depth in Candida albicans. The three polypeptides from S. cerevisiae and its counterpart from C. albicans have several conserved regions occupying the same relative positions. Studies on the function of these highly conserved enzymes are rather inconclusive.

The major exoglucanase from Candida albicans: a non-glycosylated secretory monomer related to its counterpart from Saccharomyces cerevisiae.

Exoglucanases secreted by two different strains from Candida albicans have been purified to homogeneity. The purified enzyme from each strain behaved as a non-glycosylated monomer (molecular weight 38,000) that was identical in terms of sodium dodecyl sulphate/polyacrylamide gel electrophoresis comigration, amino acid analysis and amino terminal sequence. The amino acid composition was similar to that of the major exoglucanase from Saccharomyces cerevisiae. In addition, these two enzymes displayed a 50% homology in the first 35 amino acids of the amino terminus. Antibodies against the deglycosylated exoglucanase (treated with Endo H) from S. cerevisiae were reactive with the exoglucanase from C. albicans and vice versa. Immunoblotting proved to be a semiquantitative method to detect C. albicans antigen in culture fluids. The exoglucanase from C. albicans appears to enter the secretory pathway without undergoing N-glycosylation.

Endogenous activity of cyclic nucleotide-dependent protein kinase in plasma membranes isolated from Strongylocentrotus purpuratus sea urchin sperm.

Activity of cyclic nucleotide-dependent protein kinase was investigated in flagellar plasma membranes of sea urchin sperm (S. purpuratus). Membranes incubated with [gamma-32P]ATP showed in the presence of 1 microM cAMP an increased phosphorylation in multiple polypeptides. Half maximal response was seen at 0.6 microM of cAMP. In contrast, higher concentrations (100 microM) of cGMP were required to cause the same amount of protein phosphorylation. 80% of the protein kinase activity stimulatable by cAMP was resistant to extraction by 10 mM EGTA and sonication but it was entirely recovered in a detergent-solubilized fraction. Membranes pretreated with 200 microM cAMP, ultracentrifuged and resuspended in buffer solution did not undergo cAMP-stimulated phosphorylation in their polypeptides. This study demonstrates that flagellar plasma membranes isolated from S. purpuratus sea urchin sperm have an endogenous cAMP-dependent protein kinase, which may be bound to the membrane via its regulatory subunit.