Silk fibroin hydrogels for potential applications in photodynamic therapy

In this study, we prepared translucid hydrogels with different concentrations of silk fibroin, extracted from raw silk fibers, and used them as a matrix to incorporate the photosensitizer 5-(4-aminophenyl)-10,15,20-tris-(4-sulphonatophenyl) porphyrin trisodium for application in photodynamic therapy (PDT). The hydrogels obtained were characterized by rheology, spectrophotometry, and scattering techniques to elucidate the factors involved in the formation of the hydrogel, and to characterize the behavior of silk fibroin (SF) after incorporating of the porphyrin to the matrix. The rheology results demonstrated that the SF hydrogels had a shear thinning behavior. In addition, we were able to verify that the structure of the material was able to be recovered over time after shear deformation. The encapsulation of porphyrins in hydrogels leads to the formation of self-assembled peptide nanostructures that prevent porphyrin aggregation, thereby greatly increasing the generation of singlet oxygen. Also, our findings suggest that porphyrin can diffuse out of the hydrogel and permeate the outer skin layers. This evidence suggests that SF hydrogels could be used as porphyrin encapsulation and as a drug carrier for the sustained release of photosensitizers for PDT.     

Oligopeptide Signaling through TbGPR89 Drives Trypanosome Quorum Sensing

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Absolute configuration of phomactatriene diterpenoids obtained by Wagner‐Meerwein rearrangement of epimeric verticillols

The epimeric diterpenes (+)‐(1S,3E,7E,11S,12S)‐verticilla‐3,7‐dien‐12‐ol ( 1 ), isolated from Bursera suntui, and (+)‐(1S,3E,7E,11S,12R)‐verticilla‐3,7‐dien‐12‐ol ( 2 ), isolated from Bursera kerberi, gave the same Wagner‐Meerwein rearrangement product (?)‐(1E,4Z,8Z,11S,12R)‐phomacta‐1,(15)4,8‐triene ( 3 ). The Et₂O:BF₃‐induced transformations evidence that verticillenes and phomactanes, both containing the bicyclo[9.3.1]pentadecane skeleton, are biogenetically related through the verticillen‐12‐yl cation ( A ⁺ ), which also is a key intermediate in the biosynthetic pathways to generate antitumor taxanes. Molecular modeling using the Monte Carlo protocol, followed by density functional theory (DFT) geometry optimization employing the hybrid functionals B3LYP and B3PW91, both with the DGDZVP basis set, secured the configuration of 3 as followed from the good agreement between the calculated and experimental vibrational circular dichroism spectra. Similar DFT calculations allowed determining the absolute configuration of (+)‐(1R,4R,5R,8S,9S,11S,12R,15R)‐1,15:4,5:8,9‐triepoxyphomactane ( 9 ), which surprisingly derives from epoxidation of the second minimum energy conformer of 3 .     

Studies related to progesterone-induced hamster sperm acrosome reaction

Experiments were designed to characterize the effect of progesterone on the hamster sperm acrosome reaction (AR). Progesterone stimulated exocytosis of previously capacitated spermatozoa in a dose-dependent manner Progesterone-3-(O-carboxymethyl)oxime:BSA conjugate also induced AR when added to capacitated sperm suspensions. EGTA and La³⁺, added 10 min before progesterone, completely abolished the steroid-stimulatory effect. Benzamidine, a trypsin inhibitor, also inhibited AR when added to sperm cells 10 min before progesterone. This effect was avoided when spermatozoa were treated with the Ca2+ ionophore ionomycin. Conversely, the H+ ionophore PCCP, or the Na⁺/K⁺ ionophore nigericin, did not prevent the effect of the inhibitor. Results suggest that progesterone acts on the hamster sperm plasma membrane to stimulate exocytosis, which requires external Ca2+ and presumably Ca2+ influx. In addition, a sperm trypsin like protease may be part of the mechanism by which progesterone stimulates AR. Since the ionomycin-induced AR does not require this proteolytic activity, the possible involvement of such an enzyme in the progesterone-stimulated Ca2+ influx necessary for the occurrence of AR is discussed. © 1996 Wiley-Liss, Inc.     

High prevalence of low HDL cholesterol concentrations and mixed hyperlipidemia in a Mexican nationwide survey

The prevalence of lipid abnormalities revealed in a survey done in 417 Mexican cities is described. Information was obtained on 15,607 subjects, aged 20 to 69 years. In this report, only samples obtained after a 9- to 12-h fast were included (2,256 cases: 953 men and 1,303 women). The population is representative of Mexican urban adults. Mean lipid concentrations were: cholesterol, 4.80 mmol/l; triglycerides, 2.39 mmol/l; HDL cholesterol, 1.00 mmol/l; and LDL cholesterol, 3.06 mmol/l. The most prevalent abnormality was HDL cholesterol below 0.9 mmol/l (46.2% for men and 28.7% for women). Hypertriglyceridemia (>2.26 mmol/l) was the second most prevalent abnormality (24.3%). Severe hypertriglyceridemia (>11.2 mmol/l) was observed in 0.42% of the population. Increased LDL cholesterol (> or =4.21 mmol/l) was observed in 11.2% of the sample. Half of the hypertriglyceridemic subjects had a mixed dyslipidemia or low HDL cholesterol. More than 50% of the low HDL cholesterol cases were not related to hypertriglyceridemia. Insulin resistance was found in 59% of them. In conclusion, the prevalence of hypoalphalipoproteinemia and other forms of dyslipidemia in Mexican adults is very high and it is among the highest previously reported worldwide.     

DNA breakage due to metronidazole treatment

The mutagenicity of metronidazole [1-(hidroxyethyl)-2-methyl-5-nitroimidazole] (MTZ) has been shown in different prokaryotic systems. However, data on human cells are still contradictory. In this study DNA damage was determined by the single cell gel electrophoresis (SCGE) assay, in lymphocytes from 10 healthy subjects treated with therapeutic doses of this drug. Samples were obtained before treatment, as well as 1 and 15 days after ending treatment. Results showed a significant increase of DNA strand breaks 1 day after ending treatment, although, an inverse correlation between the amount of DNA damage and plasma concentrations of MTZ was obtained. Thus, the observed damage may be induced by some MTZ metabolite rather than by the parent drug. Interestingly, the amount of DNA damage returned to basal levels 15 days after ending treatment, except in two individuals. This persistent damage should be further investigated.     

Monooxygenases involved in GA12 and GA14 synthesis in Gibberella fujikuroi

A microsomal preparation from mycelia of the gibberellin (GA)-producing fungus Gibberella fujikuroi catalyzed the first two steps in the conversion of the biosynthetic intermediate GA12-aldehyde to gibberellic acid (GA3). [14C]GA12-Aldehyde was converted to radiolabelled GA14, the major product, together with smaller amounts of non-hydroxylated GA12. The microsomal activities required reduced pyridine nucleotides and molecular oxygen. However, GA12 and GA14 synthesis differed markedly in the preferred electron source. Formation of GA12 required NADH or NADPH, while GA14 synthesis from GA12-aldehyde occurred only with NADPH. Marked differences were also found in the activating effect of FAD. When NADPH was the reductant, the rate of GA14 synthesis was enhanced 3.5 times by 5 microM FAD while this flavin nucleotide did not alter the synthesis of GA12. In contrast, GA12 synthesis was activated 3.8 times by 50 microM FAD in the presence of NADH. Both activities were inhibited by carbon monoxide and cytochrome c. These properties suggest that the 3beta-hydroxylation of GA12-aldehyde and further oxidation of carbon 7 are catalyzed by cytochrome P-450 monooxygenases in Gibberella fujikuroi.     

Modulation of endocytic traffic in polarized Madin-Darby canine kidney cells by the small GTPase RhoA

Efficient postendocytic membrane traffic in polarized epithelial cells is thought to be regulated in part by the actin cytoskeleton. RhoA modulates assemblies of actin in the cell, and it has been shown to regulate pinocytosis and phagocytosis; however, its effects on postendocytic traffic are largely unexplored. To this end, we expressed wild-type RhoA (RhoAWT), dominant active RhoA (RhoAV14), and dominant inactive RhoA (RhoAN19) in Madin-Darby canine kidney (MDCK) cells expressing the polymeric immunoglobulin receptor. RhoAV14 expression stimulated the rate of apical and basolateral endocytosis, whereas RhoAN19 expression decreased the rate from both membrane domains. Polarized basolateral recycling of transferrin was disrupted in RhoAV14-expressing cells as a result of increased ligand release at the apical pole of the cell. Degradation of basolaterally internalized epidermal growth factor was slowed in RhoAV14-expressing cells. Although apical recycling of immunoglobulin A (IgA) was largely unaffected in cells expressing RhoAV14, transcytosis of basolaterally internalized IgA was severely impaired. Morphological and biochemical analyses demonstrated that a large proportion of IgA internalized from the basolateral pole of RhoAV14-expressing cells remained within basolateral early endosomes and was slow to exit these compartments. RhoAN19 and RhoAWT expression had little effect on these postendocytic pathways. These results indicate that in polarized MDCK cells activated RhoA may modulate endocytosis from both membrane domains and postendocytic traffic at the basolateral pole of the cell.     

Genomic compatibility between two phyllotine rodent species evaluated through their hybrids

In order to investigate the genomic compatibility between allopatric rodent species, Phyllotis darwini and Phyllotis magister, we have studied several cytogenetic and reproductive features of their laboratory hybrids. Of thirty-one pairings between species, only five were successful, producing eleven newborns. Like parents, hybrids had 38 metacentric chromosomes, except for the subtelocentric Y chromosome inherited from P. magister. There was almost total C and G band correspondence between homeologous autosomes. However, parental sex chromosomes had different morphology, C and G bands. Ag-NOR bands appeared as small telomeric Ag+ regions, distributed in four chromosomal pairs of darwini, three of magister and four homeologous chromosomes of the hybrids. The three forms had similar indexes of NOR activity per cell, in spite of the variability in NOR expression which was always detected. Usually, only one member of parental homologous chromosomes showed AgNOR+; nevertheless, both homeologous chromosomes were active in many hybrid cells. The frequencies of cells that expressed their ribosomal genes in the two homologous or homeologous NOR chromosomes were similar in parental and hybrid cells. These results strongly suggest that ribosomal genes of both parental genomes would function codominantly in the hybrids. The gonad histological and morphometric analyses showed that hybrids conformed to Haldane's rule, since females were fertile and males were infertile. Our results indicate that P. darwini and P. magister genomes can function in relative harmony and compatibility when they are placed together in their laboratory generated hybrids, suggesting that these species have few genetic differences, probably because they have recently diverged.