Preparative separation and purification of bufadienolides from ChanSu by high-speed counter-current chromatography combined with preparative HPLC

Eight bufadienolides were successfully isolated and purified from ChanSu by high-speed counter-current chromatography (HSCCC) combined with preparative HPLC (prep-HPLC). First, a stepwise elution mode of HSCCC with the solvent system composed of petroleum ether - ethyl acetate - methanol - water (4:6:4:6, 4:6:5:5, v/v) was employed and four bufadienolides, two partially purified fractions were obtained from 200 mg of crude extract. The partially purified fractions III and VI were then further separated by prep-HPLC, respectively, and another four bufadienolides were recovered. Their structures were confirmed by ESI-MS and ¹H-NMR spectra.

MICROWAVE ACTIVATION OF IMMOBILIZED LIPASE FOR TRANSESTERIFICATION OF VEGETABLE OILS

This work investigated the effect of microwave irradiation (MW) on the ethanolysis rate of soybean and sunflower oils catalyzed by supported Novozyme 435 (Candida antarctica). The effects of tert-butanol, water addition and oil:ethanol molar ratio on transesterification were evaluated under conventional heating (CH), and under optimum reaction conditions (with no added water in the system, 10% tert-butanol and 3:1 ethanol-to-oil molar ratio). The reactions were monitored up to 24 h to determine the conditions of initial reaction velocity. The investigated variables under MW (50 W) were: reaction time (5.0-180 min) and mode of reactor operation (fixed power, dynamic and cycles) in the absence and presence of tert-butanol (10% (w/w). The measured response was the reaction conversion in ethyl esters, which was linked to the enzyme catalytic activity. The results indicated that the use of microwave improved the activity at fixed power mode. A positive effect of the association of tert-butanol and MW irradiation on the catalytic activity was observed. The reaction rate improved in the order of approximately 1.5 fold compared to that under CH with soybean oil. Using soybean oil, the enzymatic transesterification under MW for conversion to FAEE (fatty acid ethyl esters) reached >99% in 3h, while with the use of CH the conversions were about 57% under similar conditions.

Spontaneous rupture of renal angiomyolipoma during pregnancy

Renal angiomyolipoma is a benign tumor, composed of adipocytes, smooth muscle cells and blood vessels. The association with pregnancy is rare and related with an increased risk of complications, including rupture with massive retroperitoneal hemorrhage. The follow-up is controversial because of the lack of known cases, but the priorities are: timely diagnosis in urgent cases and a conservative treatment when possible. The mode of delivery is not consensual and should be individualized to each case. We report a case of a pregnant woman with 18 weeks of gestation admitted in the emergency room with an acute right low back pain with no other symptoms. The diagnosis of rupture of renal angiomyolipoma was established by ultrasound and, due to hemodinamically stability, conservative treatment with imaging and clinical monitoring was chosen. At 35 weeks of gestation, it was performed elective cesarean section without complications for both mother and fetus.

Do induced twin pregnancies influence the obstetric and neonatal results of multiple births born before 32 weeks? Comparison to spontaneous gestation

PURPOSE: To compare obstetric outcomes of induced preterm twin births (under 32 weeks gestation) with those spontaneously conceived. METHODS: Prospective study of twin pregnancies (25 induced and 157 spontaneously conceived) developed over a period of 16 years in a tertiary obstetric center. Demographic factors, obstetric complications, gestational age at delivery, mode of delivery, birth weight and immediate newborn outcome were compared. RESULTS: The analysis of obstetrical complications concerning urinary or other infections, hypertensive disorders of pregnancy, gestational diabetes, fetal malformations, intrauterine fetal death, intrauterine growth restriction and intrauterine discordant growth reveal no significant statistical differences between the two groups. First trimester bleeding was higher in the induced group (24 versus 8.3%, p=0.029). The cesarean delivery rate was 52.2% in spontaneous gestations and 64% in induced gestations. Gestational age at delivery, birth weight, Apgar scores at first and fifth minutes, admissions to Neonatal Intensive Care Unit and puerperal complications show no statistically significant differences between the two groups. These results were independent of chorionicity and induction method. CONCLUSION: The mode of conception did not influence obstetric and neonatal outcomes. Although induced pregnancies have higher risk of first trimester bleeding, significant differences were not observed regarding other obstetric and puerperal complications and neonatal results.

Character transformations and their functional significance as a key to the evolution of hystricognath Rodentia

Hystricognathi represent a monophyletic taxon within Rodentia. Since phylogenetically analyzed morphological systems are essential for revealing evolutionary processes, this study identifies evolutionary character transformations on the stem lineage of Hystricognathi as derived from the author's own work and the literature. Data so far indicate that evolutionary transformations in the rostral head region, the loss of tactile ability in the outer nasal skin and the mobile arrangement of the associated cartilage, were allied with a switch from omnivorous to herbivorous and fiber-rich nutrition. Additional character transformations in the skull assist in digesting such food. Structures associated with reproduction and placentation show a remarkable pro portion of derived character conditions: the chorioallantoic placenta has a ring-shaped organization and growth structure which optimizes the capacity for passive diffusion, a subplacenta occurred as a specialized region responsible for placental invasion and the inverted yolk sac facilitates substance exchange with the main placenta. Finally, precocial newborns evolved as a derived condition within Rodentia. All things considered, a mode of reproduction is indicated, which does not demand excessive additional energy intake by the mother and is in accordance with her low energetic diet. Hystricognathi possess major character transformations that represent prerequisites for their successful radiation at the time when more open ecosystems and grasslands evolved during Earth history. The analysis resulted in the reconstruction of a life-near picture of the hystricognath stem species pattern with high explanatory power in terms of changes in space and time and their interdependence with biodiversity.

Fracture modes and acoustic emission characteristics of hydrogen-assisted cracking in high-strength low-alloy steel weldment

The aim of the present paper is to study the relationship between the fracture modes in hydrogen-assisted cracking (HAC) in microalloied steel and the emission of acoustic signals during the fracturing process. For this reason, a flux-cored arc weld (FCAW) was used in a high-strength low-alloy steel. The consumable used were the commercially available AWS E120T5-K4 and had a diameter of 1.6 mm. Two different shielding gases were used (CO2 and CO2+5% H2) to obtain complete phenomenon characterization. The implant test was applied with three levels of restriction stresses. An acoustic emission measurement system (AEMS) was coupled to the implant test apparatus. The output signal from the acoustic emission sensor was passed through an electronic amplifier and processed by a root mean square (RMS) voltage converter. Fracture surfaces were examined by scanning electron microscopy (SEM) and image analysis. Fracture modes were related with the intensity, the energy and the number of the peaks of the acoustic emission signal. The shielding gas CO2+5% H2 proved to be very useful in the experiments. Basically, three different fracture modes were identified in terms of fracture appearance: microvoid coalescence (MVC), intergranular (IG) and quasi-cleavage (QC). The results show that each mode of fracture presents a characteristic acoustic signal.

Anatomical and developmental aspects of leaf galls induced by Schizomyia macrocapillata Maia (Diptera: Cecidomyiidae) on Bauhinia brevipes Vogel (Fabaceae)

Samples of healthy leaves and galls induced by Schizomyia macrocapillata Maia on Bauhinia brevipes Vogel were submitted to routine techniques to investigate gall anatomy and development. Pouch galls are induced on the abaxial surface of unfolded immature leaves, and become spheroid with long reddish hairs covering their external surface. Galls occur isolated or coalesce when in larger numbers. Gall development was divided into six phases: 1) initiation; 2) tissue re-arrangement; 3) tissue differentiation; 4) maturation; 5) growth phase; and 6) dehiscence. This last phase corresponds to gall senescence, which takes place just after the larva exits the chamber to pupate. An important developmental phase of tissue reorientation was recorded after the initiation phase. The presence of hyphae close to the covering layer characterizes this gall as an ambrosia gall and the feeding mode of the gall migde is discussed. Few hyphae were found during the first developmental phases and fungi may play an important role during gall morphogenesis. Neoformed trichomes may provide not only photoprotection but also protection against natural enemies and water loss. The neoformation of phloematic bundles suggests host manipulation and indicates the establishment of a deviating sink.

Are there evidences of a complex mimicry system among Asclepias curassavica (Apocynaceae), Epidendrum fulgens (Orchidaceae), and Lantana camara (Verbenaceae) in Southern Brazil?

The goal of this paper was to test the presence of mimicry in Asclepias curassavica L., Epidendrum fulgens Brong., and Lantana camara L. The study was carried out at the Parque Estadual de Itapeva, RS, southern Brazil, from 2004 to 2006. Flowering period of each of the three species was followed up; focal observations of butterflies visiting flowers, from fixed point and during random walks were carried out. We also estimated the frequency of pollinaria removal in the orchid, as well as its mode of reproduction. All these variables were important for testing the mimicry hypothesis. Despite some temporal coincidences in the flowering period of two plants in the system, there was no statistical association among the three plants as to flowering period. Twenty-nine species of butterflies, as potential pollinators, were recorded, particularly Agraulis vanillae maculosa, Dryas iulia alcionea, Urbanus simplicius, Tegosa claudina, and Heliconius erato phyllis, which were the more frequent visitors of the three plants. There was association between the number of visits to L. camara and E. fulgens, based on Pearson correlation (r = 0.4603; n = 19; P = 0.0473). Pollinaria removal of E. fulgens was low, as measured by the percentage of removal (range: 0 - 10%). The analysis of the mode of reproduction of this orchid showed its pollinator-dependence, since no fruits were formed by spontaneous self-pollination. In contrast, the percentage of fruit set that resulted from geitonogamy and xenogamy was, in average, 86%. The results here shown are not conclusive as to the occurrence of a mimicry system among the three plants.

A novel polymorphism in the coding region of the vasopressin type 2 receptor gene

Nephrogenic diabetes insipidus (NDI) is a rare disease characterized by renal inability to respond properly to arginine vasopressin due to mutations in the vasopressin type 2 receptor (V2(R)) gene in affected kindreds. In most kindreds thus far reported, the mode of inheritance follows an X chromosome-linked recessive pattern although autosomal-dominant and autosomal-recessive modes of inheritance have also been described. Studies demonstrating mutations in the V2(R) gene in affected kindreds that modify the receptor structure, resulting in a dys- or nonfunctional receptor have been described, but phenotypically indistinguishable NDI patients with a structurally normal V2(R) gene have also been reported. In the present study, we analyzed exon 3 of the V2(R) gene in 20 unrelated individuals by direct sequencing. A C®T alteration in the third position of codon 331 (AGC®AGT), which did not alter the encoded amino acid, was found in nine individuals, including two unrelated patients with NDI. Taken together, these observations emphasize the molecular heterogeneity of a phenotypically homogeneous syndrome

Structural basis for the pathophysiology of lipoprotein(a) in the athero-thrombotic process

Lipoprotein Lp(a) is a major and independent genetic risk factor for atherosclerosis and cardiovascular disease. The essential difference between Lp(a) and low density lipoproteins (LDL) is apolipoprotein apo(a), a glycoprotein structurally similar to plasminogen, the precursor of plasmin, the fibrinolytic enzyme. This structural homology endows Lp(a) with the capacity to bind to fibrin and to membrane proteins of endothelial cells and monocytes, and thereby to inhibit plasminogen binding and plasmin generation. The inhibition of plasmin generation and the accumulation of Lp(a) on the surface of fibrin and cell membranes favor fibrin and cholesterol deposition at sites of vascular injury. Moreover, insufficient activation of TGF-ß due to low plasmin activity may result in migration and proliferation of smooth muscle cells into the vascular intima. These mechanisms may constitute the basis of the athero-thrombogenic mode of action of Lp(a). It is currently accepted that this effect of Lp(a) is linked to its concentration in plasma. An inverse relationship between Lp(a) concentration and apo(a) isoform size, which is under genetic control, has been documented. Recently, it has been shown that inhibition of plasminogen binding to fibrin by apo(a) is also inversely associated with isoform size. Specific point mutations may also affect the lysine-binding function of apo(a). These results support the existence of functional heterogeneity in apolipoprotein(a) isoforms and suggest that the predictive value of Lp(a) as a risk factor for vascular occlusive disease would depend on the relative concentration of the isoform with the highest affinity for fibrin

Absence of linkage between MHC and a gene involved in susceptibility to human schistosomiasis

Six hundred million people are at risk of infection by Schistosoma mansoni. MHC haplotypes have been reported to segregate with susceptibility to schistosomiasis in murine models. In humans, a major gene related to susceptibility/resistance to infection by S. mansoni (SM1) and displaying the mean fecal egg count as phenotype was detected by segregation analysis. This gene displayed a codominant mode of inheritance with an estimated frequency of 0.20-0.25 for the deleterious allele and accounted for more than 50% of the variance of infection levels. To determine if the SM1 gene segregates with the human MHC chromosomal region, we performed a linkage study by the lod score method. We typed for HLA-A, B, C, DR and DQ antigens in 11 informative families from an endemic area for schistosomiasis in Bahia, Brazil, by the microlymphocytotoxicity technique. HLA-DR typing by the polymerase chain reaction with sequence-specific primers (PCR-SSP) and HLA-DQ were confirmed by PCR-sequence-specific oligonucleotide probes (PCR-SSOP). The lod scores for the different q values obtained clearly indicate that there is no physical linkage between HLA and SM1 genes. Thus, susceptibility or resistance to schistosomiasis, as defined by mean fecal egg count, is not primarily dependent on the host's HLA profile. However, if the HLA molecule plays an important role in specific immune responses to S. mansoni, this may involve the development of the different clinical aspects of the disease such as granuloma formation and development of hepatosplenomegaly.

Two variants of HIV-1 B serotype are transmitted heterosexually in São Paulo, Brazil

HIV-1 variability may have an important impact on transmission and pathogenicity. Better characterization of the HIV epidemic in Brazil is necessary for the development of vaccine trials in this country. We analyzed sera from 108 HIV-1-infected volunteers from São Paulo City to determine serotype and reactivity for V3 motifs of HIV in this population, and the relationship to transmission mode. We concluded that the HIV-1 B serotype is frequent among heterosexually infected women, even in the absence of anal sex, and that two major V3 motifs, GPGR and GWGR, had similar prevalence among women (48% and 52%, respectively) and men (56% and 44%, respectively). We also observed an equal distribution of these strains regardless of their CD4+ T cell counts, clinical status, and mode of transmission. Even though V3 serology for HIV-1 subtyping is an inexpensive tool for use in developing countries, additional methods, such as heteroduplex mobility assay and direct DNA sequencing, should be included to determine HIV-1 genetic diversity.

Six years of treatment with the HELP system of a patient with familial hypercholesterolemia

The purpose of the present report is to demonstrate the long-term efficacy and safety of heparin-induced extracorporeal lipoprotein precipitation (HELP) of LDL-c and fibrinogen in the management of familial hypercholesterolemia. From June 1992 to June 1998 a 22-year-old young male patient with familial hypercholesterolemia (double heterozygote for C660X and S305C) resistant to medication and diet and with symptomatic coronary artery disease (angina) was treated weekly with 90-min sessions of the HELP system. The patient had also been previously submitted to right coronary artery angioplasty. The efficacy of the method was evaluated by comparing the reduction of total cholesterol, LDL-c and fibrinogen before and after the sessions and before and after initiation of the study (data are reported as averages for each year). During the study, angina episodes disappeared and there were no detectable adverse effects of the treatment. Total cholesterol (TC), fibrinogen, and LDL-c decreased significantly after each session by 59.6, 66.1 and 64%, respectively. HDL-c showed a nonsignificant reduction of 20.4%. Comparative mean values pre- and post-treatment values in the study showed significant differences: TC (488 vs 188 mg/dl), LDL-c (416.4 vs 145 mg/dl), and fibrinogen (144.2 vs 57.4 mg/dl). There was no significant change in HDL-c level: 29.4 vs 23 mg/dl. These data show that the HELP system, even for a long period of time, is a safe and efficient mode of treatment of familial hypercholesterolemia and is associated with disappearance of angina symptoms.

Role of Tamm-Horsfall protein and uromodulin in calcium oxalate crystallization

One of the defenses against nephrolithiasis is provided by macromolecules that modulate the nucleation, growth, aggregation and retention of crystals in the kidneys. The aim of the present study was to determine the behavior of two of these proteins, Tamm-Horsfall and uromodulin, in calcium oxalate crystallization in vitro. We studied a group of 10 male stone formers who had formed at least one kidney stone composed of calcium oxalate. They were classified as having idiopathic nephrolithiasis and had no well-known metabolic risk factors involved in kidney stone pathogenesis. Ten normal men were used as controls, as was a group consisting of five normal women and another consisting of five pregnant women. Crystallization was induced by a fixed supersaturation of calcium oxalate and measured with a Coulter Counter. All findings were confirmed by light and scanning electron microscopy. The number of particulate material deposited from patients with Tamm-Horsfall protein was higher than that of the controls (P<0.001). However, Tamm-Horsfall protein decreased the particle diameter of the stone formers when analyzed by the mode of the volume distribution curve (P<0.002) (5.64 ± 0.55 µm compared to 11.41 ± 0.48 µm of uromodulin; 15.94 ± 3.93 µm and 12.45 ± 0.97 µm of normal men Tamm-Horsfall protein and uromodulin, respectively; 8.17 ± 1.57 µm and 9.82 ± 0.95 µm of normal women Tamm-Horsfall protein and uromodulin, respectively; 12.17 ± 1.41 µm and 12.99 ± 0.51 µm of pregnant Tamm-Horsfall protein and uromodulin, respectively). Uromodulin produced fewer particles than Tamm-Horsfall protein in all groups. Nonetheless, the total volume of the crystals produced by uromodulin was higher than that produced by Tamm-Horsfall protein. Our results indicate a different effect of Tamm-Horsfall protein and uromodulin. This dual behavior suggests different functions. Tamm-Horsfall protein may act on nucleation and inhibit crystal aggregation, while uromodulin may promote aggregation of calcium oxalate crystals.

Effects of mercury intoxication on the response of horizontal cells of the retina of thraira fish (Hoplias malabaricus)

Methyl mercury (MeHg) is highly neurotoxic, affecting visual function in addition to other central nervous system functions. The effect of mercury intoxication on the amplitude of horizontal cell responses to light was studied in the retina of the fish Hoplias malabaricus. Intracellular responses were recorded from horizontal cells of fish previously intoxicated with MeHg by intraperitoneal injection (IP group) or by trophic exposure (T group). Only one retina per fish was used. The doses of MeHg chloride administered to the IP group were 0.01, 0.05, 0.1, 1.0, 2.0, and 6.0 mg/kg. The amplitudes of the horizontal cell responses were lower than control in individuals exposed to 0.01 (N = 4 retinas), 0.05 (N = 2 retinas) and 0.1 mg/kg (N = 1 retina), whereas no responses were recorded in the 1.0, 2.0, and 6.0 mg/kg groups. T group individuals were fed young specimens of Astyanax sp previously injected with MeHg corresponding to 0.75 (N = 1 retina), 0.075 (N = 8 retinas) or 0.0075 (N = 4 retinas) mg/kg fish body weight. After 14 doses, one every 5 days, the amplitude of the horizontal cell response was higher than control in individuals exposed to 0.075 and 0.0075 mg/kg, and lower in individuals exposed to 0.75 mg/kg. We conclude that intoxication with MeHg affects the electrophysiological response of the horizontal cells in the retina, either reducing or increasing its amplitude compared to control, and that these effects are related to the dose and/or to the mode of administration.