Mutants of common bean alpha-amylase inhibitor-2 as an approach to investigate binding specificity to alpha-amylases

Despite the presence of a family of defense proteins, Phaseolus vulgaris can be attacked by bruchid insects resulting in serious damage to stored grains. The two distinct active forms of a-amylase inhibitors, a-AI1 and a-AI2, in P. vulgaris show different specificity toward a-amylases. Zabrotes subfasciatus a-amylase is inhibited by a-AI2 but not by a-AI1. In contrast, porcine a-amylase is inhibited by a-AI1 but not by a-AI2. The objective of this work was to understand the molecular basis of the specificity of two inhibitors in P. vulgaris (a-AI1 and a-AI2) in relation to a-amylases. Mutants of a-AI2 were made and expressed in tobacco plants. The results showed that all the a-AI2 mutant inhibitors lost their activity against the insect a-amylases but none exhibited activity toward the mammalian a-amylase. The replacement of His33 of a-AI2 with the a-AI1-like sequence Ser-Tyr-Asn abolished inhibition of Z. subfasciatus a-amylase. From structural modeling, the conclusion is that the size and complexity of the amylase-inhibitor interface explain why mutation of the N-terminal loop and resultant abolition of Z. subfasciatus a-amylase inhibition are not accompanied by gain of inhibitory activity against porcine a-amylase.

The stability of right- and left-handed alpha-helices as a function of monomer chirality

Poly-L-alanine forms stable right-handed alpha-helices, whereas Poly-D-alanine is stable as left-handed alpha helices.

Salivary and serum cortisol levels, salivary alpha-amylase and unstimulated whole saliva flow rate in pregnant and non-pregnant

PURPOSE: To compare salivary and serum cortisol levels, salivary alpha-amylase (sAA), and unstimulated whole saliva (UWS) flow rate in pregnant and non-pregnant women. METHOD: A longitudinal study was conducted at a health promotion center of a university hospital. Nine pregnant and 12 non-pregnant women participated in the study. Serum and UWS were collected and analyzed every trimester and twice a month during the menstrual cycle. The salivary and serum cortisol levels were determined by chemiluminescence assay and the sAA was processed in an automated biochemistry analyzer. RESULTS: Significant differences between the pregnant and non-pregnant groups were found in median [interquartile range] levels of serum cortisol (23.8 µL/dL [19.4-29.4] versus 12.3 [9.6-16.8], p<0.001) and sAA (56.7 U/L [30.9-82.2] versus 31.8 [18.1-53.2], p<0.001). Differences in salivary and serum cortisol (µL/dL) and sAA levels in the follicular versus luteal phase were observed (p<0.001). Median UWS flow rates were similar in pregnant (0.26 [0.15-0.30] mL/min) and non-pregnant subjects (0.23 [0.20-0.32] mL/min). Significant correlations were found between salivary and serum cortisol (p=0.02) and between salivary cortisol and sAA (p=0.01). CONCLUSIONS: Serum cortisol and sAA levels are increased during pregnancy. During the luteal phase of the ovarian cycle, salivary cortisol levels increase, whereas serum cortisol and sAA levels decline.

Polymorphism in the lymphotoxin-alpha gene, position +252 (rs909253), is not associated with preeclampsia development in Brazilian women

PURPOSE: To investigate the frequencies of polymorphic allele and genotypes for the LT-α gene, position +252 (rs909253), in Brazilian women with preeclampsia.METHODS: This is a case-control study, in which 30 women with preeclampsia, classified according to the criteria of the National High Blood Pressure Education Program, and 115 women in the control group, with at least two healthy pregnancies, were selected. Peripheral blood was collected, and DNA was extracted, followed by genotyping, using specific primers and restriction analysis. The genotypes obtained were AA, AG and GG. Statistical analysis was performed using the χ2association test. The Hardy-Weinberg Equilibrium was tested using the Haploview Program.RESULTS: The results showed no association between genotypes and preeclampsia development (χ2=2.0; p=0.4). When the AG and GG genotypes were grouped according to allele G presence or absence (genotype AA), the data showed that the presence of allele G was not significantly different between cases (women with preeclampsia) and controls (χ2=0.0; p=1.0). The LT-α gene polymorphism, position +252 (rs909253), seems not to be an important candidate for the development of preeclampsia. Other inflammatory genes should be researched, and studies involving gene-environment interactions should be performed, in order to reach a better understanding of the etiology of the preeclampsia.

Gross anatomical features of the oropharyngeal cavity of the ostrich (Struthio camelus)

Most descriptions of the ostrich oropharynx and oesophagus are superficial and supply little meaningful morphological data. The aim of this investigation is describe the ostrich oropharingeal cavity, in order to supply the deficiency of macroscopic data about this important animal. Five heads of 12 to 14-month-old ostriches of either sex were anatomically dissected to expose the oropharynx. The ostrich oropharynx was "bell-shaped" composed by the maxillary and mandibular ramphoteca. The roof and floor presented two distinct regions different in colour of the mucosa. The rostral region was pale pink contrasting to creamy-pink coloured caudal region. The median longitudinal ridge extended rostrally from the apex of the choana to the tip of the beak in the roof and it is clearly more prominent and rigid than the homolog in the floor that appeared thin and stretched rostrally, continuing caudally surrounding the tongue and the laryngeal mound eventually merging with the oesophageal mucosa. The floor was formed by the interramal region, tongue and laryngeal mound containing shield-shaped glottis. It can be concluded that the present study, in addition to confirming the basic features of the oropharynx previously described for the ostrich, clarified the contradictory information presented in the literature and also provided new, unreported morphological data, some of which may be important when studying nutrition and health in these birds.

Necrotizing enteritis associated with Clostridium perfringensType B in chinchillas (Chinchilla lanigera)

Four 3-4 month-old chinchillas (Chinchilla lanigera) from a commercial flock of 395 chinchillas, were found dead with evidence of previous diarrhea and prolapsed rectum. A fifth 8 month-old chinchilla died 8 hours after being found recumbent, apathetic, diarrheic and with a prolapsed rectum. Two chinchillas were necropsied and observed gross lesions consisted of extensive hemorrhagic enteritis, mild pulmonary edema and enlarged and yellow liver; this latter finding was particularly prominent in the chinchilla presenting longer clinical course. Histologically there was necrotizing enteritis associated with abundant bacterial rods aggregates in the intestinal surface epithelium and within the lamina propria. In the lungs there were small amounts of pink proteinaceous material (edema) in the interstitium and marked vacuolar hepatocellullar degeneration (lipidosis) in the liver. Anaerobic cultures from the intestinal contents of one of the affected chinchillas yielded Clostridium perfringens. Genotyping of this C. perfringens isolate was achieved by multiplex polymerase chain reaction (mPCR) as C. perfringenstype B due to detection of alpha, beta and epsilon-toxin genes. These findings suggest C. perfringens type B as an important cause of sudden or acute death in chinchillas.

Gross morphology of rhea oropharyngeal cavity

The rhea (Rhea americana americana) is an american bird belonging to Ratite's family. Studies related to its morphology are still scarce. This study aims to describe the macroscopic structures of the oropharyngeal cavity. Five heads (2 to 6 months old) formalin preserved were anatomically dissected to expose the oropharynx. The oropharynx of the rhea was "bell-shaped" composed by the maxillary and mandibular rhamphotheca. The roof and floor presented two distinct regions different in colour of the mucosa. The rostral region was pale pink contrasting to grey coloured caudal region. The median longitudinal ridge extended rostrally from the apex of the choana to the tip of the beak in the roof and it is clearly more prominent and rigid than the homolog in the floor that appeared thin and stretched merely along the rostral portion of the regio interramalis. The floor was formed by the interramal region, (regio interramalis) tongue and laryngeal mound containing glove-shaped glottis. This study confirmed the basic morphology of the oropharinx of the rhea. However, important morphological information not previously described is highlighted and contradictory information present in the literature is clarified.

alpha-Tocopherol enhances tumour growth inhibition by cis-dichlorodiammine platinum (II)

Present studies indicate that alpha-tocopherol enhances the efficacy of cisplatin as demonstrated by inoculation of Dalton's lymphoma cells incubated with either cisplatin (5 or 10 µg/ml) alone or cisplatin + alpha-tocopherol (25 or 50 µg/ml) into C3H/He mice. Tumour cells (3 x 10(6) cells/mouse) incubated with cisplatin grow slowly in syngeneic mice as indicated by the late appearance of tumour. However, mice failed to develop tumour when inoculated with tumour cells incubated with cisplatin + alpha-tocopherol. When the animals were challenged with tumour cells (3 x 10(6) cells/mouse) on the 15th day after the initial inoculation, 30-50% survived more than 60 days, with 10% tumour-free survivors being observed in some groups. Antitumour activity was higher in mice receiving lymphoma cells (3 x 10(6) cells/mouse) preincubated with cisplatin + alpha-tocopherol compared to cisplatin alone. Tumour-bearing mice receiving cisplatin in combination with different concentrations of alpha-tocopherol exhibited significantly higher (P<0.001) intratumour platinum content (123-306%) but without any change in the kidney platinum content as compared to those receiving cisplatin (5 or 10 µg/ml) alone. Enhancement of cisplatin-induced tumour growth inhibition is probably due to the modulation of tumour cell membrane permeability by alpha-tocopherol. alpha-Tocopherol might increase the influx of cisplatin into tumour cells, causing the DNA repair machinery to be less efficient due to increased efficiency of adduct formation in the DNA molecule. This effect of alpha-tocopherol can render cisplatin more effective as an antitumour agent.

alpha-Globin genes: thalassemic and structural alterations in a Brazilian population

Seven unrelated patients with hemoglobin (Hb) H disease and 27 individuals with alpha-chain structural alterations were studied to identify the alpha-globin gene mutations present in the population of Southeast Brazil. The -alpha3.7, --MED and -(alpha)20.5 deletions were investigated by PCR, whereas non-deletional alpha-thalassemia (alphaHphalpha, alphaNcoIalpha, aaNcoI, alphaIcalpha and alphaTSaudialpha) was screened with restriction enzymes and by nested PCR. Structural alterations were identified by direct DNA sequencing. Of the seven patients with Hb H disease, all of Italian descent, two had the -(alpha)20.5/-alpha3.7 genotype, one had the --MED/-alpha3.7 genotype, one had the --MED/alphaHphalpha genotype and three showed interaction of the -alpha3.7 deletion with an unusual, unidentified form of non-deletional alpha-thalassemia [-alpha3.7/(aa)T]. Among the 27 patients with structural alterations, 15 (of Italian descent) had Hb Hasharon (alpha47Asp->His) associated with the -alpha3.7 deletion, 4 (of Italian descent) were heterozygous for Hb J-Rovigo (alpha53Ala->Asp), 4 (3 Blacks and 1 Caucasian) were heterozygous for Hb Stanleyville-II (alpha78Asn->Lys) associated with the alpha+-thalassemia, 1 (Black) was heterozygous for Hb G-Pest (alpha74Asp->Asn), 1 (Caucasian) was heterozygous for Hb Kurosaki (alpha7Lys->Glu), 1 (Caucasian) was heterozygous for Hb Westmead (alpha122His->Gln), and 1 (Caucasian) was the carrier of a novel silent variant (Hb Campinas, alpha26Ala->Val). Most of the mutations found reflected the Mediterranean and African origins of the population. Hbs G-Pest and Kurosaki, very rare, and Hb Westmead, common in southern China, were initially described in individuals of ethnic origin differing from those of the carriers reported in the present study and are the first cases to be reported in the Brazilian population.

N-terminal amino acids of bovine alpha interferons are relevant for the neutralization of their antiviral activity

The structure-function relationship of interferons (IFNs) has been studied by epitope mapping. Epitopes of bovine IFNs, however, are practically unknown, despite their importance in virus infections and in the maternal recognition of pregnancy. It has been shown that recombinant bovine (rBo)IFN-alphaC and rBoIFN-alpha1 differ only in 12 amino acids and that the F12 monoclonal antibody (mAb) binds to a linear sequence of residues 10 to 34. We show here that the antiviral activities of these two IFNs were neutralized by the F12 mAb to different extents using two tests. In residual activity tests the antiviral activity dropped by more than 99% with rBoIFN-alphaC and by 84% with rBoIFN-alpha1. In checkerboard antibody titrations, the F12 mAb titer was 12,000 with rBoIFN-alphaC and only 600 with rBoIFN-alpha1. Since these IFNs differ in their amino acid sequence at positions 11, 16 and 19 of the amino terminus, only these amino acids could account for the different neutralization titers, and they should participate in antibody binding. According to the three-dimensional structure described for human and murine IFNs, these amino acids are located in the alpha helix A; amino acids 16 and 19 of the bovine IFNs would be expected to be exposed and could bind to the antibody directly. The amino acid at position 11 forms a hydrogen bond in human IFNs-alpha and it is possible that, in bovine IFNs-alpha, the F12 mAb, binding near position 11, would disturb this hydrogen bond, resulting in the difference in the extent of neutralization observed.

High prevalence of alpha-thalassemia among individuals with microcytosis and hypochromia without anemia

In order to determine the contribution of alpha-thalassemia to microcytosis and hypochromia, 339 adult outpatients seen at Unicamp University Hospital (with the exception of the Clinical Hematology outpatient clinics), who showed normal hemoglobin (Hb) levels and reduced mean corpuscular volume and mean corpuscular hemoglobin, were analyzed. Ninety-eight were Blacks (28.9%) and 241 were Caucasians (71.1%). In all cases, Hb A2 and F levels were either normal or low. The most common deletional and nondeletional forms of alpha-thalassemia [-alpha3.7, -alpha4.2, --MED, -(alpha)20.5, alphaHphIalpha, alphaNcoIalpha, aaNcoI and alphaTSAUDI] were investigated by PCR and restriction enzyme analyses. A total of 169 individuals (49.9%) presented alpha-thalassemia: 145 (42.8%) were heterozygous for the -alpha3.7 deletion (-alpha3.7/aa) and 18 (5.3%) homozygous (-alpha3.7/-alpha3.7), 5 (1.5%) were heterozygous for the nondeletional form alphaHphIalpha (alphaHphIalpha/aa), and 1 (0.3%) was a --MED carrier (--MED/aa). Among the Blacks, 56 (57.1%) showed the -alpha3.7/aa genotype, whereas 12 (12.2%) were -alpha3.7/-alpha3.7 and 1 (1.0%) was an alphaHphIalpha carrier; among the Caucasians, 89 (36.9%) were -alpha3.7/aa, 6 (2.5%) had the -alpha3.7/-alpha3.7 genotype, 4 (1.7%) presented the nondeletional form (alphaHphIalpha/aa), and 1 (0.4%) was a --MED carrier. These results demonstrate that alpha-thalassemia, mainly through the -alpha3.7 deletion, is an important cause of microcytosis and hypochromia in individuals without anemia. These data are of clinical relevance since these hematological alterations are often interpreted as indicators of iron deficiency.

alpha-Smooth muscle actin and proliferating cell nuclear antigen expression in focal segmental glomerulosclerosis: functional and structural parameters of renal disease progression

The aim of the present study was to investigate the expression of alpha-smooth muscle actin (alpha-SM-actin) and proliferating cell nuclear antigen (PCNA) in renal cortex from patients with focal segmental glomerulosclerosis (FSGS) and their correlations with parameters of renal disease progression. We analyzed renal biopsies from 41 patients with idiopathic FSGS and from 14 control individuals. The alpha-SM-actin immunoreaction was evaluated using a score that reflected the changes in the extent and intensity of staining in the glomerular or cortical area. The PCNA reaction was quantified by counting the labeled cells of the glomeruli or renal cortex. The results, reported as median ± percentile (25th; 75th), showed that the alpha-SM-actin scores in the glomeruli and tubulointerstitium from the renal cortex were 2.0 (2.0; 4.0) and 3.0 (3.0; 4.0), respectively, in patients with FSGS, and 0.5 (0.0; 1.0) and 0.0 (0.0; 0.5) in the controls. The number of PCNA-positive cells per glomerulus and graded field of tubulointerstitium from the renal cortex was 0.2 (0.0; 0.4) and 1.1 (0.3; 2.2), respectively, for patients with FSGS, and 0.0 (0.0; 0.5) and 0.0 (0.0; 0.0) for controls. The present data showed an increase of alpha-SM-actin and PCNA expression in glomeruli and renal cortex from FSGS patients. The extent of immunoreaction for alpha-SM-actin in the tubulointerstitial area was correlated with the intensity of proteinuria. However, there was no correlation between the kidney expression of these proteins and the reciprocal of plasma creatinine level or renal fibrosis. These findings suggest that the immunohistochemical alterations may be reversible.

Influences of alpha-tocopherol on cholesterol metabolism and fatty streak development in apolipoprotein E-deficient mice fed an atherogenic diet

Although the role of oxidized lipoproteins is well known in atherogenesis, the role of vitamin E supplementation is still controversial. There is also little information about cholesterol metabolism (hepatic concentration and fecal excretion) in the new models of atherosclerosis. In the present study, we evaluated the effect of moderate vitamin E supplementation on cholesterol metabolism and atherogenesis in apolipoprotein E (apo E)-deficient mice. Apo E-deficient mice were fed an atherogenic diet containing 40 or 400 mg/kg of alpha-tocopherol acetate for 6 weeks. Total cholesterol in serum and liver and 3-OH-alpha-sterols in feces, and fecal excretion of bile acids were determined and histological analyses of aortic lesion were performed. A vitamin E-rich diet did not affect body weight, food intake or serum cholesterol. Serum and hepatic concentrations of cholesterol as well as sterol concentration in feces were similar in both groups. However, when compared to controls, the alpha-tocopherol-treated mice showed a reduction of about 60% in the atherosclerotic lesions when both the sum of lesion areas and the average of the largest lesion area were considered. These results demonstrate that supplementation of moderate doses of alpha-tocopherol was able to slow atherogenesis in apo E-deficient mice and to reduce atherogenic lipoproteins without modifying the hepatic pool or fecal excretion of cholesterol and bile acids.

Uniform decrease of alpha-global field power induced by intermittent photic stimulation of healthy subjects

Nineteen-channel EEGs were recorded from the scalp surface of 30 healthy subjects (16 males and 14 females, mean age: 34 years, SD: 11.7 years) at rest and under trains of intermittent photic stimulation (IPS) at rates of 5, 10 and 20 Hz. Digitalized data were submitted to spectral analysis with fast fourier transformation providing the basis for the computation of global field power (GFP). For quantification, GFP values in the frequency ranges of 5, 10 and 20 Hz at rest were divided by the corresponding data obtained under IPS. All subjects showed a photic driving effect at each rate of stimulation. GFP data were normally distributed, whereas ratios from photic driving effect data showed no uniform behavior due to high interindividual variability. Suppression of alpha-power after IPS with 10 Hz was observed in about 70% of the volunteers. In contrast, ratios of alpha-power were unequivocal in all subjects: IPS at 20 Hz always led to a suppression of alpha-power. Dividing alpha-GFP with 20-Hz IPS by alpha-GFP at rest (R = alpha-GFP IPS/alpha-GFPrest) thus resulted in ratios lower than 1. We conclude that ratios from GFP data with 20-Hz IPS may provide a suitable paradigm for further investigations.

Bone mineral density in young women of the city of São Paulo, Brazil: correlation with both collagen type I alpha 1 gene polymorphism and clinical aspects

Osteoporosis is a multifactorial disease with great impact on morbidity and mortality mainly in postmenopausal women. Although it is recognized that factors related to life-style and habits may influence bone mass formation leading to greater or lower bone mass, more than 85% of the variation in bone mineral density (BMD) is genetically determined. The collagen type I alpha 1 (COLIA1) gene is a possible risk factor for osteoporosis. We studied a population of 220 young women from the city of São Paulo, Brazil, with respect to BMD and its correlation with both COLIA1 genotype and clinical aspects. The distribution of COLIA1 genotype SS, Ss and ss in the population studied was 73.6, 24.1 and 2.3%, respectively. No association between these genotypes and femoral or lumbar spine BMD was detected. There was a positive association between lumbar spine BMD and weight (P<0.0001), height (P<0.0156), and body mass index (BMI) (P<0.0156), and a negative association with age at menarche (P<0.0026). There was also a positive association between femoral BMD and weight (P<0.0001), height (P<0.0001), and BMI (P<0.0001), and a negative correlation with family history for osteoporosis (P<0.041). There was no association between the presence of allele s and reduced BMD. We conclude that a family history of osteoporosis and age at menarche are factors that may influence bone mass in our population.