Isoelectric focusing of human head hair proteins. Preliminary research

The examination of human hair keratin to obtain genetic information, which may be useful also in forensic sciences, has been carried out with the use of isoelectrophoretic procedure obtaining considerable evidence for the existence of specific-species patterns. In this paper the keratins extracted from hairs of 280 subjects belonging to Sardinian people (113 males, 167 females, aged from 1 to 89, belonging to 52 families) were analyzed using IEF in thin-layer polyacrylamide gel (0.5 mm) in the pH range 2.5–7.0, followed by the silver staining method. Number, position and colour of the bands were the same in all the analyzed samples but a large individual variability was revealed for the relative intensity of some bands. Differences for a long time storage were not revealed as well hair's sample as protein extract: Neither were differences in the number and position of the bands analyzing samples of hair from several sites of the head of the same individual revealed. The results obtained are a useful indication to continue this research considering the numerous fields of application of this analysis system.     

Time-frequency analysis of the electrocortical activity during maturation using wavelet transform

In this study, we introduce the wavelet transform (WT) as a method for characterizing the maturational changes in electrocortical activity in 24 fetal lambs ranging from 110–144 days gestation (term 145 days). The WT, based on multiresolution signal decomposition, is free of assumptions regarding the characteristics of the signal. The approximation of the electrocortical activity at resolutions varying from 2^j+1 to 2^j can be extracted by decomposing the signal on a wavelet orthonormal basis of L ²(R). We performed multiresolution decomposition for four sets of parameters D _2j, where −1<j<−4. The four series WT represent the detail signal bandwidths: (1) 16–32 Hz, (2) 8–16 Hz, (3) 4–8 Hz, (4) 2–4 Hz. The data were divided into three groups according to gestational age: 110–122 days (early), 123–135 days (middle), and 136–144 days (late). In the early group, the power was highest in the fourth signal bandwidth, with relatively low power in the other bands. Increase in gestational age was characterized by increased power in all four bandwidths. Comparison of the cumulative distribution function of the power in the four wavelet bands confirmed the presence of two statistically different patterns in all three age groups. These two patterns correspond to the visually identified patterns of HVSA (high-voltage slow activity) and LVFA (low-voltage fast activity). The earliest development change occurred in HVSA, with progressive increase in power in the 2–8 Hz band. Later changes occurred in LVFA, with a significant increase in power in the 16–32 Hz band. The same database was also analyzed by the short-term Fourier transform (STFT) method, the most common time-frequency analysis method. Comparison of the results clearly show that the WT provided much better time-frequency resolution than the STFT method and was superior in demonstrating maturational changes in electrocortical activity. Received: 7 July 1993/Accepted in revised form: 15 November 1993     

Essential trace and toxic element distribution in the scalp hair of Pakistani myocardial infarction patients and controls

The pathogenesis of heart disease has been associated with changes in the balance of certain trace elements. The aim of this study was to evaluate the Zn, Fe, Cu, Cr, Ni, Pb, and Cd contents in scalp hair samples of myocardial infarction (MCI) patients hospitalized in the cardiac ward of National Hospital in Hyderabad city (Pakistan). Scalp hair samples were collected from 193 patients (104 male, 89 female) of 3 age groups (46–60, 61–75, and 76–90 yr), for a comparative study, 200 normal, healthy subjects (103 male, 97 female) of the same age groups residing in the same city were selected. All metals in scalp hair samples were assessed by a flame/graphite furnace atomic absorption spectrophotometer, prior to microwave-assisted and conventional wet acid digestion methods. Results were calculated in micrograms per gram. The mean values of Fe and Zn of scalp hair samples of MCI patients were significantly reduced compared to the control subjects of both genders. The mean Fe concentrations in male patients were 19.42, 12.36, and 6.98 vs 30.69, 24.42, and 16.75 for the control patients in the three age groups (46–60, 61–75, and 76–90 yrs, respectively). The mean Zn concentration in male patients were 169.2, 149.4, and 107.7 μg/g vs 206.1, 188.0, and 154.4 μg/g for the control group (p<0.002, 0.004, and 0.001) in all three age groups, respectively. These differences were also observed in the female study groups. The mean values of Pb, Cd, and Ni were significantly high in patients compared to healthy subjects (mean Pb in male patients: 11.85, 12.89, and 14.52 those of female patients were 11.88, 12.73, and 14.21 vs the male controls patients (6.08, 7.56, and 8.56) and female controls (5.99, 7.41, and 8.25) for all three age groups, respectively. The concentration of Ni and Cd in the scalp hair samples of the heart patients of both sexes were significantly higher compared to the control; in the case of Ni the range of significant difference for males was found to be p<0.001–0.009 and for females to be p<0.0.002–0.007 and significantly high concentration of Cd were observed in hair samples of patients than in controls in the range for males (p<0.001–0.009) and in females (p<0.001–0.011). The Zn/Cu and Zn/Cd ratios in the scalp hair (p<0.01) of the diseased groups were significantly lower than that of the healthy groups. Deficiency of essential trace metals and high level of toxic metals might play a role in the development of heart disease in the subjects of this study. Toxic metals might also cause diminished absorption of essential elements.     

Microbial diversity in lake sediments detected by PCR-DGGE

In this study, PCR-denaturing gradient gel electrophoresis (DGGE) was applied to analyze the microbial communities in lake sediments from Lake Xuanwu, Lake Mochou in Nanjing and Lake Taihu in Wuxi. Sediment samples from seven locations in three lakes were collected and their genomic DNAs were extracted. The DNA yields of the sediments of Lake Xuanwu and Lake Mochou were high (10 μg/g), while that of sediments in Lake Taihu was relatively low. After DNA purification, the 16S rDNA genes (V3 to V5 region) were amplified and the amplified DNA fragments were separated by parallel DGGE. The DGGE profiles showed that there were five common bands in all the lake sediment samples indicating that there were similarities among the populations of microorganisms in all the lake sediments. The DGGE profiles of Lake Xuanwu and Lake Mochou were similar and about 20 types of microorganisms were identified in the sediment samples of both lakes. These results suggest that the sediment samples of these two city lakes (Xuanwu, Mochou) have similar microbial communities. However, the DGGE profiles of sediment samples in Lake Taihu were significantly different from these two lakes. Furthermore, the DGGE profiles of sediment samples in different locations in Lake Taihu were also different, suggesting that the microbial communities in Lake Taihu are more diversified than those in Lake Xuanwu and Lake Mochou. The differences in microbial diversity may be caused by the different environmental conditions, such as redox potential, pH, and the concentrations of organic matters. Seven major bands of 16S rDNA genes fragments from the DGGE profiles of sediment samples were further re-amplified and sequenced. The results of sequencing analysis indicate that five sequences shared 99%–100% homology with known sequences (Bacillus and Brevibacillus, uncultured bacteria), while the other two sequences shared 93%–96% homology with known sequences (Acinetobacter, and Bacillus). The study shows that the PCR-DGGE technique combined with sequence analysis is a feasible and efficient method for the determination of microbial communities in sediment samples. __________ Translated from Acta Ecologica Sinica, 2006, 26(11): 3610–3616 [译自: 生态学报]     

Six variants of HLA-1327 identified by isoelectric focusing

Six variant forms of HLA-1327 were identified among 68 unrelated 1327-positive donors by isoelectric focusing (IEF) gel analysis. Each of the six IEF variants was distinguished by charge heterogeneity of desialated B27 heavy chains immunoprecipitated with specific monoclonal antibody (MAb). Charge differences varied from single to several charge units, indicating that these variants may have substantially different amino acid compositions. Informative family study showed that three B27 variant molecules were genetically determined. The uniqueness of these variant molecules was also demonstrable using a panel of alloantisera and MAbs recognizing 1327-associated epitopes. Six distinct serological reactivity patterns were observed. Five of these serological patterns correlated with four of the IEF-defined variants, two of these patterns being associated with one IEF variant form. The sixth serological pattern was shared by the remaining two IEF variants. Combining the results of the electrophoretic and serological analyses, it is apparent that there are more than six structural variants within the B27 alloantigen family. Some B27 variant forms were found only in individuals of particular racial origin, indicating that unique genetic variations might occur in different racial groups. In a preliminary analysis of patients with ankylosing spondylitis, no apparent correlation was observed between any specific B27 variants and disease susceptibility.     

The effect of ampholytes on ferritin isoelectric focussing patterns

Ferritin was subjected to isoelectric focussing (IEF) on agarose gels containing different commercial carrier ampholytes. In two gels protein staining revealed banded patterns which differed from one another, while a third gel yielded zones rather than discrete bands, indicating that the bands may be artefacts.The differences between banded patterns were studied by isolating bands from an IEF gel and refocussing these on gels containing either the original ampholyte or a different ampholyte preparation. Striking differences were noted.Chromatofocussing of ferritin resulted in the elution of broad peaks between the same pH limits as indicated by IEF patterns.     

Genotyping of Trichophyton rubrum by analysis of ribosomal-DNA intergenic spacer regions

An attempt was made to explore the genotyping of Trichophyton rubrum (T. rubrum) and the relationship between genotype and geographical origin using ribosomal restriction endonuclease polymorphic analysis. The total DNA was extracted by cetyltrimethyl ammonium bromide (CTAB). The probe was amplified from part of the 18S, ITSI, 5.8S, and ITSII region of T. rubrum standard strain with the universal fungal primers NS5 [5′-AACTT AAAGG AATTG ACGGA AG-3′] and ITS4 [5′-TCCTC CGCTT ATTGA TATGC-3′]. The genomic DNA of 49 clinical T. rubrum isolates digested by EcoR1 were hybridized with this probe, and the hybridization patterns were used as the basis of genotyping. Of the data from 49 strains of T. rubrum studied (21 from Nanjing, 26 from Dalian, and two from Beijing), 20 individual patterns (DNA Type A–T) were identified, among which Type A–C accounted for 48.98% of all the strains. The DNA patterns of Nanjing strains were represented by three bands, those of Dalian strains were represented by four bands. The DNA typing of T. rubrum by Southern blotting was highly sensitive and highly distinguishable. The DNA patterns of Nanjing strains were obviously different from those of Dalian strains.     

Frequency of an electrophoretic variant of hair alpha keratin in human populations.

A previously reported electrophoretic variant in hair keratin was looked for in a number of different racial groups. Only Caucasian samples showed the variant with the exception of one American black and one American Indian where Caucasian admixture is likely.     

Identification of Human Age Using Trace Element Concentrations in Hair and the Support Vector Machine Method

Trace element content in hair is affected by the age of the donor. Hair samples of subjects from four counties in China where people are known to have long lifespan (“longevity counties”) were collected and the trace element content determined. Samples were subdivided into three age groups based on the age of the donors from whom these were taken: children (0–15 years); elderly (80–99 years); and centenarians (≥100 years). We compared the trace element content in hair of different age groups of subjects. Support vector machine classification results showed that a non-linear polynomial kernel function could be used to classify the three age groups of people. Age did not have a significant effect on the content of Ca and Cd in human hair. The content of Li, Mg, Mn, Zn, Cr, Cu, and Ni in human hair changed significantly with age. The magnitude of the age effect on trace element content in hair was in the order Cu > Zn > Ni > Mg > Mn > Cr > Li. Cu content in hair decreased significantly with increasing age. The hair of centenarians had higher levels of Li and Mn, and lower levels of Cr, Cu, and Ni comparing with that of the children and elderly subjects. This could be a beneficial factor of their long lifespan.     

Comparison of allergenic potency of five batches ofAlternaria alternata for preparation of reference standard

Five batches of 34-016 isolate were grown separately on synthetic revised tobacco medium for 28 days. Extracts of the mycelia were prepared and their biochemical and immunological properties were examined. The extracts had similar isoelectric focussing (IEF) patterns. In crossed-radioimmuno-electrophoresis (CRIE) using human atopic sera, each showed two dominant and two to three minor allergens. In direct radioallergosorbent (RAST) and RAST inhibition tests with human atopic sera and passive cutaneous anaphylaxis (PCA) tests with mouse IgE, all samples were of similar potency. In IgE immunoblots of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, each extract showed strongly reactive bands at 16, 18, 30, 33 and 59–100 kDa MW. These results indicate that a reference preparation ofA. alternata suitable for standardization purposes can be obtained from any batch of 34-016 isolate.     

Fast carnivores and slow herbivores: differential foraging strategies among grizzly bears in the Canadian Arctic

Categorizing animal populations by diet can mask important intrapopulation variation, which is crucial to understanding a species’ trophic niche width. To test hypotheses related to intrapopulation variation in foraging or the presence of diet specialization, we conducted stable isotope analysis (δ¹³C, δ¹⁵N) on hair and claw samples from 51 grizzly bears (Ursus arctos) collected from 2003 to 2006 in the Mackenzie Delta region of the Canadian Arctic. We examined within-population differences in the foraging patterns of males and females and the relationship between trophic position (derived from δ¹⁵N measurements) and individual movement. The range of δ¹⁵N values in hair and claw (2.0–11.0‰) suggested a wide niche width and cluster analyses indicated the presence of three foraging groups within the population, ranging from near-complete herbivory to near-complete carnivory. We found no linear relationship between home range size and trophic position when the data were continuous or when grouped by foraging behavior. However, the movement rate of females increased linearly with trophic position. We used multisource dual-isotope mixing models to determine the relative contributions of seven prey sources within each foraging group for both males and females. The mean bear dietary endpoint across all foraging groups for each sex fell toward the center of the mixing polygon, which suggested relatively well-mixed diets. The primary dietary difference across foraging groups was the proportional contribution of herbaceous foods, which decreased for both males and females from 42–76 to 0–27% and 62–81 to 0–44%, respectively. Grizzlies of the Mackenzie Delta live in extremely harsh conditions and identifying within-population diet specialization has improved our understanding of varying habitat requirements within the population.     

Purification and characterization of a novel fibrinolytic protease from <Emphasis Type="Italic">Fusarium</Emphasis> sp. CPCC 480097

A novel fibrinolytic enzyme from Fusarium sp. CPCC 480097, named Fu-P, was purified to electrophoretic homogeneity using ammonium sulfate precipitation and ion exchange and gel filtration chromatography. Fu-P, a single protein had a molecular weight of 28 kDa, which was determined by SDS-PAGE and gel filtration chromatography. The isoelectric point of Fu-P determined by isoelectric focusing electrophoresis (IEF) was 8.1, and the optimum temperature and pH value were 45°C and 8.5, respectively. Fu-P cleaved the α-chain of fibrin (ogen) with high efficiency, and the β-chain and γ-γ (γ-)-chain with lower efficiency. Fu-P activity was inhibited by EDTA and PMSF, and the enzyme exhibited a high specificity for the chymotrypsin substrate S-2586. Fu-P was therefore identified as a chymotrypsin-like serine metalloprotease. The first 15 amino acids of the N-terminal sequence of Fu-P were Q-A-S–S-G-T-P-A-T-I-R-V-L-V–V and showed no homology with that of other known fibrinolytic enzymes. This protease may have potential applications in thrombolytic therapy and in thrombosis prevention.     

Regulation of the formation of proteinases inBacillus megaterium

The exocellular proteinases of asporogenic and sporogenicBacillus megaterium KM (megaterioproteinase A and S) were found to be active enzymes of the monomer type. The electrophoretic mobility of megaterioproteinase A is higher than that of S on acrylamide gel. After mixing, the enzymes could be separated again. The molecular weight of megaterioproteinase A was found to be 20,000–23,500, that of megaterioproteinase S 16,500–20,000 daltons, according to the “molecular sieving” method. The electrophoretic mobility of both proteinases was determined at different pH and the graphically calculated isoelectric point (pI) was found to be 7.3–7.4. The pK values of the ES complex estimated by plotting the logarithm of the maximum velocity of the enzymic reaction against pH were 6.0–6.1 and 7.8–8.0 for both megaterioproteinases. The activation energy was 13,500–13,600 for both enzymes. It is concluded that the above two enzymes resemble each other in enzymic properties but differ in electrophoretic mobility and probably also in molecular weight.     

Age- and sex-related differences in zinc and lead levels in human hair

The hair of 1518 healthy subjects between newborn and 70 yr old living in Linfen city and the surrounding rural area in Shanxi province, North China, was analyzed by means of a flame atomic absorption spectrophotometer method in order to determine the concentrations of zinc (Zn) and lead (Pb). The collected samples were subdivided on the basis of age, sex, and place of residence (urban and rural). In female hair, the contents of Zn were higher than in male, but the hair Pb concentrations of males were slightly higher than those of females. Age seems to have a different influence on the two elements. The maximum amount of hair Zn was found generally in newborns. From the baby group (<1 yr) to the puberty group (15–19 yr), Zn levels in hair increased with age, and then Zn levels decreased with age from the puberty group to elderly group (61–70 yr). The hair Pb levels were the lowest in newborns, increased with age from newborn to infant (1–2 yr), and then decreased with age from infant to the elderly. It was shown that the hair Pb and Zn levels were inversely related in various age groups, suggesting that there is possibly antagonism between Pb and Zn in the human body. The results also showed the hair Pb levels of subjects living in the urban area, except for newborns, were higher than those of subjects residing in the rural area. However, no difference between the hair Zn contents of the populations residing in urban and rural areas.     

Soil acidity factors and nodulation ofTrifolium repens

Summary Effects of factors associated with soil acidity (low pH, low calcium, high aluminium and high manganese) on theTrifolium repens-Rhizobium trifolii symbiosis were investigted under laboratory conditions using an axenic solution-culture technique. 200 μM manganese increased root elongation in the range pH 4.3–5.5, but had no effect on root hair formation, the number of Rhizobium in the rhizosphere, or nodule formation. Root elongation and root hair formation were unaffected at pH 4.3 when 500 or 1000μM calcium was supplied, whereas multiplication of Rhizobium in the rhizosphere and nodulation were inhibited at pH 4.3 and 4.7.50–1000μM calcium had no effect either on the multiplication of Rhizobium in the range pH 4.3–5.5, or on nodule formation in the absence of aluminium. 50 μM aluminium inhibited, root elongation and root hair formation at pH 4.3 and 4.7; the effect on root elongation was reduced by increasing the calcium concentration from 50 to 1000μM. 50μM aluminium also inhibited Rhizobium multiplication in the rhizosphere and reduced nodule formation at pH 5.5 (at which aluminium precipitated out of solution), but root elongation and root hair formation were unaffected. These, effects of aluminium at pH 5.5 may explain the poor response to inoculation by white clover in acid mineral soils after liming.     

Evaluation of Toxic Risk Assessment of Arsenic in Male Subjects Through Drinking Water in Southern Sindh Pakistan

The arsenic (As) hazardous quotient was estimated based on concentration of As in drinking water and scalp hair of male subjects of two age groups (n = 360) consuming As contaminated water at different levels and non-contaminated drinking water. The total As concentrations in drinking water of less-exposed (LE) and high-exposed (HE) areas was found to be 3- to 30-fold higher than the permissible limit of the World Health Organization (2004) for drinking water, while the levels of As in drinking water of non-exposed (NE) areas was within the permissible limit. The levels of As in scalp hair samples of male subjects of two age groups belonging to NE, LE, and HE areas ranged from 0.01 to 0.27, 0.11–1.31, and 0.36–6.80 μg/g, respectively. A significant correlation between As contents of drinking water and As concentration in scalp hair was observed in sub-district Gambit (r = 0.825–0.852, p < 0.001) as compared to those subjects belonging to LE sub-district Thari Mirwah. A toxicity risk assessment provides a hazard quotient corresponding to <10 that indicates non-carcinogenic exposure risk of understudy areas.     

Induction and Characterization of Laccase in the Ligninolytic Fungus Pleurotus eryngii

Two protein bands with laccase activity were found after PAGE of culture liquid or mycelium extract of Pleurotus eryngii, grown on glucose–ammonium tartrate–yeast extract medium with and without inducers. A major and a minor laccase band were observed in the basal medium. The intensity of the major band (laccase I) did not change after the addition of inducers. However, the minor band (laccase II), characterized by higher electrophoretic mobility, was strongly induced by wheat–straw alkalilignin and vanillic and veratric acids. Laccase activity in the basal medium had an optimum pH of 4.5 and was stable from pH 3 to 10 during 24 h at room temperature. This enzyme had wide substrate specificity on hydroquinones, methoxy-substituted monophenols, and aromatic amines. In general, laccase activity was found only with compounds having a redox potential lower than 0.5 mV. The highest activity was obtained with methoxy- and methyl-substituted p-hydroquinones and aromatic diamines. Some activity also occurred with the aliphatic compound 3,5-cyclohexadiene-1,2-diol. Received: 22 April 1996 / Accepted: 29 June 1996     

Serial analysis of zein by isoelectric focusing and sodium dodecyl sulfate gel electrophoresis

Zein, the major storage protein of maize (Zea mays L.) endosperm, was extracted from a number of inbreds with alcohol plus a reducing agent. Isoelectric focusing (IEF) separated total zeins into 41 components, while sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separated total zeins into about 15 components. Each procedure gave characteristic patterns of zein bands for a number of maize inbreds. IEF and SDS-PAGE were used serially so that each band separated by IEF could be assayed as an individual SDS-PAGE sample. Some IEF bands revealed only a single band after SDS-PAGE, while others revealed two or more bands. A nomenclature system is presented which integrates the two separation systems with information about chromosome locations of zein genes, maize mutations which affect zein synthesis, and inbred sources for different zeins. SDS-PAGE of zein gives apparent molecular masses which vary widely according to the standards used and the properties of the gels, therefore an artificial nomenclature for identifying zein bands after SDS-PAGE is presented. The new nomenclature provides a flexible system which is useful and can be conveniently used in different laboratories.     

Determination of copper and zinc levels in human hair

The Cu and Zn levels of both 607 men (1–85 y old) and 649 women (1–92 y old) were determined by atomic absorption spectrometry. Sex does not influence Cu (14.89±0.89 μg/g and 15.26±0.79 μg/g hair for males and females, respectively) and Zn contents (200.97±9.68 μg/g for men and 209.81±9.49 μg/g hair for women). Age influences Cu and Zn concentrations, but only significantly in females: Cu levels decrease over 60 y of age; whereas Zn levels increase significantly from age groups 2–5 to 20–40 years. Hair color influences Cu concentrations in both males and females. In males, white hair containes less Cu than black hair; in females, white hair's Cu levels are significantly lower than those of dark blond, red, light brown, and brown hair. There are no significant differences in Zn concentrations with respect to different hair colors, in either males or females.     

Use of TaqMan gene probe for real-time monitoring of acidophilic hydrogen-producing bacteria

Using a TaqMan gene probe targeting the 16S rDNA of acidophilic hydrogen-producing bacteria (HPB), the abundance of this HPB in the biomass was found to increase from 0.02 to 72% in a single batch treating rice slurry waste at pH 4.5 over 130 h. The corresponding abundances were 4.4% in the batch operated at pH 5.0 and 0.01–0.02% at pH 5.5–6.5. During the growth phase, the generation time for the acidophilic HPB at pH 4.5 averaged 3.5 h.