Water deficit and abscisic acid treatments increase the expression of a glucomannan mannosyltransferase gene (GMMT) in Aloe vera Burm. F.

The main polysaccharide of the gel present in the leaves of or Aloe vera Burm.F., (Aloe barbadensis Miller) a xerophytic crassulacean acid metabolism (CAM) plant, is an acetylated glucomannan named acemannan. This polysaccharide is responsible for the succulence of the plant, helping it to retain water. In this study we determined using polysaccharide analysis by carbohydrate gel electrophoresis (PACE) that the acemannan is a glucomannan without galactose side branches. We also investigated the expression of the gene responsible for acemannan backbone synthesis, encoding a glucomannan mannosyltransferase (GMMT, EC 2.4.1.32), since there are no previous reports on GMMT expression under water stress in general and specifically in Aloe vera. It was found by in silico analyses that the GMMT gene belongs to the cellulose synthase-like A type-9 (CSLA9) subfamily. Using RT-qPCR it was found that the expression of GMMT increased significantly in Aloe vera plants subjected to water stress. This expression correlates with an increase of endogenous ABA levels, suggesting that the gene expression could be regulated by ABA. To corroborate this hypothesis, exogenous ABA was applied to non-water-stressed plants, resulting in a significant increase of GMMT expression after 48 h of ABA treatment.

Enzyme Polymorphism in Sitophilus oryzae (Linnaeus, 1763) and Sitophilus zeamais (Motschulsky, 1855) (Coleoptera, Curculionidae) in southern Brazil / Polimorfismo Enzimático em Sitophilus oryzae (Linnaeus, 1763) e Sitophilus zeamais (Motschulsky, 1855) (Coleoptera, Curculionidae) no sul do Brasil

Beetles of the species Sitophilus oryzae and S. zeamais are pests of great economic importance since they attack not only rice and maize but also several other cereals. In fact, these beetles are one of the most visible threats to sustainable food production. Current study estimated the genetic variability of S. oryzae in two samples, one from the State of Paraná (PR), Brazil, and another from the state of Rio Grande do Sul (RS), Brazil, and a sample of S. zeamais from the State of Santa Catarina (SC), Brazil. Isozyme electrophoresis in starch gel technique was employed to analyze eight enzyme systems (AAT, ACP, GDH, GPI, IDH, MDH, PGM and ME). Average heterozygosity rates were 0.0091, 0.0100 and 0.0000 and expected heterozygosity rates were 0.0419, 0.0452 and 0.0000 respectively for the samples of PR, SC and RS samples. The percentage of polymorphic loci was 30% in the PR sample, 0% in the RS sample and 30% in the SC sample. Genetic identity rates were I=0.9983 between samples from PR and RS; I = 0.6892 between PR and SC, and I = 0.6925 between SC and RS. Nei´s (1978) genetic distance rates were 0.0017, 0.3722 and 0.3675. Samples presented low genetic variability.

Os besouros Sitophilus oryzae e S. zeamais são considerados pragas de grande importância econômica. Além do arroz e do milho, eles atacam outros diversos cereais. São uma das ameaças mais visíveis para a produção sustentável de alimentos. Este trabalho teve como objetivo estimar a variabilidade genética de S. oryzae em duas amostras, uma do Estado do Paraná (PR), e outra do Rio Grande do Sul (RS) e uma amostra de S. zeamais de Santa Catarina (SC). Utilizou-se a técnica de eletroforese de isozimas em gel de amido para a análise de oito sistemas enzimáticos (AAT, ACP, GDH, GPI, IDH, MDH, ME e PGM). A heterozigosidade média observada foi de 0,0091, 0,0100 e 0,0000 e a esperada foi de 0,0419, 0,0452 e 0,0000 para as amostras do PR, SC e RS, respectivamente. A porcentagem de locos polimórficos foi de 30, 0 e 30% nas amostras do PR, RS e SC, respectivamente. Os valores para identidade genética foram de I = 0,9983 entre as amostras do PR e RS; I = 0,6892 entre PR e SC e I = 0,6925 entre SC e RS, e os valores da distância genética de Nei (1978) foram 0,0017, 0,3722 e 0,3675, respectivamente. As amostras apresentaram pouca variabilidade genética.

Transferrin polymorphism and opportunistic infections in HIV-infected women in Rwanda.

OBJECTIVE: We investigated the prevalence of opportunistic infections in HIV-infected women according to transferrin (TF) phenotype. METHODS: We conducted a cross-sectional study among 200 HIV-positive women in the Butare University Teaching Hospital in Rwanda. TF phenotypes were determined using starch gel electrophoresis. RESULTS: Phenotype frequencies of TF CD, CB and CC were 14.5, 3 and 82.5%, respectively. The homozygous TF DD phenotype was not found. Subjects with TF CD phenotype had a significantly higher prevalence of opportunistic infections than subjects with TF CC phenotype, 76 and 52%, respectively (p = 0.026). In logistic regression, there was a significant correlation between TF phenotypes and opportunistic infections (p = 0.012). Subjects with TF CD phenotype had significantly lower values for TF (p = 0.006) than TF CC subjects. Hematological parameters (RBC, RBC indices, hemoglobin, hematocrit, WBC, neutrophils, lymphocytes, platelets and erythrocyte sedimentation rate), iron, ferritin, TF saturation, C-reactive protein and CD4 count did not differ according to TF phenotype. CONCLUSION: Subjects with TF CC phenotype have a lower prevalence of opportunistic infections. Iron status may play a role in this association.

How it all began: a personal history of gel electrophoresis.

Arne Tiselius' moving boundary electrophoresis method was still in general use in 1951 when this personal history begins, although zonal electrophoresis with a variety of supporting media (e.g., filter paper or starch grains) was beginning to replace it. This chapter is an account of 10 years of experiments carried out by the author during which molecular sieving gel electrophoresis was developed and common genetic variants of two proteins, haptoglobin and transferrin, were discovered in normal individuals. Most of the figures are images of pages from the author's laboratory notebooks, which are still available, so that some of the excitement of the time and the humorous moments are perhaps apparent. Alkaline gels, acidic gels with and without denaturants, vertical gels, two-dimensional gels, and gels with differences in starch concentration are presented. The subtle details that can be discerned in these various gels played an indispensable role in determining the nature of the change in the haptoglobin gene (Hp) that leads to the polymeric series characteristic of Hp ( 2 ) /Hp ( 2 ) homozygotes. Where possible, the names of scientific friends who made this saga of gel electrophoresis so memorable and enjoyable are gratefully included.

Interactions of hemoglobin in live red blood cells measured by the electrophoresis release test.

Electrophoresis release test (ERT) is the starch-agarose mixed gel electrophoresis of live red blood cells (RBCs). Mixed gel electrophoresis used to be one of the classic methods to isolate proteins, and in our laboratory, this technique is usually performed to isolate hemoglobins. Recently, combined with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS), ERT has been used to study the interactions between hemoglobin and other proteins in live RBCs.

Inter and intra subpopulation genetic variability of roe deer (Capreolus capreolus L.) assessed by I and II class genetic markers.

The material was collected in three regions of Poland and consisted of 105 randomly chosen individuals killed during hunts (49 males, 56 females), out of which 51 were from Wielkopolska, 22 from Podkarpacie and 32 from Warmia. From each animal a blood sample was taken from the chest, stored in a probe with K2EDTA and frozen. The serum was used to establish the genotype for transferin and albumin whereas the samples with erythrocytes provided information on hemoglobin genotype. DNA was isolated from samples from each individual. Characteristics of eight (from among twelve studied) microsatellite loci and genetic distances were estimated by the use of standard computer package programs. Generally, monomorphism in blood proteins was registered. For the microsatellite loci the number of alleles ranged from 3 in the RT27-6-Fa locus (effectively two as the third allele was present only in two subpopulations with a very low frequency) to 10 in RT1-VI. Five loci showed heterozygosity of 0.5 or above which suggests their usefulness in parentage control. Considerable genetic distances (corresponding to geographical mileages) between the subpopulations were observed based on microsatellite markers.

Brewer's spent grain and corn steep liquor as alternative culture medium substrates for proteinase production by Streptomyces malaysiensis AMT-3

Brewer's spent grain and corn steep liquor or yeast extract were used as the sole organic forms for proteinase production by Streptomyces malaysiensis in submerged fermentation. The influence of the C and N concentrations, as well as the incubation periods, were assessed. Eight proteolytic bands were detected through gelatin-gel-electrophoresis in the various extracts obtained from the different media and after different incubation periods, with apparent molecular masses of 20, 35, 43, 50, 70, 100, 116 and 212 kDa. The results obtained suggest an opportunity for exploring this alternative strategy for proteinases production by actinomycetes, using BSG and CSL as economically feasible substrates.

Characterization of a novel Entamoeba histolytica strain from Burkina Faso, Africa, possessing a unique hexokinase-2 gene.

An Entamoeba histolytica strain (BF-841 cl1) that originated from Burkina Faso, Africa presented with novel, polymorphic genotypes of the serine-rich E. histolytica protein and the anodic hexokinase­2 (HXK-2) isoenzyme band, which showed less electrophoretic mobility than that of an E. histolytica reference strain [HM-1:IMSS cl6 (zymodeme (Z)-II)] by starch gel electrophoresis and isoelectric focusing (IEF). The HXK-2 gene of BF-841 cl1 had amino acid variations at four positions compared to the sequence of HM-1:IMSS cl6. These variations were absent from the sequences of four other E. histolytica strains with different zymodemes [KU27 (Z-II), SAW1627 (Z-IIa-), SAW755CR clB (Z-XIV), and KU2 (Z-XIX)]. The results of IEF showed no difference in the substrate specificity of HXK (HXK-1 and HXK-2) between BF-841 cl1 and the three reference E. histolytica strains (HM-1:IMSS cl6, SAW755 clB, and KU27). It was also confirmed that BF-841 cl1 was able to form liver abscesses in Syrian hamsters.

Adenylate kinase locus 1 polymorphism and feto-placental development.

OBJECTIVE: Recently our group has found that the correlation between birth weight and placental weight - an index of a balanced feto-placental unit development - is influenced by genetic factors. Since adenylate kinase locus 1 (AK1) is a polymorphic enzyme that plays an important role in the synthesis of nucleotides required for many metabolic functions, we have investigated the possible role of its genetic variability in the correlation between birth weight and placental weight. STUDY DESIGN: 342 consecutive healthy newborn infants from the population of Rome (Italy) and 286 puerperae from another population from Central Italy were studied. RESULTS: The correlation coefficient between birth weight and placental weight is much higher in infants with low activity AK12-1 phenotype than in those with high activity AK11 phenotype. The difference between AK1 and AK12-1 is well marked only in newborns with a gestational age greater than 38 weeks and it is not influenced by sex, maternal age and maternal smoking. A similar pattern is observed with maternal AK1 phenotype. CONCLUSIONS: These results suggest that the difference in enzymatic activity between AK1 phenotypes influencing the equilibrium among ATP, ADP, AMP and adenosine could have an important role in a balanced development of feto-placental unit.

Characterization of two closely related α-amylase paralogs in the bark beetle, Ips typographus (L.).

Ips typographus (L.), the eight-spined spruce bark beetle, causes severe damage throughout Eurasian spruce forests and suitable nuclear markers are needed in order to study its population structure on a genetic level. Two closely related genes encoding α-amylase in I. typographus were characterized and named AmyA and AmyB. Both α-amylase paralogs consisted of six exons and five introns. AmyA encodes a polypeptide of 483 amino acids, whereas AmyB has two alternative transcripts encoding polypeptides of 483 and 370 amino acids. The expression levels of both genes were high during larval stage and adulthood. The AmyB transcripts were absent in the pupal stage. A modification of the allozyme staining method allowed us to detect two clusters of bands on the electrophoretic gel that may correspond to the two α-amylase genes. There was a correlation between the lack of AmyB expression in pupa and the absence of the fast migrating isozyme cluster at this stage, suggesting that the faster migrating isoforms are products of the AmyB gene, whereas the slowly migrating bands are derived from the AmyA.

Consequences of habitat fragmentation for the prairie-endemic weevil Haplorhynchites aeneus.

Widespread destruction of tallgrass prairies in the midwestern United States has fragmented plant communities with the result that populations of endemic animal species have become geographically isolated from one another. The goal of the research summarized here was to evaluate the potential for conserving endemic prairie species of herbivorous insects by managing their host plants. Our study species was the weevil Haplorhynchites aeneus (Boehman), adults of which feed on pollen of plants in the genus Silphium (Asteraceae: Heliantheae). The female weevils clip the peduncles of flower heads and oviposit into the heads, where the larvae feed on the ovules. The research was conducted in 12 prairie sites in eastern Illinois. An allozyme analysis revealed that most populations of H. aeneus at the various prairie sites were genetically differentiated from one another, but the degree of differentiation was not associated with geographic distance between sites. Adult H. aeneus fed and oviposited on the plant species Silphium laciniatum L., S. integrifolium Michx., and S. terebinthinaceum Jacq, which differ in bloom phenology. There was no evidence of genetic differentiation of weevil populations with respect to host plant species, and adult weevils strongly preferred S. terebinthinaceum. We conclude that the oligophagous nature of the weevil assures its survival in small prairie remnants even where some of the host plant species are absent. Although H. aeneus can have a significant impact on reproduction of host plants by clipping flower heads, the perennial nature of Silphium species prevents their local extinction.

Allozyme polymorphisms in horseshoe crabs, Carcinoscorpius rotundicauda, collected from polluted and unpolluted intertidal areas in Peninsular Malaysia.

It has been widely reported that allozyme frequency variation is a potential indicator of heavy metal-induced impacts in aquatic populations. In the present study, wild populations of horseshoe crab (Carcinoscorpius rotundicauda) were collected from contaminated and uncontaminated sites of Peninsular Malaysia. By adopting horizontal starch gel electrophoresis, seven enzyme systems were used to study allozyme polymorphisms. Nine polymorphic loci were observed in C. rotundicauda. The relationships of allozyme variations with the concentrations of Cd, Cu, Ni, and Zn in sediments and in muscle tissues of horseshoe crabs were determined. Based on genetic distance, the lower mean value of Nei's D (0.017) indicated that both of the contaminated populations of Kg. Pasir Puteh and Kuala Juru were very closely related when compared to the relatively uncontaminated Pantai Lido population. Higher heterozygosities were shown by the contaminated populations when compared to the uncontaminated population. Different allelic frequencies could be observed for the aldolase (ALD; E.C. 2.7.5.1) locus between the contaminated and uncontaminated populations of C. rotundicauda. The dendrogram of genetic relationships of the three populations of C. rotundicauda showed the same clustering pattern as the dendrograms are based on heavy metals in the sediments and in the horseshoe crabs' abdominal muscles. From the F statistics, the present study showed that the three populations of horseshoe crabs were considered to have undergone moderate genetic differentiation with a mean F (ST) value of 0.092 .The current results suggest that allozyme polymorphism in horseshoe crabs is a potential biomonitoring tool for metal contamination, although further validation is required.

Genetic and morphometric evidence for the conspecific status of the bumble bees, Bombus melanopygus and Bombus edwardsii.

The taxonomic status of closely related bumble bee species is often unclear. The relationship between the two nominate taxa, Bombus melanopygus Nylander (Hymenoptera: Apidae) and Bombus edwardsii Cresson (Hymenoptera: Apidae), was investigated using genetic (enzyme electrophoretic) and morphometric analyses. The taxa differ in the color of the abdominal terga two and three, being ferruginous in B. melanopygus and black in B. edwardsii. B. edwardsii occurs throughout California, while B. melanopygus extends north through Oregon, to Alaska and Canada. They are sympatric only in southern Oregon and northern California. The taxonomic status of these taxa was questioned when Owen and Plowright (1980) reared colonies from queens collected in the area of sympatry, and discovered that pile coloration was due to a single, biallelic Mendelian gene, with the red (R) allele dominant to the black (r). Here it is shown that all the taxa, whether from California, Oregon, or Alberta, have the same electrophoretic profile and cannot be reliably distinguished by wing morphometrics. This strongly supports the conclusion that B. melanopygus and B. edwardsii are conspecific and should be synonymized under the name B. melanopygus. Hence, there is a gene frequency cline running from north to south, where the red allele is completely replaced by the black allele over a distance of about 600 km.

[Monomorphism of isoenzyme loci in natural and cultivated amaranth (Amaranthus L.) populations].

Starch gel electrophoresis was used for isozyme analysis of ADH, GDH, MDH, IDH, and ME in populations of amaranth. Experiments were performed with 93 populations and 4 cultivars. Some populations proved to be polymorphic, and this fact allowed analysis of the genetic control of the enzymes listed. The populations examined showed poor allozyme variability. Monomorphism for all loci studied was observed in 73 populations and 4 varieties. Starch gel electrophoresis was used for isozyme analysis ofADH, GDH, MDH, IDH, and ME in populations of amaranth. Experiments were performed with 93 populations and 4 cultivars. Some populations proved to be polymorphic, and this fact allowed analysis of the genetic control of the enzymes listed. The populations examined showed poor allozyme variability. Monomorphism for all loci studied was observed in 73 populations and 4 varieties. Only some populations demonstrated rare polymorphism for a single locus each: Adh, Mdh 2, Gdh, Idh 1, Idh 2, or Mod 2. The results demonstrate genetic monomorphism of amaranth for the studied loci.

Different survival of chromium-exposed Oxya chinensis among allozyme genotypes.

The goal of the present study was to compare the different survival of Oxya chinensis exposed to chromium(VI) among allozyme genotypes to gain a better understanding of the relationship between the genetic variations and environmental disturbance. This study analyzed the occurrence of genotypes in O. chinensis population exposed to Cr(VI). O. chinensis samples were collected at Yuanping, Shanxi Province, China and used in acute toxicity tests. Specimens were assigned to Cr(VI) exposure (LD50: 291.0 mg/kg) for 24 h. The genetic composition of both dead and survived specimens was analyzed with horizontal starch gel electrophoresis in four enzymes (GPI, PGM, LDH, and ME). The results indicated that under Cr(VI) exposure, specimens with different genotypes had various mortalities at the four loci in laboratory conditions, and there was a genetic basis for tolerance in O. chinensis during acute exposure to Cr(VI).

Genetic structure and gene flow of Anopheles minimus and Anopheles harrisoni in Kanchanaburi Province, Thailand.

Isozyme frequencies were compared in seven field collections of Anopheles minimus complex using starch gel electrophoresis. Mosquito collections were sampled from four districts in Kanchanaburi Province where malaria is endemic. From eight enzyme systems, nine loci and seven polymorphisms were detected, indicating limited genetic differentiation among the seven collections (F(ST) = 0.061). The highest percent polymorphic loci were observed in Bong Ti Noi (BTN) Village (55.6%), whereas the least percent polymorphism was seen in Tha Kradan (TK) Village (22.2%). Comparing villages Pra Jedee (PJ) with Pu Teuy C (PTC) and Huai Khayeng (HK) with Pra Jedee (PJ), gene flow among collections varied from 3.72 to 62.25 reproductive migrants per generation. Among the seven collections, no correlation was seen between genetic and geographical distances (P > 0.05). Anopheles minimus (former species A) and Anopheles harrisoni (former species C) from Pu Teuy fit most closely in the same cluster, possibly indicating relatively recent divergence between taxa. The genetic and epidemiological ramifications of these findings are discussed.

Population genetic structure and diversity of the Apennine endemic stream frog, Rana italica--insights on the Pleistocene evolutionary history of the Italian peninsular biota.

For most species in the Western Palaearctic region, southern Mediterranean peninsulas have been identified as major Quaternary refugia and hotspots of intraspecific diversity, and thus, as areas of particular relevance for the conservation of the evolutionary potential. We analysed the patterns of geographical variation among 26 populations of the Italian stream frog, using both nuclear (allozymes) and mitochondrial (partial cytochrome b sequences) markers. Phylogenetic, phylogeographical and population genetic analyses suggested that the species survived the last glacial-interglacial cycles in two distinct refugia, one restricted to the tip of the Calabrian peninsula, at the extreme south of the species' range, the other spanning from central Calabria to central Apennines and showing evidences for further population subdivision therein. Historical demographic tests suggested a significant population expansion from the latter, which most likely began around the last pleniglacial. This expansion would have led to the rapid colonization of the northern Apennines to the north, and to a secondary contact and population admixture with the population from the southern refugium in southern central Calabria. A comparison of the evolutionary history inferred for the Italian stream frog with the data emerging for other codistributed species suggests: (i) the generality of a multiple-refugia scenario for the Italian peninsula, (ii) the possible occurrence of at least one suture zone in southern Italy, and (iii) that for most species, this Pleistocene refugium is not only a hotspot, but also a melting pot of intraspecific genetic diversity. Finally, the conservation implications of these results are also briefly highlighted.