Purification,characterization and differential hormonal regulation of a β-1,3-glucanase and two chitinases from chickpea (Cicer arietinum L.)

Chickpea (Cicer arietinum L.) cell-suspension cultures were used to isolate one -1,3-glucanase (EC 3.2.1.29) and two chitinases (EC 3.2.1.14). The -1,3-glucanase (M_r = 36 kDa) and one of the chitinases (M_r = 32 kDa) belong to class I hydrolases with basic isoelectric points (10.5 and 8.5, respectively) and were located intracellularly. The basic chitinase (BC) was also found in the culture medium. The second chitinase (M_r = 28 kDa), with an acidic isoelectric point of 5.7, showed homology to N-terminal sequences of class III chitinases and represented the main protein accumulating in the culture medium. Polyclonal antibodies raised against the basic -1,3-glucanase (BG) and the acidic chitinase (AC) were shown to be monospecific. The anti-AC antiserum failed to recognize the BC on immune blots, confirming the structural diversity between class I and class III chitinases. Neither chitinase exhibitied lysozyme activity. All hydrolases were endo in action on appropriate substrates. The BC inhibited the hyphal growth of several test fungi, whereas the AC failed to show any inhibitory activity. Expression of BG activity appeared to be regulated by auxin in the cell culture and in the intact plant. In contrast, the expression of neither chitinase was apparently influenced by auxin, indicating a differential hormonal regulation of -1,3-glucanase and chitinase activities in chickpea. After elicitation of cell cultures or infection of chickpea plants with Ascochyta rabiei, both system were found to have hydrolase patterns which were qualitatively and quantitatively comparable. Finally, resitant (ILC 3279) and susceptible (ILC 1929) cultivars of chickpea showed no appreciable differences with regard to the time and amount of hydrolase accumulation after inoculation with spores of A. rabiei.Abbreviations AC acidic chitinase - BC basic chitinase - BG = basic -1,3-glucanase - CM-Chitin-RBV carboxymethylated-chitin-remazol brilliant violet - 2,4-D 2,4-dichlorophenoxyacetic acid - ILC international legume chickpea - M_r relative molecular mass - pI isoelectric point - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis We thank the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie for financial support and ICARDA, Aleppo, Syria, for the provision of seed material. We also thank Dr. B. Fritig (Institut de Biologie Moléculaire des Plantes, CNRS, Straßbourg, France) and Dr. F. Meins, Jr. (Friedrich-Miescher-Institut, Basel, Switzerland) for their kind gifts of antibodies.     

Physiological aspects of hydrocarbon emulsification, metal resistance and DNA profile of biodegrading bacteria isolated from oil polluted sites

The production of biosurfactants was evaluated for seven bacterial strains isolated from different oil contaminated sites by the Emulsification Index using diesel oil as the hydrocarbon source. Minimum Inhibitory Concentrations of Mg²⁺, Cr³⁺ and Cu²⁺ were determined to identify the less sensitive bacteria in order to select the best strains for bioremediation. Plasmid extraction was also performed in order to search for gene sequences involved with biosurfactant synthesis. All strains were able to emulsify diesel oil. Rhodococcus ruber AC239 presented the best index (58%), followed by other Rhodococcus strains. Pseudomonas aeruginosa, R. ruber AC239, AC87 and R. erytropolis AC272 presented smallest sensitivities to heavy metals used, being suitable for use in sites contaminated with high concentrations of them. No plasmid DNA was detected showing that biosurfactant coding genes should be in the chromosomal DNA.     

AC29: the most abundant protein of Alexandrium catenella and its potential use in encystment/excystment studies

The objective of this research was to characterize specific protein(s) from Alexandrium catenella to evaluate its use as markers for specific physiological functions. To identify such protein(s) we concentrated our efforts on characterizing proteins with a high level of expression in vegetative cells of A. catenella. The electrophoretic analysis of a total protein cell extract showed the presence of a very abundant 29 kDa protein that we have named AC29. Analysis by 2D SDS-PAGE shows that the 29 kDa band contains one abundant protein (AC29) and various less abundant polypeptides, suggesting the presence of either different proteins with similar molecular weight or isoforms of AC29 protein. Ultracytolocalization using antibodies raised against gel purified AC29 indicates that this protein localizes within the chloroplast and that it is associated with thylakoid membranes, as well as with other membranes surrounding the chloroplast. Western blot analysis of cells grown under light starvation shows that the expression of the AC29 protein is down regulated. A similar analysis shows that this protein is not expressed in natural cysts or by isolated intracellular bacterium. The amino terminus of the AC29 protein that was recovered from 2D SDS-PAGE was sequenced. The sequence shows homology to the peridinin-chlorophyll a-protein from the marine organisms Alexandrium cohorticula, Amphidinium carterae and Symbiodinium. Based on these results, we suggest that the AC29 protein has the potential of being used as a marker for A. catenella encystment and excystment processes.     

Quantitative reduction of type I adenylyl cyclase in human alcoholics.

The amounts of adenylyl cyclase type I (AC I) were examined in various parts of the postmortem brains from alcoholics who prior to death had been abstinent from alcohol for at least 6 months and compared with controls using immunoblot analysis with anti-AC I specific antibody. It was revealed that a significant reduction of AC I was observed in both frontal and temporal cortices. On the other hand, in other areas (occipital cortex, caudate nucleus, putamen, and hippocampus) the amounts were comparable between alcoholics and controls. In the next step, we examined two subtypes of human AC mRNA levels (AC I and AC VIII) in blood cells by quantitative RT-PCR using [alpha-32P]dCTP with two sets of the synthetic oligonucleotide primers based on the DNA sequences reported elsewhere (Villacres, E.C. et al., Genomics 16 (1993) 473-478; J. Parma et al., Biochem. Biophys. Res. Commun. 179 (1991) 455-462). The amounts of amplified DNAs of both AC I and AC VIII were significantly smaller in alcoholics than in controls. On the other hand, the amounts of amplified DNA of beta-actin DNA were almost equal between alcoholics and controls. It appears from these results that a reduction in the amount of AC subtypes may be a biological marker for alcoholics.     

Control of early seedling growth in varietal lines of hexaploid wheat (Triticum aestivum), durum wheat (Triticum durum), and barley (Hordeum vulgare) in response to the phthalimide growth regulant,AC 94,377

AC 94,377 caused elongation of seedlings of Triticum aestivum, Triticum durum, and Hordeum vulgare when applied to the soil, or the soil plus seed at planting. Affected were the leaf sheathes and the coleoptiles, and at high compound rates there was premature elongation of the stem internodes. As exemplified by the response of T. aestivum var. Fidel, the influence on coleoptile elongation was greatest under conditions whereby the coleoptile was naturally stimulated to elongate, i.e., when growth was in the dark and temperatures were cool (15°C). All of the stem internodes were capable of elongation except the one below the coleoptile node. The effect on leaf sheath elongation was prolonged when compared to activity of gibberellic acid.Several varieties of the three cereal species were examined in the greenhouse for sensitivity to AC 94,377 in order to evaluate the extent of the response. All of the barley varieties examined were sensitive to AC 94,377, elongating regardless of the planting conditions. Two such conditions were established, including incubation under warm (28/20°C) conditions following planting the grains 1 cm deep, and incubation under cool (22/16°C) conditions following planting the grains 6 cm deep. Wheat varieties distributed into two general categories, those which were sensitive and those which were not. The insensitivity correlated well to the presence of the reduced height (Rht) and GA-insensitive (Gai) genes in Triticum aestivum and Triticum durum, respectively. Thus, AC 94,377 can be used conveniently to evaluate varietal lines for the presence of this phenotype. This correlation also lends support to the notion that the Rht/Gai mutations in wheat are either at the level of a gibberellin receptor or at a step in the signal transduction pathway.     

Arabinogalactan-protein epitope Gal4 is differentially regulated and localized in cell lines of hybrid fir (<Emphasis Type="Italic">Abies alba</Emphasis> × <Emphasis Type="Italic">Abies cephalonica</Emphasis>) with different embryogenic and regeneration potential

Arabinogalactan proteins (AGPs) are important proteoglycans regulating somatic embryogenesis in diverse plant species. Embryogenic cells of somatic embryos are covered by special extracellular cell wall layer called extracellular surface matrix network (ECMSN) at their early developmental stages. Here we show that highly embryogenic cell line AC78 of hybrid fir (Abies alba × Abies cephalonica) differs from very low-embryogenic cell line AC77 in the abundance, subcellular localization and deposition of subset of secreted AGPs. A specific AGP epitope containing Gal residues and reacting to Gal4 antibody is secreted and deposited into ECMSN, which covers the surface of the embryogenic cells showing high embryogenic and regeneration capacity in the cell line AC78. On the other hand, this Gal4 AGP epitope was not secreted and/or found on the surface of meristematic cells showing low embryogenic and regeneration capacity in the cell line AC77, as well as on the surface of non-embryogenic suspensor cells and callus cells in both cell lines AC77 and AC78. As a positive control, we have used another AGP epitope LM2 (containing glucuronic acid) showing no significant differences in these two Abies hybrid lines. This study defines specific AGPs containing β-(1→6)-galactotetraosyl group as a first molecular component of ECMSN covering embryogenic cells in gymnosperms. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Promoting effect of Antrodia camphorata as an immunomodulating adjuvant on the antitumor efficacy of HER-2/neu DNA vaccine

It is well known that DNA vaccines induce protective humoral and cell-mediated immune responses in several animal models. Antrodia camphorata (AC) is a unique basidiomycete fungus of the Polyporaceae family that only grows on the aromatic tree Cinnamomum kanehirai Hayata (Lauraceae) endemic to Taiwan. Importantly, AC has been shown to be highly beneficial in the treatment and prevention of cancer. The goal of this study was to investigate whether AC is able to augment the antitumor immune properties of a HER-2/neu DNA vaccine in a mouse model in which p185neu is overexpressed in MBT-2 tumor cells. Compared with the mice that received the HER-2/neu DNA vaccine alone, co-treatment with AC suppressed tumor growth and extended the survival rate. This increase in the antitumor efficacy was attributed to the enhancement of the Th1-like cellular immune response by the HER-2/neu DNA vaccine–AC combination. Evidence for this came from the marked increase in the IFN-γ mRNA expression in CD4⁺ T cells in the draining inguinal lymph nodes, an increase in the number of functional HER-2/neu-specific CTLs, and the increased tumor infiltration of both CD4⁺ and CD8⁺ T cells, depletion of which abolishes the antitumor effect of the HER-2/neu DNA vaccine–AC therapy. Our results further indicate that the treatment of mice with AC enhanced DC activation and production of Th1-activating cytokines (e.g. IL-12, and IFN-α) in the draining lymph nodes, which were sufficient to directly stimulate T cell proliferation and higher IFN-γ production in response to ErbB2. Overall, these results clearly demonstrate that AC represents a promising immunomodulatory adjuvant that could enhance the therapeutic potency of HER-2/neu DNA vaccines in cancer therapy.     

基于响应面法的高抗原活性禽多杀性巴氏杆菌疫苗培养基的研制与效果评价

【背景】禽多杀性巴氏杆菌(Pasteurella multocida)引发的禽霍乱疫情造成了巨大的危害,而现有培养基存在培养菌密度较低的问题。【目的】研制高抗原活性的禽多杀性巴氏杆菌疫苗培养基。【方法】通过单因素试验、Plackett-Burman试验和响应面分析方法对禽多杀性巴氏杆菌培养基的成分进行调整,并对不同发酵阶段的菌体进行免疫原性测定。最后使用该培养基培养细菌后制备疫苗并通过动物攻毒试验评价其保护效果。【结果】使用研制的培养基培养禽多杀性巴氏杆菌,活菌密度能够在6 h达到约1.84×1010 CFU/mL,增菌效果是对照培养基的2.6倍;免疫原性测定结果显示在生长平台期菌体的抗原活性最高;攻毒试验表明制备的疫苗能够很好地抵抗禽多杀性巴氏杆菌的侵袭。【结论】研制出了高抗原活性的禽多杀性巴氏杆菌疫苗培养基,为疫苗的生产奠定了基础。     

一株维氏气单胞菌发酵培养基优化及其制备疫苗免疫效力比较

采用单因素试验、响应面试验法对维氏气单胞菌(Aeromonas veronii)发酵培养基的氮源、碳源、无机盐和磷酸盐成分及用量进行优化组合,确定优化培养基组成：胰蛋白胨10.8 g/L,葡萄糖5.0 g/L,牛肉膏3.0 g/L,磷酸二氢钾2.0 g/L,硫酸镁0.4 g/L,NaCl 5.0 g/L。并与基础培养基的发酵活菌数、制备的灭活疫苗免疫效力进行比较,经过验证试验绘制维氏气单胞菌在优化培养基条件下的7 L发酵罐生长曲线。在优化发酵培养基条件下,维氏气单胞菌活菌数为5.94×10⁹ cfu/mL,比基础培养基增幅43.13%;制备的灭活疫苗相对保护率为77.78%,比基础培养基提高了14.81%。7 L发酵罐发酵培养10 h,活菌数达到最大8.85×10⁹ cfu/mL。通过对发酵培养基的优化,可以获得低成本、优质高效的维氏气单胞菌发酵菌液,为今后维氏气单胞菌灭活疫苗规模化发酵培养提供参考。     

Positively Selected Codons in Immune-Exposed Loops of the Vaccine Candidate OMP-P1 of <Emphasis Type="Italic">Haemophilus influenzae</Emphasis>

The high levels of variation in surface epitopes can be considered as an evolutionary hallmark of immune selection. New computational tools enable analysis of this variation by identifying codons that exhibit high rates of amino acid changes relative to the synonymous substitution rate. In the outer membrane protein P1 of Haemophilus influenzae, a vaccine candidate for nontypeable strains, we identified four codons with this attribute in domains that did not correspond to known or assumed B- and T-cell epitopes of OMP-P1. These codons flank hypervariable domains and do not appear to be false positives as judged from parsimony and maximum likelihood analyses. Some closely spaced positively selected codons have been previously considered part of a transmembrane domain, which would render this region unsuited for inclusion in a vaccine. Secondary structure analysis, three-dimensional structural database searches, and homology modeling using FadL of E. coli as a structural homologue, however, revealed that all positively selected codons are located in or near extracellular looping domains. The spacing and level of diversity of these positively selected and exposed codons in OMP-P1 suggest that vaccine targets based on these and conserved flanking residues may provide broad coverage in H. influenzae. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. [Reviewing Editor: Dr. Rasmus Nielson]     

Trichinella spiralis: intranasal immunization with attenuated Salmonella enterica carrying a gp43 antigen-derived 30mer epitope elicits protection in BALB/c mice

Trichinellosis is a public health problem and is considered an emergent/re-emergent disease in various countries. The etiological agent of trichinellosis is the nematode Trichinella, which infects domestic animals such as pigs and horses, as well as wild animals and humans. A veterinary vaccine could be an option to control the disease in domestic animals. Although several vaccine candidates have shown promising results, a vaccine against trichinellosis remains unavailable to date. Attenuated Salmonella strains are especially attractive live vectors because they elicit mucosal immunity, which is known to be important for the control of Trichinella spiralis infection at the intestinal level and can be administered by oral or intranasal routes. In this study, the autotransporter ShdA was used to display, on the surface of the Salmonella enterica serovar Typhimurium SL3261, the 210–239 amino acid epitope, (designated as Ag30) derived from the 43 kDa glycoprotein of T. spiralis muscle larvae. The fusion protein elicited antibodies in BALB/c mice that were able to recognize the native epitope on the surface of T. spiralis muscle larvae. Mice immunized by intranasal route with the recombinant Salmonella induced a protective immune response against the T. spiralis challenge, reducing by 61.83% the adult burden at day eight postinfection. This immune response was characterized by the induction of antigen-specific IgG1 and of IL-5 production. This study demonstrates the usefulness of Salmonella as a carrier of nematode epitopes providing a surface display system for intestinal parasite vaccine applications.     

In vitro evaluation of the action of the nematophagous fungi Duddingtonia flagrans, Monacrosporium sinense and Pochonia chlamydosporia on Fasciola hepatica eggs

This work evaluated the in vitro action of four isolates of the nematophagous fungi Duddingtonia flagrans (AC001), Monacrosporium sinense (SF53) and Pochonia chlamydosporia (VC1 and VC4) on eggs of Fasciola hepatica. The eggs were plated on 2% water-agar with the grown isolates and control without fungus. After 7, 14 and 21 days, the eggs were removed and classified according to the following parameters: effect type 1, lytic effect with no morphological damage to eggshells; type 2, lytic effect with morphological changes in eggshells and embryos; and type 3, lytic effect with morphological changes in embryos and eggshells, with hyphal penetration and internal egg colonization. Pochonia chlamydosporia showed ovicidal activity on F. hepatica eggs in the studied intervals of the type-3 effect, of 12.8% (VC1) and 16.5% (VC4); 14.4% (VC1) and 18.7% (VC4), 20.1% (VC1) and 21.5 % (VC4), over 7, 14 and 21 days respectively. No statistical difference was found (P > 0.01) among the isolates VC1 and VC4 for effects type 1, 2 and 3 during the studied intervals. Duddingtonia flagrans (AC001) and Monacrosporium sinense fungi only showed effect type 1, with no significant difference between them, with the following results: 60.1% (AC001) and 57.5% (SF53); 62.3% (AC001) and 62.0% (SF53); 66.5% (AC001) and 73.4% (SF53), over 7, 14 and 21 days respectively. Pochonia chlamydosporia fungi negatively influenced the in vitro F. hepatica viability. Therefore it can be considered as a potential biological control agent for this helminth.     

Fine Mapping and in silico Isolation of the EUI1 Gene Controlling Upper Internode Elongation in Rice

Upper internode elongation in rice is an important agronomic trait. Well-known mutants with an elongated uppermost internode (eui) are important germplasms for developing unsheathed-panicle male-sterile lines in hybrid rice breeding. We finely mapped the eui1 gene and identified its candidate gene using in silico analysis based on previous research work and rice genomic sequence data. The rice eui1 gene was mapped to two overlapping BAC clones, OSJNBa0095J22 and OSJNBb0099O15, between the markers AC40 and AC46, that were 0.64 cM apart and spanned approximately 152 kb. A simple sequence repeat (SSR) marker AC41 that cosegregated with eui1 was located in an intron of a putative cytochrome P450-related gene. In silico analysis suggested that this encoded the cytochrome CYP714D1. Allelic sequencing confirmed that EUI1 corresponded to this P450 gene. A gamma ray-induced eui1 mutant carried a deletion in exon II of the EUI1 gene, and resulted in a frame-shift deletion that produced a truncated polypeptide. We conclude that the EUI1 gene controlling the upper internode elongation in rice is 9804 bp long, and comprises two exons and one intron. The length of the cDNA is 1931 bp containing a 1734 bp ORF, a 110 bp 5′-UTR and a 87 bp 3′-UTR. The ORF encodes an unknown 577 amino acid functional protein, that appears to be a member of the cytochrome P450 family. Hongli Ma, Shubiao Zhang: These authors contributed equally to this work     

Terminal repeat retrotransposon in miniature (TRIM) as DNA markers in Brassica relatives

We have developed a display system using a unique sequence of terminal repeat retrotransposon in miniature (TRIM) elements, which were recently identified from gene-rich regions of Brassica rapa. The technique, named TRIM display, is based on modification of the AFLP technique using an adapter primer for the restriction fragments of BfaI and a primer derived from conserved terminal repeat sequences of TRIM elements, Br1 and Br2. TRIM display using genomic DNA produced 50–70 bands ranging from 100 to 700 bp in all the species of the family Brassicaceae. TRIM display using B. rapa cDNA produced about 20 bands. Sequences of 11 randomly selected bands, 7 from genomic DNA and 4 from cDNA, begin with about 104 bp of the terminal repeat sequences of TRIM elements Br1 or Br2 and end with unique sequences indicating that all bands are derived from unique insertion sites of TRIM elements. Furthermore, 7 of the 11 unique sequences showed significant similarity with expressed gene. Most of the TRIM display bands were polymorphic between genera and about 55% (132 of 239 bands) are polymorphic among 19 commercial F1 hybrid cultivars. Analysis of phylogenetic relationships shows clear-cut lineage among the 19 cultivars. Furthermore, a combination of 11 polymorphic bands derived from only one primer combination can clearly distinguish one cultivar from the others. TRIM display bands were reproducible and inheritable through successive generations that is revealed by genetic mapping of 6 out of 27 polymorphic TRIM markers on the genetic map of Brassica napus. Collective data provide evidence that TRIM display can provide useful DNA markers in Brassica relatives because these markers are distributed in gene-rich regions, and are sometimes involved in the restructuring of genes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. GenBank accession nos.: AC190326 (TRIM display fragment gCP01); AC190327 (TRIM display fragment gCP02); AC190328 (TRIM display fragment gCP02) AC190329 (TRIM display fragment gCP04) AC190330 (TRIM display fragment gDP01); AC190331 (TRIM display fragment gDP02); AC190332 (TRIM display fragment gDP03); AC190333 (TRIM display fragment gDP04); AC190334 (TRIM display fragment gDP05); AC190335 (TRIM display fragment gDP06); AC190336 (TRIM display fragment gDP07).     

AC133-2, a novel isoform of human AC133 stem cell antigen

Human AC133 antigen, also called CD133, was recently identified as a hematopoietic stem cell marker. However, the molecular structure and function of this protein has remained unclear. Here we cloned and identified a novel isoform of AC133, which we named AC133-2. In comparison to the reported AC133 cDNA, which is referred to herein as AC133-1, a small exon of 27 nucleotides is deleted in AC133-2 by alternative mRNA splicing. Similar to the previously characterized AC133 antigen, recombinant AC133-2 expressed in 293 cells was glycosylated and transported to plasma membrane. AC133-2 mRNA was found predominant in a variety of human fetal tissue, adult tissues, and several carcinomas. In contrast, AC133-1 mRNA was more prominent in fetal brain and adult skeletal muscle but was not detected in fetal liver and kidney, adult pancreas, kidney, and placenta, suggesting different roles for the two isoforms in fetal development and mature organ homeostasis. Here, we demonstrate that AC133-2 is the isoform expressed on hematopoietic stem cells derived from fetal liver, bone marrow, and peripheral blood. The results indicate that AC133-2, not AC133-1, has been the cell surface antigen recognized by anti-AC133 monoclonal antibodies that are used for isolation of hematopoietic stem cells. To further investigate its expression in other stem cell populations, we found that AC133-2 co-expressed with beta(1) integrin in the basal layer of human neonatal epidermis. AC133-2(+)/beta(1) integrin(+) cells proliferated and differentiated in culture, which coincided with a loss of AC133-2 and gain in a terminal differentiation marker involucrin. Taken together, these results suggest that AC133-2 is expressed in multiple stem cell niches and may provide a means to isolate specific stem cell subpopulations from human tissues.     

Attempts at a peptide vaccine against paracoccidioidomycosis, adjuvant to chemotherapy

Chemotherapy is the basis of treatment of paracoccidioidomycosis in its various forms. Depending on the Paracoccidioides brasiliensis virulence, the status of host immunity, the degree of tissue involvement and fungal dissemination, treatment can be extended for long periods with an alarming frequency of relapses. Association of chemotherapy with a vaccine to boost the cellular immune response seemed a relevant project not only to reduce the time of treatment but also to prevent relapses and improve the prognosis of anergic cases. The candidate immunogen is the gp43 major diagnostic antigen of P.␣brasiliensis and more specifically its derived peptide P10, carrying the CD4⁺ T-cell epitope. Both gp43 and P10 protected Balb/c mice against intratracheal infections with virulent P. brasiliensis strain. P10 as single peptide or in a multiple-antigen-peptide (MAP) tetravalent construction was protective without adjuvant either by preimmunization and intratracheal challenge or as a therapeutic agent in mice with installed infection. P10 showed additive protective effects in drug-treated mice stimulating a Th-1 type immune response with high IFN-γ and IL-12. P10 and few other peptides in the gp43 were selected by Tepitope algorithm and actually shown to promiscuously bind several prominent HLA-DR molecules suggesting that a peptide vaccine could be devised for a genetically heterogenous population. P10 was protective in animals turned anergic, was effective in a DNA minigene vaccine, and increased the protection by monoclonal antibodies in Balb/c mice. DNA vaccines and peptide vaccines are promising therapeutic tools to be explored in the control of systemic mycoses.     

CD133 Expression Is Not Synonymous to Immunoreactivity for AC133 and Fluctuates throughout the Cell Cycle in Glioma Stem-Like Cells

A transmembrane protein CD133 has been implicated as a marker of stem-like glioma cells and predictor for therapeutic response in malignant brain tumours. CD133 expression is commonly evaluated by using antibodies specific for the AC133 epitope located in one of the extracellular domains of membrane-bound CD133. There is conflicting evidence regarding the significance of the AC133 epitope as a marker for identifying stem-like glioma cells and predicting the degree of malignancy in glioma cells. The reasons for discrepant results between different studies addressing the role of CD133/AC133 in gliomas are unclear. A possible source for controversies about CD133/AC133 is the widespread assumption that expression patterns of the AC133 epitope reflect linearly those of the CD133 protein. Consequently, the readouts from AC133 assessments are often interpreted in terms of the CD133 protein. The purpose of this study is to determine whether and to what extent do the readouts obtained with anti-AC133 antibody correspond to the level of CD133 protein expressed in stem-like glioma cells. Our study reveals for the first time that CD133 expressed on the surface of glioma cells is poorly immunoreactive for AC133. Furthermore, we provide evidence that the level of CD133 occupancy on the surface of glioma cells fluctuates during the cell cycle. Our results offer a new explanation for numerous inconsistencies regarding the biological and clinical significance of CD133/AC133 in human gliomas and call for caution in interpreting the lack or presence of AC133 epitope in glioma cells.     

Biodegradation of naphthalene-2-sulfonic acid present in tannery wastewater by bacterial isolates <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. 2AC and <Emphasis Type="Italic">Comamonas</Emphasis> sp. 4BC

Two bacterial strains, 2AC and 4BC, both capable of utilizing naphthalene-2-sulfonic acid (2-NSA) as a sole source of carbon, were isolated from activated sludges previously exposed to tannery wastewater. Enrichments were carried out in mineral salt medium (MSM) with 2-NSA as the sole carbon source. 16S rDNA sequencing analysis indicated that 2AC is an Arthrobacter sp. and 4BC is a Comamonas sp. Within 33 h, both isolates degraded 100% of 2-NSA in MSM and also 2-NSA in non-sterile tannery wastewater. The yield coefficient was 0.33 g biomass dry weight per gram of 2-NSA. A conceptual model, which describes the aerobic transformation of organic matter, was used for interpreting the biodegradation kinetics of 2-NSA. The half-lives for 2-NSA, at initial concentrations of 100 and 500 mg/l in MSM, ranged from 20 h (2AC) to 26 h (4BC) with lag-phases of 8 h (2AC) and 12 h (4BC). The carbon balance indicates that 75–90% of the initial TOC (total organic carbon) was mineralized, 5–20% remained as DOC (dissolved organic carbon) and 3–10% was biomass carbon. The principal metabolite of 2-NSA biodegradation (in both MSM and tannery wastewater) produced by Comamonas sp. 4BC had a MW of 174 and accounted for the residual DOC (7.0–19.0% of the initial TOC and 66% of the remaining TOC). Three to ten percent of the initial TOC (33% of the remaining TOC) was associated with biomass. The metabolite was not detected when Arthrobacter sp. 2AC was used, and a lower residual DOC and biomass carbon were recorded. This suggests that the two strains may use different catabolic pathways for 2-NSA degradation. The rapid biodegradation of 2-NSA (100 mg/l) added to non-sterile tannery wastewater (total 2-NSA, 105 mg/l) when inoculated with eitherArthrobacter 2AC or Comamonas 4BC showed that both strains were able to compete with the indigenous microorganisms and degrade 2-NSA even in the presence of alternate carbon sources (DOC in tannery wastewater = 91 mg/l). The results provide information useful for the rational design of bioreactors for tannery wastewater treatment.     

Comparative molecular and three-dimensional analysis of the peptide–MHC II binding region in both human and Aotus MHC-DRB molecules confirms their usefulness in antimalarial vaccine development

A vaccine against malaria is desperately needed, and Aotus monkeys are highly susceptible to experimental infection with malarial parasites. A thorough analysis of this monkey’s immune system molecules was thus undertaken in our institute. Cloning and sequencing, followed by three-dimensional analysis, has revealed high homology with some HLA-DRB1 molecules in terms of their peptide binding region pockets. Molecules such as HLA-DRB1*03, 11, 08, and HLA-DRB1*04 are so similar to Aotus MHC-DRB molecules that peptides identified as binding to these molecules and inducing protective immunity in these monkeys could be used in humans without further refinement, while small modifications seem to be needed for those binding to HLA-DRB1*07, HLA-DRB1*15, 16, and HLA-DRB1*10-like molecules, making this New World monkey an excellent model for tailor-made vaccine development, especially against malaria.     

Tissue-specific expression of the PAL3 promoter requires the interaction of two conserved cis sequences

The bean PAL2 and PAL3 promoters confer expression in overlapping sets of tissue types in transgenic tobacco. The PAL3 promoter contains motifs that resemble two AC cis elements which are required for tissue-specific expression of the PAL2 promoter. The functions of these motifs in the PAL3 promoter were determined by analysis of mutated PAL3 promoter-GUS constructs in transgenic tobacco. This revealed that the AC motifs are necessary for tissue-specific expression of the PAL3 promoter. Therefore, a key role is indicated for AC elements, which are Myb-protein binding sites, in regulating tissue-specific expression of the bean PAL gene family.