Large-scale purification and characterization of recombinant Pseudomonas ceramidase: regulation by calcium

Ceramidases (CDases) hydrolyze ceramide to sphingosine (SPH) and fatty acid. Pseudomonas CDase (pCDase) is a homolog of mammalian neutral ceramidases and may play roles in disease pathogenesis. In this study, pCDase was cloned and expressed in Escherichia coli (E. coli). The expressed recombinant pCDase was solubilized by optimizing several factors, including culture medium, the concentration of isopropyl-beta-thiogalactopyranoside (IPTG), temperature, and time of induction, which were identified to be critical for the optimal production of recombinant pCDase. The recombinant pCDase was purified using nickel-nitrilotriacetic acid affinity, phenyl-Sepharose, and Q-Sepharose column chromatography, which gave an overall yield of 0.45 mg/l purified protein of starting culture. The activity of the recombinant pCDase followed classical Michaelis-Menten kinetics, with optimum activity in the neutral pH range. Both the hydrolytic and the reverse activities of CDase were stimulated by calcium with an affinity constant (K(a)) of 1.5 microM. Kinetics studies showed that calcium caused a decrease of K(m) and an increase in V(max) of pCDase. Calcium and D-erythro-sphingosine caused significant changes in the near ultraviolet circular dichroism (CD) spectra and the changes were inhibited in the presence of EGTA. These results identify important interactions between calcium and pCDase, which may play an essential role in the interaction of pCDase and its substrate.     

Development of SCAR markers linked to powdery mildew (<Emphasis Type="Italic">Uncinula necator</Emphasis>) resistance in grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L. and <Emphasis Type="Italic">Vitis</Emphasis> sp.)

Sequence-characterized amplified regions markers (SCARs) were developed from six randomly amplified polymorphic DNA (RAPD) markers linked to the major QTL region for powdery mildew (Uncinula necator) resistance in a test population derived from the cross of grapevine cultivars “Regent” (resistant) × “Lemberger”(susceptible). RAPD products were cloned and sequenced. Primer pairs with at least 21 nucleotides primer length were designed. All pairs were tested in the F1 progeny of “Regent” × “Lemberger”. The SCAR primers resulted in the amplification of specific bands of expected sizes and were tested in additional genetic resources of resistant and susceptible germplasm. All SCAR primer pairs resulted in the amplification of specific fragments. Two of the SCAR markers named ScORA7-760 and ScORN3-R produced amplification products predominantly in resistant individuals and were found to correlate to disease resistance. ScORA7-760, in particular, is suitable for marker-assisted selection for powdery mildew resistance and to facilitate pyramiding powdery mildew resistance genes from various sources.     

Pax‐6 is expressed early in the differentiation of a corneal epithelial model system

Pax‐6 is a regulatory gene with a major role during visual system development, but its association with corneal epithelial differentiation is not clearly established. Using the RCE1‐(5T5) cell line, which mimics corneal epithelial differentiation, we analyzed Pax‐6 biological role. Immunostaining of proliferating colonies and confluent sheets showed that Pax‐6‐positive cells were also K3 keratin‐positive, suggesting that Pax‐6 is expressed in differentiating cells. Pax‐6 mRNA was barely expressed in early cell cultures; but after confluence, its levels raised up to fivefold as demonstrated by Northern blot and RT‐qPCR. The raise in Pax‐6 expression preceded for 9 h the increase in LDH‐H and LDH‐M mRNAs, previously shown as early markers of corneal epithelial cell differentiation. The full‐length mRNAs encoding for the two major Pax‐6 isoforms were found at very low levels in proliferating cells, and abundantly expressed in the confluent stratified epithelia; Pax‐6 mRNA was 2‐ to 2.5‐fold more abundant than Pax‐6(5a) mRNA. The ectopic expression of Pax‐6 or Pax‐6(5a) decreased proliferative ability leading to the formation of abortive, non‐proliferative colonies. In contrast, culture conditions that delay or block corneal epithelial cell differentiation reduced or inhibited the expression of Pax‐6. Collectively, results show that Pax‐6 is the earlier differentiation marker expressed by corneal epithelial cells, and open the possibility for a major role of Pax‐6 as the main driver of the differentiation of corneal epithelial cells. J. Cell. Physiol. 220: 348–356, 2009. © 2009 Wiley‐Liss, Inc.     

Adrenal glands and testes as steroidogenic tissue are affected by retinoylation reaction

This study was undertaken to better understand the physiological role of the retinoylation process in steroidogenic tissues. In adrenal gland mitochondria, the retinoylation extent was found equal to that of testes mitochondria but without ATP in the incubation buffer. We pointed out that the endogenous mitochondrial ATP in adrenal glands is much higher than in testes, about 1.3 x 10⁻² M and 5.2 x 10⁻⁸ M, respectively. In addition, less CoASH is required for the maximal acylation activity of the retinoyl moiety to protein(s) compared to testes. The fatty acid analysis revealed a different composition of mitochondrial membranes of these two tissues. Among the different values of fatty acids, it is important to note that adrenal glands contain a much higher amount of C18:0 and a much lower amount of C22:5 ω6 and C22:6 ω3 than testes in the mitochondrial membranes. In addition, there were also differences in arachidonic acid (ARA, C20:4 ω6) content between adrenal glands and testes mitochondria. These different values in the fatty acids composition should explain the different extent of the retinoylation process between the two organs.     

Continued proteomic analysis of Mycobacterium leprae subcellular fractions

Recently the sequence of the Mycobacterium leprae chromosome, the only known obligate intracellular mycobacterium, was completed. It has a dramatic reduction in functional genes, with a coding capacity of only 49.5%, the lowest one so far observed among bacterial genomes. The leprosy bacillus seems to preserve a minimal set of genes that allows its survival in the host. The identification of genes that are actually expressed by the bacterium is of high significance in the context of mycobacterial pathogenesis. In this current study, a proteomic approach was undertaken to identify the proteins present in the soluble/cytosol and membrane subcellular fractions obtained from armadillo derived M. leprae. Proteins from each fraction were separated by two-dimensional gel electrophoresis (2-DE) and identified by mass spectrometry. A total of 147 protein spots were identified from 2-DE patterns and shown to comprise products of 44 different genes, twenty eight of them corresponding to new proteins. Additionally, two highly basic proteins (with pI >10.0) were isolated by heparin affinity chromatography and identified by N-terminal sequencing. This study constitutes the first application of proteomics to a host-derived Mycobacterium.     

Outcrossing potential between 11 important genetically modified crops and the Chilean vascular flora

The potential impact of genetically modified (GM) crops on biodiversity is one of the main concerns in an environmental risk assessment (ERA). The likelihood of outcrossing and pollen‐mediated gene flow from GM crops and non‐GM crops are explained by the same principles and depend primarily on the biology of the species. We conducted a national‐scale study of the likelihood of outcrossing between 11 GM crops and vascular plants in Chile by use of a systematized database that included cultivated, introduced and native plant species in Chile. The database included geographical distributions and key biological and agronomical characteristics for 3505 introduced, 4993 native and 257 cultivated (of which 11 were native and 246 were introduced) plant species. Out of the considered GM crops (cotton, soya bean, maize, grape, wheat, rice, sugar beet, alfalfa, canola, tomato and potato), only potato and tomato presented native relatives (66 species total). Introduced relative species showed that three GM groups were formed having: a) up to one introduced relative (cotton and soya bean), b) up to two (rice, grape, maize and wheat) and c) from two to seven (sugar beet, alfalfa, canola, tomato and potato). In particular, GM crops presenting introduced noncultivated relative species were canola (1 relative species), alfalfa (up to 4), rice (1), tomato (up to 2) and potato (up to 2). The outcrossing potential between species [OP; scaled from ‘very low’ (1) to ‘very high’ (5)] was developed, showing medium OPs (3) for GM–native relative interactions when they occurred, low (2) for GMs and introduced noncultivated and high (4) for the grape‐Vitis vinifera GM–introduced cultivated interaction. This analytical tool might be useful for future ERA for unconfined GM crop release in Chile.     

Functional morphology of the male reproductive system in Callichirus major (Crustacea: Decapoda: Axiidea): Evidence of oocytes in the gonad

Callichirus major inhabits the intertidal region of marine ecosystems and it is frequently used as live bait for fishing. This study aimed to describe the functional anatomy of the male reproductive system by microscopic techniques. The animals were collected along the Corujão beach, Piuma—ES, Brazil, and, in laboratory, the males were classified into two phases: immature (IM) and developed (DE) based on the macroscopic characteristics of the gonads. The gonad and vas deferens were dissected for histological routine and histochemical tests. Histologically, it was noted that in both phases, the more distal region of gonads has ovarian characteristics, showing developing oocytes. Also, different male germ cells were identified: spermatogonium (SPG), spermatocytes I and II (SPTCI, SPTCII), initial and final spermatid (IS, FS) and sperm (SPZ). Accessory cells with spherical or pyramidal nuclei were also present inside the testicular lobules. According to the vas deferens structure, three regions can be characterized: proximal (PVD), middle (MVD) and distal (DVD). In the lumen of the vas deferens, a spermatophoric matrix highly reactive for histochemical tests was observed. The presence of female germ cells in males suggests the occurrence of intersexuality or hermaphroditism in this species.     

Sex matters during adolescence: testosterone-related cortical thickness maturation differs between boys and girls

Age-related changes in cortical thickness have been observed during adolescence, including thinning in frontal and parietal cortices, and thickening in the lateral temporal lobes. Studies have shown sex differences in hormone-related brain maturation when boys and girls are age-matched, however, because girls mature 1-2 years earlier than boys, these sex differences could be confounded by pubertal maturation. To address puberty effects directly, this study assessed sex differences in testosterone-related cortical maturation by studying 85 boys and girls in a narrow age range and matched on sexual maturity. We expected that testosterone-by-sex interactions on cortical thickness would be observed in brain regions known from the animal literature to be high in androgen receptors. We found sex differences in associations between circulating testosterone and thickness in left inferior parietal lobule, middle temporal gyrus, calcarine sulcus, and right lingual gyrus, all regions known to be high in androgen receptors. Visual areas increased with testosterone in boys, but decreased in girls. All other regions were more impacted by testosterone levels in girls than boys. The regional pattern of sex-by-testosterone interactions may have implications for understanding sex differences in behavior and adolescent-onset neuropsychiatric disorders.     

A mammalian artificial chromosome engineering system (ACE System) applicable to biopharmaceutical protein production, transgenesis and gene-based cell therapy

Mammalian artificial chromosomes (MACs) provide a means to introduce large payloads of genetic information into the cell in an autonomously replicating, non-integrating format. Unique among MACs, the mammalian satellite DNA-based Artificial Chromosome Expression (ACE) can be reproducibly generated de novo in cell lines of different species and readily purified from the host cells' chromosomes. Purified mammalian ACEs can then be re-introduced into a variety of recipient cell lines where they have been stably maintained for extended periods in the absence of selective pressure. In order to extend the utility of ACEs, we have established the ACE System, a versatile and flexible platform for the reliable engineering of ACEs. The ACE System includes a Platform ACE, containing >50 recombination acceptor sites, that can carry single or multiple copies of genes of interest using specially designed targeting vectors (ATV) and a site-specific integrase (ACE Integrase). Using this approach, specific loading of one or two gene targets has been achieved in LMTK⁻ and CHO cells. The use of the ACE System for biological engineering of eukaryotic cells, including mammalian cells, with applications in biopharmaceutical production, transgenesis and gene-based cell therapy is discussed.