RNA/DNA ratio as an index of physiological condition of Colossoma macropomum and Piaractus branchypomus (Pisces: Characiformes) during embryonic development

We evaluated RNA/DNA ratio as an index of physiological condition during larval development of a hybrid between the fishes Colossoma macropomum (cachama) and Piaractus brachypomus (morocoto). The samples were obtained by induced reproductive technology and the eggs were maintained in acrylic conical incubator with a continuous waterflow. Embryonic development, from egg fertilization to cell division and hatch out, took 12 hours 20 minutes at 29.5 degrees C, dissolved oxygen contents of 6.0 ppm and pH 7.5. Nucleic acids quantification was determined by fluorometry with ethidium bromide and Hoechst 33258 dyes. We observed significant changes of RNA/DNA ratios during all stages of the embryonic larval development. Therefore, RNA/DNA relation is an useful technique to evaluate physiological condition in short period and could be utilized as nutritional condition and/or instantaneous growth for routine check to verify the health status in early life of cultivated species.     

Functional expression of recombinant tumstatin in stably transformed Drosophila melanogaster S2 cells

Recombinant tumstatin was expressed in stably transformed Drosophila melanogaster S2 cells and secreted into the medium with a molecular size of 29 kDa. Recombinant endostatin was also purified to homogeneity using a simple one-step Ni²⁺ affinity fractionation. Purified recombinant tumstatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition for recombinant tumstatin was approx. 0.7 g ml^–1. A maximum production of 4.6 g recombinant tumstatin (10⁷ cells)^–1 was obtained in a T-flask culture of S2 cells, 7 d after induction with 0.5 mM CuSO₄.     

Development of a set of expression vectors in Hansenula polymorpha

Four expression vectors based on formate dehydrogenase promoter (FMDp) and methanol oxidase promoter (MOXp) from Hansenula polymorpha were developed to express heterologous genes in Hansenula polymorpha. A secretion signal sequence of the mating factor-alpha from Saccharomyces cerevisiae was inserted in the secretory expression plasmids for efficient secretion. A modified green fluorescent protein (mGFP5) was used as the marker of expression for the first time in H. polymorpha NCYC495 (leu 1.1) to determine the expression ability of these plasmids. The mGFP5 thus expressed retained its biochemical and physiological properties, such as accumulation inside cells and efficient secretion into the culture media. These results indicated that the four integrative vectors are useful expression systems which could be directly applied for production of heterologous proteins of interests in H. polymorpha.     

An approach for cloning biosynthetic genes of 2-deoxystreptamine-containing aminocyclitol antibiotics: isolation of a biosynthetic gene cluster of tobramycin from Streptomyces tenebrarius

Genes homologous to 2-deoxystreptamine (DOS) biosynthetic genes were isolated from aminoglycoside producers, Micromonospora and Streptomyces spp., using PCR primers based on the core sequences of 2-deoxy-scyllo-inosose (DOI) synthase and L-glutamine: scyllo-inosose aminotransferase (GIA). Identities of 40-45% were observed for DOI synthases, and 65-75% were observed for GIAs. The gene cluster of tobramycin biosynthesis was isolated from the genomic library of Streptomyces tenebrarius using DOI synthase as a probe. Sequencing of 33.9 kb revealed 24 putative open reading frames including the tobramycin biosynthetic gene cluster (13.8 kb) and a transport protein. This cluster encodes proteins homologous to 2-deoxystreptamine biosynthetic enzymes, glycosyltransferase and other aminocyclitols biosynthetic enzymes. Sequence analysis revealed the evolution of DOI synthases from 3-dehydroquinate (DHQ) synthases in actinomycetes. DOI synthases and GIA are therefore useful for cloning biosynthetic genes of DOS-containing aminocyclitol antibiotics or for screening such metabolites producers.     

Decolorization of synthetic dyes and production of manganese-dependent peroxidase by new fungal isolates

Two yeasts, Debaryomyces polymorphus, Candida tropicalis, and two filamentous fungi, Umbelopsis isabellina, Penicillium geastrivorus, could completely decolorize 100 mg Reactive Black 5 (RB 5) l^–1 within 16–48 h. Manganese-dependent peroxidase (MnP) activities between 60 and 424 U l^–1 were detected in culture supernatants of three of these organisms indicating the color removal by enzymatic biodegradation but with P. geastrivorus there was no ligninolytic enzyme activity in its culture and the decolorization was mainly due to biosorption to mycelium. Extensive decolorization by D. polymorphus (69–94%) and C. tropicalis (30–97%) was obtained with five other azo dyes and one anthraquinone dye. Except for Reactive Brilliant Blue KNR and Reactive Yellow M-3R, the four azo dyes, Reactive Red M-3BE, Procion Scharlach H-E3G, Procion Marine H-EXL and Reactive Brilliant Red K-2BP, induced D. polymorphus to produce MnP (105–587 U l^–1). However, MnP activities of 198–329 U l^–1 were only detected in the culture of C. tropicalis containing Reactive Red M-3BE and Reactive Brilliant Red K-2BP, respectively.     

Effects of europium ions (Eu3+) on the distribution and related biological activities of elements in Lathyrus sativus L. roots

Scanning electron microscopic and energy-dispersive X-ray analyses were used to study the distributions of different types of elements in the epidermis, exodermis, endodermis, and vascular cylinder of the fracture face in the Lathyrus sativus L. roots in the presence or absence of Eu³⁺. Some index of the biological activity related to the elements binding with protein were determined also. The results showed that the tissular distributions of elements in the fracture face are different in the presence and absence of Eu³⁺. The atomic percentages of P, S, Ca, and Mn were influenced more than those of other elements. Eu³⁺ promoted the biological activities of various kinds of element. The one possible mechanism changing the biological activities was that the reaction of Eu³⁺ Eu²⁺ would influence the electron capture or transport in elements of binding protein. Another mechanism was that CaM-Ca²⁺ becoming CaM-Eu³⁺ through Eu³⁺ instead of Ca²⁺ would affect the biological activity of elements by regulating the Ca²⁺ level in the plant cell.     

Detection and identification of Legionella pneumophila by PCR-restriction fragment length polymorphism analysis of the RNA polymerase gene (rpoB)

The partial RNA polymerase beta-subunit coding gene (rpoB) sequences of 38 Legionella species (59 reference strains) were used to select both Legionella genus-specific and Legionella pneumophila species-specific primers to amplify the 347-bp and 217-bp DNAs, respectively. Enzyme restriction sites for PCR-restriction fragment length polymorphism (PCR-RFLP) analysis were also generated by a computer program. Thirty-eight Legionella species were well differentiated by the identification scheme for Legionella genus-specific PCR-RFLP using HaeIII, AluI, CfoI, PstI, and MaeII. The most common and important pathogenic species, L. pneumophila, was differentiated into two subspecies (L. pneumophila subsp. pneumophila and L. pneumophila subsp. fraseri) by both Legionella genus-specific PCR-RFLP and L. pneumophila species-specific PCR-RFLP using BamHI. Eighty-two Korean culture isolates could also be easily identified by both PCR-RFLP methods as 68 strains of L. pneumophila subsp. pneumophila, 11 strains of L. pneumophila subsp. fraseri, and three novel strains that were separately confirmed by 16S rDNA and rpoB sequence analysis. These results suggest that the rpoB PCR-RFLP for Legionella is a simple and convenient method, not only for specific detection, but also for the rapid identification of Legionella species.     

Regulation of intestinal and hypothalamic apolipoprotein A-IV

This review discusses the regulation of the intestinal and hypothalamic apolipoprotein A-IV (apo A-IV) gene and protein expression. Apo A-IV is a glycoprotein secreted together with triglyceride-rich lipoproteins by the small intestine. Intestinal apo A-IV synthesis is stimulated by fat absorption, probably mediated by chylomicron formation. This stimulation of intestinal apo A-IV synthesis is attenuated by intravenous leptin infusion. Chronic ingestion of a high-fat diet blunts the intestinal apo A-IV in response to dietary lipid. Intestinal apo A-IV synthesis is also stimulated by members of the pancreatic polypeptide family, including peptide YY (PYY), neuropeptide Y (NPY), and pancreatic polypeptide (PP). Recently, apo A-IV was demonstrated to be present in the hypothalamus as well. Hypothalamic apo A-IV level was reduced by food deprivation and restored by lipid feeding. Intracerebroventricular administration of apo A-IV antiserum stimulated feeding and decreased the hypothalamic apo A-IV mRNA level, implying that feeding is intimately regulated by endogenous hypothalamic apo A-IV. Central administration of NPY significantly increased hypothalamic apo A-IV mRNA levels in a dose-dependent manner.     

Mass spectrometry and tandem mass spectrometry analysis of rat mitochondrial ATP synthase: up-regulation in pancreatic acinar cells treated with cerulein

Mitochondrion is a vulnerable intracellular target to reactive oxygen species (ROS). ROS have been considered to be important regulators of the pathogenesis of pancreatitis. This study aims to determine whether ROS induces mitochondrial damage by monitoring the expression level of mitochondrial ATP synthase as the key molecular component in mitochondria associated with cellular damage. Pancreatic acinar AR42J cells were treated with cerulein which induces symptoms similar to that associated with human acute pancreatitis. Proteins were separated by two-dimensional electrophoresis using pH gradients of 5-8 and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MS), quadrupole time-of-flight MS and MS/MS with nano-electrospray. Following cerulein treatment, mitochondrial ATP synthase beta chain was highly expressed compared to nontreated cell. The protein was identified by its pI of 5.2 and molecular weight (56 354 Da) with 27 matched peptides. Among the MS spectrum, precursor ions m/z 488.28, 544.81, 631.82, 693.34, 718.38, 729.41, 801.40, 809.39, 825.94, and 994.52 were further identified using MS/MS and confirmed the isolated protein to be mitochondrial ATP synthase beta chain. In conclusion, cerulein-induced oxidative injury may result in the induction of mitochondrial ATP synthase, which may act as an adaptive pathophysiological process in the pancreas.