Genes for the biosynthesis of spinosyns: applications for yield improvement in Saccharopolyspora spinosa

Spinosyns A and D are the active ingredients in an insect control agent produced by fermentation of Saccharopolyspora spinosa. Spinosyns are macrolides with a 21-carbon, tetracyclic lactone backbone to which the deoxysugars forosamine and tri-O-methylrhamnose are attached. The spinosyn biosynthesis genes, except for the rhamnose genes, are located in a cluster that spans 74 kb of the S. spinosa genome. DNA sequence analysis, targeted gene disruptions and bioconversion studies identified five large genes encoding type I polyketide synthase subunits, and 14 genes involved in sugar biosynthesis, sugar attachment to the polyketide or cross-bridging of the polyketide. Four rhamnose biosynthetic genes, two of which are also necessary for forosamine biosynthesis, are located outside the spinosyn gene cluster. Duplication of the spinosyn genes linked to the polyketide synthase genes stimulated the final step in the biosynthesis — the conversion of the forosamine-less pseudoaglycones to endproducts. Duplication of genes involved in the early steps of deoxysugar biosynthesis increased spinosyn yield significantly. Journal of Industrial Microbiology & Biotechnology (2001) 27, 399–402. Received 31 May 2001/ Accepted in revised form 09 July 2001     

Determination of antibody affinity by using antigenic and antibody immunosorbents

To determine the affinity of the active centers of antibodies, cellulose immunosorbents for antibodies and antigens have been used. The fixation of serum proteins on the sorbent, the interaction of fixed antibodies with a monovalent antigen and the graphic analysis of the results thus obtained allows one to assess not only the concentration of the effective active centers on the sorbent, but also all known characteristics of antibody affinity: the average association constant K0, the common association constant Kt, the geometric association constant Kg, the average association constants which determine the affinity of different antibody groups. The use of antigenic immunosorbent permits one to determine the value of the average internal association constant K0. The determination of antibody affinity in hyperimmune antiplague sera by means of immunosorbents and red blood cells coated with capsular antigen has resulted in obtaining similar values of affinity indices.     

Algal single cell protein from wastewater treatment and renovation process

A Source of high-quality protein for animal feed, based upon algae recovered in the process of upgrading waste oxidation pond effluents and promising to be particularly economical, is being developed at the Technion. Unlike other types of single cell protein(SCP), the algal protein does not have to return the full production cost but only that of concentration and final processing. The balance is shared by the value of waste disposal and the reclaimed water. Whereas such systems as activated sludge require considerable mechanical energy to supply the oxygen needed for aerobically degrading organics in wastewater, oxidation ponds utilize solar energy for that purpose. The sludge obtained when their effluents are clarified consists largely of algae, bacteria, fungi, and zooplankton in relative proportions varying with operating conditions, and contains 40–60%(dry basis) high-quality protein. The high rate oxidation pond (a particularly intensive type of pond) produces on the average 34 g/m²7sol;day solids, or over 100 tons/ha (hectare) annually. Two clarification routes have been found promising: centrifugation and alum flocculation followed by frothflotation. The latter route is less expensive in terms of both fixed and operating cost, and gives clarified effluent of higher quality, which can be seasonally stored with minimal eutrophication because the aluminum removes most of the phosphate from the effluent. A good product has been obtained by drum-drying the concentrate, and preliminary feeding tests have indicated that it can replace at least 1/4 of the soymeal in broiler rations and 2/3 of the fishmeal in carp feed. No ill effect of the aluminum in the product recovered by alum flocculation has been found so far a process for removing and recycling the aluminum has been developed nonetheless, in case ill effects do show up in further tests. The combined value of the benefits derived from a system centered around the high-rate oxidation pond with clarification by flocculation–flotation, in terms of waste treatment by alternative means, potable water saved, and soymeal replaced, significantly exceeds estimated cost.     

Rapid immunofluorescent determination of cells in the S phase in pea root meristems: An alternative to autoradiography

Photosystem II (PS II) activity and the localization of ribulose-l,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39) were studied in primary leaves of young maize plants ( Zea mays L. cv. Fronica) by tetra-nitro-blue-tetrazoliumchloride reduction and immunolocalization, respectively. In tissue of 3-day-old plants all chloroplasts were structurally identical. From day 4 they developed into their typical appearance of mesophyll and bundle sheath chloroplasts. First PS II-activity was present in both types of chloroplasts. From day 4 it disappeared in bundle sheath chloroplasts concomitant with the loss of grana. RuBP carboxylase on the other hand was only present in bundle sheath chloroplasts at all stages of development. Thus, the control of the development of the photosystems and the Calvin cycle enzymes seem to differ.     

Mitogenic effect of a human placental factor on astrocytes and glial precursors

We have characterized and partially purified a new 'factor' present in human placenta which strongly stimulates the in vitro proliferation of two immunocytochemically characterized subtypes of astrocytes and of bipotential precursors of putative fibrous astrocytes and oligodendrocytes. This 'factor' has an apparent Mr of 60-80 kD and exhibits physicochemical and chromatographic properties characteristic of polypeptides. Our observations suggest that placenta-derived growth factors (PDMF) control the proliferation of glial cells and glial precursors during fetal development.     

Effects of hypoxia on lung mechanics in the newborn cat

The present study was designed to investigate the effects of hypoxia on lung mechanics in the newborn cat and to determine if vagal efferent innervation to the airways is involved in the response. We studied 11 animals, aged 2-7 days, anesthetized with a mixture of chloralose-urethane administered intraperitoneally. A tracheal cannula was inserted just below the larynx and following paralysis (pancuronium bromide), mechanical ventilation was initiated. A pneumothorax was created by a midline thoracotomy and an end-expiratory load was applied to maintain functional residual capacity. Animals were placed in a flow plethysmograph from which measurements of transpulmonary pressure, flow, and volume, mean inspiratory resistance, and dynamic compliance of the lung were calculated. The experimental protocol consisted of a series of 8-min trials, each preceded by a controlled volume history. The hypoxia challenge was composed of 1 min of ventilation with 40% O2, followed by 5 min exposure to 10% O2 and 2 min of recovery. In the majority of animals (7 out of 11), hypoxia had no effect on lung mechanics compared with control trials. Four animals responded to hypoxia with an increase in resistance and a decrease in compliance. Resistance remained elevated throughout the hypoxia with an average maximal increase of 47.2 +/- 22.2% (SD). Dynamic compliance was significantly decreased at the 2nd, 3rd, and 4th min only of hypoxia. Bilateral vagotomy abolished the response in the four animals and hypoxia had no effect on mechanics postvagotomy. Our data suggest that in most cases changes in lung mechanics do not play a causal role in the biphasic ventilatory response to hypoxia seen in the newborn.     

A protein induced by NGF in PC12 cells is stored in secretory vesicles and released through the regulated pathway.

We have previously described the isolation of a cDNA clone corresponding to an mRNA rapidly induced to high levels in PC12 cells by treatment with NGF. We report here the complete amino acid sequence of the protein (named VGF8a) as deduced by nucleotide sequencing of overlapping cDNA clones. VGF8a is particularly rich in proline residues and has a conspicuous number of short stretches of basic amino acid residues which may represent potential targets for proteolytic cleavage. Antibodies directed against recombinant VGF8a-beta-galactosidase fusion proteins were used for immunofluorescent staining of the protein in PC12 cells as well as for its localization, by Western blot analysis, in subfractions of cell homogenates. We demonstrate that in PC12 cells, VGF8a protein is stored in secretory vesicles and is released in response to a variety of stimuli that are known to induce the regulated secretion of neurotransmitters.     

NG-methylarginine, an inhibitor of endothelium-derived nitric oxide synthesis, is a potent pressor agent in the guinea pig: does nitric oxide regulate blood pressure in vivo?

Nitric oxide is a major endothelium-derived vascular smooth muscle relaxing factor; its synthesis from L-arginine is selectively inhibited by L-NG-methylarginine. To assess whether basal nitric oxide release contributes to blood pressure regulation in vivo, we have investigated the cardiovascular effects of L-NG-methylarginine in the anesthetized guinea pig. L-NG-methylarginine (0.1-10 mg/kg, i.v. bolus) elicited a sustained, dose-dependent, increase in arterial pressure and a moderate bradycardia. L-arginine (30 mg/kg i.v.) prevented or reversed the pressor effect of L-NG-methylarginine, while atropine (2 mg/kg) abolished the associated bradycardia. In contrast, L-arginine did not attenuate the pressor effect of norepinephrine or angiotensin. Our findings suggest that basal nitric oxide production is sufficient to modulate peripheral vascular resistance; hence nitric oxide may play a role in arterial pressure homeostasis.