Enzymatic production of l-phenylalanine from the racemic mixture of d,l-phenyllactate

Summary The production of l-phenylalanine from the racemate d,l-phenyllactate in an enzyme membrane reactor has been examined. In a first step the racemate is dehydrogenated to the prochiral intermediate phenylpyruvate by the enzymes d-and l-hydroxyisocaproate dehydrogenase. In a second step phenylpyruvate is reductively aminated to l-phenylalanine by l-phenylalanine dehydrogenase. Both steps are dependent on coenzyme, the first one requires NAD, the second one NADH in stoichiometric amounts; in this way the coenzyme is regenerated and only required catalytically. The coenzyme is covalently bound to polyethylene glyco-20 000 and can thus be retained in the reactor analogously to the three enzymes. In order to optimize the continuous production of l-phenylalanine from d,l-phenyllactate, models of the reaction kinetics and of the reactor system have been set up. By means of the reactor model, we can calculate the optimum ratio of the three enzymes, the optimum coenzyme concentration and the optimum phenylpyruvate concentration in the feed.In this process, at a substrate concentration of 50 mM d,l-phenyllactate we reached a spacetime-yield of 28 g l-Phe/(l*d).Abbreviations PEG polyethylene glycol - d-HicDH d-hydroxyisocaproate dehydrogenase - l-HicDH l-hydroxyisocaproate dehydrogenase - PheDH l-phenylalanine dehydrogenase - V _max maximum velocity - K _M Michaelis-Menten constant - K _l inhibition constant - R₁ reaction rate of the d-HicDH forward reaction - R₂ reaction rate of the d-HicDH reverse reaction - R₃ reaction rate of the l-HicDH forward reaction - R₄ reaction rate of the l-HicDH reverse reaction - R₅ reaction rate of the PheDH forward reaction - R₆ reaction rate of the PheDH reverse reaction - d-PLac d-phenyllactate - l-PLac l-phenyllactate - PPy phenylpyruvate - l-Phe l-phenylalanine - NH₄ ammonium - residence time     

Accidental selenium poisoning of growing pigs

Chronic selenium (Se) toxicosis was diagnosed in two groups of growing pigs. Emaciation, loss of hair, necrotic areas in the skin, lesions of the coronary band and hooves, postnecrotic atrophic cirrhosis of liver, and lumbal poliomyelomalacia were the principal findings. High Se concentrations were detected in blood plasma. Addition of the calculated amounts of sodium selenite directly to the feedstuff instead to mineral premix was the cause of this intoxication.     

Inhibition of human leukocyte elastase and cathepsin G by extended peptides and subunits derived from human C-reactive protein

Summary Extended peptides that derive from the primary sequence of the acute phase reactant C-reactive protein (CRP) are shown to inhibit in vitro the enzymatic activities of human leukocyte elastase (hLE) and human leukocyte cathepsin G (hCG), which are associated with the tissue damage that occurs during the course of several chronic inflammatory conditions. Major inhibitory activity was observed in the peptides CRP_70–98 and CRP_50–98 towards hLE (K_i=4.0μ M) and hCG (K_i=1.4 μM), respectively. In contrast to the inability of intact CRP pentamers to inhibit both enzymes, CRP subunits (monomers) inhibited hLE (3.0 μM) and hCG (3.6 μM) activity.     

Interleukin 1 components in cicatricial pemphigoid. Role in intravenous immunoglobulin therapy.

Interleukin (IL-)1 is an important mediator of inflammatory responses and plays an important role in the pathogenesis of various autoimmune diseases. Cicatricial pemphigoid (CP) is a multisystem autoimmune inflammatory disease. We have studied the role of IL-1 in its pathogenesis. We have investigated the serum levels of IL-1 components (IL-1alpha, IL-1beta, and IL-1Ra), and determined the role of intravenous immunoglobulin (IVIg) therapy in patients with CP. Serum levels of IL-1alpha and beta were significantly higher in untreated patients with active disease compared to levels in patients in prolonged clinical remission and normal human controls (P<0.0001). The serum levels of IL-1Ra were higher in patients in prolonged clinical remission compared to patients with active disease (P=0.002). Hence elevated levels of IL-1alpha and beta and low levels of IL-1Ra correlate with disease activity. The levels of IL-1alpha and beta were statistically significantly higher in sera of CP patients with active disease pre-IVIg therapy compared to post-IVIg therapy (P<0.0001). Statistically significantly higher levels of IL-1Ra were present in post-IVIg treatment serum samples when compared to levels in pre-IVIg treatment (P<0.0001). In the in vitro experiments, the levels of IL-1alpha and beta produced by the peripheral blood mononuclear cells (PBMC) isolated from patients before IVIg therapy were significantly higher when compared to the PBMC isolated from post-IVIg patients (P<0.0001). Significantly higher levels of IL-1Ra were observed in the supernatants of PBMC collected from pre-IVIg patients and cultured with exogenously added IVIg, when compared to the levels of PBMC to which IVIg was not added (P<0.0001). IL-1 may be an important cytokine involved in the pathogenesis of CP. The regulation of IL-1 could be one of the mechanisms, amongst others, by which IVIg may exert its beneficial effect in the treatment of CP.     

Inhibitory effect of LY 255283 on the synthesis of leukotriene B(4) and thromboxane A(2) in human peripheral blood polymorphonuclear leukocytes and monocytes

LY 255283 [(1-(5-ethyl-2-hydroxy-4-(6-methyl-6-)1H-tetrazol-5-yl)-heptyloxy) phenyl)ethanone], a specific leukotriene B(4) (LTB(4)) receptor antagonist, inhibited the production of LTB(4) in human peripheral blood polymorphonuclear leukocytes (PMNL) and in monocytes activated by calcium ionophore A23187. In human monocytes activated by ionophore it inhibited also the production of thromboxane B(2) (TXB(2)). The effect of LY 255283 on 5-lipoxygenase (5-LO) and LTA(4) hydrolase activities which catalyse the production of LTB(4) and LTA(4) has not been studied yet. It is thought that LY 255283 may inhibit the production of LTB(4) and TXA(2) by antagonising the effect of ionophore-induced LTB(4) on 5-lipoxygenase and cyclooxygenase in human peripheral blood PMNL and monocytes.     

Introgression of Drought Tolerance into Elite Basmati Rice Variety through Marker-Assisted Backcrossing

Drought is one of the major abiotic threat to rice production in the context of climate change. Super Basmati is an elite, fine grain basmati rice variety grown in Punjab, Pakistan. Due to drought sensitive in nature, its yield has been facing an alarming situation in production because of gradual decrease in irrigated water for a couple of years. Three reported novel QTLs for drought tolerance were selected for incorporation into Super Basmati by employing marker assisted selection strategy. IR55419-04 with novel QTLs was used as a donor parent. Foreground selection was performed by applying PCR based QTL linked SSR markers followed by recombinant selection by using 2–4 flanking markers. Background selection was exercised by using polymorphic SSR markers for maximum genome recovery of the Super Basmati. The individuals homozygous at the target QTLs and with maximum background of Super Basmati at the rest of the non-target genome was selected for evaluation of drought tolerance. Under drought stress conditions, the yields of all introgressed lines (ILs) were 44.2%–125.7% higher than recurrent parent. Six superior ILs that are drought tolerant and very similar to Super Basmati in terms of agronomic and grain quality traits are marked for release as drought-tolerant varieties in arid regions or for use in breeding programs of high grain quality and drought-tolerant parents.     

Rapid identification of multi-strain HBV infection in patient by high-throughput DNA sequencing

Hepatitis B is a well-known risk factor for the development of liver cancer and is closely associated with patient morbidity and mortality. Viral mutants and variants have the potential to evade immune response and prolong infection, and thus it is crucial to develop a methodology for the rapid identification of multi-strain hepatitis infections in patients. Here we describe a method based on selective region amplification of viral genome and deep sequencing, which may be used for rapid identification of multi-strain hepatitis B virus (HBV) infection in patients. The method works even with significantly low amounts of patients’ serum samples, where the wet-lab procedures take about 1.5 days, followed by a quick bioinformatic analysis to reveal the final results. Our method can potentially be applied to the rapid and reliable identification of multi-strain HBV infection and help improve treatment regiments.