Specific substrates for spectrophotometric determination of penicillin acylase activity

Penicillin acylase substrates suitable for colorimetric determination of the enzyme activity have been tested in this study. The kinetic parameters (Km and kcat) have been elucidated for the following nine substrates: six phenylacetic acid derivatives (p-nitroanilide, p-nitrophenyl ester, p-nitro-m-carboxyanilide, p-nitro-o-carboxyanilide, p-nitro-o-hydroxyanilide, m-nitro-p-carboxyanilide), two D-phenylglycine derivatives (p-nitroanilide, p-nitro-m-carboxyanilide), and also p-nitrophenyl ester of acetic acid (p-nitrophenyl acetate). With the exception of p-nitrophenyl acetate, all the compounds studied are highly specific chromogenic substrates for penicillin acylase, but their reactivity is very variable and kcat/Km values are in a range from 0.8.10(4) to 5.10(6) M(-1).sec(-1).     

Protein-lipid interactions and enzyme requirements for light subtype generation on cycling reconstituted surfactant

Surfactant convertase is required for conversion of heavy density (H) natural surfactant to light density (L) subtype during cycling in vitro, a technique that reproduces surfactant metabolism. To study mechanisms of H to L conversion, we prepared liposomes of dipalmitoylphosphatidylcholine (DPPC) and phosphatidylglycerol (PG), or the phospholipids (PL) in combination with either surfactant protein A (SP-A), surfactant protein B (SP-B), or both SP-A and SP-B. Phospholipids alone showed time-dependent conversion from heavy to light subtype on cycling in the absence of convertase, which was decreased by adding SP-B, but not SP-A, to phospholipids (p < 0.01 for PL+SP-B, or PL+SP-A+SP-B vs. PL, or PL+SP-A). The ultrastructure, surface activity, buoyant density, and L subtype generation on cycling PL+SP-A+SP-B with partially purified convertase or with phospholipase D were similar to those of natural TM. In conclusion, a reconstituted surfactant mimics the behavior of natural surfactant on cycling, and reveals that interaction of SP-B with phospholipids decreases L subtype generation. In addition, esterase/ phospholipase D activity is required for conversion of heavy to light subtype on cycling.     

Lactic acid production from molasses by Sporolactobacillus cellulosolvens

Sporolactobacillus cellulosolvens (NCIMB 12173) isolated from an anaerobic digester and characterised biochemically is being reported for homofermentative lactic acid production from molasses in a batch culture. The effect of various process parameters on lactic acid production were optimized. A maximum lactic acid (24.2 g/l) and yield coefficient (0.79) was achieved using 3% (v/v) inoculum of 36 h old culture in molasses medium containing sugars (5%; w/v) supplemented with peptone (2.5 g/l) and (NH4)2SO4 (7.5 g/l), pH 6.5 at 40 degrees C after 72 h of fermentation.     

Anandamide synthesis is induced by arachidonate mobilizing agonists in cells of the immune system

The hypothesis that the capability of agents to mobilize arachidonic acid (AA) could predict increased anandamide (ANA) synthesis in a macrophage cell line has been examined. Lipopolysaccharide (LPS), platelet-activating factor (PAF) and cannabinoids such as Delta9-tetrahydrocannabinol (THC) and anandamide were all found to be agonists for the release of AA and led to increased ANA synthesis in RAW264.7 mouse macrophage cells. Nitric oxide, in contrast, stimulated AA release without raising ANA levels. ANA stimulation of its own synthesis indicates the existence of a positive feedback mechanism. The possible involvement of the CB2 receptor in THC-mediated AA release and ANA synthesis is addressed using the antagonist SR144528. ANA synthesis is also increased by the combination of calcium ionophore and indomethacin, suggesting that ANA is metabolized by a cyclooxygenase in this system. The data imply that ANA could play a role in the response of the immune system to cannabinoids and bacterial endotoxins and that AA mobilization is a predictor for increased ANA synthesis.     

CD28/B7 costimulation: a review

The current model of T cell activation requires two signals. The first signal is specific, requiring T cell receptor recognition and binding to MHC/Antigen presented by an antigen-presenting cell. The second signal is nonspecific, resulting from the binding of B7 ligand on the antigen-presenting cell with its receptor, CD28, on the T cell. If both signals are provided, the T cell will proliferate and secrete cytokines. Recently, it has been shown that CTLA4, another receptor for B7 that is upregulated following T cell after activation, can deliver an inhibitory signal, downregulating T cell proliferation. The B7 family of ligands has two family members, B7-1 and B7-2. They both bind to CD28 and CTLA4, but they differ in their binding affinity, structure, and temporal expression. Considerable research has been done on the CD28/B7 costimulatory pathway. Different ways of manipulating this pathway could provide insights into the mechanism and treatment of opposing pathological states. Blocking the CD28/B7 pathway could result in immunosuppression, with implications for the treatment of autoimmune diseases, organ transplantation, and graft vs. host disease. Activating the CD28/B7 pathway could be useful for including the immune system to recognize and eliminate tumors that evade the immune system. Finally, the CD28/B7 pathway could be involved with maintaining immune tolerance, as recent studies suggest the preferential binding of the B7-CTLA4 pathway results in the down-regulation of the responding T cells. Thus, the B7/CD28/CTLA4 pathway has the ability to both positively and negatively regulate immune responses.     

Maternal use of antiepileptic drugs and the risk of major congenital malformations: a joint European prospective study of human teratogenesis associated with maternal epilepsy

PURPOSE: To quantify the risks of intrauterine antiepileptic drug (AED) exposure in monotherapy and polytherapy. METHODS: Data from five prospective European studies totaling 1,379 children were pooled and reanalyzed. Data were available for 1,221 children exposed to AED during pregnancy and for 158 children of unexposed control pregnancies. RESULTS: Overall, when comparing a subgroup of 192 children exposed to AED with 158 children of matched nonepileptic controls, there was an increased risk of major congenital malformations (MCA) in children exposed to AED during gestation [relative risk (RR) 2.3; 95% confidence interval (CI): 1.2-4.7]. A significant increase in risk was found for children exposed to valproate (VPA) (RR 4.9; 95% CI: 1.6-15.0) or carbamazepine (CBZ) (RR 4.9; 95% CI: 1.3-18.0) in monotherapy. When comparing different AED regimens during all 1,221 pregnancies, risks of MCA were significantly increased for the combination of phenobarbital (PB) and ethosuximide (RR 9.8; 95% CI: 1.4-67.3) and the combination of phenytoin, PB, CBZ, and VPA (RR 11.0; 95% CI: 2.1-57.6). Offspring of mothers using > 1,000 mg VPA/day were at a significantly increased risk of MCA, especially neural tube defects, compared to offspring exposed < or =600 mg VPA/day (RR 6.8; 95% CI: 1.4-32.7). No difference in risk of MCA was found between the offspring exposed to 601-1,000 mg/day and < or =600 mg/day. CONCLUSIONS: This reanalysis shows that VPA is consistently associated with an increased risk of MCA in babies born to mothers with epilepsy. Significant associations were also observed with CBZ. Larger prospective population-based studies are needed to evaluate the risks of many other less frequently prescribed treatment regimens, including newly marketed AEDs.     

Selenium and cadmium induced pulmonary functional impairment and cytotoxicity

Ovalbumin-sensitized (50 mg/kg, i.p.) male Hartley-guinea-pigs (550-610 g; n = 6) were treated 14 days later intratracheally with saline, cadmium (Cd 0.3 mg), selenium (Se 0.3 mg or 0.06 mg) or Se (0.06 mg) with Cd (0.3 mg). After 24 h, baseline dynamic-lung-compliance (Cdynl) and pulmonary-resistance (Rp), and percent change after ovalbumin-aerosol-challenge (10 mg/ml, 60 s) were assessed. Cadmium or Se (0.3 mg), Se (0.06 mg) and/or Cd (0.3 mg) decreased Cdynl (P < 0.05). Selenium (0.3 mg) increased Rp (P < 0.05). Ovalbumin-challenge decreased Cdynl and increased Rp in all groups. Analysis of bronchoalveolar-lavage-fluid (BALF) displayed increased activities of lactate-dehydrogenase (LDH), beta-glucuronidase (beta-G), alkaline-phosphatase (AP), and protein due to 0.3 mg Se, 0.3 mg Cd alone or with 0.06 mg Se (P < 0.05). Findings indicated that, 0.3 mg Se is more detrimental than 0.3 mg Cd to lung-dynamics despite a modest protection by 0.06 mg Se against Cd illustrated by an ameliorated Cdynl and lower protein in BALF.     

Mutations in the arginine-rich protein gene (ARP) in pancreatic cancer

The ARP gene encodes a highly conserved arginine-rich protein from chromosomal band 3p21.1. At the cytogenetic level this region is frequently deleted in a variety of different solid tumors, although not in pancreatic cancer. We have reported the presence of a specific mutation (ATG50-->AGG) or deletion of codon 50 of the ARP gene in different tumor types (Shridhar et al., 1996, 1996a). In the present study, we have observed mutations involving codon 50 in 11 of 37 pancreatic tumors. The frequency of codon 50 mutation is roughly the same in pancreatic tumors as in the other types of tumors previously examined. In addition, we have detected mutations at codon 51 in multiple PCR subclones in two other pancreatic tumors. Mutations in the ARP gene are thus commonly observed in pancreatic cancer, as well as many other cancers.