Characterization of iodoacetate-mediated neurotoxicity in vitro using primary cultures of rat cerebellar granule cells

The neuroprotective efficacy of antioxidant molecules against iodoacetate (IAA) neurotoxicity in rat cerebellar granule cell (CGC) cultures was investigated. Transient exposure to IAA caused a concentration-dependent decrease in cell viability (ED50 = 9.8 microM). Dizocilpine maleate (MK-801), and 1,2,3,4-tetrahydro-6-nitro-2,3-dioxobenzo[f]quinoxaline-7-sulfonamide (NBQX), failed to prevent IAA toxicity. Certain antioxidant molecules were shown to be neuroprotective against IAA when combined with MK-801 but were ineffective when administered alone. (S)-(-)-Trolox, butylated hydroxytoluene (BHT), and U-83836E exhibited EC50 values of 78, 5.9, and 0.25 microM, respectively, in the presence of 10 microM MK-801. IAA also induced an increase in intracellular oxidative stress, which was quenched by the antioxidants (in the presence of MK-801) in cultures loaded with the oxidant sensitive dye 2'7'-dichlorodihydrofluorescein diacetate (DCFH-DA).     

Discovery and evaluation of potent, tyrosine-based alpha4beta1 integrin antagonists

Using disulphide cysteine-based inhibitors as lead structures, this communication describes our strategy for identifying more stable, potent antagonists of the alpha4beta1 integrin. These studies ultimately discovered potent, low molecular weight inhibitors based on D-thioproline-L-tyrosine.     

Quantitative trait locus mapping of genes regulating pulmonary PKC activity and PKC-alpha content

Strain A/J mice, which are predisposed to experimentally induced asthma and adenocarcinoma, have the lowest pulmonary protein kinase (PK) C activity and content among 22 inbred mouse strains. PKC in neonatal A/J mice is similar to that in other strains, so this difference reflects strain-dependent postnatal regulation. PKC activity is 60% higher in C57BL/6J (B6) than in A/J lungs, and the protein and mRNA concentrations of PKC-alpha, the major pulmonary PKC isozyme, are two- to threefold higher in B6 mice. These differences result from more than a single gene as assessed in F(1), F(2), and backcross progeny of B6 and A/J parents. Quantitative trait locus (QTL) analysis of 23 AxB and BxA recombinant inbred strains derived from B6 and A/J progenitors indicates a major locus regulating lung PKC-alpha content that maps near the Pkcalpha structural gene on chromosome 11 (D11MIT333; likelihood ratio statistic = 12.5) and a major locus controlling PKC activity that maps on chromosome 3 (D3MIT19; likelihood ratio statistic = 15.4). The chromosome 11 QTL responsible for low PKC-alpha content falls within QTLs for susceptibilities to lung tumorigenesis and ozone-induced toxicity.     

Hormonal induced lactation in transgenic goats

The aim of this study was to hormonally induce lactation in prepubertal, nulliparous, and male goats both transgenic and non-transgenic. Analysis of milk quality, recombinant protein expression levels, total amount of recombinant protein produced, and the affect on long-term reproductive capability was assessed. Fifty-one goats (Saanen, Alpine, and Toggenburg), male and non-pregnant females, 2-31 months of age, either non-transgenic or transgenic were evaluated with a total of 10 transgenes (constructs) represented. Animals were given estradiol (0.25 mg/kg, i.m.) and progesterone (0.75 mg/kg, i.m.) on days 1, 3, 5, 7, 9, 11 and 13, while prednisilone (0.4 mg/kg, i.m.) was administered on days 14-16 with mammary massage occurring daily from day 5 onward. Forty of 51 animals, (36 of 38 females and 4 of 13 males) produced milk with total volumes in the 30-day experiment, ranging from 20 microl to 530 mls per day, or approximately 500 microl to 6.8 liters total. Milk composition was analyzed for various parameters (total protein, fat content, total solids and somatic cell count) with no significant differences found between induced and natural milk. Expression levels of recombinant proteins from transgenic animals that were analyzed during the induced lactation, and subsequently during normal lactations, were found to have no significant differences. Total amount of recombinant protein produced was evaluated at different expression levels with no statistical significance seen. While over 90% of the females placed in the regimen became pregnant, there was a correlation between increased age at time of induction and an increase in number of breedings, or reproductive cycles needed to establish a pregnancy after induction. For males, 100% placed in the regimen settled females after hormonal induction of lactation. Semen quality was evaluated prior to, during, and after hormonal treatments. Semen volume and sperm number did not differ; however, for a small percentage of males, there was a decrease in sperm and post thaw motility after hormonal treatments. These levels returned to normal within 4-5 weeks. Subsequent natural lactations showed total milk volumes within breed standards. These findings indicate that hormonal induction of lactation in the caprine species is a viable alternative to pregnancy for initiating lactation and milk production, does not adversely impact reproductive performance long-term, and can benefit the early assessment of recombinant proteins produced in a transgenic founder program.     

Compositional changes in lower molecular weight flavans during grape maturation

Grapes of the Gamay Beaujolais and Siebel varieties have been sampled during the period from just before the onset of colouration to harvest. Extraction has yielded data on the composition and quantities of lower molecular weight and polymeric flavanoids present. The structure of the major flavans of these grapes has been established and the variation in their relative amounts monitored during the growth season. The basic structural unit of the grape proanthocyanidin polymer is a monomer with (?)-epicatechin stereochemistry.     

Identification and analysis of products formed from phospholipids in the free radical oxidation of human low density lipoproteins

Phospholipids reside in the surface layer of LDLs and constitute approximately 20-25% of the particle by weight. We report a study of the primary products generated from the most abundant molecular species of phosphatidylcholines present in LDL during in vitro free radical oxidations. The 13-hydroperoxides of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC) and 1-stearoyl-2-linoleoyl-sn-glycero-phosphocholine (SLPC) and the 15-hydroperoxides of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC) and 1-stearoyl-2-arachidonoyl-sn-glycero-phosphocholine (SAPC) were found to increase in a time-dependent manner and in significant amounts even in the presence of alpha-tocopherol. Phospholipid alcohols also formed during the course of the oxidations. Early in the LDL oxidations, while alpha-tocopherol was still present, the thermodynamically favored trans,trans products of PLPC and SLPC were found to form in significantly larger quantities than those formed from cholesteryl linoleate. Additionally, quantities of PAPC 11-hydroperoxide (11-OOH) decreased over time relative to PAPC 15-OOH, even while alpha-tocopherol was still present in the oxidation, presumably as a result of further oxidation of PAPC 11-OOH to form cyclic peroxide oxidation products. These results suggest that alpha-tocopherol is more closely associated with the inner cholesteryl ester-rich hydrophobic core of an LDL particle and is not as effective as an antioxidant in the outer phospholipid layer as it is in the lipid core.     

Crystal structures of recombinant human purple Acid phosphatase with and without an inhibitory conformation of the repression loop

The crystal structure of human purple acid phosphatase recombinantly expressed in Escherichia coli (rHPAP(Ec)) and Pichia pastoris (rHPAP(Pp)) has been determined in two different crystal forms, both at 2.2A resolution. In both cases, the enzyme crystallized in its oxidized (inactive) state, in which both Fe atoms in the dinuclear active site are Fe(III). The main difference between the two structures is the conformation of the enzyme "repression loop". Proteolytic cleavage of this loop in vivo or in vitro results in significant activation of the mammalian PAPs. In the crystals obtained from rHPAP(Ec), the carboxylate side-chain of Asp145 of this loop acts as a bidentate ligand that bridges the two metal atoms, in a manner analogous to a possible binding mode for a phosphate ester substrate in the enzyme-substrate complex. The carboxylate side-chain of Asp145 and the neighboring Phe146 side-chain thus block the active site, thereby inactivating the enzyme. In the crystal structure of rHPAP(Pp), the enzyme "repression loop" has an open conformation similar to that observed in other mammalian PAP structures. The present structures demonstrate that the repression loop exhibits significant conformational flexibility, and the observed alternate binding mode suggests a possible inhibitory role for this loop.