A Novel Photoaffinity Ligand for Kainate Sites Labels Two Polypeptides with Molecular Mass 45 and 33.5 kDa in Chick Cerebellum

Abstract: The synthesis of (2 S ,3 S ,4 S )-4-[1-(4-azidobenzamidomethyl)ethenyl]-2-carboxy-3-pyrrolidineacetic acid (ABCPA) is described. This novel kainic acid analogue, bearing a photolabile functionality on the isopropenyl side chain, was proven to be a good inhibitor of [³H]CNQX and [³H]kainic acid binding on chick cerebellar membranes. [³H]ABCPA was photoaffinity cross-linked on the membrane fraction of chick cerebellum. Electrophoretic analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two major radioactive bands with apparent molecular masses of 45 and 33.5 kDa. [³H]ABCPA incorporation in both bands was completely blocked by 2 m M CNQX. When photoaffinity labeling was performed in the presence of 2 m M kainic acid, incorporation of [³H]ABCPA was blocked by ∼70% in the 45-kDa band and by 18% in the 33.5-kDa band. Incorporation of radioactivity in both bands was blocked by ∼30% with 10 m M glutamate.     

Molecular characterization of a fourth isoform of the catalytic subunit of protein phosphatase 2A from Arabidopsis thaliana

We have recently reported the existence of multiple isoforms of the catalytic subunit of protein phosphatase 2A (PP2A) in Arabidopsis thaliana and the molecular cloning of cDNAs encoding three of these proteins (PP2A-1, PP2A-2, PP2A-3). The reported cDNA encoding PP2A-3 was truncated at the 5 terminus, lacking a short fragment of the N-terminal coding sequence. We have now isolated a near full-length cDNA encoding the entire PP2A-3 protein (313 residues). The clone includes 188 nucleotides of 5-untranslated region, where a 44 bp long poly(GA) track is found. We also describe the cloning of a cDNA encoding a fourth isoform of PP2A (PP2A-4). The polypeptide contains 313 residues being 98% identical to PP2A-3 and only 80% identical to both PP2A-1 and PP2A-2. The mRNA for PP2A-4 is 1.4 kb in length and, although predominantly expressed in roots, it is also found in other organs. It is concluded that in A. thaliana the isoforms of PP2A can be grouped in two extremely conserved subfamilies.     

Transfection of the mouse ICAM-1 gene into murine neuroblastoma enhances susceptibility to lysis,reduces in vivo tumorigenicity and decreases ICAM-2-dependent killing

We determined the expression of intercellular adhesion molecules (ICAM) on neuro-2a cells in order to evaluate whether they were involved in cytolysis of murine neuroblastoma. Fluorescence-activated cell sorting analysis revealed that the control neomycin-resistance-genetransduced line (neuro-2a/LN) had poor expression of ICAM-1 (mean channel fluorescence, MCF=3.7). An ICAM-1-positive transfectant of neuro-2a (neuro-2a/ICAM-1⁺) (CMF=64.3) was generated to evaluate directly the role of this adhesion molecule in cytolysis. Neuro-2a/ICAM-1⁺ was more sensitive to LAK killing (69.7% at an effector-to-target ratio of 1001) compared to neuro-2a/LN (48.6%) (P<0.001). Blocking of neuro-2a/LN and neuro-2a/ICAM-1⁺ lysis with anti-ICAM-1 monoclonal antibodies (mAbs) did not account for all the LFA-1-dependent killing. These data indicate that even in neuro-2a/ICAM-1⁺ cells, other LFA-1 ligands participated in the effector-target interaction. Therefore, we examined these cell lines for ICAM-2 expression. Both neuro-2a/LN and neuro-2a/ICAM-1⁺ lines expressed ICAM-2 (MCF=16.4 and 16.5). ICAM-2 accounted for the majority of the LFA-1-dependent killing in the ICAM-1-negative target, neuro-2a/LN, while ICAM-1 played a primary role in the cytolysis of the ICAM-1⁺ transfectant. Inhibition of lysis in the presence of anti-ICAM-1 and ICAM-2 mAbs was comparable to that seen with the addition of anti-LFA-1 mAb, indicating that other LFA-1 ligands were not involved in this system. ICAM-1 expression was associated with decreased in vivo tumorigenicity; mice inoculated with neuro-2a/ICAM-1⁺ cells had a significantly longer survival compared to those receiving neuro-2a/LN cells (median survival time 35.5 versus 24.5 days) (P<0.001). It is important to note that ICAM-1 transfection of murine neuroblastoma did not alter its metastatic potential. We conclude that transfection of mouse neuroblastome with ICAM-1 increases its sensitivity to in vitro lysis and reduces its in vivo tumorgenicity. In ICAM-1-negative murine neuroblastoma cells, ICAM-2 plays a primary role in cell-mediated lysis.This work was supported in part by the Children's Cancer Research Fund, the Minnesota Medical Foundation, the Viking Children's Fund and NIH grants PO1-CA-21737, NO1-AI-85002. E. K. is a recipient of the Irvine McQuarrie Research Scholar Award and B. R. B. a recipient of the Edward Mallinkrodt Foundation Scholar Award     

Oxidative mechanisms involved in kainate-induced cytotoxicity in cortical neurons

In our previous experiments, evidence of free radical formation has been demonstrated in gerbil brain after kainic acid (KA) administration. In the present study, the mechanisms involved in KA-induced free radical formation and subsequent cell degeneration were investigated using high density cortical neuron cultures. A free radical trapping agent,a-phenyl-N-tert-butyl-nitrone (PBN), as well as the combined action of superoxide dismutase and catalase attenuated KA neurotoxic effect. Calpain-induced xanthine oxidase (XO) activation may play an important role in KA excitotoxicity since calpain inhibitor I as well as allopurinol, a selective XO inhibitor, significantly protected the cortical neurons from KA-induced cell death. However, XO activation may not be the only source producing free radicals, other free radical generating systems such as nitric oxide synphase may also play a role in KA insult.     

Flash behavior of femalePhoturis versicolor fireflies (Coleoptera: Lampyridae) in simulated courtship and predatory dialogues

FemalePhoturis versicolor fireflies attempt to capture males by responding to heterospecific flash patterns. A mating-dependent switch occurs which affects response timing and frequency of female flashes. We examined the switch using females of known age, mating status, and flash experience to assess how accurate mimicry is, what factors influence it, and what mechanism produces it. Presentations of simulated male flash patterns before and after mating revealed elements of an entrainment mechanism controlling female responsiveness. Unmated females preferentially answered conspecific patterns with variable latencies, averaging 1 s. Mating induced changes in both response frequency and response latency: Females answered heterospecific patterns more frequently, and latencies elicited by conspecific patterns shifted away from the unmated range. Heterogeneity in mean and variance of response latency among individuals indicates that females do not share a discrete reply to a given pattern. Little correspondence exists between latencies of sympatric species andP. versicolor females, suggesting that the flash response mechanism produces entriainment to any rhythmic pattern, not a one-to-one matching between prey and predator latencies. Different selective scenarios underlie strict mimicry versus entrainment mimicry.     

Polymorphism of the tumor necrosis factor beta gene in systemic lupus erythematosus: TNFB-MHC haplotypes

We investigated the Nco I restriction fragment length polymorphism (RFLP) of the tumor necrosis factor beta (TNFB) gene in 173 patients with systemic lupus erythematosus (SLE), 192 unrelated healthy controls, and eleven panel families, all of German origin. The phenotype frequency of the TNFB*1 allele was significantly increased in patients compared to controls (63.6% vs 47.1%, RR = 1.96, p <0.002). The results of a two-point haplotype statistical analysis between TNFB and HLA alleles show that there is linkage disequilibrium between TNFB*1 and HLA-A1, Cw7, B8, DR3, DQ2, and C4A DE. The frequency of TNFB*1 was compared in SLE patients and controls in the presence or absence of each of these alleles. TNFB*1 is increased in patients over controls only in the presence of the mentioned alleles. Therefore, the whole haplotype A1, Cw7, B8, TNFB*1, C4A DE, DR3, DQ2 is increased in patients and it cannot be determined which of the genes carried by this haplotype is responsible for the susceptibility to SLE. In addition, two-locus associations were analyzed in 192 unrelated healthy controls for TNFB and class I alleles typed by serology, and for TNFB and class II alleles typed by polymerase chain reaction/oligonucleotide probes. We found positive linkage disequilibrium between TNFB*1 and the following alleles: HLA-A24, HLA-B8, DRB1*0301, DRB1*1104, DRB1*1302, DQA1*0501, DQB1*0201, DQB1*0604, and DPB1*0101. TNFB*2 is associated with HLA-B7, DRB1*1501, and DQB1*0602.This study was supported by grants from the Federal Ministry of Research and Technology (BMFT/DFVLR, 01 VM 8608/9), the German Academic Exchange Service (DAAD, 322/501/014/0), and SFB (217).This work is part of the doctoral thesis of M. P. Bettinotti.     

Environmental responses of plants and ecosystems as predictors of the impact of global change

An understanding of plant responses to fluctuations in environment is critical to predictions of plant and ecosystem responses to climate change. In the northern hemisphere, the northern limits of distribution of major biomes are probably determined by the tolerance of their dominant physiognomic types (e.g., deciduous hardwood trees) to minimum winter temperatures and can thus be predicted from long-term patterns of temperature fluctuations. At a more detailed level, the responses of functional groups of plants to altered climate can be predicted from their known responses to fluctuations in soil resources (nutrients and water) and the expected effect of climatic change on these soil resources. Laboratory and field experiments demonstrate the feasibility of this approach.     

Effects of testosterone on a sexually dimorphic frog muscle: Repeated in vivo observations and androgen receptor distribution

In the present study the sexually dimorphic, androgen-sensitive flexor carpi radialis muscle (FCR) in male Xenopus laevis was viewed repeatedly in vivo to assess the influence of testosterone on muscle fiber size over a period of up to 12 weeks. Regions of the muscle innervated by different spinal nerves responded differently to testosterone treatment. Muscle fibers innervated by spinal nerve 2 (SN2) hypertrophied within 7 days in frogs that had been castrated and given testosterone-filled implants. This initial hypertrophy was followed by a return to normal fiber size a week late, after which fiber size slowly increased again. In castrated males with empty implants, muscle fibers innervated by SN2 gradually atrophied. Fibers innervated by spinal nerve 3 (SN3) were not affected by androgen replacement or withdrawal. The sartorius, a control muscle that is neither sexually dimorphic nor particularly androgen sensitive, was also unaffected. The in vivo observations were confirmed by measurements of muscle fiber cross-sectional areas in frozen sections of whole forelimbs. At 8 and 12 weeks after castration, cross-sectional areas of fibers innervated by SN2 were significantly larger in frogs provided with testosterone than in castrates without testosterone. No difference was found in the SN2 region or in the anconeus caput scapulare (triceps), another control muscle. Immunocytochemistry employing an antibody against the androgen receptor (AR) indicated that the receptor is present in myonuclei of all muscles of the forelimb. While no difference in labeling intensity was detected, the number of AR-containing nuclei per muscle fiber cross-section was higher in fibers innervated by SN2 than in those innervated by SN3, and was yet lower in the triceps. This suggests that regulation of androgen sensitivity may occur via muscle fiber. ARs, although an influence of the nerve may also contribute. 1994 John Wiley & Sons, Inc.     

Striatal L-DOPA Decarboxylase Activity in Parkinson's Disease In Vivo: Implications for the Regulation of Dopamine Synthesis

Abstract: L-DOPA is a large neutral amino acid subject to transport out of, as well as into, brain tissue. Competition between dopamine synthesis and L-DOPA egress from striatum must favor L-DOPA egress if decarboxylation declines relatively more than transport in Parkinson's disease. To test this hypothesis, we injected patients with Parkinson's disease with a radidabeled analogue of L-DOPA and recorded regional brain radioactivity as a function of time by means of positron emission tomography. We simultaneously estimated the activity of the decarboxylating enzyme and the amino acid transport. In the striatum of patients, we found the L-DOPA decarboxylase activity to be reduced in the head of the caudate nucleus and the putamen. However, the rate of egress of the DOPA analogue was unaffected by the disease and thus inhibited dopamine synthesis more than predicted in the absence of L-DOPA egress.