Pyruvate blocks zinc-induced neurotoxicity in immortalized hypothalamic neurons

Zinc is an essential trace element and present at high concentrations in the central nervous system. Recent studies have revealed that excess amount of extracellular zinc is neurotoxic, and that the disruption of zinc homeostasis may be related to various neurodegenerative diseases. Zinc (25–100 M) caused significant death of immortalized hypothalamic neuronal cells (GT1-7 cells) in a dose- and time-dependent manner. LD₅₀ was estimated to be 34 M. The degenerated cells were TUNEL-positive and exhibited apoptosis-like characteristics. Preadministration of sodium pyruvate (1–2 mM), a downstream energy substrate, inhibited the zinc-induced neurotoxicity in GT1-7 cells. GT1-7 cells can be used as a good tool for the investigation of zinc neurotoxicity in the hypothalamus.     

Activation of AMPK is essential for AICAR-induced glucose uptake by skeletal muscle but not adipocytes

5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) reportedly activates AMP-activated protein kinase (AMPK) and stimulates glucose uptake by skeletal muscle cells. In this study, we investigated the role of AMPK in AICAR-induced glucose uptake by 3T3-L1 adipocytes and rat soleus muscle cells by overexpressing wild-type and dominant negative forms of the AMPKalpha2 subunit by use of adenovirus-mediated gene transfer. Overexpression of the dominant negative mutant had no effect on AICAR-induced glucose transport in adipocytes, although AMPK activation was almost completely abolished. This suggests that AICAR-induced glucose uptake by 3T3-L1 adipocytes is independent of AMPK activation. By contrast, overexpression of the dominant negative AMPKalpha2 mutant in muscle markedly suppressed both AICAR-induced glucose uptake and AMPK activation, although insulin-induced uptake was unaffected. Overexpression of the wild-type AMPKalpha2 subunit significantly increased AMPK activity in muscle but did not enhance glucose uptake. Thus, although AMPK activation may not, by itself, be sufficient to increase glucose transport, it appears essential for AICAR-induced glucose uptake in muscle.     

Grafting of "abbreviated" complementarity-determining regions containing specificity-determining residues essential for ligand contact to engineer a less immunogenic humanized monoclonal antibody

Murine mAb COL-1 reacts with carcinoembryonic Ag (CEA), expressed on a wide range of human carcinomas. In preclinical studies in animals and clinical trials in patients, murine COL-1 showed excellent tumor localization. To circumvent the problem of immunogenicity of the murine Ab in patients, a humanized COL-1 (HuCOL-1) was generated by grafting the complementarity-determining regions (CDRs) of COL-1 onto the frameworks of the variable light and variable heavy regions of human mAbs. To minimize anti-V region responses, a variant of HuCOL-1 was generated by grafting onto the human frameworks only the "abbreviated" CDRs, the stretches of CDR residues that contain the specificity-determining residues that are essential for the surface complementarity of the Ab and its ligand. In competition RIAs, the recombinant variant completely inhibited the binding of radiolabeled murine and humanized COL-1 to CEA. The HuCOL-1 and its variant showed no difference in their binding ability to the CEA expressed on the surface of a CEA-transduced tumor cell line. Compared with HuCOL-1, the HuCOL-1 variant showed lower reactivity to patients' sera carrying anti-V region Abs to COL-1. The final variant of the HuCOL-1, which retains its Ag-binding reactivity and shows significantly lower serum reactivity than that of the parental Ab, can serve as a prototype for the development of a potentially useful clinical reagent.     

Evolution of contest competition and its effect on host–parasitoid dynamics

In experimental populations of the cowpea bean weevil Callosobruchus maculatus (Coleoptera: Bruchidae) and a parasitic wasp Heterospilus prosopidis (Hymenoptera: Braconidae), large changes in the abundances and the fluctuations of both species occurred after approximately 20 generations. In this paper, we examine the hypothesis that this observed change in the dynamics may have been caused by an evolutionary shift in the mode of competition among the bean weevils. A Nicholson-Bailey type model is developed using parameters measured from the experiments. The host larvae can differ in the type of competitive behaviour that they exhibit, which can be either of a contest type or of a scramble type. If a bean contains one or more larvae of the contest type, only one of these will survive and any scramble-type larvae in the bean will be killed. If no contest-type larvae are present within a bean, multiple individuals of the scramble type can emerge from a single bea n. The model assumes many genotypes, differing in the fraction of offspring of the two types. If a high per capita resource availability is maintained, then the scramble type is selected for, but if resources are limited, then the contest type is selected for. The host population at the start of the experiment, taken from a stock culture, was composed mostly of the scramble type. The model is successful in explaining the initial quick increase in the host's abundance, followed by the evolutionary increase in the fraction of the contest type among hosts, resulting in the more stable population dynamics of the host–parasitoid system, as observed in the experiments. However, it predicts a parasitoid abundance much higher than that observed. We discuss alternative hypotheses to explain the observed evolutionary shift in the population dynamics. We also examine the effect of the difference in size of the beans in the stock culture and those used in the experiments.     

Free radical scavenging actions of metallothionein isoforms I and II

By employing electron spin resonance spectroscopy, we examined the free radicals scavenging effects of hepatic metallothionein (MT) isoforms I and II (MTs-I and II) on four types of free radicals. Solutions of 0.15mM of MT-I and 0.3mM of MT-II were found to scavenge the 1,1-diphenyl-2-picrylhydrazyl radicals (1.30 × 10¹⁵ spins/ml) completely. In addition, both isoforms exhibited total scavenging action against the hydroxyl radicals (1.75 × 10¹⁵ spins/ml) generated in a Fenton reaction. Similarly, 0.3mM of MT-I scavenged almost 90% of the superoxide (2.22 × 10¹⁵ spins/ml) generated by the hypoxanthine and xanthine oxidase system, while a 0.3mM MT-II solution could only scavenge 40% of it. By using 2,2,6,6-tetramethyl-4-piperidone as a “spin-trap” for the reactive oxygen species (containing singlet oxygen, superoxide and hydroxyl radicals) generated by photosensitized oxidation of riboflavin and measuring the relative signal intensities of the resulting stable nitroxide adduct, 2,2,6,6-tetramethyl-4-piperidine-1-oxyl, we observed that MT-II (0.3 mM) could scavenge 92%, while MT-I at 0.15 mM μl/ml concentrations could completely scavenge all the reactive species (2.15 × 10¹⁵ spins/ml) generated.

The results of these studies suggest that although both isoforms of MT are able to scavenge free radicals, the MT-I appears to be a superior scavenger of superoxide and 1,1 diphenyl-2-picrylhydrazyl radicals.     

A pronounced sex difference when two linked loci of the Japanese brown frogRana japonica are recombined

To investigate the sex differences of crossing-over inRana japonica, the recombination frequencies between two linked loci,LDH-B andMPI, were examined in offspring obtained from 29 matings involving either females or males heterozygous at both loci. No recombinants were found among 303 offspring bred from 6 heterozygous males, whereas 160 were parental and 138 were recombinants among 298 offspring bred from 9 heterozygous females. The recombination frequency was 46.3%. These results show that the sex difference when two loci were recombined was marked and significant inRana japonica.     

Stimulatory effect of prostaglandins on the production of hexosamine-containing substances by cultured fibroblasts (4) adenosine 3′:5′-cyclic monophosphate independent stimulation by prostaglandin F2α

The mechanism of the stimulatory effect of prostaglandin (PG) F_2α on the production of hexosamine-containing substances by cultured fibroblasts was studied with special reference to adenosine 3′:5′- cyclic monophosphate (cAMP). At the stationary phase, the cells were exposed for 6 hrs to PGF_2α, E₁, cAMP or dibutyryl-cAMP in a wide range of concentrations. cAMP itself showed a slight stimulation on the production of hexosamine-containing substances, and the effect was enhanced by using the dibutyryl derivative. PGF_2α had much a greater capacity than either the exogeneous cAMP or the dibutyryl-cAMP for enhancing the production of hexosamine-containing substances. To know whether cAMP is involved in the stimulatory effect of PGF_2α, intracellular cAMP level was concomitantly measured in both PGF_2α and PGE₁ treated cultures. Although the cellular cAMP level in PGE₁ treated cultures was much higher than that in the PGF_2α treated cultures, the stimulatory effect on the production of hexosamine-containing substances in PGE₁ treated cultures was always much smaller than that in the PGF_2α treated cultures. Moreover, PGF_2α had a significant stimulatory effect on the production of hexosamine-containing substances even at a low concentration as 100 pg/ml, which is small enough not to increase any cellular cAMP level. From these results, it was concluded that the stimulatory effect of PGF_2α on the production of hexosamine-containing substances by cultured fibroblasts is not mediated by cAMP and is caused by a mechanism different from that caused by cAMP.     

Microbody of n-alkane-grown yeast

Microbodies appearing abundantly in n-alkane-grown cells of Candida tropicalis pK 233 were isolated by means of sucrose density gradient centrifugation. Electron microscopical observation showed that the microbodies isolated were intact. Localization of catalase and d-amino acid oxidase in the isolated microbodies was confirmed. Isocitrate lyase, malate synthase and NADP-linked isocitrate dehydrogenase were also located in the microbody, but malate dehydrogenase, citrate synthase, aconitase and NAD-linked isocitrate dehydrogenase were not. Neither cytochrome P-450 nor NADPH-cytochrome c reductase, the components involved in the n-alkane hydroxylation system of the yeast, were detected in the microbody fraction.