The formation of chlorophyll from chlorophyllide in leaves containing proplastids is a four-step process

The time course of the different esters of chlorophyllide (Chlide) during the formation of chlorophyll a (Chl) in embryonic bean leaves containing proplastids was investigated by HPLC. After the reduction of photoactive Pchlide (Pchlide) to Chlide, three intermediates, i.e. Chlide geranylgeraniol, Chlide dihydrogeranylgeraniol and Chlide tetrahydrogeranylgeraniol were detected before the formation of Chlide phytol, i.e. authentic Chl. The transformation of Chlide to Chl was found to be much faster in leaves containing proplastids than in etiolated leaves with etioplasts.     

Neuronal nicotinic acetylcholine receptors from Drosophila: two different types of alpha subunits coassemble within the same receptor complex

Although neuronal nicotinic acetylcholine receptors from insects have been reconstituted in vitro more than a decade ago, our knowledge about the subunit composition of native receptors as well as their functional properties still remains limited. Immunohistochemical evidence has suggested that two alpha subunits, alpha-like subunit (ALS) and Drosophila alpha2 subunit (Dalpha2), are colocalized in the synaptic neuropil of the Drosophila CNS and therefore may be subunits of the same receptor complex. To gain further understanding of the composition of these nicotinic receptors, we have examined the possibility that a receptor may imbed more than one alpha subunit using immunoprecipitations and electrophysiological investigations. Immunoprecipitation experiments of fly head extracts revealed that ALS-specific antibodies coprecipitate Dalpha2, and vice versa, and thereby suggest that these two alpha subunits must be contained within the same receptor complex, a result that is supported by investigations of reconstituted receptors in Xenopus oocytes. Discrimination between binary (ALS/beta2 or Dalpha2/beta2) and ternary (ALS/Dalpha2/beta2) receptor complexes was made on the basis of their dose-response curve to acetylcholine as well as their sensitivity to alpha-bungarotoxin or dihydro-beta-erythroidine. These data demonstrate that the presence of the two alpha subunits within a single receptor complex confers new receptor properties that cannot be predicted from knowledge of the binary receptor's properties.     

Expression of GABA Receptor ρ Subunits in Rat Brain

Abstract: The GABA receptor ρ1, ρ2, and ρ3 subunits are expressed in the retina where they form bicuculline-insensitive GABA_C receptors. We used northern blot, in situ hybridization, and RT-PCR analysis to study the expression of ρ subunits in rat brains. In situ hybridization allowed us to detect ρ-subunit expression in the superficial gray layer of the superior colliculus and in the cerebellar Purkinje cells. RT-PCR experiments indicated that (a) in retina and in domains that may contain functional GABA_C receptors, ρ2 and ρ1 subunits are expressed at similar levels; and (b) in domains and in tissues that are unlikely to contain GABA_C receptors, ρ2 mRNA is enriched relative to ρ1 mRNA. These results suggest that both ρ1 and ρ2 subunits are necessary to form a functional GABA_C receptor. The use of RT-PCR also showed that, except in the superior colliculus, ρ3 is expressed along with ρ1 and ρ2 subunits. We also raised an antibody against a peptide sequence unique to the ρ1 subunit. The use of this antibody on cerebellum revealed the rat ρ1 subunit in the soma and dendrites of Purkinje neurons. The allocation of GABA_C receptor subunits to identified neurons paves the way for future electrophysiological studies.     

Bax-alpha promotes apoptosis induced by cancer chemotherapy and accelerates the activation of caspase 3-like cysteine proteases in p53 double mutant B lymphoma Namalwa cells

Stable transfected human p53 (mt/mt) B lymphoma Namalwa variant lines showing differential expression of the Bax-alpha protein were derived under hygromycin selection. Overexpression of Bax-alpha in these variant cells accelerates cell death induced by short or continuous treatments with various concentrations of camptothecin, etoposide, vinblastine and shows no accelerating cell death activity in cis-platinum and paclitaxel-treated cells. Activation of apoptosis and oligonucleosome-sized DNA fragmentation was observed in the variant lines with more pronounced effect in cells containing high level of Bax-alpha protein. These results suggest that increased cell death mediated by anticancer drugs correlates with Bax-alpha level of expression and that Bax-alpha sensitizes Namalwa cells treated at low drug concentrations. The extent of DNA synthesis inhibition following DNA topoisomerase inhibitor treatments was similar in control and all transfected Namalwa cells suggesting that Bax-alpha acts downstream of DNA topoisomerase-mediated DNA strand breaks. To define further the relation between Bax-alpha expression and apoptosis activation, kinetics of caspase activation was measured in drug-treated cells. Caspase activities were measured using specific fluorogenic peptide derivatives DABCYL-YVADAPV-EDANS and Ac-DEVD-AMC, substrates of the caspase 1-like and caspase 3-like families, respectively. In control and Bax-alpha transfected Namalwa cells no increase in caspase 1-like activity was detected following camptothecin and etoposide treatments. In contrast, a significant difference in Ac-DEVD-AMC hydrolysis activity was observed in Bax-alpha transfected Namalwa cells compared to that of control Namalwa cells after camptothecin and etoposide treatment. Increased caspase 3-like activity correlated also with poly(ADPribosyl) polymerase cleavage. Taken together, these results suggest that Bax-alpha sensitize B lymphoma cells to series of anticancer drugs and accelerates the activation of apoptotic protease cascade.     

Hyaluronectin is produced by oligodendrocytes and Schwann cells in vitro

A hyaluronectin (HN)-like antigen was found in rat O-2A progenitors and oligodendrocytes, as well as in Schwann cells and in their culture medium. The HN-like antigen secreted in culture supernatants had a higher molecular mass than HN extracted from rat brain at acidic pH. In vitro the secreted HN-like antigen was spontaneously and slowly degraded into species whose Mr was close to that of HN found in acidic brain extract. In brain or nerve neutral pH extracts, both HN-like antigen and HN were present. The high Mr of the secreted antigen, the homology in amino acid sequences between HN and N-terminal domain of PG-M/versican, in addition to a positive hybridization between Schwann cell RNAs and a probe obtained with primers derived from HN sequences also found in versican suggested that HN is closely related to the large proteoglycan PG-M/versican. The presence in Schwann cell extract of a HN mRNA whose Mr was compatible with the size expected for HN showed that HN may be directly secreted by cells and not only the consequence of a proteolytic cleavage. The similarity of HN with PG-M (V3) suggested that HN found in vivo could be the result of an alternative splicing of a single gene. We conclude that HN as other members of the PG-M/versican family is a marker of oligodendrocytes and Schwann cells in culture.