Monoclonal Antibody NM2 Recognizes the Protein Kinase C Phosphorylation Site in B-50 (GAP-43) and in Neurogranin (BICKS)

Abstract: Mouse monoclonal B-50 antibodies (Mabs) were screened to select a Mab that may interfere with suggested functions of B-50 (GAP-43), such as involvement in neurotransmitter release. Because the Mab NM2 reacted with peptide fragments of rat B-50 containing the unique protein kinase C (PKC) phosphorylation site at serine-41, it was selected and characterized in comparison with another Mab NM6 unreactive with these fragments. NM2, but not NM6, recognized neurogranin (BICKS), another PKC substrate, containing a homologous sequence to rat B-50 (34–52). To narrow down the epitope domain, synthetic B-50 peptides were tested in ELISAs. In contrast to NM6, NM2 immunoreacted with B-50 (39–51) peptide, but not with B-50 (43–51) peptide or a C-terminal B-50 peptide. Preabsorption by B-50 (39–51) peptide of NM2 inhibited the binding of NM2 to rat B-50 in contrast to NM6. NM2 selectively inhibited phosphorylation of B-50 during endogenous phosphorylation of synaptosomal plasma membrane proteins. Preabsorption of NM2 by B-50 (39–51) peptide abolished this inhibition. In conclusion, NM2 recognizes the QASFR peptide in B-50 and neurogranin. Therefore, NM2 may be a useful tool in physiological studies of the role of PKC-mediated phosphorylation and calmodulin binding of B-50 and neurogranin.     

TEMPERATURE RESPONSES AND EVOLUTION OF THERMAL TRAITS IN CLADOPHOROPSIS MEMBRANACEA (SIPHONOCLADALES,CHLOROPHYTA)1

Temperature tolerances and relative growth rates were determined for different isolates of the tropical to warm temperate seaweed species Cladophoropsis membranacea (C. Agardh) Boergesen (Siphonodadales, Chlorophyta) and some related taxa. Most isolates of C membranacea survived undamaged at 18° C for at least 8 weeks. Lower temperatures (5°–15°C) were tolerated for shorter periods of time but caused damage to cells. All isolates survived temperatures up to 34° C, whereas isolates from the eastern Mediterranean and Red Sea survived higher temperatures up to 36°C. Growth occurred between 18° and 32° C, but an isolate from the Red Sea had an extended growth range, reaching its maximum at 35°C. Struvea anastomosans (Harvey) Piccone & Grunow, Cladophoropsis sundanensis Reinbold, and an isolate of C. membranacea from Hawaii were slightly less cold- tolerant, with damage occurring at 18°C. Upper survival temperatures were between 32° and 36° C in these taxa. Temperature response data were mapped onto a phylogenetic tree. Tolerance for low temperatures appears to be a derived character state that supports the hypothesis that C. membranacea originated from a strictly tropical ancestor. Isolates from the Canary Islands, which is near the northern limit of distribution, are ill adapted to local temperature regimes. Isolates from the eastern Mediterranean and Red Sea show some adaptation to local temperature stress. They are isolated from those in the eastern Atlantic by a thermal barrier at the entrance of the Mediterranean.     

Catabolism of 4-hydroxybenzoate in Candida parapsilosis proceeds through initial oxidative decarboxylation by a FAD-dependent 4-hydroxybenzoate 1-hydroxylase

Abstract The first two steps in the catabolism of 4-hydroxybenzoate by the ascomycetous yeast Candida parapsilosis CBS604 were investigated. In contrast to the well-known bacterial pathways and to what was previously assumed, metabolism of 4-hydroxybenzoate in C. parapsilosis proceeds through initial oxidative decarboxylation to give 1,4-dihydroxybenzene. This reaction is catalyzed by a NAD(P)H and FAD-dependent 4-hydroxybenzoate 1-hydroxylase. Further metabolism of 1,4-dihydroxybenzene to the ring-fission substrate 1,2,4-trihydroxybenzene is catalyzed by a NADPH-specific FAD-dependent aromatic hydroxylase acting on phenolic compounds. ¹⁹F-NMR experiments with cell extracts and 2-fluoro-4-hydroxybenzoate as the model compound confirm this metabolic pathway and exclude the alternative pathway proceeding through initial 3-hydroxylation followed by oxidative decarboxylation in the second step.     

Expression of giant silkmoth cecropin B genes in tobacco

Cecropin B is a small antibacterial peptide from the giant silkmothHyalophora cecropia. To reveal the potential of this peptide for engineering bacterial disease resistance into crops, several cecropin B gene constructs were made either for expression in the cytosol or for secretion. All constructs were cloned in a plant expression vector and introduced in tobacco viaAgrobacterium tumefaciens. A cDNA-derived cecropin B gene construct lacking the amino-terminal signal peptide was poorly expressed in transgenic plants at the mRNA level, whereas plants harbouring a full-length cDNA-derived construct containing the insect signal peptide, showed increased cecropin B-mRNA levels. Highest expression was found in plants harbouring a construct with a plant-gene-derived signal peptide. In none of the transgenic plants could the cecropin B peptide be detected. This is most likely caused by breakdown of the peptide by plant endogenous proteases, since a chemically synthesized cecropin B peptide was degraded within seconds in various plant cell extracts. This degradation could be prevented by the addition of specific protease inhibitors and by boiling the extract prior to adding the peptide. In addition, anionic detergents, in contrast to cationic, zwitter-ionic or non-ionic detergents, could prevent this degradation. Nevertheless, transgenic tobacco plants were evaluated for resistance toPseudomonas solanacearum, the causal agent of bacterial wilt of many crops, andP. syringae pv.tabaci, the causal agent of bacterial wildfire, which are highly susceptible to cecropin Bin vitro. No resistance was found. These experiments indicate that introduction and expression of cecropin B genes in tobacco does not result in detectable cecropin B protein levels and resistance to bacterial infections, most likely due to degradation of the protein by endogenous proteases.     

Quantification of an C-labelled β-adrenoceptor ligand, S-(−)CGP 12177, in plasma of humans and rats

β-Adrenoceptors in human lungs and heart can be imaged with the radioligand 4-[3-[(1,1-dimethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-¹¹C-one (CGP 12177, [¹¹C]I). For quantification of receptor density with compartment models by adjustment of rate constants, an ‘input function’ is required which consists of the integral of the concentration of unmodified ligand in arterial plasma over time. A discrepancy in the literature regarding metabolic stability of [¹¹C]I prompted us to study metabolism in rats by reversed-phase HPLC (RP-HPLC) of trichloroacetic acid extracts of arterial plasma after i.v. injection of [¹¹C]I (> 11.1 TBq/mmol, 11 MBq/kg). Some plasma samples were also directly applied to an internal-surface reversed-phase (ISRP) column. In parallel experiments, tritiated [¹¹C]I was employed and methanol extracts of arterial plasma were analyzed by straight-phase TLC. The three methods were in excellent agreement. Unmodified [¹¹C]I decreased from > 98.5% (³H) or > 99.9% (¹¹C) initially to 57 ± 7% at 80 min post injection to formation of two polar metabolites. Using the RP-HPLC method, no metabolism was detectable in humans up to 30 min after injection of [¹¹C]I (1851 MBq). Deproteinization of plasma with acetonitrile resulted in the formation of a radioactive species (artifact) which eluted immediately after the void volume in RP-HPLC and which could be mistakenly interpreted as a metabolite. Plasma protein binding was low (ca. 30%) in both humans and rats. Association of the radioligand to blood cells suggested that equilibrium between receptor-bound and free radioligand was reached within 15 min after high-specific-activity injections, but only after more than 30 min after low-specific-activity injections.     

A two-element Enhancer-Inhibitor transposon system in Arabidopsis thaliana

The Enhancer-Inhibitor (En-I), also known as Suppressor-mutator (Spm-dSpm), transposable element system of maize was modified and introduced into Arabidopsis by Agrobacterium tumefaciens transformation. A stable En/Spm transposase source under control of the CaMV 35S promoter mediated frequent transposition of I/dSpm elements. Transposition occurred continuously throughout plant development over at least seven consecutive plant generations after transformation. New insertions were found at both linked and unlinked positions relative to a transposon donor site. The independent transposition frequency was defined as a transposition parameter, which quantified the rate of unique insertion events and ranged from 7.8% to 29.2% in different populations. An increase as well as a decrease in I/dSpm element copy number was seen at the individual plant level, but not at the population level after several plant generations. The continuous, frequent transposition observed for this transposon system makes it an attractive tool for use in gene tagging in Arabidopsis.     

Expression of biologically active hordothionins in tobacco. Effects of pre- and pro-sequences at the amino and carboxyl termini of the hordothionin precursor on mature protein expression and sorting

Hordothionins (HTHs) are small anti-bacterial proteins present in barley endosperm which are processed from larger precursor proteins, consisting of an amino-terminal signal peptide (SP), the mature highly basic HTH and a carboxy-terminal acidic peptide (AP). Different HTH precursor proteins were expressed in tobacco to study the effects of the pre-sequences (SP) and pro-sequences (AP) on expression, processing, sorting and biological activity and hence the feasibility of engineering bacterial disease resistance into crops which lack these proteins. Maximum HTH expression levels of approximately 0.7% (11 mol/kg) of total soluble protein in young tobacco leaves were obtained using a semi-synthetic gene construct encoding a complete chimaeric HTH precursor protein. Tenfold lower HTH expression levels (maximum 1.3 mol/kg) were obtained using synthetic gene constructs without the AP-coding sequence and no expression was found in plants containing synthetic HTH gene constructs without SP-and AP-coding sequences. In both cases where expression was found, the precursors were apparently correctly processed, although the HTH produced in plants containing a construct without AP sequence appeared to be slightly modified. No effect on plant phenotype was observed. Localization studies indicated that the HTH was in identical fractions of plants expressing the two different precursors, albeit at a different ratio, and was not secreted into the intercellular spaces of leaves or culture medium by protoplasts. Our results indicated that the AP is not involved in sorting and suggested that it might facilitate transport through membranes. The in vitro toxicity of HTH isolated from transgenic tobacco plants expressing the two different precursor proteins for the bacterial plant pathogen Clavibacter michiganensis subsp. michiganensis appeared similar to that of the HTH purified from barley endosperm.     

Genetics of lactose utilization in lactic acid bacteria

Abstract: Lactose utilization is the primary function of lactic acid bacteria used in industrial dairy fermentations. The mechanism by which lactose is transported determines largely the pathway for the hydrolysis of the internalized disaccharide and the fate of the glucose and galactose moieties. Biochemical and genetic studies have indicated that lactose can be transported via phosphotransferase systems, transport systems dependent on ATP binding cassette proteins, or secondary transport systems including proton symport and lactose-galactose antiport systems. The genetic determinants for the group translocation and secondary transport systems have been identified in lactic acid bacteria and are reviewed here. In many cases the lactose genes are organized into operons or operon-like structures with a modular organization, in which the genes encoding lactose transport are tightly linked to those for lactose hydrolysis. In addition, in some cases the genes involved in the galactose metabolism are linked to or co-transcribed with the lactose genes, suggesting a common evolutionary pathway. The lactose genes show characteristic configurations and very high sequence identity in some phylogenetically distant lactic acid bacteria such as Leuconostoc and Lactobacillus or Lactococcus and Lactobacillus . The significance of these results for the adaptation of lactic acid bacteria to the industrial milk environment in which lactose is the sole energy source is discussed.