Distribution of δ-aminolevulinic acid biosynthetic pathways among phototrophic bacterial groups

Two biosynthetic pathways are known for the universal tetrapyrrole precursor, -aminolevulinic acid (ALA). In the ALA synthase pathway which was first described in animal and some bacterial cells, the pyridoxal phosphate-dependent enzyme ALA synthase catalyzes condensation of glycine and succinyl-CoA to form ALA with the loss of C-1 of glycine as CO₂. In the five-carbon pathway which was first described in plant and algal cells, the carbon skeleton of glutamate is converted intact to ALA in a proposed reaction sequence that requires three enzymes, tRNA^Glu, ATP, Mg²⁺, NADPH, and pyridoxal phosphate. We have examined the distribution of the two ALA biosynthetic pathways among various genera, using cell-free extracts obtained from representative organisms. Evidence for the operation of the five-carbon pathway was obtained by the measurement of RNase-sensitive label incorporation from glutamate into ALA, using 3,4-[³H]glutamate or 1-[¹⁴C]glutamate as substrate. ALA synthase activity was indicated by RNase-insensitive incorporation of label from 2-[¹⁴C]glycine into ALA. The distribution of the two pathways among the bacteria tested was in general agreement with their previously established phylogenetic relationships and clearly indicates that the five-carbon pathway is the more ancient process, whereas the pathway utilizing ALA synthase probably evolved much later. The five-carbon pathway is apparently the more widely utilized one among bacteria, while the ALA synthase pathway seems to be limited to the subgroup of purple bacteria.Abbreviations ALA -aminolevulinic acid - DTT dithiothreitol - PALP pyridoxal phosphate - SDS sodium dodecyl sulfate - tricine N-tris-(hydroxymethyl)methylglycine     

Classification and ordination of macroinvertebrate assemblages to predict stream acidity in upland Wales

Macroinvertebrates were collected from riffles at 104 sites in upland Wales during April and July 1984. Species assemblages were ordinated by DECORANA, classified by TWINSPAN and related to stream chemistry and other environmental factors using correlation and multiple discriminant analysis. DECORANA axis 1 was most strongly correlated with pH and aluminium concentration whilst axis 2 correlated with stream gradient and flow. Four TWINSPAN site groups established in each season were also principally related to pH and aluminium concentration, and reflected overall taxon-richness; differences between groups were most apparent during spring, when catchment forest cover and taxon-richness were also related. A dichotomous key based on indicator species was established for each season with the coleopteran Hydraena gracilis Germar and the Ephemeroptera, including Baetis rhodani Pictet, important indicators at Level 1. We propose that these indicator systems may be used for the rapid detection and assessment of acid waters throughout Wales, and that the methodology is applicable generally.     

Cell cycle analysis of asynchronous cell populations by flow cytometry using bromodeoxyuridine label and Hoechst-propidium iodide stain.

Continuous labelling of cells with deoxybromouridine (BrdUrd) followed by staining with a bis-benzimidazole (Hoechst 33258) and a phenanthridinium (propidium iodide or ethidium bromide) allows the cells to be separated by flow cytometry according to the extent of their DNA replication. This BrdUrd-Hoechst/PI method has been used mainly to observe perturbations of the cell cycle in synchronously growing cells. In this paper we demonstrate that, when the method is applied to asynchronously dividing cells, more extensive information can be derived about the effects of cytotoxic and other treatments on the kinetics of the cell cycle. The interpretation of the data is explained, the effects of different types of cytotoxic agent are described, and the method is compared briefly to other methods for following cell cycle kinetics.     

蚕豆叶片发育与衰老过程中超氧物歧化酶活性与丙二醛含量变化

蚕豆植株叶片随茎节自上而下表现出明显的发育与衰老顺序,可作为衰老特征的是叶绿素和蛋白质含量明显下降。蚕豆叶中SOD活性主要定位于12 000× g离心后所得的上清液和叶绿体组分。衰老叶片的SOD总活性和叶绿体组分的相对活性都有所下降,SOD同工酶谱也发生了改变。O_2~ 产生速率随叶龄增大而稍上升;而MDA含量在叶片外观表现枯黄衰老征兆前就急剧上升。可能因为衰老叶片过氧化氢酶活性大幅度下降与SOD之间的不平衡,致使O_2~ 代谢中间产物累积而引起膜的损伤.     

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

Biosynthesis of chlorosome proteins is not inhibited in acetylene-treated cultures of Chlorobium vibrioforme

The composition, abundance and apparent molecular masses of chlorosome polypeptides from Chlorobium tepidum and Chlorobium vibrioforme 8327 were compared. The most abundant, low-molecular-mass chlorosome polypeptides of both strains had similar electrophoretic mobilities and abundances, but several of the larger proteins were different in both apparent mass and abundance. Polyclonal antisera raised against recombinant chlorosome proteins of Cb. tepidum recognized the homologous proteins in Cb. vibrioforme, and a one-to-one correspondence between the chlorosome proteins of the two species was confirmed. As previously shown [Ormerod et al. (1990) J Bacteriol 172: 1352–1360], acetylene strongly suppressed the synthesis of bacteriochlorophyll c in Cb. vibrioforme strain 8327. No correlation was found between the bacteriochlorophyll c content of cells and the cellular content of chlorosome proteins. Nine of ten chlorosome proteins were detected in acetylene-treated cultures, and the chlorosome proteins were generally present in similar amounts in control and acetylene-treated cells. These results suggest that the synthesis of chlorosome proteins and the assembly of the chlorosome envelope is constitutive. It remains possible that the synthesis of bacteriochlorophyll c and its insertion into chlorosomes might be regulated by environmental parameters such as light intensity.This revised version was published online in October 2005 with corrections to the Cover Date.     

`Every dogma has its day'*: a personal look at carbon metabolism in photosynthetic bacteria

Dogmas are unscientific. What is perhaps the greatest biological dogma of all time, the `unity of biochemistry' is, in the main, still having its day. According to present knowledge, the exceptions to this dogma are mere details when seen in relation to the biosystem as a whole. Nevertheless the exceptions are scientifically interesting and the understanding of them has led to a better comprehension of photosynthesis and ecology. Until the discovery of ¹⁴C, photosynthetic CO₂ fixation was like a slightly opened black box. With ¹⁴C in hand scientists mapped out the path of carbon in green plant photosynthesis in the course of a few years. The impressive reductive pentose phosphate cycle was almost immediately assumed to be universal in autotrophs, including anoxygenic phototrophs, in spite of the odd observation to the contrary. A new dogma was born and held the field for about two decades. Events began to turn when green sulfur bacteria were found to contain ferredoxin-coupled ketoacid-oxidoreductases. This led to the formulation of a novel CO₂-fixing pathway, the reductive citric acid cycle, but its general acceptance required much work by many investigators. However, the ice had now been broken and after some years a third mechanism of CO₂ fixation was discovered, this time in Chloroflexus, and then a fourth in the same genus. One consequence of these discoveries is that it has become apparent that oxygen is an important factor that determines the kind of CO₂-fixing mechanism an organism uses. With the prospect of the characterization of hordes of novel bacteria forecast by molecular ecologists we can expect further distinctive CO₂ fixation mechanisms to turn up. This revised version was published online in August 2006 with corrections to the Cover Date.     

Spatial working memory and hippocampal size across pregnancy in rats

The present experiments investigated the effects of pregnancy on performance in the Morris water maze and on hippocampal volume. In the first study, pregnant rats (in between the first and second trimester) outperformed nonpregnant rats on the Morris water maze on 1 day of testing. In the second study, rats were tested in a working memory variation of the maze in which the spatial location of the platform varied. Pregnant females traveled shorter distances than nonpregnant females during the first two trimesters, but performed worse than nonpregnant females during the third trimester. Latency measures showed a similar profile. Group differences in performance were not related to changes in swim speed. However, changes in performance in pregnant females may be related to estrogen, progesterone, and/or corticosterone levels during pregnancy, with low levels of estradiol and high levels of progesterone being associated with better performance. There were no significant differences between pregnant and nonpregnant animals on any of the brain measures, although pregnant animals tended to have a smaller hippocampus than nonpregnant animals. These results indicate that pregnancy can affect performance, possibly related to the hormonal changes that accompany pregnancy.