Polypeptide composition,assembly and phosphorylation patterns of the photosystem II antenna system of Chlamydomonas reinhardtii

In recent years major progress has been made in describing the gene families that encode the polypeptides of the light-harvesting antenna system of photosystem II (PSII). At the same time, advances in the biochemical characterization of these antennae have been hampered by the high degree of similarity between the apoproteins. To help interpret the molecular results, we have re-examined the composition, the assembly and the phosphorylation patterns of the light-harvesting antenna of PSII (LHCII) in the green alga Chlamydomonas reinhardtii Dang, using a non-Tris SDS-PAGE system capable of resolving polypeptides that differ by as little as 200 daltons. Research to date has suggested that in C. reinhardtii the LHCII comprises just four polypeptides (p11, p13, p16 and p17), and CP29 and CP26 just one polypeptide each (p9 and p10, respectively), i.e. a total of six polypeptides. We report here that these antenna systems contain at least 15 polypeptides, 10 associated with LHCII, 3 with CP29, and 2 with CP26. All of these polypeptides have been positively identified by means of appropriate antibodies. We also demonstrate substantial heterogeneity to the pattern of in-vitro phosphorylation, with major differences found among members of closely spaced and immunologically related polypeptides. Most intriguing is the fact that the polypeptides that cross-react with the anti-type 2 LHCII antibodies of higher plants (p16, and to a lesser extent p11) are not phosphorylated, whereas in higher plants these are the most highly phosphorylated polypeptides. Also, unlike in higher plants, CP29 is heavily phosphorylated. Phosphorylation does not appear to have any effect on the mobility of polypeptides on fully denaturing SDS-PAGE gels. To learn more about the accumulation and organization of the light-harvesting polypeptides, we have also investigated a chlorophyll b-less mutant, cbn1-48. The LHCII is almost completely lost in this mutant, along with at least some LHCI. But the accumulation of CP29 and CP26 and their binding to PSII core complexes, is relatively unaffected. As expected, the loss of antenna polypeptides is accompanied by a reduction of the size of large reaction-center complexes. Following in-vitro phosphorylation the number of phosphorylated proteins is greatly increased in the mutant thylakoids compared to wildtype thylakoids. We present a model of the PSII antenna system to account for the new polypeptide complexity we have demonstrated.This work was supported by National Institute of Health grant GM22912 to L.A.S. We would like to thank Anastasios Melis for helpful discussions.     

Isolation and genetic structure of IS112, an insertion sequence responsible for the inactivation of the SalI restriction-modification system of Streptomyces albus G.

Summary IS112 is a transposable element identified in Streptomyces albus G by its frequent mutagenic insertion into the genes for the SalI restriction-modification system. IS112 is present in several copies in the genome of S. albus G. Homologous sequences were detected in other Streptomyces strains. Sequence analysis revealed that IS112 has a length of 883 by with a GC content of 67.4%. The copy that was isolated contained imperfect inverted repeats (16/20 match) at its ends and was flanked by a 2 by duplication at the target site, which was located within the gene (salIR) for the Sall endonuclease. A long open reading frame (ORF) encoding a putative polypeptide of 256-253 amino acids spans almost the entire sequence. Significant homology was detected between this polypeptide and that corresponding to ORFB of IS493, an insertion sequence recently isolated from Streptomyces lividans 66.

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An individual-level selection model for the apparent altruism exhibited by cellular slime moulds

In Dictyostelium discoideum, cells that become part of the stalk or basal disc display behaviour that can be interpreted as altruistic. Atzmony et al. (Curr Sci 72:142–145, 1997) had hypothesised that this behaviour could be the outcome of an adaptive strategy based on differing intrinsic quality as reflected by phenotypes that indicate differences in potential for survival and reproduction, followed by intercellular competition among amoebae of differing qualities. Low-quality amoebae would have a poor chance of succeeding in the competition to form spores; they could enhance their chances of survival by adopting a presumptive stalk strategy. Here we extend the hypothesis by making use of recent findings. Our approach is based on the view that an evolutionary explanation for the apparent altruism of stalk cells in D. discoideum must apply broadly to other cellular slime moulds (CSMs) that exhibit stalk cell death. Further, it must be capable of being modified to cover social behaviour in CSMs with an extracellular stalk, as well as in sorocarpic amoebae whose stalk cells are viable. With regard to D. discoideum, we suggest that (a) differentiation-inducing factor, thought of as a signal that inhibits amoebae from forming spores and induces them to differentiate into basal disc cells, is better viewed as a mediator of competition among post-aggregation amoebae and (b) the products of the ‘recognition genes’, tgrB and tgrC, allow an amoeba to assess its quality relative to that of its neighbours and move to a position within the aggregate that optimises its reproductive fitness. From this perspective, all cells behave in a manner that is ‘selfish’ rather than ‘altruistic’, albeit with different expectations of success.     

The fluidity of the nuclear envelope lipid does not affect the rate of nucleocytoplasmic RNA transport in mammalian liver

The effects of in vitro and in vivo modifications of nuclear envelope lipid on DNA leakage and on ATP-stimulated RNA release from isolated rat liver nuclei were investigated. The modifications included corn-oil feeding of the animals to alter the fatty acid composition of the lipids, phospholipase treatment of the isolated nuclei, and extraction of the total lipid with Triton X-100. Significant changes in lipid composition and approximate order parameter values of the spin-label 5-doxylstearate resulted, but there was no significant effect on RNA transport rate. It was concluded that the nuclear envelope lipid does not play any important part in nucleocytoplasmic RNA transport in mammalian liver.     

A rapid procedure for the construction of PCR cDNA libraries from small amounts of plant tissue

We describe a general method for the preparation of λZAP II cDNA libraries from very small amounts (<50 mg) of plant tissue. We have achieved this by combining an efficient method for RNA extraction with a modified PCR protocol for the synthesis and amplification of cDNA. Using this protocol we have found it possible to generate cDNA libraries containing more than 10⁶ clones from as little as 1 μg of total RNA.     

More than a store: regulatory roles for glycogen in skeletal muscle adaptation to exercise

The glycogen content of muscle determines not only our capacity for exercise but also the signaling events that occur in response to exercise. The result of the shift in signaling is that frequent training in a low-glycogen state results in improved fat oxidation during steady-state submaximal exercise. This review will discuss how the amount or localization of glycogen particles can directly or indirectly result in this differential response to training. The key direct effect discussed is carbohydrate binding, whereas the indirect effects include the metabolic shift toward fat oxidation, the increase in catecholamines, and osmotic stress. Although our understanding of the role of glycogen in response to training has expanded exponentially over the past 5 years, there are still many questions remaining as to how stored carbohydrate affects the muscular adaptation to exercise.     

ICAM-1-mediated, Src- and Pyk2-dependent vascular endothelial cadherin tyrosine phosphorylation is required for leukocyte transendothelial migration

Leukocyte transendothelial migration (TEM) has been modeled as a multistep process beginning with rolling adhesion, followed by firm adhesion, and ending with either transcellular or paracellular passage of the leukocyte across the endothelial monolayer. In the case of paracellular TEM, endothelial cell (EC) junctions are transiently disassembled to allow passage of leukocytes. Numerous lines of evidence demonstrate that tyrosine phosphorylation of adherens junction proteins, such as vascular endothelial cadherin (VE-cadherin) and beta-catenin, correlates with the disassembly of junctions. However, the role of tyrosine phosphorylation in the regulation of junctions during leukocyte TEM is not completely understood. Using human leukocytes and EC, we show that ICAM-1 engagement leads to activation of two tyrosine kinases, Src and Pyk2. Using phospho-specific Abs, we show that engagement of ICAM-1 induces phosphorylation of VE-cadherin on tyrosines 658 and 731, which correspond to the p120-catenin and beta-catenin binding sites, respectively. These phosphorylation events require the activity of both Src and Pyk2. We find that inhibition of endothelial Src with PP2 or SU6656 blocks neutrophil transmigration (71.1 +/- 3.8% and 48.6 +/- 3.8% reduction, respectively), whereas inhibition of endothelial Pyk2 also results in decreased neutrophil transmigration (25.5 +/- 6.0% reduction). Moreover, overexpression of the nonphosphorylatable Y658F or Y731F mutants of VE-cadherin impairs transmigration of neutrophils compared with overexpression of wild-type VE-cadherin (32.7 +/- 7.1% and 38.8 +/- 6.5% reduction, respectively). Our results demonstrate that engagement of ICAM-1 by leukocytes results in tyrosine phosphorylation of VE-cadherin, which is required for efficient neutrophil TEM.     

Estrogen and Progestogen Correlates of the Structure of Female Copulation Calls in Semi-Free-Ranging Barbary Macaques (<Emphasis Type="BoldItalic">Macaca sylvanus</Emphasis>)

Females of many Old World primates produce conspicuous vocalizations in combination with copulations. Indirect evidence exists that in Barbary macaques (Macaca sylvanus), the structure of these copulation calls is related to changes in reproductive hormone levels. However, the structure of these calls does not vary significantly around the timing of ovulation when estrogen and progestogen levels show marked changes. We here aimed to clarify this paradox by investigating how the steroid hormones estrogen and progesterone are related to changes in the acoustic structure of copulation calls. We collected data on semi-free-ranging Barbary macaques in Gibraltar and at La Forêt des Singes in Rocamadour, France. We determined estrogen and progestogen concentrations from fecal samples and combined them with a fine-grained structural analysis of female copulation calls (N = 775 calls of 11 females). Our analysis indicates a time lag of 3 d between changes in fecal hormone levels, adjusted for the excretion lag time, and in the acoustic structure of copulation calls. Specifically, we found that estrogen increased the duration and frequency of the calls, whereas progestogen had an antagonistic effect. Importantly, however, variation in acoustic variables did not track short-term changes such as the peak in estrogen occurring around the timing of ovulation. Taken together, our results help to explain why female Barbary macaque copulation calls are related to changes in hormone levels but fail to indicate the fertile phase.     

Differential expression of cell-wall-related genes during the formation of tracheary elements in the Zinnia mesophyll cell system

Plants, animals and some fungi undergo processes of cell specialization such that specific groups of cells are adapted to carry out particular functions. One of the more remarkable examples of cellular development in higher plants is the formation of water-conducting cells that are capable of supporting a column of water from the roots to tens of metres in the air for some trees. The Zinnia mesophyll cell system is a remarkable tool with which to study this entire developmental pathway in vitro. We have recently applied an RNA fingerprinting technology, to allow the detection of DNA fragments derived from RNA using cDNA synthesis and subsequent PCR-amplified fragment length polymorphisms (cDNA-AFLP), to systematically characterize hundreds of the genes involved in the process of tracheary element formation. Building hoops of secondary wall material is the key structural event in forming functional tracheary elements and we have identified over 50 partial sequences related to cell walls out of 600 differentially expressed cDNA fragments. The Zinnia system is an engine of gene discovery which is allowing us to identify and characterize candidate genes involved in cell wall biosynthesis and assembly.     

An in vitro demonstration of peroxisome proliferation and increase in peroxisomal β-oxidation system mRNAs in cultured rat hepatocytes treated with ciprofibrate

Using the normal adult rat hepatocytes, plated on rat tail collagen-coated dishes and fed a chemically defined medium, we demonstrate here that ciprofibrate at 0.1 mM concentration, increases significantly the mRNA levels of fatty acyl-CoA oxidase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and thiolase (the three enzymes of the β-oxidation system), and causes peroxisome proliferation. Increase in mRNA levels of these genes was evident within 1 h and was maximal 24 h after the addition of ciprofibrate. In hepatocytes cultured in the absence of ciprofibrate, the basal levels of these enzymes were low and further declined with time. Concomitant treatment of hepatocytes with cycloheximide did not inhibit or superinduce the mRNA levels, indicating that this induction may represent a primary (direct) effect of this compound on the expression of these genes and does not apparently involve short-lived repressor protein(s).