14-3-3 isoforms and pattern formation during barley microspore embryogenesis

The members of the 14-3-3 isoform family have been shown to be developmentally regulated during animal embryogenesis, where they take part in cell differentiation processes. 14-3-3 isoform-specific expression patterns were studied in plant embryogenic processes, using barley (Hordeum vulgare L.) microspore embryogenesis as a model system. After embryogenesis induction by stress, microspores with enlarged morphology showed higher viability than non-enlarged ones. Following microspore culture, cell division was only observed among the enlarged microspores. Western blot and immunolocalization of three barley 14-3-3 isoforms, 14-3-3A, 14-3-3B and 14-3-3C were carried out using isoform-specific antibodies. The level of 14-3-3C protein was higher in enlarged microspores than in non-enlarged ones. A processed form of 14-3-3A was associated with the death pathway of the non-enlarged microspores. In the early embryogenesis stage, 14-3-3 subcellular localization differed among dividing and non-dividing microspores and the microspore-derived multicellular structures showed a polarized expression pattern of 14-3-3C and a higher 14-3-3A signal in epidermis primordia. In the late embryogenesis stage, 14-3-3C was specifically expressed underneath the L(1) layer of the shoot apical meristem and in the scutellum of embryo-like structures (ELSs). 14-3-3C was also expressed in the scutellum and underneath the L(1) layer of the shoot apical meristem of 21 d after pollination (DAP) zygotic embryos. These results reveal that 14-3-3A processing and 14-3-3C isoform tissue-specific expression are closely related to cell fate and initiation of specific cell type differentiation, providing a new insight into the study of 14-3-3 proteins in plant embryogenesis.     

A whole-genome shotgun optical map of Yersinia pestis strain KIM

Yersinia pestis is the causative agent of the bubonic, septicemic, and pneumonic plagues (also known as black death) and has been responsible for recurrent devastating pandemics throughout history. To further understand this virulent bacterium and to accelerate an ongoing sequencing project, two whole-genome restriction maps (XhoI and PvuII) of Y. pestis strain KIM were constructed using shotgun optical mapping. This approach constructs ordered restriction maps from randomly sheared individual DNA molecules directly extracted from cells. The two maps served different purposes; the XhoI map facilitated sequence assembly by providing a scaffold for high-resolution alignment, while the PvuII map verified genome sequence assembly. Our results show that such maps facilitated the closure of sequence gaps and, most importantly, provided a purely independent means for sequence validation. Given the recent advancements to the optical mapping system, increased resolution and throughput are enabling such maps to guide sequence assembly at a very early stage of a microbial sequencing project.     

Two new proteins preferentially associated with membrane immunoglobulin D.

The IgM and IgD classes of antigen receptor can perform different functions on B cells. However, so far no class-specific components communicating with the cytoplasm have been found in the two antigen receptors. We have employed a new biotinylation protocol to search for intracellular membrane Ig-associated proteins. Here we describe two proteins of 29 and 31 kDa that are associated with membrane IgD and to some extent with membrane IgM. The membrane IgM molecule is associated specifically with three proteins of 32, 37 and 41 kDa. The purification and sequencing of the two mIgD-associated proteins revealed that they are novel proteins which are related to each other. These proteins may be the missing link between the antigen receptor and the cytoskeleton and may contribute to functional differences between membrane IgM and membrane IgD.     

Stimulation of canine pancreatic polypeptide, gastrin, and gastric acid secretion by ranatensin, litorin, bombesin nonapeptide and substance P

Four dogs with chronic gastric fistulas were give intravenous bombesin nonapeptide (B9), ranatensin, and litorin by constant infusion for 90 min at 1.2 micrograms x kg-1 on separate days. A dose response study with substance P (1.5, 3.0, 60, 18 and 54 micrograms x kg-1 x h-1) was also carried out and all tests compared to a standard protein meal (10g x kg-1). Plasma gastrin and PP were measured by radioimmunoassay and gastric acid by autobiuret titration. Substance P failed to stimulate gastric acid secretion or release either pancreatic polypeptide (PP) or gastrin. Basal gastrin levels were 8 +/-2 fmol/ml. The peak increment of gastrin released by bombesin was 95 +/- 16, ranatensin 22 +/- 6, litorin 18 +/- 4, and meal 39 +/- 5 fmol/ml. Bombesin caused significantly greater release of gastrin than a meal, litorin or ranatensin (P less than 0.01). Basal gastric secretion was 23 +/- 4 microequiv./min. B9 produced a peak acid secretion of 356 +/- 124 muequiv./min. There was no significant difference between the bombesin-like peptides (P less than 0.01). Basal plasma PP was 38 +/- 12 fmol/ml. B9 produced a peak PP increment of 600 +/- 50, litorin 137 +/- 36, ranatensin 98 +/- 11, and a meal 305 +/- 58 fmol/ml. B9 released significantly more PP than either litorin of ranatensin (P less than 0.01). The different amino acid sequences of the peptides are probably responsible for their potency. The substitution of a penultimate phenylalanine residue in litorin and ranatensin for leucine in bombesin does not prevent PP or gastrin release by bombesin-like peptides. Since bombesin-like peptides are widely distributed in the gastrointestinal tract of man and stimulate both acid and gut hormone secretion, it is possible that they might play a physiological role in the modulation of gastrointestinal function.     

Occurrence and functions of the phosphatidylinositol cycle in the myocardium

In the last decade a great deal of attention was awarded to a signal transduction pathway which is utilized primarily by Ca²⁺ mobilizing signal molecules and which involves the hydrolysis of a quantitatively minor phospholipid, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) by a PtdIns-specific phospholipase C (PLC). The evidence for the existence of receptor-mediated GTP binding protein-coupled PLC in myocardium and its possible functions are briefly summarized. The minireview is concentrated on the following aspects: 1) cellular localization and synthesis of polyphospho-PtdIns from PtdIns, 2) desensitization of the ₁-adrenergic agonist and endothelin-1 mediated PtdIns responses, 3) oscillatory Ca²⁺ transients initiated by Ptdlns(4,5)P₂ hydrolysis, 4) polyunsaturated fatty acids as constituents of polyphospho-PtdIns and of the protein kinase C activator 1,2-diacylglycerol (DAG), 5) source other than Ptdlns(4,5)P₂ contributing to the stimulated DAG, 6) role of the PtdIns pathway in cardiomyocyte growth and gene expression during the hypertrophic response. (Mol Cell Biochem116: 59–67, 1992)Abbreviations Phosphatidylinositol 4,5-bisphosphate PtdIns(4,5)P₂ - Phosphatidylinositol 4-monophosphate PtdIns(4)P₄ - Phosphatidylinositol PtdIns - Inositol 1,4,5-triphosphate Ins(1,4,5)P₃ - Inositol 1,3,4,5-tetrakisphosphate Ins(1,3,4,5)P₄ - Inositol 1-monophosphate Ins(1)P - Inositol 1,4-bisphosphate Ins(1,4)P₂ - Inositol Ins - Inositolphosphates InsP_n - Guanine 5'-triphosphate GTP - GTP binding protein G-protein - Phosphatidylinositolspecific phospholipase C PLC - Protein kinase C PKC - 1,2-Diacylglycerol DAG - Monoacylglycerol MAG - cytidyldiphoshate-diacylglycerol CDP-DAG - Sarcolemma SL - Sarcoplasmic reticulum SR - Stearic acid 18:0 - Polyunsaturated fatty acids PUFA - Arachidonic acid 20:4n-6 - Linoleic acid 18:2n-6 - Eicosapentaenoic acid 20:5n-3 - Docosahexaenoic acid 22:6n-3 - Phosphatidic acid PtdOH - Phospholipase D PLD - Phosphatidylcholine PtdChol     

L-propionylcarnitine and myocardial performance in stunned porcine myocardium

Recently, we showed that L-propionylcarnitine did not affect recovery of regional contractile function of porcine myocardium subjected to 1 h of low-flow ischemia followed by 2 hr of reperfusion. In that study, ischemia may have been too severe and/or the duration of reperfusion too short to detect a beneficial effect of the compound. Therefore, in the present study we investigated the effects of saline (control group; n = 14) or pretreatment with L-propionylcarnitine (3 days of 50 mg/kg p.o. b.i.d. + 50 mg/kg i.v. prior to the experiment; n = 13) on recovery of regional contractile function of the myocardium in open-chest anesthetized pigs, subjected to two cycles of 10 min of left anterior descending coronary artery (LADCA) occlusion, each followed by 30 min of reperfusion. In the control animals, at the end of the second reperfusion period, systemic vascular resistance had increased by 18%, which, however, was not observed in the L-propionylcarnitine-treated pigs. In the control group, during the first occlusion, systolic segment length shortening (SSLS) of the LADCA-perfused area decreased from 18.5 ± 5.5% to - 3.7 = 3.2%. After 30 min of reperfusion, SSLS of the LADCA-perfused area had only partially recovered to 6.2 ±5.9%. During the second occlusion-reperfusion cycle similar values for SSLS were observed. In the treated animals., SSLS of the LADCA-perfused area was slightly improved after the second occlusion-reperfusion cycle (p = 0.056). This effect did not result in an overall improvement in cardiac pump function. We conclude that in a model of myocardial stunning, L-propionylcarnitine prevents systemic vasoconstriction in response to ischemia and reperfusion and, possibly as a result of this effect, slightly ameliorates post-ischemic hypofunction. (Mol Cell Biochem116: 147–153, 1992)     

Rapid changes in the concentration of phosphoenolpyruvate carboxykinase mRNA in rat liver and kidney. Effects of insulin and cyclic AMP

Starvation and diabetes both caused a marked increase in the concentration of hepatic phosphoenolpyruvate caroboxykinase mRNA while the administration of insulin to diabetic rats or refeeding glucose to starved animals caused a marked reduction in the levels of enzyme mRNA as measured by hybridization using a cDNA probe.l The Administration of dibutyryl cAMP to a starved-refed cat caused an 8-fold induction of phosphoenolpyruvate carboxykinase mRNA in 1 h. Triamcinolone plus acidosis induced the levels of enzyme mRNA in kidney 3-fold within 6 h, however, starvation for 24h had only marginal effects. In all of the above conditions, the levels of phosphoenolpyruvate carboxykinase mRNA measured by hybridization assay agreed well with the relative levels of translatable mRNA for the enzyme. The half-time of phosphoenolpyruvate carboxykinase mRNA, determined after the administration of either alpha-amanitin or cordycepin to starved animals, was approximately 40 min. However, cycloheximide either alone or together with cordycepin, not only prevented the decrease in phosphoenolpyruvate carboxykinase mRNA sequence abundance, but induced it 2-fold. Cycloheximide itself, when injected into 21-day fetal rats in utero caused an induction of enzyme mRNA equal to that noted when dibutyryl cAMP was administered. The mRNA for phosphoenolpyruvate carboxykinase is approximately 2.8 kb in length, but nuclei from the livers of diabetic rats contain a number of putative precursor RNA species for the enzyme, up to 6.5 kb in size, all containing a poly(A) tail. Two hours after refeedng glucose to a starved rat, these nuclear RNA species could no longer be detected by hybridization to our cDNA probe.     

Presence of functional sarcoplasmic reticulum in the developing heart and its confinement to chamber myocardium

During development fast-contracting atrial and ventricular chambers develop from a peristaltic-contracting heart tube. This study addresses the question of whether chamber formation is paralleled by a matching expression of the sarcoplasmic reticulum (SR) Ca(2+) pump. We studied indo-1 Ca(2+) transients elicited by field stimulation of linear heart tube stages and of explants from atria and outflow tracts of the prototypical preseptational E13 rat heart. Ca(2+) transients of H/H 11+ chicken hearts, which constitute the prototypic linear heart tube stage, were sensitive to verapamil only, indicating a minor contribution of Ca(2+)-triggered SR Ca(2+) release. Outflow tract transients displayed sensitivity to the inhibitors similar to that of the linear heart tube stages. Atrial Ca(2+) transients disappeared upon addition of ryanodine, tetracaine, or verapamil, indicating the presence of Ca(2+)-triggered SR Ca(2+) release. Quantitative radioactive in situ hybridization on sections of E13 rat hearts showed approximately 10-fold higher SERCA2a mRNA levels in the atria compared to nonmyocardial tissue and approximately 5-fold higher expression in compact ventricular myocardium. The myocardium of atrioventricular canal, outflow tract, inner curvature, and ventricular trabecules displayed weak expression. Immunohistochemistry on sections of rat and human embryos showed a similar pattern. The significance of these findings is threefold. (i) A functional SR is present long before birth. (ii) SR development is concomitant with cardiac chamber development, explaining regional differences in cardiac function. (iii) The pattern of SERCA2a expression underscores a manner of chamber development by differentiation at the outer curvature, rather than by segmentation of the linear heart tube.