Preferential Release of Newly Synthesized Insulin Assessed by a Multi-Label Reporter System Using Pancreatic β-Cell Line MIN6

Newly synthesized hormones have been suggested to be preferentially secreted by various neuroendocrine cells. This observation indicates that there is a distinct population of secretory granules containing new and old hormones. Recent development of fluorescent timer proteins used in bovine adrenal chromaffin cells revealed that secretory vesicles segregate into distinct age-dependent populations. Here, we verify the preferential release of newly synthesized insulin in the pancreatic β-cell line, MIN6, using a combination of multi-labeling reporter systems with both fluorescent and biochemical procedures. This system allows hormones or granules of any age to be labeled, in contrast to the timer proteins, which require fluorescence shift time. Pulse-chase labeling with different color probes distinguishes insulin secretory granules by age, with younger granules having a predominantly intracellular localization rather than at the cell periphery.     

Polysaccharide-polynucleotide complexes (15): thermal stability of schizophyllan (SPG)/poly(C) triple strands is controllable by alpha-amino acid modification

Schizophyllan (SPG), a beta-1,3-glucan polysaccharide which is known to form macromolecular complexes with certain polynucleotides, was modified by a reductive amination method with alpha-amino acids (Arg, Lys, and Ser). The thermal stability of the complexes as estimated by T(m) was enhanced in SPG-Arg and SPG-Lys conjugates which have pI values higher than the pH of the medium (8.0). The T(m) shift increased with the increase in the percentage of alpha-amino acid introduced and the highest T(m) values attained were 64 degrees C for SPG-Arg conjugate and 62 degrees C for SPG-Lys conjugate, which are higher by 13 and 11 degrees C, respectively, than those of the unmodified SPG+poly(C) complex. In the SPG-Ser conjugate with a pI lower than the medium pH (8.0), the T(m) values decreased with an increase in the percentage of Ser. Formation of the macromolecular complex was no longer detected above 13.2% Ser. The findings indicate that the T(m) values are easily controllable by the type and percentage of the introduced alpha-amino acids. We believe, therefore, that the present conjugates, consisting of naturally originated SPG and alpha-amino acids, provide an important lead for developing nontoxic artificial vectors and to control the affinity with polynucleotides in response to medium pH and temperature.     

Excitation energy relaxation in a symbiotic cyanobacterium, Prochloron didemni, occurring in coral-reef ascidians, and in a free-living cyanobacterium, Prochlorothrix hollandica

The marine cyanobacterium Prochloron is a unique photosynthetic organism that lives in obligate symbiosis with colonial ascidians. We compared Prochloron harbored in four different host species and cultured Prochlorothrix by means of spectroscopic measurements, including time-resolved fluorescence, to investigate host-induced differences in light-harvesting strategies between the cyanobacteria. The light-harvesting efficiency of photosystems including antenna Pcb, PS II-PS I connection, and pigment status, especially that of PS I Red Chls, were different among the four samples. We also discuss relationships between these observed characteristics and the light conditions, to which Prochloron cells are exposed, influenced by distribution pattern in the host colonies, presence or absence of tunic spicules, and microenvironments within the ascidians' habitat.     

Structural and mutational studies of anthocyanin malonyltransferases establish the features of BAHD enzyme catalysis

The BAHD family is a class of acyl-CoA-dependent acyltransferases that are involved in plant secondary metabolism and show a diverse range of specificities for acyl acceptors. Anthocyanin acyltransferases make up an important class of the BAHD family and catalyze the acylation of anthocyanins that are responsible for most of the red-to-blue colors of flowers. Here, we describe crystallographic and mutational studies of three similar anthocyanin malonyltransferases from red chrysanthemum petals: anthocyanidin 3-O-glucoside-6'-O-malonyltransferase (Dm3MaT1), anthocyanidin 3-O-glucoside-3', 6'-O-dimalonyltransferase (Dm3MaT2), and a homolog (Dm3MaT3). Mutational analyses revealed that seven amino acid residues in the N- and C-terminal regions are important for the differential acyl-acceptor specificity between Dm3MaT1 and Dm3MaT2. Crystallographic studies of Dm3MaT3 provided the first structure of a BAHD member, complexed with acyl-CoA, showing the detailed interactions between the enzyme and acyl-CoA molecules. The structure, combined with the results of mutational analyses, allowed us to identify the acyl-acceptor binding site of anthocyanin malonyltransferases, which is structurally different from the corresponding portion of vinorine synthase, another BAHD member, thus permitting the diversity of the acyl-acceptor specificity of BAHD family to be understood.     

Spermine modulates the expression of two probable polyamine transporter genes and determines growth responses to cadaverine in Arabidopsis

Key message

Two genes, LAT1 and OCT1 , are likely to be involved in polyamine transport in Arabidopsis. Endogenous spermine levels modulate their expression and determine the sensitivity to cadaverine.

Abstract

Arabidopsis spermine (Spm) synthase (SPMS) gene-deficient mutant was previously shown to be rather resistant to the diamine cadaverine (Cad). Furthermore, a mutant deficient in polyamine oxidase 4 gene, accumulating about twofold more of Spm than wild type plants, showed increased sensitivity to Cad. It suggests that endogenous Spm content determines growth responses to Cad in Arabidopsis thaliana. Here, we showed that Arabidopsis seedlings pretreated with Spm absorbs more Cad and has shorter root growth, and that the transgenic Arabidopsis plants overexpressing the SPMS gene are hypersensitive to Cad, further supporting the above idea. The transgenic Arabidopsis overexpressing L-Amino acid Transporter 1 (LAT1) absorbed more Cad and showed increased Cad sensitivity, suggesting that LAT1 functions as a Cad importer. Recently, other research group reported that Organic Cation Transporter 1 (OCT1) is a causal gene which determines the Cad sensitivity of various Arabidopsis accessions. Furthermore, their results suggested that OCT1 is involved in Cad efflux. Thus we monitored the expression of OCT1 and LAT1 during the above experiments. Based on the results, we proposed a model in which the level of Spm content modulates the expression of OCT1 and LAT1, and determines Cad sensitivity of Arabidopsis.

     

Identification of novel pyrrolopyrazoles as protein kinase C β II inhibitors

A novel series of pyrrolopyrazole-based protein kinase C β II inhibitors has been identified from high-throughput screening. Herein, we report our initial structure-activity relationship studies with a focus on optimizing compound ligand efficiency and physicochemical properties, which has led to potent inhibitors with good cell permeability.     

Studies on the plant cytoskeleton using miniprotoplasts of tobacco BY-2 cells

Vacuoles in plant cells can be eliminated by centrifugation of protoplasts through a density gradient. In this review, properties of evacuolated protoplasts, named ‘miniprotoplasts’, and the significant roles in plant cytoskeleton studies are described. Miniprotoplasts, prepared from tobacco BY-2 cells whose cell-cycle had been synchronized at late anaphase, continued to divide to form two daughter cells. In the presence of cytochalasin B cytokinetic cleavage was enhanced, suggesting a role of actin filaments in plant cytokinesis. In the cytoplasmic extract of miniprotoplasts both tubulin and actin could be polymerized to form microtubules (MTs) and actin filaments (AFs), respectively. A purification method for tubulin, actin and related proteins was developed using the extract. To investigate the interaction between cortical microtubules and the plasma membrane, an experimental system in which MTs were reconstructed on membrane ghosts was developed by combination of membrane ghosts and the extract.     

Quantifying Interactions Within the NADP(H) Enzyme Network in Drosophila melanogaster

In this report, we use synthetic, activity-variant alleles in Drosophila melanogaster to quantify interactions across the enzyme network that reduces nicotinamide adenine dinucleotide phosphate (NADP) to NADPH. We examine the effects of large-scale variation in isocitrate dehydrogenase (IDH) or glucose-6-phosphate dehydrogenase (G6PD) activity in a single genetic background and of smaller-scale variation in IDH, G6PD, and malic enzyme across 10 different genetic backgrounds. We find significant interactions among all three enzymes in adults; changes in the activity of any one source of a reduced cofactor generally result in changes in the other two, although the magnitude and directionality of change differs depending on the gene and the genetic background. Observed interactions are presumably through cellular mechanisms that maintain a homeostatic balance of NADPH/NADP, and the magnitude of change in response to modification of one source of reduced cofactor likely reflects the relative contribution of that enzyme to the cofactor pool. Our results suggest that malic enzyme makes the largest single contribution to the NADPH pool, consistent with the results from earlier experiments in larval D. melanogaster using naturally occurring alleles. The interactions between all three enzymes indicate functional interdependence and underscore the importance of examining enzymes as components of a network.IN traits determined by a network of gene products, the phenotype is a function of the alleles present and of the relative contributions of individual network member genes. Since selection is on phenotype, the total composite genotype, not just individual loci, determines the fitness of an organism. In establishing the connection between genotype and phenotype for such networks, the first challenge is to quantify the relative contribution of each member of the network to the endpoint phenotype. By addressing function on a network-wide basis, interactions and interconnections that may not be apparent in individual gene examinations can be determined (Proulx et al. 2005).In most organisms, reduction of the cofactor nicotinamide adenine dinucleotide phosphate, or NADP, to NADPH is primarily the function of four enzymes: cytosolic malic enzyme (MEN), cytosolic isocitrate dehydrogenase (IDH), and the two oxidative enzymes of the pentose shunt, glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate (6PGD; recently reviewed in Ying 2008). In larval Drosophila melanogaster, MEN produces ∼30% of the available NADPH, IDH ∼20%, and G6PD and 6PGD the remaining ∼40% (Geer et al. 1979a,b). It is believed that these four enzymes interact to maintain the NADP/NADPH balance and supply of reducing power for lipogenesis and antioxidation (Geer et al. 1976, 1978, 1981; Wilton et al. 1982; Bentley et al. 1983; Geer and Laurie-Ahlberg 1984; Merritt et al. 2005; Pollak et al. 2007; Singh et al. 2007; Ying 2008). Dietary induction studies and observations of natural genetic variation have found connections between MEN activity and the activities of the pentose shunt enzymes to be generally straightforward and compensatory; reductions in one lead to increases in the other. The interactions involving IDH activity, however, have been found to be more complicated and at times counterintuitive; reductions in reducing power sometimes lead to decreases in IDH activity.In an earlier study (Merritt et al. 2005), we quantified the impact of genetic variation in Men activity on IDH and G6PD activities and triglyceride (a strong correlate with total lipid; Clark and Keith 1989) concentration. 6PGD was not independently assayed because earlier works suggest that G6PD and 6PGD activities are highly correlated, likely because of their coupled function in the pentose shunt (Wilton et al. 1982). We examined both naturally occurring Men alleles and synthetic alleles created by P-element excision and found significant associations between MEN activity and induction of the activities of both IDH and G6PD. The apparent interactions between MEN and IDH and G6PD across these 10 different third chromosome lines were quantified as mean elasticity coefficients: = −0.76 ± 0.236 and = −0.88 ± 0.208. Because MEN activity was reduced by 20%, both IDH and G6PD activity varied in a compensatory direction, increasing almost 1:1 with the decrease in MEN.The significant change in enzyme activity of two members of the NADPH network in response to our genetic reduction of the activity of a third strongly suggests that a physiological mechanism coregulates the three enzymes. Such functional interdependence would mean that individual members of the network do not act in isolation and should be examined collectively, not as isolated units. In this study, we characterize the effects of the independently varying activity levels of IDH, G6PD, and MEN on the activity of each other and triglyceride concentration in adult flies. We found significant responses to changes in all three enzymes, although the responses to genetic changes in IDH and G6PD were generally small; variation in MEN caused the greatest changes in the other enzymes.     

Oscillatory membrane currents paradoxically induced via NO-activated pathways in detrusor cells

Oscillatory inward membrane currents (I(oscil-in)) reflecting intracellular Ca(2+) ([Ca(2+)](i)) activity in detrusor cells, are thought to play an important role in producing tonic bladder contractions during micturition. The present patch clamp study revealed a new activation mechanism: sodium nitroprusside (SNP), a nitric oxide (NO) donor induced I(oscil-in) in a subpopulation of detrusor cells. The inhibitory effect of niflumic acid on SNP-induced I(oscil-in) suggests that Ca(2+)-activated Cl(-) channels are responsible for this current. In addition, SNP-induced I(oscil-in) required the cooperation of Ca(2+) influx through SK&F96365-sensitive channels and intracellular Ca(2+) release channels sensitive to ryanodine but insensitive to xestospongin C (XeC). This is also true for muscarinic agonist (carbachol: CCh)-induced I(oscil-in). However, 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a guanylyl cyclase inhibitor, suppressed SNP-induced I(oscil-in) but not CCh-induced I(oscil-in). The results suggest that a subpopulation of detrusor cells employ the NO/cGMP cascade to potentiate bladder contraction. Mechanisms underlying NO-induced I(oscil-in) are likely to contribute not only to the physiology but also to the pathophysiology of the lower urinary tract.