Quantal transmission at purinergic junctions: stochastic interaction between ATP and its receptors.

The time course of most quantal currents recorded with a small diameter electrode placed over visualized varicosities of sympathetic nerve terminals that secrete ATP was determined: these had a time to reach 90% of peak of 1.3-1.8 ms and a time constant of decay of 12-18 ms; they were unaffected by blocking ectoenzymes or the uptake of adenosine. Monte Carlo methods were used to analyze the stochastic interaction between ATP, released in a packet from a varicosity, and the underlying patch of purinoceptors, to reconstitute the time course of the quantal current. This leads to certain restrictions on the possible number of ATP molecules in a quantum (about 1000) and the density of purinoceptors at the junctions (about 1000 microns-1), given the known geometry of the junction and the kinetics of ATP action. The observed quantal current has a relatively small variability (coefficient of variation < 0.1), and this stochastic property is reproduced for a given quantum of ATP. Potentiation effects (of about 12%) occur if two quanta are released from the same varicosity because the receptor patch is not saturated even by the release of two quanta. The simulations show that quantal currents have a characteristically distinct shape for varicosities with different junctional cleft widths (50-200 nm). Finally, incorporation of an ectoenzyme with the known kinetics of ATPase into the junctional cleft allows for a quantal current of the observed time course, provided the number of ATP molecules in a quantum is increased over the number in the absence of the ATPase.     

Variations in random amplified polymorphic DNA profile and toxin production among isolates of Colletotrichum falcatum Went from sugarcane in Tamil Nadu,India

Red rot, caused by Colletotrichum falcatum Went, is one of the most important diseases of sugarcane (Saccharum officinarum L.). The pathogen shows a great diversity in virulence as a number of pathotypes are known to occur in nature. In the present study, the toxin producing ability and genetic variability among isolates of C. falcatum collected from major sugarcane growing areas of Tamil Nadu, India were analysed. The C. falcatum isolates differed significantly in their ability to produce toxin in vitro. The toxin from C. falcatum isolate Cf 671a induced the maximum electrolyte leakage (300 μS) from sugarcane leaf tissues. The genetic relatedness of the isolates of C. falcatum differing in toxin production potential was investigated by using RAPD analysis. Analysis of the genetic coefficient matrix derived from the scores of RAPD profiles showed that minimum and maximum percent similarities among the tested C. falcatum isolates were in the range of 19 to 95% respectively. The phylogenetic analysis by the UPGMA identified two main clusters. Cluster A contains only one isolate (Cf 98061) and all the other isolates were placed in Cluster B confirming high genetic diversity among the isolates. No correlation was observed between clustering of the C. falcatum isolates in the dendrogram and their toxin producing abilities.     

Behavioral and electrophysiological analysis of general anesthesia in 3 background strains of Drosophila melanogaster

General anesthetics achieve behavioral unresponsiveness via a mechanism that is incompletely understood. The study of genetic model systems such as the fruit fly Drosophila melanogaster is crucial to advancing our understanding of how anesthetic drugs render animals unresponsive. Previous studies have shown that wild-type control strains differ significantly in their sensitivity to general anesthetics, which potentially introduces confounding factors for comparing genetic mutations placed on these wild-type backgrounds. Here, we examined a variety of behavioral and electrophysiological endpoints in Drosophila, in both adult and larval animals. We characterized these endpoints in 3 commonly used fly strains: wild-type Canton Special (CS), and 2 commonly used white-eyed strains, isoCJ1 and w¹¹¹⁸. We found that CS and isoCJ1 show remarkably similar sensitivity to isoflurane across a variety of behavioral and electrophysiological endpoints. In contrast, w¹¹¹⁸ is resistant to isoflurane compared to the other 2 strains at both the adult and larval stages. This resistance is however not reflected at the level of neurotransmitter release at the larval neuromuscular junction (NMJ). This suggests that the w¹¹¹⁸ strain harbors another mutation that produces isoflurane resistance, by acting on an arousal pathway that is most likely preserved between larval and adult brains. This mutation probably also affects sleep, as marked differences between isoCJ1 and w¹¹¹⁸ have also recently been found for behavioral responsiveness and sleep intensity measures.     

Regulation of mat responses by a differentiation MAPK pathway in Saccharomyces cerevisiae

Fungal species exhibit diverse behaviors when presented with extracellular challenges. Pathogenic fungi can undergo cell differentiation and biofilm formation in response to fluctuating nutrient levels, and these responses are required for virulence. In the model fungal eukaryote Saccharomyces cerevisiae, nutrient limitation induces filamentous growth and biofilm/mat formation. Both responses require the same signal transduction (MAPK) pathway and the same cell adhesion molecule (Flo11) but have been studied under different conditions. We found that filamentous growth and mat formation are aspects of a related response that is regulated by the MAPK pathway. Cells in yeast-form mats differentiated into pseudohyphae in response to nutrient limitation. The MAPK pathway regulated mat expansion (in the plane of the XY-axis) and substrate invasion (downward in the plane of the Z-axis), which optimized the mat's response to extracellular nutrient levels. The MAPK pathway also regulated an upward growth pattern (in the plane of the Z-axis) in response to nutrient limitation and changes in surface rigidity. Upward growth allowed for another level of mat responsiveness and resembled a type of colonial chemorepulsion. Together our results show that signaling pathways play critical roles in regulating social behaviors in which fungal cells participate. Signaling pathways may regulate similar processes in pathogens, whose highly nuanced responses are required for virulence.     

The foxtail millet (Setaria italica) terpene synthase gene family

Terpenoid metabolism plays vital roles in stress defense and the environmental adaptation of monocot crops. Here, we describe the identification of the terpene synthase (TPS) gene family of the panicoid food and bioenergy model crop foxtail millet (Setaria italica). The diploid S. italica genome contains 32 TPS genes, 17 of which were biochemically characterized in this study. Unlike other thus far investigated grasses, S. italica contains TPSs producing all three ent‐, (+)‐ and syn‐copalyl pyrophosphate stereoisomers that naturally occur as central building blocks in the biosynthesis of distinct monocot diterpenoids. Conversion of these intermediates by the promiscuous TPS SiTPS8 yielded different diterpenoid scaffolds. Additionally, a cytochrome P450 monooxygenase (CYP99A17), which genomically clustered with SiTPS8, catalyzes the C19 hydroxylation of SiTPS8 products to generate the corresponding diterpene alcohols. The presence of syntenic orthologs to about 19% of the S. italica TPSs in related grasses supports a common ancestry of selected pathway branches. Among the identified enzyme products, abietadien‐19‐ol, syn‐pimara‐7,15‐dien‐19‐ol and germacrene‐d ‐4‐ol were detectable in planta, and gene expression analysis of the biosynthetic TPSs showed distinct and, albeit moderately, inducible expression patterns in response to biotic and abiotic stress. In vitro growth‐inhibiting activity of abietadien‐19‐ol and syn‐pimara‐7,15‐dien‐19‐ol against Fusarium verticillioides and Fusarium subglutinans may indicate pathogen defensive functions, whereas the low antifungal efficacy of tested sesquiterpenoids supports other bioactivities. Together, these findings expand the known chemical space of monocot terpenoid metabolism to enable further investigations of terpenoid‐mediated stress resilience in these agriculturally important species.     

The effects of staged intra-articular injection of cultured autologous mesenchymal stromal cells on the repair of damaged cartilage: a pilot study in caprine model

Introduction

Treatment of chondral injuries remains a major issue despite the many advances made in cartilage repair techniques. Although it has been postulated that the use of marrow stimulation in combination with cell-based therapy may provide superior outcome, this has yet to be demonstrated. A pilot study was thus conducted to determine if bone marrow derived mesenchymal stromal cells (BM-MSCs) have modulatory effects on the repair outcomes of bone marrow stimulation (BMS) techniques.

Methods

Two full-thickness chondral 5 mm diameter defects were created in tandem on the medial condyle of left stifle joints of 18 Boer caprine (N = 18). Goats were then divided equally into three groups. Simultaneously, bone marrow aspirates were taken from the iliac crests from the goats in Group 1 and were sent for BM-MSC isolation and expansion in vitro. Six weeks later, BMS surgery, which involves subchondral drilling at the defect sites, was performed. After two weeks, the knees in Group 1 were given autologous intra-articular BM-MSCs (N = 6). In Group 2, although BMS was performed there were no supplementations provided. In Group 3, no intervention was administered. The caprines were sacrificed after six months. Repairs were evaluated using macroscopic assessment through the International Cartilage Repair Society (ICRS) scoring, histologic grading by O’Driscoll score, biochemical assays for glycosaminoglycans (GAGs) and gene expressions for aggrecan, collagen II and Sox9.

Results

Histological and immunohistochemical analyses demonstrated hyaline-like cartilage regeneration in the transplanted sites particularly in Group 1. In contrast, tissues in Groups 2 and 3 demonstrated mainly fibrocartilage. The highest ICRS and O’Driscoll scorings was also observed in Group 1, while the lowest score was seen in Group 3. Similarly, the total GAG/total protein as well as chondrogenic gene levels were expressed in the same order, that is highest in Group 1 while the lowest in Group three. Significant differences between these 3 groups were observed (P <0.05).

Conclusions

This study suggests that supplementing intra-articular injections of BM-MSCs following BMS knee surgery provides superior cartilage repair outcomes.     

Recruitment of rpd3 to the telomere depends on the protein arginine methyltransferase hmt1

In the yeast Saccharomyces cerevisiae, the establishment and maintenance of silent chromatin at the telomere requires a delicate balance between opposing activities of histone modifying enzymes. Previously, we demonstrated that the protein arginine methyltransferase Hmt1 plays a role in the formation of yeast silent chromatin. To better understand the nature of the Hmt1 interactions that contribute to this phenomenon, we carried out a systematic reverse genetic screen using a null allele of HMT1 and the synthetic genetic array (SGA) methodology. This screen revealed interactions between HMT1 and genes encoding components of the histone deacetylase complex Rpd3L (large). A double mutant carrying both RPD3 and HMT1 deletions display increased telomeric silencing and Sir2 occupancy at the telomeric boundary regions, when comparing to a single mutant carrying Hmt1-deletion only. However, the dual rpd3/hmt1-null mutant behaves like the rpd3-null single mutant with respect to silencing behavior, indicating that RPD3 is epistatic to HMT1. Mutants lacking either Hmt1 or its catalytic activity display an increase in the recruitment of histone deacetylase Rpd3 to the telomeric boundary regions. Moreover, in such loss-of-function mutants the levels of acetylated H4K5, which is a substrate of Rpd3, are altered at the telomeric boundary regions. In contrast, the level of acetylated H4K16, a target of the histone deacetylase Sir2, was increased in these regions. Interestingly, mutants lacking either Rpd3 or Sir2 display various levels of reduction in dimethylated H4R3 at these telomeric boundary regions. Together, these data provide insight into the mechanism whereby Hmt1 promotes the proper establishment and maintenance of silent chromatin at the telomeres.