Efficient plant regeneration of Mesembryanthemum crystallinum via somatic embryogenesis

 An efficient plant regeneration procedure has been established from hypocotyl explants of the common ice plant, Mesembryanthemum crystallinum L, a halophytic leaf succulent that exhibits a stress-induced switch from C3 photosynthesis to crassulacean acid metabolism (CAM). Somatic embryos were initiated and developed up to globular and heart stages in Murashige and Skoog (MS) media supplemented with 3% sucrose, 0.6% bacto-agar, 80 mM NaCl, 5 μM 2,4-D and 1 μM kinetin. High frequency regeneration occurred when somatic embryos were germinated on media that lacked 2,4-D. High cytokinin treatment suppressed normal growth of embryos and favored abnormal embryo proliferation. Without growth regulators, regenerated plants rooted on MS medium with 100% efficiency. Mature, regenerated plants were fertile and morphologically identical to seed-derived plants. Received: 29 April 1999 / Revision received: 2 July 1999 · Accepted: 12 July 1999     

The Role of the δ GABA(A) Receptor in Ovarian Cycle-Linked Changes in Hippocampus-Dependent Learning and Memory

The δ subunit of the GABA_AR is highly expressed in the dentate gyrus of the hippocampus where it mediates a tonic extrasynaptic inhibitory current that is sensitive to neurosteroids. In female mice, the expression level of the δ subunit within the dentate gyrus is elevated in the diestrous relative to estrous phase of the estrous cycle. Previous work in our lab found that female δ-GABA_AR KO mice showed enhanced hippocampus-dependent trace but normal hippocampus-independent delay fear conditioning. Wild-type females in this study showed a wide range of freezing levels, whereas δ-GABA_AR KO mice expressed only high levels of fear. We hypothesized that the variability in the wild-type mice may have been due to estrous cycle-mediated changes in the expression of the δ-GABA_AR, with low levels of freezing in mice that were in the diestrous phase when dentate gyrus tonic inhibition is high. In the present study we tested this hypothesis by utilizing contextual, delay, and trace fear conditioning protocols in mice that were trained and tested in either the diestrous or estrous phases. Consistent with our hypothesis, we found a significant impairment of hippocampus-dependent learning and memory during diestrus relative to estrus in wild-type mice and this impairment was absent in δ-GABA_AR mice. These findings argue that the δ-GABA_AR plays an important role in estrous cycle-mediated fluctuations in hippocampus-dependent learning and memory.     

Drugs inhibiting the renin - angiotensin system

There are several approaches for interfering with the renin-angiotensin system. Antibodies against renin angiotensins I and II (AI and AII) have not been consistently successful in the past, probably because of nonspecific effects; however, recent purification of renin now makes this approach more promising. Renin inhibitors include pepstatin and analogs, lipids and phospholipids, and renin-substrate analogs. Pepstatin and analogs are the most potent and specific but they are not orally active. The phospholipids are the most effective in vivo but their specificity is yet to be established. No renin-substrate analogs have been developed that have biologically significant effects. Some of the most potent and specific agents available for interfering with the renin-angiotensin system are the AII-receptor antagonists. While these compounds effectively prevent the actions of AII, they suffer from several severe deficiencies: partial agonist activity, short duration of action, and lack of oral activity. The recent development of angiotensin-converting enzyme ACE) inhibitors that are orally active has provided the greatest degree of clinical success for inhibitors of the renin-angiotensin system and, consequently, the impetus to develop still better compounds. Captopril (SQ 14,225) is the prototype ACE inhibitor, being highly potent and specific with no other demonstrated pharmacological activity. Captopril is effective in all forms of human and animal models of hypertension except mineralocorticoid hypertension, which requires concomitant diuretic therapy. Because ACE is the same enzyme as kininase II, the enzyme that degrades kinins, the possibility exists that kinins are involved in the cardiovascular action of captopril, although this prospect is unlikely.     

Cell surface labeling of glucose transporter isoform GLUT4 by bis-mannose photolabel. Correlation with stimulation of glucose transport in rat adipose cells by insulin and phorbol ester

A new impermeant photoaffinity label has been used for identifying cell surface glucose transporters in isolated rat adipose cells. This compound is 2-N-4(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4- yloxy)-2- propylamine. We have used this reagent in combination with immunoprecipitation by specific antibodies against the GLUT4 and GLUT1 glucose transporter isoforms to estimate the relative abundance of these two transporters on the surface of the intact adipose cell following stimulation by insulin and phorbol 12-myristate 13-acetate (PMA). In the basal state, GLUT4 and GLUT1 are both present at the cell surface but GLUT4 is more abundant than GLUT1. In response to insulin, GLUT4 increases 15-20-fold and GLUT1 increases approximately 5-fold while 3-O-methyl-D-glucose transport is stimulated 20-30-fold. By contrast, PMA only induces a approximately 4-fold increase in GLUT4 while GLUT1 increases approximately 5-fold to the same level as seen with insulin. In addition, PMA stimulates 3-O-methyl-D-glucose transport approximately 3-fold to only 13% of the insulin-stimulated state. Thus GLUT4 is the major glucose transporter isoform under all conditions, and it is selectively and markedly enriched in response to insulin but not PMA which increases GLUT1 and GLUT4 equally. Furthermore, stimulation of glucose transport activity correlates closely with the appearance of GLUT4 on the cell surface in response to both insulin and PMA but does not correlate with the sum of GLUT1 and GLUT4 appearance. These results suggest that GLUT4 may be inherently more active than GLUT1 due to a higher TK (turnover/Km).     

Determinations of adipose cell size and number in suspensions of isolated rat and human adipose cells

The osmic acid fixation-Coulter electronic counter method described for determining adipose cell size and number in intact adipose tissue fragments has been modified for use with suspensions of isolated rat and human adipose cells. Mean cell sizes in tissue fragments and isolated cell suspensions prepared from the same tissue are virtually identical in rats of various weights. No statistically significant difference in mean adipose cell size between tissue and isolated cell suspension was observed in human adipose tissue although the variability was much greater than in rat tissue. The distribution of cell sizes among replicate samples is more uniform in the isolated cell preparations, possibly reflecting the considerably larger quantities of tissue used in preparing isolated cells than in determining cell size and number directly from tissue fragments. An example of the utility of the modified method during routine metabolic studies with isolated rat epididymal adipose cells is described; isolated cells of increasing size can be obtained from rats of increasing body weight, or from the separated distal and proximal portions of the fat pads of rats of the same weight.     

Isolation by distance,resistance and/or clusters? Lessons learned from a forest‐dwelling carnivore inhabiting a heterogeneous landscape

Landscape genetics provides a valuable framework to understand how landscape features influence gene flow and to disentangle the factors that lead to discrete and/or clinal population structure. Here, we attempt to differentiate between these processes in a forest‐dwelling small carnivore [European pine marten (Martes martes)]. Specifically, we used complementary analytical approaches to quantify the spatially explicit genetic structure and diversity and analyse patterns of gene flow for 140 individuals genotyped at 15 microsatellite loci. We first used spatially explicit and nonspatial Bayesian clustering algorithms to partition the sample into discrete clusters and evaluate hypotheses of ‘isolation by barriers’ (IBB). We further characterized the relationships between genetic distance and geographical (‘isolation by distance’, IBD) and ecological distances (‘isolation by resistance’, IBR) obtained from optimized landscape models. Using a reciprocal causal modelling approach, we competed the IBD, IBR and IBB hypotheses with each other to unravel factors driving population genetic structure. Additionally, we further assessed spatially explicit indices of genetic diversity using sGD across potentially overlapping genetic neighbourhoods that matched the inferred population structure. Our results revealed a complex spatial genetic cline that appears to be driven jointly by IBD and partial barriers to gene flow (IBB) associated with poor habitat and interspecific competition. Habitat loss and fragmentation, in synergy with past overharvesting and possible interspecific competition with sympatric stone marten (Martes foina), are likely the main factors responsible for the spatial genetic structure we observed. These results emphasize the need for a more thorough evaluation of discrete and clinal hypotheses governing gene flow in landscape genetic studies, and the potential influence of different limiting factors affecting genetic structure at different spatial scales.     

Landscape genetics and limiting factors

Population connectivity is mediated by the movement of organisms or propagules through landscapes. However, little is known about how variation in the pattern of landscape mosaics affects the detectability of landscape genetic relationships. The goal of this paper is to explore the impacts of limiting factors on landscape genetic processes using simulation modeling. We used spatially explicit, individual-based simulation modeling to quantify the effects of habitat area, fragmentation and the contrast in resistance between habitat and non-habitat on the apparent strength and statistical detectability of landscape genetic relationships. We found that landscape genetic effects are often not detectable when habitat is highly connected. In such situations landscape structure does not limit gene flow. We also found that contrast in resistance values between habitat and non-habitat interacts with habitat extensiveness and fragmentation to affect detectability of landscape genetic relationships. Thus, the influence of landscape features critical to providing connectivity may not be detectable if gene flow is not limited by spatial patterns or resistance contrast of these features. We developed regression equations that reliably predict whether or not isolation by resistance will be detected independently of isolation by distance as a function of habitat fragmentation and contrast in resistance between habitat and non-habitat.     

A sister group contrast using untargeted global metabolomic analysis delineates the biochemical regulation underlying desiccation tolerance in Sporobolus stapfianus

Understanding how plants tolerate dehydration is a prerequisite for developing novel strategies for improving drought tolerance. The desiccation-tolerant (DT) Sporobolus stapfianus and the desiccation-sensitive (DS) Sporobolus pyramidalis formed a sister group contrast to reveal adaptive metabolic responses to dehydration using untargeted global metabolomic analysis. Young leaves from both grasses at full hydration or at 60% relative water content (RWC) and from S. stapfianus at lower RWCs were analyzed using liquid and gas chromatography linked to mass spectrometry or tandem mass spectrometry. Comparison of the two species in the fully hydrated state revealed intrinsic differences between the two metabolomes. S. stapfianus had higher concentrations of osmolytes, lower concentrations of metabolites associated with energy metabolism, and higher concentrations of nitrogen metabolites, suggesting that it is primed metabolically for dehydration stress. Further reduction of the leaf RWC to 60% instigated a metabolic shift in S. stapfianus toward the production of protective compounds, whereas S. pyramidalis responded differently. The metabolomes of S. stapfianus leaves below 40% RWC were strongly directed toward antioxidant production, nitrogen remobilization, ammonia detoxification, and soluble sugar production. Collectively, the metabolic profiles obtained uncovered a cascade of biochemical regulation strategies critical to the survival of S. stapfianus under desiccation.