Advances in cereal protoplast research

Beginning in 1986, plants have been regenerated from protoplasts of all of the important cereal species, including wheat, rice, maize, and barley, and grasses such as sugarcane. In addition, somatic hybrids/cybrids as well as transgenic plants with introduced useful agronomic traits have been obtained in several instances. This rapid and impressive progress in the genetic manipulation of cereals has been made possible by two critical technical advances during the past decade: the establishment of embryogenic suspension cultures as a source of totipotent protoplasts and the direct delivery of DNA into protoplasts for genetic transformation.     

Curcumin Intake Affects miRNA Signature in Murine Melanoma with mmu-miR-205-5p Most Significantly Altered

Melanoma is the most aggressive form of skin cancer with estimated 48,000 deaths per year worldwide. The polyphenol curcumin derived from the plant Curcuma longa is well known for its anti-inflammatory and anti-cancerogenic properties. Accordingly, dietary intake of this compound may be suitable for melanoma prevention. However, how this compound affects basic cellular mechanisms in developing melanoma still remains elusive. Therefore, the aim of this study was to investigate for the first time the impact of oral curcumin administration on the miRNA signature of engrafting melanoma. For this purpose, the effects of a 4% curcumin diet were tested on melanoma, which were established by injection of murine B78H1 cells in the flank of C57BL/6 mice. Curcumin diet or standard chow (control) was administered two weeks prior to injection of tumor cells until termination of the experiment. High throughput chip-based array analysis was deployed to detect alterations in the miRNA signature of the tumors. Curcumin treatment significantly reduced the growth of the flank tumors. Furthermore the miRNA expression signature in tumors was substantially altered by curcumin intake with mmu-miR-205-5p over 100 times higher expressed when compared to controls. The expression levels of identified key miRNAs in the tumor samples were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). A comparable expression pattern of these miRNAs was also detected in other curcumin-treated melanoma cell lines under in vitro conditions. Putative targets of curcumin-induced up-regulated miRNAs were enriched in ‘o-glycan biosynthesis’, ‘endoplasmatic reticulum protein processing’ and different cancer-related pathways. Western Blot analyses revealed that of these targets anti-apoptotic B-cell CLL/lymphoma 2 (Bcl-2) and proliferating cell nuclear antigen (PCNA) were significantly down-regulated in curcumin-treated tumors. These findings demonstrate a profound alteration of the miRNA expression signature in engrafting curcumin-treated melanoma with mmu-miR-205-5p being up-regulated most significantly.     

Patterns of morphological and genetic diversity of Valeriana jatamansi Jones in different habitats and altitudinal range of West Himalaya,India

Importance to know and understand diversity of Himalayan plants is increasingly recognized considering the fact that various natural and anthropogenic pressures might bring about serious influences to morphological and genetic diversity of the vegetation in the region. In this context, Valeriana jatamansi was investigated in detail, taking into account its importance in various Ayurvedic and modern medicines. Randomly selected mature plants from twenty five different populations (located between 1215 m to 2775 m asl) of V. jatamansi were analysed for their morphological attributes. Further, ISSR markers were used to detect genetic variation among 151 plants of selected 25 populations. Use of 20 primers yielded 125 reproducible polymorphic loci which were used to estimate different parameters of genetic diversity. These parameters were in turn applied to develop relationships with habitat types and altitude range. Significant variation (p < 0.05) in above ground dry weight (AGDW) and below ground dry weight (BGDW) across the populations was observed. Nei's genetic diversity index (He) ranged from 0.25 to 0.37 across the populations, with a mean of 0.31. Genetic diversity exhibited a decreasing trend with increasing altitude, and maximum diversity (He = 0.325) was observed in the range of 1201–1500 m asl. Among the different habitat conditions, highest genetic diversity (He = 0.334; Pp = 84.38) was observed in grassland habitats while minimum in mixed forest habitats (He = 0.285; Pp = 72.433). The genetic diversity (He) had significant negative relationships with AGDW, BGDW and rhizome diameter (Pearson r = −0.359, −0.424 and −0.317, respectively; p < 0.05). The genetic characterization of V. jatamansi from the western Himalaya by this study suggests influences of habitat types and the altitudinal range upon genetic diversity, and based on these proposals for conservation strategies in favour of the species are made.     

Ca2+ block and flickering both contribute to the negative slope of the IV curve in BK channels

Single-channel current–voltage (IV) curves of human large-conductance, voltage- and Ca²⁺-activated K⁺ (BK) channels are quite linear in 150 mM KCl. In the presence of Ca²⁺ and/or Mg²⁺, they show a negative slope conductance at high positive potentials. This is generally explained by a Ca²⁺/Mg²⁺ block as by Geng et al. (2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210955) in this issue. Here, we basically support this finding but add a refinement: the analysis of the open-channel noise by means of β distributions reveals what would be found if measurements were done with an amplifier of sufficient temporal resolution (10 MHz), namely that the block by 2.5 mM Ca²⁺ and 2.5 mM Mg²⁺ per se would only cause a saturating curve up to +160 mV. Further bending down requires the involvement of a second process related to flickering in the microsecond range. This flickering is hardly affected by the presence or absence of Ca²⁺/Mg²⁺. In contrast to the experiments reported here, previous experiments in BK channels (Schroeder and Hansen. 2007. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.200709802) showed saturating IV curves already in the absence of Ca²⁺/Mg²⁺. The reason for this discrepancy could not be identified so far. However, the flickering component was very similar in the old and new experiments, regardless of the occurrence of noncanonical IV curves.     

N‐glycan maturation is crucial for cytokinin‐mediated development and cellulose synthesis in Oryza sativa

To explore the physiological significance of N‐glycan maturation in the plant Golgi apparatus, gnt1, a mutant with loss of N‐acetylglucosaminyltransferase I (GnTI) function, was isolated in Oryza sativa. gnt1 exhibited complete inhibition of N‐glycan maturation and accumulated high‐mannose N‐glycans. Phenotypic analyses revealed that gnt1 shows defective post‐seedling development and incomplete cell wall biosynthesis, leading to symptoms such as failure in tiller formation, brittle leaves, reduced cell wall thickness, and decreased cellulose content. The developmental defects of gnt1 ultimately resulted in early lethality without transition to the reproductive stage. However, callus induced from gnt1 seeds could be maintained for periods, although it exhibited a low proliferation rate, small size, and hypersensitivity to salt stress. Shoot regeneration and dark‐induced leaf senescence assays indicated that the loss of GnTI function results in reduced sensitivity to cytokinin in rice. Reduced expression of A‐type O. sativa response regulators that are rapidly induced by cytokinins in gnt1 confirmed that cytokinin signaling is impaired in the mutant. These results strongly support the proposed involvement of N‐glycan maturation in transport as well as in the function of membrane proteins that are synthesized via the endomembrane system.     

Nerve growth factor stimulates axon outgrowth through negative regulation of growth cone actomyosin restraint of microtubule advance

Nerve growth factor (NGF) promotes growth, differentiation, and survival of sensory neurons in the mammalian nervous system. Little is known about how NGF elicits faster axon outgrowth or how growth cones integrate and transform signal input to motor output. Using cultured mouse dorsal root ganglion neurons, we found that myosin II (MII) is required for NGF to stimulate faster axon outgrowth. From experiments inducing loss or gain of function of MII, specific MII isoforms, and vinculin-dependent adhesion-cytoskeletal coupling, we determined that NGF causes decreased vinculin-dependent actomyosin restraint of microtubule advance. Inhibition of MII blocked NGF stimulation, indicating the central role of restraint in directed outgrowth. The restraint consists of myosin IIB- and IIA-dependent processes: retrograde actin network flow and transverse actin bundling, respectively. The processes differentially contribute on laminin-1 and fibronectin due to selective actin tethering to adhesions. On laminin-1, NGF induced greater vinculin-dependent adhesion–cytoskeletal coupling, which slowed retrograde actin network flow (i.e., it regulated the molecular clutch). On fibronectin, NGF caused inactivation of myosin IIA, which negatively regulated actin bundling. On both substrates, the result was the same: NGF-induced weakening of MII-dependent restraint led to dynamic microtubules entering the actin-rich periphery more frequently, giving rise to faster elongation.     

Variation in number and differentiation of the abdominal infrared receptors in the Australian 'fire-beetle' Merimna atrata (Coleoptera, Buprestidae)

Most individuals of the Australian ‘fire-beetle’ Merimna atrata have two pairs of IR receptors which are located ventrolaterally on the second and third abdominal sternite. An IR receptor consists of a specialized IR absorbing area, which is innervated by a neural complex. This complex contains one thermoreceptive multipolar neuron with a unique terminal dendritic mass (TDM) and two scolopidia and was termed ‘sensory complex’. However, also individuals with one pair of IR receptors on the second sternite and beetles with three pairs on the second, third, and fourth sternites were found. Additionally, beetles having one or two pairs of IR receptors may have preliminary stages of IR receptors on the third and fourth sternite, respectively. We found two kinds of preliminary stages, both of which are characterized by a much less pronounced absorbing area. In all five abdominal sternites segmental nerves are attached to the cuticle with a neural complex. Investigation of complexes of non-IR sternites suggests that the sensory cells inside the sensory complex of an IR receptor have developed from common internal stretch receptors. From our results it can be hypothesized that the IR sensory system in Merimna atrata has not yet reached a stage, which can be regarded as evolutionary stable.     

Novel peptide inhibitors of angiotensin-converting enzyme 2

Angiotensin-converting enzyme 2 (ACE2), a recently identified human homolog of ACE, is a novel metallocarboxypeptidase with specificity, tissue distribution, and function distinct from those of ACE. ACE2 may play a unique role in the renin-angiotensin system and mediate cardiovascular and renal function. Here we report the discovery of ACE2 peptide inhibitors through selection of constrained peptide libraries displayed on phage. Six constrained peptide libraries were constructed and selected against FLAG-tagged ACE2 target. ACE2 peptide binders were identified and classified into five groups, based on their effects on ACE2 activity. Peptides from the first three classes exhibited none, weak, or moderate inhibition on ACE2. Peptides from the fourth class exhibited strong inhibition, with equilibrium inhibition constants (K(i) values) from 0.38 to 1.7 microm. Peptides from the fifth class exhibited very strong inhibition, with K(i) values < 0.14 microm. The most potent inhibitor, DX600, had a K(i) of 2.8 nm. Steady-state enzyme kinetic analysis showed that these potent ACE2 inhibitors exhibited a mixed competitive and non-competitive type of inhibition. They were not hydrolyzed by ACE2. Furthermore, they did not inhibit ACE activity, and thus were specific to ACE2. Finally, they also inhibited ACE2 activity toward its natural substrate angiotensin I, suggesting that they would be functional in vivo. As novel ACE2-specific peptide inhibitors, they should be useful in elucidation of ACE2 in vivo function, thus contributing to our better understanding of the biology of cardiovascular regulation. Our results also demonstrate that library selection by phage display technology can be a rapid and efficient way to discover potent and specific protease inhibitors.