Effects of permeant ion concentrations on the gating of L-type Ca2+ channels in hair cells

We determined the gating and permeation properties of single L-type Ca(2+) channels, using hair cells and varying concentrations (5-70 mM) of the charge carriers Ba(2+) and Ca(2+). The channels showed distinct gating modes with high- and low-open probability. The half-activation voltage (V(1/2)) shifted in the hyperpolarizing direction from high to low permeant ion concentrations consistent with charge screening effects. However, the differences in the slope of the voltage shifts (in VM(-1)) between Ca(2+) (0.23) and Ba(2+) (0.13), suggest that channel-ion interaction may also contribute to the gating of the channel. We examined the effect of mixtures of Ba(2+) and Ca(2+) on the activation curve. In 5 mM Ca(2+), the V(1/2) was, -26.4 +/- 2.0 mV compared to Ba(2+), -34.7 +/- 2.9 mV, as the charge carrier. However, addition of 1 mM Ba(2+) in 4 mM Ca(2+), a molar ratio, which yielded an anomalous-mole fraction effect, was sufficient to shift the V(1/2) to -34.7 +/- 1.5 mV. Although Ca(2+)-dependent inactivation of the L-type channels in hair cells can yield the present findings, we provide evidence that the anomalous gating of the channel may stem from the closed interaction between ion permeation and gating.     

Determination of the ovulatory mechanism of the grasscutter (Thryonomys swinderianus)

The objective of this study was to determine whether gonadotrophin-releasing hormone (GnRH), oxytocin (OT) and vasoactive intestinal polypeptide (VIP) modulate beta-endorphin-like immunoreactivity (beta-END-LI) secretion by dispersed anterior pituitary cells of pigs and in vivo priming with steroid hormones, estradiol benzoate (EB) and progesterone (P(4)), influences the cell reactivity to peptide hormones tested. Additionally, the aim of this research was to examine the involvement of cyclic nucleotides (cAMP and cGMP) in transduction of signals induced by GnRH, OT and VIP in porcine pituitary cells. Pituitaries were collected from ovariectomized (OVX) gilts that were divided into four experimental groups. Animals of group 1 (OVX) received 1ml corn oil (placebo)/100 kg body weight (b.w.), group 2 (OVX+EB I) and group 3 (OVX+EB II) were treated with EB at the dose 2.5mg/100 kg b.w., 30-36 and 60-66 h before slaughter, respectively. Animals of group 4 (OVX+P(4)) were injected with P(4) at the dose 120 mg/100 kg b.w. for 5 subsequent days before slaughter. Anterior pituitaries were dispersed with trypsin and then pituitary cells were cultured (10(6) per well) in McCoy's 5A medium containing horse serum (10%) and fetal calf serum (2.5%) for 3 days at 37 degrees C under an atmosphere of 95% air and 5% CO(2). Subsequently, plates were rinsed with fresh McCoy's 5A medium and pituitary cells were treated with one of the following agents: GnRH (100 ng/ml), OT (10(-6)M) or VIP (10(-7)M) and incubated for 3.5h at 37 degrees C.GnRH did not affect beta-END-LI secretion by pituitary cells of OVX (group 1) and OVX+P(4) (group 4) gilts. When the pituitary cells were incubated in the presence of OT and VIP, significant increases were observed. After priming of OVX gilts with EB, 30-36 h before slaughter (group 2), we noted a significant increase in beta-END-LI release from pituitary cells only in the presence of VIP. Pituitary cells from gilts treated with EB, 60-66 h before slaughter (group 3), produced markedly elevated amounts of beta-END-LI after GnRH, OT or VIP addition.GnRH markedly stimulated cGMP release from cultured pituitary cells in all experimental groups and significantly increased cAMP production by the cells from OVX, OVX+EB II and OVX+P(4) animals. The addition of OT enhanced both cAMP and cGMP output in all experimental groups of pigs. VIP stimulated cAMP release from pituitary cells derived from OVX, OVX+EB I and OVX+EB II animals. cGMP output was markedly elevated under the influence of VIP from pituitary cells of OVX, OVX+EB II and OVX+P(4) gilts.In conclusion, our results suggest that GnRH, OT and VIP can modulate beta-endorphin release from porcine pituitary cells and imply the involvement of cAMP and cGMP in transduction of signals induced by studied peptides in the cells.     

Construction of 2 intraspecific linkage maps and identification of resistance QTLs for Phytophthora capsici root-rot and foliar-blight diseases of pepper (Capsicum annuum L.).

Two linkage maps of pepper were constructed and used to identify quantitative trait loci (QTLs) conferring resistance to Phytophthora capsici. Inoculations were done with 7 isolates: 3 from Taiwan, 3 from California, and 1 from New Mexico. The first map was constructed from a set of recombinant inbred lines (RILs) of the PSP-11 (susceptible) x PI201234 (resistant) cross; and the second map was from a set of F(2) lines of the Joe E. Parker' (susceptible) x 'Criollo de Morelos 334' (resistant) cross. The RIL map covered 1466.1 cM of the pepper genome, and it consisted of 144 markers -- 91 amplified fragment length polymorphisms (AFLPs), 34 random amplified polymorphic DNA (RAPDs), 15 simple sequence repeats (SSRs), 1 sequence characterized amplified region (SCAR), and 3 morphological markers -- distributed over 17 linkage groups. The morphological markers mapped on this population were erect fruit habit (up), elongated fruit shape (fs(e)), and fasciculate fruit clusters (fa). The F(2) map consisted of 113 markers (51 AFLPs, 45 RAPDs, 14 SSRs, and 3 SCARs) distributed in 16 linkage groups, covering a total of 1089.2 cM of the pepper genome. Resistance to both root rot and foliar blight were evaluated in the RIL population using the 3 Taiwan isolates; the remaining isolates were used for the root-rot test only. Sixteen chromosomal regions of the RIL map contained single QTLs or clusters of resistance QTLs that had an effect on root rot and (or) foliar blight, revealing a complex set of genetics involved in resistance to P. capsici. Five QTLs were detected in the F(2) map that had an effect on resistance to root rot.     

Involvement of a Plasmid in Production of Ropiness (Mucoidness) in Milk Cultures by Streptococcus cremoris MS

Curing and genetic transfer experiments showed that lactose-fermenting ability (Lac⁺) and the ability to produce mucoidness in milk cultures (Muc⁺) in Streptococcus cremoris MS were coded on plasmids. The Lac⁺ phenotype was associated with a 75.8-megadalton plasmid, pSRQ2201. The Muc⁺ phenotype was associated with a 18.5-megadalton plasmid, pSRQ2202. The Lac plasmid, pSRQ2201, was first conjugatively transferred from S. cremoris MS to Lac⁻S. lactis ML-3/2.2. Later, the Muc plasmid, pSRQ2202, was conjugatively transferred from Lac⁻ Muc⁺S. cremoris MS04 to Lac⁺ nonmucoid S. lactis transconjugant ML-3/2.201. Subsequently, pSRQ2201 and pSRQ2202 were cotransferred from Lac⁺ Muc⁺S. lactis transconjugant ML-3/2.202 to Lac⁻, nonmucoid, malty S. lactis 4/4.2 and S. lactis subsp. diacetylactis SLA3.25. Transconjugants showing pSRQ2201 were Lac⁺; those containing pSRQ2202 were Muc⁺. With the transfer of pSRQ2202, the transconjugants S. lactis ML-3/2.202 and S. lactis subsp. diacetylactis SLA3.2501 not only acquired the Muc⁺ phenotype but also resistance to bacteriophages, which were lytic to the respective parent strains S. lactis ML-3/2.201 and S. lactis subsp. diacetylactis SLA3.25.     

Archeal lectins: An identification through a genomic search

Forty‐six lectin domains which have homologues among well established eukaryotic and bacterial lectins of known three‐dimensional structure, have been identified through a search of 165 archeal genomes using a multipronged approach involving domain recognition, sequence search and analysis of binding sites. Twenty‐one of them have the 7‐bladed β‐propeller lectin fold while 16 have the β‐trefoil fold and 7 the legume lectin fold. The remainder assumes the C‐type lectin, the β‐prism I and the tachylectin folds. Acceptable models of almost all of them could be generated using the appropriate lectins of known three‐dimensional structure as templates, with binding sites at one or more expected locations. The work represents the first comprehensive bioinformatic study of archeal lectins. The presence of lectins with the same fold in all domains of life indicates their ancient origin well before the divergence of the three branches. Further work is necessary to identify archeal lectins which have no homologues among eukaryotic and bacterial species. Proteins 2016; 84:21–30. © 2015 Wiley Periodicals, Inc.     

AbiQ, an Abortive Infection Mechanism from Lactococcus lactis

Lactococcus lactis W-37 is highly resistant to phage infection. The cryptic plasmids from this strain were coelectroporated, along with the shuttle vector pSA3, into the plasmid-free host L. lactis LM0230. In addition to pSA3, erythromycin- and phage-resistant isolates carried pSRQ900, an 11-kb plasmid from L. lactis W-37. This plasmid made the host bacteria highly resistant (efficiency of plaquing <10⁻⁸) to c2- and 936-like phages. pSRQ900 did not confer any resistance to phages of the P335 species. Adsorption, cell survival, and endonucleolytic activity assays showed that pSRQ900 encodes an abortive infection mechanism. The phage resistance mechanism is limited to a 2.2-kb EcoRV/BclI fragment. Sequence analysis of this fragment revealed a complete open reading frame (abiQ), which encodes a putative protein of 183 amino acids. A frameshift mutation within abiQ completely abolished the resistant phenotype. The predicted peptide has a high content of positively charged residues (pI = 10.5) and is, in all likelihood, a cytosolic protein. AbiQ has no homology to known or deduced proteins in the databases. DNA replication assays showed that phage c21 (c2-like) and phage p2 (936-like) can still replicate in cells harboring AbiQ. However, phage DNA accumulated in its concatenated form in the infected AbiQ⁺ cells, whereas the AbiQ⁻ cells contained processed (mature) phage DNA in addition to the concatenated form. The production of the major capsid protein of phage c21 was not hindered in the cells harboring AbiQ.     

Silencing of H4R inhibits the production of IL-1β through SAPK/JNK signaling in human mast cells

Context: Mast cell (MC) activation through H4R releases various inflammatory mediators which are associated with allergic asthma.

Objectives: To investigate the siRNA-mediated gene silencing effect of H4R on human mast cells (HMCs) functions and the activation of stress-activated protein kinases (SAPK)/jun amino-terminal kinases (JNK) signaling pathways for the release of ineterleukin-1β (IL-1β) in HMCs.

Materials and methods: H4R expression was analyzed by RT-PCR and western blotting in human mast cell line-1 (HMC-1) cells and H4RsiRNA transfected cells. The effect of H4RsiRNA and H4R-antagonist on H4R mediated MC functions such as intracellular Ca²⁺ release, degranulation, IL-6 and IL-1β release, and the activation SAPK/JNK signaling pathways were studied. HMC-1 cells were stimulated with 10?μM of histamine (His) and 4-methylhistamine (4-MH) and pretreated individually with H4R-antagonist JNJ7777120 (JNJ), histamine H1 receptor (H1R)-antagonist mepyramine, and signaling molecule inhibitors SP600125 (SP) and Bay117082.

Results: We found that the HMC-1 cells expressed H4R and H4RsiRNA treatment down regulated the H4R expression in HMC-1 cells. Both His and 4-MH induced the intracellular Ca²⁺ release and degranulation whereas; H4R siRNA and JNJ inhibited the effect. Furthermore, the activation of H4R caused the phosphorylation of SAPK/JNK pathways. H4R gene silencing and pretreatment with SP and JNJ decreased His and 4-MH induced phosphorylation of SAPK/JNK. We found that the activation of H4R caused the release of IL-1β (124.22?pg/ml) and IL-6 (122.50?pg/ml) on HMC-1 cells. Whereas, SAPK/JNK inhibitor (68.36?pg/ml) inhibited the H4R mediated IL-1β release.

Conclusions: Taken together, the silencing of H4R inhibited the H4R mediated MC functions and SAPK/JNK phosphorylation. Furthermore, the H4R activation utilized SAPK/JNK signaling pathway for IL-1β release in HMC-1 cells.     

Carbon stocks in the Guinea savanna of Ghana: estimates from three protected areas

Savannas are widespread in sub‐Saharan Africa (SSA) and play a major role in the global carbon balance. Extensive quantification of savanna carbon stocks in SSA will therefore contribute to better accounting of the global carbon budget in the era of climate change. In this study, we investigated the spatial distribution of carbon stocks of different soil fractions and aboveground biomass within three forest reserves in the Guinea savanna zone of Ghana. Soil carbon stocks (SCSs) ranged from 4.80 to 12.61 Mg C/ha in surface soils (0–10 cm depth). Higher SCSs were associated with the silt +clay fraction than microaggregates and small macroaggregates in all three reserves. Relative to the dominant tree species (Vitellaria paradoxa), the highest SCSs were recorded under the sub‐canopy (SC), drip line (DL), and interspace (2 * SC + DL) zones for the Klupene, Sinsablegbinni, and Kenikeni forest reserves, respectively. The highest tree carbon stock was 60.01 Mg C/ha in Kenikeni. Sinsablegbinni had an average stock of 26.74 Mg C/ha and had the highest tree density. Average carbon capture by a single tree ranged from 0.04 to 0.34 Mg C. Aboveground grass carbon stock ranged from 0.08 to 0.47 Mg C/ha, while the belowground carbon stock ranged from 0.03 to 0.44 Mg C/ha. Accumulation of carbon in the aboveground grass biomass was greater at Klupene with low forest cover.     

Lack of genetic and cytogenetic effects in mice fed on irradiated wheat.

Experiments were conducted to test the mutagenicity of wheat irradiated with 20 and 200 krad gamma-rays by feeding male and female mice for various periods starting from weaning time. The results obtained from dominant lethal tests, specific locus mutation test, studies on chromosome rearrangements in males and gonadal cell survival studies indicated no positive evidence for genetic and cytogenetic effects.     

Efficient TDZ-induced regeneration from capitulum explants of Gerbera jamesonii Bolus ex Hooker F. - an ornamental plant with high aesthetic value

Gerbera jamesonii Bolus ex Hooker F. (Asteraceae), a most prominent ornamental plant ranking fifth in the world cut flower market has gained huge demand across the globe for its high aesthetic value. In the present study, an efficient plant regeneration protocol was developed using capitulum explants from the field grown mature plants of G. jamesonii. A successful surface sterilization procedure was established using a series of sterilants without affecting explant regenerability. The culture of capitulum explants on Pre-culture Induction Medium [(PIM), Murashige and Skoog (MS) medium fortified with 0.5?mg/L thidiazuron (TDZ) in combination with low levels of auxins] for a period of 2 weeks in dark conditions was crucial for inducing morphogenetic response. Upon transfer onto Shoot Regeneration Medium [(SRM), MS +4.0?mg/L 6-benzyladenine (BA) and 0.2?mg/L NAA], explants has significantly produced highest number of shoots (16?±?0.33). Incubation for 18–21?days on Shoot Multiplication and Elongation Medium [(SMEM), MS +2.0?mg/L BA +0.2?mg/L NAA] resulted in the highest number of multiple shoots (22.00?±?0.93) with increased mean shoot length (3.49?±?0.08). In vitro elongated shoots were rooted (95%) on ½ MS +1.0?mg/L indole-3-acetic acid (IAA) within 10–12?days and fully rooted plants were transferred to poly-house for hardening with 90% survival rate.