Generation and Identification of Monoclonal Antibody Against Porcine Adipocyte Plasma Membrane Proteins

Production of monoclonal antibody against porcine adipocyte plasma membrane proteins to explore a new way of controlling body fat deposition and improving carcass quality is discussed in this article. Membrane proteins of pig adipocyte plasma membrane proteins were extracted with the help of sucrose density gradient centrifugation, and two kinds of proteins were obtained. The monoclonal antibody （designated 3B2 and 3F3） of IgG1 and IgG2b subclass against adipocyte membrane proteins were produced by immunization, with adipocyte membrane proteins as an antigen, and its titer was 1：105 detected by enzyme-linked immunoadsorbent assay （ELISA）. The cell strains were identified by analyzing the number of chromosomes, the heat stability, the acid and alkali, the types and subtypes of immnoglobulin, and its peculiarities and affinities. Through identification, the chromosome number of hybridoma cell strains was from 80 to 100 and the strains formed good hybridomas colonies. The strains＇ affinity constants were 4.63 × 10^9 and 3.75 × 10^9 （mol L^-1）-1, respectively. At the same time, the McAb secreted was stable to environmental factors, such as, temperature, acid, alkali and so on. The monoclonal antibodies had been obtained and their specificity to porcine adipocyte plasma membrane proteins had been identified.

Water relations and an expression analysis of plasma membrane intrinsic proteins in sensitive and tolerant rice during chilling and recovery

A symptom of chilling injury is development of water deficit in shoots, resulting from an imbalance of water transport and transpiration. In this work, two rice varieties （Oryza sativa L. var. Wasetoitsu and Somewake） seedlings were chilled at 7 ℃, followed by recovery at 28 ℃. Based on the growth phenotype and electrolyte leakage tests, Somewake was shown to be a chilling-tolerant variety, and Wasetoitsu a chilling-sensitive one. The chilling stress reduced markedly the relative water content （RWC） of leaves, accumulative transpiration and osmotic root hydraulic conductivity （Lp） in both varieties. But when retumed to 28 ℃, the water relation balance of Somewake recovered better. The mRNA expression profile of all the 11 plasma membrane intrinsic proteins （PIPs）, a subgroup of aquaporins, was subsequently determined by real-time reverse transcription （RT）-PCR with TaqMan-minor grove binder （MGB） probes derived from rice var. Nipponbare during chilling treatment and recovery. Most of the PIP genes was down-regulated at the low temperature, and recovered at the warm temperature. The relative expression of some PIPs in both Somewake and Wasetoitsu decreased in parallel during the chilling. However during the recovery, the relative expression of OsPIP1;1, OsPIP2;1, OsPIP2;7 in shoots and OsPIP1：1, OsPIP2：1 in roots were significantly higher in Somewake than Wasetoitsu. This supports the role of PIPs in re-establishing water balance after chilling conditions. We discuss the diversified roles played by members of the aquaporin PIP subfamily in plant chilling tolerance depending on aquaporin isoforms, plant tissue and the stage of chilling duration.

Norepinephrine transporter (NET) is expressed in cardiac sympathetic ganglia of adult rat

The sympathetic nervous system plays a cardinal role in regulating cardiac function through releasing the neurotransmitter norepinephrine (NE). In comparison with central nervous system, the molecular mechanism of NE uptake in myocardium is not clear. In present study, we proved that in rat the CNS type of NE transporter (NET) was also expressed in middle cervical-stellate ganglion complex (MC-SG complex) which is considered to control the activity of heart, but not expressed in myocardium. The results also showed that NET expression level in right ganglion was significantly higher than in the left, rendering the greater capacity of NE uptake in right ventricle, a fact which may contribute to the maintenance of right ventricular function under pathologic state.

GABA transporter 1 transcriptional starting site exhibiting tissue specific difference

GABA transporter 1(GAT1) takes important roles in multiple physiological processes through the uptake and release of GABA, but the regulation of GAT1 gene expression in different tissues is rarely known. To address the question, first, 5’ Rapid amplification of cDNA end (RACE) was used to determine GAT1 transcriptional starting sites in neonatal mouse cerebral cortex and intestine, adult mouse brain and adult rat testis. The products of 5’RACE were confirmed by DNA sequencing. We found that the transcript of GAT1 in neonatal mouse cerebral cortex and adult mouse brain starts at the same site (inside of exon 1), while in mouse intestine, GAT1 starts transcription in intron 1, and in rat testis, the transcript of GAT1 has an additional untranslation exon to the 5’ direction.

Identification and characterization of plasma membrane aquaporins isolated from fiber cells of Calotropis procera

Calotropis procera, commonly known as "milkweed", possesses long seed trichomes for seed dispersal and has the ability to survive under harsh conditions such as drought and salinity. Aquaporins are water channel proteins expressed in all land plants, divided into five subfamilies plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), NOD26-like proteins (NIPs), small basic intrinsic proteins (SIPs), and the unfamiliar X intrinsic proteins (XIPs). PIPs constitute the largest group of water channel proteins that are involved in different developmental and regulatory mechanisms including water permeability, cell elongation, and stomata opening. Aquaporins are also involved in abiotic stress tolerance and cell expansion mechanisms, but their role in seed trichomes (fiber cells) has never been investigated. A large number of clones isolated from C. procera fiber cDNA library showed sequence homology to PIPs. Both expressed sequence tags (ESTs) and real-time polymerase chain reaction (PCR) studies revealed that the transcript abundance of this gene family in fiber cells of C. procera is greater than that of cotton. Full-length cDNAs of CpPIP1 and CpPIP2 were isolated from C. procera fiber cDNA library and used for constructing plant expression vectors under constitutive (2×35S) and trichome-specific (GhLTP3) promoters. Transgenic tobacco plants were developed via Agrobacterium-mediated transformation. The phenotypic characteristics of the plants were observed after confirming the integration of transgene in plants. It was observed that CpPIP2 expression cassette under 2×35S and GhLTP3 promoter enhanced the numbers of stem and leave trichomes. However, 2×35S::CpPIP2 has a more amplified effect on trichome density and length than GhLTP3::CpPIP2 and other PIP constructs. These findings imply the role of C. procera PIP aquaporins in fiber cell elongation. The PIPs-derived cell expansion mechanism may be exploited through transgenic approaches for improvement of fiber staple length in cotton and boosting of defense against sucking insects by enhancing plant pubescence.

Enquiry into the Topology of Plasma Membrane- Localized PIN Auxin Transport Components

Auxin directs plant ontogenesis via differential accumulation within tissues depending largely on the activity of PIN proteins that mediate auxin efflux from cells and its directional cell-to-cell transport. Regard- less of the developmental importance of PINs, the structure of these transporters is poorly characterized. Here, we present experimental data concerning protein topology of plasma membrane-localized PINs. Utilizing approaches based on pH-dependent quenching of fluorescent reporters combined with immuno- localization techniques, we mapped the membrane topology of PINs and further cross-validated our results using available topology modeling software. We delineated the topology of PIN1 with two transmembrane （TM） bundles of five m-helices linked by a large intracellular loop and a C-terminus positioned outside the cytoplasm. Using constraints derived from our experimental data, we also provide an updated position of helical regions generating a verisimilitude model of PIN1. Since the canonical long PINs show a high degree of conservation in TM domains and auxin transport capacity has been demonstrated for Arabidopsis representatives of this group, this empirically enhanced topological model of PIN1 will be an important starting point for further studies on PIN structure-function relationships. In addition, we have established protocols that can be used to probe the topology of other plasma membrane proteins in plants.

Molecular Cloning and Sequence Analysis of an Aquaporin Gene Related to Tapping Panel Dryness in Rubber Tree( Hevea brasiliensis Muell. Arg. )

As a syndrome with tapping incision blocked partly or entirely during latex exploiting in rubber tree （Hevea brasiliensis ）, ＂Tapping panel dryness （TPD）＂ causes great yield losses, thereby becoming the most important factor limiting rubber production. On the basis of an EST down-regulated in TPD-affected rubber trees, a 903 bp cDNA denoted HbPIP2;2 was isolated from the bark tissue with a combination of in silico cloning and RT-PCR. The cDNA contains an 867 hp ORF, 13 bp 5＇UTR and 23 bp 3＇ UTR. Sequence analysis indicated that HbPIP2;2 encodes 288 amino acids with a theoretical molecular weight （Mw） of 30. 71 kDa and isolectric point （Pi） of 8.20. Bioinformatics analysis suggested that the deduced protein is predicted to have six transmembrane helices located to the plasma membrane and harbor one conserved MIP domain that can be grouped into plasma membrane intrinsic proteins （PIPs） of aquaporin （AQP） family. Homolo- gy search revealed that the protein shares a similarity of more than 90% with the homologues in Ricinus communis, Popttlus trichocarpa, Juglans regia and Theobro- ma cacao, suooorting a hie.hly conserved evolution. This study provided basis for further uncovering the regulatory role of AOPs in TPD occurrence.

Activation effect of extracellular calmodulin on heterotrimeric G protein in pollen plasma membrane

IN recent years, calmodulin （CaM）, an important Ca2+ receptor and constituent of cellular signal transduction systems, has been found extracellularly. We have verified that CaM is presented extracellularly in all of plant species we have examined. In addition, we have reported that extracellular CaM has some biological significance, such as stimulation of cell proliferation, cell wall regeneration, initiation of pollen germination and tube growth and inducement of rbcS gene expression. The role of heterotrimeric G proteins in pollen germination, tube growth and signal transduction of extracellular CaM has been examined in Lily pollen, and two kinds of antibodies against animal Gzα internal sequence and N-terminal

Retinal dopamine transporter in experimental myopia

OBJECTIVE: To investigate the distribution, changes and a possible role for retinal dopamine transporter (DAT) in experimental myopia in chickens. METHODS: Two-day-old chickens were divided into four groups. Chicken eyes were fitted with lenses of -10D,-20D and translucent goggles unilaterally. Normal eyes were used as controls. After 3 wk, all chickens were given an intramuscular injection of (125)I-beta-CIT 2beta-carbomethoxy-3beta-(4-iodophenyl)tropane and sacrificed two hours post injection. Retinal pigment epithelium (RPE) and the neural retina were obtained together or RPE was dissected out from the neural retina. Radioactive DAT from each specimen was assayed by gamma-counter. RESULTS: Retinal DAT was detected in RPE specimens rather than in the neural retina in all eyes. Radioactive DAT in myopic eyes was higher, compared with control eyes. CONCLUSIONS: Retinal DAT is mainly located in the RPE and may be involved in the formation of lens induced myopia (LIM) and form deprivation myopia (FDM). These methods may provide a new approach for further studying the role of the dopamine system in experimental myopia.

GEX3, Expressed in the Male Gametophyte and in the Egg Cell of Arabidopsisthaliana, Is Essential for Micropylar Pollen Tube Guidance and Plays a Role during Early Embryogenesis

Double fertilization in flowering plants occurs when the two sperm cells, carried by the pollen tube, are released in a synergid cell of the embryo sac and then fertilize the egg and the central cell. Proteins on the surfaces of the sperm, egg, central, and synergid cells might be important for guidance and recognition/fusion of the gametes. Here, we present functional analyses ofArabidopsis GEX3, which encodes a plasma membrane-localized protein that has homologs in other plants. GEX3 is expressed in both the vegetative and sperm cells of the male gametophyte and in the egg cell of the female gametophyte. Transgenic lines in which GEX3 was down-regulated or overexpressed, using the Arabidopsis GEX2 promoter, had reduced seed set. Reciprocal crosses and imaging after pollination with a reporter line showed that, in both cases, the defect causing reduced seed set occurred in the female. In the antisense lines, micropylar pollen tube guidance failed, In the overexpression lines, fertilization of mutant ovules was mostly blocked because pollen tube guidance failed, although, occasionally, non-viable embryos were formed. We conclude that properly regulated expression of GEX3 in the egg cell of Arabidopsis is essential for pollen tube guidance.

Chaihushugan decoction exerts antiepileptic effects by increasing hippocampal glutamate metabolism in pentylenetetrazole-kindled rats

OBJECTIVE: To investigate the antiepileptic effects of Chaihushugan decoction(CHSGD) in rats with pentylenetetrazole(PTZ)-induced seizures and to discuss the impact of CHSGD on glutamate metabolism, a hypothesized underlying mechanism of seizure reduction.METHODS: Fifty Wistar rats were divided randomly into either control(n = 10) or experimental(n = 40)groups. Rats in the control group were administered physiological saline intraperitoneally. A subconvulsive dose of PTZ(35 mg/kg) was administered intraperitoneally to rats in the experimental group to induce seizures. The fully PTZ-kindled rats were then randomly divided into five subgroups(n = 8 each) based on the following treatment categories: physiological saline, VPA(200 mg/kg), CHSGD(2.5 g/kg), CHSGD(5 g/kg), or CHSGD(10 g/kg),administered orally once per day, respectively. On day 28 following initiation of drug treatment, seizures were monitored. The rats were then sacrificed, and hippocampal dissections were performed for subsequent studies.RESULTS: CHSGD significantly prolonged the latency of myoclonic, clonic, and tonic seizures, while decreasing overall seizure rates in the kindled rats.The measured concentrations of 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose(2-NBDG) and glutamate were significantly lower in the hippocampi of kindled rats in groups treated with CHSGD compared with those treated with PTZ alone. In addition, CHSGD was found to up-regulate both the expression of glutamate transporter-1(GLT-1) protein and the activity of glutamine synthetase(GS) in the hippocampi of kindled rats.CONCLUSION: These results suggest that CHSGD has antiepileptic effects on PTZ-induced seizures.The results further suggest an increase in glutamate metabolism at the synaptic cleft is a putative underlying mechanism of seizure reduction.

TOLs Function as Ubiquitin Receptors in the Early Steps of the ESCRT Pathway in Higher Plants

Protein abundance and localization at the plasma membrane(PM)shapes plant development and mediates adaptation to changing environmental conditions.It is regulated by ubiquitination,a post-translational modification crucial for the proper sorting of endocytosed PM proteins to the vacuole for subsequent degradation.To understand the significance and the variety of roles played by this reversible modification,the function of ubiquitin receptors,which translate the ubiquitin signature into a cellular response,needs to be elucidated.In this study,we show that TOL(TOM1-like)proteins function in plants as multivalent ubiquitin receptors,governing ubiquitinated cargo delivery to the vacuole via the conserved Endosomal Sorting Complex Required for Transport(ESCRT)pathway.TOL2 and TOL6 interact with components of the ESCRT machinery and bind to K63-linked ubiquitin via two tandemly arranged conserved ubiquitin-binding domains.Mutation of these domains results not only in a loss of ubiquitin binding but also altered localization,abolishing TOL6 ubiquitin receptor activity.Function and localization of TOL6 is itself regulated by ubiquitination,whereby TOL6 ubiquitination potentially modulates degradation of PM-localized cargoes,assisting in the fine-tuning of the delicate interplay between protein recycling and downregulation.Taken together,our findings demonstrate the function and regulation of a ubiquitin receptor that mediates vacuolar degradation of PM proteins in higher plants.