Arg/Ser-rich (RS) proteins play a crucial role in splicing and are implicated in splice site selection in metazoa. In plants, intron recognition seems to differ from the one in animals due to specific factor requirements. Here we describe a new plant-specific RS-rich protein, atRSZ33, with a unique domain structure consisting of an RNA recognition motif (RRM), two zinc knuckles embedded in a basic RS region, and an acidic C-terminal domain. atRSZ33 was found to be a phosphoprotein that concentrates in nuclear speckles and is predominantly present in roots and flowers. In a yeast two-hybrid screen, atRSZ33 interacted with splicing factors atSRp34/SR1, an Arabidopsis ortholog of human SF2/ ASF; atRSZp21 and atRSZp22, which are similar to the human 9G8; and three novel SC35-like splicing factors termed atSCL28, atSCL30, and atSCL33/SR33. Two further members of the SCL family, namely SCL30a and the ortholog of mammalian SC35, atSC35, were also found to interact with atRSZ33. These interactions were verified by in vitro binding assays; furthermore, the transcriptional activity of atRSZ33 was found to overlap with the ones of its interacting partners. These specific interactions coupled with the many similarities of atRSZ33 to SR proteins suggest that its main activity is in spliceosome assembly. Mapping of regions necessary for protein-protein interaction between atRSZ33 and atSCL33/ SR33 revealed that both zinc knuckles together with a small part of the RS and the RRM domain are required for efficient binding. However, the interacting domain is relatively small, allowing binding of additional proteins, a feature that is consistent with the proposed role of atRSZ33 in spliceosome assembly.Eukaryotes have enlarged their protein-coding potential by the combinatorial removal of intervening sequences during the splicing process. Nuclear pre-mRNA processing takes place in the spliceosome, a large complex, consisting of five small nuclear ribonucleoproteins (U1, U2, U4/U6 and U5 snRNPs) 1 and numerous non-snRNP proteins. The precise excision of an intron is accomplished by complementary binding of U1 and U2 snRNP to the 5Ј and 3Ј splice sites, respectively. A network of proteins then assembles and brings the two splice sites into close proximity (reviewed in Refs. 1 and 2). The consecutive transesterification steps then generate the mature mRNA. The rearrangements necessary for these two steps make the spliceosome a dynamic particle with varying composition of structural proteins and enzymes (reviewed in Ref.3).Investigation of proteins involved in the splicing process has shown that many of them have a modular structure consisting of one or two RNA binding domains, which could be the RNA recognition motif (RRM), K homology domain, zinc knuckle domain, or RGG box, and auxiliary domains, which include Arg/Ser (RS)-rich regions. There is a growing family of RS proteins; the first ones identified were a group of proteins termed SR (serine/arginine) proteins, which contain one or two RRMs, and usually a C-terminal RS domain wi...

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Several recent studies have elucidated the structure of the mammalian LH/hCG receptor; as reported in the present work, we have developed a series of monoclonal antibodies (mAbs) against the rat ovarian LH/hCG receptor using highly purified receptor as immunogen and by screening hybridomas with purified LH/hCG receptors. The mAbs were able to specifically immunoprecipitate LH/hCG receptors from solubilized preparations of rat ovarian membranes as well as from partially purified preparations. Western blotting with mAb P1B4 detected a probable receptor dimer and a receptor fragment in rat and porcine ovarian tissue but not in other tissues. This mAb also partially inhibited hCG binding to rat and porcine ovarian tissues. The receptor mAbs were able to inhibit hCG-induced progesterone synthesis in cultured human and porcine granulosa cells without affecting cAMP- and FSH-induced progesterone synthesis. The mAb P1B4 was used to demonstrate that the majority of ovarian receptors are internalized after hCG treatment and that in pseudopregnant rats receptors are present in the rough endoplasmic reticulum and in microvesicles. Bovine corpus luteal cells also contained P1B4 binding sites, as detected by immunohistochemical technique. Taken together, these results suggest that the mAbs are specific for the LH/hCG receptor, mAb P1B4 recognizes an epitope that is highly conserved among mammals, and this epitope is probably in the extracellular domain.

The aim of this study was to investigate the effects of pomegranate (Punica granatum L., Punicaceae) seed extract on uterine contractility. Pomegranate seeds were methanolic extracted and their constituents analyzed using gas chromatography and mass spectrometry. Isometric force was measured in strips of longitudinal rat myometrium and the effects of pomegranate seed extract studied. We found beta-sitosterol to be the main constituent of the extract (16%) and its effects were also investigated. Pomegranate seed extract and beta-sitosterol increased spontaneous contractions in a concentration-dependent manner with a maximum effect at 250 mg/100 mL and 1 mg/100 mL, respectively. The amplitude and frequency of the phasic contraction were significantly increased along with basal tension. The effects of pomegranate seed extract were very similar to those of beta-sitosterol. Force produced in the presence of pomegranate seed extract was abolished by the inhibition of L-type calcium channels or myosin light chain kinase (MLCK). Contractions were not potentiated by pomegranate extract following the inhibition of K channels or inhibition of the sarcoplasmic reticulum calcium ATPase (SERCA). The actions of beta-sitosterol and the extract were not blocked by the estrogen receptor blocker, fulvestrant. We conclude that pomegranate seed extract is a potent stimulator of phasic activity in rat uterus. Our data suggest that the uterotonic effect is due to nonestrogenic effects of beta-sitosterol acting to inhibit K channels and SERCA and thereby increasing contraction via calcium entry on L-type calcium channels and MLCK. We suggest that pomegranate extract and beta-sitosterol may be a useful uterine stimulant.

Distinct luteinizing hormone receptor (LHR) protein variants exist due to the posttranslational modifications. Besides ovaries, LHR immunoreactivity (LHRI) was also found in other tissues, such as the brain, fallopian tube, endometrium, trophoblast and resident tissue macrophages. The 3B5 mouse monoclonal antibody was raised against purified rat LHR. In rat, porcine and human ovaries, the 3B5 identified six distinct LHR bands migrating at ~92, 80, 68, 59, 52 and 48 kDa. Characteristic LHRI was detected in rat, human and porcine corpora lutea. During cellular differentiation, subcellular LHR distribution changed from none to granular cytoplasmic, perinuclear, surface, nuclear and no staining. There were also differences in vascular LHR expression – lack of LHRI in ovarian vessels and strong staining of vessels in other tissues investigated. In normal human term placentae, villous LHRI was associated with blood sinusoids and cytotrophoblast cells, and rarely detected in trophoblastic syncytium. In all abnormal placentae, the LHRI of sinusoids was absent, and syncytium showed either enhanced (immature placental phenotypes) or no LHRI (aged placental phenotype). LHRI in human brain was identified in microglial cells (CD68+ resident macrophages). Protein extracts from human vaginal wall and levator ani muscle and fascia showed strong ~92 and 68 kDa species, and LHRI was detected in smooth muscle cells, fibroblasts, resident macrophages and nuclei of skeletal muscle fibers. Our observations indicate that, in contrast to the theory on the role of vascular hormone receptors in preferential pick up of circulating hormones, there is no need to enhance selective pick up rather only prevent LH/CG transport to inappropriate sites. Abnormal placental LHR expression may play a role in the development of abnormal pregnancy. Expression of LHR in the pelvic floor compartments suggests that high LH levels in postmenopausal women may contribute to the pelvic floor relaxation and increased incidence of pelvic floor disorders. Since chorionic gonadotropin increases secretion of a variety of cytokines by monocytes, and induces their inflammatory reaction and phagocytic activity, high LH levels in aging individuals may also activate microglia (mononuclear phagocyte system in the central nervous system) and contribute to the development of Alzheimer's disease and other inflammation-mediated neurodegenerative diseases.