Proteomic characterization of the small subunit of Chlamydomonas reinhardtii chloroplast ribosome: identification of a novel S1 domain-containing protein and unusually large orthologs of bacterial S2, S3, and S5

To understand how chloroplast mRNAs are translated into functional proteins, a detailed understanding of all of the components of chloroplast translation is needed. To this end, we performed a proteomic analysis of the plastid ribosomal proteins in the small subunit of the chloroplast ribosome from the green alga Chlamydomonas reinhardtii. Twenty proteins were identified, including orthologs of Escherichia coli S1, S2, S3, S4, S5, S6, S7, S9, S10, S12, S13, S14, S15, S16, S17, S18, S19, S20, and S21 and a homolog of spinach plastid-specific ribosomal protein-3 (PSRP-3). In addition, a novel S1 domain-containing protein, PSRP-7, was identified. Among the identified proteins, S2 (57 kD), S3 (76 kD), and S5 (84 kD) are prominently larger than their E. coli or spinach counterparts, containing N-terminal extensions (S2 and S5) or insertion sequence (S3). Structural predictions based on the crystal structure of the bacterial 30S subunit suggest that the additional domains of S2, S3, and S5 are located adjacent to each other on the solvent side near the binding site of the S1 protein. These additional domains may interact with the S1 protein and PSRP-7 to function in aspects of mRNA recognition and translation initiation that are unique to the Chlamydomonas chloroplast.     

Effects of dehydration on renal aminopeptidase activities in adult male and female rats

Orexin A injected into the lateral hypothalamus (LH) stimulates feeding and activates neurons in brain sites regulating feeding and arousal. The feeding effects of orexin A have been demonstrated during the light cycle, a time when rats are normally resting, and the effect of orexin A on activity after injection into the LH has not been previously measured. Thus, it is unclear whether LH orexin A-induced feeding is secondary to enhanced arousal. To address this, LH-cannulated rats habituated to a running wheel were injected with either orexin A (1000 pmol) or vehicle during light and dark cycles. Food intake and running wheel rotations were measured for 2 h. Spontaneous physical activity (SPA) was also measured during the dark cycle. During the light cycle, orexin A in the LH stimulated feeding in the presence and absence of a running wheel and increased number of running wheel rotations in the presence and absence of food. During the dark cycle, orexin A in the LH induced SPA (+/- presence of food), but had no effect on feeding. These data show that LH orexin A stimulation of feeding is not always coincident with increased activity, suggesting that feeding induced by LH-injected orexin A is not consequent to enhanced arousal.     

Expression and distribution of pituitary adenylate cyclase-activating peptide in human prostate and prostate cancer tissues

The therapeutic potential of the intestinotrophic mediator glucagon-like peptide-2 (1-33) [GLP-2 (1-33)] has increased interest in the pharmacokinetics of the peptide. This study was undertaken to investigate whether the primary degradation product GLP-2 (3-33) interacts with the GLP-2 receptor. Functional (cAMP) and binding in vitro studies were carried out in cells expressing the transfected human GLP-2 receptor. Furthermore, a biologic response of GLP-2 (3-33) was tested in vivo. Mice were allocated to groups treated for 10 days (twice daily) with: (1) 5 microg GLP-2 (1-33), (2) 25 microg GLP-2 (3-33), (3) 5 microg GLP-2 (1-33)+100 microg GLP-2 (3-33), or (4) 5 microg GLP-2 (1-33)+500 microg GLP-2 (3-33). The intestine was investigated for growth changes. GLP-2 (3-33) bound to the GLP-2 receptor with a binding affinity of 7.5% of that of GLP-2 (1-33). cAMP accumulation was stimulated with an efficacy of 15% and a potency more than two orders of magnitude lower than that of GLP-2 (1-33). Increasing doses of GLP-2 (3-33) (10(-7)-10(-5) M) caused a shift to the right in the dose-response curve of GLP-2 (1-33). Treatment of mice with either GLP-2 (1-33) or (3-33) induced significant growth responses in both the small and large intestines, but the response induced by GLP-2 (3-33) was much smaller. Co-administration of 500 microg of GLP-2 (3-33) and 5 microg GLP-2 (1-33) resulted in a growth response that was smaller than that of 5 microg GLP-2 (1-33) alone. Consistent with the observed in vivo activities, our functional studies and binding data indicate that GLP-2 (3-33) acts as a partial agonist with potential competitive antagonistic properties on the GLP-2 receptor.     

Apoptotic rate: a new indicator for the quantification of the incidence of apoptosis in cell cultures

BACKGROUND: Late apoptotic cells divide into apoptotic bodies and are missed by current detection methods. This results in an artificially low apoptotic index (AI). METHODS: This study proposes a flow cytometry-based ratiometric method that uses an internal reference standard of microbeads combined with fluorescein-annexin V binding and 7-aminoactinomycin D to enumerate viable, necrotic, and early and late apoptotic cells within specific subsets of a heterogeneous culture. RESULTS: In the absence of cell growth, the number of apoptotic cells that undergo fragmentation into apoptotic bodies in culture can also be determined accurately by this method. This information can then be used to obtain the apoptotic rate (AR), a new indicator of apoptosis that calculates the proportion of cells that have undergone apoptosis with respect to the total number of seeded cells. The main limitation of the method is that the AR is only suitable for the study of apoptosis in noncycling cells. CONCLUSIONS: This study reveals the superiority of the proposed method over the widely used Nicoletti method and current annexin-V binding methods. The AI did not reflect the true incidence of lymphocyte apoptosis, neither in response to lectins or phorbol esters, nor to serum deprivation. AR was more sensitive than AI, detecting apoptosis at lower concentrations of cell death inducers in all the subsets studied.     

Altered proteinogram in short term portal vein stenosed rats

The electrophoretic pattern of serum proteins has been studied in short-term prehepatic portal hypertensive rats since atrophy is produced in the liver, which is the main origin of most of these proteins, during this postoperative period. After 28 days of evolution, rats (n = 9) with triple stenosing ligated portal vein showed hypoalbuminemia, hypo-alpha-globulinemia, hyper-alpha2-globulinemia and hyper-gamma-globulinemia, the albumin/globulin ratio decreased with respect to the control animals (n = 8). These alterations are associated with hepatic atrophy, portosystemic and portohepatic (44.4%) collateral circulation. The proteinogram alterations found in rats with short-term prehepatic portal hypertension suggest that hepatic failure exists in spite of potential portohepatic revascularization which is frequently originated by the development of portohepatic collateral circulation.     

Temporal localization of porcine reproductive and respiratory syndrome virus in reproductive tissues of experimentally infected boars

Porcine reproductive and respiratory syndrome virus (PRRSV) has been reported to be shed in the semen of infected boars. To determine whether the reproductive tissues could be a persistent source of virus and the possible origin of PRRSV found in semen of infected boars, 20 PRRSV-seronegative boars were intranasally inoculated with 5 x 10(6) median tissue culture infective doses (TCID50) of PRRSV and necropsied at different times post-inoculation (p.i.) from Day 2 to Day 37 p.i. Blood samples were collected before experimental inoculation, at necropsy and at different times p.i. At necropsy, epididymal semen and reproductive tissues were collected and the presence of the virus determined by virus isolation. The infection of the boars was demonstrated by the isolation of the virus from the sera of all inoculated boars and by seroconversion. PRRSV was detected in serum samples from Day 2 to Day 23 p.i., although the viremic period was largely dependent on the individual response to infection. Viral replication was proven within different reproductive tissues from Day 2 to Day 23 p.i., being most consistently found in the epididymus. In addition, PRRSV was isolated in semen from Day 4 to Day 10 p.i. The correlation of a diminished viremia and the inability to isolate PRRSV from semen or reproductive tissues may be due to one of two possibilities. First, viremia is responsible for most of the virus isolated from reproductive tissues due to the movement of PRRSV-infected cells out of the blood and into the tissues. Second, viremia may initially seed the reproductive tissues with PRRSV, and then the virus is produced into the reproductive tract and shed into semen at low levels.     

Characterization of the low molecular weight human serum proteome

Serum potentially carries an archive of important histological information whose determination could serve to improve early disease detection. The analysis of serum, however, is analytically challenging due to the high dynamic concentration range of constituent protein/peptide species, necessitating extensive fractionation prior to mass spectrometric analyses. The low molecular weight (LMW) serum proteome is that protein/peptide fraction from which high molecular weight proteins, such as albumin, immunoglobulins, transferrin, and lipoproteins, have been removed. This LMW fraction is made up of several classes of physiologically important proteins such as cytokines, chemokines, peptide hormones, as well as proteolytic fragments of larger proteins. Centrifugal ultrafiltration of serum was used to remove the large constituent proteins resulting in the enrichment of the LMW proteins/peptides. Because albumin is known to bind and transport small molecules and peptides within the circulatory system, the centrifugal ultrafiltration was conducted under solvent conditions effecting the disruption of protein-protein interactions. The LMW serum proteome sample was digested with trypsin, fractionated by strong cation exchange chromatography, and analyzed by microcapillary reversed-phase liquid chromatography coupled on-line with electrospray ionization tandem mass spectrometry. Analysis of the tandem mass spectra resulted in the identification of over 340 human serum proteins; however, not a single peptide from serum albumin was observed. The large number of proteins identified demonstrates the efficacy of this method for the removal of large abundant proteins and the enrichment of the LMW serum proteome.     

Mitochondrial,sarcoplasmic membrane integrity and protein degradation in heart and skeletal muscle in exercised rats

Several different exercise regimens varied in the severity of tissue damage induced. Therefore, this study investigated the effects of a single bout of exercise versus endurance training in heart and skeletal muscles with different predominant fiber types on indices of mitochondrial, endoplasmic reticulum (ER) integrity and protein degradation. Male Wistar rats performed different treadmill exercise protocols: exhaustive, maximal exhaustive, eccentric, training and exhaustive exercise after training. The maximal and eccentric exercises resulted in a significant loss of integrity of the sarcoplasmic and ER muscle, while no changes were observed in cardiac muscle. Mitochondrial membrane fluidity measured by the fluorescence polarization method was significantly increased post-acute exercises in heart and oxidative muscles. Regular exercise can stabilize and preserve the viscoelastic nature of mitochondrial membranes in both tissues. The highest increase in carbonyl content was obtained in heart after exhaustive exercise protocol, from 1+/-0.1 to 3.6+/-0.14 nmol mg protein(-1), such increase were not found after regular exercise with values significantly decreased. Nitrate heart levels showed attenuated generation of nitric oxide after training. Muscle protein oxidation was produced in all exhaustive exercises including eccentric exercise.     

Ginseng increases intestinal elimination of albendazole sulfoxide in the rat

Herbal products show potential drug interactions, some of them with adverse effects. The main aim of this work was to study the effect of Panax ginseng on the intestinal elimination of the benzimidazole derivative albendazole sulfoxide (ABZSO). An upper small intestine segment was isolated and perfused in situ with saline, while ABZSO solution (10 mg/kg i.v.) was administered intravenously. Blood samples and intestinal secretion were collected over 60 min and analysed by HPLC. The intestinal clearance of ABZSO was 0.106+/-0.010 ml/min. Systemic co-administration of ginseng (10 mg/kg i.v.) increased significantly (P<0.05) the clearance of ABZSO (0.132+/-0.005 ml/min). The increase in ABZSO elimination could be the result of the effect of ginseng on metabolic pathways. These results highlight the interactions between herbal products (sometimes dietary constituents) and drugs such as benzimidazoles, since ginseng modifies the luminal clearance of this anthelminthic drug and could potentially interfere with drugs that undergo the same intestinal processes.     

VIP and PACAP receptors coupled to adenylyl cyclase in human lung cancer: a study in biopsy specimens

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are important neuropeptides in the control of lung physiology. Both of these commonly bind to specific G protein coupled receptors named VPAC(1)-R and VPAC(2)-R, and PAC(1)-R (with higher affinity for PACAP). VIP and PACAP have been implicated in the control of cell proliferation and tumor growth. This study examined the presence of VIP and PACAP receptors in human lung cancer samples, as well as the functionality of adenylyl cyclase (AC) stimulated by both peptides. Results from RT-PCR and immunoblot experiments showed the expression of VPAC(1)-, VPAC(2)- and PAC(1)-R in lung cancer samples. Immunohistochemical studies showed the expression of VPAC(1) and VPAC(2) receptors. These receptors were positively coupled to AC, but the enzyme activity was impaired as compared to normal lung. There were no changes in Galpha(s) or Galpha(i) levels. Present results contribute to a better knowledge of VIP/PACAP actions in lung cancer and support the interest for the development of VIP/PACAP analogues with therapeutic roles.