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PubMed1996

Expression of the rat BFGF antisense RNA transcript is tissue-specific and developmentally regulated.

The basic fibroblast growth factor (bFGF) gene locus is transcribed into a number of mRNA transcripts including an antisense mRNA derived from the opposite DNA strand of the bFGF gene. Expression of this natural antisense RNA has been implicated in regulation of the bFGF sense mRNA expression and turnover. In the present study we examined the developmental pattern of expression of the bFGF antisense transcript in fetal and postnatal rat tissues. Northern hybridization with a strand-specific cRNA probe detected a 1.5-kb polyadenylated antisense RNA in all tissues examined except brain, in which two transcripts were detected as a doublet of approximately 1.3-1.5 kb in size. The level of antisense transcript expression was markedly tissue- and age-dependent. In the developing brain, both sense and antisense transcripts were detected by Northern hybridization, but the pattern of their expression was inversely related. The 6.0-kb bFGF sense transcript increased in an age-dependent manner from days 3-30 of postnatal development while the antisense transcript decreased to nearly undetectable levels over the same period. In embryonic (days 15-19) liver, kidney, heart and intestine bFGF antisense RNA expression was low but increased dramatically at parturition, rising 5-10-fold over fetal levels by 10 days of age, then declined slowly to a new steady-state level in adult tissues. The level of antisense RNA in these tissues was much higher than that of bFGF sense mRNA, which was undetectable by Northern analysis. Sense and antisense trancripts were also detected in midgestational (11.5 days) embryos by RT-PCR. Antisense expression did not increase when embryos were explanted and cultured for 48 h (9.5-11.5 days). The apparent reciprocal relationship between the abundance of sense and antisense bFGF transcripts in developing brain supports the possibility of a regulatory role for the antisense transcript in this tissue. There was no evidence for a reciprocal relationship between sense and antisense expression in the other tissues examined, indicating that the relationship between sense and antisense RNA expression may be tissue-specific.

Adrenal Glands↗

PubMed1994

Evidence for the presence of pro-oxytocin/neurophysin-converting enzyme in the human ovary.

The activity of pro-oxytocin/neurophysin (pro-OT/Np)-processing enzymes was determined in human granulosa cells, follicular fluids and purified secretory granules of corpora lutea. We detected the presence of an endoprotease which releases OT-Gly10-Lys11-Arg12 on cleavage of the synthetic pro-OT/Np(1-20) peptide after the dibasic Lys11-Arg12 doublet. This endoprotease was inhibited by EDTA, but was not affected by phenylmethanesulfonyl fluoride and pepstatin. Enzymatic activity was markedly reduced by replacement of L-Arg by D-Arg in the basic amino acid doublet of the substrate. The molecular weight of this enzyme was estimated to be 60 kDa. These features closely resembled those of the endoprotease identified in the bovine pituitary. The endoprotease is a metalloenzyme, specific for the Lys-Arg doublet. We also detected a carboxypeptidasic activity, inhibited by guanidinoethane-mercaptosuccinic acid. In the light of our previous detection of the processing intermediates, OT-Gly-Lys-Arg, OT-Gly-Lys and OT-Gly, in human ovary, these observations are in favour of a pro-OT/Np-processing pathway in the human ovary comparable with that in the bovine ovary. Moreover, these results confirm that oxytocin post-translational processing occurs in the human ovary and strongly suggest that pro-OT/Np is synthesized locally.

Cell Membrane↗

PubMed1988

Characterization of proteins that associate with an unglycosylated form of the transferrin receptor in A431 cells.

A protein doublet (Mr = 135,000/130,000) was found to coprecipitate with an unglycosylated form of the transferrin receptor in tunicamycin-treated A431 cells. This doublet is not detected with either a monoclonal or polyclonal antibody to the transferrin receptor on Western blots indicating that these proteins do not interact directly with transferrin receptor antibody. Proteolytic digestion patterns of the individual proteins of the Mr = 135,000/130,000 doublet suggest that they are related to one another and are distinct from the transferrin receptor. Further characterization of these proteins indicates that they form a high molecular weight complex with the unglycosylated but not the glycosylated form of the transferrin receptor. Pulse-chase experiments demonstrate that the proteins post-translationally associate with the receptor.

Blotting, Western↗

PubMed1979

Immunochemical characterization of tumor-associated surface antigens on a Moloney leukemia virus-lymphoma, MBL-2.

Tumor-associated surface antigens (TASA) on a Moloney leukemia virus (M-MuLV)-induced lymphoma, MBL-2, in C57BL/6 mice (B6) were characterized. The surface proteins of MBL-2 cells were selectively radioiodinated and then extracted by Nonidet P40. The solubilized materials were then reacted with a variety of antisera: monospecific antisera to murine leukemia viral proteins (anti-gp69/71, anti-p30, anti-p15, anti-p12 and anti-p10), sera from B6 which regressed murine sarcoma tumors induced by murine sarcoma virus (anti-MSV) and a rabbit anti-MBL-2 antiserum. The resulting radioimmune precipitates were analyzed and compared in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The following results were obtained. (1) Among all anti-viral protein antisera tested only anti-gp69/71 was active and detected a protein doublet of gp69/71 and its degradation fragments of 42,000 and 35,000 daltons. (2) Radioimmune precipitates prepared with anti-MSV showed a SDS-PAGE pattern similar to that seen with anti-gp69/71. This result indicated that the surface antigen detected by the anti-MSV serum on MBL-2 tumor cell was probably a viral envelope antigen. (3) The rabbit anti-MBL-2 serum detected on the cell membrane an antigen of approximately 95,000 daltons which was tumor-associated and did not appear to be related to virion components. The anti-MBL-2-serum still reacted with the 95,000 dalton antigen after absorption with disrupted M-MuLV virus and with gp69/71 and p30 purified from the virus.

Animals↗

PubMed2006

Rapid and efficient MALDI-TOF MS peak detection of 2-nitrobenzenesulfenyl-labeled peptides using the combination of HPLC and an automatic spotting apparatus.

In this paper, we report MALDI-TOF ms analysis of 2-nitrobenzenesulfenyl (NBS) labeled peptides with the powerful aid of an LC-automatic spotting system. using this approach we analyzed mammalian sera (rat and mouse) as biological samples to demonstrate performance. The labeling was carried out using a binary set of 2-nitrobenzenesulfenyl chloride (heavy and light), which modified tryptophan residues in sample proteins. Approximately 1600 doublet peaks were detected in the mass spectrum, some of which had more than threefold differences in their intensities. systematic separation/spotting followed by mass analysis of the NBS-labeled peptides derived from biological samples is described for the first time. This method has proved to be an effective application of NBS-labeled peptides and can be a powerful technique for quantitative analysis of proteins expressed in biological systems.

Animals↗

PubMed1997

Identification of extractable growth factors from small intestinal submucosa.

When implanted as a biomaterial for tissue replacement, selected submucosal layers of porcine small intestine induce site-specific tissue remodeling. Small intestinal submucosa (SIS), as isolated, is primarily an acellular extracellular matrix material. In an attempt to discover the components of small intestinal submucosa which are able to induce this tissue remodeling, the material was extracted and extracts were tested for the ability to stimulate Swiss 3T3 fibroblasts to synthesize DNA and proliferate. Each of the four different extracts of small intestinal submucosa had measurable cell-stimulating activity when analyzed in both a whole cell proliferation assay (alamarBlue dye reduction) and a DNA synthesis assay ([3H]-thymidine incorporation). Proteins extracted from SIS with 2 M urea induced activity profiles in the two assays which were very similar to the activity profiles of basic fibroblast growth factor (FGF-2) in the assays. As well, the changes in cell morphology in response to the extracted proteins mimicked the changes induced by FGF-2. Neutralization experiments with specific antibodies to this growth factor confirmed the presence of FGF-2 and indicated that it was responsible for 60% of the fibroblast-stimulating activity of the urea extract of small intestinal submucosa. Western blot analysis with a monoclonal antibody specific for FGF-2 detected a reactive doublet at approximately 19 kDa and further confirmed the presence of FGF-2. Cell stimulating activity of proteins extracted from SIS with 4 M guanidine was neutralized by an antibody specific for transforming growth factor beta (TGF beta). Changes in the morphology of the fibroblasts exposed to this extract were nearly identical to changes induced by TGF beta. Although no reactive protein band was detected at 25 kDa in nonreduced western blot analysis, several bands were reactive at higher molecular weight. The identity of this TGF beta-related component of small intestinal submucosa is unknown. Identification of FGF-2 and TGF beta-related activities in SIS, two growth factors known to significantly affect critical processes of tissue development and differentiation, provides the opportunity to further elucidate the mechanisms by which this extracellular matrix biomaterial modulates wound healing and tissue remodeling.

3T3 Cells↗

PubMed1987

Progesterone receptor regulation by 17 beta-estradiol in human endometrial carcinoma grown in nude mice.

Regulation of progesterone receptor (PR) in human endometrial carcinoma was investigated in vivo in a multisite nude mouse tumor experimental system by estrogen administration and withdrawal. The cytosolic PR concentration was low in tumors grown in the absence of 17 beta-estradiol, but increased rapidly upon estrogen administration, reaching a maximal receptor concentration of 1.4-1.6 pmol/mg cytosol protein within 7 days. Protein blot analysis using a monoclonal antibody (hPRa 1) raised against PR from EnCa 101 showed no immunoreactivity in tumors grown in the absence of estrogen. Immunoreactive proteins of mol wt 116,000 and 81,000 were detectable 8 h after estrogen administration and increased in intensity as the cytosolic PR concentration increased. Interestingly, the protein of mol wt 116,000 was composed of mol wt isoforms and was detectable as a doublet 8 h after estrogen administration and finally as a triplet. The effect of estrogen withdrawal on EnCa 101 PR concentration and structure was determined by removal of 17 beta-estradiol pellets (200 pg/ml plasma) from EnCa 101-bearing animals after achievement of maximal tumor PR concentrations. The PR concentration in tumor cytosols decreased in a biphasic manner after estrogen removal, with the initial rapid phase having a half-life of around 2 days. Cytosolic PR was still detectable 21 days after estrogen withdrawal. Protein blot analysis showed that immunoreactive proteins of mol wt 116,000 and 81,000 were also detectable up to that time. Photoaffinity labeling with [3H]R5020 demonstrated that the 81,000 mol wt protein, as well as each of the triplet proteins at mol wt 116,000, was specifically photoaffinity labeled. The 116,000-mol wt protein was detected as a triplet on protein blots until 13 days after estrogen withdrawal, when diminution in the intensity of the highest mol wt triplet protein was noted.

Animals↗

PubMed1994

Characterization of the gap junction protein connexin56 in the chicken lens by immunofluorescence and immunoblotting.

PURPOSE: To characterize the chicken lens gap junction protein, connexin56 (Cx56). METHODS: The methods used were immunoblotting, immunofluorescence, alkaline phosphatase treatment, in vitro translation, and primary tissue culture. RESULTS: Connexin56 translated in vitro showed a single band with an electrophoretic mobility of approximately 66 kd. Multiple bands of 67 to 90 kd were detected in immunoblots of chicken lens homogenates using antibodies raised against a peptide from Cx56. Most, if not all, of these bands represented different phosphorylated forms of Cx56, because immunoreactive Cx56 was detected as a doublet of 65 to 67 kd after treatment of lens homogenates with alkaline phosphatase. Indirect immunofluorescence demonstrated that Cx56 was localized at membrane appositions between lens fibers and bow region cells. Levels of Cx56 increased from embryonic days 4 to 15; thereafter, levels remained fairly constant until hatching, after which they declined. Before embryonic day 9, the slowest migrating bands were not as abundant as they were at later ages. After embryonic day 20, less Cx56 was observed by immunofluorescence in the nucleus than in the cortex; however, both regions had similar levels of Cx56 as measured by immunoblotting. The pattern of bands differed between the two lens regions, suggesting differential protein modification. Immunoreactive Cx56 bands of 35 to 38 kd were detected unless homogenates were prepared in the presence of ethylenediaminetetraacetic acid (EDTA). Cx56 was also detected in lentoid-containing primary cultures derived from chicken lens. CONCLUSIONS: Cx56 is a phosphoprotein. Its appearance and modification by phosphorylation, as detected by immunoblotting, correlate with lens fiber differentiation.

Amino Acid Sequence↗

PubMed1997

Immunoblot studies of the acid-labile subunit (ALS) in biological fluids, normal human serum and in children with GH deficiency and GH receptor deficiency before and after long-term therapy with GH or IGF-I respectively.

OBJECTIVE: The aims of this investigation were (a) to study the presence of immunoreactive forms of the acid-labile subunit (ALS) in different human biological fluids, (b) to define the age dependence of serum ALS in normal children and adults and (c) to compare the regulation of ALS by GH or IGF-I in children with GH deficiency (GHD) and GH receptor deficiency (GHRD) before and after 1 year of therapy with GH or IGF-I, respectively. DESIGN AND PATIENTS: Selected human biological fluids from different consenting volunteers and serum from 68 normal children and 5 adults were analysed. Four children diagnosed as GHD and 7 children diagnosed as GHRD were treated with recombinant human (rh) GH at a dosage of 0.05 mg/kg/day s.c. or rhIGF-I at a dosage of 120 micrograms/kg twice daily s.c., respectively, for 12 months. MEASUREMENTS: Immunoreactive forms of ALS were studied by Western immunoblot using a specific rabbit antiserum derived against synthetic human ALS and quantified by laser densitometry analysis. Serum from children with GHD or GHRD were sampled before and at 6 and 12 months of therapy; serum from these patients had been also assayed at baseline for determination of IGF-I and IGF binding protein (IGFBP)-3 by radioimmunoassay and immunoradiometric assay, respectively. RESULTS: An immunoreactive 85 kDa doublet of ALS was detected in serum, plasma, follicular, peritoneal and synovial fluid, but not in urine, seminal plasma, amniotic or extra-embryonic coelomic fluids. Assessment of serum from newborns to adults revealed an age dependence; the ALS doublet was low, but detectable, in newborns, increased during adolescence and remained constant in adulthood. ALS levels were significantly lower in GHD (P = 0.02) and in GHRD children (P = 0.001) than in age-matched controls. Treatment with rhGH in GHD children produced a 2.7-fold increase in serum ALS concentrations at 6 months of therapy (P = 0.01), which was maintained after 1 year of treatment (P = 0.006), leading to normalization of ALS concentrations. In contrast, administration of rhIGF-I to GHRD children failed to increase and normalize serum ALS levels either at 6 or 12 months of therapy. CONCLUSIONS: Immunoreactive forms of acid-labile subunit are present in serum and plasma, as well as in follicular, peritoneal and synovial fluids, suggesting that acid-labile subunit can either cross the capillary barrier or be secreted locally. Acid-labile subunit concentrations are age-dependent with a sharp increase during adolescence, and are reduced in GH deficient and GH receptor deficient children. While treatment with rhGH is able to increase and normalize acid-labile subunit concentrations in GH deficient children, therapy with rhIGF-I fails to increase serum acid-labile subunit levels in GH receptor deficient patients. These data suggest that acid-labile subunit is directly GH-regulated, and that IGF-I cannot increase acid-labile subunit levels, as assessed by Western immunoblot.

Adolescent↗

PubMed2006

Complex glutamate labeling from [U-13C]glucose or [U-13C]lactate in co-cultures of cerebellar neurons and astrocytes.

Glutamate metabolism was studied in co-cultures of mouse cerebellar neurons (predominantly glutamatergic) and astrocytes. One set of cultures was superfused (90 min) in the presence of either [U-(13)C]glucose (2.5 mM) and lactate (1 mM) or [U-(13)C]lactate (1 mM) and glucose (2.5 mM). Other sets of cultures were incubated in medium containing [U-(13)C]lactate (1 mM) and glucose (2.5 mM) for 4 h. Regardless of the experimental conditions cell extracts were analyzed using mass spectrometry and nuclear magnetic resonance spectroscopy. (13)C labeling of glutamate was much higher than that of glutamine under all experimental conditions indicating that acetyl-CoA from both lactate and glucose was preferentially metabolized in the neurons. Aspartate labeling was similar to that of glutamate, especially when [U-(13)C]glucose was the substrate. Labeling of glutamate, aspartate and glutamine was lower in the cells incubated with [U-(13)C]lactate. The first part of the pyruvate recycling pathway, pyruvate formation, was detected in singlet and doublet labeling of alanine under all experimental conditions. However, full recycling, detectable in singlet labeling of glutamate in the C-4 position was only quantifiable in the superfused cells both from [U-(13)C]glucose and [U-(13)C]lactate. Lactate and alanine were mostly uniformly labeled and labeling of alanine was the same regardless of the labeled substrate present and higher than that of lactate when superfused in the presence of [U-(13)C]glucose. These results show that metabolism of pyruvate, the precursor for lactate, alanine and acetyl-CoA is highly compartmentalized.

Alanine↗

PubMed1996

Protein-protein interactions between Epstein-Barr virus nuclear antigen-LP and cellular gene products: binding of 70-kilodalton heat shock proteins.

The EBNA-LP protein encoded by the open reading frame in the leader exons of the Epstein-Barr nuclear antigen messages is essential for efficient immortalization of B lymphocytes. Protein-protein interaction studies using affinity precipitation of proteins from [35S]methionine-labeled cell lysates and bacterially expressed maltose binding protein EBNA-LP fusions were performed. A cellular 68/72-kDa doublet protein was detected. This banding pattern was shown to be identical to that obtained in affinity precipitations with fusions of glutathione-S-transferase and Sp1 (a basal transcription factor). For both EBNA-LP and Sp1 the specific interacting cellular proteins have been identified as heat shock proteins (HSP) 72/73. Affinity precipitation of HSP 72/73 with deletion mutants of EBNA-LP maps the interaction domain on EBNA-LP to exon Y2 which is required for immortalization. Immunoprecipitation of EBNA-LP from EBV-positive lymphoblastoid cell lines coprecipitated the HSP 72/73 proteins, indicating that the interaction occurs in vivo as well as in vitro. The association of HSPs with a widening range of nuclear proteins involved in gene expression and proliferation control now includes Sp1 and EBNA-LP and suggests that there is a central role for molecular chaperones in these processes.

Antigens, Viral↗

PubMed1993

The relationship between L-arginine-dependent nitric oxide synthesis, nitrite release and dinitrosyl-iron complex formation by activated macrophages.

We identified the source of the nitrogen included into nitric oxide (NO) and studied the relationship between formation of NO, intracellular dinitrosyl ferrous iron complex (DNIC) and release of nitrite by murine bone-marrow-derived macrophages stimulated with E. coli lipopolysaccharide (LPS). NO was trapped in the cell membrane by iron-diethyldithiocarbamate complex (FeDETC) and was detected as a paramagnetic NOFe(DETC)2 complex by electron paramagnetic resonance (EPR) spectroscopy. Macrophages stimulated for 7 h up to 48 h with LPS and then incubated for 2 h with DETC exhibited an anisotropic EPR signal of axial symmetry with g-factor values g perpendicular = 2.035, g parallel = 2.02 and a triplet hyperfine structure (hfs) at g perpendicular characteristic for NOFe(DETC)2. In cells incubated with [15NG]L-arginine instead of [14NG]L-arginine the EPR signal of [15N]OFe(DETC)2 was detected with a doublet hfs at g perpendicular, indicating that NO was generated exclusively from the terminal guanidino-nitrogen of extracellular L-arginine. The ratio of NO formation and of nitrite release changed with time of exposure to LPS, nitrite exceeding NO at early stages of macrophage activation, and NO exceeding nitrite at later stages. DNIC with thiolate ligands (0.5 nmol/10(7) cells) was observed in stimulated macrophages not loaded with DETC. Furthermore, DNIC released from macrophages was trapped in the extracellular medium by bovine serum albumin (BSA) (1 nmol/10(7) cells per 2 h) by formation of a paramagnetic DNIC with BSA. DNIC release not only provides a route for iron loss from activated macrophages, but may also play a role in the cytotoxic and microbiostatic activity of macrophages.

Animals↗

PubMed1995

The presence of phorbol ester responsive and non-responsive forms of the zeta isozyme of protein kinase C in mouse epidermal cells.

The possible involvement of zeta isozyme of protein kinase C (PKC zeta) in phorbol ester-induced signal transduction was investigated in mouse epidermal cells. Western blot analysis of RESOURCE Q column chromatography eluates obtained from 105,000 g supernatants and particulate fractions of epidermal cells was performed using anti-PKC zeta specific antibody. Anti-PKC zeta antibody recognised proteins in low salt range corresponding to 25-125 mM NaCl (low salt-eluted PKC zeta; 1-PKC zeta) as well as high salt range corresponding to 175-300 mM NaCl (high salt-eluted PKC zeta; h-PKC zeta) in both subcellular fractions. 1-PKC zeta and h-PKC zeta were detected as a doublet protein of 79,000 and 85,000 M(r) in 105,000 g supernatants, but as a 79,000 M(r) protein in particulate fractions. Immunoprecipitated 1-PKC zeta and h-PKC zeta with anti-PKC zeta specific antibody possessed phosphatidylserine (PS)-dependent protein kinase activity, but neither 1-PKC zeta nor h-PKC zeta were further activated by 40 nM phorbol 12-myristate 13-acetate (PMA) in the presence of PS. Furthermore, 1-PKC zeta and h-PKC zeta can be autophosphorylated, indicating that both 1-PKC zeta and h-PKC zeta are PKC zeta. Treatment of intact epidermal cells with PMA or other PKC activators caused the apparent shift of 79,000 M(r) 1-PKC zeta to the 85,000 M(r) from in particulate fractions. Prolonged treatment of the cells with PMA induced the downregulation of both forms of 1-PKC zeta in particulate fractions. Under the same condition, 1-PKC zeta in 105,000 g supernatants and h-PKC zeta in both fractions did not respond to PMA. This apparent shift was reversible and the content ratio of 85,000 to 75,000 M(r) 1-PKC zeta was decreased by acid phosphatase treatment, indicating that the apparent shift results at least in part from phosphorylation of 79,000 M(r) 1-PKC zeta. Total activity of 1-PKC zeta was increased in association with the apparent shift from the 79,000 to 85,000 M(r) form in response to PMA treatment of intact epidermal cells. All of these results indicate that PKC zeta is present as multiple forms in mouse epidermal cells, and that especially 1-PKC zeta in particulate fractions play a significant role(s) in PMA-induced signal transduction in mouse epidermal cells.

Amino Acid Sequence↗

PubMed1994

Thallium-205 and carbon-13 NMR studies of human sero- and chicken ovotransferrin.

We have examined the binding of Tl3+ to human serotransferrin and chicken ovotransferrin in the presence of carbonate and oxalate by 205Tl and 13C NMR spectroscopy. With carbonate as the synergistic anion, one observes two 205Tl NMR signals due to the bound metal ion in the two high-affinity iron-binding sites of each protein. When the same adducts are prepared with 13C-labeled carbonate, one finds two closely spaced doublets in the carbonyl region of the 13C NMR spectrum of serotransferrin; these correspond to the labeled anion directly bound to the metal ion in both sites of the protein. The analogous resonances in ovotransferrin are completely degenerate, and only one doublet can be detected. The magnitudes of the spin-spin coupling between the bound metal ion and carbonate range from 2J(205Tl-13C) approximately 270 to 290 Hz. We have used the proteolytic half-molecules of ovotransferrin and the recombinant N-terminal half-molecule of serotransferrin to assign the 205Tl and 13C NMR signals due to the bound metal ion and anion in both proteins. From titration studies, we found that Tl3+ is bound with a greater affinity at the C-terminal site of serotransferrin, whereas no site preference can be noted for ovotransferrin. When oxalate is used as the anion instead of carbonate, the 205Tl NMR signals arising from the bound metal ion in the sites of ovotransferrin are shifted downfield and become almost degenerate. A very complex pattern of resonances is observed for bound 13C2O4(2-) in the 13C NMR spectra of both proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1997

1H NMR investigation of toxic effects of lindane and paraquat on Hep 3B and Hep G2 human hepatoma cell lines.

Lindane and paraquat induce biochemical changes in the liver. In order to specify their molecular impact at the cellular level, a 300 MHz 1H NMR investigation of hepatoma cell lines Hep 3B and Hep G2 responses was performed. Cells were exposed over 24 h to 50 mg/L lindane (0.178 mM) or to 100 mg/L (0.389 mM) paraquat concentrations. The main observation following exposure to lindane was a decrease in betaine methyl groups (3.26 ppm) which could be related to the steatosis reported by some authors. Specifically, in Hep G2 cells with this pesticide, the glycine peak (3.56 ppm) was lowered, thus confirming that the glycine synthesis pathway involving methionine, choline, and betaine was disturbed by lindane. Moreover, in this hepatoma cell line, the p-chlorobenzoate ion could be detected as a doublet at 7.55 ppm. In Hep 3B cells, paraquat increased betaine and methionine levels, suggesting disturbance in glycine biosynthesis. Possibilities of cellular uptake were considered, and the presence of this herbicide in cells was revealed by spectrophotometric and NMR measurements after chlorhydric hydrolysis, suggesting interaction with cellular components. The impact of paraquat on Hep G2 cells appeared to be located on mitochondrial function, as indicated by the observed decrease in succinate and pyruvate levels.

Adenosine Triphosphate↗

PubMed2002

Guadeloupean parkinsonism: a cluster of progressive supranuclear palsy-like tauopathy.

An unusually high frequency of atypical Parkinson syndrome has been delineated over the last 5 years in the French West Indies. Postural instability with early falls, prominent frontal lobe dysfunction and pseudo-bulbar palsy were common and three-quarters of the patients were L-dopa unresponsive. One-third of all patients seen had probable progressive supranuclear palsy (PSP). This new focus of atypical parkinsonism is reminiscent of the one described in Guam and may be linked to exposure to tropical plants containing mitochondrial complex I inhibitors (quinolines, acetogenins, rotenoids). Two hundred and twenty consecutive patients with Parkinson's syndrome seen by the neurology service at Pointe à Pitre, Guadeloupe University Hospital were studied. Currently accepted operational clinical criteria for Parkinson's syndromes were applied. The pathological findings of three patients who came to autopsy are reported. Fifty-eight patients had probable PSP, 96 had undetermined parkinsonism and 50 had Parkinson's disease, 15 had amyotrophic lateral sclerosis with parkinsonism and one had probable multiple system atrophy. All three PSP patients in whom post-mortem study was performed had early postural instability, gaze palsy and parkinsonian symptoms, followed by a frontolimbic dementia and corticobulbar signs. Neuropathological examination showed an accumulation of tau proteins, predominating in the midbrain. There was an exceptionally large accumulation of neuropil threads in Case 1. Biochemical studies detected a major doublet of pathological tau at 64 and 69 kDa in brain tissue homogenates. All cases were homozygous for the H1 tau haplotype, but no mutation of the tau gene was observed. Clinical, neuropathological and biochemical features were compatible with the diagnosis of PSP, although some unusual pathological features were noted in Case 1. A cluster of cases presenting with atypical parkinsonism is reported. Guadeloupean parkinsonism may prove to be a tauopathy identical or closely related to PSP.

Aged↗

PubMed2002

Glutathione S-transferase alpha expressed in porcine Sertoli cells is under the control of follicle-stimulating hormone and testosterone.

Glutathione S-transferases (GSTs) are a family of detoxification isoenzymes present in different tissues including the testis and that conjugate many toxic substrates to glutathione. Among these substrates are carcinogens, mutagens and products of oxidative processes. In the present report we show that GSTalpha is expressed in somatic testicular Leydig cells and Sertoli cells. GSTalpha expression in Sertoli cells is under the hormonal control of FSH, testosterone, and estradiol. In Leydig cells, immunoreactive GSTalpha was present at the neonatal, pubertal, and adult periods. In Sertoli cells, GSTalpha was predominant in pubertal and adult testes (but not in neonatal testes), suggesting that its expression is controlled by gonadotropins. The regulatory action and the mechanisms of action of FSH and testosterone on GSTalpha mRNA and protein levels were studied by using a model of primary cultures of porcine testicular Sertoli cells. FSH increased GSTalpha mRNA levels in a dose-dependent manner (ED50 = 18.5 nm/ml) with a maximal effect observed after 48 h of exposure (a 3-fold increase; P < 0.001). In addition, FSH increased GSTalpha protein, which was detected as a doublet of 28 kDa. Treatment with testosterone enhanced GSTalpha mRNA levels in a dose-dependent (ED50 = 1.4 ng/ml) and time-dependent manner with a maximal effect delayed at 8 h of exposure (a 2-fold increase; P < 0.001). Similarly, Sertoli cell treatment with testosterone metabolites, dihydrotestosterone (DHT) and estradiol, led to an increase in GSTalpha mRNA levels. Because stimulatory effects of FSH and androgens were also observed on GSTalpha protein, we therefore had to determine whether the different hormones were affecting GSTalpha gene transcriptional activity, or GSTalpha mRNA stability, or both. FSH and 8-Br-cAMP (but not testosterone) increased the stability of GSTalpha mRNA. The effects of FSH and testosterone on GSTalpha protein were additive, confirming that both hormones act through distinct mechanisms on the expression of the enzyme. Taken together, the present observations indicate that Sertoli cell GSTalpha is targeted by FSH, testosterone, and its metabolites, and they reinforce the concept that Sertoli cells exert a protective role and are under endocrine control to ward against toxic agents in the context of Sertoli-germ cell interactions during spermatogenesis.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2006

Splice variants of human FOXP3 are functional inhibitors of human CD4+ T-cell activation.

FOXP3 has been identified as a key regulator of immune homeostasis. Mutations within the FOXP3 gene result in dysregulated CD4+ T-cell function and elevated cytokine production, leading to lymphoproliferative disease. FOXP3 expression in CD4+ T cells is primarily detected with the CD4+ CD25+ regulatory T-cell population. In humans the protein is detected as a doublet following immunoblot analysis. The lower band of the doublet has been identified as a splice isoform lacking a region corresponding to exon 2. The aim of this study was to investigate whether the splice variant form lacking exon 2 and a new novel splice variant lacking both exons 2 and 7, were functional inhibitors of CD4+ T-cell activation. The data generated showed that full-length FOXP3 and both splice variant forms of the protein were functional repressors of CD4+ T-cell activation.

Base Sequence↗