Biomedical subjects

O Tsolas

Publications and source records attributed to O Tsolas.

At least 37 records · Page 2Linked to original sources

PubMed1996

Regulation of prothymosin alpha during the cell cycle.

A number of studies have indicated that the small nuclear acidic protein prothymosin alpha is associated with cellular-proliferation events. For example, c-myc causes immediate transcriptional activation of prothymosin alpha, and prothymosin alpha antisense oligonucleotides inhibit myeloma cell division. To investigate the regulation of prothymosin alpha, we examined its mRNA and protein levels during the cell cycle of mononuclear cells and fibroblastic cells. We isolated immunoreactive material from cellular extracts and immunolocalized the protein to the nucleus during the cell cycle. We reported here that the material present in the cells is prothymosin alpha rather than the amino-terminal peptide thymosin alpha 1. [3H]Thymidine-incorporation studies associate maximum accumulation of mRNA and protein with the S/G2 phase of the cell cycle. This induction of prothymosin alpha mRNA seems to resemble cyclin B expression and is more pronounced in fibroblasts. Moreover, transient-transfection experiments indicate that transcription factor E2F is a strong positive regulator of the prothymosin alpha gene. Our results are consistent with the hypothesis that prothymosin alpha is involved in proliferation checkpoints of the cell cycle.

Animals↗

PubMed1995

Purification and properties of human placental ATP diphosphohydrolase.

ATP diphosphohydrolase activity (ATP-DPH) has been previously identified in the particulate fraction of human term placenta [Papamarcaki, T. & Tsolas, O. (1990) Mol. Cell. Biochem. 97, 1-8]. In the present study we have purified to homogeneity and characterized this activity. A 260-fold purification has been obtained by solubilization of the particulate fraction and subsequent chromatography on DEAE Sepharose CL-6B and 5'-AMP Sepharose 4B. The preparation has been shown to be free of alkaline phosphatase even though the placental extract is rich in this activity. The purified enzyme is a glycoprotein and migrates as a single broad band of 82 kDa on SDS/PAGE. The same band is obtained after photoaffinity labeling of the enzyme with 8-azido-[alpha-32P]ATP. The enzyme has a broad substrate specificity, hydrolyzing triphosphonucleosides and diphosphonucleosides but not monophosphonucleosides or other phosphate esters. The activity is dependent on the addition of divalent cations Ca2+ or Mg2+. The Km values for ATP and ADP were determined to be 10 microM and 20 microM, respectively. Maximum activity was found at pH 7.0-7.5 with ATP as substrate, and pH 7.5-8.0 with ADP. The enzymic activity is inhibited by NaN3, NaF, adenosine 5'-[beta,gamma-imido]triphosphate and adenosine 5'-[alpha,beta-methylene]triphosphate. Protein sequence analysis showed ATP-DPH to be N-terminally blocked. Partial internal amino acid sequence information was obtained after chymotryptic cleavage and identified a unique sequence with no significant similarity to known proteins. ATP-DPH activity has been reported to be implicated in the prevention of platelet aggregation, hydrolysing ADP to AMP and thus preventing blood clotting.

Affinity Labels↗

PubMed1995

Prothymosin alpha mRNA levels vary with c-myc expression during tissue proliferation, viral infection and heat shock.

Expression of prothymosin alpha, an acidic nuclear protein implicated in cellular proliferation, has been reported to be regulated by c-myc in vitro. We have studied the correlation of expression levels between prothymosin alpha and c-myc, using three different in vivo systems, viz. normal ontogenic process of placental development, lytic viral infection and heat shock treatment. The two genes have been found to share a similar expression pattern, although prothymosin alpha mRNA remains always detectable, indicating the existence of yet another mechanism, in addition to c-myc, which regulates its expression in vivo.

Adenoviridae↗

PubMed1995

Superoxide anion generation by human peripheral blood mononuclear cells in response to prothymosin alpha.

The ability of human peripheral blood mononuclear cells to respond to highly purified prothymosin alpha by generating superoxide anion was investigated. The generation of superoxide anion was detected by measuring the superoxide dismutase-inhibitable reduction of oxidized cytochrome C. Prothymosin alpha was shown to stimulate weakly these cells. The dose-response curve displayed a biphasic bell-shaped superoxide generation profile with two specific concentration optima for each individual blood donor, but with variations in optimal concentrations between the donors. By using a counter current centrifugation (elutriation) system, the mononuclear cell population was separated into several fractions according to their volume and density. Selective stimulation of these fractions with prothymosin alpha revealed that different cell populations were responsible for the generation of superoxide at higher and lower concentrations of stimulant, respectively. The response to the stimulus was immediate and lasted for a time period of about 4 to 8 min during which approximately 0.7 nmol O2- per min/10(6) cells were generated. The superoxide generation was cell-number-dependent with an optimum at 1 x 10(6) cells and lower rates for both smaller and larger cell numbers. Staurosporine, a potent inhibitor of protein kinase C, at concentrations sufficient to inhibit totally PMA-induced O2- generation, failed to affect the response of the cells to prothymosin alpha, while chelation of the extracellular Ca2+ abolished the lower but not the higher peak of O2- generation. Finally, simultaneous addition of prothymosin alpha and PMA resulted in a approximately 40% decrease of the O2- generation induced by PMA alone. A putative role as cell injury indicator is proposed for prothymosin alpha.

Cell Separation↗

PubMed1995

Role of the spleen in lipid metabolism.

The frequency of ischaemic heart disease observed after splenectomy for trauma and the low cholesterol levels found in patients with hypersplenism are observations that suggest a possible role for the spleen in lipid metabolism and in the aetiology of atherosclerosis. The present study was designed to examine this role in experimental animals. Serum levels of total cholesterol, triglyceride and high-density lipoprotein (HDL) cholesterol were determined in 32 rats. The spleen was removed in 16 rats and the remaining 16 were sham operated. Half of the splenectomized and half of the remaining rats were fed on a diet rich in fat and the two other subgroups were fed normal chow. Blood tests were performed before, and 3 and 6 months after operation. A significant increase in serum triglyceride and decrease in serum HDL cholesterol levels was observed after splenectomy in rats fed normal chow compared with sham-operated rats. An increase in serum triglyceride and a decrease in serum HDL cholesterol levels was observed in both groups of rats fed normal chow plus high-fat cheese. However, these changes were more pronounced in splenectomized rats. These findings suggest that the spleen has a role in lipid metabolism in rats and may therefore influence atherosclerosis.

Animals↗

PubMed1995

The follicle-stimulating hormone threshold level for follicle maturation in superovulated cycles.

OBJECTIVE: To compare the FSH threshold level for follicle growth up to the preovulatory stage between superovulated and natural cycles. DESIGN: Normally cycling women were investigated during an untreated spontaneous cycle (control) and three cycles treated with FSH. SETTING: Infertility clinic in a university hospital. PATIENTS: Thirteen normally ovulating women with unexplained infertility. INTERVENTIONS: Follicle-stimulating hormone was injected at the single dose of six ampules on cycle day 2 without extra FSH (FSH + 0 cycle) or with extra FSH from cycle day 4 onward at the daily dose of either one ampule (FSH + 1 cycle) or two ampules (FSH + 2 cycle). MAIN OUTCOME MEASURES: Serum FSH and E2 during the follicular phase. Number of follicles and growth rate by ultrasound. RESULTS: Multiple follicular development occurred in all FSH treated cycles. In these cycles the growth rate of the first (dominant) follicle was similar to that in the control cycles. In the presence of physiological concentrations of FSH (control and FSH + 0 cycles), only one follicle was > 15 mm at midcycle. When serum FSH values increased (FSH + 1 and FSH + 2 cycles), the growth rate of the second and third in order follicles and subsequently the number of the preovulatory follicles (> 15 mm) increased in proportion to the FSH increment. CONCLUSIONS: Follicles recruited by exogenous FSH in the early follicular phase of the cycle require supraphysiological concentrations of FSH throughout the follicular phase to become preovulatory. The extra FSH acts through an increase in the growth rate of these follicles.

Estradiol↗

PubMed1995

Primary idiopathic hypomagnesemia in two female siblings.

Two female siblings with primary idiopathic hypomagnesemia, born to consanguineous parents, are described. Both presented at 6 weeks of age, with convulsions and persistent hypocalcemia (calcium 1.5 adn 1.6 mmol/l; normal range (NR) 2.2-2.6 mmol/l), which could not be controlled with anticonvulsants and/or calcium gluconate. On further investigation they were also found to have hypomagnesemia (magnesium 0.17 mmol/l and 0.22 mmol/l; NR 0.65-1.05 mmol/l). Convulsions and the low serum calcium and magnesium levels were first managed by im and then by oral administration of magnesium supplements. A burst in circulating parathyroid hormone levels to well above the physiological range was observed at the start of therapy. Serum magnesium values of the mother and father were just below the normal range, with normal serum calcium. This type of infantile primary hypomagnesemia appears to be a hereditary disease with autosomal recessive characteristics, although a partially penetrant X-linked or autosomal dominant trait cannot be excluded.

Calcium↗

PubMed1995

Amniotic fluid prealbumin as a potential marker of fetal abnormalities.

Normal amniotic fluid prealbumin (PAB) levels between 16 and 41 weeks of pregnancy are demonstrated in 175 samples from various gestational ages. PAB was measured quantitatively by immunonephelometry and qualitatively estimated by electrophoresis while total protein content was measured by colorimetry. The percent of PAB values in the total protein content is inversely correlated to the gestational age. Fetal abnormalities and abnormal conditions in pregnancy diagnosed in the second trimester and early third trimester (16-32 weeks) show significantly (p < 0.01) higher than expected PAB values (25.9 +/- 4.6 mg/l, range 19.5-35.6, n = 23) in comparison to normal fetuses and normal pregnancies of the same age (13.6 +/- 3.6 mg/l, range 4.3-19.8, n = 87). The remaining 65 samples of normal third trimester amniotic fluid were evaluated in comparison to the second trimester normal values, and inverse correlation in respect to advancing pregnancy was observed. PAB values were corrected for the total protein content and the resulting ratio had the lowest variation compared to PAB and total protein values. It is proposed that PAB, although not specific for a particular disorder, expressed as a ratio of the total protein content is a potential biochemical marker for certain fetal defects and complications of pregnancy.

Amniotic Fluid↗

PubMed1995

Gamma-chain heterogeneity in Greek (delta beta)zero-thalassemia.

A molecular and biochemical population study of (delta beta)zero thalassemia in central Greece is described. The molecular study was focused on the type of the deletion and the status of G gamma-XmnI polymorphism, whereas the biochemical approach was centered on the G gamma/A gamma ratio as well as the frequency of the A gamma T chain in the fetal hemoglobin of 19 delta beta-thalassemia heterozygotes and 3 homozygotes. This study includes individuals from the mountainous district of Epirus (northwestern Greece) where the trait was found to be concentrated along the river Arachthos. The Sicilian (delta beta)zero thalassemia deletion was found in all subjects tested by direct PCR. The levels for the G gamma-chain presented values ranging from 29 to 83% of the total gamma-chain content. Thirteen heterozygotes had the adult G gamma/A gamma ratio (mean G gamma: 35% +/- 10) of whom 10 were XmnI-negative (- / -), 6 had the newborn ratio (mean G gamma: 70% +/- 9) and were XmnI-positive, while homozygotes had equal amounts of G gamma and A gamma. Five of the 19 heterozygotes were A gamma T-positive with low levels of this A gamma-chain variant, suggesting an in-trans to the delta beta-thalassemia determinant production.

Adolescent↗

PubMed1995

Interaction of BiP with substance P and nucleotides.

A rapid and simple spin column assay has been used to study interactions of BiP with substance P (SP) and ATP. At 4 degrees C, the binding of SP to BiP requires ATP and a stable SP-BiP.ATP complex is formed. Nonhydrolyzable ATP analogues or ADP cannot replace ATP. Although ATP converts BiP dimers to monomers, the requirement for ATP for SP binding is not solely due to BiP dissociation, because purified BiP monomers also require ATP for peptide binding. At 37 degrees C, there is rapid binding of SP to BiP even in the absence of ATP and, in fact, ATP at concentrations above 5 microM causes release of SP from BiP. At this higher temperature, there is also rapid hydrolysis of ATP bound to BiP. These results extend our previous results (Brot et al., 1994) that indicated the formation, at low ATP concentrations, of a labile SP.BiP.ATP complex that, after ATP hydrolysis, resulted in a stable SP.BiP.ADP complex.

Adenosine Triphosphate↗

PubMed1994

Large-scale chromatofocusing-based method for isolating thymosin beta 4 and thymosin beta 9 from bovine tissues.

A large-scale method for the isolation of thymosin beta 4 (up to 120 mg) and thymosin beta 9 (up to 40 mg) from bovine lung (up to 2 kg) was developed. The isolation protocol included tissue homogenization in 0.4 M HClO4, centrifugation, solid-phase extraction through LiChroprep RP-18 material, chromatofocusing on polybuffer exchanger PBE 94-modified Sepharose and dialysis against water. The isolated products were characterized by analytical isoelectric focusing, reversed-phase HPLC, electrospray ionization mass spectrometry and amino acid analysis. The method developed is rapid and convenient, requires no expensive equipment and can be used for the isolation of thymosin beta 4 and homologous peptides from various animal tissues.

Amino Acid Sequence↗

PubMed1994

Prothymosin alpha binds to histone H1 in vitro.

Previous studies have shown that prothymosin alpha (ProT alpha) is a nuclear acidic protein implicated in cell proliferation. To identify proteins that interact with ProT alpha we have used ligand-blotting assays. We report here that purified ProT alpha binds specifically to histone H1 in a dose dependent manner. Polyglutamic acid, an analog of the central acidic domain of ProT alpha, strongly inhibits the above interaction, suggesting that the binding of ProT alpha to histone H1 is mediated through its acidic domain.

Amino Acid Sequence↗

PubMed1994

Autoantibodies to HMG-17 nucleosomal protein in patients with scleroderma.

Autoantibodies to HMG-17, a non-histone nucleosomal protein, were found in systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD) and ANA positive pauciarticular juvenile rheumatoid arthritis (JRA), but not in rheumatoid arthritis (RA). Using highly purified HMG-17 derived from porcine thymus, we tested sera from 50 patients with scleroderma for antibodies to HMG-17 by enzyme-linked immunosorbent assay (ELISA). There were 16 patients with diffuse cutaneous systemic sclerosis (dSSc) and 34 with limited cutaneous systemic sclerosis (1SSc) with age at disease onset 40.25 +/- 11.68 and 39.94 +/- 15.68 years, respectively, and disease duration 6.03 +/- 4.98 and 13.34 +/- 11.80 years, respectively (P < 0.0001). Anticentromere antibodies (ACA) were found in 65% of 1SSc patients but not in dSSc (P < 0.0001) while the prevalence of antinuclear antibodies (ANA) with other than ACA patterns did not differ in the two groups. Anti-HMG-17 antibodies occurred in 20 patients (40%), five with dSSc (31%) and 15 with 1SSc (41%). Twelve of the 20 HMG-17 positive patients were also positive for ACA (60%) but this association was not significant. No association was found between anti-HMG-17 and other antibody patterns. In conclusion, anti-HMG-17 antibodies occur in one third of scleroderma patients, do not discriminate scleroderma variants and are not associated with other autoantibodies.

Adult↗

PubMed1994

Induction of lymphokine-activated killer activity in mice by prothymosin alpha.

We have recently demonstrated that prothymosin alpha (ProT alpha) when administered intraperitoneally (i.p.) protects DBA/2 mice against the growth of syngeneic leukemic L1210 cells through the induction of tumoricidal peritoneal cells producing high levels of tumor necrosis factor alpha (TNF alpha) [Papanastasiou et al. (1992) Cancer Immunol Immunother 35: 145]. In this report we tested further immunological alterations that may be caused by the administration of ProT alpha in vivo. We demonstrate that i.p. injections of ProT alpha enhance natural killer (NK) cell activity and induce lymphokine-activated (LAK) activity in vivo. Thus, splenocytes from ProT alpha-treated DBA/2 animals exhibited significantly higher cytotoxic activity (up to threefold) against the NK-sensitive YAC cell line and the NK-resistant P815 and L1210 syngeneic tumor cells, as compared to splenocytes from syngeneic control mice. The enhancement of the cytotoxic profile of DBA/2 splenocytes was associated with increased percentages of CD8+ cells, NK cells and activated CD3+ cells. The ProT alpha-induced effect persisted for 30 days after the end of the ProT alpha treatment period and returned to normal levels 20 days later. Splenocytes from non-treated DBA/2 animals generated high NK and LAK activities in response to ProT alpha in vitro. The ProT alpha-induced NK and LAK activities reached 84% and 75% respectively of what was obtained with interleukin-2 (IL-2). High concentrations of TNF alpha and IL-2 were generated in response to ProT alpha in LAK cultures. These findings suggest that ProT alpha may provide an overall protective effect against tumor growth in vivo through induction of NK and LAK activities possibly indirectly via the production of IL-2 and TNF alpha in the spleen, peritoneal cavity and probably other lymphoid organs.

Animals↗

PubMed1994

Lipid parameters including Lp(a) in hemodialysis patients.

Chronic hemodialysis (CHD) patients have a high incidence and prevalence of atherosclerotic disease which may be related to numerous atherosclerotic risk factors. Among them dyslipidemia plays a significant role. Elevated Lp(a) levels, which are strongly associated with atherosclerosis, have been reported recently in uremic patients. The aim of our study was the determination of the levels of lipid parameters including Lp(a) in 151 CHD patients (76 male) aged 57 (12-81) years, who were on hemodialysis for a mean of 44.3 (range 1 to 189) months. Eighty-four normal individuals age and sex matched were used as controls. The median serum Lp(a) concentration in hemodialysis patients was 13 mg/dL compared with 6.5 mg/dL in healthy controls, p < 0.001 by distribution-free Mann-Whitney test. The prevalence of subjects with Lp(a) levels above 25 mg/dL was significantly higher in CHD patients compared to normal subjects (30% vs. 8%, p < 0.001). Even if CHD patients were matched for fasting lipid levels, they showed Lp(a) levels significantly higher than controls. No significant correlation was found between Lp(a) levels and either the age of the patients or the duration of hemodialysis. The etiology of primary renal disease did not influence the Lp(a) levels.

Arteriosclerosis↗

PubMed1993

Autoantibodies to HMG-17 nucleosomal protein in autoimmune rheumatic diseases. Correlation with systemic lupus erythematosus clinical activity and with antibodies to double-stranded DNA.

OBJECTIVE: Previous studies have shown that serum from patients with systemic lupus erythematosus (SLE) contains antibodies directed against HMG-17, a nucleosomal nonhistone high mobility group (HMG) protein found in chromatin. The aim of the present study was to investigate any associations between the presence of antibodies to HMG-17 and clinical and serologic features of SLE. METHODS: Using porcine thymus as a source, HMG-17 was purified by Sephacryl S-200 chromatography followed by high performance liquid chromatography. An enzyme-linked immunosorbent assay utilizing the purified HMG-17 as antigen was developed and was used to evaluate sera from patients with autoimmune rheumatic diseases, for the presence of autoantibodies. RESULTS: Anti-HMG-17 antibodies were found in the serum of 34.8% of the patients with SLE, compared with 11.5% of patients with primary Sjögren's syndrome, 4.4% of patients with rheumatoid arthritis, and 4.1% of normal blood donors. Analysis of the clinical features of SLE using 2 different lupus activity indices revealed that anti-HMG-17 antibodies were more frequently found in patients with active disease. A positive correlation was also observed between anti-HMG-17 and anti-dsDNA levels, and levels of both of these autoantibodies demonstrated a negative correlation with C4. Analysis of sequentially obtained serum samples from 4 SLE patients revealed that, in 2 patients, anti-HMG-17 levels fluctuated in parallel with both disease activity and anti-dsDNA levels, and in the remaining 2, anti-HMG-17 levels fluctuated in parallel with disease activity. CONCLUSION: Antibodies to HMG-17 are found in patients with many different autoimmune rheumatic diseases, although they are more frequently observed in those with SLE. Their presence appears to be associated with lupus disease activity as well as with anti-dsDNA and C4 levels.

Adolescent↗

PubMed1993

Isolation by a new method and sequence analysis of chromosomal HMG-17 protein from porcine thymus.

Nonhistone chromosomal protein HMG-17 from porcine thymus has been isolated by extraction in boiling water, gel filtration and HPLC, and its complete primary structure (89 residues) has been determined. Peptides derived from enzymatic hydrolysis with trypsin, Staphylococcus aureus V-8 protease, Arg-C, and Glu-C proteinases were purified by HPLC and sequenced by the 4-(N,N-dimethylamino)azobenzene-4'-isothiocyanate/phenylisothiocyanate double coupling method. Porcine HMG-17 has a molecular mass of 9248 Da and a pI > 9.8. No glycosylation or methylation has been detected. The primary structure of this protein is almost identical to the sequence deduced from a cDNA clone derived from a human cell line. Porcine thymus HMG-17 differs from the human protein by only a single conservative substitution at position 64 (aspartic acid instead of glutamic acid). As in other HMG-17 proteins, the sequence is characterized by a lysine- and proline-rich central region, which has been implicated in DNA binding.

Amino Acid Sequence↗

PubMed1992

A sensitive and specific competition microELISA for the immunoactive polypeptide parathymosin and detection of this peptide in porcine tissues.

A sensitive and specific microELISA assay is described for the immunoactive polypeptide parathymosin. Antibodies against a synthetic peptide corresponding to the rat parathymosin sequence 5-30 were raised in rabbits immunised with this peptide conjugated to keyhole limpet hemocyanin (KLH). The useful range of the assay was 0.25-30 pmol (3-330 ng) of parathymosin and the assay was specific. The related immunoactive polypeptides prothymosin alpha or thymosin alpha 1 showed no cross-reactivity. In spiking experiments the recovery of the assay was found to be greater than 92% at all concentrations tested. The intra-assay variation was 17%, whereas the inter-assay variation was 26%. Using this assay the highest concentration of parathymosin was found in porcine liver, followed by kidney, lung, thymus and spleen. This assay compares favorably with one microELISA and two RIA methods already published, in that it is more sensitive by at least an order of magnitude, and it is simpler and quicker.

Animals↗