Quercetin suppresses heat shock-induced nuclear translocation of Hsp72

The effect of quercetin and heat shock on the Hsp72 level and distribution in HeLa cells was studied by Western blotting, indirect immunofluorescence and immunogold electron microscopy. In control cells and after quercetin treatment, Hsp72 was located both in the cytoplasm and in the nucleus in comparable amounts. After hyperthermia, the level of nuclear Hsp72 raised dramatically. Expression of Hsp72 in cytoplasm was also higher but not to such extent as that observed in the nucleus. Preincubation of heated cells with quercetin inhibited strong Hsp72 expression observed after hyperthermia and changed the intracellular Hsp72 distribution. The cytoplasmic level of protein exceeded the nuclear one, especially around the nucleus, where the coat of Hsp72 was noticed. Observations indicating that quercetin was present around and in the nuclear envelope suggested an involvement of this drug in the inhibition of nuclear translocation. Our results indicate that pro-apoptotic activity of quercetin may be correlated not only with the inhibition of Hsp72 expression but also with suppression of its migration to the nucleus.     

31P MRS analysis of the phospholipid composition of normal human peripheral blood mononuclear cells (PBMC)

The aim of this investigation was to characterize the phospholipid composition of normal human blood mononuclear cells using 31P NMR spectroscopy. Mononuclear cells of peripheral blood were obtained from 10 volunteers. Phospholipid extracts were prepared from 60x10(6) cells according to modified Folch's method. An AMX 300 Bruker spectrometer 7.05 T was used. The 31P spectrum of phospholipid extracts from normal human PBMC consisted of 9 peaks, with one each for phosphatidylcholine (PC), plasmalogen of phosphatidylcholine (CPLAS), lysophosphatidylcholine (LPC), sphingomyelin (SM), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS) and cardiolipin (CL), and another one due to the external reference substance, methylenediphosphonic acid (MDPA). The concentrations of these phospholipids (PL), based on the integral intensities, were as follows: 0.398 +/- 0.078 mmole/l for PC; 0.033 +/- 0.019 mmole/l for CPLAS; 0.155 +/- 0.043 mmole/l for SM; 0.266 +/- 0.104 mmole/l for PI+PE; 0.101 +/- 0.040 mmole/l for PS, and 0.026 +/- 0.033 mmole/l for CL. The results of this study confirmed that 31P MRS is a convenient tool for measuring the phospholipid concentrations of biological samples.     

Correlation of endostatin and tissue inhibitor of metalloproteinases 2 (TIMP2) serum levels with cardiovascular involvement in systemic sclerosis patients

Fibrosis of oesophagus, lungs, heart, and kidney in the course of systemic sclerosis (SSc) may lead to dysfunction of the above organs or even patients death. Recent studies point out the role of angiogenesis and fibrosis disturbances in the pathogenesis of SSc. Heart fibrosis is one of the most important prognostic factors in SSc patients. So, the aim of our study was to examine cardiovascular dysfunction in SSc patients and its correlation with serum levels of vascular endothelial growth factor (VEGF), endostatin, and tissue inhibitor of metalloproteinase 2 (TIMP2). The study group comprised 34 patients (19 with limited scleroderma (lSSc) and 15 with diffuse scleroderma (dSSc)). The control group consisted of 20 healthy persons, age and sex matched. Internal organ involvement was assessed on the basis of specialist procedures. Serum VEGF, endostatin, and TIMP2 levels were evaluated by ELISA. We found cardiovascular changes in 15 patients with SSc (8 with lSSc and 7 with dSSc). The observed symptoms were of different characters and also coexisted with each other. Higher endostatin serum levels in all systemic sclerosis patients in comparison to the control group were demonstrated (P < .05). Also higher serum levels of endostatin and TIMP2 were observed in patients with cardiovascular changes in comparison to the patients without such changes (P < .05). The obtained results support the notion that angiogenesis and fibrosis disturbances may play an important role in SSc. Evaluation of endostatin and TIMP2 serum levels seems to be one of the noninvasive, helpful examinations of heart involvement in the course of systemic sclerosis.     

Acetylcholinesterase--apoptosis induction, role in neurological diseases and leukemia

Acetylcholinesterase (AChE - EC. 3.1.1.7) plays an essential role in acetylcholine-mediated neurotransmission. Unfortunately, an AChE-peptide exhibits pathophysiological activity via an apoptotic pathway that could play an important role in neuronal development and neurodegeneration. It was found that a peptide derived from AChE may induce neuronal death and acetylcholinesterase may induce neurological changes in the development of Alzheimer's disease. It was also stated that complex of AChE with beta-amyloid is much more toxic than amyloid and causes stronger neurological changes. AChE promotes the generation of amyloid by accelerating the expression of peptide precursor (beta-APP) in glial cells. The essential role is also played by AChE in induction of hematological disease. It is well known that phospho-organic compounds cause inhibition of AChE precursors what is related to decrease of hemoglobin concentration, number of erythrocytes and hematocrit level. The article is an attempt to explain the role of acetylcholinesterase in neuronal apoptosis, Alzheimer's disease and Myasthenia gravis as well as in leukemia.     

N-glycodiversity of the Pregnancy-Associated Glycoprotein family (PAG) produced in vitro by trophoblast and trophectoderm explants during implantation, placentation and advanced pregnancy in the pig

The PAG family is encoded by distinct genes expressed in extra-embryonic chorionic membranes (TR--trophoblast, TRD--trophectoderm) of various pregnant mammals. The objective of our study was to determine N-glycodiversity of porcine PAG protein family (pPAG) produced in vitro by TR or TRD explants of gilts (n=26) throughout pregnancy (16-77 dpc). Explants were cultured for over 1200 h (TR, 16 dpc) or for 8 h (TRD, 17-77 dpc). Released proteins were isolated from media by separating ultra-filtration (>10 kDa). A deglycosylation (removal of N-linked carbohydrate side chains) of proteins was performed by glycopeptidase F, and compared to non-deglycosylated forms by PAGE-Western blotting with anti-pPAG sera and additionally to polypeptide pPAG precursors, coded by ORF of their cloned cDNAs. We demonstrated gestation-stage dependent diversity of deglycosylated/glycosylated forms of the pPAG proteins produced in vitro in the pig. TR explants harvested on 16 dpc during long term culture released 43 kDa pPAG proteins. These proteins were deglycosylated to approximately 36.9 and approximately 39.6 kDa (16 dpc). Tissue harvested on 17 dpc in vitro secreted 65-68 kDa pPAG proteins which were reduced to three forms, 50.6, 58.7 and 63.5 kDa. In addition, approximately 73.3 kDa major pPAG proteins (77 dpc) were reduced to at least three forms: approximately 39.6, approximately 36.9 and approximately 33.4 kDa. Such N-deglycosylation was not detected on days 25-61. N-deglycosylation of native pPAG proteins clearly corresponded to three N-glycosylation sites of asparagines (N-x-S/T) found in ORF of the pPAG2-like precursors, identified by their in silico translated cDNAs. Thus, the pregnancy-stage dependent N-glycodiversity of the pPAG protein family, containing an average 9.66% of N-linked oligosaccharides, may play some role(s) in porcine conceptus attachment, successful implantation and during advanced pregnancy.     

Primary antisera against selected steroids or proteins and secondary antisera against gamma-globulins--an available tool for studies of reproductive processes

This paper presents characteristics of different polyclonal antisera raised against several steroid and protein antigens: 1/ primary antisera against steroid hormones: estradiol-17beta (anti-E2), estrone (anti-E1), testosterone (anti-T), androstendione (anti-A4), cortisol (anti-F) and corticosterone (anti-B); 2/ primary antisera against porcine luteinizing hormone (anti-pLH) and against different forms of porcine pregnancy associated glycoproteins (anti-pPAG) - proteins produced by chorionic tissue; 3/ secondary monovalent antisera raised against rabbit gamma-globulins (Sm-r); 4/ secondary polyvalent antisera against rabbit, pig and quinea pig gamma-globulins mixed at a ratio 1:1:1 (Sp-rpq). All antisera described in the paper present sufficient quality to be routinely used in various RIA, ELISA or Western determinations in physiological and clinical studies of reproductive processes. The antisera against steroid hormones and pLH are available on request.     

Protein A of quinolinate synthetase is the site of oxygen poisoning of pyridine nucleotide coenzyme synthesis in Escherichia coli.

De novo biosynthesis of pyridine nucleotide coenzymes in Escherichia coli is initiated by an enzyme complex (quinolinate synthetase) containing protein B which converts -aspartate into iminoaspartate protein A, which then generates quinolinate on the pathway to the coenzymes. This complex has been shown to be poisoned by hyperbaric oxygen. ^7,8 We performed assays made dependent on both proteins B and A versus only protein A, using cell-free extracts of hyperbaric-oxygen poisoned and aerobically grown cells. The specific activities were produced by a similar amounts of 68% and 60%, respectively, when measured in assays made dependent on enzymes B and A versus only protein A that was derived from oxygen-poisoned extract. Thus, protein A is the oxygen-sensitive component.     

Peroxidase activity in non-embryogenic and embryogenic calli and in developing somatic embryos of white fir (Abies concolor Gord. et Glend)

ABSTRACT

Peroxidase activity was monitored during somatic embryogenesis of white fir (Abies concolor Gord. et Glend) starting from a non-embryogenic callus. Results revealed profound differences between non-embryogenic and embryogenic calli with an elevated level of enzyme activity in non-embryogenic ones. Precotyledonary, early cotyledonary and late cotyledonary stages of somatic embryogenesis were characterized by a substantially reduced peroxidase activity compared to callus tissues and regenerated plantlets. Changes in peroxidase activity are as a rule paralleled by variation in isoenzyme composition. The utility of the enzyme in the induction stage of somatic embryogenesis in white fir is proposed.