Mitochondrial respiration of adipocytes differentiating from human mesenchymal stem cells derived from adipose tissue.

Burden of obesity is increasing in the contemporary world. Although multifactorial in origin, appropriate mitochondrial function of adipocytes emerges as a factor essential for healthy adipocyte differentiation and adipose tissue function. Our study aimed to evaluate mitochondrial functions of human adipose-derived mesenchymal stem cells committed to adipogenesis. On days 0, 4, 10, and 21 of adipogenesis, we have characterized adipocyte proliferation and viability, quantified lipid accumulation in maturing cells, performed qualitative and quantitative analysis of mitochondria, determined mitochondrial respiration of cells using high-resolution respirometry, and evaluated mitochondrial membrane potential. In the course of adipogenesis, mitochondrial oxygen consumption progressively increased in states ROUTINE and E (capacity of the electron transfer system). State LEAK remained constant during first days of adipogenesis and then increased probably reflecting uncoupling ability of maturing adipocytes. Citrate synthase activity and volume of mitochondrial networks increased during differentiation, particularly between days 10 and 21. In addition, lipid accumulation remained low until day 10 and then significantly increased. In conclusion, during first days of adipogenesis, increased mitochondrial respiration is needed for transition of differentiating cells from glycolytic to oxidative metabolism and clonal expansion of preadipocytes and then more energy is needed to acquire typical metabolic phenotype of mature adipocyte.

Polychlorinated biphenyl 153 in lipid medium modulates differentiation of human adipocytes.

Emerging evidence indicates that polychlorinated biphenyls (PCBs) are involved in the development of diabetes mellitus in the obese. The purpose of this study was to determine mechanisms by which PCB 153 (2,2´,4,4´,5,5´-hexachloro-biphenyl) could influence diet-induced obesity and insulin resistance during adipogenesis. Lineage of h-ADMSCs was differentiated either as control (differentiation medium only), or with lipid vehicle modeling high fat nutrition (NuTRIflex) or lipid free vehicle (dimethylsulfoxide) for 28 days with or without PCB 153 daily co-exposure (in three concentrations 0.1, 1, and 10 microM). Gene expression analyses were performed using RT-qPCR at days 4, 10, 21, 24, 28; protein levels Akt and phosphorylated Akt (Phospho-Akt) by Western blot at days 4, and 21. PCB 153 treatment of h-ADMSCs only in lipid vehicle was associated with down regulation of key master genes of adipogenesis: PPARgamma, SREBP-1, PPARGC1B, and PLIN2 during the whole process of differentiation; and with increased Akt and decreased Phospho-Akt protein level at day 21. We have shown that PCB 153, in concentration 0.1 microM, has a potential in lipid rich environment to modulate differentiation of adipocytes. Because European and U.S. adults have been exposed to PCB 153, this particular nutrient-toxicant interaction potentially impacts human obesity and insulin sensitivity.

Effect of long-term treatment on subjective problems of patients with common thyroid gland disorders in the Czech Republic.

In this study, a total of 364 patients of the Endocrinology outpatient clinic who had undergone thyreopathy treatment of various lengths were investigated. The objective of the study was to find out the consequences of thyroid gland disorder on a person's life, and whether the treatment length has any effect on subjective problems of patients. Common problems reported by patients were evaluated; the final number of such "common problems" in the final stage of the research was 56. In order to achieve better orientation in the processing of results, the patient-described types of disorders were classified according to the patients' own opinions into the following groups: eufunctional thyroid gland, inflammation, hypothyroidism, hyperthyroidism, tumour and surgery. The fact is that in the first year of treatment, almost half of all patients with a thyroid gland disorder experienced some problems caused by the disease. It is not a favourable finding that in the upcoming years, i.e. from 2 to 10 years, the number of those with problems will increase to as much as 61%. The period of treatment between 11 and 15 years means a decrease in the occurrence of problems to 50%, but with the number of patients with problems increasing, their number becomes even higher than that in the period of 2 to 10 years. Influence of aging processes should be also taken into account.

The prevalence of metabolic syndrome and its components in two main types of primary aldosteronism.

Metabolic syndrome (MS) is frequent clinical condition in patients with hypertension. Primary aldosteronism (PA) is a common form of secondary hypertension. This study was aimed at investigating the prevalence of the MS and its components in the two major forms of PA, in unilateral aldosterone-producing adenoma (APA) and bilateral aldosterone overproduction because of idiopathic hyperaldosteronism (IHA). The diagnosis of the particular form of PA was based on adrenal venous sampling and/or successful surgery confirmed by histopathological examination. We analyzed clinical and laboratory data from 100 patients with PA (50 patients with IHA and 50 patients with APA) and from 90 patients with essential hypertension (EH). Metabolic profiles of patients with bilateral form of PA (because of IHA) were similar to EH, but differed from those in patients with unilateral form of PA (APA). The prevalence of the MS (62% in IHA, 34% in APA and 56% in EH), the body mass index value (30±4 kg m(-2) in IHA, 27±5 kg m(-2) in APA and 29±5 kg m(-2) in EH) and triglycerides levels (1.9±0.9 mmol l(-1) in IHA,1.4±0.8 mmol l(-1) in APA and 2.01±1.39 mmol l(-1) in EH) were all significantly (P<0.05) higher in IHA compared with APA patients. Metabolic profile of patients with bilateral form of PA (because of IHA) is similar to EH in contrast to unilateral form of PA (APA).

Moderate hyperhomocysteinemia in patients treated for epilepsy.

Homocystein (Hcy) is regarded as a neuroexcitatory substance, which is therefore used as an epileptogenic agent in experimental epileptology. Experiments "in vivo" as well as "in vitro" revealed its relation to NMDA glutamate receptors, and its potential neurotoxicity. From the clinical aspect, hyperhomocysteinemia (HHcy), mostly as a marker of the risk factor in the vascular damage, was often studied in patients treated with antiepileptic drugs (AE). However, the neuroexcitatory influence of mild HHcy (up to 30 micromol/l) was rarely discussed. Out of a group of 123 adult patients on long-term conventional AE we analyzed 8 patients (7 men and one woman) with moderate to severe HHcy (30.7-109.0 micromol/l) retrospectively and 2-5 years after HHcy normalization. All of them suffered from partial and/or secondary generalized seizures accompanied by neuropsychological impairment depending on the aetiology of the disease. The patients were characterized by a concurrence of several factors: (1) All of them received conventional AEs inducing the cytochrome P 450 at the time HHcy was diagnosed. (2) Molecular-genetic tests showed enzymopathic impairment (methylentetrahydrofolate reductase-MTHFR mutation of the gene C677 T) also in all eight, homozygous in 7 cases and heterozygous in 1 case. (3) All patients were found to have a vitamin deficit or marginal values of at least one of the vitamins under study, especially folate and/or vitamin B6 and 812. With reference to clinical and EEG features, the potential neuroexcitatory influence of Hcy is discussed taking into account its effect on pathogenetic factors.

[Pharmacological aspects of Lyme borreliosis]. / Farmakologické aspekty lymeské borreliózy.

Clinical signs of Lyme boreliosis in humans are versatile and in their whole scope they finally affect the nervous system, heart, and joints. The therapeutic effect of antibiotics is maximal in the first acute stage of the disease when doxycycline and amoxiciline are administered. These antibiotics possess a comparable in vitro effect, tissue penetration, pharmacokinetics, and therapeutic effect. The treatment of disseminated infections in the second stage, such as neuroborreliosis, carditis, and iritis, is difficult and with relative success they are treated with large doses of penicillin G, or cefriaxon, and doxycycline. The treatment of the third stage of borreliosis aims at chronic inflammatory changes in the affected organs. Antibiotics, however, are successfully effective only in 50% of cases. Administration of antibiotics, such as tetracycline, cefuroxim, doxycycline, or large doses of penicillin is a long-term one, coming up to four weeks. A special therapeutic regimen is used in pregnant women and children.

Levels of apolipoprotein A-II in cerebrospinal fluid in patients with neuroborreliosis are associated with lipophagocytosis.

Levels of most of the examined proteins in cerebrospinal fluid (CSF) of 107 patients with neuroborreliosis were associated with cytological findings, the status of hematoencephalic barrier as evaluated by Qalb (cerebrospinal fluid to serum quotient) and the intrathecal synthesis of immunoglobulins. Cytological findings consisted of normal cytology, or both oligocytosis and pleocytosis of monocytes or lymphocytes. The lipophagic elements were present in 20% of samples. Concentrations of apolipoproteins A-I and A-II in the CSF were correlated with the concentration of albumin without regard to the CSF cytology. The levels of apolipoprotein B were increased only in samples with lymphocytic pleocytosis and Qalb > 7.4. The presence of lipophages in the CSF was significantly associated with the CSF concentration of apolipoprotein A-II.

[Homocysteinemia--its significance in gynecology and obstetrics]. / Homocysteinemie--její význam v gynekologii a porodnictví.

OBJECTIVE: Metabolic study on plasmatic levels of homocysteine (Hcy) in healthy women during normal or pathological pregnancy accompanied with corresponding levels of Hcy in amniotic fluid and foetal sera. Increased levels of Hcy--hyperhomocysteinaemia is respected as an independent risk factor accelerating the early development of vessel damage and causing the neural tube defects (NTD). DESIGN: Basic study to get our own data about Hcy in Czech healthy and population at risk of pregnant and non-pregnant women. SETTING: Department of Obstetrics and Gynaecology, 1st Faculty of Medicine, Charles University, Prague. METHODS: Total homocysteine in plasma, amniotic fluid and foetal sera was estimated by chromatographic method with use of fluorescence detection. RESULTS: Normal homocysteine in preclimacteric healthy nonpregnant women is: 9.7 +/- 1.6 mumol/l with evident age-dependence. In healthy climacteric women are higher levels of Hcy (corresponding to the men values): 11.8 +/- 2.6 mumol/l. After use of hormonal contraceptives the plasmatic levels of Hcy decrease: 7.2 +/- 2.0 mumol/l. In physiological pregnancies Hcy reachs the lowest values: 4.4 +/- 1.7 mumol/l with any evident oscillations during pregnancy. In women in childbed period was Hcy 8.4 +/- 2.1 mumol/l observed. In pathological pregnanciesare its levels slightly elevated: 6.3 +/- 2.1 mumol/l, most evident in placental abruptions: 7.5 +/- 1.7 mumol/l. In pregnant women with susp. results of screening on M. Down only unsignificant increase of Hcy was observed: 6.12 +/- 2.4 mumol/l. In amnial fluids of healthy pregnant women are levels of Hcy are quite low: 4.1 +/- 1.2 mumol/l with any oscillations during pregnancy. In foetal sera of pregnancies at risk (NTD, susp., trisomy, inborn errors of metabolism): 3.6 +/- 1.4 mumol/l of Hcy was detected. The foetoplacental quotient for Hcy is 0.62. CONCLUSION: Average values for Hcy were established in physiological as well as in pathological pregnancies and till now only limited diagnostic significance has been observed. The hyperhomocysteinaemia mentioned in previous papers was not in NTD observed because our pregnant patients were regularly supplemented with all critical vitamins (folate, B6, B12).

Formation of myo-inositol phosphates by Aspergillus niger 3-phytase.

Kinetics of phytate hydrolysis by Aspergillus niger phytase and correlation between the amount of released phosphate and creation of lower myo-inositol phosphates were investigated. Phytase was able to hydrolyze myo-inositol hexakis-, pentakis-, tetrakis-, and trisphosphates. Finally, about 56% of total phosphate were released and myo-inositol bisphosphate was detected as the end-product.

Phytase: sources, preparation and exploitation.

This review deals with phytase (myo-inositol hexakisphosphate phosphohydrolase) and covers microbiological sources, phytase occurrence in plants and animals, its purification, physico-chemical and molecular properties. Protein engineering of phytase and potential enzyme applications are discussed.

NKR-P1A protein, an activating receptor of rat natural killer cells, binds to the chitobiose core of uncompletely glycosylated N-linked glycans, and to linear chitooligomers.

NKR-P1 represent a family of activating receptors in rodent natural killer cells related to C-type animal lectins. We identify here the elements involved in the reactivity of the major receptor of rat, NKR-P1A, with N-linked oligosaccharides of glycoproteins. Plate inhibition assays with isolated, structurally defined N-glycans as inhibitors of binding of NKR-P1A to GlcNAc16-BSA revealed that the removal of both the external sialic acids and the penultimate galactose residues resulted in attaining of significant inhibitory activities. Surprisingly, additional plate inhibition and glycoprotein overlay experiments brought evidence that the core chitobiose, depending on its substitution, can per se support the interaction with NKR-P1A. In a series of linear chitooligomers (n = 2-7), the inhibitory activities reached a maximum for the chitotetraose. The ability of NKR-P1 to recognize both the periphery and the core region of complex type oligosaccharides may define its dual specificity towards carbohydrate components of eukaryotic (e.g., tumor) cell surfaces, but also reflect an evolutionarily conserved reactivity with microbial saccharides important in immune recognition and signaling functions.

Characterization of phytase produced by Aspergillus niger.

The extracellular activity of Aspergillus niger phytase at the end of the growth phase was 132 nkat/mL in a laboratory bioreactor. The purified enzyme has molar mass approximately 100 kDa, pH optimum at 5.0, temperature optimum at 55 degrees C and high pH and temperature stability. The Km for dodecasodium phytate, calcium phytate and 4-nitrophenyl phosphate are 0.44, 0.45 and 1.38 mmol/L, respectively. The enzyme is noncompetively inhibited by inorganic monophosphate (Ki = 2.85 mmol/L) and by Cu2+, Zn2+, Hg2+, Sn2+, Cd2+ ions and strongly by F- ones; it is activated by Ca2+, Mg2+ and Mn2+ ions. The substrate specificity of phytase is broad with the highest affinity to calcium phytate.

beta-Glucosylation of chitooligomers by galactosyltransferase.

Galactosyltransferase from bovine milk was found to be able to utilise UDP-Glc to transfer Glc onto GlcNAc and chitooligomers[-beta-GlcNAc-(1-->4)-]n, n = 2-4. beta-Glucosylated products were used in binding studies with NKR-P1A protein cloned from rat natural killer cells.

[Hyperhomocysteinemia. A risk factor for the development of vascular diseases not associated with lipid levels]. / Hyperhomocysteinémie. Rizikový faktor pro vznik cévních onemocnení nezávislý na hladinách lipidu.

BACKGROUND: Elevated total homocysteine plasma levels are considered a significant factor of vascular damage. As they are encountered in more than half the patients with atherosclerotic vascular damage the importance as a lipid-dependent or lipid-independent risk factor in the promotion of pathophysiological processes is discussed. METHODS AND RESULTS: In a group of 100 healthy subjects and 529 patients with indication for an aortocoronary or peripheral arterial bypass and in patients from the lipid clinic the mutual relation between total plasma homocysteine levels and selected indicators of the lipid metabolism was investigated. The following results more obtained: for total cholesterol a correlation coefficient of r = 0.26, for HDL-cholesterol r = 0.20, for LDL-cholesterol r = 0.21, for triacylglycerols r = 0.29, apolipoprotein A-I r = 0.06, apolipoprotein B r = -0.12 and for Lp(a) r = -0.03. To ensure correct evaluation of the homocysteine levels simultaneously also folate levels were examined (correlation coefficient r = 0.28), vitamin B12 r = (0.03) and fibrinogen r = (0.09). CONCLUSIONS: The authors did not detect an unequovical relationship between the total homocysteine level and selected lipid indicators in any of the patient groups (p < 0.05).

Lack of evidence for the interaction between renin-angiotensin-aldosterone system and endothelin in vivo.

Aim of the study was to reveal the possible factors regulating plasma endothelin (ET) levels in vivo in patients with essential hypertension (EH) by the simultaneous determination of plasma renin activity (PRA) and plasma aldosterone (ALD). In addition, the possible relationship between ET and circulating endothelial cells as a marker of endothelial damage was also investigated. The postural test revealed a significant increase of ET levels (26.7 +/- 9 vs 11.5 +/- 3 fmol/ml, p < 0.05) in the upright position. Captopril administration did not change plasma ET levels. No significant correlation was found between ET and PRA or ALD. Although a tendency to a positive correlation between ET and circulating endothelial cells (as the marker of endothelial perturbation) was found, it did not attain statistical significance. Our data do not support the suggestion that the renin-angiotensin-aldosterone system plays a major role in the regulation of ET secretion in vivo in EH. Postural stimulation of ET secretion may be caused by other factors than renin-angiotensin-aldosterone system.

Phytase from Aspergillus niger.

132 microorganisms, isolates from soil and decayed fruits, were tested for phytase production. All isolates intensively producing active extracellular phytase were of fungal origin. The most active fungal isolates with phytase activity were identified as Aspergillus niger. At the end of the growth phase, the extracellular phytase activity produced by A. niger strain 92 was 132 nkat/mL, with strain 89 it was 53 nkat/mL. In both strains the extracellular enzyme activity exhibited two marked activity optima at pH 1.8 and 5.0 and a temperature optimum at 55 degrees C.

Pharmacokinetics and tolerance of 7-methoxytacrine following the single dose administration in healthy volunteers.

7-methoxy-tetrahydroaminoacridine (7-MEOTA) is a new reversible cholinesterase inhibitor. Forty-eight young male volunteers divided into six dosage groups were included into a single-dose pharmacokinetic study with either oral (p.o.) or intramuscular (i.m.) administration. The dose of 7-MEOTA was 2, 4 or 8 mg/kg body weight p.o. or 0.5, 1 or 2 mg/kg body weight i.m. in the respective six dosage groups. The plasma levels data were fitted to an open one-compartmental model. The compound showed cholinomimetic adverse effects in 2 subjects with the blood levels exceeding 1,500 micrograms/l. The red blood cells levels paralleled those in plasma and were 2.5 times higher. The tmax was 4 hours and 1 h, t1/2 8.7 +/- 3.9 hours and 6.5 +/- 5.8 hours in case of p.o. and i.m. administration, respectively. The apparent clearance (D/AUC) was 5 times higher following p.o. administration, reflecting the differences in bioavailability.

[Relation between levels of atrial natriuretic factor and circulating plasma volume]. / Závislost koncentrace atriálního natriuretického faktoru na objemu cirkulující plazmy.

The authors investigated in a group of 50 subjects the influence of changes in the volume of circulating plasma on the feedback regulation of plasmatic concentrations of the atrial natriuretic factor (ANF). They proved a fairly close direct correlation between the plasma concentration of ANF and the plasma volume in litres (r = 0.465, p less than 0.01) and when expressed in percent of body weight (r = 0.546, p less than 0.01). The rising concentration of ANF with the patients' age (r = 0.381, p less than 0.01) may be due to actual age conditioned changes or the more frequent incidence of pathological conditions in more advanced age groups, leading to an increase of the ANF plasma level. The results indicate that in addition to pressure changes and distension of the atria in the regulation of the plasma concentration of ANF also changes in the circulating plasma and blood volume participate which thus influence by a feedback mechanism the volume homeostasis.