Genistein-mediated inhibition of glycosaminoglycan synthesis, which corrects storage in cells of patients suffering from mucopolysaccharidoses, acts by influencing an epidermal growth factor-dependent pathway

<p>Abstract</p> <p>Background</p> <p>Mucopolysaccharidoses (MPS) are inherited metabolic disorders caused by mutations leading to dysfunction of one of enzymes involved in degradation of glycosaminoglycans (GAGs). Due to their impaired degradation, GAGs accumulate in cells of patients, which results in dysfunction of tissues and organs. Substrate reduction therapy is one of potential treatment of these diseases. It was demonstrated previously that genistein (4', 5, 7-trihydroxyisoflavone) inhibits synthesis and reduces levels of GAGs in cultures of fibroblasts of MPS patients. Recent pilot clinical study indicated that such a therapy may be effective in MPS III (Sanfilippo syndrome).</p> <p>Methods</p> <p>To learn on details of the molecular mechanism of genistein-mediated inhibition of GAG synthesis, efficiency of this process was studied by measuring of incorporation of labeled sulfate, storage of GAGs in lysosomes was estimated by using electron microscopic techniques, and efficiency of phosphorylation of epidermal growth factor (EGF) receptor was determined by using an ELISA-based assay with fluorogenic substrates.</p> <p>Results</p> <p>Effects of genistein on inhibition of GAG synthesis and accumulation in fibroblasts from patients suffering from various MPS types were abolished in the presence of an excess of EGF, and were partially reversed by an increased concentration of genistein. No such effects were observed when an excess of 17β-estradiol was used instead of EGF. Moreover, EGF-mediated stimulation of phsophorylation of the EGF receptor was impaired in the presence of genistein in both wild-type and MPS fibroblasts.</p> <p>Conclusion</p> <p>The results presented in this report indicate that the mechanism of genistein-mediated inhibition of GAG synthesis operates through epidermal growth factor (EGF)-dependent pathway.</p

Risk factors in ischemic stroke (preliminary study)

The aim of the study was the epidemiological analysis of patients with ischemic stroke hospitalised in the Department of Neurology Medical Academy in Gdańsk in 2000 year. We especially investigated the risk factors for ischemic stroke in order to improve our ability to prevent stroke. We studied 222 patients with ischemic stroke. There were 79 (35.5%) women and 143 (64.4%) men. The mean age of the patients was 68.05 &plusmn; 13.4 years and it was statistically significantly higher in women (72.05 &plusmn; 12.4) than in men (65.9 &plusmn; 13.4) (p = 0.001). Stroke was more severe in women (mostly TACS), than in men (mostly PACS) and it was statistically significant. The statistical analysis were made using computer program StatView version 5.5. The most often observed risk factors were hypertension (68%), morbus coronarius (50.8%), hypercholesterolemia (27.9%). Diabetes mellitus and alcoholism were recognised in lower percent of the cases. In 60 (26.1%) patients the presence of a few risk factors in one patients were observed. The patients arrival to the hospital was: within 3 h from onset of stroke - 24.6%, within 6 h - 43.7% of patients. Early presentation of patients with acute stroke to the hospital is important for immediate treating within limited therapeutic time window. The CT examination was made in 94.9% of patients and in 53.7% within 24 h of symptom onset. During hospitalisation 38.7% patients significantly improved and 13.8% patients died within 30 days of stroke beginning. Comparison of the examination of the patients using Rankin scale showed improvement of our patients after treatment.Celem pracy jest analiza epidemiologiczna chorych z udarem niedokrwiennym mózgu hospitalizowanych w Klinice Neurologii Dorosłych AMG. Szczególną uwagę zwrócono na analizę czynników ryzyka udaru mózgu pod kątem pierwotnej i wtórnej profilaktyki udaru. Badania przeprowadzono w ramach Narodowego Programu Udaru Mózgu. Analizy statystyczne przeprowadzono na podstawie komputerowego programu Statistica. Badano 222 chorych z udarem niedokrwiennym, w tym 79 (35,5%) kobiet i 143 (64,4%) mężczyzn hospitalizowanych w Klinice Neurologii Dorosłych AMG w 2000 roku. Wiek chorych wynosił średnio 68,05 &plusmn; 13,4 lata i był statystycznie znamiennie wyższy wśród kobiet (72,05 &plusmn; 12,4) niż wśród mężczyzn (65,9 &plusmn; 13,4) (p = 0,001). Udar mózgu jest cięższy w przebiegu u kobiet niż u mężczyzn (p = 0,001). Do najczęstszych czynników ryzyka udaru mózgu należały: nadciśnienie tętnicze (68%), choroba wieńcowa (50,8%), hipercholesterolemia (27,9%), stosunkowo rzadko występowała cukrzyca (19,5%). Znamienne było współwystępowanie kilku czynników ryzyka u 1 chorego. Chorzy docierali do Kliniki stosunkowo szybko: do 3 h od wystąpienia udaru - 24,6% chorych, do 6 h - 43,7% chorych. Tomografię komputerową (CT) głowy wykonano u 94,9% chorych, w tym u 53,7% w 1. dobie hospitalizacji. Zgon stwierdzono u 13,8% pacjentów, a wyraźną poprawę - u 38,7% chorych. Porównując stan neurologiczny chorych w momencie przyjęcia i wypisu w skali Rankina, stwierdzono znamienną poprawę stanu chorych po leczeniu

Effect of silicone on the collagen fibrillogenesis and stability

Collagen, the most abundant protein in mammals, is able to form fibrils, which have central role in tissue repair, fibrosis, and tumor invasion. As a component of skin, tendons, and cartilages, this protein contacts with any implanted materials. An inherent problem associated with implanted prostheses is their propensity to be coated with host proteins shortly after implantation. Also, silicone implants undergoing relatively long periods of contact with blood can lead to formation of thrombi and emboli. In this paper, we demonstrate the existence of interactions between siloxanes and collagen. Low-molecular-weight cyclic siloxane (hexamethylcyclotrisiloxane—D3) and polydimethylsiloxanes (PDMS) forming linear chains, ranging in viscosity from 20 to 12,000 cSt, were analyzed. We show that D3 as well as short-chain PDMS interact with collagen, resulting in a decrease in fibrillogenesis. However, loss of collagen native structure does not occur because of these interactions. Rather, collagen seems to be sequestered in its native form in an interlayer formed by collagen–siloxane complexes. On the other hand, silicone molecules with longer chains (i.e., PDMS with viscosity of 1000 and 12,000 cSt, the highest viscosity analyzed here) demonstrate little interaction with this protein and do not seem to affect collagen activity

Hyperhomocysteinemia - important risk factor for ischemic stroke

Udary mózgu są trzecią co do częstości przyczyną śmierci oraz najczęstszą przyczyną inwalidztwa w populacji osób dorosłych. Etiologia udarów często pozostaje nieustalona. Wśród licznych czynników etiopatogenetycznych udaru mózgu należy uwzględnić hiperhomocysteinemię, która odgrywa istotną rolę w patogenezie udaru mózgu jako czynnik wywołujący proces miażdżycowy i wpływający na zaburzenia krzepnięcia. Metabolizm homocysteiny zależy od wielu czynników, takich jak: stężenia kwasu foliowego, witamin B6 i B12 lub mutacji genów kodujących metabolizm homocysteiny: reduktazy N5, N10-metylenotetrahydrofolianowej (MTHFR, methylenetetrahydrofolate reductase), &#946;-syntazy cystationinowej (CBS, cystationine &#946; syntase) i syntazy metioninowej (MS, metionine syntase). Ocena stężenia homocysteiny u pacjentów z chorobami naczyniowymi, w tym u chorych z udarem mózgu, nie tylko poszerza wiedzę o etiologii i mechanizmach udarów mózgu, ale w sposób praktyczny wpływa na ich leczenie i zapobieganie im.Hyperhomocysteinemia is emerging as possible risk factor for cardiovascular disease, including cerebral stroke. Stroke is one of the leading causes of mortality and disability in Poland. The etiology of stroke is often unknown; it has been estimated that etiology and pathophysiology in more that 40% of strokes remains unexplained. Hyperhomocysteinemia is considered a modifiable risk factor for stroke, possibly because of atherogenic and prothrombotic mechanism. Both, genetics and environmental (e.g. dietary intake of folic acid and B vitamins) factors affects homocysteine level. One of the most common genetics defects of homocysteine metabolism is a mutation in the enzyme methylenetetrahydrofolate reductase (MTHFR), metionine syntase (MS) and cystationine beta syntase (CBS). Identification of the role of hyperhomocysteinemia as the modifiable risk factor for stroke may lead to more effective prevention of stroke through dietary and pharmacological modification of homocysteine level

Lipophagy and lipolysis status in lipid storage and lipid metabolism diseases

This review discusses how lipophagy and cytosolic lipolysis degrade cellular lipids, as well as how these pathway ys communicate, how they affect lipid metabolism and energy homeostasis in cells and how their dysfunction affects the pathogenesis of lipid storage and lipid metabolism diseases. Answers to these questions will likely uncover novel strategies for the treatment of aforementioned human diseases, but, above all, will avoid destructive effects of high concentrations of lipids—referred to as lipotoxicity—resulting in cellular dysfunction and cell death

Metal and antibiotic resistance of bacteria isolated from the Baltic Sea

The resistance of 49 strains of bacteria isolated from surface Baltic Sea waters to 11 antibiotics was analyzedand the resistance of selected strains to three metal ions (Ni2+, Mn2+, Zn2+) was tested. Most isolates belonged to Gammaproteobacteria (78 %), while Alphaproteobacteria (8 %), Actinobacteria (10 %), and Bacteroidetes (4 %) were lessabundant. Even though previous reports suggested relationships between resistance and the presence of plasmids or the abilityto produce pigments, no compelling evidence for such relationships was obtained for the strains isolated in this work. In particular, strains resistant to multiple antibiotics did not carry plasmids more frequently than sensitive strains. A relationbetween resistance and the four aminoglycosides tested (gentamycin, kanamycin, neomycin, and streptomycin), but not tospectinomycin, was demonstrated. This observation is of interest given that spectinomycin is not always classified as anaminoglycoside because it lacks a traditional sugar moiety. Statistical analysis indicated relationships between resistance tosome antibiotics (ampicillin and erythromycin, chloramphenicol and erythromycin, chloramphenicol and tetracycline, erythromycinand tetracycline), suggesting the linkage of resistance genes for antibiotics belonging to different classes. The effectsof NiSO4, ZnCl2 and MnCl2 on various media suggested that the composition of Marine Broth might result in low concentrationsof Mn2+ due to chemical interactions that potentially lead to precipitation. [Int Microbiol 2012; 15(3):131-139

Nonsteroidal anti-inflammatory drugs modulate cellular glycosaminoglycan synthesis by affecting EGFR and PI3K signaling pathways

In this report, selected non-steroidal anti-inflammatory drugs (NSAIDs), indomethacin and nimesulide, and analgesics acetaminophen, alone, as well as in combination with isoflavone genistein as potential glycosaminoglycan (GAG) metabolism modulators were considered for the treatment of mucopolysaccharidoses (MPSs) with neurological symptoms due to the effective blood-brain barrier\ud (BBB) penetration properties of these compounds. We found that indomethacin and nimesulide, but not acetaminophen, inhibited GAG synthesis in fibroblasts significantly, while the most pronounced impairment of glycosaminoglycan production was observed after exposure to the mixture of nimesulide and genistein. Phosphorylation of the EGF receptor (EGFR) was inhibited even more effective in the presence of indomethacin and nimesulide than in the presence of genistein. When examined the activity of phosphatidylinositol-3-kinase (PI3K) production, we observed its most significant decrease in the case of fibroblast exposition to nimesulide, and afterwards to indomethacin and genistein mix, rather than indomethacin used alone. Some effects on expression of individual GAG metabolism-related and lysosomal function genes, and significant activity modulation of a number of genes involved in intracellular signal transduction pathways and metabolism of DNA and proteins were detected. This study documents that NSAIDs, and their mixtures with genistein modulate cellular glycosaminoglycan synthesis by affecting EGFR and PI3K signaling pathways

Synthetic genistein derivatives as modulators of glycosaminoglycan synthesis

Background: Mucopolysaccharidoses (MPS) are severe metabolic disorders caused by 26 accumulation of undegraded glycosaminoglycans (GAGs) in lysosomes due to defects in certain 27 lysosomal hydrolases. Substrate reduction therapy (SRT) has been proposed as one of potential 28 treatment procedures of MPS. Importantly, small molecules used in such a therapy might 29 potentially cross the blood-brain barrier (BBB) and improve neurological status of patients, as 30 reported for a natural isoflavone, 5, 7-dihydroxy-3- (4-hydroxyphenyl)-4H-1-benzopyran-4-one, 31 also known as genistein. Although genistein is able to cross BBB to some extent, its delivery to 32 the central nervous system is still relatively poor (below 10% efficiency). Thus, we aimed to 33 develop a set of synthetically modified genistein molecules and characterize physicochemical as 34 well as biological properties of these compounds. Methods: Following parameters were 35 determined for the tested synthetic derivatives of genistein: cytotoxicity, effects on cell 36 proliferation, kinetics of GAG synthesis, effects on epidermal growth factor (EGF) receptor’s 37 tyrosine kinase activity, effects on lysosomal storage, potential ability to cross BBB. Results: We 38 observed that some synthetic derivatives inhibited GAG synthesis similarly to, or more 39 efficiently than, genistein and were able to reduce lysosomal storage in MPS III fibroblasts. The 40 tested compounds were generally of low cytotoxicity and had minor effects on cell proliferation. 41 Moreover, synthetic derivatives of genistein revealed higher lipophilicity (assessed in silico) than 42 the natural isoflavone. Conclusion: Some compounds tested in this study might be promising 43 candidates for further studies on therapeutic agents in MPS types with neurological symptoms

The model homologue of the partially defective human 5,10-methylenetetrahydrofolate reductase, considered as a risk factor for stroke due to increased homocysteine level, can be protected and reactivated by heat shock proteins

The A222V substitution in the human MTHFR gene product (5,10-methylenetetrahydrofolate reductase) is responsible for a decreased activity of this enzyme. This may cause an increased homocysteine level, considered as a risk factor for arteriosclerosis and stroke. The bacterial homologue of the human enzyme, MetF, has been found to be a useful model in genetic and biochemical studies. The similarity of Escherichia coli MetF and human MTHFR proteins is so high that particular mutations in the corresponding human gene can be reflected by the bacterial mutants. For example, the A222V substitution in MTHFR (caused by the C667T substitution in the MTHFR gene) can be ascribed to the A117V substitution in MetF. Here, it is reported that a temperature-sensitive MetF117 (A117V) protein can be partially protected from a thermal inactivation by the heat shock proteins from the Hsp70/100 systems. Moreover, activity of the thermally denatured enzyme can be partially restored by the same heat shock proteins. High temperature protein G (HtpG) had no effect on MetF117 activity in both experimental systems. The presented results indicate that functions of heat shock proteins may be required for maintenance of the MetF117 function. This may have implications for the mechanisms of arteriosclerosis and stroke, especially in the light of previous findings that the A222V MTHFR polymorphism may be a risk factor for stroke, as well as recently published results which demonstrated the increased levels of antibodies against heat shock proteins in stroke patients