Parkin, PINK1 and DJ1 as possible modulators of mTOR pathway in ganglioglioma.

PURPOSE: Ganglioglioma (GG) is a non-malignant tumor classified as G1 by the WHO. Although we currently know that the neoplasm may result from the hyperactivity of protein kinase B (PKB or Akt) or extracellular-regulated kinase (Erk), which upregulates mammalian target of rapamycin kinase (mTOR) and leads to translation of proteins responsible for cell cycle regulation, there are still many questions to be answered. In the current paper we try to analyze the link between GG formation and activity of three proteins known to play a role in neuroprotection (parkin, PINK1 and DJ1). MATERIALS AND METHODS: In our paper, we review the current information on the involvement of these proteins in the transmission of information in the cell and triggering various cell signals, like survival or apoptosis. We also review current literature data on involvement of parkin, DJ1 and PINK1 in the regulation of mTOR, the pathway probably contributing to the development of GG. RESULTS: Parkin is an E3 ubiquitin ligase, shown to trigger proteasome-dependent degradation and autophagy, necessary for the maintenance of homeostasis in neurons. PINK1, a mitochondrial protein kinase, is required for mitochondrial maintenance and neuronal survival. DJ1 is a sensor of reactive oxygen species, and protects the cells against oxidative stress. Mutations in the genes encoding these three proteins are known to underlie autosomal recessive forms of Parkinson's disease, as well as other neurodegenerative and neuroinflammatory disorders. CONCLUSION: It appears that mutations of parkin, PINK1 and DJ1 may result in the development of both neurodegeneration and tumors. Also, these proteins might be used as markers of disease, thus allowing better diagnosis and therapy.

Prevalence of Urogenital Mycoplasmas Among Men with NGU in Upper Silesia, Poland. Preliminary Study.

The prevalence of urogenital mycoplasmas in men with NGU in Upper Silesia (Poland) was studied. Mycoplasmas were detected in 36.7% men (Ureaplasma parvum and Mycoplasma genitalium were found in 30% and 16.7% respectively). Urealyticum urealyticum was not detected. We suggest including M. genitalium in the diagnostic scheme for nongonococcal urethritis (NGU).

Angiomodulatory properties of some antibiotics and Tolpa Peat Preparation.

UNLABELLED: Deterioration of the immune system due to antibiotic therapy can be restored by immunomodulator application. In this paper we estimate the effect of ampicillin, amikacin, doxycycline, rifampicin, rifamycine and immunomodulator Tolpa Peat Preparation (TPP) on neovascular reaction induced in murine skin by human mononuclear cells (MNC) injection. MNC originating from 15 healthy volunteers were injected intradermally to Balb/c mice. Antibiotics (3, 15, or 75 mg/kg of body weight) alone or with TPP (10 mg/kg of body weight) were administrated subcutaneously to mice on three consecutive days. The number of newly formed blood vessels was measured in dissection microscope 72 hours after cell injection. RESULTS: TPP stimulated angiogenic activity of MNC at the dose 5 and 10 mg/kg. Rifamycine exerted strong stimulatory action, ampicillin slightly stimulated immune response, while doxycycline and rifampicin downregulated it. Amikacin did not influence the results of angiogenesis tests. Studied antibiotics (15 mg/kg), except rifamycine, inhibit the angiostimulatory effect of the tested immunomodulator. TPP should be applied after antibiotic therapy to maintain its stimulatory effect and restore proper host immune function.

A natural herbal remedy modulates angiogenic activity of bronchoalveolar lavage cells from sarcoidosis patients.

Sarcoidosis is a systemic inflammatory disease with abnormally high angiogenic activity of inflammatory cells. Reumaherb preparation consisting of three herbs: Echinacea purpurea, Harpagophytum procumbens, and Filipendula ulmaria, and it exerts anti-inflammatory, antioxidant, and analgesic activity and stimulates regenerative and immunological processes. The aim of this paper was to estimate the effect of Reumaherb on immunological angiogenesis induced by bronchoalveolar lavage (BAL) cells collected from six patients with sarcoidosis and grafted into Balb/c mice skin. After grafting, the animals were fed for three days with 0.6 or 1.2 mg of Reumaherb (calculated from recommended human daily dose) daily, suspended in 40 µl of water, or 40 µl of water alone (control group). A significant reduction of newly formed blood vessels was obtained in four cases for 1.2 mg and in three cases for 0.6 mg daily dose of this remedy. Thus, we hypothesise that Reumaherb promotes anti-angiogenic activity and may potentially be used in diseases associated with excessive blood vessel formation.

Angiomodulatory properties of Rhodiola spp. and other natural antioxidants.

Disturbances of angiogenesis and oxidative stress can lead to many serious diseases such as cancer, diabetes or ischemic heart disease. Substances neutralizing oxidative stress are known as antioxidants. They can affect angiogenesis process also, and thus, they modulate therapy results. Antioxidants become more and more frequently used in order to maintain homeostasis of the organism and diminish the risk of disease. Herein, we introduce some antioxidant preparations of natural plant origin (Rhodiola, Aloe vera, Resveratrol, Echinacea, Plumbagin) and antioxidant supplements (Padma 28, Reumaherb, Resvega). Analyses of their angiogenic properties, their multidirectional molecular effect on angiogenesis as well as medical application are within the scope of this review. Most of presented preparations down regulate neovascularization. They can be safely administered to patients with abnormally high angiogenesis. Rhodiola modulates, and Echinacea, Aloe vera and Plumbagin inhibit tumour-related angiogenesis in vitro and in vivo (animal models). Resveratrol and Resvega reduce neovascularization in the eye and may be applicable in eye disorders. Padma 28 preparation exhibits angioregulatory activity, decreasing high angiogenesis of cancer cells and increasing physiological angiogenesis, therefore can be used in therapy of patients with various disturbances of angiogenesis. Antioxidant application in the case of angiogenesis-related diseases should take into consideration angiogenic status of the patient.

Fluvastatin influences hair color in C57BL/6 mice.

Our recent in vitro experiments suggest that fluvastatin may influence tyrosinase (key enzyme of melanogenesis) synthesis. The aim of the present study was to verify those findings in experiments, in vitro, in melanoma cell line, and in vivo, in mice. The expression of tyrosinase in B16F10 melanoma cell line, after induction of melanogenesis by UVB irradiation, was examined by Western blot analysis. Afterwards, the effect of fluvastatin on melanin synthesis in hair follicles of C57Bl/6 mice was investigated. The expression of tyrosinase was reduced in the presence of fluvastatin. In mice after anagen induction over the dorsal skin, gel containing fluvastatin in various concentrations was injected subcutaneously, while in part of control groups of mice, gel with placebo was injected. In addition, gel with fluvastatin was injected to four week-old mice (mice in first postnatal anagen) without anagen induction. In extension, injections of gel with fluvastatin or placebo were performed in mice without anagen induction (but after first postnatal anagen). In part of study group of mice (mice after anagen induction and injection of fluvastatin) regrowth of depigmented hair was observed, while in all control groups (mice after injection of placebo), such hair depigmentation over the skin area was not found. In conclusion, this study, for the first time, shows that fluvastatin might affect melanin synthesis in vivo.

Fecal lactoferrin and Clostridium spp. in stools of autistic children.

Stools from autistic and healthy children were studied for fecal lactoferrin, Clostridium difficile toxins, Clostridium perfringens enterotoxin and cultured for Clostridium spp. Elevated level of FLA was demonstrated in 24.4% stools, all from boys (31.25%). No toxins were detected. Clostridium spp. was isolated with similar frequency from all samples. C. perfringens were isolated significantly often from the autistic stools, intermediate sensitive strains to penicillin 19%, to clindamycin 11.3%, and to metronidazole 7.5% were detected. Further studies on fecal microflora and inflammatory mediators, with larger groups of patients, are required in order to explain their role in neurological deficits.

Mycoplasma genitalium: a significant urogenital pathogen ?

Detection of mycoplasma infections, especially those caused by Mycoplasma genitalium (M. genitalium) is not a part of routine microbiological diagnostics, as commercial strip tests based on microculture in test wells do not include M. genitalium in their identification spectrum. Also classical methods of culture are especially demanding in the case of M. genitalium strains. PCR technique with application of specific starter oligonucleotides is currently used in the diagnostics of M. genitalium. In view of growing number of reports on the significance of this mycoplasma in etiopathogenesis of mainly urogenital disorders, an increasing number of laboratories uses molecular methods in diagnostics of M. genitalium infection. In this review we summarize epidemiological, clinical and microbiological research data of M. genitalium in the light of recent publications and our own observations.

Fibroblasts from normal skin of a tuberous sclerosis patient show upregulation of mTOR pathway.

Skin lesions are one of the characteristic features in tuberous sclerosis (TS), a neurocutaneous disorder caused by mutation of 1 of 2 tumor suppressor genes, encoding hamartin and tuberin. So far, however, studies on skin abnormalities present in TS patients are very few and do not contribute to the knowledge of the disease. In our current work, we cultured fibroblasts from healthy skin of a TS patient and evaluated upregulation of pathways found to be implicated in progression of TS tumors. We found that even healthy skin fibroblasts show upregulation of S6 ribosomal protein.

Occurrence of Ureaplasma parvum and Ureaplasma urealyticum in women with cervical dysplasia in Katowice, Poland.

The aim of this study was to evaluate the occurrence of genital mycoplasmas, especially Ureaplasma parvum and Ureaplasma urealyticum, in women with atypical squamous cells of undetermined significance (ASCUS), low grade squamous intraepithelial lesions (LSIL) and high grade squamous intraepithelial lesions (HSIL), compared to women with normal cytology living in Katowice, Poland. Two sterile swabs were used to obtain material from the posterior vaginal fornix of 143 women with squamous intraepithelial lesions and 39 healthy women: first for general bacteriology, second for detection of urogenital mycoplasmas using Mycoplasma IST2 kit. From each positive Mycoplasma IST2 culture DNA was isolated and PCR was performed for identification of U. parvum and U. urealyticum. Mycoplasma IST was positive in 34.1% cases. Urogenital mycoplasmas were demonstrated in women with HSIL significantly more often compared to women with LSIL, ASCUS, and with normal cytology. DNA of U. parvum was demonstrated in majority of Mycoplasma IST2-positive cases, U. urealyticum DNA-only in 9 (4.9%). Predominance of 3/14 serovars of U. parvum was demonstrated. U. urealyticum biovar 2 was present more often in women with squamous intraepithelial lesions.

Possible mechanisms of disease development in tuberous sclerosis.

The two-hit hypothesis presented by Knudson in 1971 explains the development of tumours deficient in anti-oncogenes. Hamartomas in patients with tuberous sclerosis usually fit into this model, the first hit is a congenital lesion of either of the tuberous sclerosis genes (TSC1 or TSC2), and the second hit is loss of heterozygosity of this gene. Although this mechanism is true for most tumours associated with tuberous sclerosis, only 30-60% of brain and cardiac tumours show loss of heterozygosity--the remaining tumours develop despite the presence of an intact allele. Tumours in which loss of heterozygosity is rare, such as subependymal giant-cell astrocytoma, might all share a common feature that mimics loss of heterozygosity either by inactivation of the TSC complex or by direct activation of mammalian target of rapamycin (mTOR) or its downstream targets. Because phosphorylation of the TSC complex can inactivate it, expression and activation patterns of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK), two potent protein kinases that are activators of the mTOR pathway, have been implicated. AKT activation is detected only in few samples, whereas ERK is hyperactive in all subependymal giant-cell astrocytomas. We postulate that ERK activation consistently detected in different tuberous-sclerosis-associated tumours is a molecular trigger for the development of these neoplasms.

Tuberin-heterozygous cell line TSC2ang1 as a model for tuberous sclerosis-associated skin lesions.

Tuberous sclerosis (TS), neurological disorder manifesting with the formation of tumors in numerous organ systems, is a disease associated with the upregulation of mammalian target of rapamycin (mTOR) pathway. It has been found that in healthy individuals two tumor suppressor genes, TSC1 and TSC2, encoding proteins called hamartin and tuberin, respectively, are responsible for the control over mTOR kinase. Loss of one of these genes constitutes the genetic background of TS. In the current study, we aimed at evaluating the fitness of the only TS-associated sarcoma cell line deposited in American Tissue Culture Collection, TSC2ang1, for the in vitro studies on TS. We found that the line shows a stable chromosome pattern with typical Robertsonian translocations. Similarly to primary tumors from TS patients, TSC2ang1 cells respond to rapamycin-induced mTOR inhibition. The cells demonstrate activation of both Akt and Erk pathways, but inhibition of neither of them is as effective as mTOR suppression when considering proliferation potential. Based on these results we propose TSC2ang1 as a good and stable model for pathophysiological and pharmacological studies on skin lesions in TS.

Implication of active Erk in the classic type of human medulloblastoma.

Molecular pathways underlying medulloblastoma (MB), the most common malignant brain tumour in children, are still under scrutiny. The mammalian target of the rapamycin (mTOR) pathway is one of the kinases that was recently found to be implicated in a number of human tumours. Also in the case of MB it is suspected that mTOR dysregulation may play an important role in pathogenesis. Active mTOR leads to translation of several proteins, some of which affect cellular proliferation. On the other hand, Akt/PKB (protein kinase B) and Erk (extracellular signal-regulated kinase, also called mitogen-activated protein kinase, MAPK) are two protein kinases whose hyperactivity leads to a number of downstream effects, including activation of mTOR. In our previous report we found that indeed Akt and Erk are variably activated in human MBs. However, because MBs are a highly heterogeneous group of tumours, we were unable to associate Akt or Erk activation with all the cases of MB. In this paper we evaluated six cases of MB, only of the classic subtype. We found that elements of the Erk pathway are hyperactive in all six tumours. Thus, we postulate that in classic type of MB, growth factor stimulation may lead to Erk upregulation and mTOR-dependent protein translation, causing malignant growth.

Molecular implications of skin lesions in tuberous sclerosis.

Tuberous sclerosis (TS), neurocutaneous disorder resulting from the mutation of 1 of 2 genes, TSC1 or TSC2, is often associated with the formation of hamartomatous lesions in various organ systems, including the skin. TS patients may present with hypomelanic macules, confetti-like spots, facial angiofibromas, ungual fibromas, shagreen patches, forehead plaques, and other dermatological signs. Some of these manifestations are pathognomic for TS and thus should be carefully evaluated when TS diagnosis is suspected. Little is known however on molecular links connecting disease pathogenesis and formation of such hamartomas. In the current review, we describe molecular pathways thought to be responsible for the development of the disease and show how their upregulation may affect the skin. Special attention is paid to protein kinase B (PKB/Akt), extracellular signal-regulated kinase, and mammalian target of rapamycin, which have recently been found to participate in the control of melanin biosynthesis through microphthalmia-associated transcription factor and tyrosinase transcription.

[Statins and their pleiotropic effects]. / Pozalipidowe dzialanie statyn.

Statins are well-established and effective drugs in the treatment of hyperlipidemias and coronary heart disease. However the effects of statins extend beyond their lipid-lowering actions, due to their capacity to inhibit prenylation of some intracellular regulatory proteins. Recent studies have shown that statins could modulate inflammantory response, improve endothelial function, exert antiarrhytmic properties, have beneficial effects on renal function and bone tissue. Statins may exert effect in the treament and prevention of dementia and some autoimmune disorders. Although statins therapy is generally well-tolerated, sometimes they lead to objective adverse effects, mainly in muscular system. Combination therapy with fibrates, coenzyme Q and other substances may increase and even extend therapeutic effects of statins. Further studies will help to clarify the clinical role of statins.

[Factors affecting melanogenesis and methods used for identification of pigmentation disorders]. / Czynniki modulujace melanogeneze oraz metody identyfikacji zaburzen barwnikowych.

Melanins are pigments widely distributed in living organisms. In humans melanin is synthetised in the retina, choroid and epidermis. Melanins fulfil a protective function and are responsible for the color of eyes, skin and hair. Melanogenesis is a process undergoing in specialized organelles of melanocytes--melanosomes. After synthesis, melanin is transported to target cells--keratinocytes. The key enzyme of the melanin synthesis pathway is tyrosinase. Many agents used in the treatment of hyperpigmentation act as tyrosinase inhibitors. Depigmenting agents and those increasing melanin synthesis are used in medicine and cosmetology. Also natural plant melanins extracted from e.g. Nigella sativa, have interesting effects on mammalian cells. In the current review paper we present methods used for identification of melanin and melanocytes in diagnostics of disorders affecting melanogenesis process.

Brain tumor formation in tuberous sclerosis depends on Erk activation.

Tuberous sclerosis (TS) is an autosomal dominant disease associated with the formation of usually benign tumors or hamartomas. The disease is connected with upregulation of mammalian target of rapamycin, central regulator of protein translation, which is usually regarded to be activated by Akt kinase. Here, we show for the first time that in all four brain lesions and one angiomyolipoma from TS patients both extracellular signal-regulated kinase (Erk) and p90 ribosomal S6 kinase 1 activation as well as Erk-dependent phosphorylation of p70 ribosomal S6 kinase 1 are markedly elevated whereas Akt, participating in the classical pathway of mammalian target of rapamycin activation is not always activated. Erk activation is also present in TS-derived cell lines. Importantly, Erk inhibition leads to the decrease of proliferation potential of such lines. These results show that Erk is specifically implicated in the pathogenesis of hamartomas.

Lethal factor of Clostridium histolyticum kills cells by apoptosis.

In this study, for the first time, Clostridium histolyticum lethal factor was purified by ammonium sulfate precipitation of culture broth, centrifugation through an Amicon filter device and hydrophobic interaction chromatography. The purified lethal factor was devoid of proteolytic activity. At a concentration of 150 ng mL(-1) the lethal factor killed 50% of HeLa cells within 24 h of exposure. Abrogation of actin filaments, activation of caspases, fragmentation of nuclear DNA as well as ultrastructural changes indicated that the cell death occurred by apoptosis. The apoptotic action of the lethal factor is in agreement with changes induced in animal tissues by administration of C. histolyticum culture medium.

Upregulation of the WNT pathway in tuberous sclerosis-associated subependymal giant cell astrocytomas.

Tuberous sclerosis (TS), autosomal dominant disorder manifested by the formation of usually benign tumors in the brain, heart, kidneys and skin, results from an inactivating mutation in one of two tumor suppressor genes TSC1 or TSC2. Protein products of these genes, hamartin and tuberin, respectively, have been shown to participate in the mTOR pathway controlling translation of approx. 10-15% of all proteins. In the current paper, we aimed at verifying whether hamartin and tuberin may also be implicated in the control of gene transcription. Very recently it has been hypothesized that the pathway triggered by WNT, one of embryonic growth factors involved in cell differentiation and migration, could be disturbed in TS. In order to test this hypothesis we evaluated samples of four subependymal giant cell astrocytomas (SEGAs), brain tumors developing in the progress of TS. We found that beta-catenin, transcription factor and mediator of WNT pathway activity is indeed present and active in SEGAs. mRNA transcripts for c-Myc and N-Myc, proteins whose transcription is regulated by beta-catenin, were upregulated in two of four SEGAs, while cyclin D1 mRNA was significantly higher in three SEGAs. At the same time, c-Myc and N-Myc proteins were detected in the same two samples. Thus, we show for the first time that aberrant WNT signaling may contribute to the pathogenesis of TS-associated SEGAs.

The influence of trkB deficiency on long-term outcome of peripheral nerve injury in mice.

The long-term outcome of peripheral nerve injury is often unsatisfactory, especially if the injury resulted in a gap between transected nerve stumps. Brain-derived neurotrophic factor and its receptor, trkB, are strongly implicated in the early phase of axonal regeneration after injury. We examined the role of trkB in long-term functional and morphological outcome of peripheral nerve injury. The sciatic nerve was transected in wild-type and heterozygous trkB-deficient mice. The nerve was either left cut or immediately sewn up or the gap injury model was performed. The gap was provided with autologous or cross (obtained from other genetic group) graft. Sciatic nerve function as well as autotomy was assessed during 16-week follow-up. The long-term functional outcome of nerve cut or immediately rejoined did not differ between wild-type and trkB-deficient mice. Gap injury provided with nerve graft resulted in better functional outcome in trkB-deficient mice than wild-type animals. Sixteen weeks after the surgery, the animals were sacrificed and histological evaluations were performed. The number of nerve fibres regenerating into the distal stump of transected and rejoined nerves did not differ between wild-type and trkB-deficient animals. TrkB deficiency markedly increased the number of Schwann cells as well as mast cells at the injury site and in the distal stump of the regenerating nerve. TrkB deficient nerves also showed higher expression of bcl-2 protein but lower of trkA and NGF than wild-type ones. Our results show for the first time the possible deleterious role of trkB receptor in long-term outcome of peripheral nerve injury.