MULTIFUNCTIONAL NANO-BIO MATERIALS WITHIN CELLULAR MACHINERY.

Functional nanoscale materials that possess specific physical or chemical properties can leverage energy transduction in vivo. Once these materials integrate with biomolecules they combine physical properties of inorganic material and the biorecognition capabilities of bio-organic moieties. Such nano-bio hybrids can be interfaced with living cells, the elementary functional units of life. These nano-bio systems are capable of bio-manipulation or actuation via altering intracellular biochemical pathways. Thus, nano-bio conjugates are appealing for a wide range of applications from the life sciences and nanomedicine to catalysis and clean energy production. Here we highlight recent progress in our efforts to develop smart nano-bio hybrid materials, and to study their performance within cellular machinery under application of external stimuli, such as light or magnetic fields.

Tumor suppression in Xiphophorus by an accidentally acquired promoter.

Melanoma formation in the teleost Xiphophorus is caused by a dominant genetic locus, Tu. This locus includes the Xmrk oncogene, which encodes a receptor tyrosine kinase. Tumor induction is suppressed in wild-type fish by a tumor suppressor locus, R. Molecular genetic analyses revealed that the Tu locus emerged by nonhomologous recombination of the Xmrk proto-oncogene with a previously uncharacterized sequence, D. This event generated an additional copy of Xmrk with a new promoter. Suppression of the new Xmrk promoter by R in parental fish and its deregulation in hybrids explain the genetics of melanoma formation in Xiphophorus.

Activation of the Xmrk proto-oncogene of Xiphophorus by overexpression and mutational alterations.

Xmrk is a receptor tyrosine kinase closely related to the human EGF receptor. In the teleost fish Xiphophorus two versions of the Xmrk gene exist, an oncogene (ONC) and a proto-oncogene (INV). While ONC-Xmrk is the melanoma-inducing gene, INV-Xmrk appears not to be involved in transformation of pigment cells. To elucidate the mechanism that converts the proto-oncogene into a transforming oncogene a comparative analysis of the structure, expression and function of both versions of the gene was performed. In contrast to ONC-Xmrk which is expressed at high levels in melanoma cells, the proto-oncogene INV-Xmrk is ubiquitously expressed at very low levels indicating overexpression as one possible reason for tumorigenicity by ONC-Xmrk. As sequence comparison of the proto-oncogene and the oncogene revealed a number of amino acid changes, a possible effect of these mutations on the activation of the ONC-Xmrk receptor was determined. A constitutive activation of the oncogenic receptor was found and ectopic expression of INV-Xmrk after microinjection into medakafish embryos did not lead to the high tumour rate in transgenic fish as observed for the oncogene. Our data therefore suggest that overexpression of the receptor alone is not sufficient for melanoma induction, but that in addition activating mutations in ONC-Xmrk are responsible for its full tumorigenic potential.

Digestive system damage caused by substance abuse.

Substance abuse and addiction represent a worldwide problem and cause a number of family, social and health problems. Digestive system damage caused by substance intake is an increasing problem amoung drug addicts. Many studies show that substances can cause cancer of all parts of the digestive system. Alcohol consumption was significantly associated with colon and rectal cancer. For rectal cancer, the risk was increased in association with drinking of alcoholic beverages, specialy for beer consumption. Sinthetic drugs such as ecstasy may lead also to digestive and hepatic damage, as well as vascular complications of the stomach. Many studies show the existance of supstance associated enterocolitis as well as ishemic colitis. Diagnosis of ishemic colitis is based on the presence of rectal bleeding, abdominal pain, a history of substance use, supportive endoscopic and histopathologic findings, and the absence of other etiologic mechanisms of ischemic colitis. Great damage to the digestive system is also produced by smuggling narcotics packed into small pages that are afterwards been swallowed or implemented on other sorts of ways inside the smugglers natural body spaces as the rectum or vagina. In the paper authors reviewed literature conserning digestive system damage caused by substance abuse and drug smuggling.

Effects of cocaine in 5-lipoxygenase-deficient mice.

5-Lipoxygenase (5-LOX), along with 12-lipoxygenase and cyclooxygenases, metabolizes arachidonic acid into eicosanoids. In rodents, 12-lipoxygenase deficiency alters behavioral responses to cocaine. We used 5-LOX-deficient mice and their controls to investigate cocaine's actions. After repeated cocaine injections, the increase in locomotor activity was greater in 5-LOX-deficient mice. Since the 5-LOX pathway may regulate the levels/metabolism of arachidonoylethanolamide (AEA) we assayed the AEA levels in the striatum, the binding of the endogenous AEA to the cannabinoid receptor CB1R, and anandamide hydrolase (FAAH) activity in the striatum, hippocampus, and cortex. Striatal AEA levels decreased after repeated cocaine injections. Cocaine also decreased CB1R binding in all brain regions studied and the only significant differences between 5-LOX-deficient and control mice was the greater hippocampal FAAH activity in 5-LOX-deficient mice. Our results demonstrated that a 5-LOX deficiency alters sensitivity to repeated cocaine. It should be investigated whether a human 5-LOX gene polymorphism affects cocaine's actions.

Intra-abdominal injection of double-stranded RNA into anesthetized adult Drosophila triggers RNA interference in the central nervous system.

RNA interference (RNAi) is a gene silencing mechanism that can be triggered by introducing double-stranded RNA (dsRNA) into cells expressing the appropriate molecular machinery, which then degrades the corresponding endogenous mRNA. RNAi can be used for determining gene function and creating functional "knockout" organisms. Here we show for the first time that RNAi can be induced in adult fruit flies by injecting dsRNA into the abdomen of anesthetized Drosophila, and that this method can also target genes expressed in the central nervous system (CNS). Two genes were targeted to investigate the effects of dsRNA injection on their mRNA content; lacZ transgene (expressed either in the gut or in the CNS), and GM06434, the Drosophila homologue of the C. elegans gene nrf (nose resistant to fluoxetine). Both the transgene and the endogenous gene were successfully silenced in adult Drosophila by intra-abdominal injection of their respective dsRNA. We propose that our method of RNAi in adult flies can be used to characterize gene functioning in the CNS without the typical interference in development found in most gene mutation studies.

Evolutionary origin and molecular biology of the melanoma-inducing oncogene of Xiphophorus.

Melanoma formation in platyfish/swordtail hybrids of genus Xiphophorus is due to overexpression of the receptor tyrosine kinase oncogene Xmrk. This gene is the molecular equivalent to the Tu-locus of platyfish formerly identified by Mendelian genetics. The supposed evolutionary origin of the Xmrk oncogene is a nonhomologous recombination event in the 5' region of the corresponding Xmrk protooncogene with an anonymous sequence, D. This event led to a gene duplication of Xmrk, whereby the new copy obtained a novel promoter derived from D. Inactivity of this promoter in parental fish warrants lack of tumorigenicity of the Xmrk oncogene in wild playfish. In hybrids, however, the promoter is active. This leads to the pigment cell transforming overexpression of Xmrk.

Ligand-independent dimerization and activation of the oncogenic Xmrk receptor by two mutations in the extracellular domain.

Overexpression of the oncogenic receptor tyrosine kinase ONC-Xmrk is the first step in the development of hereditary malignant melanoma in the fish Xiphophorus. However, overexpression of its proto-oncogene counterpart (INV-Xmrk) is not sufficient for the oncogenic function of the receptor. Compared with INV-Xmrk, the ONC-Xmrk receptor displays 14 amino acid changes, suggesting the presence of activating mutations. To identify such activating mutations, a series of chimeric and mutant receptors were studied. None of the mutations present in the intracellular domain was found to be involved in receptor activation. In the extracellular domain, we found two mutations responsible for activation of the receptor. One is the substitution of a conserved cysteine (C578S) involved in intramolecular disulfide bonding. The other is a glycine to arginine exchange (G359R) in subdomain III. Either mutation leads to constitutive dimer formation and thereby to activation of the ONC-Xmrk receptor. Besides, the presence of these mutations slows down the processing of the Xmrk receptor in the endoplasmic reticulum, which is apparent as an incomplete glycosylation.

Hyponatremic hypertensive syndrome.

We report on a 4-year-old girl with hyponatremic-hypertensive syndrome (HHS), a rare entity in childhood. The girl was referred to us from a local hospital with a history of recurrent fever, vomiting, and seizures. On admission she was markedly dehydrated. Initial investigations revealed severe hyponatremia (serum Na 120 mmol/l), hypochloremia (serum Cl 68 mmol/l), and mild hypokalemia (serum K 3.3 mmol/l), while serum calcium and magnesium were normal. Serum urea was 5 mmol/l and serum creatinine was 62 mumol/l. Despite hyponatremic dehydration, her urine output was high (2050 ml/24 h), as was her urinary sodium (168 mmol/24 h). She had massive transient proteinuria (maximal 1642 mg/24 h) while being severely hypertensive (blood pressure 210/160 mmHg). Further investigations revealed right kidney scarring, hyper-reflexive bladder dysfunction, massive brain infarcts, and myocardial left ventricular hypertrophy. Renal arteries were normal on arteriography. Blood pressure control resulted in normalization of serum and urinary electrolytes and decrease of proteinuria. Hyponatremia and transient massive proteinuria in this patient seem to be caused by high-pressure-forced diuresis due to malignant renoparenchymal hypertension.

[Hemodialysis in children and beta 2-microglobulin]. / Hemodijaliza kod dece i mikroglobulina beta-2.

Retention of beta-2-microglobulin, due to loss of excretory renal function inevitably occurs in uremia. A significant correlation between the duration of hemodialysis and the incidence of carpal tunnel syndrome or destructive cystic lesions of bone has been reported. Amyloid deposition has been found to directly cause these lesions. This amyloid protein has been identified to be beta-2-microglobulin. The aim of this report was to study plasma beta-2-microglobulin levels in hemodialysed children and to detect clinical manifestations or carpal tunnel syndrome and of bone lesions. 12 children aged 9-24 years were studied. The average duration of dialysis was 51.9 ae 32 months. Beta-2-microglobulin plasma levels were studied before and after hemodialysis treatment. All patients were examined for carpal tunnel syndrome using clinical methods including nerve conduction studies, and for destructive cystic lesions using X-rays of bone. Plasma beta-2-microglobulin concentrations in our patients were found to be sevrefold time higher (73.8 ae 22.1 mg/l) than control values (1.6 ae 0.5 mg/l). The increase of beta-2-microglobulin concentration during hemodialysis tratment was due probably by hemoconcentration. We noted poor correlation between plasma beta-2-microglobulin and duration of hemodialysis. Any signs of carpal tunnel syndrome was found to our patients. Radiological signs of amyloid osteopathy found to be present in 3 children, was not verified by bone biopsy.

Thrombus precursor protein (TpP): marker of thrombosis early in the pathogenesis of myocardial infarction.

We have developed an enzyme-linked immunosorbent immunoassay for quantifying the immediate precursor proteins to intravascular thrombi. This thrombus precursor protein (TpP) assay identifies active thrombosis in several clinical conditions, including early myocardial infarction (MI). In a study of patients recruited for the GUSTO intervention study, MI patients had concentrations of TpP 4-20-fold that of controls; patients diagnosed without MI had concentrations similar to the control subjects. In a separate study of subjects presenting at the emergency room with chest pain, MI patients who presented early after the onset of chest pain had TpP concentrations significantly (P <0.01) higher than controls. Patients presenting late or diagnosed with other chest pain had concentrations within the reference range. The potential utility of the TpP assay as an aid for the diagnosis of thrombotic MI and other thrombotic conditions is described.

Proteome analysis of rat hepatomas: carcinogen-dependent tumor-associated protein variants.

Proteome analysis led to the identification and characterization of tumor-associated protein variants by two-dimensional electrophoresis and mass spectrometry. We focused on comparing the influence of genotoxic nitroso compounds N-methyl-N-nitrosourea, diethylnitrosamine and N-nitrosomorpholine and the nongenotoxic peroxisome proliferator Nafenopin as tumor-inducing agents on the protein pattern of rat hepatomas. We found several tumor-associated variants that represent members of the aldo-keto reductase superfamily. Their induction and/or inhibition was specifically related to the carcinogen used for tumor induction. The most prominent tumor-associated protein, rat aldose reductase-like protein-1 (rARLP-1) (69% sequence identity to lens aldose reductase) and three additional types of rARLP-1 were detected in nitroso compound-induced rat hepatomas, while rat aldo-keto reductase protein-c (Rak-c), a novel tumor-associated variant (65% sequence identity with 3alpha-hydroxysteroid dehydrogenase) was discovered in N-methyl-N-nitrosourea-induced hepatomas only. 3Alpha-hydroxysteroid dehydrogenase and delta4-3-ketosteroid-5beta-reductase, both liver-specific enzymes, were reduced in amount in all hepatomas investigated, independent of their mode of induction. We conclude, that detoxification enzymes like 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) and delta4-3-ketosteroid-5beta-reductase (5beta-Red) might be replaced in hepatomas by tumor-associated proteins that are often present in the embryonal state, like the rARLPs or the Rak-c protein. Their induction appears to reflect an altered constitutive pattern of detoxification enzymes, detoxifying toxic aldehydes being induced by nitroso compounds. In contrast, members of the aldo-keto reductase superfamily have not been found in Nafenopin-induced hepatomas. The pattern of tumor-associated protein variants is apparently characteristic for a given group of initiating carcinogens. The hypothesis is proposed that carcinogens leave specific fingerprints at the proteome level of manifest liver tumors.

[The Lesch-Nyhan syndrome]. / Les-Nijanov (Lesch-Nyhan) sindrom.

Deficiency of hypoxanthine phosphoribosyltransferase (HPRT) has a broad spectrum of clinical manifestations, from the complete enzyme defect, the Lesch-Nyhan syndrome with severe neurological deficiency to the partial defect associated only with uric acid overproduction and its consequences. We present a 5-year old boy with Lesch-Nyhan syndrome. He came to our hospital because of abdominal pain, vomiting and gross haematuria. At the age of 8 months he was categorized as a "cerebral palsy" patient due to involuntary movements and high degree of spastically and tonic spasms. He remained incapable of sitting or standing alone. The patient's brother and two uncles were also categorized as "cerebral palsy" cases and died at the age of 8-14 years. Clinical examination revealed hyperuricaemia and hyperuricosuria, radiolucent renal and urinary bladder stones. HPRT enzyme activity was totally absent, while adenine phosphoribosyl transferase activity was increased compared to control. The patient was treated with allopurinol, urinary alkalization, low-purine diet and adequate hydration while he was in hospital. However, his parents refused further treatment and follow-up. The most important issue is whether the healthy sisters of the patients are heterozygotes for HPRT deficiency. This DNA analysis is now in progress.