Distribution and function of polycystin-2 in mouse retinal ganglion cells.

The polycystin family of transient receptor potential (TRP) channels form Ca(2+) regulated cation channels with distinct subcellullar localizations and functions. As part of heteromultimeric channels and multi-protein complexes, polycystins control intracellular Ca(2+) signals and more generally the translation of extracellular signals and stimuli to intracellular responses. Polycystin-2 channels have been cloned from retina, but their distribution and function in retinal ganglion cells (RGCs) have not yet been established. In the present study, we determined cellular and subcellular localization as well as functional properties of polycystin-2 channels in RGCs. Polycystin-2 expression and distribution in RGCs was assessed by immunohistochemistry on vertical cryostat section of mouse retina as well as primary cultured mouse RGCs, using fluorescence microscopy. Biophysical and pharmacological properties of polycystin-2 channels isolated from primary cultured RGCs were determined using planar lipid bilayer electrophysiology. We detected polycystin-2 immunoreactivity both in the ganglion cell layer as well as in primary cultured RGCs. Subcellular analysis revealed strong cytosolic localization pattern of polycystin-2. Polycystin-2 channel current was Ca(2+) activated, had a maximum slope conductance of 114 pS, and could be blocked in a dose-dependent manner by increasing concentrations of Mg(2+). The cytosolic localization of polycystin-2 in RGCs is in accordance with its function as intracellular Ca(2+) release channel. We conclude that polycystin-2 forms functional channels in RGCs, of which biophysical and pharmacological properties are similar to polycystin-2 channels reported for other tissues and organisms. Our data suggest a potential role for polycystin-2 in RGC Ca(2+) signaling.

Agmatine transport in brain mitochondria: a different mechanism from that in liver mitochondria.

The diamine agmatine (AGM), exhibiting two positive charges at physiological pH, is transported into rat brain mitochondria (RBM) by an electrophoretic mechanism, requiring high membrane potential values and exhibiting a marked non-ohmic force-flux relationship. The mechanism of this transport apparently resembles that observed in rat liver mitochondria (RLM), but there are several characteristics that strongly suggest the presence of a different transporter of agmatine in RBM. In this type of mitochondria, the extent of initial binding and total accumulation is higher and lower, respectively, than that in liver; saturation kinetics and the flux-voltage relationship also exhibit different trends, whereas idazoxan and putrescine, ineffective in RLM, act as inhibitors. The characteristics of agmatine uptake in RBM lead to the conclusion that its transporter is a channel with two asymmetric energy barriers, showing some characteristics similar to those of the imidazoline receptor I(2) and the sharing with the polyamine transporter.

Transport of amino acids through the placenta and their role.

Amino acids are transported across the human placenta mediated by transporter proteins that differ in structure, mechanism and substrate specificity. Some of them are Na+-dependent systems, whereas others are Na+-independent. Among these there are transporters composed of a heavy chain, a glycoprotein, and a light chain. Moreover, they can be differently distributed in the two membranes forming the syncytiotrophoblast. The transport mechanisms involved and their regulation are only partially known. In the placenta itself, part of the amino acids is metabolized to form other compounds important for the fetus. This occurs for instance for arginine, which gives rise to polyamines and to NO. Interconversion occurs among few other amino acids Transport is altered in pregnancy complications, such as restricted fetal growth.

S-adenosylmethionine and its products.

S-adenosylmethionine is involved in many processes, mainly methylation, polyamine synthesis and radical-based catalysis. It is synthesised through the catalysis of differently regulated enzyme forms. When it is used, the compounds formed are reutilized in different ways: in case of methylation, its end product is homocysteine, which can be remethylated to methionine, give rise to cysteine in the so-called transsulphuration pathway, or be released; in the case of polyamine synthesis, the methylthioadenosine formed is cleaved and gives rise to compounds which can be reutilized; during radical-based catalysis, 5-deoxyadenosine is formed and this, too, is cleaved and reutilized.

Advanced glycation end-products (AGEs): involvement in aging and in neurodegenerative diseases.

Advanced glycation end-products (AGEs) are formed from the so-called Amadori products by rearrangement followed by other reactions giving rise to compounds bound irreversibly. The structure of some of them is shown and the mechanism of formation is described. Several AGE binding molecules (Receptors for AGE, RAGE) are known and it is thought that many of the effects caused by AGEs are mediated by RAGE. Some of these were shown to be toxic, and called TAGE. The mechanism of detoxification of glyoxal and methylglyoxal by the glyoxalase system is described and also the possibility to eliminate glycated proteins by deglycation enzymes. Compounds able to inhibit AGEs formation are also taken into consideration.

Inhibition of agmatine transport in liver mitochondria by new charge-deficient agmatine analogues.

The charge of the agmatine analogues AO-Agm [N-(3-aminooxypropyl)guanidine], GAPA [N-(3-aminopropoxy)guanidine] and NGPG [N-(3-guanidinopropoxy)guanidine] is deficient as compared with that of agmatine and they are thus able to inhibit agmatine transport in liver mitochondria. The presence of the guanidine group is essential for an optimal effect, since AO-Agm and NGPG display competitive inhibition, whereas that of GAPA is non-competitive. NGPG is the most effective inhibitor (K(i)=0.86 mM). The sequence in the inhibitory efficacy is not directly dependent on the degree of protonation of the molecules; in fact NGPG has almost the same charge as GAPA. When the importance of the guanidine group for agmatine uptake is taken into account, this observation suggests that the agmatine transporter is a single-binding, centre-gated pore rather than a channel.

Potassium channel blockers quinidine and caesium halt cell proliferation in C6 glioma cells via a polyamine-dependent mechanism.

Potassium channels are ubiquitous in cells and serve essential functions in physiology and pathophysiology. Potassium channel blockers have been shown to block tumour growth by arresting cells at the G(0)/G(1) checkpoint of the cell cycle. We investigated the effect of quinidine and caesium (Cs(+)) on cell proliferation, LDH (lactate dehydrogenase) release, free internal calcium, membrane potential, polyamine concentration, ODC (ornithine decarboxylase) activity and polyamine uptake in C6 glioma cells. The EC(50) for reducing cell proliferation was 112 microM for quinidine, whereas Cs(+) was less effective with an EC(50) of 4.75 mM. KCl or sucrose did not affect proliferation. LDH release was augmented by quinidine. Quinidine caused a transient increase in free internal calcium but decreased calcium after a 48 h incubation period. Further 300 microM quinidine depolarized the cell membrane in a similar range as did 30 mM KCl. Quinidine decreased cellular putrescine beyond detection levels while spermidine and spermine remained unaffected. ODC activity was reduced. Addition of putrescine could not override the antiproliferative effect owing to a reduced activity of the polyamine transporter. Our study indicates that the antiproliferative effect of quinidine is not due to a simple membrane depolarization but is caused by a block of ODC activity.

Different behavior of agmatine in liver mitochondria: inducer of oxidative stress or scavenger of reactive oxygen species?

Agmatine, at concentrations of 10 microM or 100 microM, is able to induce oxidative stress in rat liver mitochondria (RLM), as evidenced by increased oxygen uptake, H(2)O(2) generation, and oxidation of sulfhydryl groups and glutathione. One proposal for the production of H(2)O(2) and, most probably, other reactive oxygen species (ROS), is that they are the reaction products of agmatine oxidation by an unknown mitochondrial amine oxidase. Alternatively, by interacting with an iron-sulfur center of the respiratory chain, agmatine can produce an imino radical and subsequently the superoxide anion and other ROS. The observed oxidative stress causes a drop in ATP synthesis and amplification of the mitochondrial permeability transition (MPT) induced by Ca(2+). Instead, 1 mM agmatine generates larger amounts of H(2)O(2) than the lower concentrations, but does not affect RLM respiration or redox levels of thiols and glutathione. Indeed, it maintains the normal level of ATP synthesis and prevents Ca(2+)-induced MPT in the presence of phosphate. The self-scavenging effect against ROS production by agmatine at higher concentrations is also proposed.

S-adenosylmethionine and radical-based catalysis.

S-adenosylmethionine is the major methyl donor in all living organisms, but it is also involved in many other reactions occurring through radical-based catalysis. The structure and function of some of these enzymes, including those involved in the synthesis of the molybdenum cofactors, biotin, lipoate, will be discussed.

S-adenosylmethionine and protein methylation.

The enzymes responsible for protein methylation by S-adenosylmethionine, both at the carboxyl groups and at the nitrogen groups, are reviewed. The possibility that the reactions involved may be reversible is also considered.

Arginine revisited: minireview article.

Arginine is a precursor of proteins and employed in urea synthesis. It is also the precursor of many other compounds, such as creatine, nitric oxide, polyamines, agmatine, proline. In this review, its transport and that of other basic amino acids are examined, along with its transformation into nitric oxide, agmatine and proline, and the mutual regulation of the individual pathways.

Metabolism and function in animal tissues of agmatine, a biogenic amine formed from arginine.

Recently agmatine, decarboxylated arginine, has been shown to be an important biological compound in several animal tissues. This paper summarizes the known information regarding the transport of arginine, its decarboxylation and the effects of the agmatine formed mainly on NO and polyamine synthesis.

Dermolipectomia Abdominal em âncora após cirurgia bariátrica / Abdominal dermolipectomy in anchor after bariatric surgery

A obesidade mórbida é reconhecida atualmente como um grande problema para a saúde. Os tratamentos clínicos desta patologia são falhos quando avaliados a longo prazo. Isto levou os cirurgiões a desenvolverem técnicas que resultam em efeitos restritivos ou malabsortivos à ingestão de alimentos. Após alguns meses da cirurgia bariátrica, existe uma perda de peso maciça resultando em grande flacidez da pele do abdômen, mamas, coxas e braços. Neste estudo avalia-mos a técnica de Correa-Iturraspe para o tratamento da região abdominal em dezessete pacientes que apresentaram grande perda ponderal após cirurgia bariátrica.

Morbid obesity is increasingly recognized as a major health concern. Drug treatments of the morbid obese have entirely failed in the long run. These circumstances have constituted incentives for surgeons to develop techniques resulting in malabsorptive or restrictive effects on food intake. Some months after the bariatric surgery there is a massive weight loss and a great skin laxity mainly at the abdomen, breast, tights and arms. In this study we evaluate the Correa-Iturraspe technique to treat the abdominal region in seventeen patiens with massive weigth loss after bariatric surgery.

Tratamento cirúrgico da gigantomastia / Surgical Treatment of Gigantomastia

Mastoplastia redutora é um procedimento cirúrgico bastante requisitado por pacientes que apresentam mamas grandes associadas a dores lombres. As grandes hipertrofias impõem técnicas que permitam ressecções associadas ao tratamento da ptose que, na maior parte dos casos, é acentuada. Neste estudo analisamos as técnicas empregadas em 231 casos de mastoplastia redutora, realizados no Serviço de Cirurgia Plástica do Hospital de Clínicas da UFPR no período de 1998 a 2002, das quais 13,8 foram considerados casos de gigantomastia.

Women with big breasts and back pain claim for a reduction mammaplasty. Large volume breast reductions need special atention for the nipple areolar complex migration. In this study we analise the techniques used in 231 reduction mammaplasties performed at “Hospital de Clínicas da Universidade Federal do Parana” from 1998 to 2002. 13,8 of the cases where considered with very large hipertrophies.

Microssomia craniofacial: experiência de 105 casos em 7 anos / Hemifacial microsomy: experience with 105 cases during 7 years

A Microssomia Craniofacial é deformidade com grande variedade de apresentações clínicas. Tratamos 105 casos em 7 anos, entre 1996 a 2002. Os casos foram revisados enfocando as suas alterações anatômicas. A idade de início do tratamento foi entre 0 e 5 anos em 39, 6 a 10 anos em 27,6 e após 10 anos em 33,4. Em 44,8 o lado comprometido foi o direito, o esquerdo em 38 e bilateral em 17,2. O pavilhão auricular era normal em 10; microtia correspondeu a 81 das deformidades auriculares, anotia a 12,6, orelha constricta a 6,3, question ear a 4,2, duplicidade a 4,2 e ausência do lóbulo a 1. Em 65,7 dos pacientes o meato acústico estava ausente e em 2,8 havia atresia. De acordo com a classificação de Pruzanski, foram de 35,2 as alterações mandibulares de Grau I, 21,9 de Grau IIA, 16,1 de Grau IIB e 3,8 de Grau III. Em 22,8 a mandíbula apresentava-se normal. Paralisia facial foi encontrada em 11,4 e macrostomia em 22,8. Em 10 dos pacientes apresentavam alterações oftalmológicas, dentre elas, 3 casos dermóide epibulbar. Em 21,9 foram identificadas outras deformidades faciais, com 5 casos de hipoplasia de partes moles, 9 de fissuras lábiopalatais e 2 de atrofia do masséter.

Hemifacial Microsomia is a deformity with a great variety of clinical feature. We have been treating 105 cases in last 7 years. The revision is focusing over the anatomic disorders. The patients looked for treatment between 0 – 5 y.old in 39, 5 – 10 in 27,6 and after 10 years old in 33,4. In 44,8 of the patients the deformities were at right side and 38 in the left, and in 17,2 was bilateral. Normal ear was seen in 10. In patients with auricular deformities microtia was seen in 81, anotia 12,6, lop and cup ear in 6,3, question ear in 4,2, duplicity in 4,2 and absence of lobulus in 1. In 65,7 of the patients the meatus was absence and 2,8 with atresic meatus. According to classification of Pruzanski, 35,2 of mandibular deformities were grade I, 21,9 grade IIA, 16,1 grade IIB and 3,8 grade III. Mandible was normal in 22,8. Facial palsy was found in 11,4, and macrostomy in 22,8. Around 10 of our patients had ocular deformities, and 3 of them had epibulbar dermoids. Others facial malformation occurred in 21,9, included: soft tissue hipoplasia in 5 cases, cleft lip and/or palate in 9 cases, and masseter atrophy in 2 cases .

Estudo de doppler fluxometria em retalho de abdominoplastia / Study of Eco-Doppler fluxometry in abdominoplasty flap

A abdominoplastia tem-se difundido mundialmente, tanto esteticamente como de forma reparadora, e novas técnicas estão surgindo com o objetivo de minimizar as complicações isquêmicas do retalho e melhorar o contorno corporal. No período de janeiro a julho de 2002, vinte e uma pacientes, do sexo feminino, com idade variando entre 39 e 54 anos, apresentando alteração no abdome do tipo III e IV de BOZOLA, foram submetidas a abdominoplastia associada à lipoaspiração do abdome e flancos e descolamento apenas da porção central para plicatura da diástase muscular de acordo com a técnica de Saldanha(2001). Foi realizado um estudo com doppler fluxometria colo- rida dos vasos epigástricos superiores, inferiores e suas perfurantes músculo cutâneas, no pré-operatório e no décimo quinto dia de pós-operatório, avaliando a vascularização da parede abdominal superior e inferior, contabilizando os ramos perfurantes, calibre e fluxo sangüíneo dos vasos. Este estudo comprovou, através de doppler fluxometria, a preservação dos vasos perfurantes na região periumbilical e hipocôndrio e o aumento do calibre dos vasos em 9 e do fluxo sangüíneo em 56. A manutenção da irrigação sangüínea do retalho foi demonstrada clinicamente, através da boa evolução da cicatriz e ausência de complicações, como necrose da borda do retalho e seroma.

Abdominoplasty surgery has spread universally, both as aesthetic and reconstructive procedures, and new techniques are appearing with the goal of minimizing ischemic complications of the flap and bettering body contour. From January 2002 to July 2002, twenty one female patients, range of 30 to 54 years, presenting abdominal alterations classified as type III and IV of Bozola, were submitted to abdominoplasty combined with abdominal and flank lipossuction and central portion undermining for muscular diastasis plication according to Saldanha’s technique. Eco-doppler fluxometry of the superior and inferior epigastric vessels and of their cutaneous muscular perfurans was done preoperatively and 15 days after the surgery, assessing the superior and inferior abdominal wall circulation, counting the number of perfurans vessels, the diameter and vessels flow. This study showed, through eco-doppler fluxometry, perfurans vessel preservation at the periumbelical and hypocondric areas and enhancement of vessels diameter (9) and vessels flow (56). Blood flow maintenance was demonstrated clinically through the good scar evolution and absence of complications, as flap necrosis and seroma.

Agmatine induces apoptosis in rat hepatocyte cultures.

BACKGROUND/AIMS: Agmatine, the compound formed by decarboxylation of arginine, is believed to be an endogenous neurotransmitter through interaction with the imidazoline receptors. However, it also appears to regulate rat hepatocyte polyamines by modifying both their synthesis and their catabolism. As the decrease in polyamine content has been correlated with apoptosis, we examined the possibility that agmatine has an effect on this phenomenon. METHODS: Apoptotic cells were detected by visualizing nuclear shrinkage/fragmentation in hepatocytes cultured at 21 and 5% oxygen tension. Caspase-3 activity, cleavage of PARP, release of cytochrome c and mitochondrial swelling were therefore measured in the two conditions and in the presence or not of agmatine. RESULTS: In rat hepatocytes agmatine promoted apoptosis, procaspase 3 processing and increase of caspase-3 like activity. This occurred through mitochondria swelling and release of cytochrome c. Cyclosporin A and catalase blocked the swelling. CONCLUSIONS: Our experiments show that agmatine, besides all the known biological effects, has also part, at least in hepatocytes, in the modulation of programmed cell death.

Transport and metabolism of agmatine in rat hepatocyte cultures.

Rat hepatocytes in culture take up [14C]-agmatine by both a high-affinity transport system [KM = 0.03 mM; Vmax = 30 pmol x min x (mg protein)-1] and a low-affinity system. The high-affinity system also transports putrescine, but not cationic amino acids such as arginine, and the polyamines spermidine and spermine. The rate of agmatine uptake is increased in cells deprived of polyamines with difluoromethylornithine. Of the agmatine taken up, 10% is transformed into polyamines and 50% is transformed into 4-guanidinobutyrate, as demonstrated by HPLC and MS. Inhibition by aminoguanidine and pargyline shows that this is due to diamine oxidase and an aldehyde dehydrogenase. 14C-4-aminobutyrate is also accumulated in the presence of an inhibitor of 4-aminobutyrate transaminase.

Oxygen regulation of rat hepatocyte iNOS gene expression.

BACKGROUND/AIMS: The human iNOS promoter contains a consensus sequence for binding the hypoxia inducible factor. The aim of this study was to see whether iNOS gene expression is triggered by oxygen tension in rat hepatocytes exposed in vivo to high (periportal) and low (perivenous) oxygen tension. METHODS: Hepatocytes transfected or not with a plasmid containing rat iNOS promoter linked to chloramphenicol acetyltransferase were cultured at 21% and 5% oxygen tension. In normal hepatocytes, iNOS protein, mRNA and activity were detected. In transfected cells, chloramphenicol acetyltransferase activity was measured. RESULTS: In cells cultured in a hypoxic environment, both iNOS protein and mRNA increased, whereas the nitrite level in the medium decreased. However, electron paramagnetic resonance analysis and in vitro iNOS activity indicated that iNOS was active. Transfection experiments showed that the expression of chloramphenicol acetyltransferase driven by iNOS promoter was increased in cells maintained at low oxygen tension. CONCLUSIONS: Our experiments show that in rat hepatocytes: 1) iNOS is induced by low oxygen tension; 2) the modification occurs at the transcriptional level; 3) the enzyme at 5% oxygen is able to catalyze the synthesis of NO, although no nitrites are accumulated in the medium. These findings could have physiopathological relevance, e.g. in determining the resistance of perivenous hepatocytes to ischemia injury.

Long chain diamines inhibit growth of C6 glioma cells according to their hydrophobicity. An in vitro and molecular modeling study.

A series of diamines with the general structure NH2(CH2)xNH2, x=2-12, was tested for their potential effects on cell proliferation of cultured rat C6 glioma cells in comparison to natural polyamines. Long chain diamines reduced cell number after 48 h in culture with a sequence of 1,12-diaminododecane (1,12-DD) >1,10-diaminodecane >1,9-diaminononane. Polyamines (putrescine, spermidine and spermine) as well as diamines up to a CH2-chain length of x=8 were found to be ineffective. The spermine analogue 1,12-DD was the most effective molecule in reducing cell number in an irreversible, dose-dependent manner (EC50=3 microM under serum-free conditions). In further experiments we investigated the mechanisms of action of 1,12-DD. The compound had only a minor effect on cell cycle and did not affect free internal calcium concentration. Under physiological conditions 1,12-DD interacts with triplex DNA but not with duplex DNA. Ornithine decarboxylase activity as well as the concentration of internal polyamines were found to be reduced by 1,12-DD. Polyamine application, however, was not able to reverse the effect of 1,12-DD, indicating a polyamine-independent or non-competitive mechanism of action. 1,12-DD reduced cell number by induction of apoptosis as well as necrosis. In molecular modeling studies it was found that a minimal hydrophobic intersegment of at least 4 A was required to make a diamine an effective drug in respect to cellular growth. A hydrophobic gap of this size fits the minimum requirement expected from molecular modeling to provide space for hydrophobic interactions with parts of proteins like a CH3-group. Our results show that 1,12-DD acts as a potent drug, reducing the number of C6 glioma cells, and suggest that its spatial and hydrophobic properties are responsible for its mechanism of action.