Control of aversion by glycine-gated GluN1/GluN3A NMDA receptors in the adult medial habenula.

The unconventional N-methyl-d-aspartate (NMDA) receptor subunits GluN3A and GluN3B can, when associated with the other glycine-binding subunit GluN1, generate excitatory conductances purely activated by glycine. However, functional GluN1/GluN3 receptors have not been identified in native adult tissues. We discovered that GluN1/GluN3A receptors are operational in neurons of the mouse adult medial habenula (MHb), an epithalamic area controlling aversive physiological states. In the absence of glycinergic neuronal specializations in the MHb, glial cells tuned neuronal activity via GluN1/GluN3A receptors. Reducing GluN1/GluN3A receptor levels in the MHb prevented place-aversion conditioning. Our study extends the physiological and behavioral implications of glycine by demonstrating its control of negatively valued emotional associations via excitatory glycinergic NMDA receptors.

Plantas medicinais utilizadas por idosos com diagnóstico de Diabetes mellitus no tratamento dos sintomas da doença / Medicinal plants used by elderly people with Diabetes mellitus in the treatment of the disease symptoms

Este trabalho teve como objetivo investigar as plantas medicinais utilizadas por idosos assistidos em uma Unidade Básica de Saúde de Pelotas-RS, com diagnóstico de Diabetes mellitus, como terapia complementar no tratamento dos sintomas da doença. Caracterizou-se por pesquisa qualitativa, realizada em julho de 2009, no município de Pelotas-RS. Foram entrevistados 18 idosos com idade entre 60 e 77 anos, sendo 14 do sexo feminino. Os participantes citaram 20 plantas medicinais utilizadas como terapia complementar no tratamento do Diabetes mellitus. Entre estas, as mais citadas utilizadas para diminuir os níveis de glicose no sangue foram Sphagneticola trilobata, Bauhinia spp. e Syzygium cumini, sendo que para as duas últimas há comprovação científica do efeito hipoglicemiante. A infusão foi a forma de preparo predominante. Considera-se importante a realização de estudos farmacológicos que investiguem os efeitos das plantas utilizadas pela população, a fim de que o uso proporcione os benefícios desejados e não cause danos à saúde.

This study aimed to investigate medicinal plants used as complementary therapy by the elderly with diabetes mellitus, assisted by a Health Basic Unit in Pelotas (RS, Brazil). This qualitative research was conducted in July 2009. Eighteen 60 to 77 years old elderly (14 of them female) were interviewed. The participants identified 20 medicinal plants used as complementary therapies in the treatment of diabetes mellitus. Among these, the most used ones to reduce the levels of glucose in the blood were Sphagneticola trilobata, Bauhinia spp. and Syzygium cumini. For the last two plants, there are scientific proofs of hypoglycemic effects. The infusion was the predominant form of preparation. Studies to investigate the pharmacological effects of plants used by the population have become very important, for providing scientific information on their possible health benefits and side effects.

Lesions in the central nucleus of the amygdala: discriminative avoidance learning, discriminative approach learning, and cingulothalamic training-induced neuronal activity.

The amygdala is critically involved in discriminative avoidance learning. Large lesions of the amygdala block discriminative avoidance learning and abolish cingulothalamic training-induced neuronal activity. These results indicated that amygdalar processing is critical for cingulothalamic plasticity. The larger lesions did not allow differentiation of the specific functioning of various amygdalar nuclei. Anatomical analysis showed that damage in the central (CE) nucleus of the amygdala was correlated with the severity of the behavioral deficit. The present study was carried out to determine whether smaller lesions, centered in the CE nucleus, would impair discriminative avoidance learning and block cingulothalamic plasticity. In addition, the possible role of the CE nucleus in appetitively motivated discriminative approach learning was examined for the first time. New Zealand White rabbits with CE nuclear lesions were first trained in the discriminative approach task. After attaining asymptotic performance, discriminative avoidance training sessions were alternated with continuing approach training sessions, one session each day. The rabbits with lesions were severely impaired in avoidance learning but showed no impairment of approach learning. Surprisingly, the attenuating effects of the lesions on cingulothalamic training-induced neuronal activity were more prevalent during approach learning than during avoidance learning. These results indicated that avoidance learning can be impaired by lesions centered in the CE nucleus that leave cingulothalamic plasticity largely intact and that the CE nucleus is involved in extra-cingulothalamic learning processes.

Recurrence of the R408W mutation in the phenylalanine hydroxylase locus in Europeans.

The relative frequency of the common phenylalanine hydroxylase (PAH) mutation R408W and its associations with polymorphic RFLP, VNTR, and short-tandem-repeat (STR) sites in the PAH gene were examined in many European populations and one representative North American population of defined European descent. This mutation was found to cluster in two regions: in northwest Europe among Irish and Scottish peoples, and in eastern Europe, including the Commonwealth of Independent States. This allele was significantly less frequent in intervening populations. In eastern European populations, the R408W mutation is strongly associated with RFLP haplotype 2, the three-copy VNTR allele (VNTR 3), and the 240-bp STR allele. In northwestern European populations, it is strongly associated with RFLP haplotype 1, the VNTR allele containing eight repeats (VNTR 8), and the 244-bp STR allele. An examination of the linkage between the R408W mutation and highly polymorphic RFLP, VNTR, and STR haplotypes suggests that recurrence is the most likely mechanism to account for the two different major haplotype associations of R408W in Europe.

Potentiation of the antitumor activity of 5-fluorouracil in colon carcinoma cells by the combination of interferon and deoxyribonucleosides results from complementary effects on thymidine phosphorylase.

alpha-Interferon (IFN alpha) potentiates the cytotoxicity of 5-fluorouracil (FUra) in vitro, and the combination has clinical efficacy in advanced colorectal cancer. We have reported previously an IFN alpha-mediated elevation in cellular FdUMP levels accompanied by the stimulation of thymidine phosphorylase (TP) activity in extracts from HT-29 human colon carcinoma cells treated with IFN alpha. We have now found that this effect of IFN alpha can be measured in vivo as an increase in thymine incorporation in intact cells. The increase was only 3-fold, however, compared to the 12-fold increase seen in TP activity in cell extracts. This suggested that the cosubstrate for TP, deoxyribose-1-phosphate, was rate limiting in the cells. Since the synthetic pathway of TP can also proceed via a transferase reaction, natural and modified deoxyribonucleosides were tested as deoxyribosyl donors. TP activity was measurable in cell extracts using deoxyinosine as cosubstrate with either thymine or FUra, although activity was only 10% of that measured with deoxyribose-1-phosphate. The pyrimidine analogue 5-propynyloxy-2'-deoxyuridine (PO-dUrd) had 15% of the maximal TP activity in cell extracts and also increased thymine incorporation in intact cells 10-fold. Both 2'-deoxyinosine and PO-dUrd potentiated the cytotoxicity of FUra by 8-11-fold. IFN alpha potentiated the cytotoxicity of FUra by 1.8-fold, and the combination of IFN alpha and PO-dUrd produced a 25-fold increase in the cytotoxicity of FUra. Neither the corresponding analogue riboside, 5-propynyloxyuridine, nor the analogue base, 5-propynyloxyuracil, had any effect on FUra cytotoxicity. There was a significant correlation between the ability of a nucleoside and/or IFN alpha combination to increase thymine incorporation and to reduce the 50% inhibitory concentration for FUra. IFN alpha and PO-dUrd also potentiated the inhibition by FUra of thymidylate synthase activity. These findings suggest that the use of a deoxyribonucleoside to provide the rate limiting cosubstrate would complement the stimulation of TP by IFN alpha, and together they should further enhance the antitumor activity of FUra.

Erythrocytapheresis therapy to reduce iron overload in chronically transfused patients with sickle cell disease.

Chelation therapy with deferoxamine is effective in preventing the risk of transfusional iron overload, but treatment failure is common because of noncompliance. To reduce the transfusional iron load, we have evaluated longterm erythrocytapheresis in 14 subjects with sickle cell disease and stroke (11) or other complications (3) as an alternative to simple transfusion. Subjects were treated with erythrocytapheresis using the Haemonetics V50 (Haemonetics Corp, Braintree, MA) to maintain the target pretransfusion hemoglobin S (Hb S) level less than 50% for 6 to 71 months. The transfusional iron load and the donor blood usage were analyzed for a 6- to 36-month study period and were compared with similar data from a subset of 7 subjects previously treated with conventional (target Hb S < 30%) and modified (target Hb S < 50%) simple transfusion protocols. The effect of erythrocytapheresis on iron accumulation was determined by assessment of serum ferritin levels in the absence of iron chelation. The mean transfusional iron load and donor blood usage with erythrocytapheresis were 19 +/- 14 mg iron/kg/yr (range, 6 to 50) and 188.4 +/- 55.2 mL packed-red blood cells (RBC)/kg/yr (range, 107 to 281), respectively. Of 6 subjects receiving no iron chelation therapy, 5 maintained normal or nearly normal serum ferritin levels during 11 to 36 months of erythrocytapheresis. In comparison with conventional simple transfusion and modified simple transfusion, erythrocytapheresis reduced iron loading by 87% (P < .01) and 82% (P < .01), respectively, but increased donor blood usage by 23% and 73%, respectively. Subjects with pre-erythrocytapheresis Hb levels > or = 8.0 g/dL had lower iron accumulation (P < .001) and less donor blood usage (P < .005) than subjects with Hb levels < or = 8.0 g/dL. Although donor blood usage is increased in comparison with simple transfusion, long-term erythrocytapheresis markedly reduces or prevents iron accumulation. This form of transfusion therapy allows the cessation of iron chelation in well-chelated subjects and, if used as the initial form of transfusion therapy, may prevent long-term complications of sickle cell disease without risk of iron overload and the need for chelation therapy.

Elastin-associated microfibrils (10 nm) in a three-dimensional fibroblast culture.

The purpose of this study is to present a three-dimensional dermal fibroblast model. Skin fibroblasts cultured in this system deposit large amounts of collagen and microfibrils. Fibroblasts were seeded onto a nylon filtration mesh and incubated in the presence or absence of ascorbic acid. Collagen fibril formation was found in the presence of ascorbic acid whereas microfibril formation was seen independent of ascorbic acid supplementation. Immunoelectron microscopy revealed that microfibrils were labeled with fibrillin at 67 nm periodicity. Isolated microfibrils studied by rotary shadowing had a beaded appearance consisting of beads linked to each other by a filamentous structure. The spaces between the beads ranged from 10.00-33.33 nm, suggesting that these microfibrils may have an extension-contraction mechanism. Furthermore, the size and spacing of the beads were similar to that seen in microfibrils from tissues (measured after rotary shadowing). Fibroblasts cultured in a three-dimensional mesh represent an effective in vitro model with which to study microfibril formation.

Vision and hearing during deferoxamine therapy.

To determine the frequency of eye and auditory complications and their relationship to drug dosage and iron stores in patients receiving deferoxamine, we studied 52 regularly transfused patients who received deferoxamine by subcutaneous or intravenous infusion in doses from 26 to 136 mg/kg/day, and whose serum ferritin levels of 185 to 17,775 micrograms/L reflected a wide range of iron stores. Forty-nine patients (94%) had no evidence of drug-induced visual or auditory abnormalities. Symptomatic loss of vision and hearing developed in one patient; both problems improved when chelation therapy was stopped. Of the 51 symptom-free patients, one had a mild degree of macular stippling and one had a mild, bilateral, high-frequency sensorineural hearing loss. Eye and ear abnormalities in the symptom-free patients did not progress despite continuation or resumption of chelation therapy at the same dosage. Patients with ophthalmologic and audiologic abnormalities did not receive higher doses of deferoxamine and did not have lower serum ferritin levels than patients without such abnormalities. These findings demonstrate that eye and ear abnormalities during chelation therapy with deferoxamine may not occur uniformly at as high a frequency as previously reported, even in patients who receive large doses of the chelating agent or who have only modest amounts of excessive iron.

Ascorbate-induced changes in gangliosides of calf aortic smooth muscle cells in culture: possible influence of extracellular matrix.

The ganglioside composition of calf aortic smooth muscle cells, cultured in the presence and absence of ascorbate, was analyzed. Previous work has shown that ascorbate supplementation leads to the formation of an extracellular matrix consisting primarily of collagen and that this matrix influences the biosynthetic capabilities of the cell. Cell cultures were supplemented with ascorbate for 3 wk and labeled with [14C]glucosamine for 3 d before harvesting. Ascorbate supplementation resulted in increased ganglioside sialic acid levels and a change in chromatographic profile involving both absolute and relative increases in GD1a. The latter, along with polysialo species, showed increased incorporation of [14C]glucosamine. These findings are interpreted in relation to the proposed role of gangliosides as mediators in the interaction of various cells with extracellular matrix.

Rapid removal of excessive iron with daily, high-dose intravenous chelation therapy.

We investigated the value of high-dose intravenous iron chelation therapy with deferoxamine as an alternative to conventional subcutaneous therapy in eight patients receiving regular transfusions who had massive iron stores, including two with clinical heart disease. Six to twelve grams of deferoxamine was infused daily for 12 hours over 12 to 25 months through externalized central venous catheters or implanted reservoirs. Serum ferritin levels decreased by 56% to 99%. Liver iron concentrations, measured by magnetic susceptibility in two patients, were 1234 and 2438 micrograms/gm wet weight (22.1 and 43.6 mumol/gm wet weight) after treatment for 17 and 25 months, respectively. A patient with congestive heart failure and a patient with severe ventricular dysrhythmias no longer required cardiac medication after 12 to 24 months of chelation therapy. Three episodes of bacteremia and three episodes of cellulitis accounted for a catheter-related infection rate of 0.14 per 100 patient-days. The catheter removal rate was 0.20 per 100 patient-days. No patient experienced serious visual, auditory, or other toxicities. We conclude that in some patients receiving regular erythrocyte transfusions, high-dose intravenous chelation therapy with deferoxamine is superior to conventional subcutaneous treatment.

Variable regulation of sensitivity to retinoic acid-induced differentiation in wild-type and retinoic acid-resistant HL-60 cells.

The initial cell association and metabolic conversion of retinoic acid (RA) by HL-60 cells in serum-free, transferrin/insulin-supplemented, RPMI 1640 medium was greater than or equal to 10-fold greater than in RPMI 1640 medium containing 10% fetal bovine serum (FBS). This was paralleled under the serum-free conditions by 10-fold greater sensitivity to RA-induced differentiation, which was partially reversed by the addition of purified bovine serum albumin to the same concentration present in 10% FBS. In serum-free HL-1 medium, HL-60 cell sensitivity to RA-induced differentiation was approximately 250-fold less than in serum-free RPMI 1640 medium but, in this comparison, there was little difference in RA cell association or metabolism. A greater than 200-fold RA-resistant HL-60 subline had RA cell-association and metabolism rates similar to those of wild-type cells under all culture conditions. No significant qualitative differences in the high performance liquid chromatography elution patterns of polar metabolites were observed under any circumstances. These results indicate that inherent cellular properties, not associated with gross differences in RA uptake or metabolism, primarily determined the relative sensitivity or insensitivity of HL-60 cells to RA-induced differentiation but that RA responsiveness was markedly regulated by extracellular factors, one of which, serum albumin, appeared to act by decreasing the initial cell association and metabolism of RA, whereas other, as yet unidentified exogenous factors, may have acted independently of these functions.

Overview of the beta thalassemias: genetic and clinical aspects.

There has been much progress in basic studies of the beta-thalassemia disorders in recent years, as well as in the practical aspects of prenatal diagnosis and clinical management. Alterations in a single or a few nucleotides account for most of the types of beta-thalassemia syndromes that have been characterized, and a variety of deletions have been found associated with beta O thalassemia, delta beta thalassemia, gamma delta beta thalassemia, and hereditary persistence of fetal hemoglobin (HPFH). Studies of nondeletional types of HPFH have revealed several single base changes 5' to the G gamma and A gamma genes, suggesting that these regions may be of major importance in the switchover from fetal to adult hemoglobin synthesis and in the relative production of the G gamma and A gamma globin chains during development. Evaluation of restriction enzyme polymorphisms and the assignment of haplotypes in the beta-like globin region of chromosome 11 have allowed delineation of the origin and distribution of thalassemia mutations, and have provided an important means for prenatal diagnosis. These studies have accumulated much new information about the function and expression of eukaryotic genes, and have served as a model for the investigation of human genetic disorders. The clinical management of patients with Cooley's anemia has benefited greatly from changes in transfusion practice, clear guidelines for splenectomy, and the availability of effective chelation therapy for iron overload. Current basic and clinical studies may lead to new approaches to management, treatment and eventual cures for this disease, including safe bone marrow transplantation, oral chelation treatment, and gene therapy.

Thalassemia syndromes. Recent advances.

Laboratory and clinical investigators in the past several years have provided many advancements in the understanding of the thalassemia syndromes and in the care of affected patients. The diversity of genetic defects causing thalassemia has been extensively explored, with major benefits to our knowledge of normal globin gene function and of the consequences of specific mutations. In addition, the use of molecular biology methods in these studies has provided major advances in population genetics, gene transfer, and prenatal diagnosis of thalassemia. In the clinical area, guidelines for transfusion, splenectomy, prevention of postsplenectomy infection, and effective iron chelation have been considerably improved, and bone marrow transplantation is now available as an alternative means of treatment. Despite these advances, a great deal remains to be done in the areas of understanding the developmental regulation of globin gene expression, effective gene transfer, oral chelation therapy, safer blood products, and other important areas that will benefit patients afflicted with thalassemia.

Proportionate mortality ratio analysis of automobile mechanics and gasoline service station workers in New Hampshire.

A proportionate mortality ratio (PMR) analysis of all deaths recorded from 1975 to 1985 among New Hampshire white male residents (age 20 years or older) was performed using death certificate information. Among automobile mechanics, the analysis revealed increases in mortality from leukemia (PMR = 178, N = 6); cancers of the oral cavity (PMR = 163, N = 4), lung (PMR = 112, N = 36), bladder (PMR = 169, N = 5), rectum (PMR = 182, N = 4), and lymphatic tissues (PMR = 200, N = 6); and cirrhosis of the liver (PMR = 140, N = 13) and suicide (PMR = 177, N = 22; p less than 0.05). Workers in the gasoline service station industry experienced a leukemia mortality excess (PMR = 328, N = 3; p less than 0.05) as well as increases in deaths from suicide (PMR = 162, N = 4), emphysema (PMR = 245, N = 4), and mental and psychoneurotic conditions (PMR = 394, N = 3). These workers are potentially exposed to a variety of substances including gasoline vapor, benzene, solvents, lubricating oils and greases, and asbestos (from brake and clutch repair) as well as welding fumes and car and truck exhaust. Despite limitations regarding the small number of deaths and methodologic constraints, the results of this analysis suggest that one or more of the exposures experienced by these workers poses a significant carcinogenic risk. More definitive epidemiologic studies are required to determine if the leukemia excess is associated with exposure to benzene, gasoline, or other workplace substances.