Estradiol cypionate treatment in suckling/weaning and estrous cycling/anestrous beef cows subjected to fixed-time artificial insemination.

This study was conducted to evaluate the interaction between dose of estradiol cypionate (ECP) and ovarian status in beef cows on which different weaning/suckling regimens were imposed before fixed-time artificial insemination (FTAI). A total of 8070 estrous cycling and anestrous cows were subjected to three experiments, when calves were weaned early (Experiment 1), anti-suckling nose plates were applied for 9 or 10 days (Experiment 2), or there was continued suckling (Experiment 3). The cows were administered an estradiol/progesterone-based treatment regimen for FTAI and were treated with 0.5 or 1.0 mg of ECP im at the time of progesterone intravaginal device removal. Artificial insemination was performed from 46 to 56 h after the time of ECP treatment. Pregnancy per artificial insemination (P/AI) was affected by dose of ECP differentially in early-weaned and suckled cows. Whereas P/AI percentage was greater in early-weaned cows treated with 0.5 than 1.0 mg ECP (P < 0.05), P/AI percentage was greater for suckled cows treated with 1.0 than 0.5 mg ECP (P < 0.05). Although there were greater P/AI percentages in estrous cycling than anestrous cows (P < 0.05) when there was nose plate weaning and continuation of suckling, there was no difference between estrous cycling and anestrous cows (P = NS) when there was early weaning. Overall results indicate ECP administration affects fertility in a dose-dependent manner, suggesting an interaction between suckling and estrous cycling effects. As more critical the condition was (i.e., suckling anestrous cows), larger dose of estradiol was required.

Retinoblastoma and mental retardation microdeletion syndrome: clinical characterization and molecular dissection using array CGH.

We describe three patients with retinoblastoma, dysmorphic features and developmental delay. Patients 1 and 2 have high and broad forehead, deeply grooved philtrum, thick anteverted lobes and thick helix. Patient 1 also has dolicocephaly, sacral pit/dimple and toe crowding; patient 2 shows intrauterine growth retardation and short fifth toe. Both patients have partial agenesis of corpus callosum. Patient 3 has growth retardation, microcephaly, thick lower lip and micrognathia. Using array-comparative genomic hybridization (CGH), we identified a 13q14 de novo deletion in patients 1 and 2, while patient 3 had a 7q11.21 maternally inherited deletion, probably not related to the disease. Our results confirm that a distinct facial phenotype is related to a 13q14 deletion. Patients with retinoblastoma and malformations without a peculiar facial phenotype may have a different deletion syndrome or a casual association of mental retardation and retinoblastoma. Using array-CGH, we defined a critical region for mental retardation and dysmorphic features. We compared this deletion with a smaller one in a patient with retinoblastoma (case 4) and identified two distinct critical regions, containing 30 genes. Four genes appear to be good functional candidates for the neurological phenotype: NUFIP1 (nuclear fragile X mental retardation protein 1), HTR2A (serotonin receptor 2A), PCDH8 (prothocaderin 8) and PCDH17 (prothocaderin 17).

The Italian XLMR bank: a clinical and molecular database.

Mental retardation (MR) is a nonprogressive condition characterized by a significant impairment of intellectual capabilities with deficit of cognitive and adaptive functioning and onset before 18 years. Mental retardation occurs in about 2 to 3% of the general population and it is estimated that 25 to 35% of the cases may be due to genetic causes. Among these "genetic" MR, 25 to 30% are probably due to mutations in a gene on the X chromosome (X-linked mental retardation, XLMR). Given the genetic heterogeneity of XLMR, the availability of a considerable number of patients with accurate phenotypic classification is a crucial factor for research. The X-linked Mental Retardation Italian Network, which has been active since 2003, has collected detailed clinical information and biological samples from a vast number of MR patients. Collected samples and clinical information are inserted within the XLMR bank, a comprehensive molecular and clinical web-based database available at the address http://xlmr.unisi.it. The database is organized in three distinct parts. Part I and II contain several electronic schedules to register information on the family, the phenotypic description, the photographs, and a 20 sec movie of the patient. Part III allows the registration of molecular analyses performed on each case; samples and clinical data are usable via password-restricted access. Clinical and molecular centers interested in joining the network may request a password by simply contacting the Medical Genetics of the University of Siena. The XLMR bank is an innovative biological database that allows the collection of molecular and clinical data, combines descriptive and iconographic resources, and represents a fundamental tool for researchers in the field of mental retardation.

Immunohistochemical visualization of newly formed quinolinate in the normal and excitotoxically lesioned rat striatum.

Intracerebral infusion of 3-hydroxyanthranilate (3HANA) rapidly increases the brain content of the endogenous excitotoxin quinolinate (QUIN). QUIN formation from 3HANA is readily prevented by coadministration of the specific 3-hydroxyanthranilate oxygenase inhibitor 4-chloro-3HANA (4-Cl-3HANA). This experimental paradigm was used to identify the cell populations which are responsible for the rapid de novo production of QUIN in the rat striatum in vivo. Rats received an intrastriatal infusion of 3HANA, alone or together with equimolar 4-Cl-3HANA, for 1 h. Striatal QUIN immunoreactivity (ir) was assessed immunohistochemically, using an antibody against protein-conjugated QUIN. This antibody displayed no significant crossreactivity with compounds structurally or functionally related to QUIN. QUIN-ir cells were detected after infusion with > or =300 microM 3HANA, but not in naïve striata or after co-infusion of 4-Cl-3HANA. Cellular staining was also abolished by preabsorption of the antibody with protein-conjugated QUIN. In the normal striatum, QUIN-ir was detected exclusively in cells of an apparent microglial morphology. When examined in the excitotoxically lesioned striatum, 3HANA-induced QUIN-ir localized exclusively to OX42-ir cells of an activated microglial/macrophage morphology. These data indicate that microglia and macrophages are the major source of QUIN in the rat striatum when hyperphysiological concentrations of 3HANA are used to drive QUIN synthesis. Comparison with earlier biochemical and immunohistochemical studies suggests that the enzyme responsible for microglial QUIN production is a distinct 3-hydroxyanthranilate oxygenase with high capacity and low affinity for 3HANA.

[Nursing: idealism and realism. Perspectives of nursing students on the nursing profession]. / Enfermagem: idealismo x realismo. Perspectivas de formandos do curso de graduação de enfermagem sobre a profissão de enfermagem.

Exploratory and descriptive study with the following objectives: to identify the nursing students' feelings related to the undergraduate course and their perception towards the nursing profession. After the four-year course, 57.4% of students perceived nursing in a positive way as a profession for the future, valued, recognised, compensating even with some limitations. However, 25% of students still perceived nursing as a mechanical, manual and sacrificed profession, with a limited scientific vision and with a gap between theory and practice and as a consequence a lower recognition by the society. If current trends are maintained, the nurse's value would be much greater in the next decades within the Brazilian society according to 61% of respondents.

Kynurenergic manipulations influence excitatory synaptic function and excitotoxic vulnerability in the rat hippocampus in vivo.

Competing enzymatic mechanisms degrade the tryptophan metabolite L-kynurenine to kynurenate, an inhibitory and neuroprotective compound, and to the neurotoxins 3-hydroxykynurenine and quinolinate. Kynurenine 3-hydroxylase inhibitors such as PNU 156561 shift metabolism towards enhanced kynurenate production, and this effect may underlie the recently discovered anticonvulsant and neuroprotective efficacy of these drugs. Using electrophysiological and neurotoxicological endpoints, we now used PNU 156561 as a tool to examine the functional interplay of kynurenate, 3-hydroxykynurenine and quinolinate in the rat hippocampus in vivo. First, population spike amplitude in area CA1 and the extent of quinolinate-induced excitotoxic neurodegeneration were studied in animals receiving acute or prolonged intravenous infusions of L-kynurenine, PNU 156561, (L-kynurenine+PNU 156561) or kynurenate. Only the latter two treatments, but not L-kynurenine or PNU 156561 alone, caused substantial inhibition of evoked responses in area CA1, and only prolonged (3h) infusion of (L-kynurenine+PNU 156561) or kynurenate was neuroprotective. Biochemical analyses in separate animals revealed that the levels of kynurenate attained in both blood and brain (hippocampus) were essentially identical in rats receiving extended infusions of L-kynurenine alone or (L-kynurenine+PNU 156561) (4 and 7microM, respectively, after an infusion of 90 or 180min). However, addition of the kynurenine 3-hydroxylase inhibitor resulted in a significant decrement in the formation of 3-hydroxykynurenine and quinolinate in both blood and brain. These data suggest that the ratio between kynurenate and 3-hydroxykynurenine and/or quinolinate in the brain is a critical determinant of neuronal excitability and viability. The anticonvulsant and neuroprotective potency of kynurenine 3-hydroxylase inhibitors may therefore be due to the drugs' dual action on both branches of the kynurenine pathway of tryptophan degradation.

Pyrrolo[3,2-c]quinoline derivatives: a new class of kynurenine-3-hydroxylase inhibitors.

A series of pyrrolo[3,2-c]quinoline derivatives were synthesised and evaluated as inhibitors of selected enzymes of the kynurenine pathway. 7-Chloro-3-methyl-1H-pyrrolo[3,2-c]quinoline-4-carboxylic acid (7a) was found to be a relatively potent and selective inhibitor of kynurenine-3-hydroxylase (KYN-3-OHase). A molecular modelling study showed a good superimposition of 7a with PNU-156561 and kynurenine the natural substrate of KYN-3-OHase.

Temporal and spatial changes of quinolinic acid immunoreactivity in the gerbil hippocampus following transient cerebral ischemia.

Quinolinic acid (QUIN) is an endogenous neurotoxin which originates from the kynurenine pathway of tryptophan metabolism. An increase of brain QUIN level occurs in several degenerative and inflammatory disorders, but the cellular source of QUIN is still a matter of controversy. In the present study, the gerbil model of transient global ischemia was used to investigate the time course and the cellular localization of QUIN immunoreactivity. Neurodegeneration was evident in the subiculum and in the CA1 area of the hippocampus 4, 7 and 14 days after ischemia. QUIN positive cells, with microglia-like morphology, appeared in the subiculum and in the CA1, 4 days after ischemia. At 7 days post-ischemia they extended to the whole CA1, disappearing at 14 days. Neither neurodegeneration nor QUIN positive cells could be detected in ischemic gerbils sacrificed at 1 and 2 days after ischemia and in sham-operated animals. These findings suggest that microglia-like cells infiltrating the degenerating areas of the hippocampus represent the major source of QUIN following transient ischemia in the gerbil. Thus, in situ production of QUIN in vulnerable brain regions may contribute to the pathophysiological mechanisms of delayed brain injury.

4-Phenyl-4-oxo-butanoic acid derivatives inhibitors of kynurenine 3-hydroxylase.

Kynurenine 3-hydroxylase (KYN 3-OHase) is a key enzyme in the kynurenine pathway of tryptophan degradation and its inhibition may be an effective mechanism for counteracting neuronal excitotoxic damage. We present here a new class of inhibitors derived from a structure-activity relationship (SAR) study of the benzoylalanine side-chain of 1. 2-hydroxy-4-(3,4-dichlorophenyl)-4-oxobutanoic acid (8) and 2-benzyl-4-(3,4-dichlorophenyl)-4-oxo-butanoic acid (10) emerged as the most interesting derivatives. Enantiospecific synthesis for both enantiomers of 8 and diastereomeric salt resolution for those of 10 were successfully applied.

Cloning and recombinant expression of rat and human kynureninase.

Kynureninase [E.C.3.7.1.3.] is one of the enzymes involved in the biosynthesis of NAD cofactors from tryptophan through the kynurenine pathway. By tryptic and CNBr digestion of purified rat liver kynureninase, we obtained about 28% of the amino acid sequence of the enzyme. The rat kynureninase cDNA, isolated by means of reverse-transcribed polymerase chain reaction and hybridization screening, codes for a polypeptide of 464 amino acids. Northern blot analysis revealed the synthesis of a 2.0 kb rat kynureninase mRNA. A cDNA encoding human liver kynureninase was also isolated. The deduced amino acid sequence is 85% identical to that of the rat protein. COS-1 cells were transfected with both cDNAs. The Km values of the rat enzyme, for L-kynurenine and DL-3-hydroxykynurenine, were 440 +/- 20 microM and 32 +/- 5 microM and of the human enzyme 440 /- 20 microM and 49 +/- 6 microM, respectively. Interestingly, COS-1 cells transfected with the cDNA coding for rat kynureninase also display cysteine-conjugate beta-lyase activity.

(R,S)-3,4-dichlorobenzoylalanine (FCE 28833A) causes a large and persistent increase in brain kynurenic acid levels in rats.

Kynurenic acid is an endogenous excitatory amino-acid receptor antagonist with neuroprotective and anticonvulsant properties. We demonstrate here that systemic administration of the new and potent kynurenine 3-hydroxylase inhibitor (R,S)-3,4-dichlorobenzoylalanine (FCE 28833A) causes a dose-dependent elevation in endogenous kynurenine and kynurenic acid levels in rat brain tissue. In hippocampal microdialysates, peak increases of 10- and 80-fold above basal kynurenic acid concentrations, respectively, were obtained after a single oral or intraperitoneal administration of 400 mg/kg FCE 28833A. After intraperitoneal treatment with FCE 28833A, extracellular brain kynurenic acid levels remained significantly elevated for at least 22 h, rendering this compound a far more effective enhancer of kynurenic acid levels than the previously described kynurenine 3-hydroxylase blocker m-nitrobenzoylalanine. FCE 28833A and similar molecules may have therapeutic value in diseases which are linked to a hyperfunction of excitatory amino-acid receptors.

Alternative splicing at the C-terminal but not at the N-terminal domain of the NMDA receptor NR1 is altered in the kindled hippocampus.

Several lines of evidence suggest that N-methyl-D-aspartate (NMDA) receptors significantly contribute to the development of kindling. In addition, a lasting enhancement of the NMDA receptor function has been suggested to play a significant role in the chronic hyperexcitability occurring in the hippocampus after kindling epileptogenesis. We have investigated whether hippocampal kindling induces changes in the NMDA receptor at the molecular level by assessing the expression of mRNAs of the different spliced variants at the N-terminal (exon 5) and C-terminal (exon 21) position of the NMDA receptor 1 (NR1) gene by means of the reverse transcription-polymerase chain reaction. Alternative splicing at exon 5 confers different sensitivity of the NMDA receptor to polyamines while exon 21 encodes a 37-amino acid insert containing the major phosphorylation sites for protein kinase C. One week after the acquisition of stage 5 of kindling in rats (generalized tonic-clonic seizures), the relative abundance of the two alternatively spliced forms at the C-terminal domain, respectively containing (+) or lacking (-) exon 21, was reversed compared to controls (implanted with electrodes but not stimulated) in the dorsal hippocampus ipsilateral and contralateral to the electrical stimulation. The exon 21+/exon 21- mRNA ratio for controls was 1.3 +/- 0.04 (mean +/- SE); for ipsilaterally kindled rats it was 0.64 +/- 0.05 (P < 0.05), and for contralaterally kindled rats it was 0.48 +/- 0.07 (P < 0.01). Similar bilateral effects were observed in the ventral hippocampus (temporal pole). No changes were found 4 weeks after stage 5 seizures and 1 week after the induction of a single afterdischarge. No significant alterations were induced by kindling in the relative abundance of the spliced variants containing or lacking exon 5. Our findings show selective changes in alternative splicing of the NR1 gene after repeated application of an epileptogenic stimulus. This may generate receptors with different functional properties, which may contribute to the increased sensitivity for the induction of generalized seizures during kindling.

Molecular cloning of rat kynurenine aminotransferase: identity with glutamine transaminase K.

The enzyme kynurenine aminotransferase (KAT) catalyses the conversion of L-kynurenine to kynurenic acid. A combination of polymerase chain reaction techniques and hybridization screening was used to isolate a cDNA clone encompassing the entire coding region of KAT from rat kidney. Identification of the cDNA as coding for KAT was based both on the comparison of amino acid sequences obtained from purified rat KAT and on the expression of KAT activity in COS-1 cells transfected with the cDNA. RNA blot analysis indicated that KAT mRNA is widely expressed in rat tissues. Cultured cells transfected with the cDNA for KAT also showed glutamine transaminase K activity. Based mainly on sequence data, these results demonstrate that rat kidney KAT is identical with glutamine transaminase K.