Agrin defines polarized distribution of orthogonal arrays of particles in astrocytes

Accumulating evidence indicates that agrin, a heparan sulphate proteoglycan of the extracellular matrix, plays a role in the organization and maintenance of the blood-brain barrier. This evidence is based on the differential effects of agrin isoforms on the expression and distribution of the water channel protein, aquaporin-4 (AQP4), on the swelling capacity of cultured astrocytes of neonatal mice and on freeze-fracture data revealing an agrin-dependent clustering of orthogonal arrays of particles (OAPs), the structural equivalent of AQP4. Here, we show that the OAP density in agrin-null mice is dramatically decreased in comparison with wild-types, by using quantitative freeze-fracture analysis of astrocytic membranes. In contrast, anti-AQP4 immunohistochemistry has revealed that the immunoreactivity of the superficial astrocytic endfeet of the agrin-null mouse is comparable with that in wild-type mice. Moreover, in vitro, wild-type and agrin-null astrocytes cultured from mouse embryos at embryonic day 19.5 differ neither in AQP4 immunoreactivity, nor in OAP density in freeze-fracture replicas. Analyses of brain tissue samples and cultured astrocytes by reverse transcription with the polymerase chain reaction have not demonstrated any difference in the level of AQP4 mRNA between wild-type astrocytes and astrocytes from agrin-null mice. Furthermore, we have been unable to detect any difference in the swelling capacity between wild-type and agrin-null astrocytes. These results clearly demonstrate, for the first time, that agrin plays a pivotal role for the clustering of OAPs in the endfoot membranes of astrocytes, whereas the mere presence of AQP4 is not sufficient for OAP clustering.

Syntaxin 3B is essential for the exocytosis of synaptic vesicles in ribbon synapses of the retina.

Ribbon synapses of the vertebrate retina are specialized synapses that release neurotransmitter by synaptic vesicle exocytosis in a manner that is proportional to the level of depolarization of the cell. This release property is different from conventional neurons, in which the release of neurotransmitter occurs as a short-lived burst triggered by an action potential. Synaptic vesicle exocytosis is a calcium regulated process that is dependent on a set of interacting synaptic proteins that form the so-called SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex. Syntaxin 3B has been identified as a specialized SNARE molecule in ribbon synapses of the rodent retina. However, the best physiologically-characterized neuron that forms ribbon-style synapses is the rod-dominant or Mb1 bipolar cell of the goldfish retina. We report here the molecular characterization of syntaxin 3B from the goldfish retina. Using a combination of reverse transcription (RT) polymerase chain reaction (PCR) and immunostaining with a specific antibody, we show that syntaxin 3B is highly enriched in the plasma membrane of bipolar cell synaptic terminals of the goldfish retina. Using membrane capacitance measurements we demonstrate that a peptide derived from goldfish syntaxin 3B inhibits synaptic vesicle exocytosis. These experiments demonstrate that syntaxin 3B is an important factor for synaptic vesicle exocytosis in ribbon synapses of the vertebrate retina.

Expression, induction and regulation of the cytochrome P450 monooxygenase system in the rat glioma C6 cell line

The cytochrome P450 monooxygenase system consists of NADPH- cytochrome P450 reductase (P450 reductase) and cytochromes P450, which can catalyze the oxidation of a wide variety of endogenous and exogenous compounds, including steroid hormones, fatty acids, drugs, and pollutants. The functions of this system are as diverse as the substrates. P450 reductase transfers reducing equivalents from NADPH to P450, which in turn catalyzes metabolic reactions. This enzyme system has the highest level of activity in the liver. It is also present in other tissues, including brain. The functions of this enzyme system in brain seem to include: neurotransmission, neuroendocrinology, developmental and behavioral modulation, regulation of intracellular levels of cholesterol, and potential neurotoxicity.^ In this study, we have set up the rat glioma C6 cell line as an in vitro model system to examine the expression, induction, and tissue-specific regulation of P450s and P450 reductase. Rat glioma C6 cells were treated with P450 inducers phenobarbital (PB) or benzo(a)anthracene (BA). The presence of P450 reductase and of cytochrome P450 1A1, 1A2, 2A1, 2B1/2, 2C7, 2D1-5 and 2E1 was detected by reverse transcription followed by polymerase chain reaction (RT-PCR) and confirmed by restriction digestion. The induction of P450 1A1 and 2B1/2 and P450 reductase was quantified using competitive PCR. Ten- and five-fold inductions of P450 1A and 2B mRNA after BA or PB treatments, respectively, were detected. Western blot analysis of microsomal preparations of glioma C6 cells demonstrated the presence of P450 1A, 2B and P450 reductase at the protein level. ELISAs showed that BA and PB induce P450 1A and 2B proteins 7.3- and 13.5-fold, respectively. Microsomes prepared from rat glioma C6 cells showed cytochrome P450 CO difference spectra with absorption at or near 450 nm. Microsomes prepared from rat glioma C6 cells demonstrated much higher levels of ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-dealkylase (PROD) activity, when treated with BA or PB, respectively. These experiments provide further evidence that the rat glioma C6 cell line contains an active cytochrome P450 monooxygenase system which can be induced by P450 inducers. The mRNAs of P450 1A1 and 2B1/2 can not bind to the oligo(dT) column efficiently, indicating they have very short poly(A) tails. This finding leads us to study the tissue specific regulation of P450s at post-transcriptional level. The half lives of P450 1A1 and 2B1/2 mRNA in glioma C6 cells are only 1/10 and 1/3 of that in liver. This may partly contribute to the low expression level of P450s in glial cells. The induction of P450s by BA or PB did not change their mRNA half lives, indicating the induction may be due to transcriptional regulation. In summary of this study, we believe the presence of the cytochrome P450 monooxygenase system in glial cells of the brain may be important in chemotherapy and carcinogenesis of brain tumors. ^

Analysis of primary Wilms tumors for WT1 RNA processing alterations

Wilms tumor (WT) is an embryonal renal tumor with a heterogeneous genetic etiology that serves as a valuable model for studying tumorigenesis. Biallelic inactivation of the tumor suppressor gene WT1, a zinc-finger transcriptional regulator located at 11p13, is critical for the development of some Wilms tumors. Interestingly, WT1 genomic analysis has demonstrated mutations in less than 20% of WT cases. This suggests either other genes play a more major role in Wilms tumorigenesis or WT1 is functionally altered by mechanisms other than DNA mutation. Previous observations in rat and in WT xenograft cell lines have suggested that abnormal WT1 RNA processing (exon 6 RNA editing and aberrant exon 2 splicing, respectively) is a potential mechanism of altering WT1 function in the absence of a WT1 DNA mutation. However, the role of this abnormal RNA processing has not previously been assessed in primary Wilms tumors. ^ To test the hypothesis that abnormal WT1 RNA processing is a mechanism of WT1alteration during tumor development, WT1 RNA from 85 primary tumors was analyzed using reverse transcription and polymerase chain reaction amplification (RT-PCR). Although no evidence for WT1 RNA editing was observed, variable levels (5% to 50%) of aberrant WT1 exon 2 splicing were detected for 11 tumors in the absence of a detectable WT1 DNA mutation. Also, alteration of normal WT1 alternative splicing, observed as RNA isoform loss, was detected in five tumors with no apparent WT1 genomic alteration, although no consistent pattern of RNA isoform loss was detected. This abnormal WT1 splicing, detected by either loss of exon 2 from some of the transcripts or loss of RNA isoforms, is statistically correlated with relapse (p = 0.005). These studies demonstrate that abnormal WT1 RNA processing is not a common mechanism of abrogating normal WT1 function in primary tumors. However, in those cases in which abnormal WTI splicing is present, these data indicate that it may serve as a useful prognostic marker for relapse in WT patients. ^

Amplification of the full-length hepatitis A virus genome by long reverse transcription-PCR and transcription of infectious RNA directly from the amplicon.

The genetic study of RNA viruses is greatly facilitated by the availability of infectious cDNA clones. However, their construction has often been difficult. While exploring ways to simplify the construction of infectious clones, we have successfully modified and applied the newly described technique of "long PCR" to the synthesis of a full-length DNA amplicon from the RNA of a cytopathogenic mutant (HM 175/24a) of the hepatitis A virus (HAV). Primers were synthesized to match the two extremities of the HAV genome. The antisense primer, homologous to the 3' end, was used in both the reverse transcription (RT) and the PCR steps. With these primers we reproducibly obtained a full-length amplicon of approximately 7.5 kb. Further, since we engineered a T7 promoter in the sense primer, RNA could be transcribed directly from the amplicon with T7 RNA polymerase. Following transfection of cultured fetal rhesus kidney cells with the transcription mixture containing both the HAV cDNA and the transcribed RNA, replicating HAV was detected by immunofluorescence microscopy and, following passage to other cell cultures, by focus formation. The recovered virus displayed the cytopathic effect and large plaque phenotype typical of the original virus; this result highlights the fidelity of the modified long reverse transcription-PCR procedure and demonstrates the potential of this method for providing cDNAs of viral genomes and simplifying the construction of infectious clones.

Bidirectional uncoating of the genomic RNA of a helical virus.

An essential step in the initiation of a virus infection is the release of the viral genome from the other constituents of the virus particle, a process referred to as uncoating. We have used reverse transcription and polymerase chain reaction amplification procedures to determine the rate and direction of in vivo uncoating of the rod-shaped tobacco mosaic virus. The virus particles contain a single 6.4-kb RNA molecule that lies between successive turns of a helical arrangement of coat protein subunits. When the particles are introduced into plant cells, the subunits are removed via a bidirectional uncoating mechanism. Within 2-3 min, the part of the viral RNA from the 5' end to a position >70% toward the 3' end has been freed of coat protein subunits. This is followed by removal of subunits from the 3' end of the RNA and sequential uncoating of the RNA in a 3'-to-5' direction. An internal region of the viral RNA is the final part to be uncoated. Progeny virus particles are detected in the cells 35-40 min after inoculation.

Extrapituitary expression of the rat V1b vasopressin receptor gene.

[Arg8]vasopressin (AVP) stimulates adrenocorticotropic hormone release from the anterior pituitary by acting on the V1b AVP receptor. This receptor can be distinguished from the vascular/hepatic V1a and renal V2 AVP receptors by its differential binding affinities for structural analogous of AVP. Recent studies have shown that the cloned V1a and V2 receptors are structurally related. We have isolated a clone encoding the V1b receptor from a rat pituitary cDNA library using polymerase chain reaction (PCR)-based methodology. The rat V1b receptor is a protein of 421 amino acids that has 37-50% identity with the V1a and V2 receptors. Homology is particularly high in the seven putative membrane-spanning domains of these guanine nucleotide-binding protein-coupled receptors. Expression of the recombinant receptor in mammalian cells shows the same binding specificity for AVP agonists and antagonists as the rat pituitary V1b receptor. AVP-stimulated phosphotidylinositol hydrolysis and intracellular Ca2+ mobilization in Chinese hamster ovary or COS-7 cells expressing the cloned receptor suggest second messenger signaling through phospholipase C. RNA blot analysis, reverse transcription PCR, and in situ hybridization studies reveal that V1b receptor mRNA is expressed in the majority of pituitary corticotropes as well as in multiple brain regions and a number of peripheral tissues, including kidney, thymus, heart, lung, spleen, uterus, and breast. Thus, the V1b receptor must mediate some of the diverse biological effects of AVP in the pituitary as well as other organs.

Efeitos do ramipril sobre a doença periodontal induzida experimentalmente em ratos

A doença periodontal (DP) corresponde a um grupo de doenças inflamatórias que acomete as estruturas periodontais de proteção e de suporte e pode levar à perda dentária. A etiologia está relacionada à placa dentobacteriana que leva à produção de grande quantidade de citocinas pró-inflamatórias importantes na destruição tecidual. A angiotensina (Ang) II também pode contribuir para a inflamação e destruição tecidual no periodonto agindo como mediador chave. A utilização de drogas que atuem na cascata do sistema renina-angiotensina (SRA) poderia interferir no estado de saúde ou inflamação do tecido mole, na perda óssea alveolar e na expressão gênica dos componentes do SRA e mediadores inflamatórios. Portanto, o objetivo do presente trabalho foi investigar se o ramipril, um inibidor da enzima conversora de angiotensina (ECA), altera a progressão da DP induzida experimentalmente em ratos. Foi utilizado o modelo de indução da DP por colocação de ligadura ao redor do primeiro molar inferior direito de ratos. Os grupos com 10 animais cada, foram divididos em tratados com ramipril (via gavagem 10 mg/kg/dia) ou água (veículo) durante 14 e 21 dias e o grupo Sham submetido à indução fictícia da DP. Outros quatro grupos foram submetidos ao pré-tratamento com ramipril durante os períodos de 7 e 14 dias e após a indução da DP e tratados por 14 ou 21 dias. As metodologias de avaliação foram: extração de RNA total, transcrição reversa seguida de reação em cadeia da polimerase quantitativa (RTqPCR), análises histológica e da perda óssea alveolar. Os dados foram analisados por meio de gráficos e os resultados foram submetidos à análise unidirecional de variância (ANOVA) e representaram médias e respectivos desvios-padrão. Diferenças entre os grupos foram consideradas estatisticamente significativas quando p < 0,05. Com base nos resultados obtidos pode-se concluir que o ramipril foi capaz de reduzir a progressão da perda óssea no grupo tratado por 21 dias (DP-21d-Rami), entretanto houve aumento do processo inflamatório, além de alteração da expressão de RNAm de ECA-2 e do receptor Mas, alguns mediadores do processo inflamatório, como COX2 e VEGF, e os receptores VEGF-R1 e VEGF-R2.

The inhibitory effect of pentobarbitone on reverse transcription-PCR

Pentobarbitone sodium (Sodium 5-ethyl-5[1-methylbutyl]-pentobarbitone) is a short-acting barbiturate that is commonly used to euthanase animals. As part of our studies into the molecular genetics of copper toxicosis in Bedlington terrier dogs, reverse-transcription (RT)-PCR was noted to always fail on RNA samples collected from livers of dogs sacrificed by pentobarbitone injection. When samples were collected without pentobarbitone, however, RTPCR was always successful. We suspected the possible inhibition by pentobarbitone sodium of either reverse transcriptase or Taq polymerase. In vitro studies showed that pentobarbitone interference of PCR occurred at >4 mug/mul. To identify if pentobarbitone produced competitive inhibition, each components (Taq polymerase, MgCl2, dNTP, etc.) of the PCR was individually altered. However, inhibition still persisted, suggesting that multiple PCR components may be affected. Also it was shown that pentobarbitone interference was not dependent on the PCR product size. Simple dilution of pentobarbitone contaminated DNA solutions, and the addition of bovine serum albumin (BSA) to the PCR mix overcame pentobarbitone interference. In vivo, PCR by pentobarbitone was found to be compounded by high DNA concentration and pentobarbitone contamination. In addition, both high DNA concentration and pentobarbitone contamination could be overcome through dilution and the addition of BSA. Further work is required to quantify pentobarbitone concentration in the liver-extracted DNA and RNA samples before this inhibition effect on PCR can be fully elucidated. (C) 2004 Elsevier B.V. All rights reserved.

Cholecystokinin (CCK) and CCK receptor expression by human gliomas:evidence for an autocrine/paracrine stimulatory loop

Cholecystokinin (CCK) is a gut-brain peptide has been described to be able to induce mitosis according to recent studies. Additionally, conflicting data has been published on whether tumours of the central and peripheral nervous system in general, and gliomas in particular, express CCK receptors. In the present in vitro study we employed reverse transcription followed by the polymerase chain reaction (RT-PCR) to investigate whether mRNA for CCK-A and CCK-B receptors as well as CCK peptide itself is present in primary human gliomas and the U-87 MG GBM cell line. The data show that 14/14 (100%) of the primary gliomas exhibited mRNA expression for the CCK peptide gene and the B receptor including the U-87 MG cells, whereas, only 2/14 (14%) showed presence of the CCK-A receptor. The presence of CCK receptors together with CCK peptide expression itself suggests presence of an autocrine loop controlling glioma cell growth. In support of this conclusion, a neutralizing antibody against the CCK peptide exhibited a dose dependent inhibition of cell growth whereas, antagonists to CCK caused a dose depend inhibition of exogenous stimulated glioma cell growth in vitro, via the CCK-B receptor which is PKC activated. Assessment of apoptosis and proteasome activity were undertaken and we report that treatment with CCK antagonists decreased proteasome and increased caspase-3 activity. These data indicate that CCK peptide and CCK-B are abundant in human gliomas and they act to stimulate cell growth in an autocrine manner, primarily via the high affinity CCK-B receptor, which was blocked by antagonists to CCK, perhaps via apoptosis.

Meningiomas expressing and responding to cholecystokinin (CCK)

The effect of cholecystokinin (CCK) on cultured human meningioma derived cells was investigated. Exposure of meningioma cells for 6-12 days to CCK-8s (2-200 nM) resulted in a dose dependent stimulation of cell growth to a maximum of 1.1-fold over basal controls. A time course study showed stimulation of cell growth at day 3 followed by increase throughout day 6. The stimulatory effect of CCK on meningioma cell growth was completely abolished by a CCK-B specific receptor antagonist, L-365,260. Reverse-transcription of meningioma-derived RNA into cDNA followed by amplification by the polymerase chain reaction using specific primers for CCK peptide and its CCK-A and/B receptor revealed 100% presence of CCK peptide and CCK-B receptors mRNA whereas CCK-A receptor was expressed in 66% of the meningiomas. These results provide evidence that human meningioma cells possess CCK peptide and its receptors the activation of which leads to increase of cell growth possibly via an autocrine/paracrine mechanism. © Springer 2005.

Colorimetric Assessment of BCR-ABL1 Transcripts in Clinical Samples Via Gold Nanoprobes

Gold nanoparticles functionalized with thiolated oligonucleotides (Au-nanoprobes) have been used in a range of applications for the detection of bioanalytes of interest, from ions to proteins and DNA targets. These detection strategies are based on the unique optical properties of gold nanoparticles, in particular, the intense color that is subject to modulation by modification of the medium dieletric. Au-nanoprobes have been applied for the detection and characterization of specific DNA sequences of interest, namely pathogens and disease biomarkers. Nevertheless, despite its relevance, only a few reports exist on the detection of RNA targets. Among these strategies, the colorimetric detection of DNA has been proven to work for several different targets in controlled samples but demonstration in real clinical bioanalysis has been elusive. Here, we used a colorimetric method based on Au-nanoprobes for the direct detection of the e14a2 BCR-ABL fusion transcript in myeloid leukemia patient samples without the need for retro-transcription. Au-nanoprobes directly assessed total RNA from 38 clinical samples, and results were validated against reverse transcription-nested polymerase chain reaction (RT-nested PCR) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The colorimetric Au-nanoprobe assay is a simple yet reliable strategy to scrutinize myeloid leukemia patients at diagnosis and evaluate progression, with obvious advantages in terms of time and cost, particularly in low- to medium-income countries where molecular screening is not routinely feasible. Graphical abstract Gold nanoprobe for colorimetric detection of BCR-ABL1 fusion transcripts originating from the Philadelphia chromosome.

Survival, differentiation, and neuroprotective mechanisms of human stem cells complexed with neurotrophin-3-releasing pharmacologically active microcarriers in an ex vivo model of Parkinson's disease

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Chilean IPNV isolates: Robustness analysis of PCR detection

Background: The genomes of several infectious pancreatic necrosis viruses (IPNVs) isolated in Chile were sequenced with a single amplification approach for both segments A and B. The resulting sequences were then used to determine the conservation of the primer-binding regions used in polymerase chain reaction (PCR)-based diagnostic methods proposed in the literature. Thus, the robustness of each technique was studied, particularly the eventual effect of further mutations within the primer-binding sites. Results: On analysis, most methods currently used to detect Chilean IPNV varieties were deemed adequate. However, the primers were designed to be genogroup specific, implying that most detection methods pose some risk of detecting all strains prevalent in the country, due to the coexistence of genogroups 1 and 5. Conclusions: Negative resultsmust be interpreted carefully given the high genomic variability of IPNVs. Detection techniques (quantitative reverse transcription (qRT)-PCR) based on degenerate primers can be used to minimize the possibilities of false-negative detections.