Design and characterization of microstrip based E-field sensor for GSM and UMTS frequency bands.

An Electric (E-) field sensor based on coplanar waveguide-fed microstrip antenna to measure E-field strength for dual-band operation at 914 MHz and 2.1 GHz is proposed, designed, and characterized. The parametric optimization of the design has been performed to obtain resonance at global system for mobile communication and universal mobile telecommunication system frequency band. Low return loss (-17 dB and -19 dB), appropriate gain (0.50 dB and 1.55 dB), and isotropic behaviour (directivity ∼ 1 dB), respectively, at 914 MHz and 2.1 GHz, are obtained for probing application. Antenna factor (AF) is used as an important parameter to characterize the performance of the E-field sensor. The AF measurement is explained in detail and results are reported. Finally, using the designed E-field sensor, the E-field strength measurements are carried out in a transverse electromagnetic cell. The key sources of uncertainties in the measurement are identified, evaluated, and incorporated into the final results. The measurement results are compared with theoretical values, which are found in good agreement. For comparative validation, the results are evaluated with reference to an already calibrated commercially available isotropic probe.

Development of a real-time PCR for Escherichia coli based on gadE, an acid response regulatory gene.

Increasingly, molecular methods have become important in identification and confirmation of bacteria at the species level. Rapid molecular methods provide sensitivity and specificity while reducing cost and resources. The primary goal of this study was to develop a real-time PCR assay for identification of Escherichia coli from an agar plate. GadE (gadE) directly regulates the glutamate-dependent acid response system (GDAR) in E. coli and is responsible for survival of at pH 2. Based on gene sequence data, a real-time PCR assay targeting gadE was developed for this purpose. Seventy bacterial isolates recovered from ground beef enrichments and 714 isolates from caecal contents were identified biochemically and tested with the real-time PCR assay developed in this study. The PCR assay and the biochemical identification had 100% agreement on the tested isolates. The gadE real-time PCR assay was demonstrated in this study to be an inexpensive, reliable method for confirming E. coli colonies within 1.5 h from an agar plate, thereby saving on final identification time.

Control of life, death, and differentiation in cultured midgut cells of the lepidopteran, Heliothis virescens.

Differentiated cells in the insect midgut depend on stem cells for renewal. We have immunologically identified Integrin beta1, a promotor of cell-cell adhesion that also induces signals mediating proliferation, differentiation, and apoptosis on the surfaces of cultured Heliothis virescens midgut cells; clusters of immunostained integrin beta1-like material, indicative of activated integrin, were detected on aggregating midgut columnar cells. Growth factor-like peptides (midgut differentiation factors 1 and 2 [MDF1 and MDF2]), isolated from conditioned medium containing Manduca sexta midgut cells, may be representative of endogenous midgut signaling molecules. Exposing the cultured midgut cells to Bacillus thuringiensis (Bt) toxin caused large numbers of mature differentiated cells to die, but the massive cell death simultaneously induced a 150-200% increase in the numbers of midgut stem and differentiating cells. However, after the toxin was washed out, the proportions of cell types returned to near-control levels within 2 d, indicating endogenous control of cell-population dynamics. MDF1 was detected immunologically in larger numbers of Bt-treated columnar cells than controls, confirming its role in inducing the differentiation of rapidly produced stem cells. However, other insect midgut factors regulating increased proliferation, differentiation, as well as inhibition of proliferation and adjustment of the ratio of cell types, remain to be discovered.

Apoptosis in cultured midgut cells from heliothis virescens larvae exposed to various conditions.

We exposed midgut cells from primary cultures of Heliothis virescens larvae to cell-free previously used medium, the Vaughn X and HyQ SFtrade mark media used for serum-free culture of insect cell lines which do not support H. virescens midgut cells, and to toxin from Bacillus thuringiensis. A statistically significant increase in the percent of dying cells was counted in cell populations in Vaughn X medium. Use of the TUNEL method to detect apoptosis indicated a low rate (7.2%) of apoptosis in control cultures grown in Heliothis medium, an increase to approximately 20% in previously used and HyQ SFtrade mark media, and to approximately 45% of cells remaining after exposure to and initial destruction by B. thuringiensis toxin. Apoptotic nuclei were predominant (approximately 6%) in mature columnar cells in control cultures. Approximately 1% of goblet, stem, and differentiating cells were apoptotic. However, apoptosis rose to 12% in stem and differentiating cells exposed to used and unsuitable medium. B. thuringiensis exposure to toxin for 2-3 days resulted in visible membrane damage and necrosis, causing the death of 84% of the cells as measured by both the TUNEL and Annexin methods. Some of the columnar cells and stem and differentiating cells that remained also contained apoptotic nuclei. Stem and differentiating cells normally replace dying mature cells in the midgut. Thus, exposure of cultures of H. virescens midgut cells to adverse environments such as unsuitable or poisonous media appeared to induce down-regulation of the cell populations by apoptosis.

Felbamate increases [3H]glycine binding in rat brain and sections of human postmortem brain.

The anticonvulsant compound felbamate (2-phenyl-1,3-propanediol dicarbamate; FBM) appears to inhibit the function of the N-methyl-D-aspartate (NMDA) receptor complex through an interaction with the strychnine-insensitive glycine recognition site. Since we have demonstrated previously that FBM inhibits the binding of [3H]5, 7-dichlorokynurenic acid (DCKA), a competitive antagonist at the glycine site, we assessed the ability of FBM to modulate the binding of an agonist, [3H]glycine, to rat forebrain membranes and human brain sections. In contrast to its ability to inhibit [3H]5,7-DCKA binding, FBM increased [3H]glycine binding (20 nM; EC50 = 485 microM; Emax = 211% of control; nH = 1.8). FBM, but not carbamazepine, phenytoin, valproic acid or phenobarbital, also increased [3H]glycine binding (50 nM; EC50 = 142 microM; Emax = 157% of control; nH = 1.6) in human cortex sections. Autoradiographic analysis of human brain slices demonstrated that FBM produced the largest increases in [3H]glycine binding in the cortex, hippocampus and the parahippocampal gyrus. Because various ions can influence the binding of glycine-site ligands, we assessed their effects on FBM-modulation of [3H]glycine binding. FBM-enhanced [3H]glycine binding was attenuated by Zn++ and not inhibited by Mg++ in human brain. These results suggest that FBM increases [3H]glycine binding in a manner sensitive to ions which modulate the NMDA receptor. These data support the hypothesis that FBM produces anticonvulsant and neuroprotective effects by inhibiting NMDA receptor function, likely through an allosteric modulation of the glycine site.

Localization of dopamine receptors and associated mRNA in transplants of human fetal striatal tissue in rodents with experimental Huntington's disease.

Huntington's Disease (HD) is characterized by deficits in motor and cognitive functions. This neurodegenerative disease shows an extensive loss of medium-sized spiny projection neurons (GABAergic) within the neostriatum. With the loss of these neurons, there is a concomitant loss of associated receptors, such as those for GABA, glutamate, and dopamine. In the present study, we have addressed the question of whether dopamine receptors are re-established in the lesioned rodent striatum following the transplantation of human striatal cells. Human striatal cell suspension or saline (transplant controls) was injected into the striatum of rats previously lesioned with quinolinic acid (QA). Three nine months following transplantation, the animals were sacrificed and the brains were processed for receptor autoradiography and in situ hybridization of dopamine D1 and D2 receptor subtypes. Our results demonstrate that animals transplanted with human striatal cells show a significant increase in D1 receptors following transplantation when compared to the lesion area in control animals, while D1 receptor mRNA remains unchanged. In contrast to D1 receptor binding, D2 receptor levels are not increased in the lesioned and transplanted area of the striatum when compared to controls; however, D2 receptor mRNA levels are significantly increased. These results demonstrate that at the times the animals were examined, D1 and D2 receptors were differentially regulated. Our results further indicate that human striatal primordium will survive following transplantation and will express D1 receptors and D2 receptor mRNA that are depleted in the QA lesioned rodent striatum. This study compliments and extends previous findings on human striatal cell transplantation in rodent models of HD.

The temporal evolution of striatal dopamine receptor binding and mRNA expression following hypoxia-ischemia in the neonatal rat.

Neonatal hypoxic-ischemic (HI) brain injury in the rat alters dopamine receptors. To determine whether such changes are permanent, dopamine receptors and corresponding mRNA were examined at various time points after neonatal HI using receptor autoradiography and in situ hybridization. Rat pups underwent ligation of the left common carotid artery followed by hypoxic exposure (8.5% O2 for 3 h). Controls underwent sham surgery alone. Animals surviving for 2-80 days following HI were studied. Striatal D1 receptors (labeled by [3H]SCH23390) were reduced as early as 2 days following HI, remained depressed for 21 days, but recovered to control levels by young adulthood (3 months of age). D2 receptors (labeled by [125I] iodosulpride) did not decline until 10 days after HI, and remained uniformly depressed throughout the caudate-putamen thereafter. Changes in D1 receptor mRNA transcripts closely paralleled alterations in receptors: early reductions in D1 mRNA signal recovered by young adulthood. D2 mRNA exhibited a unique temporal profile with an early decrease (2 days following HI), and prompt, persistent recovery. Dopamine receptors and transcripts are differentially affected by HI injury early in development. Whereas D1 receptor expression recovers from neonatal HI injury, D2 receptors remain permanently affected despite the presence of normal levels of D2 receptor transcripts. A persistent, post-transcriptional effect of HI on D2 receptor expression is suggested.

Biological behaviour of moderate dysplasia--a prospective study.

The present communication reports the biological behaviour of women with moderate dysplastic lesions of uterine cervix based on a long term prospective study. Two hundred and thirty nine women with moderate dysplasia by cervical cytology who satisfied the criteria for registration were longitudinally followed up at 3 +/- 1 monthly intervals along with age and parity matched controls for a period ranging from 4 to 132 months. The cumulative rate of progression from moderate dysplasia to malignancy (CIS) was observed to be 23.0% at the end of 72 months of follow up with mean transition interval of 24.2 months. Out of 239 cases, 142 women who had more than 24 months of follow up were considered for studying the biological behaviour of the lesion. It was observed that during a follow up of 132 months, 14(9.9%) and 15(10.6%) women progressed to carcinoma in-situ and severe dysplasia respectively. The persistence of lesion was observed in 21(14.8%) women while 11(7.3%) and 81(57.0%) regressed to mild dysplasia and normalcy respectively.

Age-related loss of cholinergic-muscarinic coupling to PLC: comparison with changes in brain regional PLC subtypes mRNA distribution.

Activation of phospholipase C (PLC) coupled to phosphoinositide (PtdIns) hydrolysis occurs through one of the two pathways. One of the major pathways for the neurotransmitter signaling involves phosphoinositide (PtdIns) specific and G-protein dependent PLC-beta, which stimulates the formation of inositol triphosphate (IP3) and inositol tetraphosphate (IP4). Another pathway through the stimulation of calcium influx can directly activate all of the PLC isozymes. At least three isozymes of PLC have been characterized in the brain; PLC-A (alpha), PLC-I (beta) and PLC-II (gamma), which are shown to be localized differentially in brain regions. Muscarinic-cholinergic signals are mediated in large part through the hydrolysis of PtdIns by PLC. To investigate changes in muscarinic coupling to PLC during aging, we examined carbachol stimulated and calcium stimulated PtdIns hydrolysis in cerebral cortical membranes in young, middle aged and old rats. In order to determine whether PtdIns hydrolysis changes correspond to PLC isozyme expression in these animals, we examined three subtypes of PLC mRNA expression in brain sections of young and old rats using in situ hybridization technique. Our study indicated decreased carbachol-induced PLC activity in the cerebral cortex and, in contrast, increased PLC-beta mRNA in the frontal cortex and superficial cortical layer of aged rats. PLC-alpha mRNA was decreased in hippocampal regions of older rats. These studies suggest that during aging there is an uncoupling of muscarinic stimulated PtdIns hydrolysis, which is accompanied by an increased PLC-beta mRNA and decreased PLC-alpha mRNA that may represent compensatory changes in PLC expression.

Age does not alter Protein kinase C isozymes mRNA expression in rat brain.

Calcium and phospholipid dependent Protein kinase C (PKC) may play a role in memory function and pathogenesis of many neurodegenerative disorders such as Alzheimer's disease (AD). Abnormal phosphorylation by PKC as well as reduced levels of PKC has been implicated in the neurodegeneration associated with AD and aging. Recently, many subtypes of PKC isozymes have been identified by molecular biology techniques which are expressed differentially in various regions of the brain. The reduction and alterations in the activities and distribution of these subtypes of PKC isozymes may be accountable for the decline of selective neurons during aging. In order to investigate the role of PKC isozymes during aging, we examined the distribution of PKC-alpha, beta, and gamma mRNA expressions between young (4 months) and old (25 months) rat brains using in situ hybridization histochemistry. Our studies showed that signals of three isoforms of PKC mRNA vary in cortical and hippocampal regions. However, no change was detected in any of the PKC isoforms mRNA expression in aged animals.

Time dependent changes in DA uptake sites, D1 and D2 receptor binding and mRNA after 6-OHDA lesions of the medial forebrain bundle in the rat brain.

Quantitative receptor autoradiography and in situ hybridization techniques were used to examine the temporal pattern of changes in dopamine uptake sites, D1 and D2 receptors and their transcripts in the striata of animals lesioned with 6-hydroxydopamine. Animals were unilaterally lesioned in the medial forebrain bundle and the brains were analyzed at 1, 2, 4, 6, 8, and 16 weeks postlesion. Degeneration of the nigrostriatal pathway induced a significant loss of dopamine uptake sites in the ipsilateral caudate putamen of all lesioned animals. D1 receptor binding was significantly increased in the caudate putamen on the lesioned side from 1 week to 16 weeks postlesion, whereas the expression of D1 receptor mRNA did not show any change during this period. There was a significant upregulation of D2 receptor binding as well as D2 mRNA from 2 weeks to 8 weeks postlesion. However, at 16 weeks postlesion, D2 receptor binding continued to increase, whereas the mRNA appeared to compensate. These studies show that a different regulatory mechanism may exist between these two DA receptor subtypes. D1 receptor changes occur at the post-transcriptional or translational level, whereas D2 alterations occur by both transcriptional and translational processes. These studies also indicate that the postsynaptic supersensitivity observed in D1 receptors may not be accompanied by a corresponding increase in D1 receptor mRNA.

In situ determination of M1 and M2 muscarinic receptor binding sites and mRNAs in young and old rat brains.

Changes in M1, M2 receptor binding and mRNA in aged (25-26 months) rat brains were examined to determine whether decreases in receptors are due to declines in expression of corresponding mRNA levels. With aging, the M2 muscarinic receptor binding sites and m2 receptor mRNA were significantly decreased in the medial septum and diagonal band of Broca. In addition, M2 receptor binding was also reduced in the basal ganglia, CA3 field of the hippocampus, deeper layers of cortex, medial and central nuclei of amygdala, and thalamic nuclei. However, M1 binding was decreased in the basal ganglia, superficial layers of cortex, CA3 field of hippocampus and lateral nuclei of amygdala. There was no change in m1 receptor mRNA expression between any brain region of young and old rats. These studies suggest the reduction of the M2 receptor subtype during the transcriptional process, and alterations of M1 subtypes during translational or post-transcriptional periods.

Interaction of felbamate with [3H]DCKA-labeled strychnine-insensitive glycine receptors in human postmortem brain.

The dicarbamate felbamate has been shown to be capable of competing for the binding of 5,7-[3H]dichlorokynurenic acid ([3H]DCKA) to strychnine-insensitive glycine receptors in sections of human postmortem brain. The IC50 for this interaction was 305.8 microM and the inhibition was complete at 1 mM. Autoradiographic localization of [3H]DCKA binding revealed many regions of human brain in which strychnine-insensitive glycine receptors are manifest. The specific binding in most of these areas was markedly reduced in the presence of 625 microM felbamate. In many regions, [3H]DCKA binding was reduced to background in the presence of felbamate, but some areas retained binding by as much as 41% (i.e., the CA2 region of the hippocampus). This is in contrast to the binding of [3H]DCKA in the presence of carbamazepine, phenytoin, or valproic acid. The binding of the glycine receptor antagonist was not affected by any of these latter agents to the same degree as felbamate. Strychnine-insensitive glycine receptors represent a site of action of felbamate in the human brain.

Dopamine deficiency in a genetic mouse model of Lesch-Nyhan disease.

We have examined several aspects of neurotransmitter function in the brains of mice carrying a deletion mutation in the gene encoding the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). During the first 6 weeks of postnatal development, dopamine levels in whole-brain extracts from the mutant mice (HPRT-) failed to increase at rates comparable to normal animals, resulting in 40% lower dopamine levels throughout adulthood. Regional analysis in adult animals showed the caudoputamen to be the most severely affected region, with dopamine deficits of 48-64%. Dopamine levels in other regions were normal or less severely affected. The decrease in dopamine was accompanied by a decrease in tyrosine hydroxylase (TH) activity, the rate-limiting step in dopamine synthesis. Kinetic analysis of TH extracted from the caudoputamen of normal and HPRT- mice demonstrated a 45% decrease in Vmax with an increased affinity for the tetrahydropterin cofactor in the mutants. Labeling of midbrain dopamine neurons using TH immunohistochemistry revealed no obvious deficits in the number of midbrain dopamine neurons, but quantitative autoradiographic studies revealed significant reductions in the binding of 3H-N-[1-(2-benzo(beta)thiophenyl)cyclohexyl]piperidine (3H-BTCP) to dopamine uptake sites in the forebrain of the mutants. In contrast to these abnormalities of the dopamine systems in the mutant mice, other neurotransmitter systems appeared relatively unaffected. Norepinephrine, 5-HT, tryptophan hydroxylase, and glutamic acid decarboxylase were present at normal levels in the brains of the mutants. ChAT activity was slightly lower than normal in the caudoputamen of the mutant animals, but was normal in all other brain regions examined. These results indicate that HPRT deficiency is associated with a relatively specific deficit in basal ganglia dopamine systems that emerges during the first 2 months of postnatal development.

Use of [3H]5,7 dichlorokynurenic acid to identify strychnine-insensitive glycine receptors in human postmortem brain.

[3H]5,7 Dichlorokynurenic acid ([3H]DCKA) was used to define conditions for obtaining selective binding to strychnine-insensitive glycine receptors. The parameters were established in sections of human brain prior to localizing the receptors sites by autoradiography. The binding of [3H]DCKA was of high affinity (Kd = 14.5 nM), readily reversible (K-1 = 0.216 min-1), and specific (60% specific binding determined by inhibition with 100 microM glycine or D-serine). High levels of strychnine-insensitive glycine receptors were identified in several brain areas including portions of the cerebral cortex (Bmax in middle temporal gyrus: 174.0 fmol/mg tissue), basal ganglia, hippocampal formation, and midbrain. These results identify regions where glycine receptors may be involved in modulating NMDA-mediated channel activity.

A sensitive and rapid HPLC-ECD method for the simultaneous analysis of norepinephrine, dopamine, serotonin and their primary metabolites in brain tissue.

This report details a very sensitive, rapid and accurate ion-pair HPLC-ECD method for the analysis of biogenic amines and their metabolites in brain tissue. The method described enables detection of picogram amounts of 3-methoxy-4-hydroxy phenethyleneglycol (MHPG) (37 pg), 3,4-dihydroxy phenylacetic acid (DOPAC) (18 pg), norepinephrine (NE) (12 pg), epinephrine (E) (6 pg), 5-hydroxyindoleacetic acid (5-HIAA) (18 pg), 3,4-dihydroxyphenylethylamine (DA) (6 pg), 3-methoxy-4-hydroxyphenylacetic acid (HVA) (12 pg) and 5-hydroxytryptamine (5-HT) (12 pg). The linearity of the method is from 18.75 ng/mL to 300 ng/mL for MHPG; 9.37 to 150 ng/mL for DOPAC and 5-HIAA; 6.25 to 100 ng/mL for NE, HVA and 5-HT; and from 3.12 to 100 ng/mL for E and DA. The reproducibility, expressed as coefficient of variance (CV%) within-run and between-run groups, was 2.25% and 19.49% for MHPG; 3.84% and 29.84% for DOPAC; 0.89% and 8.97% for NE; 1.26% and 5.61% for E; 1.07% and 28.77% for 5-HIAA; 2.65% and 10.65% for DA; 5.97% and 24.38% for HVA; and 4.44% and 9.45% for 5-HT.

Reduction in striatal D2 dopamine receptor mRNA and binding following AF64A lesions.

Unilateral lesions by a cholinotoxin, receptor autoradiography, and in situ hybridization techniques were employed to determine if dopaminergic receptors are located on cholinergic interneurons in the caudate-putamen (CPu). Lesion of the CPu with small amounts of the cholinotoxin AF64A resulted in a significant decrease in D2 receptor mRNA and D2 receptor binding. The loss was more pronounced in lateral and central portions of the CPu. Results obtained using [3H] SCH23390 binding to D1 receptors indicated that there was no change in this dopamine receptor subtype in the AF64A-lesioned CPu. A decrease in D2 receptor mRNA and receptor binding in AF64A-lesioned animals indicates that a population of postsynaptic D2 receptors is associated with the cholinergic interneurons. Lack of any change in [3H]SCH23390 binding in the AF64A-lesioned animals suggests that D1 receptors are not located on cholinergic neurons. These results provide evidence to support the selectivity of the lesion when used as indicated.

Alterations in the dopaminergic receptor system after chronic administration of cocaine.

Several studies suggest that one of the most important factors contributing to cocaine dependence is an alteration in the actions of the neurotransmitter dopamine in the central nervous system. In order to understand some of the neuroreceptor consequences of cocaine administration, groups of rats were injected with cocaine (2 daily doses of 15 mg/kg) for 1 to 21 days. Binding of [3H]cocaine, [3H]SCH23390, [3H]raclopride, and [3H]BTCP in striatal and cortical tissue from the treated animals was compared to controls. [3H]Cocaine binding was increased by the drug in the striatum and cortex at days 14 and 21, respectively. The binding of [3H]SCH23390 to D1 dopamine receptors was significantly increased at day 3 of cocaine exposure. In striatal membranes, [3H]BTCP binding to dopamine uptake sites was significantly increased after day 7, whereas binding in cortical membranes was increased from day 1. [3H]Raclopride binding to D2 dopamine receptors remained unchanged throughout the study in both cortical and striatal tissues. These results indicate that repeated exposure to cocaine produces an upregulation (possible supersensitivity) in cortical D1, cocaine, and DA-uptake sites which occurs in a time-dependent manner. These increases are coupled with an upregulation in striatal D1, cocaine, and DA-uptake sites, without simultaneous changes in D2 receptors. Thus, cocaine's effects are not uniformly distributed across all brain regions, but rather are focused within areas of the dopamine system.

Radio-label and mass determinations of inositol 1,3,4,5-tetrakisphosphate formation in rat cerebral cortical slices: differential effects of myo-inositol.

To investigate the effects of increasing concentrations of myo-inositol (inositol) on receptor stimulated [3H]inositol polyphosphate formation in the absence of lithium, slices of rat cerebral cortex were incubated with various concentrations of [3H]inositol (1 to 30 microM). Carbachol stimulated formation of [3H]inositol trisphosphate (InsP3) and [3H]inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) increased several fold when the inositol concentration was increased reaching a plateau at approximately 12 microM inositol. Time course studies revealed that in the presence of low concentrations of inositol (1 microM), [3H]InsP3 and [3H]Ins(1,3,4,5)P4 formation in response to carbachol stimulation increased slowly over a 10 to 20 min time period, whereas in the presence of 4 and 12 microM inositol, carbachol stimulated [3H]InsP3 and [3H]Ins(1,3,4,5)P4 formation was rapid and essentially complete within 3 to 5 min after carbachol addition. Although the carbachol dose response in 12 microM inositol had a much greater maximal efficacy, there was no change in potency. Similar to the effects of carbachol on [3H]Ins(1,3,4,5)P4 formation from prelabeled phosphoinositides, muscarinic receptor stimulation increased Ins(1,3,4,5)P4 mass formation by seven fold. Furthermore, Li+ (8 mM) completely inhibited carbachol stimulated increases in Ins(1,3,4,5)P4 mass formation. In contrast to the effects of increasing inositol on carbachol stimulated formation of radiolabeled inositol phosphates, increasing inositol had no effect upon mass formation of Ins(1,3,4,5)P4. These results show that when measuring inositol polyphosphate formation by the radiolabeling technique in the absence of Li+, increasing the inositol concentration greatly increases the stimulated component of [3H]InsP3 and [3H]Ins(1,3,4,5)P4 formation. However, this inositol induced increase in agonist stimulated Ins(1,3,4,5)P4 formation is not reflected as an increase in mass formation.