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PubMed1998

An atypical cytochrome b in the colorless alga Polytomella spp.: the high potential bH heme exhibits a double transition in the alpha-peak of its absorption spectrum.

Polytomella spp. is a colorless alga of the family Chlamydomonadaceae that lacks chloroplasts and cell wall. A highly active ubiquinol-cytochrome c oxidoreductase (bc1 complex), sensitive to antimycin and myxothiazol, has been purified and characterized from this alga (Gutiérrez-Cirlos et al., 1994, J. Biol. Chem. 269, 9147-9154). Both in mitochondrial membranes and in the isolated complex, the visible spectrum of cytochrome b from Polytomella spp. exhibits an atypical alpha-band with a maximum at 567 nm. This maximum is shifted 3-4 nm to the red when compared with b-type cytochromes from other organisms. Analysis of the b hemes of the bc1 complex by high performance liquid chromatography revealed no differences in the retention time and in the absorption spectra of the b-type hemes from Polytomella spp. and hemin, indicating that the prosthetic group in this alga is protoheme and thus ruling out the possibility that the red-shift could be due to different chemical substitutions in the porphyrin rings of the bL or bH hemes. The two b hemes were characterized by electrochemical redox titration; at pH 7.8-8.0, the midpoint potential for bL was-143 mV and for bH +25 mV. The spectra of the two b-type hemes were recorded in the presence of different reductants, at selected electrochemical potentials, and in the presence of antimycin A, to distinguish between the contribution of bL and bH to the visible spectrum. Both hemes bL and bH of the algal cytochrome b contribute to the observed bathochromic absorption maximum in the alpha-band of the spectrum. The data also show that the low potential bL heme from Polytomella spp. is spectroscopically similar to that of other organisms, with two transitions in the alpha-peak at 558.7 and 568.4 nm. The high-potential heme bH also exhibits a spectrum with two transitions at 557.2 and 568.9 nm, which surprisingly differs from the spectra of cytochrome bH of mammals, plants, yeasts, and bacteria, which all exhibit a single transition centered around 560 nm.

Animals↗
PubMed2001

Different Na, K-ATPase mRNA(beta1) species exhibit unique translational efficiencies.

We have previously identified five Na, K-ATPase beta1-mRNA species that are expressed in the rat heart, kidney, and brain. These mRNAs which are unequal in their abundance have an identical coding region but differ in the length of their 5'- and 3'-untranslated regions (UTRs). In this study we examined the possibility that the beta1-mRNA species exhibit differential translational efficiencies. We constructed expression plasmids encoding each of the five mRNAs and transcribed and translated them in vitro. Using rabbit reticulocyte system we determined the translation of the different mRNAs under conditions optimized for each beta1-cRNA and under an equivalent (standard) condition. The longest beta1-cRNA species (initiating at the first transcription start site and ending at the last [fifth] poly(A) site) exhibited the lowest relative translational efficiency averaging 0.2 +/- 0.05 units/mol of cRNA compared to the shortest beta1-cRNA species initiating at the first transcription start site and ending at the first poly(A) signal (with an assigned relative value of 1.0). These results suggested that the different translation rates of beta1-mRNAs may be due to their 3'-UTRs. To further define the role of beta1-3'-UTR, chimeric luciferase constructs containing different segments of the beta1-3'-UTR were transiently transfected into Clone 9 cells. Compared to the chimeric construct containing the shortest beta1-3'-UTR segment (ending at the first poly(A) site), the construct containing the full-length beta1-3'-UTR exhibited a luciferase expression of 0.23 +/- 0.04. To control for potential changes in the abundance of the expressed chimeric mRNAs which may lead to differences in luciferase expression, luciferase activity was normalized against chimeric luciferase-mRNA content measured in mixtures of cells stably transfected with the above constructs. The ratio of luciferase activity/chimeric luciferase-mRNA content in cells expressing the construct containing the entire beta1-3'-UTR region was 0.17 that in cells expressing chimeric luciferase mRNA containing beta1-3'-UTR up to the first poly(A) signal (P < 0.05). We conclude that the translational efficiency of the different beta1-mRNA species is negatively regulated by the 3'-UTR of the mRNA and that a regulating region appears to be localized between the second and fifth poly(A) signals of beta1-mRNA.

3' Untranslated Regions↗
PubMed1995

The 33-kDa C-terminal domain of Raf-1 protein kinase exhibits a Ras-independent serum- and phorbol ester-induced shift in gel mobility.

Experiments were carried out to determine Raf-1 protein kinase domain fragments which exhibit a characteristic electrophoretic mobility shift noted with Raf-1 protein kinase in response to serum and phorbol ester (PMA) treatment of serum-deprived NIH 3T3 cells. Epsilon-epitope tagged 84 kDa Raf-1 holoenzyme (HR-epsilon), as well as the epsilon-epsilon pitope tagged 35 kDa N-terminal (RI-epsilon), 33 kDa mid-portion (RII-epsilon), and 33 kDa C-terminal (RIII-epsilon) fragments of Raf-1 were overexpressed in NIH 3T3 cells. The overexpressed HR-epsilon exhibited a serum- and PMA-induced shift in gel mobility similar to that noted with endogenous Raf-1. The C-terminal RIII-epsilon fragment exhibited a similar shift in gel mobility while the electrophoretic mobility of the N-terminal RI-epsilon fragment remained unchanged. These results suggest that modification(s) within the 33 kDa C-terminal portion of Raf-1 which occur independently of association with Ras may be responsible for the band shift observed with serum and PMA treatment of serum-deprived NIH 3T3 cells.

3T3 Cells↗
PubMed1999

Neural precursor cells differentiating in the absence of Rb exhibit delayed terminal mitosis and deregulated E2F 1 and 3 activity.

The severe neurological deficit in embryos carrying null mutations for the retinoblastoma (Rb) gene suggests that Rb plays a crucial role in neurogenesis. While developing neurons undergo apoptosis in vivo neural precursor cells cultured from Rb-deficient embryos appear to differentiate and survive. To determine whether Rb is an essential regulator of the intrinsic pathway modulating terminal mitosis we examined the terminal differentiation of primary cortical progenitor cells and bFGF-dependent neural stem cells derived from Rb-deficient mice. Although Rb -/- neural precursor cells are able to differentiate in vitro we show that these cells exhibit a significant delay in terminal mitosis relative to wild-type cells. Furthermore, Rb -/- cells surviving in vitro exhibit an upregulation of p107 that is found in complexes with E2F3. This suggests that p107 may partially compensate for the loss of Rb in neural precursor cells. Functional ablation of Rb family proteins by adenovirus-mediated delivery of an E1A N-terminal mutant results in apoptosis in Rb-deficient cells, consistent with the interpretation that other Rb family proteins may facilitate differentiation and survival. While p107 is upregulated and interacts with the putative Rb target E2F3 in neural precursor cells, our results indicate that it clearly cannot restore normal E2F regulation. Rb-deficient cells exhibit a significant enhancement of E2F 1 and 3 activity throughout differentiation concomitant with the aberrant expression of E2F-inducible genes. In these studies we show that Rb is essential for the regulation of E2F 1 and 3 activity as well as the onset of terminal mitosis in neural precursor cells.

Adenoviridae↗
PubMed1998

Localization of human cadherin genes to chromosome regions exhibiting cancer-related loss of heterozygosity.

This report presents the chromosomal localization of cadherin genes. Cadherins are cellular adhesion molecules. Since disturbance of intracellular adhesion is important for invasion and metastasis of tumor cells, cadherins are considered prime candidates for tumor suppressor genes. A variety of solid tumors show loss of heterozygosity of the long arm of chromosome 16, which is indicative of the potential location of tumor suppressor genes. Refined and new localizations of six cadherin genes (CDH3, 5, 8, 11, 13, and 15) to the long arm of chromosome 16 are shown. CDH15 was localized to 16q24.3, in a region that exhibits loss of heterozygosity in a number of sporadic breast cancer tumors. Previous localization of CDH13 (H-cadherin) to 16q24 suggested this gene as a tumor suppressor candidate in the 16q24.3 loss of heterozygosity region; however, refined mapping presented in this report localizes CDH13 proximal to this region. A human EST homologous to the chicken cadherin-7 was partially sequenced and found to represent a new human cadherin. This cadherin mapped to chromosome 18q22-q23, a region that exhibits loss of heterozygosity in head and neck squamous cell carcinomas. CDH16 was localized to 8q22.1, a region exhibiting loss of heterozygosity in adult acute myeloid leukemia.

Breast Neoplasms↗
PubMed1993

A DNA dodecamer containing an adenine tract crystallizes in a unique lattice and exhibits a new bend.

The structure of d(CGCGAAAAAACG)/d(CGTTTTTTCGCG) was determined at 2.3 A resolution in order to deduce the local structural features that give rise to DNA bending by adenine tracts. Whereas all previously reported B-DNA dodecamers have crystallized isomorphously (spacegroup P2(1)2(1)2(1) with unit cell dimensions of a = 24.5 A, b = 40.3 A, c = 65.9 A), the duplex reported here crystallizes in a different lattice (spacegroup P2(1)2(1)2 with unit cell dimensions of a = 44.8 A, b = 66.1 A, c = 42.9 A). The DNA exhibits a 30 degree bend in the helix axis that is 180 degrees away from the 20 degree bend exhibited by the adenine tract DNA crystal structures that have been previously determined. This bend is 90 degrees away from the bend predicted for an adenine tract by solution and gel experiments. The adenine tract is straight and bending occurs in the G+C-rich regions. Comparison of the various adenine tract DNA crystal structures reveals that in all cases the adenine tracts have nearly identical structures, even though the overall bends of the helix axes are quite different. This implies that the structure of the adenine tract is robust, at least under the conditions of crystallization. The base-pairs in the adenine tracts exhibit a large propeller twist that leads to the formation of bifurcated hydrogen bonds and a narrow minor groove. In the crystal structure of d(CGCGAAAAAACG)/d(CGTTTTTTCGCG), a minor groove spine of hydration is observed that probably stabilizes the straight structure of the adenine tract. This straight structure of the A-tract is not consistent with the results of fiber diffraction, gel experiments, and NMR studies. Although this may imply that the results of solution experiments need to be reinterpreted, the conditions under which the crystals were grown are different from those under which the solution experiments were done. The possibility remains that the 2-methyl-2,4-pentanediol necessary for crystal growth may facilitate formation of the spine of hydration that stabilizes the straight A-tract, although recent NMR results show the presence of the spine of hydration in aqueous solution. We have also extended our previously reported observation that non-self-complementary DNA structures can exist in the crystal lattice in two orientations. A dodecamer brominated on one strand provides experimental evidence that d(CGCAAAAAAGCG)/d(CGCTTTTTTG CG) is positioned in two orientations in the crystal lattice that are related by a 180 degree rotation around the pseudo-dyad axis of the sequence.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenine↗
PubMed1997

Partitioning of plasmid R1. The ParM protein exhibits ATPase activity and interacts with the centromere-like ParR-parC complex.

The parA system of plasmid R1 consists of two genes, parM and parR, and a cis-acting centromere-like site parC. The ParM protein exhibits similarity with a superfamily of ATPases that includes actin, hsp70 and hexokinase. ParM was purified to near-homogeneity and assayed for in vitro ATPase activity. The wild-type ParM protein was found to posses ATPase activity. Mutant ParM derivatives that exhibited decreased in vitro ATPase activity were non-functional in vivo, indicating that the ATP turnover by ParM is essential for correct plasmid partitioning. The mutant ParM proteins exhibited trans-dominance, suggesting that ParM participates as a structural component of the partitioning apparatus. The ATPase activity of ParM was activated slightly by the presence of ParR and activated to a much greater extent when ParR was bound to the centromere-like parC region. An analysis using the yeast two-hybrid system indicated that ParM and ParR interact, and demonstrated that ParR interacts with itself. Thus our results suggest a direct interaction of ParM and ParR at the natural partition site parC, and that the ATPase activity of ParM is specifically stimulated by this interaction.

Adenosine Triphosphatases↗
PubMed1999

COOH-terminal truncated cardiac myosin-binding protein C mutants resulting from familial hypertrophic cardiomyopathy mutations exhibit altered expression and/or incorporation in fetal rat cardiomyocytes.

Mutations in human cardiac myosin-binding protein C (cMyBP-C) gene are associated with familial hypertrophic cardiomyopathy (FHC), and most of them are predicted to produce COOH-truncated proteins. To understand the molecular mechanism(s) by which such mutations cause FHC, we analyzed (i) the accumulation of human cMyBP-C mutants in fetal rat cardiomyocytes, and (ii) the protein sequence of the human wild-type (wt) cMyBP-C by hydrophobic cluster analysis with the aim of identifying new putative myosin-binding site(s). Accumulation and sarcomeric localization of the wt protein and of four FHC-mutant cMyBP-Cs (E542Q and three COOH-truncated proteins) were studied in cardiomyocytes by immunostaining and confocal microscopy after transfection with myc-tagged constructs. We found that: (i) 10 % of the cells expressing COOH-truncated mutants exhibit an incorporation into the A-band of the sarcomere without any alteration of the myofibrillar architecture versus 76 % of those expressing the wt or E542Q mutant cMyBP-Cs (p<0.001); (ii) 90 % of the cells expressing the truncated mutants show a diffuse localization of these proteins in the cardiomyocytes, out of which 45 % exhibit a significant alteration of the sarcomeric structure (p<0.0001 versus wt); and (iii) the two shortest mutant cMyBP-Cs accumulate at very low levels in fetal rat cardiomyocytes as compared to the wt (p<0.008). Protein sequence analysis indicated that a 45-residue sequence in the NH2-terminal C0 domain of cMyBP-C exhibits a consistent homology (sequence similarity score of 42 %) with a segment of the NH2-terminal domain of myomesin, another myosin-binding protein. This result suggests that the C0 domain of human cMyBP-C contains a novel putative myosin-binding site that could account for the A-band incorporation of the truncated mutants. In addition, the faint accumulation and the diffuse localization of truncated mutants could probably be explained by a low affinity of the C0 domain for myosin. We conclude that COOH-truncated cMyBP-Cs may act as poison polypeptides that disrupt the myofibrillar architecture and result in the defects observed in FHC.

Amino Acid Motifs↗
PubMed1998

Human ventricular myocytes in vitro exhibit both early and delayed preconditioning responses to simulated ischemia.

Myocardial tissue has been demonstrated to exhibit, in response to brief periods of ischemia, both an immediate period of cytoprotection [i.e. early or "first window" preconditioning response (EPR)], and a later period of cytoprotection [i.e. delayed or "second window" preconditioning response (DPR)], when exposed to a subsequent prolonged hypoxic insult. EPR has been documented in vitro in isolated cardiac myocytes, as well as in situ in intact hearts or trabeculae, for a number of vertebrate species, including humans. However, there are no reports to date of DPR in human cardiac myocytes. To address this question, human ventricular myocytes (HVM) primary isolates were prepared from fetal ventricular muscle, grown to confluency, and studied in primary culture in serum-free medium (> 90%) ventricular myocytes as determined by immunohistochemical analysis with an anti-myosin chain antibody). Using cell viability as determined by trypan blue exclusion, an EPR response could readily be detected following 15, 30, or 60 min of simulated ischemia (SI) in a hypoxic (< 1 tau pO2) buffer containing 11 mmol/l 2-deoxyglucose, followed by a prolonged (c. 17 h) SI challenge. In addition, HVM exposed to 60 min of SI, followed after 24 h by a period of SI, also exhibited a "second window" DPR (80 +/- 10% compared to 71 +/- 11% survival, in preconditioned and non-preconditioned cultures; P < 0.05; n = 18 independent experiments). Thus, in response to short periods of SI, human ventricular myocytes in vitro exhibit both "first window" and "second window" cytoprotective responses to subsequent, prolonged ischemic stress.

Analysis of Variance↗
PubMed2001

Fetal hippocampal CA3 cell grafts transplanted to lesioned CA3 region of the adult hippocampus exhibit long-term survival in a rat model of temporal lobe epilepsy.

Intracerebroventricular administration of kainic acid in the adult rat, a widely used model for studying human temporal lobe epilepsy, results in widespread degeneration of CA3-pyramidal neurons. Transplantation of specific fetal hippocampal CA3 cell grafts into the lesioned CA3-region at a prolonged post lesion delay of 45-day leads to 31% graft cell survival at 1 month postgrafting and significantly facilitates appropriate recovery of the lesioned host hippocampus. However, the capability of hippocampal CA3 cell grafts for enduring survival in this model is unknown. We hypothesize that a significant fraction of fetal CA3 cells grafted into the lesioned CA3 region of the adult hippocampus at 45-days postlesion exhibit long-term survival. We measured the extent of cell survival within 5'-bromodeoxyuridine-labeled CA3 cell grafts at 1 year postgrafting, following their transplantation at 45 days postlesion into the lesioned CA3-region. Quantification of absolute graft cell survival using BrdU immunostaining and the optical fractionator counting method revealed survival of 36% of grafted cells at 1 year postgrafting. Thus, over a third of fetal hippocampal CA3 cells transplanted to the lesioned CA3-region at 45 days postlesion exhibit long-term survival. Further, the extent of cell survival in these grafts is highly analogous to the degree of cell survival in CA3 grafts analyzed earlier at 1 month postgrafting, suggesting that specific fetal cells that survive the first month of grafting into the lesioned CNS area are capable of exhibiting enduring survival.

Aging↗
PubMed1995

Nucleotide sequence analysis of a 30-kb region of the bovine herpesvirus 1 genome which exhibits a colinear gene arrangement with the UL21 to UL4 genes of herpes simplex virus.

We report the nucleotide sequence of the 19-kb HindIII fragment B of bovine herpesvirus 1 (BHV-1) DNA and adjacent parts of the HindIII A and L fragments, which together span a still completely uncharted 30-kb region located between the glycoprotein H gene and the right end of the unique long segment. The analysis revealed 17 complete open reading frames (ORFs) and 2 ORFs that were interrupted by potential splice donor and acceptor sites. All of these ORFs exhibited strong amino acid sequence homology to the gene products of other alphaherpesviruses. The BHV-1 ORFs were arranged colinearly with the prototype sequence of herpes simplex virus 1 in the range of the UL21 to UL4 genes. Colinearity was also observed with the genes of betaherpesviruses and gamma herpesviruses, although not all ORFs exhibited clear sequence homology. The possible functions of the proteins encoded within the sequenced region are assessed and features found are discussed. Unexpected findings include the following: high amino acid sequence conservation among alphaherpesviruses despite large differences in G + C content, ranging from 45% for varicella zoster virus to 72% for BHV-1; high similarity with other UL20 proteins at the predicted structural level in spite of relatively low amino acid homology; and a 2-kb open reading frame overlapping UL19 in the opposite sense and exhibiting high amino acid similarity to the same area of pseudorabies virus.

Amino Acid Sequence↗
PubMed1994

Transmembrane distribution of lipophilic cations in response to an electrochemical potential in reconstituted cytochrome c oxidase vesicles and in vesicles exhibiting a potassium ion diffusion potential.

It has been shown previously that biogenic amines and a number of pharmaceutical agents can redistribute across vesicle membranes in response to imposed potassium ion or proton gradients. Surprisingly, drug accumulation is observed for vesicles exhibiting either a pH gradient (interior acidic) or a membrane potential (interior negative), implying that these compounds can traverse the lipid bilayer as either the neutral or charged species. This interpretation, however, is complicated by the fact that vesicles exhibiting a membrane potential (interior negative) accumulate protons in response to this potential, thereby creating a pH gradient (interior acidic). This raises the possibility that in both vesicle systems drug redistribution occurs in response to the proton gradient present. We have therefore compared the uptake of several lipophilic cations by reconstituted cytochrome c oxidase vesicles and by similar vesicles exhibiting a potassium ion diffusion potential. While turnover of the oxidase generates a membrane potential of comparable magnitude to the potassium ion diffusion system, it is associated with a proton gradient of opposite polarity (interior basic). Both systems show rapid uptake of the permanently charged lipophilic cation, tetraphenylphosphonium, but only the potassium ion diffusion system accumulates the lipophilic amines doxorubicin and propranolol. This provides compelling evidence that such weak bases redistribute only in response to pH gradients and not membrane potential.

Cations↗
PubMed1982

The effect of duration of intravenous infusion on maximum and threshold blood concentration for drugs exhibiting biexponential elimination kinetics.

The relationships among peak blood concentration (Cmax), the time (tD) during which blood concentrations are maintained above the minimum effective concentration (Cmin), and the duration of a constant-rate intravenous infusion (T) of a drug exhibiting biexponential pharmacokinetics were simulated by digital computer using Newton iterative procedures. These simulations showed that, in contrast with our previous findings for drugs with monoexponential pharmacokinetics, the relationships are more complex due to the larger number of variables. Therefore, investigation of these relationships for biexponential drugs should be done on a drug by drug basis. Accordingly, meperidine and sulfamethoxazole were chosen as examples of drugs which exhibit biexponential kinetics and were used to determine what errors were involved in using the simpler guidelines for drugs which exhibit monoexponential kinetics as an approximation. These simulations showed the following . (a) The effect of T on Cmax may be adequately estimated by using the guidelines for monoexponential kinetics with the elimination half-life (t1/2) taken as t1/2 lambda1, provided that lambda1 is 20 to 30 times lambda2 and that the AUC of the distribution phase (i.e., C1/lambda1) comprises greater than about 20% of the total AUC. (b) As with monoexponential drugs, it is possible to obtain a larger tD by infusing the dose compared to that obtained with a bolus, if the value of C(0)/Cmin less than 2.5. (c) Using th approximation of monoexponential pharmacokinetics to estimate the effect of T on tD will underestimate both tD and the maximum infusion time, which will just attain the Cmin unless the AUC of the distribution phase comprises only a very small proportion of the total AUC. (d) The simulations with meperidine also showed that the nature of the relationship between tD and T varies depending on whether Cmin is maintained in the distribution phase or in both the distribution and elimination phases.

Computers↗
PubMed1990

Isolation of temperature-sensitive CHO-K1 cell mutants exhibiting chromosomal instability and reduced DNA synthesis at nonpermissive temperature.

Twenty-five temperature-sensitive (ts) mutants were isolated from Chinese hamster CHO-K1 cells after mutagenization with N-methyl-N'-nitro-N-nitrosoguanidine. Of 13 complementation groups identified, nine exhibited chromosomal instability at a nonpermissive temperature. They were classified into three major classes according to inducibility of sister chromatid exchange (SCE) and/or chromosomal aberration (CA): class 1 resulted in predominant SCEs, class 2 manifested both SCEs and CAs, and class 3 exhibited higher induction of CAs. Flow cytometric analysis of the mutants exhibiting chromosomal instability indicated that many of the mutants were arrested in the S or S to G2 phases of the cell cycle at the nonpermissive temperature, accompanied by a decrease in the rate of DNA synthesis. These results imply that ts defects are related to some points in DNA replication and might be responsible for the induction of SCEs and/or CAs at the nonpermissive temperature.

Animals↗
PubMed2002

Children as a special population at risk--quinolones as an example for xenobiotics exhibiting skeletal toxicity.

The sensitivity of the immature skeletal system during postnatal mammalian development was discussed during the workshop "Children as a Sensitive Subgroup and their Role in Regulatory Toxicology" (Schwenk et al. 2002, Arch Toxicol, in press). As a typical example of xenobiotics exhibiting this extraordinary type of toxicity, the quinolone-induced effects on cartilage, bone growth, and tendons were considered in depth. Animal experiments indicate that chondrotoxicity of quinolones can affect articular cartilage depending on the developmental stage. Results from studies with pipemidic acid in dogs at various developmental stages from 1 week old to 17 months old showed pronounced effects in the most sensitive age groups: 3- to 6-month-old dogs exhibited gait alterations and lameness, but signs of toxicity were not observed in 12- or 17-month-old dogs. Similarly, experiments performed in rats indicate that they exhibit the highest sensitivity for articular cartilage defects at a certain stage of development between 3 and 6 weeks postnatally; mature articular cartilage does not seem to be a target for quinolone-induced toxicity. Some very limited data indicate that in animals at an early developmental phase the epiphyseal growth plate can also be damaged by quinolones, and that these effects are associated with irreversible bone damage and growth inhibition. Another manifestation of toxic effects of quinolones on connective tissue structures are tendopathies. For this effect, it is not quite clear whether juveniles are more susceptible than adults because clinical and experimental data seem to point in different directions. Pathogenesis of both effects can probably be explained by the magnesium-chelating properties of these drugs, leading to a deficit of functionally available magnesium and, subsequently, to radical formation and irreversible connective tissue lesions. However, unequivocal explanations for the phase-dependency of the effects are lacking.

Adolescent↗
PubMed2006

Distribution of NADPH diaphorase-exhibiting primary afferent neurons in the trigeminal ganglion and mesencephalic trigeminal nucleus of the rabbit.

1. Nitric oxide (NO) is highly reactive gaseous molecule to which many physiological and pathological functions have been attributed in the central (CNS) and peripheral (PNS) nervous system. The present investigation was undertaken to map the distribution pattern of the enzyme responsible for the synthesis of NO, nitric oxide synthase (NOS), and especially its neuronal isoform (nNOS) in the population of primary afferent neurons of the trigeminal ganglion (TG) and mesencephalic trigeminal nucleus (MTN) of the rabbit. 2. In order to identify neuronal structures expressing nNOS we applied histochemistry to its specific histochemical marker nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd). 3. We found noticeable amount of NADPHd-exhibiting primary afferent neurons in TG of the rabbit under physiological conditions. The intensity of the histochemical reaction was highly variable reaching the maximum in the subpopulation of small-to-medium-sized neurons. The large-sized neurons were only weakly stained or actually did not posses any NADPHd-activity. In addition, NADPHd-positive nerve fibers were detected between clusters of the ganglionic cells and in the peripheral branches of the trigeminal nerve (TN). NADPHd-exhibiting MTN neurons were noticed in the whole rostrocaudal extent of the nucleus even though some differences were found concerning the ratio of NADPHd-positive versus NADPHd-negative cell bodies. Similarly, we observed striking diversity in the intensity of NADPHd histochemical reaction in the subpopulations of small-, medium-, and large-sized MTN neurons. 4. The predominant localization of NADPHd in the subpopulation of small-to-medium-sized TG neurons which are generally considered to be nociceptive suggests that NO probably takes part in the modulation of nociceptive inputs from the head and face. Furthermore, we tentatively assume that NADPHd-exhibiting MTN neurons probably participate in transmission and modulation of the proprioceptive impulses from muscle spindles of the masticatory muscles and mechanoreceptors of the periodontal ligaments and thus provide sensory feedback of the masticatory reflex arc.

Animals↗
PubMed1996

Expressed human hippocampal ASCT1 amino acid transporter exhibits a pH-dependent change in substrate specificity.

In mammalian cells, the basal Na+-dependent uptake for many of the neutral amino acids is mediated by a transport activity designated System ASC. A cloned human brain cDNA sequence, ASCT1, encodes a Na+-dependent neutral amino acid transport activity that exhibits a substrate specificity similar to that commonly associated with System ASC. However, the characteristics of ASC activity varies significantly between cell types and not all tissues contain detectable levels of ASCT1 mRNA. A unique property of System ASC activity is an altered substrate selectivity such that at pH values below 7.4 anionic amino acids function as inhibitors and substrates. The experiments in this report were designed to determine if the cloned ASCT1 transporter exhibited this pH-dependent anionic transport. Following transfection of HeLa cells with the ASCT1 cDNA, transport strongly favored neutral zwitterionic) amino acids when uptake was measured at a physiologic pH value of 7.5. However, lowering the assay pH to 5.5 significantly enhanced the interaction of the ASCT1 carrier with anionic amino acids such as cysteate, in a pH-dependent manner. The apparent pK for the titratable group was in the range of 6.5-7.0. These results provide evidence that the human brain ASCT1 transporter exhibits the most distinguishing characteristic known for System ASC and provides a model system to investigate the molecular basis for this shift in substrate acceptance.

Amino Acid Transport Systems↗
PubMed1995

Naringenin: a weakly estrogenic bioflavonoid that exhibits antiestrogenic activity.

Treatment of immature 21-day-old female Sprague-Dawley rats with 17 beta-estradiol (E2) (0.5 microgram/rat) caused a significant increase in uterine wet weight, DNA synthesis, progesterone receptor (PR) binding, and peroxidase activity. At doses as high as 40 mg/rat, the bioflavonoid naringenin did not cause a significant increase in any of these E2-induced responses. However, in rats cotreated with E2 (0.5 microgram/rat) plus naringenin (30 mg/rat); there was a significant decrease in E2-induced uterine wet weight, DNA synthesis, PR binding, and peroxidase activity, indicating that naringenin exhibits antiestrogenic activity in the immature rodent uterus. The binding of uterine nuclear extracts to a 32P-labeled estrogen responsive element (ERE) or progesterone responsive element (PRE) was determined using gel electrophoretic band shift assays. Incubation of [32P]ERE with uterine nuclear extracts from rats treated with naringenin or E2 resulted in the formation of estrogen receptor (ER):ERE complexes; a higher mobility complex was prominent in the extracts from E2-treated rats, whereas a lower mobility complex was observed using nuclear extracts from naringenin-treated animals. There was a significant decrease in the intensity of the E2-induced complex using nuclear extracts from rats treated with E2 plus naringenin. In contrast, transformed cytosol from control rats gave an intense ER:ERE complex, whereas the intensity of the band was decreased markedly using transformed uterine cytosol from treated rats. Formation of a PR:PRE complex was also determined using transformed uterine cytosol. Cytosol from E2-treated rats gave an intense retarded band, whereas only weak bands were observed using cytosols from DMSO- (solvent), naringenin-, or naringenin plus E2-treated cells. The results of in vitro studies showed that 1 nM E2 increased (3- to 4-fold) the growth of MCF-7 human breast cancer cells, whereas 1-1000 nM naringenin had no effect on cell proliferation. In cells cotreated with 1 nM E2 plus 1000 nM naringenin, there was a significant decrease in E2-induced cell growth. In MCF-7 cells transiently transfected with a pS2 promoter-regulated luciferase reporter gene, naringenin exhibited weak estrogenic activity. In cells cotreated with 0.1 or 1.0 microM naringenin plus 1 nM E2, naringenin inhibited E2-induced luciferase activity. The results of these studies confirmed that naringenin is a weak estrogen that also exhibits partial antiestrogenic activity in the female rat uterus and MCF-7 human breast cancer cells.

Animals↗