A Qualitative and Quantitative Correlation Study of Lumbar Intervertebral Disc Degeneration Using Glycosaminoglycan Chemical Exchange Saturation Transfer, Pfirrmann Grade, and T1-ρ.

BACKGROUND AND PURPOSE: Glycosaminoglycan chemical exchange saturation transfer (gagCEST) imaging allows the direct measurement and mapping of glycosaminoglycans. In this study, we aimed to evaluate the usefulness of gagCEST imaging in the quantitative assessment of intervertebral disc degeneration in a comparison with Pfirrmann grade and T1-ρ measurements. MATERIALS AND METHODS: Ninety-six lumbar intervertebral discs in 24 volunteers (36.0 ± 8.5 years of age, 21 men and 3 women) were examined with both gagCEST imaging and T1-ρ measurements. The gagCEST imaging was performed at 3T with a saturation pulse with 1.0-second duration and the B1 amplitude of 0.8 µT followed by imaging by a 2D fast spin-echo sequence. The Z-spectra were obtained at 25 frequency offsets from -3 to +3 ppm (step, 0.25 ppm). A point-by-point B0 correction was performed with a B0 map. The gagCEST signal and T1-ρ values were measured in the nucleus pulposus in each intervertebral disc. The Pfirrmann grades were assessed on T2-weighted images. RESULTS: The gagCEST signal at grade I (5.36% ± 2.79%) was significantly higher than those at Pfirrmann grade II (3.15% ± 1.40%, P = .0006), grade III (0.14% ± 1.03%, P < .0001), grade IV (-1.75% ± 2.82%, P < .0001), and grade V (-1.47% ± 0.36%, P < .0001). The gagCEST signal at grade II was significantly higher than those of grade III (P < .0001), grade IV (P < .0001), and grade V (P < .0001). The gagCEST signal was significantly correlated negatively with Pfirrmann grade (P < .0001) and positively correlated with T1-ρ (P < .0001). CONCLUSIONS: GagCEST imaging could be a reliable and quantitative technique for assessing intervertebral disc degeneration.

Role of endogenous pituitary adenylate cyclase-activating polypeptide in adult hippocampal neurogenesis.

Hippocampal neurogenesis occurs throughout life in mammals and has pivotal roles in brain functions. An enriched environment stimulates hippocampal neurogenesis, but the exact mechanisms are still unclear. The present study investigated the role of pituitary adenylate cyclase-activating polypeptide (PACAP) in adult hippocampal neurogenesis under standard or enriched rearing conditions. Rearing in the enriched conditions from 4-weeks old for 4-weeks increased the survival of newly divided cells in the subgranular zone and granule cell layer of the dentate gyrus of wild-type and PACAP-knockout (PACAP-/-) mice. The increase in the survival in the granule cell layer was less in PACAP-/- mice than in the wild-type mice. In contrast, the proliferation of newly divided cells in mice reared in the standard and enriched conditions did not differ between the wild-type and PACAP-/- mice. Regarding the differentiation of newborn cells in the dentate gyrus, most of the newly divided cells exhibited the neuronal phenotype in both the wild-type and PACAP-/- mice under standard and enriched conditions. These findings suggest that endogenous PACAP is partly involved in the survival of the enriched environment-induced generation, but not in the basal rate, of newborn cells in the dentate gyrus of the adult hippocampus.

Effect of maternal stress on fetal heart rate assessed by vibroacoustic stimulation.

This study was designed to determine whether maternal stress levels, state and trait anxiety levels, and stress hormones affect fetal heart rate (FHR) patterns after vibroacoustic stimulation (VAS) at 30 weeks of gestation. A total of 24 healthy pregnant women with a single fetus pregnancy were enrolled. Corticotropin releasing hormone (CRH) and adrenocorticotropic hormone in maternal plasma and cortisol, and chromogranin A in saliva were measured. The FHR patterns after VAS were divided into three types: type I, a long period of acceleration or one acceleration lasting > 1 min or at least two accelerations lasting > 15 s; type II, a biphasic response with acceleration followed by deceleration; and type III, no response or prolonged deceleration. In the high trait anxiety group, CRH levels were significantly higher than in the low trait anxiety group, and FHR patterns after VAS showed mostly a type II response pattern. These findings suggest that stress in pregnant women with high trait anxiety may influence FHR patterns after VAS.

Regulation of antiviral innate immune responses by RIG-I family of RNA helicases.

The recognition of viral nucleic acids with pattern recognition receptors (PRRs) is the first step in inducing the innate immune system. Type I interferons (IFNs), central mediators in antiviral innate immunity, along with other cytokines and chemokines, disrupt virus replication. Recent studies indicated at least two distinct pathways for the induction of type I IFN by viral infection. Toll-like receptors (TLRs) are extracellular or endosomal PRRs for microbial pathogens, whereas retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are novel intracellular PRRs for the viral dsRNA. In this review, we describe the distinct mechanisms inducing type I IFNs through TLRs and RIG-I/MDA5 pathways.

Chlorpropham-induced splenotoxicity and its recovery in rats.

To determine the reversibility of hematological and pathological changes in spleen induced by sub-chronic administration of chlorpropham (CIPC), male F344 rats were given CIPC in the diet at 0, 600, 3000 or 15,000 ppm for 13 weeks (administration period) and then were given standard (0 ppm) diet for 10 weeks (recovery period). At 0, 1, 2, 4 or 10 weeks in the recovery period, 5 rats in each groups were examined for hematology and pathology. At the end of CIPC administration, dose-dependent and significant methemoglobinemia, anemia, splenomegaly and pathological lesions indicating hemolytic anemia were observed in all the treated groups. The hematological changes, congestion of red pulp, lymphoid atrophy, increased extramedullary hematopoiesis in spleen and hematopoietic cell hyperplasia in bone marrow were diminished during the 10 weeks recovery period. However, increased hemosiderin deposition and capsular fibrosis in spleen of the treated groups remained at the end of recovery period. The results indicated that hematological changes induced by sub-chronic administration of CIPC were reversible but hemosiderin deposition and fibrosis in spleen were not reversible in the recovery period examined, suggesting the significance of splenic lesion in CIPC-toxicity.

Cyclic thrombocytopenia and polycythemia vera.

A periodic fall of platelet number characterizes an acquired pathological condition named cyclic thrombocytopenia. We describe an unusual case of polycythemia vera in which the episodes of thrombocytopenia were followed regularly by thrombocytosis. The period of platelet count fluctuation was about 50 days, with the counts ranging from 34 to 820 x 10(9)/l. Bone marrow megakaryocytes were decreased in number during platelet nadir. Circulating thrombopoietin levels fluctuated out of phase with the platelet count. We suggest that at least some cases of polycythemia vera may have an unstable hematopoietic stem cell pool in nature, which could contribute to the development of unprovoked cyclic thrombocytopenia.

Cardiac electrophysiologic and hemodynamic effects of sildenafil, a PDE5 inhibitor, in anesthetized dogs.

A recent in vitro study demonstrated that supratherapeutic concentrations of sildenafil, a phosphodiesterase type 5 (PDE5) inhibitor, blocked I(Kr) and prolonged cardiac repolarization. This study assessed the in vivo cardiohemodynamic and electrophysiologic effects of sildenafil using a halothane-anesthetized, closed-chest canine model (n = 5) to bridge the gap between basic observation and clinical experience. Intravenous administration of sildenafil citrate in doses of 0.03, 0.3, and 3.0 mg/kg for 10 min, which provided sub-to supratherapeutic plasma drug concentrations, did not affect the monophasic action potential duration or effective refractory period of the right ventricle during the sinus rhythm as well as the ventricular pacing at the cycle length of 400 and 300 ms. However, sildenafil decreased the total peripheral resistance, simultaneously inducing positive chronotropic and inotropic effects at the top dose, which gave plasma concentrations at least 10 times higher than the therapeutic range. This cardiohemodynamic profile of sildenafil can be largely explained by reflex sympathetic activation associated with its vasodilator effect. Meanwhile, the lack of prolongation of the ventricular repolarization phase at the therapeutically relevant to moderately supratherapeutic sildenafil concentrations supports the earlier clinical studies that indicate that sildenafil has no effect on electrocardiogram.

Chronic toxicity of thiabendazole (TBZ) in CD-1 mice.

Male and female CD-1 mice (50 mice per group) were administered thiabendazole (TBZ) in diet at levels of 0 (control), 0.031, 0.125 and 0.5% for 78 weeks. A life time study was terminated after 78 weeks due to enhanced strain specific mortality. There were no significant differences in mortality between the control and treated groups. Mean body weights of high-dose groups showed significant decreases compared with the controls. The bladder weights of male and female mice of the 0.5% group were significantly higher than those of the control mice. Gross findings in treated mice included the renal atrophy, hydronephrosis, calculi in renal pelvis and/or bladder and ovarian atrophy. Microscopic findings in the kidneys of treated mice included the nephrosis, hydronephrosis or hyperplasia of transitional epithelium of renal pelvis or papilla. In the bladder of treated mice, hyperplasia or squamous metaplasia of transitional epithelium and one transitional cell papilloma were observed. Dose-dependent decreases in the incidence of spontaneous lesion in the male or female reproductive system were recognized. It is concluded that TBZ is not carcinogenic to CD-1 mice of both sexes. However, caution should be exercised in the long-term application of high TBZ doses.

Degradation of c-Fos protein expressed by N-methyl-D-aspartic acid in nuclear fractions of murine hippocampus.

In both nuclear and cytosolic fractions of murine hippocampus, constitutive expression was seen with Fra-2 protein, but not with other Fos family members tested including c-Fos, Fos-B and Fra-1 proteins. Fos-B protein was only detected in nuclear fractions. The systemic administration of N-methyl-D-aspartic acid (NMDA) induced marked and transient expression of c-Fos protein, but not other family members, in both hippocampal fractions 2 h later. In vitro incubation at 30 degrees C led to more rapid degradation of inducible c-Fos protein than constitutive Fra-2 protein in nuclear fractions obtained 2 h after the administration of NMDA, without significantly affecting that of both member proteins in cytosolic fractions. The addition of phosphatase inhibitors significantly delayed the initial degradation rate of inducible c-Fos protein, with concomitant facilitation of that of constitutive Fra-2 protein, in nuclear fractions. The addition of protease inhibitors also delayed the initial degradation of constitutive Fra-2 protein, without markedly altering that of inducible c-Fos protein, in nuclear fractions. Immunoprecipitation analysis revealed that NMDA induced phosphorylation of c-Fos protein on tyrosine residues in nuclear fractions to a lesser extent than that on serine residues 2 h after administration. These results suggest that NMDA signals may be propagated to the nucleus to induce both expression and degradation of c-Fos protein through a molecular mechanism associated with phosphorylation on serine and/or tyrosine residues in murine hippocampus.

PACT, a double-stranded RNA binding protein acts as a positive regulator for type I interferon gene induced by Newcastle disease virus.

Virus infection triggers innate responses to host cells including production of type I interferon (IFN). Since IFN production is also induced by treatment with poly(I:C), viral double-stranded (ds) RNA has been postulated to play a direct role in the process. In the present study, we investigated the effect of dsRNA binding proteins on virus-induced activation of the IFN-beta gene. We found that PACT, originally identified as protein activator for dsRNA-dependent protein kinase (PKR) and implicated in the regulation of translation, augmented IFN-beta gene activation induced by Newcastle disease virus. Concomitantly with the augmented activity of IFN-beta enhancer, increased activity of NF-kappaB and IRF-3 and IRF-7 was observed. For the observed effect, the dsRNA-binding activity of PACT was essential. We identified residues of PACT that interact with a presumptive target molecule to exert its function. Furthermore, PACT colocalized with viral replication complex in the infected cells. Thus the observed effect of PACT is novel and PACT is involved in the regulation of viral replication and results in a marked increase of cellular IFN-beta gene expression.

Induction of IRF-3/-7 kinase and NF-kappaB in response to double-stranded RNA and virus infection: common and unique pathways.

BACKGROUND: Infection by virus or treatment with double-stranded RNA (dsRNA) results in the activation of transcription factors including IRF-3, IRF-7 and a pleiotropic regulator NF-kappaB by specific phosphorylation. These factors are important in triggering a cascade of antiviral responses. A protein kinase that is yet to be identified is responsible for the activation of these factors and plays a key role in the responses. RESULTS: The signal cascade was analysed using sensitive assays for the activation of IRF-3 and NF-kappaB, and various inhibitors. We found that the activation of IRF-3 and NF-kappaB by dsRNA or virus involves a process that is sensitive to Geldanamycin. Although the induction of NF-kappaB by dsRNA/virus and TNF-alpha involves common downstream pathways including IKK activation, the upstream, Geldanamycin-sensitive process was unique to the dsRNA/virus-induced signal. By an in vitro assay using cell extract, we found an inducible protein kinase activity with physiological specificity of IRF-3 phosphorylation. Furthermore, the same extract specifically phosphorylated IRF-7 in a similar manner. CONCLUSIONS: Double-stranded RNA or virus triggers a specific signal cascade that results in the activation of the IRF-3/-7 kinase we detected, which corresponds to the long-sought signalling machinery that is responsible for triggering the early phase of innate response. The signal branches to a common NF-kappaB activation cascade, thus resulting in the activation of a set of critical transcription factors for the response.

Thiabendazole induces urinary tract toxicity in male ICR mice.

Male ICR mice were administered thiabendazole (TBZ) in the diet at concentration of 0 (control), 0.8, 1.2 and 1.6% for 44 weeks. The mortality was 10, 6, 40 or 90% in control, 0.8, 1.2 or 1.6% TBZ group, respectively. In dead mice, the gross findings included the abnormalities of kidney such as atrophy, hydronephrosis or swelling in 2, 67, 95 or 96% of the 0, 0.8, 1.2 or 1.6% TBZ group, respectively. In surviving mice at the end of study, the right kidney weight of treated groups was significantly lower than that of control group. The urinary bladder weight of treated groups was significantly higher than that of control group. Gross findings in treated mice included the renal atrophy, hydronephrosis, calculi in renal pelvis or urinary bladder and thickening of the bladder wall. Microscopic findings in the kidneys of treated mice included nephrosis, hydronephrosis and hyperplasia of transitional epithelium of renal pelvis and/or papilla. In the urinary bladder, hyperplasia or squamous metaplasia of transitional epithelium were found in treated mice. Administration of TBZ in the diet for 44 weeks results in nephrosis and calculus formation in the renal pelvis and urinary bladder of male ICR mice, and is associated with hyperplasia of transitional epithelium of renal pelvis or urinary bladder.

Effects of chlorpropham (CIPC) on the hemopoietic system of rats.

Male F344 rats were given 0 or 3% chlorpropham in the diet and at 2, 4, 6, 8 or 13 weeks of administration, five rats in each group were examined for hematology, plasma clinical chemistry and pathology. Marked splenomegaly and hepatomegaly were observed in treated rats at 2-13 weeks of administration. Red blood cell counts, hemoglobin concentration, packed cell volume and platelet counts were significantly decreased and methemoglobin level, mean corpuscular volume and white blood cell counts were significantly increased in treated rats at 2-13 weeks of administration. The covalent binding of m-chloraniline m-CA, (the hydrolytic metabolite of chlorpropham) was observed in hemoglobin or splenic protein of treated rats, but only small amounts of free m-CA were present in blood or spleen. Congestion, hemosiderin deposits, extramedullary hemopoiesis and lymphoid atrophy in spleen and hyperplasia of hemopoietic cells in bone marrow were observed in treated rats at 2-13 weeks and fibrosis in splenic capsule were observed in treated rats at 4-13 weeks. The pathological changes in spleen rather than hematological changes progressed during administration, suggesting splenotoxicity of CIPC in rats.

Analyses of virus-induced homomeric and heteromeric protein associations between IRF-3 and coactivator CBP/p300.

Cellular genes including the type I interferon genes are activated in response to viral infection. We previously reported that IRF-3 (interferon regulatory factor 3) is specifically phosphorylated on serine residues and directly transmits a virus-induced signal from the cytoplasm to the nucleus, and then participates in the primary phase of gene induction. In this study, we analyzed the molecular mechanism of IRF-3 activation further. The formation of a stable homomeric complex of IRF-3 between the specifically phosphorylated IRF-3 molecules occurred. While virus-induced IRF-7 did not bind to p300, the phosphorylated IRF-3 complex formed a stable multimeric complex with p300 (active holocomplex). Competition using a synthetic phosphopeptide corresponding to the activated IRF-3 demonstrated that p300 directly recognizes the structure in the vicinity of the phosphorylated residues of IRF-3. These results indicated that the phosphorylation of serine residues at positions 385 and 386 is critical for the formation of the holocomplex, presumably through a conformational switch facilitating homodimer formation and the generation of the interaction interface with CBP/p300.

Subchronic toxicity of chlorpropham (CIPC) in ICR mice.

Male and female ICR mice were given 0, 1875, 7500 or 30,000 ppm of chlorpropham (CIPC) in the diet for 13 weeks. Methemoglobin levels of male and female mice in the 7500 and 30,000 ppm groups were significantly elevated. Hemoglobin concentration, packed cell volume, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration and white blood cell count of male and female mice in the 30,000 ppm group were significantly increased. Dose-dependent splenomegaly was observed in male and female mice in the 7500 and 30,000 ppm group. Congestion, increased hemosiderin deposition and increased extramedullary hematopoiesis in the spleen, hematopoietic cell hyperplasia and hemosiderin deposition in bone marrow was observed dose dependently in male and female mice in the 7500 or 30,000 ppm group. Eosinophilic granular cytoplasm of hepatocytes, sinusoidal dilatation, hemosiderin deposition, extramedullary hematopoiesis and necrosis of hepatocytes were observed in the liver of male and female mice in the 30,000 ppm group. Hemosiderin deposition was increased in the kidney of male and female mice in the 30,000 ppm group. Administration of CIPC in diet for 13 weeks caused methemoglobinemia and splenomegaly in ICR mice.

Flow cytometric detection and analysis of tailless sperm caused by sonication or a chemical agent.

Flow cytometric analysis has been developed to detect tailless sperm with heads detached from the tails at the neck position. When isolated tailless sperm suspension was subjected to flow cytometry, a second sperm population appeared alongside the normal sperm population on light scatter-histogram. The percentage of this second sperm population (85.2%) was in good agreement with that for the tailless sperm (88.7%) determined microscopically, indicating that the second sperm population would correspond to tailless sperm population in the light scatter-histogram. Rates for tailless sperm determined by flow cytometry significantly correlated with those estimated microscopically following exposure of sperm to either sonication (r = 0.94, P < 0.01), or nitrobenzene (r = 0.80, P < 0.01). The results indicated the utility of the light scatter-histogram in flow cytometry as a simple and convenient procedure for the detection of tailless sperm induced by chemical compounds.

Effects of thiabendazole (TBZ) on mitochondrial function in renal cortex of ICR mice.

The effects of thiabendazole (TBZ) on mitochondrial function of the renal cortex were investigated in ICR mice. Mice were given 1000 or 2000 mg TBZ/kg body weight by gavage and mitochondria were isolated from the renal cortex for the measurement of respiratory rates. The state 3 and DNP-uncoupled respiratory rates of renal cortical mitochondria were dose-dependently depressed at 6 hours after dosing. The depression of these respiratory rates of renal cortical mitochondria was more marked at 16 hours after dosing. There was no depression in these respiratory rates of renal cortical mitochondria at 3 hours after dosing, although renal cortical concentrations of TBZ were higher than those at 6 or 16 hours after dosing. Histochemical examination revealed that NAD-linked isocitrate dehydrogenase, a marker enzyme of mitochondria, was inhibited in renal cortical tubules at 16 hours after dosing of 1000 or 2000 mg TBZ/kg body weight. Furthermore, renal cortical ATP level was significantly decreased at 16 hours after dosing of 1000 or 2000 mg TBZ/kg body weight. The results indicate that administration of TBZ caused mitochondrial dysfunction in renal cortical tubules of mice.

Combinatorial approach to development of peptides that recognize DNA tetraloops.

We report recognition system for DNA tetraloops (dA1G2G3C4T5T6C7G8G9C10C11T12 (core) and dAGGCTTCGGCCT (AP2); X = abasic nucleotide) by peptides selected with combinatorial chemistry. As a result, peptides with Thr/Glu/His and Gln/Asp were obtained in binding of DNA core and AP2, respectively.