Characterizing Adversity of Lysosomal Accumulation in Nonclinical Toxicity Studies: Results from the 5th ESTP International Expert Workshop.

Lysosomes have a central role in cellular catabolism, trafficking, and processing of foreign particles. Accumulation of endogenous and exogenous materials in lysosomes represents a common finding in nonclinical toxicity studies. Histologically, these accumulations often lack distinctive features indicative of lysosomal or cellular dysfunction, making it difficult to consistently interpret and assign adverse dose levels. To help address this issue, the European Society of Toxicologic Pathology organized a workshop where representative types of lysosomal accumulation induced by pharmaceuticals and environmental chemicals were presented and discussed. The expert working group agreed that the diversity of lysosomal accumulations requires a case-by-case weight-of-evidence approach and outlined several factors to consider in the adversity assessment, including location and type of cell affected, lysosomal contents, severity of the accumulation, and related pathological effects as evidence of cellular or organ dysfunction. Lysosomal accumulations associated with cytotoxicity, inflammation, or fibrosis were generally considered to be adverse, while those found in isolation (without morphologic or functional consequences) were not. Workshop examples highlighted the importance of thoroughly characterizing the biological context of lysosomal effects, including mechanistic data and functional in vitro readouts if available. The information provided here should facilitate greater consistency and transparency in the interpretation of lysosomal effects.

Assessing consumer expectations about pizza: A study on celiac and non-celiac individuals using the word association technique.

The word association (WA) technique was used to investigate the perception of two groups of consumers (72 celiac and 78 non-celiac individuals; 150 in total) to pizza dough (thick or thin) and the raw material used at the manufacture (cassava flour or rice flour). Different perceptions of the four stimuli were detected by Chi-square test (X2=314.393, p<0.0001) for both groups. Seven categories were used for both groups: food/composition, health, doubt/uncertainty, novelty, negative feelings, positive feelings, and sensory aspects. The stimulus 'pizza dough made with cassava flour' was associated with the category "food/composition" and the stimuli 'pizza made with rice flour', 'pizza made with cassava flour' and 'thin dough' were associated with "positive feelings". The stimulus 'thick dough' was related only to the category "negative feelings". WA indicated that gluten-free pizza should have thin dough and us cassava flour or rice flour as the raw material.

Landau renormalizations of superfluid density in the heavy-fermion superconductor CeCoIn5.

The formation of heavy-fermion bands can occur by means of the conversion of a periodic array of local moments into itinerant electrons via the Kondo effect and the huge consequent Fermi-liquid renormalizations. Leggett predicted for liquid (3)He that Fermi-liquid renormalizations change in the superconducting state, leading to a temperature dependence of the London penetration depth Λ quite different from that in BCS theory. Using Leggett's theory, as modified for heavy fermions, it is possible to extract from the measured temperature dependence of Λ in high quality samples both Landau parameters F(0)(s) and F(1)(s); this has never been accomplished before. A modification of the temperature dependence of the electronic specific heat C(el), related to that of Λ, is also expected. We have carefully determined the magnitude and temperature dependence of Λ in CeCoIn(5) by muon spin relaxation rate measurements to obtain F(0)(s) = 36 ± 1 and F(1)(s) = 1.2 ± 0.3, and we find a consistent change in the temperature dependence of C(el). This, the first determination of F(1)(s) with a value ≪ F(0)(s) in a heavy-fermion compound, tests the basic assumption of the theory of heavy fermions, that the frequency dependence of the self-energy is much more important than its momentum dependence.

Distribution of Taenia saginata metacestodes: a comparison of routine meat inspection and carcase dissection results in experimentally infected calves.

A comparison of techniques for detecting the presence of Cysticercus bovis in bovine carcasses was made by using carcass dissection and routine beef inspection guidelines. In the study, 28 calves were used after they were tested and found to be negative for the presence of anti-C. bovis serum antibodies and were inoculated orally with aliquots containing 6×10(4) Taenia saginata eggs. One hundred and twenty days after inoculation, the animals were slaughtered and a post mortem evaluation was done following Brazilian Federal Beef Inspection guidelines. This routine meat inspection was able to identify 71·42% of the assessed infected carcasses as being parasitized. This result implies that 28·58% of the infected carcasses would have been released as fit for human consumption since they would have been considered as free of C. bovis infection when using this method for carcass assessment. Only 3·07% of the total 2311 metacestodes present in the carcasses were identified by the conventional procedures of sanitary inspection. The assessment of different parts of the carcasses showed high infestation rates in shoulder clod (14·37%), head (11·21%), neck+chuck roll (8·05%), heart (7·75%) and top (inside) round (7·18%) which, together, were responsible for housing 48·51% of all the cysts found in the 24 beef cuts assessed. These numbers contrasted to the low incidence of cysts found in organs such as tongue (3·12%), diaphragm (1·69%) and esophagus (1·60%) which are usually described as predilection sites for the parasite.

Thoracic mass in a cynomolgus macaque (Macaca fascicularis).

A male cynomolgus macaque at the age of 3 years and 11 months suffered sudden cardiac arrest during a surgical operation. This animal had been clinically asymptomatic for 6 months from the acclimatization period to death. At necropsy, a white mass approximately 5 cm in diameter was found at the base of the heart. Histopathologically, the mass consisted of a granuloma with a number of multinucleated giant cells and multiple necrotic foci. Fungal hyphae characterized by parallel cell walls, distinct septa, and branching were observed in the lesion. The granuloma extended into the thoracic lymph nodes and the subepicardium of the left atrium, compressed the bronchioli, and was separated from the pulmonary parenchyma by a thick fibrous layer. The hyphal morphology and results of polymerase chain reaction assays demonstrated that the pathogen was Aspergillus sp.

Gel phase formation at resin-modified glass-ionomer/tooth interfaces.

Ionic bonding between polyalkenoic acid and hydroxyapatite may explain the excellent bonding retention of glass-ionomers in clinical trials. We have here investigated the extent to which the self-adhesiveness of resin-modified glass-ionomers (RMGIs) can be attributed to this chemical bonding capacity. Therefore, the interaction of 3 RMGIs with tooth substrates was comprehensively characterized, with electron and atomic force microscopy correlated with x-ray photoelectron spectroscopy (XPS). Interfacial ultrastructural analysis for 2 RMGIs disclosed a shallow hybridization of hydroxyapatite-coated collagen, on which a submicron gel phase was deposited through reaction of the polyalkenoic acid with calcium extracted from the dentin surface. One RMGI, however, bonded to dentin without hybrid layer or gel phase formation. XPS indicated that polycarboxylic acids included in the RMGIs electrostatically interacted with hydroxyapatite. We conclude that the self-adhesiveness of RMGIs should be attributed to ionic bonding to hydroxyapatite around collagen, and to micro-mechanical interlocking for those RMGIs that additionally hybridize dentin.

Comparative study on adhesive performance of functional monomers.

Mild self-etch adhesives demineralize dentin only partially, leaving hydroxyapatite around collagen within a submicron hybrid layer. We hypothesized that this residual hydroxyapatite may serve as a receptor for chemical interaction with the functional monomer and, subsequently, contribute to adhesive performance in addition to micro-mechanical hybridization. We therefore chemically characterized the adhesive interaction of 3 functional monomers with synthetic hydroxyapatite, using x-ray photoelectron spectroscopy and atomic absorption spectrophotometry. We further characterized their interaction with dentin ultra-morphologically, using transmission electron microscopy. The monomer 10-methacryloxydecyl dihydrogen phosphate (10-MDP) readily adhered to hydroxyapatite. This bond appeared very stable, as confirmed by the low dissolution rate of its calcium salt in water. The bonding potential of 4-methacryloxyethyl trimellitic acid (4-MET) was substantially lower. The monomer 2-methacryloxyethyl phenyl hydrogen phosphate (phenyl-P) and its bond to hydroxyapatite did not appear to be hydrolytically stable. Besides self-etching dentin, specific functional monomers have additional chemical bonding efficacy that is expected to contribute to their adhesive potential to tooth tissue.

Voxel-based analysis of MRI reveals anterior cingulate gray-matter volume reduction in posttraumatic stress disorder due to terrorism.

MRI studies using the manual tracing method have shown a smaller-than-normal hippocampal volume in patients with posttraumatic stress disorder (PTSD). However, these studies have yielded inconsistent results, and brain structures other than the hippocampus have not been well investigated. A recently developed, fully automated method called voxel-based morphometry enables an exploration of structural changes throughout the brain by applying statistical parametric mapping to high-resolution MRI. Here we first used this technology in patients with PTSD. Participants were 9 victims of the Tokyo subway sarin attack with PTSD and 16 matched victims of the same traumatic event without PTSD. The voxel-based morphometry showed a significant gray-matter volume reduction in the left anterior cingulate cortex (ACC) in trauma survivors with PTSD compared with those without PTSD. The severity of the disorder was negatively correlated with the gray-matter volume of the left ACC in PTSD subjects. There were no significant differences in other gray-matter regions or any of the white-matter regions between two groups. The present study demonstrates evidence for structural abnormalities of ACC in patients with PTSD. Together with previous functional neuroimaging studies showing a dysfunction of this region, the present findings provide further support for the important role of ACC, which is pivotally involved in attention, emotional regulation, and conditioned fear, in the pathology of PTSD.

Bonding efficacy of polyalkenoic acids to hydroxyapatite, enamel and dentin.

Previously, we introduced a methodology to determine the chemical bonding potential of polyalkenoic acids to mineralized tissues through quantification of the degree of ionic bond formation between the carboxyl groups of a polyalkenoic acid with calcium of hydroxyapatite. In a continuation of that study, we now investigated in how far the chemical bonding potential to synthetic hydroxyapatite is influenced by the molecular structure of the polyalkenoic acid and if this also may affect the self-adhesiveness to enamel and dentin. X-ray photoelectron spectroscopy and inductively coupled plasma atomic emission spectrometry were used to quantitatively analyze the chemical bonding efficacy of a polyalkenoic acid consisting of acrylic acid units (PAA) to synthetic hydroxyapatite (HAp) in comparison to the chemical bonding potential of the previously investigated synthesized polyalkenoic acid (s-PA) co-polymer consisting of 90w/w% acrylic and 10w/w% maleic acid units. In addition, the analysis was carried out for enamel and dentin samples. PAA revealed a significantly lower bonding effectiveness with only half of its carboxyl groups bonded to HAp versus about two-third of the carboxyl groups of s-PA. The difference in bonding potential was confirmed by the considerably lower adhesiveness of PAA to enamel and dentin as compared to that of s-PA The present findings indicate that the molecular structure of the polyalkenoic acid significantly influences the chemical bonding efficacy to Hap-based substrates.

1H-MR spectroscopy and gray matter volume of the anterior cingulate cortex in schizophrenia.

Schizophrenic and normal control subjects were examined using both H-magnetic resonance spectroscopy (MRS) and structural MR imaging, in order to accurately assess the partial volume within the spectroscopic volume of interest (VOI) in the anterior cingulate cortex. The gray matter volume within VOI correlated positively with the N-acetyl-aspartate (NAA) to choline (Cho) ratio in schizophrenics only, not in controls. Schizophrenic patients had a reduced NAA/Cho ratio and an elevated Cho/creatine ratio compared to controls after the partial volume effect was eliminated. There was a significant negative correlation between the NAA/Cho ratio and the severity of blunted affect symptom in schizophrenics. These results provide further support to the idea that the measures of H-MRS indicate not only neuronal loss but also neuronal dysfunction in schizophrenia.

Activation of prostaglandin E2-receptor EP2 and EP4 pathways induces growth inhibition in human gastric carcinoma cell lines.

The effect of prostaglandin E2 (PGE2) on the proliferation of gastric cancer cells is still unclear. PGE2 receptors are divided into four subtypes - EP1, EP2, EP3, and EP4 - which are coupled to three different intracellular signal-transduction systems. Stimulation of EP2 and EP4 is linked with cyclic adenosine 3', 5'-monophosphate (cAMP)-dependent protein kinase A (PKA). In some human gastric cancer cells, PGE2 has been suggested to have an antiproliferative effect by way of increased cAMP production. Expression of EP2 and EP4 in human gastric carcinoma cells, however, has not been examined. We examined the expression of EP2 and EP4 and the antiproliferative effects of specific EP2 and EP4 agonists on four different human gastric cancer cell lines. Our data clarified that all the cell lines investigated in this study expressed EP2 and EP4 and that the specific agonists of these receptors induced growth inhibition with an accompanying increase in cAMP production. In summary, gastric cancer cells have EP2 and EP4 receptors, and their selective activation is linked with the decreased cell proliferation.

Association study of C825T polymorphism of the G-protein b3 subunit gene with schizophrenia and mood disorders.

Alterations of G proteins have been implicated in major psychiatric illnesses. A C825T polymorphism of a gene encoding the beta3 subunit of heterotrimeric G proteins (GNB3) was reported to be associated with several pathological conditions, such as hypertension and depressive disorder. We examined whether this polymorphism is associated with functional psychoses in a Japanese sample of 370 schizophrenics, 164 bipolars, 68 depressive patients, and 198 controls. We obtained no evidence for an association of the polymorphism with any diagnostic group.

Time-course changes of ECL cell markers in acetic acid-induced gastric ulcers in rats.

BACKGROUND AND AIM: Enterochromaffin-like (ECL) cells are the major source of histamine for the regulation of gastric acid secretion, and also contain histidine decarboxylase (HDC), vesicular monoamine transporter 2 (VMAT2), and chromogranin A (CgA). Although gastric acid secretion is suppressed during ulcer healing, the role of ECL cells in that process is not yet fully understood. In the present study, we investigated the changes in ECL cell number during healing of experimental ulcers in rats. MATERIALS AND METHODS: Seven-week-old male Wistar rats were used. Acetic acid-induced ulcers were caused by an application of 100% acetic acid to the serosal surface of the rat stomachs. At different time points following the induction (12 h-15 days), time-course changes of HDC, VMAT2, and CgA mRNA expression were investigated by Northern blot analysis. The expressions of HDC, VMAT2, and CgA were immunostained on gastric mucosal sections with ulcers. RESULTS: HDC, VMAT2, and CgA mRNA in gastric mucosa each showed an initial marked transient decrease, followed by an increase on day 10 back to the initial value. HDC, VMAT2, and CgA-immunoreactive cells at the ulcer margin were reduced in number on day 3, compared with those in distant areas. On day 10, however, they returned to levels similar to those in distant areas. CONCLUSION: The present study revealed a local down-regulation of HDC, VMAT2, and CgA in ECL cells at the ulcer margin. As a result, we concluded that a suppression of ECL cell activity during ulcer healing may be involved in suppressed gastric acid secretion.

Identification of tyrosine residues involved in ligand recognition by the phosphatidylinositol 3-kinase Src homology 3 domain: circular dichroism and UV resonance Raman studies.

Src homology 3 (SH3) domains are small noncatalytic protein modules capable of mediating protein-protein interactions. We previously demonstrated that the association of a ligand peptide RLP1 (RKLPPRPSK) causes environmental and structural changes of Trp55 and some of seven Tyr residues in the phosphatidylinositol 3-kinase (PI3K) SH3 domain by circular dichroism (CD) and 235-nm excited UV resonance Raman (UVRR) spectroscopies [Okishio, N., et al. (2000) Biopolymers 57, 208-217]. In this work, the affected Tyr residues were identified as Tyr12, Tyr14, and Tyr73 by the CD analysis of a series of mutants, in which every single Tyr residue was replaced by a Phe residue. Among these three residues, Tyr14 was found to be a main contributor to the UVRR spectral change upon the RLP1 binding. Interestingly, CD and UVRR analyses revealed that RLP1 associates with the Y14F and Y14H mutants in different ways. These results suggest that Tyr14 plays a crucial role in the ligand recognition, and the amino acid substitution at Tyr14 affects the mode of PI3K SH3-ligand interaction. Our findings give an insight into how SH3 domains can produce diversity and specificity to transduce signaling within cells.

Functional magnetic resonance imaging of odor identification: the effect of aging.

BACKGROUND: Sense of smell declines with age and impairment in olfaction has been observed in some neurodegenerative disorders such as Alzheimer's disease. Functional neuroimaging techniques enable researchers to observe brain regions activated by olfactory stimuli. METHODS: We gave three mainly olfactory-mediated odors (limonene, methylsalicylate, and eugenol) to six young and six elderly subjects and observed the areas activated by using blood oxygen level dependent contrast functional magnetic resonance imaging. RESULTS: The group mapping of young subjects showed extensive activation in the orbitofrontal cortex, commonly believed to be the olfactory cortex, some limbic areas (the hippocampus and the thalamus), regions involved with gustatory sensation (the anterior insula and the inferior postcentral gyrus), superior and inferior temporal gyri, and cerebellum. In the elderly group, only the left inferior temporal gyrus and the primary visual cortex reached accepted significance levels. CONCLUSIONS: We have therefore confirmed previous reports of brain regions involved in olfactory processing in young volunteers and demonstrated decreased activation in elderly volunteers.

Clinical relevance of precore and basal core promoter variants of hepatitis B virus during natural hepatitis B e antigen seroconversion may be overstated.

BACKGROUND: Clinical relevance of nucleotide changes in precore and basal core promoters in the hepatitis B virus genome during hepatitis B e antigen seroconversion may be overstated. The authors investigated the existence and changes in the relative proportion of variants to wild virus that occur with seroconversion. METHODS: Sera from 30 school-aged long-term hepatitis B virus carriers, including 11 tested before and after seroconversion during 1 to 8 years of follow-up, were evaluated for variations in nucleotide sequences of the basal core promoter (T1762 and A1764), precore region (A1869), and carboxyl-terminus of the X region of the hepatitis B virus genome using an amplification refractory mutation detection system with mutant-specific primers. RESULTS: All variants were found to already exist before seroconversion at various wild-type/mutant ratios. The positive rates of these variants were not changed with loss of hepatitis B e antigen. Although there was a relative increase in the concentration of these mutants in wild-type/mutant mixed populations, most patients with only a wild-type population maintained the same pattern after loss of hepatitis B e antigen. CONCLUSIONS: Our results indicate that hepatitis B virus exists as a quasi species, and correlations of nucleotide sequences with clinical and serologic findings must be done with caution.

Intragastric distribution of Helicobacter pylori during short-term omeprazole therapy: study using Carnoy's fixation and immunohistochemistry for detection of bacteria.

BACKGROUND: There is controversy about the effect of acid-suppressive therapy on Helicobacter pylori density and the severity of histological gastritis in the corpus. AIM: To evaluate the precise distribution of H. pylori, both on the surface mucus cells and in the surface mucus gel layer, by using Carnoy's fixation and immunostaining for the detection of bacteria. METHODS: A total of 19 peptic ulcer patients with H. pylori infection were studied. All patients received a 6-week course of treatment with omeprazole (20 mg/day). Before and after the therapy, H. pylori density in Carnoy-fixed tissue sections was examined immunohistochemically. The effect of omeprazole therapy on the severity of gastritis was also evaluated. RESULTS: H. pylori density and the grade of gastritis significantly decreased in the antrum after omeprazole therapy. In the corpus, however, there were no significant changes in H. pylori density or the severity of gastritis after omeprazole therapy. CONCLUSION: Carnoy's fixation and immunostaining was found to be useful for the detection of H. pylori in the surface mucus gel layer as well as on the surface mucus cells in biopsy tissue sections. By using this method, H. pylori density decreased in the antrum, but remained unchanged in the corpus after a 6-week course of omeprazole therapy.

Activation of the Ras-cAMP signal transduction pathway inhibits the proteasome-independent degradation of misfolded protein aggregates in the endoplasmic reticulum lumen.

Many kinds of misfolded secretory proteins are known to be degraded in the endoplasmic reticulum (ER). Dislocation of misfolded proteins from the ER to the cytosol and subsequent degradation by the proteasome have been demonstrated. Using the yeast Saccharomyces cerevisiae, we have been studying the secretion of a heterologous protein, Rhizopus niveus aspartic proteinase-I (RNAP-I). Previously, we found that the pro sequence of RNAP-I is important for the folding and secretion, and that Deltapro, a mutated derivative of RNAP-I in which the entire region of the pro sequence is deleted, forms gross aggregates in the yeast ER. In this study, we show that the degradation of Deltapro occurs independently of the proteasome. Its degradation was not inhibited either by a potent proteasome inhibitor or in a proteasome mutant. We also show that neither the export from the ER nor the vacuolar proteinase is required for the degradation of Deltapro. These results raise the possibility that the Deltapro aggregates are degraded in the ER lumen. We have isolated a yeast mutant in which the degradation of Deltapro is delayed. We show that the mutated gene is IRA2, which encodes a GTPase-activating protein for Ras. Because Ira2 protein is a negative regulator of the Ras-cAMP pathway, this result suggests that hyperactivation of the Ras-cAMP pathway inhibits the degradation of Deltapro. Consistently, down-regulation of the Ras-cAMP pathway in the ira2 mutant suppressed the defect of the degradation of Deltapro. Thus, the Ras-cAMP signal transduction pathway seems to control the proteasome-independent degradation of the ER misfolded protein aggregates.