Construction and validation of a scoring system to predict resistance to chemotherapeutic drugs using gene expression profiles in canine lymphoma.

The present study aimed to verify the changes in the expression levels of 13 candidate genes associated with chemotherapy resistance and to construct a scoring system to predict resistance to these drugs. The expression levels of the 13 candidate genes were compared between 20 dogs with lymphoma that were sensitive to drugs used in CHOP-based protocol and 16 dogs with lymphoma that were resistant to these drugs. The expression levels of six genes; ASNS, CCR3, CALCA, FCER1A, LOC448801, and EDNRB were significantly different between the two groups. A scoring system to predict resistance to cyclophosphamide, doxorubicin and vincristine, which are used in CHOP-based protocol, was constructed based on expression levels of the six genes in these 36 dogs using logistic regression models. After internal validation, sensitivity and specificity of the scoring system were 0.759 and 0.853, respectively. External validation was conducted in another cohort of 33 dogs with lymphoma, and sensitivity and specificity of the scoring system were 0.800 and 0.696, respectively. In conclusion, this study identified six genes associated with resistance to drugs used in CHOP-based protocol in canine lymphoma and proposed a novel scoring system to predict resistance to these drugs. This system might be beneficial in selecting the most appropriate chemotherapy protocol for individual dogs with lymphoma.

Periodontal therapy for patients before and after radiotherapy: A review of the literature and topics of interest for clinicians.

BACKGROUND: To review and discuss important topics regarding periodontal treatment pre- and post-radiotherapy for head and neck cancer in human patients; to discuss the references for adequate techniques, the appropriate moment for tooth extractions and periodontal management; and to discuss the prevention of osteoradionecrosis. MATERIAL AND METHODS: Thirty-nine studies including original studies, randomized clinical trials (RCTs) and reviews were searched in online databases MEDLINE (PubMed) and the Cochrane library. No year of publication restriction was applied. RESULTS: Language was restricted to English, and the following Medical Subject Heading terms were used: radiotherapy, radiation therapy and periodontal treatment. Studies regarding periodontal treatment and tooth extraction that involved clinical management of irradiated patients were selected. CONCLUSIONS: The treatment of periodontal diseases before radiotherapy is mainly required to avoid future dental extraction and to reduce the development of osteoradionecrosis. Periodontal treatment in irradiated patients mostly includes scaling and root planing, extraction of condemned teeth and topical and systemic antimicrobial therapy. Tooth removal should be planned at least 14 days before the first day of radiation treatment. Particular care and mouthwashes should be taken during and after radiation. CLINICAL SIGNIFICANCE: The management of irradiated patients represents a challenge for health professionals, including dentists. It is important to establish recommendations for clinicians concerning dental and periodontal management in irradiated patients before, during and after treatment.

Therapeutic Effect of Sirolimus for Lymphangioleiomyomatosis Remaining in the Abdominopelvic Region After Lung Transplantation: A Case Report.

PURPOSE: Sirolimus (SRL) is used to treat pulmonary lymphangioleiomyomatosis (P-LAM). There is limited evidence that SRL has systemic efficacy for the patients with extrapulmonary lymphangioleiomyomatosis (E-LAM) remaining after lung transplantation (LT) for P-LAM. This report examines the efficacy of SRL treatment for the patient with E-LAM remaining after an LT for P-LAM. CASE SUMMARY: The course of the patient's recovery from an LT for P-LAM was complicated by lymphedema in the left femoral region that was caused by two E-LAM lesions remaining in the left pelvic cavity and in the retroperitoneal area. After the LT was performed, the patient started SRL treatment to reduce the E-LAM lesions. The daily SRL dose, selected based on the standard SRL dose for P-LAM, was initiated at 1 mg/d and was maintained at 2 mg/d. The remaining E-LAM lesions and lymphedema in the left femoral region improved in approximately 9 months after the LT with the administration of both SRL and the standard immunosuppressive therapy used by Okayama University Hospital, including tacrolimus, mycophenolate mofetil, and prednisolone. The SRL and tacrolimus trough concentrations in whole blood were maintained within the therapeutic window for the next 1.5 years after initiation of SRL treatment. The patient experienced no severe adverse events that required discontinuation of the SRL treatment during this time. CONCLUSION: The patients with remaining E-LAM lesions may receive SRL treatment to improve the quality of life after LT for P-LAM as effective therapy in cases where the patient's recovery is complicated by E-LAM lesions.

Relationship between serotonin transporter occupancies and analgesic effects of AS1069562, the (+)-isomer of indeloxazine, and duloxetine in reserpine-induced myalgia rats.

Serotonin (5-HT) and norepinephrine (NE) have been implicated in the mediation of endogenous analgesic mechanisms via the descending inhibitory pain pathway in the brain, and dysfunction in both the 5-HT and NE systems has been suggested as an etiology of fibromyalgia (FM). Given that 5-HT reuptake inhibition in the brain appears to be associated with pain reduction, this mechanism might exert an analgesic effect also on pain associated with FM. In this case, it would be of interest to investigate the correlation of 5-HT transporter (SERT) occupancy with in vivo analgesic effect on pain associated with FM. Here, we investigated the relationship between SERT occupancies and the analgesic effects of AS1069562, the (+)-isomer of indeloxazine, and duloxetine, which are both 5-HT and NE reuptake inhibitors (SNRIs), on muscular pain in reserpine-induced myalgia (RIM) rats, an animal model of FM-like chronic pain. We also investigated the SERT occupancy level necessary for AS1069562 and duloxetine to exert analgesic effects on muscular pain. AS1069562 and duloxetine attenuated muscular hyperalgesia in RIM rats, representing the first findings to be reported regarding the analgesic effect of AS1069562 on pain associated with FM. SERT occupancy levels of AS1069562 and duloxetine increased in both dose- and plasma and brain concentration-dependent manners. SERT occupancy levels of AS1069562 and duloxetine were significantly correlated with efficacy on muscular pain thresholds in RIM rats. This finding concerning the precise correlation of SERT occupancy with in vivo analgesic effect on pain associated with FM is reported here for the first time. SERT occupancy level above 70% was necessary for AS1069562 and duloxetine to exert significant analgesic effects on muscular pain. These results suggest that SERT occupancy level is useful in determining appropriate analgesic doses of AS1069562 and duloxetine for treating pain symptoms in FM patients.

Levels of the oxidative stress marker γ-glutamyltranspeptidase at different stages of nonalcoholic fatty liver disease.

OBJECTIVES: This study investigated oxidative stress in the liver, by determining hepatic expression and serum levels of γ-glutamyltranspeptidase (GGT) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in different stages of nonalcoholic fatty liver disease (NAFLD), and assessed whether GGT can differentiate between the various stages of NAFLD. METHODS: Expression of GGT and 8-OHdG was examined in biopsy specimens by immunohistochemistry, and serum GGT and 8-OHdG levels were measured by enzyme-linked immuno sorbent assays in patients with simple fatty liver (n = 10), nonalcoholic steatohepatitis (NASH; n = 10) and, as a control, in alcoholic liver disease (ALD; n = 10). RESULTS: Hepatic tissue expression of GGT and 8-OHdG was seen in ALD, NASH and fatty liver patients. The percentage of hepatocytes positive for 8-OHdG expression and serum 8-OHdG levels was significantly higher in patients with NASH than simple fatty liver. Serum GGT levels were increased in all cases with ALD, NASH and fatty liver, and correlated significantly with serum levels of 8-OHdG in ALD and NASH, but not in simple fatty liver. CONCLUSIONS: Levels of GGT in fatty liver patients may compensate for mild oxidative stress by repressing 8-OHdG levels and preventing progression to NASH; however further oxidative stress leads to increased levels of 8-OHdG and the development of NASH.

Assessment of wastewater-irrigated soil containing heavy metals and establishment of specific biomarkers.

Irrigation with treated wastewater (TWW) is a vital alternative for arid and semi-arid lands but it poses pollution-risk to soil, vegetation and groundwater. Therefore, in the present study, in vitro bioassays were used to evaluate the adverse effects of TWW and irrigated-soil extract sample, on mammalian cells, with respect to heavy metal--Ni, Cd, Pb, Fe, Al-content. The heat shock protein (HSP) 47, E-screen, and transepithelial electrical resistance (TEER) assays served to investigate the stress response of treated-HSP47-transfected Chinese hamster ovary (CHO) cells, the estrogenic activity of the samples in MCF-7 breast cancer cells, and the barrier function (BF) of Caco-2 cells. Furthermore, proteomics analyses were performed to shed light on involved mechanisms and to establish pollution biomarkers. Results showed that the TWW elicited a stress response on HSP cells from 0.1% concentration while soil extract samples exhibited a stress at 1%. TWW induced an estrogenic activity at 10%; up-regulating cell proliferation and tumor-related proteins. Soil extract triggered the enhanced expression of HSP70 family proteins as survival mechanisms against their cytotoxicity toward MCF-7 cells. Moreover, depending on the concentration, 1% of soil extract from 20 cm depth (T20) resulted in a disruption of BF in Caco-2 cells involving cell metabolism, protein synthesis and tumor marker proteins, whereas, 5% of T20 induced the expression of BF-related proteins associated to heat shock, oxidative stress, cell proliferation and glycolytic metabolic pathway. These biological techniques were found to be extremely useful to evaluate the impact of wastewater reuse and to establish specific biomarkers that are common proteins for humans, other mammals and plants. Future studies should focus on exposure quantifications.

Characteristic genotypes of vascular endothelial growth factor are susceptible to ascites in patients with cirrhosis.

This study was designed to investigate whether different vascular endothelial growth factor (VEGF) genotypes are associated with ascites formation in cirrhotic patients. Seventy cirrhotic patients were included in the study: 25 cirrhotic patients with ascites and 45 cirrhotic patients without ascites. Patient characteristics were investigated and compared between the two groups. With regard to VEGF genotype, 42 patients were C/C and 28 patients were T/T or C/T. The genotypes T/T or C/T were observed in 23 cases (51%) among the non-ascites group, but in only five cases (20%) among the ascites group. Serum levels of albumin and creatinine, and the VEGF genotypes were significantly different between the two groups. Multiple regression analysis showed that serum levels of creatinine and the VEGF genotypes were significantly correlated with ascites formation. Thus, it can be concluded that VEGF genotyping might be a valuable susceptibility marker for ascites formation in cirrhotic patients.

Ultrastructure and function of long and short sperm in Cicadidae (Hemiptera).

The cicada, Graptopsaltria nigrofuscata, produces two distinct sizes of sperm, as determined by either nuclear volume of early spermatids or nuclear length of mature sperm. Between both sperm, there is no difference in location of the acrosome and flagellum during spermiogenesis. The acrosome is covered by an anteacrosomal bleb, which is inserted in a common mass, spermatodesm, derived from cyst cells. Both kinds of sperm linked to the spermatodesm form sperm bundles, respectively. During copulation, the sperm bundles are transported from the vesicula seminalis of the male to the bursa copulatrix of the female. Morphometric analyses of the nuclear length revealed that the two kinds of sperm reach the bursa copulatrix in the same condition as that found in the vesicula seminalis. Once transferred inside the latter, the sperm bundles disintegrated to individual sperm within a few hours, and the tail components, such as the axoneme and mitochondrial derivatives, become separated from each other over time. The tail completely splits from the sperm nucleus 24 h after copulation. Fertile sperm accumulate in the spermatheca, the final storage organ, where only long sperm survived for any length of time. Fertilized eggs examined by vital staining contain only sperm with long nuclei.

Results of a phase I clinical trial of vaccination of glioma patients with fusions of dendritic and glioma cells.

Several reports of clinical trials of immunotherapy using dendritic cells have been published to date. In this study, we investigated the safety and clinical response of immunotherapy with fusions of dendritic and glioma cells for the treatment of patients with malignant glioma. Eight patients with malignant glioma, ranging in age from 4 to 63 years old, participated in this study. Dendritic cells were generated from peripheral blood. Cultured autologous glioma cells were established from surgical specimens in each case. Fusion cells of dendritic and glioma cells were prepared with polyethylene glycol, and the fusion efficiency ranged from 9.2 to 35.3% (mean, 21.9%). All patients received the fusion cells every three weeks for a minimum of 3, and a maximum of 7, immunizations. Fusion cells were injected intradermally, close to a cervical lymph node. The percentage of CD16- and CD56-positive cells in peripheral blood lymphocytes slightly increased after immunization in 4 out of 5 cases investigated. Peripheral blood mononuclear cells were incubated with irradiated autologous glioma or U87MG cells and supernatants were harvested. In 6 cases analyzed, the concentration of interferon-gamma in the supernatant increased after immunization. Clinical results showed that there were no serious adverse effects and two partial responses. Although the results of the phase I clinical trial of fusion cells indicated that this treatment safely induced immune responses. we were unable to establish a statistically significant treatment-associated response rate, due to the limited sample population. Therefore, further evaluation of the role of adjuvant cytokines is necessary.

Effect of modification of the carboxyl groups of the sialic acid binding lectin from bullfrog (Rana catesbeiana) oocyte on anti-tumor activity.

The sialic acid binding lectin from bullfrog Rana catesbeiana oocyte (cSBL) is known to have anti-tumor activity. In order to investigate the relationship between the net charge of cSBL and its anti-tumor effect, cSBL was modified with a water-soluble carbodiimide (EDC) in the presence of three kinds of nucleophiles, taurine, glycine methylester and ethylenediamine. cSBL having four carboxyl groups was partially modified (ca. 2 residues). The anti-tumor activity of modified cSBLs was in the order of ethylenediamine-modified cSBL > glycine methylester-modified cSBL > taurine modified cSBL > or = native cSBL. The results suggested that anti-tumor activity seems to increase with the increase in positive net charge, possibly enhancing the interaction of cSBL with sialoglycoprotein on the surface of tumor cells. The ribonuclease activity of ethylenediamine-modified cSBL decreased with the progress of the reaction, but the number of internalized molecules in the tumor cell increased. Thus, for antitumor activity, a higher incorporation of cSBL with reasonable RNase activity seems to be more important than total RNase activity.

Relationships between perceived workload, stress and oxidative DNA damage.

OBJECTIVES: The present study was performed to investigate the relationship between work-related factors, including psychological stress, and the formation of a type of oxidative DNA damage, 8-hydroxydeoxyguanosine (8-OH-dG), in order to examine their possible risk factor for occupational carcinogenesis. METHODS: A total of 54 healthy workers (27 male and 27 female, aged 41.2 +/- 12.5 years) in a company were investigated for 8-OH-dG levels in the peripheral blood leukocytes at the time of a questionnaire survey regarding several factors, such as working hours, workload, fatigue, sleep, psychological stress and the prospect of alleviating it. Subjects were limited to non-smoking and non-drinking workers to exclude the influence of cigarette smoking and alcohol drinking, which have been reported to have associations with the formation of 8-OH-dG. RESULTS: The levels of 8-OH-dG in female subjects were significantly related to the perceived workload (F = 5.56, P = 0.010), the perceived psychological stress (F = 6.15, P = 0.007), and the impossibility of alleviating stress (F = 3.82, P = 0.048). No associations were observed in male subjects. CONCLUSIONS: Psychological stress and perceived over-work appear to be related to the pathogenesis of cancer via the formation of 8-OH-dG, particularly in female workers.

Psychosocial factors as a potential trigger of oxidative DNA damage in human leukocytes.

Although numerous studies have been carried out on the stress-cancer linkage, the results are still inconclusive. One of the useful, but rarely applied, methods to assess this linkage is to examine the relationship between psychosocial stress and cancer-predisposing genetic alterations simultaneously. We investigated whether various psychosocial factors can be associated with the levels of 8-hydroxydeoxyguanosine (8-OH-dG), a biomarker of cancer-related oxidative DNA damage, in peripheral blood leukocytes in 362 healthy workers (276 males and 86 females). After adjustments for age, body mass index, cigarette smoking, and alcohol use, female subjects showed positive relationships between the amount of 8-OH-dG and the Tension-Anxiety, Depression-Rejection, Anger-Hostility, Fatigue, and Confusion scores of the Profile of Mood States, respectively. The levels of 8-OH-dG also increased reliably in the female subjects who had poor stress-coping behaviors, particularly wishful thinking strategy, in the NIOSH general job stress instrument. There were positive relationships of the 8-OH-dG levels to average working hours, a self-blame coping strategy, and recent loss of a close family member in male subjects. These findings in a nonclinical sample of healthy adults not only provide evidence of a stress-cancer linkage, but also suggest possible sex differences in the mechanisms of stress-related cancer initiation.

Amino acid sequence of a trypsin inhibitor from a Spirometra (Spirometra erinaceieuropaei).

A trypsin inhibitor that is highly homologous with bovine pancreatic trypsin inhibitor (BPTI) was co-purified along with RNase from Spirometra (Spirometra erinaceieuropaei). The amino acid sequence of this inhibitor (SETI) and the nucleotide sequence of the cDNA encoding this protein were determined by protein chemistry and gene technology. SETI contains 68 amino acid residues and has a molecular mass of 7,798 Da. SETI has 31 amino acid residues that are identical with BPTI's sequence, including 6 half-cystine and 5 aromatic amino acid residues. The active site Lys residue in BPTI is replaced by an Arg residue in SETI. SETI is an effective inhibitor of trypsin and moderately inhibits a-chymotrypsin, but less inhibits elastase or subtilisin. SETI was expressed by E. coli containing a PelB vector carrying the SETI encoding cDNA; an expression yield of 0.68 mg/l was obtained. The phylogenetic relationship of SETI and the other BPTI-like trypsin inhibitors was analyzed using most likelihood inference methods.

Enzymatic properties of sialic acid binding lectin from Rana catesbeiana modified with a water-soluble carbodiimide in the presence of various nucleophiles.

The anti-tumor activity of sialic acid binding lectin from Rana catesbeiana (cSBL) was increased by chemical modification with a water-soluble carbodiimide (EDC) in the presence of nucleophiles such as ethylenediamine and glycine methylester. Investigations on ribonuclease (RNase) activities of the modified cSBLs were conducted to elucidate the fundamental mechanisms underlying enhancement of the anti-tumor activity conferred by these modifications. The following three characteristics were observed with modification. (i) RNase activity of the modified cSBL was enhanced towards double stranded RNA and RNA-oligo dA hybrids. The activity increase was observed even under physiologic ionic strength conditions; (ii) RNase activity of the modified cSBL towards single stranded RNA and poly U decreased, while the activity towards poly C was unaffected; (iii) the base preference of the B2 base recognition site of modified cSBL decreased for guanine. On the contrary, the preference for cytosine and adenine increased. This result may explain why the RNase activity towards poly C was not affected by EDC-modification as mentioned above.

Structural requirements for determining the substrate affinity of peptide transporters PEPT1 and PEPT2.

Peptide transporters PEPT1 and PEPT2 transport numerous compounds including small peptides, peptide-like drugs and nonpeptidic compounds such as valacyclovir. PEPT1 and PEPT2 show low and high affinity for most substrates, respectively, but beta-lactam antibiotics without an alpha-amino group are the only known substrates that prefer PEPT1 to PEPT2. The aim of this study was to compare the recognition and affinity of various substrates between rat PEPT1 and rat PEPT2, and to determine the structural requirements influencing the substrate affinity. [14C]Glycylsarcosine uptake by PEPT1- or PEPT2-expressing transfectant was inhibited by di- and tripeptides, but not by amino acids, tetrapeptides or most cyclic dipeptides. All dipeptides and tripeptides examined showed more potent inhibition of [14C]glycylsarcosine uptake via PEPT2 than via PEPT1, irrespective of their charge and structure. Modification of the alpha-amino group of dipeptides reduced their substrate affinity to both transporters, as compared to unmodified dipeptides, but these dipeptides still showed potent inhibitory effects on PEPT2. Among the nonpeptidic substrates tested, only the eight-amino-octanoic acid displayed stronger inhibition of [14C]glycylsarcosine uptake in PEPT1 than in PEPT2. These findings suggest that alpha- or beta-amino carbonyl function is the key structure responsible for the higher affinity for PEPT2 than for PEPT1.

Amino acid sequence of a nuclease (nuclease Le1) from Lentinus edodes.

The fruit bodies of Lentinus edodes produce two acid nucleases, nucleases Le1 and Le3, both of which are thought to be candidates for the enzymes producing a tasty substance, 5'-GMP. To obtain the basic information on the mechanism of production of 5'-GMP, and structure-function relationship of these nucleases, the primary structure of nuclease Le1 was estimated by both protein chemistry and gene cloning. Nuclease Le1 is a glycoprotein and consists of 290 amino acid residues, and about 2 and 6 residues of hexosamine and neutral sugar, respectively. The nucleotide sequence of cDNA and genomic DNA encoding nuclease Le1 indicated the presence of 20 amino acid residues of a signal peptide. Nuclease Le1 has 115 and 108 residues of identical amino acid residues with nucleases P1 and S, respectively. The amino acid residues concerning the coordination with Zn2+ in nuclease P1 are all conserved in nuclease Le1. Nuclease Le1 contains 8 half-cystine residues and 4 of them are located at the same places as those of nucleases P1 and S.

Serum concentrations of 20K human growth hormone in normal adults and patients with various endocrine disorders. Study Group of 20K hGH.

The 20K hGH isoform is produced by alternative splicing of GH mRNA, and comprises approximately 10% of all GH in the pituitary. The physiological role of 20K hGH remains to be determined partly because of the lack of a simple and specific assay. We have established sensitive enzyme-linked immunoadsorbent assays (ELISAs) specific to 20K and 22K hGH. The serum levels of 20K hGH after overnight fasting was 118 +/- 178 pg/mL (N=282) in normal women, significantly higher than in normal men (64 +/- 170 pg/mL, N=226). However, there was no difference in the proportion of 20K hGH to 20K plus 22K hGH between men (6.3 +/- 2.6%, N=176) and women (6.3 +/- 2.1%, N = 263). No correlation was detected between the ratio of 20K hGH and age, body height, body weight or body fat mass in normal subjects. The proportion of 20K hGH was significantly (P < 0.001) higher in patients with active acromegaly (9.2 +/- 2.2%, N=33) and in patients with anorexia nervosa (9.0 +/- 1.9, N=8), both of which are characterized by chronic elevation of circulating GH levels. The proportion of the 20K hGH in successfully treated acromegalic patients did not differ from that in normal subjects, suggesting that GH-producing pituitary tumors secrete a higher proportion of 20K hGH, or chronic excess of 22K hGH altering the metabolic clearance rate of 20K hGH. The values in patients with adult growth hormone deficiency (GHD), hyperthyroidism, primary hypothyroidism, or GH-independent short stature did not differ from those in normal subjects. The 20K ratio did not change after acute GH provocative tests such as insulin tolerance test and GRH test. These results suggest that secretion of 20K hGH from the pituitary is under the same control as that of 22K hGH.

Possible interference between tissue-non-specific alkaline phosphatase with an Arg54-->Cys substitution and acounterpart with an Asp277-->Ala substitution found in a compound heterozygote associated with severe hypophosphatasia.

Tissue-non-specific alkaline phosphatase (TNSALP) with an Arg(54)-->Cys (R54C) or an Asp(277)-->Ala (D277A)substitution was found in a patient with hypophosphatasia [Henthorn,Raducha, Fedde, Lafferty and Whyte (1992) Proc. Natl. Acad. Sci. U.S.A.89, 9924-9928]. To examine effects of these missense mutations onproperties of TNSALP, the TNSALP mutants were expressed ectopically inCOS-1 cells. The wild-type TNSALP was synthesized as a 66-kDa endo-beta-N-acetylglucosaminidase H (Endo H)-sensitive form, and processed to an 80-kDa mature form, which is anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). Although the mutant proteins were found to be modified by GPI, digestion with phosphatidylinositol-specific phospholipase C, cell-surface biotinylation and immunofluorescence observation demonstrated that the cell-surface appearance of TNSALP (R54C) and TNSALP (D277A) was either almost totally or partially retarded respectively. The 66-kDa Endo H-sensitive band was the only form, and was rapidly degraded in the cells expressing TNSALP (R54C). In contrast with cells expressing TNSALP(R54C), where alkaline phosphatase activity was negligible, significant enzyme activity was detected and, furthermore, the 80-kDa mature form appeared on the surface of the cells expressing TNSALP (D277A). Analysis by sedimentation on sucrose gradients showed that a considerable fraction of newly synthesized TNSALP (R54C) and TNSALP(D277A) formed large aggregates, indicating improper folding and incorrect oligomerization of the mutant enzymes. When co-expressed with TNSALP (R54C), the level of the 80-kDa mature form of TNSALP (D277A)was decreased dramatically, with a concomitant reduction in enzyme activity in the co-transfected cell. These findings suggest that TNSALP(R54C) interferes with folding and assembly of TNSALP (D277A) intrans when expressed in the same cell, thus probably explaining why a compound heterozygote for these mutant alleles developed severe hypophosphatasia.

Classical conditioning of oxidative DNA damage in rats.

This study investigated whether the formation of 8-hydroxydeoxyguanosine (8-OH-dG), a known oxidative DNA modification relevant to carcinogenicity, can be classically conditioned to a novel taste in order to clarify the possible role of the central nervous system (CNS) or psychological stress on cancer initiation via a classical conditioning mechanism. Male Wistar rats underwent one or two conditioned taste aversion (CTA) experiments in which ferric nitrilotriacetate (Fe-NTA), which has renal toxicity and can induce renal cell carcinoma, served as a visceral unconditioned stimulus (US), and a saccharin solution (SAC) was used as a conditioned stimulus (CS). The 8-OH-dG levels in the group conditioned with the combination of SAC and Fe-NTA significantly increased as compared to those of the uncombined groups by two repeats of the conditioning procedure (P=0.013). The rats that showed a painful response at the Fe-NTA administration had significantly higher values of 8-OH-dG than those without pain (P=0. 003). These results not only provide the first evidence regarding classical conditioning of oxidative DNA damage using the CTA procedure, but also suggest the involvement of the CNS and psychological stress in the pathogenesis of cancer via oxidative DNA damage.

Inefficient function of the signal sequence of PTHrP for targeting into the secretory pathway.

Parathyroid hormone-related peptide (PTHrP) is not only secreted out of cells, but also targeted to the nucleoli due to a nucleolar targeting signal (NTS). We assessed the molecular mechanism underlying the dual targeting of PTHrP by constructing a series of truncated forms of rat PTHrP cDNA and expressing them in CHO cells. Immunostaining was observed in both the Golgi apparatus and nucleoli in the same cell expressing PTHrP with the N-terminal full-length signal sequence. When PTHrP molecules were translated from CUGs downstream of the AUG-initiator codon in the signal sequences, potential alternative initiators of the translation, they were exclusively localized in the nucleoli. In contrast, when a construct containing only the ATG-initiator codon was expressed, PTHrP was found to localize in both the nucleolus and the Golgi apparatus. No nucleolar staining of PTHrP was observed in the CHO cells transfected with PTH/PTHrP receptors even after incubating with a conditioned medium containing PTHrP, ruling out a possibility that PTHrP is, once secreted, internalized via receptor-mediated endocytosis and subsequently conveyed to nucleoli. Compatible with these morphological observations, a preproform of PTHrP was found in the cells expressing PTHrP in addition to proPTHrP, indicative of molecules along the secretory pathway. These results strongly indicate that the signal sequence of PTHrP is not sufficient to direct all the newly synthesized molecules across the endoplasmic reticulum, resulting in part of it being delivered to the nucleoli due to the NTS.