Presynaptic cell type-dependent regulation of GABAergic synaptic transmission by nitric oxide in rat insular cortex.

Nitric oxide (NO) is a key retrograde messenger that regulates synaptic transmission in the cerebral cortex. However, little is known about NO-induced modulatory effects and their mechanisms relative to inhibitory synaptic transmission. The present study aimed to examine the effects of NO on unitary inhibitory postsynaptic currents (uIPSCs) and to postulate the synaptic location of NO action. We performed multiple whole-cell patch-clamp recordings from rat insular cortex and divided recorded cells into three subtypes: pyramidal cells (Pyr), fast-spiking interneurons (FS), and non-FS GABAergic interneurons. In the connections from FS to Pyr (FS→Pyr), the application of S-nitroso-N-acetyl-dl-penicillamine (SNAP, 100 µM), an NO donor, suppressed uIPSC amplitudes in 31% of the connections, whereas 39% of the connections showed uIPSC facilitation. The remaining FS→Pyr connections showed little effect of SNAP on uIPSCs. An analysis of paired-pulse ratio (PPR) implied the involvement of presynaptic mechanisms in SNAP-induced effects on uIPSCs. Similar effects of SNAP were observed in FS→FS/non-FS connections; 33%, 54%, and 13% of the connections were facilitated, suppressed, and unchanged, respectively. In contrast, non-FS→Pyr or FS/non-FS showed constant uIPSC suppression by SNAP. PPR analysis supports the hypothesis that these SNAP-induced effects are mediated by presynaptic mechanisms in FS→FS/non-FS and non-FS→Pyr/FS/non-FS connections. The NO scavenger, 2-phenyl-4,4,5,5-tetramethylimidazolineoxyl-1-oxyl-3-oxide (PTIO), or the inhibitor of guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), abolished the SNAP-induced uIPSC modulation. These results suggest that NO regulation of inhibitory synaptic transmission is dependent on presynaptic cell subtypes and that, at least in part, the effects are mediated by presynaptic mechanisms.

Humanized mice for human retrovirus infection.

Inbred mice with specific genetic defects have greatly facilitated the analysis of complex biological events. Several humanized mouse models using the C.B.-17 scid/scid mouse (referred to as the SCID mouse) have been created from two transplantation protocols, and these mice have been utilized for the investigation of human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus type I (HTLV-I) pathogenesis and the evaluation of antiviral compounds. To generate a more prominent small animal model for human retrovirus infection, especially for examination of the pathological process and the immune reaction, a novel immunodeficient mouse strain derived from the NOD SCID mouse was created by backcrossing with a common gamma chain (gamma(c))-knockout mouse. The NOD-SCID gamma(c)null (NOG) mouse has neither functional T and B cells nor NK cells and has been used as a recipient in humanized mouse models for transplantation of human immune cells particularly including hematopoietic stem cells (HSC). From recent advances in development of humanized mice, we are now able to provide a new version of the animal model for human retrovirus infection and human immunity.

Increased serotonergic innervation of lumbosacral motoneurons of rolling mouse Nagoya in correlation with abnormal hindlimb extension.

Rolling Mouse Nagoya (RMN) carries a mutation in a gene encoding for alpha(1A) subunit of P/Q-type Ca(2+) channel (Ca(v)2.1). In addition to ataxia, this mutant mouse exhibits abnormal hindlimb extension, which is characterized by a sustained excessive tone of hindlimb extensor muscles. This study aimed to clarify whether serotonergic (5-HTergic) innervation of the spinal motoneurons was altered in RMN in relation to the abnormal hindlimb extension. The density of 5-HT immunoreactive fibres in the ventral horn of lumbar and sacral regions of spinal cord was significantly greater in RMN than in controls. Retrograde wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) labelling combined with 5-HT immunostaining revealed that the number of 5-HT immunoreactive terminals adjoining femoris quadriceps motoneurons was about 2.5-fold greater in RMN than in controls. Furthermore, 5-HT immunostaining in the lumbar cord ventral horn was examined in three other Ca(v)2.1 mutant mice (tottering, leaner and pogo) as to whether or not they showed the abnormal hindlimb extension. Among these mutants, the increased density of 5-HT immunoreactive fibres was observed in correlation with the presence of the abnormal hindlimb extension. The results suggest an increased 5-HTergic innervation of the lumbosacral motoneurons in correlation with the abnormal hindlimb extension in RMN and other Ca(v)2.1 mutant mice. As 5-HT is known to induce the sustained membrane depolarizations without continuous excitatory synaptic inputs (plateau potentials) in spinal motoneurons, the increased 5-HTergic innervation may cause the sustained excitation of hindlimb extensor motoneurons, resulting in the abnormal hindlimb extension.

The clinical significance of sialyl Lewis antigen expression in the spread of gastric cancer. Flow cytometric DNA analysis.

We previously reported significant relationships between sialyl Lewis antigen expression on gastric cancer cells and both hepatic metastasis and clinical prognosis. The purpose of this study was to compare the expression of sialyl Lewis antigens on gastric cancer cells to elucidate the possible role of sialyl Lewis antigens in predicting the spread of a tumor with regard to histological findings. Subjects consisted of 38 patients with gastric cancer. For comparison we measured the values for sialyl Lewisa (sLea) and sialyl Lewisx (sLex) expression on the surface of about 10,000-30,000 cancer cells. Monoclonal antibodies CA19-9 and KM-93 were used to determine the frequency (%) and quantity (channel) of the expression by flow cytometry. We assessed the correlation of sLea and sLex expression with histological findings (depth of tumor invasion (pT), lymphatic invasion (ly), venous invasion (v), and lymph node metastasis (pN)), by comparing sLea and sLex expression in relation to the grade of histological findings. A significant relationship was found between lymphatic invasion and the frequency of sLea expression (r = 0.40, p<0.05). The mean values of sLea frequency in cases categorized as ly 2 (36.30) and ly 3 (31.81) were statistically higher than those in ly 1 cases (12.74) (p<0.05). A significant relationship was also observed between lymph node metastasis and the frequency of sLea expression (r = 0.46, p<0.01). The mean value of sLea frequency in pN 3 cases (44.14) was statistically higher than those in pN 0 (17.11) and pN 1 (19.03) cases (p<0.05). Neither the depth of tumor invasion nor venous invasion showed any correlation with the expression of sialyl Lewis antigens. In conclusion the frequency of sLea expression on the surface of gastric cancer cells was greater in those patients who developed lymphatic invasion and lymph node metastasis. However, the mechanism by which sialyl Lewisa expression is upregulated remains unclear.

Cell cycle-regulated factors in esophageal cancer.

Alterations of cell cycle-regulated genes play an important role in the process of carcinogenesis, and some of them are thought to be prognostic factors in esophageal cancer. The expressions of p53, p16, pRB and Cyclin D1 proteins were evaluated immunohistochemically in 144 patients who underwent curative esophagectomy without any adjuvant therapy before surgery. p53 overexpression was observed in 99 (69%) out of the 144 patients. No significant correlation was noted between p53 and any other gene expression. p16 expression was observed in 12 (8.3%) out of all cases. A negative correlation was recognized between p16 and Cyclin D1 expression (P=0.0004). pRB expression was observed in 130 (90.3%) out of all cases, whereas pRB expression was not observed in 11 out of the 12 patients with p16-positive tumors. A negative correlation was also found between p16 and pRB (P < 0.0001). A positive correlation was noted between pRB and Cyclin D1 expression (P=0.0009). The cumulative survival rate of patients without pRB expression was significantly lower than that of patients with positive expression (P=0.003). In the multivariate survival analysis, pRB expression was an independent prognostic factor. In 98% of all patients with esophageal cancer, impairment of the G1 checkpoint is due to a loss of function by p16, pRB or Cyclin D1, which showed a negative correlation in each factor. In addition, aberrant expression of pRB is useful as a prognostic factor in esophageal cancer.

Temporary restoration of immune response against Toxoplasma gondii in HIV-infected individuals after HAART, as studied in the hu-PBMC-SCID mouse model.

We studied immune reconstitution against the parasite T. gondii in HIV-infected patients treated for 1 years with highly active antiretroviral therapy (HAART). We used SCID mice, humanized with peripheral blood mononuclear cells (PBMC) from patients, which were then infected with T. gondii cysts. Mice humanized with PBMC from patients before the start of HAART were highly susceptible to infection. In contrast, mice humanized with PBMC from patients who had received HAART for 6 months displayed higher survival rates, correlating with lower intracerebral parasite loads. However, this resistance was lost during follow up because mice humanized with PBMC from patients treated with HAART for 12 months survived for no longer than mice that had not been humanized. Specific lymphocyte proliferation assays showed that the increase in proliferative response depended on treatment duration and that HAART induced changes in IFN-gamma secretion in the presence of Toxoplasma antigens. Thus, our results indicate partial immune reconstitution against T. gondii in HIV-infected patients following HAART, possibly due to changes in the patterns of specific IFN-gamma production and redistribution of functional memory CD4+ cells.

Is frozen section effective for diagnosis of unsuspected gallbladder cancer during laparoscopic cholecystectomy?

BACKGROUND: Although frozen section is recommended to prevent tumor dissemination following laparoscopic cholecystectomy (LC) for unsuspected gallbladder cancer, there are no reports concretely demonstrating its effectiveness and outcome. METHODS: Frozen section during LC was performed in 990 patients with gallstones. The sensitivity, specificity of frozen section, and false-negative cases were evaluated in comparison with postoperative entire cross sections. RESULTS: In frozen section, 983 cases were diagnosed as benign and 7 cases as malignant. Of the benign cases, cancer was discovered in 4 patients postoperatively in which frozen section was diagnosed as regenerative epithelial severe atypia. Sensitivity was 64% and specificity was 100%. Concerning the results of frozen section by p-TNM classification, cancer was diagnosed in 40% of Tis lesions, whereas it was found in 83% of T2 or T3 lesions. CONCLUSION: Frozen section is effective in cases with T2 or greater lesions for which conversion to radical surgery should be required.

Effects of single and repeated administration of 1,2,3,4-tetrahydroisoquinoline analogs on the binding of [11C]raclopride to dopamine D2 receptors in the mouse brain.

We investigated the effects of intraperitoneal injection of 1,2,3,4-tetrahydroisoquinoline (TIQ) analogs and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on the binding of [11C]raclopride to striatal dopamine D2 receptors in mice. The binding of [11C]raclopride, but not of [11C]N-methylspiperone or [11C]nemonapride with higher affinity, to the receptors was significantly decreased immediately after TIQ injection. Neither a dopamine transporter blocker induced such effect nor TIQ affected the dopamine transporter-radioligand binding. Among the compounds investigated, including parkinsonism-inducing TIQ and (R/S)-1-benzyl-TIQ, parkinsonism-preventing (R)- and (S)-1-methyl-TIQ, and probable N-methylated metabolites of TIQ and 1-methyl-TIQ, TIQ and (S)-1-methyl-TIQ had the strongest effect on the binding of [11C]raclopride, and N-methylated derivatives showed less of an effect than the respective parent compounds. The decrease in the binding of [11C]raclopride continued for 7 hours and was followed by an increase until 10 days after the single and subchronic administration of TIQ. These findings suggest that TIQ analogs profoundly stimulated dopamine release which resulted in the competitive inhibition of the binding of [11C]raclopride to dopamine D2 receptors, but did not induce degeneration of the receptors.

Evaluation of neurotoxicity of TIQ and MPTP and of parkinsonism-preventing effect of 1-MeTIQ by in vivo measurement of pre-synaptic dopamine transporters and post-synaptic dopamine D(2) receptors in the mouse striatum.

Parkinsonism-inducing neurotoxicity of 1,2,3,4-tetrahydroisoquinoline (TIQ), as contrasted to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and parkinsonism-preventing effect of 1-methyl-1,2,3,4-tetrahydroisoquinoline (1-MeTIQ) have been investigated in mice by measuring their effects on the in vivo binding of radioligand to pre-synaptic dopamine transporters (DATs) or to dopamine D(2) receptors (D2R) in the striatum. A significant reduction of the ligand-DATs binding was found in the mice treated with MPTP, but not with TIQ, under the dosage inducing behavioral abnormality and loss of tyrosine hydroxylase-positive cells in the substantia nigra. A slight decrease in the ligand-DATs binding was observed in the mice given a larger dose of TIQ. Compensatory up-regulation in the post-synaptic D2Rs was found in the MPTP-treated mice. Pre-treatment with (S)-enantiomer, but not (R)-enantiomer, of 1-MeTIQ prevented the degeneration of DATs to some extent. We concluded that the TIQ-induced parkinsonism model is different from the MPTP-induced model as evaluated by the radioligand-DATs binding and that (S)-1-MeTIQ has a preventing effect for the degeneration of the DATs to a certain extent.

Improved nonpenetrating trabeculectomy with trabeculotomy.

PURPOSE: To examine surgical effects and complications of improved nonpenetrating trabeculectomy with trabeculotomy in glaucoma patients. METHODS: Glaucoma patients in two medical institutions underwent nonpenetrating trabeculectomy with sinusotomy with or without trabeculotomy, and the results were compared retrospectively in the two groups by evaluation of final intraocular pressure, drug score, and occurrence of postsurgical complications. RESULTS: Of the 63 eyes of 51 patients in this study, 31 were treated with nonpenetrating trabeculectomy with sinusotomy without trabeculotomy and 32 eyes were treated with nonpenetrating trabeculectomy with sinusotomy and trabeculotomy. The mean follow-up period was 17.0 months. The clinical features in both groups were similar in terms of age, presurgical intraocular pressure (P = 0.96), and presurgical drug score. The eyes treated with nonpenetrating trabeculectomy with sinusotomy without trabeculotomy had significantly reduced intraocular pressures from 21.0 +/- 4.3 (mean +/- SD) to 15.8 +/- 6.3 mm Hg (P = 0.0003) and drug scores from 2.4 +/- 1.2 to 1.6 +/- 1.1 without postsurgical complications. The eyes treated with nonpenetrating trabeculectomy with sinusotomy and trabeculotomy had significantly reduced intraocular pressures from 22.3 +/- 7.5 to 12.5 +/- 2.3 mm Hg (P < 0.0001) and drug scores from 2.5 +/- 1.9 to 0.9 +/- 1.3 without postsurgical complications. Thus, the eyes treated with nonpenetrating trabeculectomy with sinusotomy and trabeculotomy had significantly lower intraocular pressures (P = 0.016) and drug scores than did those treated with nonpenetrating trabeculectomy with sinusotomy without trabeculotomy. CONCLUSION: The authors obtained satisfactory results in reducing intraocular pressure by the combination of nonpenetrating trabeculectomy, sinusotomy, and trabeculotomy.

Mammary ductoscopy for diagnosis and treatment of intraductal lesions of the breast.

BACKGROUND: Mammary ductoscopy (mammoscopy) is an ideal diagnostic method for intraductal lesions. The usefulness of mammoscopy for intraductal lesions was evaluated. METHODS: Mammoscopy was performed in 315 cases with nipple discharge. The mammoscopic findings of 46 breast cancer cases (47 lesions) and 109 intraductal papilloma cases (119 lesions) were compared with pathological findings. RESULTS: Carcinoma was recognized by mammoscopy in 38 of 47 lesions (80.9%). Intraductal masses were detected by mammoscopy in 115 of 119 intraductal papilloma lesions. The shape of the mass was classified as hemispheric, papillary, or flat protrusion. The hemispheric and papillary shapes were most common in cases of intraductal papilloma and the flat protrusion type was most common in cases of carcinoma. The amount of material collected by intraductal biopsy under mammoscopic observation was smaller in carcinoma than in intraductal papilloma because the carcinoma lesions were usually located in peripheral duct-lobular units and had weak tissue cohesion compared with that of intraductal papilloma. Of 133 intraductal biopsies performed for 69 intraductal papillomas, 17 biopsies yielded material insufficient for diagnosis in. The effectiveness of treatment by intraductal biopsy was recognized in 38 of 46 intraductal papillomas in which clinical follow-up continued for more than two years (82.6%). The therapeutic results of biopsy were poor in cases of multiple intraductal masses in multiple duct-lobular units. CONCLUSIONS: Mammoscopy contributes not only the diagnosis in cases of nipple discharge, but is also of benefit in the treatment of intraductal papilloma.

[Experience of gamma-detecting probe for the survey of sentinel node in gastroesophageal malignancies].

Gamma-detecting probe NAVIGATOR GPS (Autosuture Japan) was evaluated by using of 99mTc. Linearlity of counting in radioactivity was fairly good, but the sensitivity of the prove is so low, that sentinel node (SN) should contain the 3.7 x 10(-3) MBq (0.1 microCi) of 99mTc or more for the most effective use of it. And the count rate of the gamma-detecting probe was influenced by the distance and angle from the 99mTc source variously. It is important for the operator to know such characteristics of the gamma-detecting probe in order to obtain the correct SN judging.

Dye-staining stereomicroscopic examinations for fine mucosal structures of the gallbladder.

BACKGROUND: In order to diagnose an unsuspected gallbladder carcinoma and to examine whether a differential diagnosis could be made between cancer and noncancerous lesions during surgery, we evaluated the findings of fine structures of various types of gallbladder mucosa. METHODS: We used stereomicroscopy with a dye-contrast technique under water and measured the maximum blood vessel diameters of the gallbladder mucosa: normal gallbladder, chronic cholecystitis, and carcinoma. RESULTS: All normal gallbladders showed fine-reticular-type findings. In chronic cholecystitis, 5.8% of the specimens (n = 69) had fine reticular type, 87.0% had rough reticular type, and 7.2% had atrophic type. All the cases of adenomyomatosis (n = 16) showed rough reticular type. In eight specimens of pancreaticobiliary maljunction, 75% of them showed high reticular type, and the other 25% showed papillary type. The two adenoma specimens showed fine granular type. In five gallbladder carcinomas, the lattice-like pattern completely disappeared and showed rough granular type. The average of maximum vessel diameters in the gallbladder mucosa were 41.0 microm in normal gallbladders, 99.1 microm in patients with chronic cholecystitis, and 614.8 microm in patients with a carcinoma. There were significant differences among them (p < 0.05). CONCLUSION: This study showed that differential diagnosis between cancer and noncancerous lesion is possible by dye-staining mucosal pattern and measurement of maximum vessel diameters by stereoscopic examination.

OX40 stimulation by gp34/OX40 ligand enhances productive human immunodeficiency virus type 1 infection.

OX40 is a member of the tumor necrosis factor (TNF) receptor superfamily and known to be an important costimulatory molecule expressed on activated T cells. To investigate the role of costimulation of OX40 in human immunodeficiency virus type 1 (HIV-1) infection by its natural ligand, gp34, the OX40-transfected ACH-2 cell line, ACH-2/OX40, chronically infected with HIV-1, was cocultured with paraformaldehyde (PFA)-fixed gp34-transfected mouse cell line, SV-T2/gp34. The results showed that HIV-1 production was strongly induced. This was followed by apparent apoptosis, and both processes were specifically inhibited by the gp34-specific neutralizing monoclonal antibody 5A8. Endogenous TNF alpha (TNF-alpha) and TNF-beta production were not involved in the enhanced HIV-1 production. Furthermore, enhanced HIV-1 transcription in gp34-stimulated ACH-2/OX40 cells was dependent on the kappa B site of the HIV-1 long terminal repeat, and the OX40-gp34 interaction activated NF-kappa B consisting of p50 and p65 subunits. When primary activated CD4(+) T cells acutely infected with HIV-1(NL4-3) (CXCR4-using T-cell-line-tropic) were cocultured with PFA-fixed gp34(+) human T-cell leukemia virus type 1-bearing MT-2 cells or SV-T2/gp34 cells, HIV-1 production was also markedly enhanced. The enhancement was again significantly inhibited by 5A8. The present study first shows that OX40-gp34 interaction stimulates HIV-1 expression and suggests that OX40 triggering by gp34 may play an important role in enhancing HIV-1 production in both acutely and latently infected CD4(+) T cells in vivo.

[A case of advanced gastric cancer treated with neoadjuvant chemotherapy of low-dose CPT-11 + CDDP].

Side effects due to administration of anti-cancer drugs often cause the treatment to be abandoned or a decrease in the amount of anti-cancer drugs. Recently, the anti-tumor effects of "low-dose CPT-11", which can be administered at the outpatient clinic, are reported. We performed "low-dose CPT-11 + CDDP" as a neoadjuvant chemotherapy to a patient with advanced gastric cancer. CPT-11 and CDDP combination chemotherapy caused very few side effects, so we could continue the treatment and achieve anti-tumor effects. Consequently, surgery could be performed, but disseminated metastasis was found so that the surgery ended as a non-curative operation. However, it was considered that this method of "low-dose CPT-11 + CDDP" was very effective as the neoadjuvant chemotherapy in a patient with advanced gastric cancer.

Two basic regions of NCp7 are sufficient for conformational conversion of HIV-1 dimerization initiation site from kissing-loop dimer to extended-duplex dimer.

Nucleocapsid (NC) protein possesses nucleotide-annealing activities, which are used in various processes in retroviral life cycle. As conserved characters, the NC proteins have one or two zinc fingers of CX(2)CX(4)HX(4)C motif surrounded by basic amino acid sequences. Requirement of the zinc fingers for the annealing activities of NC protein remains controversial. In this study, we focused the requirement in the process of maturation of dimeric viral RNA. Discrimination between immature and mature dimers of synthetic RNA corresponding to the dimerization initiation site of human immunodeficiency virus type 1 (HIV-1) genomic RNA was performed based on their Mg(2+)-dependent stability in gel electrophoreses and on their distinct signal pattern from NMR analysis of imino protons. Chaperoning activity of the HIV-1 NC protein, NCp7, and its fragments for maturation of dimeric RNA was investigated using these experimental systems. We found that the two basic regions flanking the N-terminal zinc finger of NCp7, which are connected by two glycine residues instead of the zinc finger, were sufficient, although about 10 times the amounts of peptide were needed in comparison with intact NCp7. Further, it was found that the amount of basic residues rather than the amino acid sequence itself is important for the activity. The zinc fingers may involve the binding affinity and/or such a possible specific binding of NCp7 to dimerization initiation site dimer that leads to the maturation reaction.