Restoration by VIP of the carbachol-stimulated Cl- secretion in TTX-treated guinea pig distal colon.

To determine if vasoactive intestinal peptide (VIP) restores neural activity from tetrodotoxin (TTX) blockade, we studied the effects of VIP and related agents on carbachol (Cch)-induced Cl(-) secretion in control-isolated guinea pig distal colon and in that treated with TTX. The short circuit current (I(sc)) increased dose-dependently after serosal applications of Cch (10(-6) - 2 x 10(-5) M) and VIP (5 x 10(-9) - 10(-7) M). But no additive or synergistic increase in I(sc) was observed. Cch- and VIP-induced I(sc) was completely abolished by a serosal application of TTX (10(-6) M). However, a serosal application, not mucosal, of VIP (10(-7) M) and 8-bromo-cAMP (10(-3) M) restored the Cch-stimulated, TTX-inhibited I(sc) by 113% and 75.8%, respectively. Furthermore, mucosal and serosal applications of forskolin (aden late cyclase activator) restored the I(sc) by 43.9% and 65.3%, respectively. The restored I(sc) was completely abolished by atropine (muscarinic receptor antagonist). These results suggest that VIP may restore the cholinergic activity by increasing the level of intracellular cAMP, and that cholinergic neuron is very likely to be responsible for the regulation of Cl(-) secretion at neuroepithelial junctions. The exact mechanism of VIP's effect on the TTX-inhibited epithelial Cl(-) secretion, and its possible usefulness in the treatment of TTX-induced pathophysiological conditions, remain to be determined.

Combination of tumor necrosis factor alpha and interferon alpha induces apoptotic cell death through a c-myc-dependent pathway in p53 mutant H226br non-small-cell lung cancer cell line.

We investigated the role of wild-type p53 and c-myc activity in apoptosis induced by a combination of natural human tumor necrosis factor alpha (TNF-alpha) and natural human interferon alpha (IFN-alpha). Studies were performed with two human non-small-cell lung cancer cell lines, H226b, which has wild-type p53, and H226br, which has a mutant p53. The combination of IFN-alpha and TNF-alpha significantly inhibited cell growth and induced apoptotic cell death of both H226b and H226br, compared with IFN-alpha or TNF-alpha alone. Treatment with one or both cytokines did not affect the expression level of p53 in both cell lines. These results suggest that the combination of IFN-alpha/TNF-alpha induces apoptotic cell death through a p53- independent pathway. The c-myc oncogene is known to be involved in apoptosis induced by TNF. Antisense c-myc oligonucleotides have been reported to modulate cell growth or apoptosis in several cell lines. Antisense oligodeoxynucleotides were added to the culture of H226br cells before the addition of IFN-alpha/TNF-alpha. Antisense c-myc inhibited IFN-alpha/TNF-alpha cytotoxicity and apoptotic cell death. In conclusion, this study provides support for the speculation that TNF-alpha/IFN-alpha induce apoptosis through a c-myc-dependent pathway rather than a p53-dependent pathway. (c)2001 Elsevier Science.

New approach to cell lineage analysis in mammals using the Cre-loxP system.

The Cre-loxP site-specific recombination system was used for cell lineage analysis in mammals. We constructed an expression plasmid, pCETZ-17, which consists of cytomegalovirus enhancer/chicken beta-actin promoter (CAG), a portion of the rabbit beta-globin gene, loxP-flanked DNA sequence (containing enhanced green fluorescent protein (EGFP) cDNA), and lacZ gene encoding E. coli beta-galactosidase (beta-gal). When circular pCETZ-17 plasmid DNA was microinjected into the pronuclei of fertilized eggs and these eggs were allowed to develop to two-cell stage, 62.8% (59/94) of the two-cell embryos exhibited distinct fluorescence in one or both blastomeres, but never expressed lacZ protein, as evaluated by histochemical staining with X-Gal, a substrate for beta-gal. When both circular plasmids, pCETZ-17 and pCAG/NCre (containing CAG and DNA sequences encoding nuclear location signal and Cre), were co-injected into fertilized eggs, almost all (87.0%, 47/54) embryos exhibited low or no fluorescence, but 51.9% (27/52) exhibited positive staining for beta-gal activity. This indicates that transient expression of the Cre recombinase gene removed the loxP-flanked DNA sequence in pCETZ-17 and then caused expression of the downstream lacZ sequence. We next microinjected pCETZ-17 into the pronuclei of fertilized eggs, cultured these injected eggs for 1 day, and collected only two-cell embryos expressing EGFP in both blastomeres. One blastomere of the EGFP-expressing two-cell embryos was microinjected with pCAG/NCre, and these treated embryos were cultured for 1 day up to four-cell stage. When the developing four-cell embryos were subjected to staining with X-Gal, cell lineage-related staining pattern for beta-gal activity was observed in most (77.8%, 7/9) embryos. These findings were further confirmed using two-cell embryos derived from a transgenic mouse line carrying CETZ-17 transgene. Thus, our system, which is based on transient expression of the Cre recombinase gene directly introduced into nuclei of embryonic cells by microinjection, is a powerful means for cell lineage analysis in mammals.

In vivo influence of p53 status on proliferation and chemoradiosensitivity in non-small-cell lung cancer.

Alteration of the p53 gene product is a frequent event in the progression of lung cancer. However, its importance to proliferation and response to chemoradiotherapy remains unclear. Thus, to assess its influence directly in vivo, we implanted into nude mice two kinds of human non-small-cell lung cancer (NSCLC) cells: H226br having a homozygous gene mutation in p53 (mt-p53) and H226b with intact p53 (wt-p53). We found that mt-p53 tumors grew substantially faster than wt-p53 tumors. Furthermore, treatment with cisplatin and radiation did not reduce the size of mt-p53 tumors, while wt-p53 tumors regressed by approximately 60%. Terminal-deoxytransferase-mediated dUTP-biotin nick-end labeling assay revealed apoptosis to be the mechanism responsible for the regression. Interestingly, apoptosis occurred in mt-p53 tumors although only at high doses of cisplatin and not at the magnitude detected in wt-p53 tumors. Cell labeling by staining with bromodeoxiuridine indicated that p53 is an important factor in modulating growth in NSCLC tumors. Our results are consistent with the notion that correction of a single genetic lesion enhances the therapeutic effect of chemotherapy.

Apoptosis in cultured human colon cancer cells induced by combined treatments with 5-fluorouracil, tumor necrosis factor-alpha and interferon-alpha.

Biochemical analysis using nick end-labeling was performed to investigate the effect of various combinations of 5-fluorouracil, natural human tumor necrosis factor-alpha and natural human interferon-alpha on the induction of apoptosis in RPMI 4788 human colon cancer cells. After treatment with 5-fluorouracil (1 mM) for 48 h, the number of nick end-positive cells was significantly increased in comparison to the situation without treatment. When tumor cells were treated with 1 mM 5-fluorouracil, 2.86 Japan Reference Units (JRU)/ml natural human tumor necrosis factor-alpha and 1 x 10(3) IU/ml natural human interferon-alpha in combination for 48 h, the number of nick end-positive cells was significantly higher than that after treatment with 5-fluorouracil alone. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay revealed a significant decrease of relative viability, as compared to treatment with 5-fluorouracil (1 mM), 5-fluorouracil + natural human tumor necrosis factor-alpha, or 5-fluorouracil + natural human interferon-alpha for 48 h. Pretreatment with 5-fluorouracil (1 mM) for 24 h prior to treatment with natural human tumor necrosis factor-alpha (2.86 JRU/ml) and natural human interferon-alpha (10(3) IU/ml) for 24 h resulted in a significant increase of nick end-positive cells compared to pretreatment with natural human tumor necrosis factor-alpha and natural human interferon-alpha prior to treatment with 5-fluorouracil for 24 h (p < 0.05). These results suggest that 5-fluorouracil alone can induce apoptosis in RPMI 4788 tumor cells and that this effect can be enhanced by combination with natural human tumor necrosis factor-alpha and natural human interferon-alpha.

[Four cases of carotid body tumor--especially the usefulness of CT and MRI in preoperative diagnosis].

Four cases of carotid body tumor are reported. Surgical removal of the tumor was performed in all cases. The internal carotid artery (ICA) was preserved in only two cases. In the other two cases ICA was unavoidably replaced by an EPTFE (expanded polytetrafluoroethylene) graft. In addition to the size and mobility in the anterior and posterior direction of the tumor, we concluded that CT and MRI were useful in decision of the surgical method. In case the ICA cannot be distinguished from the tumor at the bifurcation of the carotid artery on enhanced CT, it should be considered to be difficult to remove the tumor. MRI is suitable for discerning the inner surface of the carotid artery, but it is significant that MRI cannot necessarily reveal tumor invasion of the carotid artery wall.

Effect of thyroid-stimulating hormone on cultured thyrocytes obtained from patients with Graves' disease and inhibitive effect by sodium iodide: a functional study.

Thyrocytes obtained from patients with Graves' disease were cultured for 3 days. This was followed by culture with 10 mU/mL thyroid stimulating hormone (TSH) (TSH group), TSH and sodium iodide (Nal group), or without (control group) for 3 additional days. On the 8th culture day, the amounts of intra- and extra-cellular cyclic adenosine monophosphate (cAMP), extracellular cAMP and thyroglobulin (TG), peroxidase (PO) activity, and cell numbers were measured. The amounts of intra- and extra-cellular cAMP correlated well. TSH increased the values of cAMP, TG and PO to levels higher than those of the control group. As the amount of Nal added to the medium increased, these values decreased. Addition of 10(-5) mol/L Nal lowered the value of cAMP only. When 10(-4) mol/L Nal was added, these three levels were lower than those of the TSH group and the value of cAMP was almost equal to that of the control group. On cell number, no difference was found between the cells cultured with TSH, TSH and Nal, and without TSH or Nal. When the thyrocytes were cultured with 1 mmol/L dibutyryl cAMP sodium salt or 8-bromoadenosine 3',5'-cyclic monophosphate instead of TSH, 10(-4) mol/L Nal did not lower the values of thyroglobulin and peroxidase activity. These results suggest that the Nal blocks the intracellular signal transduction provoked by TSH, only at the cAMP production level.

Two cases of nasopharyngeal branchial cyst.

Two cases of nasopharyngeal cyst, in a two-year-old girl and a six-year-old boy, are described. The cysts were located in the right lateral wall of the nasopharynx in both cases. Histopathological examinations revealed that the cyst walls were lined with columnar epithelium. The positions of the cysts and pathological features indicated that they were of branchial origin, and they were assumed to originate in the second branchial pouch because of their anatomic location. They differed from previously reported cases in that they extended nearly to the base of the skull, occupying the parapharyngeal space. It was considered that they might have originated from the dorsal part of the second branchial pouch or the layer of endodermal cells cut off from the lower part of the eustachian tube.

[Statistical observations of clinical cases with frontal, ethmoid and sphenoid sinus mucoceles].

Ninety six patients with paranasal mucoceles (or pyoceles) were admitted and treated at Gunma University Hospital from 1977 to 1986 (age range 15-82 years; mean 46.4 years). We statistically investigated these cases paying a special attention to 25 patients with visual disturbance. 1) In 71 cases mucocele was located in the frontal or fronto-ethmoid sinus (FE group), in 12 it was found in the ethmoid sinus (E group), and in 13 it was situated in the sphenoid or spheno-ethmoid sinus (SE group). 2) Sixty three cases had histories of previous surgery of paranasal sinuses and 29 cases were considered to have primary mucoceles. 3) The mean age of patients with primary mucocele was 56.4, while that with postoperative mucocele was 41.2. 4) In the FE group recurrence was so frequent that some device for surgical treatment should be considered (i.e., mucous membrane grafting, prolonged insertion of silicon tubes). 5) Visual disturbance was frequently observed in both the E group (7/12) and the SE group (9/13). 6) Visual disturbance may occur when mucocele compress the optic nerve and the globe at retrobulbar portion as well as at the optic canal. Twelve cases out of 25 with visual disturbance belonged in the former type. 7) Optic atrophy was most commonly seen in the SE group, but was recognized in 1 case even in the E group and the FE group respectively. 8) In patients with sudden visual disturbance, if only they were operated within a week after the onset, their visual acuity was remarkably improved except for serious cases. 9) In the cases presented with gradually attacked or varying visual disturbance, there was no correlation between the recovery of the vision and the duration of the disturbance.