High-speed video analysis of acoustically oscillated guinea pig stapes.

OBJECTIVE: We investigated the ossicular movement in the near-intact middle ear in response to acoustic stimulation using a high-speed video camera and video analysis software program. DESIGN: We have designed a good visual access to the middle ear of the guinea pig by opening the ventral wall of the otic capsule, without injuring the sound-conducting structures, from the external auditory canal to the oval window. The high-speed video camera could record analysable ossicular motion up to 4000 frames per second. RESULTS: The stapes showed reciprocal movement in the same frequency as the stimulating tone, and with an amplitude proportional to the stimulating sound intensity. Injury to the tympanic membrane attenuated the stapedial motion, which was recovered to that of the control level by patch repair of the perforation. CONCLUSION: Our experimental set-up was capable of evaluating the conductive hearing, regardless of the status of the animal's sensorineural hearing or even life. Such a video analysis may provide a powerful tool to investigate the physiology of the middle ear.

[Preoperative laparoscopy by local anesthesia for advanced gastric cancer].

Preoperative laparoscopy by local anesthesia was performed in 8 patients with advanced gastric cancer, whose lesions had been diagnosed to be more than T3 or suspected to have peritoneal seeding, and its usefulness was assessed. After insertion of the trocars, the abdominal cavity was inspected, and biopsy and/or abdominal lavage sampling was performed. Three patients out of 8 were diagnosed as P3, and 5 patients were diagnosed as P0 and CY0. Based on these results, 6 patients underwent operation. The accuracy rate of diagnosis was 83% in P category, and 100% in CY category. In conclusion, it is considered that laparoscopy by local anesthesia is a useful preoperative examination for advanced gastric cancer.

A retinoic acid-inducible modular protease in budding ascidians.

Retinoic acid-treated mesenchyme cells of the budding ascidian Polyandrocarpa misakiensis acquire an organizer activity to induce a secondary body axis when implanted into developing buds. We identified several different mRNAs that were upregulated in the mesenchyme cells after retinoic acid treatment. We isolated a cDNA clone corresponding to one of these mRNAs. The C-terminal region of the predicted protein product is homologous to the catalytic domain of serine proteases that belong to the trypsin family. The N-terminal region contains several types of protein-protein interaction domains. We therefore named this protein tunicate retinoic acid-inducible modular protease (TRAMP). Expression of the TRAMP mRNA in mesenchyme cells during budding and its upregulation by retinoic acid were demonstrated by reverse transcription-PCR and in situ hybridization. A glutathione S-transferase-TRAMP fusion protein showed a protease activity with trypsin-like substrate specificity and stimulated proliferation of the cell line established in this species.

[The use of predeposited autologous blood transfusion for radical prostatectomy and total cystectomy].

BACKGROUND: To avoid homologus blood transfusion, we performed the operation for intra-pelvic malignancy with predeposited autologus blood using recombinant human erythropoietin (rH-EPO). MATERIALS AND METHODS: The seven cases of radical prostatectomy and the 15 cases of total cystectomy were analyzed retrospectively. All cases were given ferrons sulfate/200 mg of iron orally every day to the day before the operation and treated with 24,000 unit of rH-EPO subcutaneously every week. The target volumes of preoperative autologus blood collection were 800 to 1,000 ml and 800 to 1,200 ml for radical prostatectomy and total cystectomy, respectively. For each case, 400 ml blood was collected once a week. RESULTS: In cases of radical prostatectomy, the preserved blood volume was 885.7 +/- 157.4 ml and 6 out of 7 operations were successfully performed without additional homologus blood transfusion (85.7%). In total cystectomy, the preserved blood volume was 1,033.3 +/- 167.6 ml and 14 out of 15 operations were successfully performed without additional homologus blood transfusion (93.3%). The bleeding volume during operation showed no significant difference compared to control group where used homologus blood transfusion. Postoperative courses were uneventful and there encountered no severe side effects and complications in all our procedures. CONCLUSIONS: Our study indicates that in cases of radical prostatectomy and total cystectomy, the operation with predeposited autologus blood using rH-EPO is possible to be performed in safe. The effect on long term prognosis of malignancy is not clear, however, this technique is helpful able to avoid hazardous issues related to homologus blood transfusion during the operation.

Expression and function of a retinoic acid receptor in budding ascidians.

Retinoic acid is thought to induce transdifferentiation of multipotent epithelial stem cells in the developing buds of the ascidian Polyandrocarpa misakiensis. We isolated a cDNA clone from this species, named PmRAR, encoding a retinoic acid receptor (RAR) homologue. PmRAR clusters with other RARs on phylogenetic trees constructed by three different methods. Within the cluster, PmRAR is on a separate branch from all the subtypes of RARs, suggesting that RAR subtypes arose in the ancestral vertebrates after divergence of vertebrates and urochordates. The embryos of another ascidian species Ciona intestinalis were co-electroporated with a mixture of a PmRAR expression vector and a lacZ reporter plasmid containing vertebrate-type retinoic acid response elements. The expression of lacZ depended on the presence of both retinoic acid and PmRAR, suggesting that PmRAR is a functional receptor. PmRAR mRNA is expressed in the epidermis and mesenchyme cells of the Polyandrocarpa developing bud. The mRNA is not detectable in the mesenchyme cells in the adult body wall, but its expression can be induced by retinoic acid in vitro. These results suggest that the PmRAR is a mediator of retinoic acid signalling in transdifferentiation during asexual reproduction of protochordates.

Effects of tibolone (Org OD14) treatment for 3 months on ovariectomy-induced osteopenia in 8-month-old rats on a low-calcium diet: preventive testing for 3 months.

Tibolone (Org OD14), (7alpha, 17alpha)-17-hydroxy-7-methyl-19-norpregn-5(10)-en-20-yn++ +-3-one, is a synthetic steroid with weak estrogenic, progestational, and androgenic properties. We investigated the prophylactic effects of tibolone on bone loss, bone strength, and plasma and urinary parameters in 8-month-old ovariectomized rats on a low-Ca diet. Oral administration of tibolone (0.03-3 mg/kg/day) was started immediately after ovariectomy (ovx) and continued for 3 months. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry. Oral administration of tibolone (1 or 3 mg/kg/day) significantly prevented a decrease in BMD and bone ash density (bone ash weight/volume) of the global femur, and BMDs in the femoral distal and proximal regions. Also in the lumbar vertebrae, the ovx-induced reduction in BMD was prevented by tibolone (1 and 3 mg/kg/ day) treatment, resulting in a significantly higher lumbar vertebral (L-2) bone compression strength compared to the ovx control group. Neither ovx alone nor supplemented with tibolone affected the BMD or bending strength of the femoral mid-diaphysial region. Tibolone (0.03-3 mg/kg/day) significantly reduced the ovx-induced increases in serum osteocalcin level. Furthermore, tibolone inhibited an increase in the urinary hydroxyproline/creatinine, pyridinoline/creatinine, and deoxypyridinoline/creatinine ratios induced by ovx. Tibolone also reduced body weight gain and serum cholesterol level, as has been reported for estrogen. These findings indicate that tibolone prevents reduction in bone mass associated with osteopenia by reducing increased trabecular bone resorption induced by a combination of ovx and a low-Ca diet.

Expression cloning and intracellular localization of a human ZF5 homologue.

We isolated a cDNA encoding a human homologue of ZF5 (hZF5), which has five Kruppel-like C2H2 type zinc fingers at carboxyl terminus and the BTB/POZ (poxvirus and zinc finger) at the amino terminus, using autoimmune sera from a patient with overlap syndrome (dermatomyositis and scleroderma). Sequencing of the entire cDNA revealed an open reading frame (ORF) of 1349 bp with a deduced protein sequence of 449 amino acid residues and a calculated molecular weight of 51.3 kDa. The deduced amino acid sequence of hZF5 is highly homologous to mouse ZF5 (99.3% identity). Immunofluorescence studies revealed that HA-tagged hZF5 transiently expressed in COS-7 cells showed the nuclear dot pattern in the BTB/POZ domain-dependent manner.

Enhancement of interleukin-1 alpha mediated autocrine growth of cultured human keratinocytes by sho-saiko-to.

We investigated the effects of Sho-saiko-to, the most commonly used herbal medicine in Japan, on the production of interleukin (IL)-1 alpha by cultured human epidermal keratinocytes. IL-1 alpha production was significantly promoted by treatment with 100 or 500 micrograms/ml Sho-saiko-to for 24 or 48 hr. Expression of IL-1 alpha receptors was the most markedly upregulated after treatment with 500 micrograms/ml Sho-saiko-to for 24 hr and with 100 or 500 micrograms/ml for 48 hr; these cells showed the characteristics of multilayered differentiated keratinocytes. The presence of an anti-IL-1 alpha antibody during the treatment with 500 micrograms/ml of Sho-saiko-to for 24 or 48 hr or with 100 micrograms/ml for 48 hr significantly down-regulated the synthesis by the keratinocytes and induced damages in them. Keratinocytes treated with Sho-saiko-to might produce IL-1 alpha and express IL-1 alpha receptors. IL-1 alpha may regulate the proliferation and differentiation of keratinocytes after Sho-saiko-to treatment. These findings suggest that Sho-saiko-to enhances the autocrine growth mediated by IL-1 alpha.

Vibration-induced finger flexion reflex and inhibitory effect of acupuncture on this reflex in cervical spinal cord injury patients.

The vibration-induced finger flexion reflex (VFR) and the inhibitory effect of acupuncture on this reflex were studied in five cervical spinal cord injury patients (C-SCIs). VFR, which is a tonic finger flexion reflex induced by vibratory stimulation on the finger tip, was induced before and after acupuncture was carried out on the same hand. A stainless steel needle was inserted to the Hoku point. As in healthy subjects, VFR was performed and it was significantly inhibited by acupuncture in the C-SCIs; mean maximum VFR was 204.2 +/- S.E. 68.6 g before and 119.8 +/- S.E. 42.2 g after acupuncture. The present results suggest that at least part of the reflex center for VFR is located in the spinal cord and that part of VFR inhibition by acupuncture may be mediated via the spinal cord.

Chemical nature of the light emitter of the Aequorea green fluorescent protein.

The jellyfish Aequorea victoria possesses in the margin of its umbrella a green fluorescent protein (GFP, 27 kDa) that serves as the ultimate light emitter in the bioluminescence reaction of the animal. The protein is made up of 238 amino acid residues in a single polypeptide chain and produces a greenish fluorescence (lambda max = 508 nm) when irradiated with long ultraviolet light. The fluorescence is due to the presence of a chromophore consisting of an imidazolone ring, formed by a post-translational modification of the tripeptide -Ser65-Tyr66-Gly67-. GFP has been used extensively as a reporter protein for monitoring gene expression in eukaryotic and prokaryotic cells, but relatively little is known about the chemical mechanism by which fluorescence is produced. To obtain a better understanding of this problem, we studied a peptide fragment of GFP bearing the chromophore and a synthetic model compound of the chromophore. The results indicate that the GFP chromophore consists of an imidazolone ring structure and that the light emitter is the singlet excited state of the phenolate anion of the chromophore. Further, the light emission is highly dependent on the microenvironment around the chromophore and that inhibition of isomerization of the exo-methylene double bond of the chromophore accounts for its efficient light emission.

Effects of guizhi-fuling-wan on male infertility with varicocele.

Thirty-seven infertile patients with varicocele were treated with Guizhi-Fuling-Wan (7.5 g/day) for at least 3 months. Before and after the administration, semen qualities such as sperm concentration and motility were examined, and the varicocele was graded. A varicocele disappearance rate of 80% was obtained with 40 out of 50 varicocele, and improvement of sperm concentration and motility were found in 71.4% and 62.1% of patients, respectively. From these results, Guizhi-Fuling-Wan is considered to be effective for circulation disorders in varicocele as well as semen quality.

alpha-Tocopherol protects against radical-induced injury in cultured neurons.

The effect of alpha-tocopherol on neurons in protecting against radical-induced injuries caused by cumene hydroperoxide was assessed using a culture system. Structural and biochemical deteriorations in both clonal neuroblastoma cells and primary cultured neurons were induced by cumene hydroperoxide. These deteriorative changes were prevented prevented by pretreatment of alpha-tocopherol. Since simultaneous supplementation of alpha-tocopherol and cumene hydroperoxide had limited protective effects, prophylactic administration of alpha-tocopherol for risk patients with brain diseases concerned with damages induced by free radical is recommended to hamper the progression of neuronal impairment after attacks.

Mass spectrometric evidence for a disulfide bond in aequorin regeneration.

Tryptic digests of purified recombinant apoaequorin were analyzed, before and after reduction with DTT, by fast atom bombardment mass spectrometry. The results showed that apoaequorin contains a disulfide bond between Cys145 and Cys152 and that the reduction of this bond is involved in the regeneration of aequorin.

Effect of ibudilast, a novel antiasthmatic agent, on anaphylactic bronchoconstriction: predominant involvement of endogenous slow reacting substance of anaphylaxis.

The effect of ibudilast on anaphylactic bronchoconstriction was studied in guinea pigs sensitized actively with ovalbumin (OA). Animals were treated with indomethacin, tripelennamine and propranolol prior to the antigen challenge. Anaphylactic bronchoconstriction was prevented by ibudilast (1-4 mg/kg i.v. and 5-20 mg/kg p.o.) dose-dependently. FPL55712 and phenidone were also effective. Even when administered at the maximum development of bronchoconstriction, ibudilast (0.5 and 2 mg/kg i.v.) and FPL 55712 caused significant reduction of the increased airway tone, while phenidone did not. Ibudilast (1-4 mg/kg i.v.) and FPL55712 inhibited leukotriene D4-induced airway responses in nonsensitized guinea pigs pretreated with indomethacin and propranolol. Ibudilast (1.6 and 4 mg/kg i.v.) inhibited platelet-activating-factor (PAF)-induced airway responses in nonsensitized guinea pigs pretreated with indomethacin and propranolol, however, FPL 55712 inhibited PAF-induced airway responses only at a high dose such as 10 mg/kg i.v. Ibudilast (4 mg/kg i.v.) did not inhibit acetylcholine-induced airway response. Ibudilast showed inhibition of the release of slow-reacting substance of anaphylaxis (SRS-A) from guinea pig chopped lung sensitized with OA, which was significantly diminished by indomethacin. The drug little affected the activity of phospholipase A2 and 5-lipoxygenase in guinea pig polymorphonuclear leukocytes. These results indicate that ibudilast inhibits anaphylactic bronchoconstriction which is considered to be largely mediated by endogenously released SRS-A. The inhibitory effect of ibudilast on anaphylactic bronchoconstriction in the presence of indomethacin is considered to be exerted through its antagonism to SRS-A.

[Neoplastic angioendotheliosis: a case of T cell lymphoma positive for S-100 protein].

A 63-year-old woman presented with painless, firm, subcutaneous nodules on her legs and trunk. The lesions were annular or serpiginous and their surface was livid-red to pale-red. Superficial lymph nodes were not palpable. Upper gastrointestinal series, barium enema, Ga scintigraphy, and CT findings were negative. Histological examination revealed deep dermal vessels occupied by neoplastic cells and fibrin thrombi. We diagnosed her as having neoplastic angioendotheliosis. Electron microscopically, tumor cells lacked Weibel-Palade bodies. Immunohistochemically, the tumor cells were positive for leukocyte common antigen, T11, MT-1, HLA-DR and S-100 protein. They were negative for Factor VIII-related antigen, epithelial membrane antigen, carcinoembryonic antigen, B-1, and B-2. Immunoelectron microscopic study showed that their nuclei, cytoplasma, and cell membranes were positive for S-100 protein. Based on these findings, we diagnosed her as having T cell lymphoma. The patient rejected chemotherapy and was discharged. Three months after discharge, right hemiplegia developed. Brain CT scan revealed enhanced masses in the left frontal, temporal, and occipital lobes. Craniotomy showed only gliosis and hemorrhage. She died of cerebral bleeding three months after craniotomy.