Two cases of BRAF-mutated, bulbar conjunctival melanoma, and review of the published literature.

We describe two patients with BRAF-mutated melanoma of the epithelioid cell type arising from primary acquired melanosis with severe atypia of the right bulbar conjunctiva. Patient 1 was a 71-year-old Japanese man. After adjuvant cryotherapy and enucleation of the right eyeball, therapy with vemurafenib was administered for a distant metastasis to a lumbar vertebra, accompanied by erythema multiforme and two keratinous tumours. The patient died due to metastases to the liver and multiple vertebrae, despite therapy with nivolumab and combination therapy with dabrafenib plus trametinib. Patient 2 was a 72-year-old Japanese man. After adjuvant cryotherapy, periodic mitomycin C eye drops, and excision of the superficial portion of the right parotid gland and the dissection of cervical lymph nodes, he was treated with adjuvant combination therapy with dabrafenib plus trametinib. Dermatologists should be familiar with BRAF-mutated conjunctival melanoma, which is usually located on the bulbar conjunctiva and associated with more frequent distant metastasis.

Indications for partial parotidectomy using retrograde dissection of the marginal mandibular branch of the facial nerve for benign tumours of the parotid gland.

The aims of this study were to evaluate the efficacy of partial parotidectomy using retrograde dissection of the marginal mandibular branch of the facial nerve for benign tumours of the parotid gland and to establish the indications for its use. We examined 106 consecutive patients with previously untreated benign tumours in the lower portion of the parotid gland who were treated by parotidectomy. The first group (anterograde group, n=52) consisted of those who had standard anterograde parotidectomy. The remaining patients, who underwent retrograde parotidectomy, were further divided into two groups: those in whom the upper edge of the tumour was located below the mastoid tip (below mastoid group, n=46) or those in whom it was above the mastoid tip (above mastoid group, n=8). The operating time was significantly shorter in the below mastoid group (141.2, 127.5, and 98.1minutes, respectively) as was intraoperative blood loss (41.1, 53.0, and 24.4ml, respectively), compared with the other two groups. There was a higher incidence of facial nerve dysfunction in the above mastoid group postoperatively (4/8) than in the other two groups. The results suggested that the presence of a tumour of any size located below the mastoid tip is a good indication for parotidectomy using retrograde dissection of the marginal mandibular branch of the facial nerve.

Isolation of equine herpesvirus-1 lacking glycoprotein C from a dead neonatal foal in Japan.

We isolated a variant equine herpesvirus-1 (EHV-1), strain 5089, from the lung of a dead neonatal foal in Japan and characterized the biological nature of the virus. The virus spread in cultured cells mainly by cell-to-cell infection, unlike wild-type EHV-1, which spreads efficiently as a cell-free virus. The virus titer in cultured supernatant and the intracellular virus titer were low compared to those of wild-type EHV-1. Heparin treatment of the virus had no effect on viral infectivity in cell culture. Glycoprotein C (gC) was not detected by Western blotting and fluorescent antibody tests in 5089 virions and 5089-infected cells, respectively. RT-PCR analysis revealed that the expression level of 5089 gC mRNA was reduced considerably compared to that of wild-type EHV-1. Sequencing analysis of the 5089 gC coding region showed a point mutation in the promoter region of the gC open reading frame. However, the mutation did not affect the promoter activity. These results suggested that the lack of gC in 5089 virions might be one of the reasons for spread of the virus by cell-to-cell infection and that gC mRNA expression might not be activated efficiently due to factors other than the mutation in the gC promoter region.

Absence of arabinan in the side chains of the pectic polysaccharides strongly associated with cell walls of Nicotiana plumbaginifolia non-organogenic callus with loosely attached constituent cells.

When leaf disks from haploid plants of Nicotiana plumbaginifolia Viv. were transformed with T-DNA and cultured on shoot-inducing medium, nonorganogenic callus. designated nolac (for non-organogenic callus with loosely attached cells), appeared on approximately 7% of leaf disks. In contrast, normal callus was generated on T-DNA-transformed leaf disks from diploid plants and on non-transformed leaf disks from haploid and diploid plants. Transmission electron microscopy revealed that the middle lamellae and the cell walls of one line of mutant callus (nolac-H14) were barely stained by ruthenium red. even after demethylesterification with NaOH, whereas the entire cell wall and the middle lamella were strongly stained in normal callus. In cultures of nolac-H14 callus, the level of sugar components of pectic polysaccharides in the hemicellulose fraction was reduced and that in the culture medium was elevated, as compared with cultures of normal callus. These results indicate that pectic polysaccharides are not retained in the cell walls and middle lamellae of nolac-H14 callus. In nolac-H14, the ratio of arabinose to galactose was low in the pectic polysaccharides purified from all cell wall fractions and from the medium, in particular, in the hemicellulose fractions. The low levels of arabinofuranosyl (T-Araf, 5-Araf, 2,5-Araf, and 3,5-Araf) residues in the pectic polysaccharides of the hemicellulosic fraction of nolac-H,14 indicated that no neutral-sugar side chains, composed mainly of linear arabinan. were present in nolac-H14. Arabinose-rich pectins. which are strongly associated with cellulose-hemicellulose complexes, might play an important role in intercellular attachment in the architecture of the cell wall.

Interferon-gamma and tumor necrosis factor-alpha levels in sera and whey of cattle with naturally occurring coliform mastitis.

Concentrations of interferon-gamma (lFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) were determined in serum and whey samples from cattle with naturally occurring coliform mastitis for two weeks after onset using bovine INF-gamma and TNF-alpha-specific ELISA. In serum and whey samples from healthy cows. IFN-gamma was almost undetectable and TNF-alpha was detected at low levels. At the onset of illness, INF-gamma in sera and whey and TNF-alpha in whey from the mastitic cows were significantly higher than their respective values in healthy cows. Concentrations of IFN-gamma and TNF-alpha in whey from mastitic cattle decreased significantly as the cows recovered.

New method for thyroid transplantation across major histocompatibility complex barriers using allogeneic bone marrow transplantation.

BACKGROUND: It has been shown that allogeneic bone marrow transplantation (BMT) after lethal irradiation elicits donor-specific tolerance for organ or tissue transplantation across major histocompatibility complex (MHC) barriers. Recently, we have demonstrated that the portal venous (p.v.) administration of donor bone marrow cells (BMCs) elicits donor-specific tolerance across MHC barriers by only two administrations of an immunosuppressant (CsA or FK-506). In our study, using the central and intrahepatic tolerance-inducing system, we have established a new method for thyroid transplantation with BMT that would be more applicable to humans. METHODS: In addition to sublethal (6-5 Gy) irradiation, recipient B6 (H-2b) mice received injections i.p. with the myeloablative drug busulfan (BU) on day -2 to provide a sufficient "space" for the donor hematopoietic cells to expand in the recipients. To induce the intrahepatic tolerance, donor BALB/c (H-2d) BMCs were treated with neuraminidase (Neu), which enhances the trapping of i.v. injected BMCs in the liver. After the injection of Neu-treated BMCs, the thyroid organs from the BALB/c mice were engrafted under the renal capsules. RESULTS: A 90% graft survival rate was obtained over 100 days by a combination of BU administration, 6 Gy irradiation, and i.v. injection of Neu-treated BMCs [BU+6 Gy+(Neu) i.v.], and a 70% graft survival rate was obtained by [BU+5 Gy+(Neu) i.v.]. However, the graft survival rate significantly decreased when either the BU or Neu treatment was omitted. T cells collected from the tolerant recipients suppressed the proliferative responses to donor alloantigens. CONCLUSIONS: Using both BU and Neu treatments, we have succeeded in inducing long-term tolerance and preventing the rejection of thyroid allografts by the single-day protocol.

Prevention of accelerated presbycusis by bone marrow transplantation in senescence-accelerated mice.

A substrain of the senescence-accelerated mouse (SAM), the SAMP1 mouse, is an animal model for accelerated senescence including the age-related acceleration of both immunological dysfunction and hearing loss caused by the impairment of spiral ganglion cells. In the present study, we examine whether the accelerated presbycusis can be prevented by allogeneic BMT. Young SAMP1 (H-2(k)) mice were irradiated with 9 Gy and then reconstituted with bone marrow cells from normal BALB/c (H-2(d)) mice. Allogeneic BMT was found to prevent the development of immunological dysfunction, hearing loss, and apoptosis of spinal ganglion cells in SAMP1 mice. These findings indicate that some types of accelerated presbycusis do not result from defects in the cochlea, but do from defects in the hematopoietic stem cells (HSC) and immunocompetent cells derived from the HSC. If this is the case, either allogeneic BMT, which replaces abnormal HSC with normal HSC and reconstructs a normal immune system in the recipients, or autologous BMT using genetically modified bone marrow cells, could become a new strategy for the treatment of presbycusis.

Protective effect of OK-432 on mice against endotoxemia and infection with Pseudomonas aeruginosa and Salmonella enteritidis.

OK-432 has been used clinically as a biological response modifier for cancer therapy. We investigated here the protective effects of OK-432 against endotoxic shock and infectious death caused by Pseudomonas aeruginosa and Salmonella enteritidis in mice and proposed a possible mechanism. Pretreatment of OK-432 reduced the lethality of lipopolysaccharide (LPS)-induced endotoxic shock in D-(+)-galactosamine-sensitized C3H/HeN mice. OK-432 did not affect the TNFalpha production in blood, but it did decrease the susceptibility to TNFalpha. Furthermore, an acceleration of LPS clearance from blood was detected. The pretreatment of OK-432 also decreased the lethality of mice in bacterial infection caused by P. aeruginosa and S. enteritidis. The rapid decrease of the viable bacteria from the circulating blood and in spleen and liver in mice was observed in a manner similar to LPS clearance. These findings indicate that the protective effect of OK-432 against the endotoxemia and bacteremia may depend on an up-regulation of clearance of LPS and bacteria and the augmented resistance to TNFalpha.

[A case of sarcoidosis characterized by a nasopharyngeal tumor and neurological lesions].

A 20-year-old man was admitted to a hospital complaining a slight fever lasting for 3 months associated with a dull headache and weight loss. A tumor was found in the nasopharynx of which biopsy specimen revealed granulomas with Langhans' giant cells. He was given antituberculous agents without symptomatic improvement, and transferred to our hospital. Serum levels of soluble IL-2 receptor and lysozyme were increased, and a significant uptake was observed by Ga scintigraphy at the nasopharynx and bilateral hilar lymphnodes. Furthermore, spinal fluid contained increased number of mononuclear cells, and T2-weighted MRI scans showed an enhanced lesion at the pituitary stalk. The specimen of both TBLB and repeated biopsy of the nasopharyngeal tumor showed granulomas without caseous necrosis. Taken together with these findings, a diagnosis of sarcoidosis with CNS involvement was finally made, and he made a favorable progress by treatment with prednisolone. This is an unique case which emphasizes importance of differential diagnosis of nasopharyngeal tumors with neurological manifestations in the clinicalsetting of rheumatology.

Concentrations of IL-6 in serum and whey from healthy and mastitic cows.

Inflammatory cytokines, such as interleukin (IL)-6, have been shown to reflect clinical signs in certain conditions in diseased animals. In this study, we quantified the IL-6 concentrations in the serum and milk whey from 94 dairy cows with acute clinical mastitis and 55 healthy lactating cows. The IL-6 concentrations in serum from mastitic cows were significantly higher on the first day of illness compared to those of normal cows. Higher concentrations of IL-6 were also detected in the whey from mastitic cows, whereas low concentrations of IL-6 were detected in both serum and whey samples from normal cows. IL-6 concentrations in the serum taken at the onset of illness from cows that later required euthanasia were significantly higher than those in samples from cows that later recovered. These results suggest that serum IL-6 concentrations may be of prognostic value in identifying cows with severe mastitis.

Prevention of autoimmune hearing loss in MRL/lpr mice by bone marrow transplantation.

We examined the effects of bone marrow transplantation (BMT) on immune-mediated inner ear diseases in MRL/Mp-lpr/lpr (MRL/lpr) mice, which manifest not only lupus nephritis but also sensorineural hearing loss (SNHL) at the age of 20 weeks. These mice were treated with cyclophosphamide (CY) and irradiation (5 Gy x 2), followed by the transplantation of bones plus bone marrow cells from allogeneic normal C57BL/6 mice at the age of 12 weeks. Hematolymphoid cells were reconstituted with donor-derived cells 3 months after BMT. Thus-treated MRL/lpr mice showed neither lupus nephritis nor SNHL even 24 weeks after BMT. No pathological findings were observed in either glomeruli or cochleae. These findings suggest that BMT can be used to prevent the development of autoimmune SNHL in MRL/lpr mice.

Syndrome of inappropriate secretion of antidiuretic hormone (SIADH) and adrenal insufficiency induced by rathke's cleft cyst: a case report.

We report a case of a seventy-year-old woman with syndrome of inappropriate secretion of antidiuretic hormone (SIADH) and adrenal insufficiency induced by Rathke's cleft cyst. She experienced nausea, vomiting, diarrhea, and headache and disturbance of consciousness induced by hyponatremia at a serum sodium level of 100 mEq/l. In spite of severe hyponatremia, urinary sodium excretion was not suppressed and serum osmolality (270 mOsm/kg) was lower than urine osmolality (304 mOsm/kg), and arginine vasopressin (AVP) remained within normal range. SIADH was diagnosed because she was free from other diseases known to cause hyponatremia such as dehydration, cardiac dysfunction, liver dysfunction, renal dysfunction, hypothyroidism, and adrenal insufficiency. Cranial computed tomographic (CT) scan and cranial magnetic resonance (MR) imaging showed a cystic lesion of approximately 2 cm in diameter in the pituitary gland. These images suggested that the cystic lesion was a Rathke's cleft cyst, which was the cause of SIADH. Water restriction therapy normalized her serum sodium concentration and improved her symptoms. After one year, she suffered from general fatigue, appetite loss, fever, and body weight loss (5 kg/2 months). She had neither hypotension nor hypoglycemia, but her serum sodium level was low and serum cortisol, ACTH, and urine free cortisol were very low. Therefore, secondary adrenal insufficiency was suspected and diagnosed by stimulation tests. After start of hydrocortisone replacement therapy (10 mg/day), her symptoms disappeared. In conclusion, Rathke's cleft cyst should be kept in mind as a potential cause in a patient with SIADH, hypopituitarism, and/or adrenal insufficiency.

High levels of serum interleukin-10 and tumor necrosis factor-alpha are associated with fatality in fulminant hepatitis.

Serum pro- and anti-inflammatory mediators in patients with acute liver diseases were assessed to clarify the clinical significance of these measurements in relation to disease severity. Concentrations of circulating tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-10, IL-12, and soluble TNF receptors (sTNFR) p55 and p75 were measured at admission in patients with fulminant hepatitis (FH; n=19), severe acute hepatitis (AHS, n=15), or acute hepatitis (AH, n=7). Serum concentrations of TNF-alpha, IL-10, and sTNFR-55 were significantly higher in patients with FH than in those with AHS (P<.05, <.05, and <.01, respectively) or AH (P<.05). Serum IL-10 and TNF-alpha levels were higher in patients who died of FH (n=13) than in FH survivors (n=6; P<.05). The ratios between TNF-alpha and IL-10 and sTNFR-55 or sTNFR-75 were not valuable in predicting mortality and disease severity. However, both proinflammatory cytokine TNF-alpha and anti-inflammatory cytokine IL-10 levels at admission were associated with fatal outcome among patients with FH.

Influence of the pK(a) value of the buried, active-site cysteine on the redox properties of thioredoxin-like oxidoreductases.

Thioredoxin constitutes the prototype of the thiol-disulfide oxidoreductase family. These enzymes contain an active-site disulfide bridge with the consensus sequence Cys-Xaa-Xaa-Cys. The more N-terminal active-site cysteine is generally a strong nucleophile with an abnormal low pK(a) value. In contrast, the more C-terminal cysteine is buried and only little is known about its effective pK(a) during catalysis of disulfide exchange reactions. Here we have analyzed the pK(a) values of the active-site thiols in wild type thioredoxin and a 400-fold more oxidizing thioredoxin variant by NMR spectroscopy, using selectively (13)C(beta)-Cys-labeled proteins. We find that the effective pK(a) of the buried cysteine (pK(b)) of the variant is increased, while the pK(a) of the more N-terminal cysteine (pK(N)) is decreased relative to the corresponding pK(a) values in the wild type. We propose two empirical models which exclusively require the knowledge of pK(N) to predict the redox properties of thiol-disulfide oxidoreductases with reasonable accuracy.

High-dose-rate interstitial brachytherapy of Bartholin's gland: A case report.

OBJECTIVE: To preserve the organs and function of the anus and ureter, radiotherapy was chosen for an advanced vulvar tumor originating from Bartholin's gland. METHODS: The patient was a 74-year-old female with stage III (FIGO) vulvar cancer derived from the left Bartholin's gland. She received 63 Gy to the vulvar lesion with external beam irradiation followed by 30 Gy of high-dose-rate interstitial brachytherapy. RESULTS: One year after radiotherapy, simple vulvectomy and reconstructive surgery were performed. The anal and ureteral functions were preserved. Histological examination revealed no evident malignant cells. CONCLUSION: Owing to the technological development of radiotherapy and improved reconstructive surgery, radiotherapy in treatment of the vulvar area has become effective and safe and could be indicated more as a treatment option for the sake of organ preservation.

Quinolactacins A, B and C: novel quinolone compounds from Penicillium sp. EPF-6. I. Taxonomy, production, isolation and biological properties.

Quinolactacins A (1), B (2) and C (3), novel quinolone antibiotics have been found from the cultured broth of a fungal strain isolated from the larvae of the mulberry pyralid Margaronia pyloalis Welker). The fungal strain, EPF-6 was identified as Penicillium sp. from its morphological characteristics. Quinolactacins were obtained from the culture medium by solvent extraction and chromatographic purification. Compound 1 showed inhibitory activity against tumor necrosis factor (TNF) production induced by murine peritoneal macrophages and macrophage-like J774.1 cells stimulated with lipopolysaccharide (LPS).

[Evaluation of frozen section diagnosis of parotid gland tumors].

A retrospective study is presented comparing the results of 167 frozen section diagnoses of surgical extirpated parotid gland tumors with permanent-section diagnoses. Percentages of correct diagnosis for malignancy (cases correctly classified as benign or malignant tumors) and of correct diagnosis for histopathology (cases in which frozen section diagnosis and permanent-section diagnosis were identical) were calculated. Percentages of correct diagnosis for malignancy in all cases, benign cases, and malignant cases were 98.8%, 99.3%, and 95.8%, respectively. Percentages of correct diagnosis for histopathology in all cases, benign cases, and malignant cases were 94.0%, 97.2%, and 75.0%, respectively. These results are superior to the previous reports both of frozen section diagnosis and of fine-needle aspiration biopsy diagnosis although the data for histopathological diagnosis in malignant tumors are average compared to previous reports. We conclude that diagnoses of most parotid gland tumors based on frozen section examination are reliable and accurate, but caution should be exercised in malignant tumors diagnosis.

Circular beta-lactamase: stability enhancement by cyclizing the backbone.

We have cyclized the polypeptide backbone of beta-lactamase with a short peptide loop as a novel method for protein stabilization, using intein-mediated protein ligation. Successful cyclization was proven by mass spectrometry and subsequent re-linearization by proteolytic cleavage, as well as by resistance against carboxypeptidase. Under the conditions of the experiment, no disulfide bond is present. The circular form of beta-lactamase was found to be significantly more stable against irreversible aggregation upon heating than the linear form. The circular form could be purified from the linear one either by this heat treatment or by a his-tag which became exopeptidase-resistant by cyclization. The increased stability of the circular form is probably due to the decreased conformational entropy in the unfolded state and in the intermediate states. While the introduction of additional disulfide bonds for protein stabilization follows the same rationale, the cyclization strategy may disturb the structure less and thus constitute a general method for stabilizing those proteins with N- and C-termini in close proximity.

[Clinical study of parotid tumors: a 20 year statistical analysis of 633 cases].

During the 20 years from 1977 to 1996, 633 cases underwent excisions of parotid tumors (539 benign and 94 malignant) in the department of Otolaryngology of Kansai Medical University. The incidence of Warthin's tumors in the years from (1987 to 1996) was higher than that in the years from 1977 to 1986. On the other hand, the incidence of mucoepidermoid carcinomas was less than in previous reports. Tumors accompanied with spontaneous pain or facial nerve palsy and with invasion to both lobes frequently indicated malignancy. Facial nerve palsy caused by either benign or malignant tumors before operation showed no improvement after surgery. The incidences of facial nerve palsy (1.0% in benign, and 18.3% in malignant) and Frey's syndrome (17.8% in benign, and 18.3% in malignant) after surgery were lower than those in other reports of both benign and malignant tumors. Five-year mortality was 76.1%. All deaths that occurred five years after operation involved highly grade malignant tumors.