Calcification during bone healing in a standardised rat calvarial defect assessed by micro-CT and SEM-EDX.

OBJECTIVE: The study was designed to investigate the process of calcification during bone healing in a standardized rat calvarial bone defect model, measured by bone mineral density and the concentrations and distributions of calcium, phosphorus and carbon in the bone matrix. MATERIALS AND METHODS: A standard defect was made on the parietal bone of 12-week-old rats under anaesthesia. The rats were fixed in weeks 1, 2, 4 and 8,and the calvaria were resected and examined with microcomputed tomography, then frozen and sectioned for histology and analysed with energy-dispersive X-ray spectroscopy (EDX). Parietal bone of 12-week-old control rats was processed similarly. RESULTS: The mineral density of healing bone increased with time. The healing bone became thicker and denser with time in histology. The distributions of Ca and P expanded over the bone matrix, whereas that of C became localised and complemented that of C and P. The Ca/P concentration ratio increased, whereas the C/Ca and C/P ratios decreased in the healing bone matrix. CONCLUSION: Healing bone is immaturely calcified initially and proceeds calcification gradually, that is, as the bone volume increases, mineral increases in density and matures in quality, while organic components decrease.

In vitro activity of multiple antibiotic combinations against Nocardia: relationship with a short-term treatment strategy in heart transplant recipients with pulmonary nocardiosis.

BACKGROUND/OBJECTIVES: Pulmonary nocardiosis (PN) chiefly affects immunocompromised patients, particularly transplant recipients. Cotrimoxazole is still the mainstay of treatment, but it is associated with nephro- and myelo-toxicity, and can show unpredictable activity against Nocardia isolates. METHODS: Over a 20-year period, Nocardia isolates were identified from 12 heart transplant (HTx) recipients with PN. The in vitro activity of various antibacterials, alone or in combination, was assessed using disk-diffusion, minimal inhibitory concentration (MIC), and time-kill methodology. The in vitro results were compared with the clinical outcome of the patients. RESULTS: Seven different Nocardia strains were identified. Disk diffusion and MIC determinations showed that all isolates were susceptible to amikacin, netilmicin, and linezolid, and that moxifloxacin was the most active of the fluoroquinolones. All but 1 of the isolates were susceptible to imipenem. Time-kill studies showed that imipenem/amikacin and imipenem/moxifloxacin combinations were bactericidal for most isolates. Of 12 patients who received 3-4 weeks' intravenous (IV) treatment with amikacin or ciprofloxacin in combination with a beta-lactam, followed by 1-3 months' oral cotrimoxazole, moxifloxacin, or linezolid, 11 were cured; 1 patient died, but not related to Nocardia. CONCLUSION: Initial PN treatment in HTx recipients can be successfully carried out with bactericidal combinations such as imipenem plus amikacin or moxifloxacin, administered IV for 3-4 weeks. Within 1 month, a significant clinical and radiological improvement may be observed. In our experience, a <3 month oral regimen with cotrimoxazole, moxifloxacin, or doxycycline may then be used. This may allow a reduction of side effects and treatment-related burden, without any recurrence.

Inducing peripheral sympathetic nerve activity by therapeutic electrical stimulation.

PURPOSE: To examine whether the activity of peripheral sympathetic nerves in animals with spinal cord injury can be controlled using therapeutic electrical stimulation. METHODS: The spinal cords of 6 Wistar rats were severed at T12/T13 disk level and were given continuous therapeutic electrical stimulation. Microneurography was used to record sympathetic nerve activity at 24, 48, and 72 hours after severing the spinal cord. RESULTS: Integrated values of muscle sympathetic nerve activity after 72 hours of therapeutic electrical stimulation revealed significantly larger potentials on the stimulated side than the non-stimulated side. Skin sympathetic nerve activity showed no difference between the 2 sides. CONCLUSION: Therapeutic electrical stimulation was found to have a facilitatory effect on the muscle sympathetic nerve activity, whereas regulatory function was activated by the sympathetic nerves.

[Effect of combination chemotherapy with nedaplatin and 5-FU for head and neck squamous cell carcinoma].

Nedaplatin (254-S), which is a cisplatin (CDDP) analog, is an effective agent for head and neck squamous cell carcinoma (HNSCC). 254-S is expected to play an important role in neo-adjuvant chemotherapy (NAC) for HNSCC in place of CDDP. We have been using combination chemotherapy including CDDP, 5-FU, MTX and LV. The response rate and CR rate of this 4-drug combined chemotherapy are 87% and 33%. Thirty-six patients with HNSCC were treated with 5-FU, 800 mg/m2/day for 5 days and 254-S, 100 mg/m2 on day 4. Chemotherapy was discontinued in one patient because of allergic shock. Three patients showed a CR and 10 patients showed a PR. The response rate and CR rate of 254-S plus 5-FU chemotherapy were 37.1% and 8.6%. These were inferior to those with the 4-drug combined chemotherapy. Fourteen percent of patients showed grade 3 leukocytopenia, and 17% showed more than grade 3 thrombocytopenia. The effect of combination chemotherapy of 254-S and 5-FU was inferior to that of the previous chemotherapy including CDDP, 5-FU, MTX and LV. Further study or another combination therapy including 254-S will be essential for improving efficacy against HNSCC.

Gene for a protein capable of enhancing lateral root formation.

Analysis of genes preferentially expressed in hairy roots caused by infection with Agrobacterium rhizogenes has provided insights into the regulation of lateral root formation. A hairy root preferential cDNA, HR7, has been cloned from hairy roots of Hyoscyamus niger. HR7 encodes a novel protein partially homologous to a metallocarboxypeptidase inhibitor and is expressed exclusively in the primordium and base of lateral roots in hairy roots. Overexpression of HR7 in transgenic roots of H. niger dramatically enhances the frequency of lateral root formation. The results of this study indicate that expression of HR7 plays a critical role in initiating lateral root formation.

Antifungal susceptibility of Aspergillus species isolated from invasive oral infection in neutropenic patients with hematologic malignancies.

OBJECTIVE: The aim of this study was to evaluate the relevance of in vitro antifungal susceptibility to clinical response in neutropenic patients with invasive oral aspergillosis. STUDY DESIGN: Nine isolates of Aspergillus species were obtained from invasive oral infections in 9 patients with hematologic malignancies and tested for their in vitro susceptibility to amphotericin B, fluconazole, miconazole, 5-fluorocytosine, and itraconazole. Minimal inhibitory concentration values of the 5 drugs were obtained for each fungus through use of a microdilution broth method. The patients were treated with intravenous amphotericin B (30-50 mg/day) in combination with oral 5-fluorocytosine (3000-6000 mg/day) and/or oral itraconazole (200 mg/day). RESULTS: Amphotericin B and itraconazole were found to be very active, with minimal inhibitory concentration values of 0.861 and 0.194 microg/mL, respectively. Miconazole and 5-fluorocytosine showed minimal inhibitory concentration values of 1.72 and 3.56 microg/mL, respectively. On the other hand, fluconazole FCZ showed low activity, with a minimal inhibitory concentration value in excess of 64.0 microg/mL. During neutropenia, combined antifungal chemotherapy stabilized oral aspergillosis and prevented the spread of oral lesions in 8 patients in whom neutrophil counts eventually recovered. CONCLUSIONS: The results imply that in vitro susceptibility testing may serve as an informative parameter with respect to the efficacy of these antifungals in the treatment of invasive oral aspergillosis, inducing fungal stasis until the neutrophils recover.

Oligodendroglioma with signet-ring cell morphology: a case report with an immunohistochemical and ultrastructural study.

A case of oligodendroglioma with signet-ring cell (SRC) morphology arising in the right thalamic region in a 12-year-old boy is described. Histopathologically, the tumor was a composite neoplasm consisting of typical oligodendroglioma and anaplastic components with aggregates of SRC. Immunohistochemically the SRC were negative for glial fibrillary acidic protein (GFAP) but surrounded by GFAP-positive anaplastic cells with high-grade nuclear features. Typical oligodendrogliomatous components were negative for GFAP. The Ki-67 labeling index evaluated with MIB-1 antibody was 1.3% in the SRC component, 9.2% in the GFAP-positive anaplastic cell component, and 0.8% in the typical oligodendrogliomatous component. Ultrastructurally, the cytoplasm of the SRC was filled with irregularly and widely dilated cisternae of rough endoplasmic reticulum containing granular material. Intermediate filaments and a small number of other organelles were distributed in the perinuclear and peripheral areas. Both the SRC and anaplastic cells had slender cytoplasmic processes, although those of the SRC were short and few in number. These findings are distinct from those of SRC hitherto described in oligodendrogliomas to date, and suggest that there is a morphological heterogeneity in SRC rarely seen in oligodendrogliomas and that some examples of SRC are related to the anaplastic cells with astrocytic features in their origin.

Atomic model of a pyrimidine dimer excision repair enzyme complexed with a DNA substrate: structural basis for damaged DNA recognition.

T4 endonuclease V is a DNA repair enzyme from bacteriophage T4 that catalyzes the first reaction step of the pyrimidine dimer-specific base excision repair pathway. The crystal structure of this enzyme complexed with a duplex DNA substrate, containing a thymine dimer, has been determined at 2.75 A resolution. The atomic structure of the complex reveals the unique conformation of the DNA duplex, which exhibits a sharp kink with a 60 degree inclination at the central thymine dimer. The adenine base complementary to the 5' side of the thymine dimer is completely flipped out of the DNA duplex and trapped in a cavity on the protein surface. These structural features allow an understanding of the catalytic mechanism and implicate a general mechanism of how other repair enzymes recognize damaged DNA duplexes.

[A case of gastric cancer with Borrmann type 4 responding to 5-FU-MMC combined chemotherapy].

A 70-year-old man with advanced Borrmann type 4 was preoperatively treated with 5-FU-MMC combined chemotherapy. The primary tumor including a palpable mass in the abdomen was diminished, and eventually the patient underwent curative resection. This preoperative regimen for advanced Borrmann type 4 gastric cancer might be recommended from the standpoint of less adverse effects of chemotherapy. The patient died suddenly of cardiac infarction in 9 months without recurrent signs.

Report of a study using phantom materials, and clinical experience with simultaneous radio-hyperthermotherapy.

Simultaneous radiohyperthermotherapy (SRH) is a combined hyperthermia-radiation therapy in which irradiation is given during heating. Mutual interference between the high energy radiotherapy system (Toshiba LMR-15A) and the 13.56 MHz capacitive heating system (Omron HEH-500C) was tested with phantom materials prior to a clinical trial with SRH. The energy and flatness of irradiation were not affected by the heating system within the range of clinical use. The high energy radiotherapy system did not affect the increase or distribution of temperature during simultaneous treatment. The results of this phantom study indicated that these apparatuses would not produce clinically significant mutual interference during SRH. A clinical trial was performed on a 57-year-old woman with postoperative recurrence of rectal cancer. This is the first reported clinical case treated with true SRH in which external irradiation was administered during mid capacitive heating. Twelve SRH treatments were performed on the recurrent lesion at a frequency of twice a week for six weeks using the apparatuses described above. There was a significant reduction in pain after treatment. The tumor marker carcinoembryonic antigen (CEA) level decreased after treatment. On CT images taken after treatment, the tumor site became a low density area which indicated necrosis. There were no side effects. These results suggest that further clinical study of SRH should be performed to clarify its advantages.

[Continuous hyperthermic peritoneal perfusion (CHPP) therapy in advanced gastric cancer].

In CHPP therapy for advanced gastric cancer, a perfusate containing 20 mg CDDP and 8 mg MMC in 1,000 ml physiologic saline warmed at 47 degrees C was infused at a constant rate of 200 ml/min into the pouch of Douglas. The intraperitoneal temperature at the supra-pancreatic region was around 39.0 degrees C. To obtain a more stable and higher intraperitoneal temperature, the infusing rate was increased to 400 ml/min. This yielded a 3 degrees C higher temperature (42 degrees C) at the same measuring site. However, the temperature recorded at various intraperitoneal sites did not always reach such an effective range. The maximal plasma concentrations of MMC determined during CHPP at the 200 and 400 ml/min infusion were 0.09 +/- 0.03 and 0.11 +/- 0.03 microgram/ml, and those of CDDP 1.6 +/- 0.4 and 1.7 +/- 0.3 microgram/ml, respectively, all of which were not significantly different. When an intraperitoneal dosage of 20 mg MMC was given to 3 patients, the portal venous blood, at 10 min after the administration, produced a 1.7 times higher concentration of the agent than did the peripheral venous blood. This discrepancy between the two concentrations was much smaller than found by other investigators in animal experiments.

[Total body hyperthermia and continuous hyperthermic peritoneal perfusion as an adjuvant therapy in advanced gastric cancer].

Between September 1986 and July 1989, adjuvant hyperthermic therapy, consisting of either total body hyperthermia (TBHT) or continuous hyperthermic peritoneal perfusion (CHPP), was given to a total of 41 patients immediately following gastric resection for cancer. TBHT was performed in 1 curative- and 11 noncurative-gastrectomized patients (1 stage III and 11 stage IV), and CHPP in 18 curative- and 11 noncurative-gastrectomized patients (6 stage I/II, 10 stage III and 13 stage IV). For TBHT, the blood was warmed and maintained at 42 degrees C for 3 hours by means of a V-V bypass connected to an extracorporeal heater-pumping system. When the hyperthermic condition was established, anti-cancer drugs were administered intravenously. In CHPP, 46 degrees C saline containing anti-cancer drugs were infused at a constant rate through a tube placed at the Douglas fossa. The perfusate was drained out through another tube positioned at an uppermost part of the abdominal cavity. The hyperthermic condition was monitored by measuring the outflow temperature. Complications encountered were bone marrow depression, liver damage and pyrexia, and were more frequently experienced by the TBHT patients. Patients under 65 years of age who had had an absolute noncurative gastrectomy but with TBHT survived significantly longer than those without TBHT. When the patients who had undergone gastrectomy with CHPP for a cancer of more than se penetration were compared with those without CHPP, there was no significant difference in survival found between these two populations. This unsatisfactory result could be partly attributable to difficult maintenance of appropriate (sufficiently high) and constant perfusate temperature.

Human diabetes associated with a mutation in the tyrosine kinase domain of the insulin receptor.

Insulin receptor complementary DNA has been cloned from an insulin-resistant individual whose receptors have impaired tyrosine protein kinase activity. One of this individual's alleles has a mutation in which valine is substituted for Gly996, the third glycine in the conserved Gly-X-Gly-X-X-Gly motif in the putative binding site fo adenosine triphosphate. Expression of the mutant receptor by transfection into Chinese hamster ovary cells confirmed that the mutation impairs tyrosine kinase activity.

Directed biosynthesis of new saframycin derivatives with resting cells of Streptomyces lavendulae.

Saframycin A is an antitumor antibiotic produced by Streptomyces lavendulae 314 which falls into the category of the N-heterocyclic quinone group. Biosynthetically the quinone ring is derived from two tyrosine molecules which condense to generate the basic ring system of saframycin A. The side chain also has been found to derive from two amino acids, i.e., glycine and alanine. Supplementation by various amino acid analogs of the side chain produced three new saframycin derivatives with a replaced side chain. These three saframycins, designated Yd-1, Yd-2, and Y3, contained 2-amino-n-butyric acid, glycine, and alanine residues, respectively. in place of the normal N-terminal pyruvic acid in the side chain of saframycin A. Feeding experiments with 13C-labeled dipeptide indicated that the amino acids are probably incorporated in the side chain as a dipeptide unit. It was also found that saframycin A is produced from saframycin Y3 by an enzymatic deamination reaction. Based on these results, saframycin biosynthesis in S. lavendulae is discussed.

Chromogenicity of Streptomyces.

A simplified technique to detect polyphenol oxidase and melanin formation by Streptomyces culture filtrates was developed. The procedure involves the direct assay of pigment formation by the culture filtrate with 3-(3,4-dihydroxyphenyl)-L-alanine (L-dopa) as a substrate. Among cultures of the International Streptomyces Project, 34 failed to produce a diffusible dark brown pigment on peptone-yeast extract-iron-agar and synthetic tyrosine-agar and gave a negative reaction to the melanin formation test. Sixteen cultures produced a diffusible dark brown pigment on both peptone-yeast extract-iron-agar and synthetic tyrosine-agar and gave positive reactions to the test with either L-tyrosine or L-dopa as substrate. Twenty-one cultures produced a diffusible dark brown pigment on peptone-yeast extract-iron-agar, but failed to do so on synthetic tyrosine-agar. Most of these cultures gave a positive reaction to the test when L-dopa was used as the substrate. The correlation between chromogenicity on complex organic media and melanin formation was more clearly established with L-dopa as substrate than with synthetic tyrosine-agar in the present test. The melanin formation test by the present technique, instead of chromogenicity on complex organic media, is recommended as a key feature for the classification of Streptomyces.