Germline transgenesis and insertional mutagenesis in the ascidian Ciona intestinalis.

Stable transgenesis is a splendid technique that is applicable to the creation of useful marker lines, enhancer/gene traps, and insertional mutagenesis. Recently, transposon-mediated transformation using a Tc1/mariner transposable element Minos has been reported in two ascidians: Ciona intestinalis and C. savignyi. The transposon derived from an insect, Drosophila hydei, has high activity for excision in Ciona embryos and transposition in their genome. As much as 37% of Minos-injected C. intestinalis transmitted transposon insertions to the subsequent generation. Minos-mediated germline transgenesis has also been achieved by means of electroporation method. Minos techniques have been applied to enhancer traps and insertional mutagenesis in Ciona. For those reasons, Minos offers the high potential for use as a powerful tool for future genetic studies. This review specifically addresses recent achievements of transformation techniques in Ciona, as exemplified using the Minos system.     

Optimizing a novel regional chemotherapeutic agent against melanoma: hyperthermia-induced enhancement of temozolomide cytotoxicity.

PURPOSE: Previous preclinical studies have shown that regional temozolomide therapy via isolated limb infusion is more effective than melphalan, the current drug of choice for regional chemotherapy for advanced extremity melanoma. The aim of this study was to determine whether hyperthermia could further augment the efficacy of temozolomide, an alkylating agent, against melanoma and improve its therapeutic index in a rat model of isolated limb infusion. EXPERIMENTAL DESIGN: Athymic rats bearing s.c. human melanoma xenografts (DM6) in their hind limbs were randomized to a 15-minute isolated limb infusion procedure with or without temozolomide at room temperature, normothermic (37.5 degrees C), or hyperthermic (43 degrees C) conditions. RESULTS: The concomitant administration of hyperthermia during an infusion with temozolomide led to the greatest increase in tumor growth delay, decreased proliferative index, and increased cell death. Isolated limb infusion treatment with a low dose (350 mg/kg) of temozolomide was ineffective at producing tumor growth delay (P = 0.07). Similarly, temozolomide infusion under normothermia yielded minimal tumor growth delay (P = 0.08). In contrast, the combination of hyperthermia plus temozolomide treatment produced marked tumor growth delay of 10.4 days (P = 0.02) with minimal toxicity. The addition of heat to temozolomide treatment yielded the smallest proliferative index (P = 0.001), while markedly increasing the level of apoptosis 48 hours after isolated limb infusion. CONCLUSION: This study, the first to examine the interaction between hyperthermia and temozolomide, shows a strong, synergistic antitumor effect when hyperthermia is combined with temozolomide for regional treatment of melanoma confined to an extremity. The mechanism of this synergy seems to be through an augmentation, by hyperthermia, of the antiproliferative and proapoptotic effects of temozolomide.     

Successful treatment of a bronchial inflammatory pseudotumor by bronchoplasty in an 8-year-old boy: Report of a case

(Received for publication on Mar. 29, 1999; accepted on Nov. 11, 1999)     

Hepatocellular carcinoma in an orthotopic mouse model metastasizes intrahepatically in cirrhotic but not in normal liver

Prognosis of hepatocellular carcinoma (HCC) still remains poor mainly because of intrahepatic metastasis. In the majority of cases, HCC is found in conjunction with liver cirrhosis. It is, therefore, of great importance to investigate the invasive and metastatic behavior of HCC in cirrhotic liver. To examine this, a liver cirrhosis model was produced by injecting thioacetamide i.p. into mice. Murine HCC cells were labeled with the fluorescent carbocyanine dye, DiI, and implanted directly under the capsule of cirrhotic and normal livers of syngeneic mice. DiI‐labeled HCC cells in the liver were observed under fluorescent and confocal microscopy. Histological analysis of cirrhotic and normal livers revealed that implanted HCC cells migrated to and invaded the adjacent periportal regions, but not the adjacent centrolobular areas. This characteristic behavior of HCC was more evident in cirrhotic liver than in normal liver. Furthermore, intrahepatic metastasis to unimplanted hepatic lobes was observed in cirrhotic liver as early as 7 days after implantation, while it was not detected in normal liver even 4 weeks later. Thus, an orthotopic animal model for HCC with cirrhosis described here may be suitable for investigating the invasive and metastatic behavior of HCC. Importantly, labeling tumor cells with a fluorescent dye before orthotopic implantation may be a convenient and useful method to investigate the invasive and metastatic behavior of various types of cancer. Int. J. Cancer 80:471–476, 1999. © 1999 Wiley‐Liss, Inc.     

Distribution of herpes simplex virus types 1 and 2 genomes in human spinal ganglia studied by PCR and in situ hybridization

Clinical data indicate that the recurring herpes simplex virus (HSV) from oro-labial lesions is HSV subtype 1 and that the virus from genital lesions is HSV-2. This suggests that HSV-1 and HSV-2 reside in latent forms in the trigeminal ganglia and sacral ganglia, respectively. However, the distribution of latent HSV-1 and HSV-2 infections in human spinal ganglia has not been fully examined. This report concerns the application of polymerase chain reaction (PCR) and in situ hybridization (ISH) to such a study. By using PCR and employing the respective primers, HSV-1 and HSV-2 DNAs were detected in 207 of 524 samples from 262 spinal ganglia (from the cervical to the sacral ganglia) examined on both sides. The percentages of HSV-1 and HSV-2 detected in a given set of ganglia were similar, indicating an absence of site preference. By ISH, few but positive hybridization signals were detected evenly in sacral ganglia sections. The data suggest that regional specificity of recurrent HSV infections is not due to regional distribution of latent virus, but that local host factors may be important for recurrences. J. Med. Virol. 52:136–142, 1997. © 1997 Wiley-Liss, Inc.     

Efficacy of combination therapy with gabapentin and guanfacine for paroxysmal sympathetic hyperactivity following hypoxic encephalopathy: a case report

Paroxysmal sympathetic hyperactivity (PSH) is a clinical syndrome of episodic sympathetic hyperactivities following severe acquired brain injury. It is characterized by paroxysmal hyperthermia, tachycardia, hypertension, tachypnea, excessive diaphoresis, and specific posturing. Although the persistence of PSH increases the risk of several adverse events and worsens the prognosis, pharmacological treatments for PSH have not yet been clearly established. We report the valuable case of a 60-year-old man who developed PSH following hypoxic encephalopathy, which was effectively treated with a combination therapy of gabapentin and guanfacine. The present case suggests that combination therapy with gabapentin and guanfacine may be a therapeutic option for PSH.     

CD43 expression in a B cell lymphoma,WEHI 231, reduces susceptibility to G1 arrest and extends survival in culture upon serum depletion

CD43 is a major surface sialoprotein on hemopoietic cells, whose extracellular domain is heavily O-glycosylated. The functional role of CD43 in the hemopoietic system is not fully understood; however, it has been suggested that CD43 may have a role in cell-cell repulsion and in modifying T cell proliferation and activation. CD43 is expressed in immature B cells in the bone marrow, but not by peripheral B cells, except for B-1 B cells and plasma cells. To analyze the biological effect of CD43 in B-lineage cells, we transfected mouse CD43 cDNA into a CD43⁻ B cell lymphoma, WEHI 231, and the growth and survival in culture were compared to those of a parental cell line, human CD8 transfectants, and CD43⁻ revertants established from CD43⁺ clones. We observed that CD43 expression supported cell growth in culture upon serum reduction, whereas growth of CD43⁻ cell lines was barely detected under this condition. CD43⁻ cell lines accumulated in G₁ phase of the cell cycle, and the numbers of viable cells were greatly reduced during culture upon serum depletion, whereas expression of CD43 reduced the susceptibility to G₁ arrest and temporarily retarded the apoptotic process, which, in turn, resulted in an increase and maintenance of the number of viable cells in culture. The results suggest that CD43 may have some role in the survival and expansion of B-lineage cells. The biological effect of CD43 was initiated without stimulation by cross-linking and was significantly impaired by replacement of the extracellular domain by the human CD8 extracellular domain. The basis of these regulatory processes is discussed.     

What prevents Japan from establishing a clinical service of sleep medicine: Experience gained at a local hospital

We opened a mini-sleep disorders clinic in a local general hospital in rural Japan, and have been operating it for more than 3 years. We encountered many difficulties during this period when we tried to provide proper clinical service. Problems ranged from lack of sleep specialists and polysomnography technicians, shortage of funding, non-existence of an educational system to obtain comprehensive knowledge about sleep medicine, and also lack of cooperation among different departments necessary for maintaining clinical service. Through our experience, we analyzed various factors that prevent Japan from establishing a clinical service of sleep medicine.