Primary structure of dihydrofolate reductase and mitochondrial ribosomal protein L36 genes from the basidiomycete Coprinus cinereus.

We amplified and sequenced the dihydrofolate reductase (DHFR) gene of the basidiomycete Coprinus cinereus. Downstream of the DHFR coding region, a mitochondrial (mt) ribosomal protein L36 (RPL36) gene was discovered in the opposite orientation to DHFR gene. Putative polyadenylation signals of the two genes overlapped, both containing the 8-bp palindrome 5'-aatatatt-3'. The finding that C. cinereus DHFR gene is closely clustered with a mt protein gene strongly suggests that C. cinereus DHFR is closely related to mt function and evolution. The amino acid sequence of C. cinereus DHFR is most homologous to eukaryotic proteins such as Cryptococcus neoformans and Pneumocystis carinii DHFRs. However, the sequence of C. cinereus mt RPL36 closely resembles RPL36 of bacteria and cyanobacteria such as Synechocystis sp. and Escherichia coli. This result strongly supports the serial endosymbiotic theory of the development of ancestral eukaryotes, and suggests that C. cinereus mt RPL36 gene originated from the ancestral eubacterial genome.     

A single intratumoral injection of a fiber-mutant adenoviral vector encoding interleukin 12 induces remarkable anti-tumor and anti-metastatic activity in mice with Meth-A fibrosarcoma

Cytokine-encoding viral vectors are considered to be promising in cancer gene immunotherapy. Interleukin 12 (IL-12) has been used widely for anti-tumor treatment, but the administration route and tumor characteristics strongly influence therapeutic efficiency. Meth-A fibrosarcoma has been demonstrated to be insensitive to IL-12 treatment via systemic administration. In the present study, we developed an IL-12-encoding fiber-mutant adenoviral vector (AdRGD-IL-12) that showed enhanced gene transfection efficiency in Meth-A tumor cells, and the production of IL-12 p70 in the culture supernatant from transfected cells was confirmed by ELISA. In therapeutic experiments, a single low-dose (2 x 10(7) plaque-forming units) intratumoral injection of AdRGD-IL-12 elicited pronounced anti-tumor activity and notably prolonged the survival of Meth-A fibrosarcoma-bearing mice. Immunohistochemical staining revealed that the IL-12 vector induced the accumulation of T cells in tumor tissue. Furthermore, intratumoral administration of the vector induced an anti-metastasis effect as well as long-term specific immunity against syngeneic tumor challenge.     

Paranuclear blue inclusions of small cell carcinoma of the stomach: report of a case with cytologic presentation in peritoneal washings

BACKGROUND: Primary gastric small cell carcinoma is a rare but important entity. We describe a case that we diagnosed by peritoneal washing cytology. CASE: A 70-year-old male presented with upper abdominal discomfort and underwent endoscopic evaluation. Gastric endoscopy revealed a diffuse, infiltrating tumor from the body to the antrum. Total gastrectomy with lymph node dissection and intraoperative peritoneal washing cytology were carried out. Peritoneal washing cytology showed the presence of many undifferentiated malignant small cells with a necrotic background. The tumor cells were small and round, with naked, hyperchromatic nuclei and finely granular chromatin. Some tumor cells contained paranuclear blue inclusions (PBls) in the cytoplasm. The tumor cells were positive for neuron-specific enolase and synaptophysin on immunocyto-chemistry. Carcinoembryonic antigen, alpha-fetoprotein (AFP) and leukocyte common antigen were negative. Pathologic diagnosis after the operation was moderately to poorly differentiated adenocarcinoma and small cell carcinoma containing AFP-positive cells. CONCLUSION: The prognosis of primary gastric small cell carcinoma is usually poor. Our patient died of multiple liver metastases and peritonitis carcinomatosa 69 days after surgery. When a gastric small cell carcinoma is suspected in peritoneal washings, immunocytochemical demonstration of neuroendocrine differentiation is required to arrive at the final diagnosis.     

Augmentation of antigen-specific immune responses using DNA-fusogenic liposome vaccine

In an attempt to enhance the immunological efficacy of genetic immunization, we investigated a new biological means for delivering antigen gene directly to the cytoplasm via membrane fusion. In this context, we investigated fusogenic liposome (FL) encapsulating DNA as a possible genetic immunization vehicle. RT-PCR analysis indicated that a FL could introduce and express encapsulating OVA gene efficiently and rapidly in vitro. Consistent with this observation, an in vitro assay showed that FL-mediated antigen-gene delivery can induce potent presentation of antigen via the MHC class I-dependent pathway. Accordingly, immunization with FL containing the OVA-gene induced potent OVA-specific Th1 and Th2 cytokine production. Additionally, OVA-specific CTL responses and antibody production were also observed in systemic compartments including the spleen, upon immunization with the OVA-gene encapsulating FL. These findings suggest that FL is an effective genetic immunization carrier system for the stimulation of antigen-specific immune responses against its encoding antigen.     

Optimization of antitumor efficacy and safety of in vivo cytokine gene therapy using RGD fiber-mutant adenovirus vector for preexisting murine melanoma

We previously reported that RGD fiber-mutant adenovirus vector (AdRGD) was a very useful vector system for in vivo cytokine gene therapy for established murine B16BL6 melanoma. However, intratumoral administration of AdRGD expressing tumor necrosis factor alpha (AdRGD-TNFalpha) at high dose revealed not only the dramatic reinforcement of anti-tumor effect but also serious adverse effects, such as body weight reduction and sudden death, caused by high-level TNF-alpha leakage from the tumor into circulation. These results strongly suggested that the determination of 'limiting dose', which demonstrated therapeutic effectiveness without adverse effect, against each vector was important for the development of appropriate cytokine gene therapy. In the present study, we investigated the efficacy and the safety of AdRGD expressing interleukin-12 (AdRGD-IL12) in murine melanoma model, and determined its limiting dose. Moreover, we demonstrated that combination therapy using AdRGD-IL12 and AdRGD-TNFalpha at limiting doses or less could achieve more effective tumor regression without adverse effects. Therefore, we conclude that a combination of multiple AdRGD expressing cytokines having distinct anti-tumor mechanisms can contribute to the establishment of in vivo cytokine gene therapy for melanoma, which possesses both excellent efficacy and high safety.     

Design and synthesis of a peptide-PEG transporter tool for carrying adenovirus vector into cells

The adenovirus vector is a promising carrier for the efficient transfer of genes into cells via the coxackie-adenovirus receptor (CAR) and integrins (alphavbeta3 and alphavbeta5). The clinical use of the adenovirus vector remains problematic however. Successful administration of this vector is associated with side effects because antibodies to this vector are commonly found throughout the human body. To make the adenovirus vector practicable for clinical use, it is necessary to design an auxiliary transporter. The present study describes the use of Arg-Gly-Asp(RGD)-related peptide, a peptide that binds to integrins, as an auxiliary transporter to aid efficient transport of adenovirus vector. Furthermore, poly(ethylene glycol) (PEG) was also used as a tool to modify the adenovirus such that the risk of side effects incurred during clinical application was reduced. The present study describes the design, preparation and use of (acetyl-Tyr-Gly-Gly-Arg-Gly-Asp-Thr-Pro-(beta)Ala)(2)Lys-PEG-(beta)Ala-Cys-NH(2)[(Ac-YGGRGDTP(beta)A)(2)K-PEG-(beta)AC] as an efficient peptide-PEG transporter tool for carrying adenovirus vector into cells. (Ac-YGGRGDTP(beta)A)(2)K-PEG-(beta)AC was coupled with 6-maleimidohexanoic acid N-hydroxysuccinimide ester and the resulting 6-[(Ac-YGGRGDTP(beta)A)(2)K-PEG-(beta)AC-succinimido]hexanoic acid N-hydroxysuccinimide ester reacted with adenovirus. The modified adenovirus with the peptide-PEG hybrid exhibited high gene expression even in a CAR-negative cell line, DC2.4.     

A novel method for making nested deletions and its application for sequencing of a 300 kb region of human APP locus.

We developed a novel in vitro method for making nested deletions and applied it to a large-scale DNA sequencing. A DNA fragment to be sequenced (up to 15 kb long) was cloned with a new vector possessing two unique Sfi I sites, digested by Sfi I and ligated to generate a large head-to-tail concatemer. The large concatemer was randomly fragmented by sonication and then redigested by Sfi I to separate insert and vector DNAs. The fragments of various length were then cloned into the other vector(s) specifically designed for selective cloning of insert-derived DNA fragments to generate a library of nested deletions. This method allowed a single person to generate >20 nested deletion libraries sufficient to cover 100 kb in a few days. We applied the method for sequencing of P1 clones and successfully determined the complete sequence of approximately 300 kb of the human amyloid precursor protein (APP) locus on chromosome 21 with a redundancy of 3.8, reasonably low cost and very few gaps remaining to be closed. Development of some new instruments and software is also described which makes this method more applicable for large-scale sequencing.     

Transient Increase of Cyclic AMP Induced by Glutamate in Cultured Neurons from Rat Spinal Cord

Abstract: We demonstrated that glutamate increased the cyclic AMP level in cultured neurons from rat spinal cord. A bath application of glutamate (300 µ M ) elicited a rapid increase of the cyclic AMP concentration reaching a level three times as high as the basal level in ∼3 min, and its content then decreased to the control level in 15 min. The increase was not observed in a Ca²⁺-free medium and was inhibited by an antagonist of NMDA receptors or a voltage-sensitive Ca²⁺ channel blocker. Preincubation with W7 also inhibited the glutamate-evoked cyclic AMP increase. NMDA, aspartate, and high-K⁺ conditions also induced a cyclic AMP increase; however, a decreasing phase did not follow. The decreasing phase was observed when (2 S ,1' S ,2' S )-2-(carboxycyclopropyl)-glycine, a potent agonist for metabotropic glutamate receptors, was combined with NMDA. These results suggest that the cyclic AMP increase is mediated by a Ca²⁺ influx via both NMDA receptors and voltage-sensitive Ca²⁺ channels followed by an activation of the Ca²⁺/calmodulin system, and the decreasing phase observed in the case of glutamate exposure is due to the activation of the metabotropic glutamate receptors.     

Study of polysaccharides by the fluorescence method. III. Chain length dependence of the micro-Brownian motion of amylose in an aqueous solution

The fluorescence polarization method was applied to the investigation of the micro-Brownian motion of amylose chains having a wide range of degree of polymerization (DP). We prepared two types of fluorescent conjugates of amylose: amylose conjugated with fluorescein randomly throughout the chain (F-amylose) and amylose conjugated locally on a terminal segment (t-F-amylose). The degree of fluorescence polarization of these conjugates was measured by changing the solvent viscosity at a constant temperature (25°C). The data obtained were analyzed by a Perrin-type equation to calculate the mean rotational relaxation time, 〈ρ〉. By examination of the plots of 〈ρ〉 vs DP, and by comparison of 〈ρ〉 with the theoretical rotational relaxation time of the whole molecule at a given DP, it was found that 〈ρ〉 mainly reflects the segmental motion of the amylose chain in the high-DP range. Thus, the fact that 〈ρ〉 for t-F-amylose is much smaller than that for F-amylose at a sufficiently high DP shows that a terminal segment undergoes a more rapid micro-Brownian motion than interior segments. In the low-DP range, we felt that the rotational diffusion of the whole molecule contributes significantly to the relaxation process. We also examined, for comparison, the segmental motion of dextran and pullulan in a similar manner and found that these segmental motions are more rapid than those of amylose. Based on the results obtained, the segmental mobility and conformation of the amylose molecule are discussed in relation to its chain length.     

Cell Cycle-Dependent Nuclear Localization of Exogenously Added Fibroblast Growth Factor-1 in BALB/c 3T3 and Human Vascular Endothelial Cells

Previous studies have shown that the presence of a functional nuclear targeting sequence in the primary structure of fibroblast growth factor (FGF)-1 correlates with its activity as a mitogen, but not with its potential for inducing receptor tyrosine phosphorylation, suggesting the presence of a yet undefined function of FGF-1 as a nuclear protein. In the present study we have investigated the cytosolic and nuclear localization of exogenously added FGF-1. FGF-1-specific monoclonal antibodies were raised. By an extensive screening, highly specific antibody clones were isolated. For both BALB/c 3T3 and human umbilical vein endothelial (HUVE) cells, immunofluorescence studies performed with those clones delineated that during G1 stage of cell cycle, FGF-1 transits from cytosol to nucleus. This was followed by a shift to the perinuclear and juxtanuclear region just prior to the onset of S-phase in BALB/c 3T3 cells. Confocal microscopical examinations confirmed that the nuclear staining resides throughout the nuclear matrix with some enrichment at the envelope boundary and in the nucleoli. Immunoblot analysis of the fractionated BALB/c 3T3 cells that had been induced to proliferate by serum and pulsed with exogenous FGF-1 at various timings revealed that the incorporation of exogenous FGF-1 into cytosol took place constantly, whereas the nuclear translocation significantly increased after 5 h following stimulation of the quiescent cells. The cytosolic form of FGF-1 is indicated to be present in soluble cytosolic fraction rather than membrane-enveloped compartments, endosomes, by the microinjection of anti FGF-1 antibody to HUVE cells cultured in the presence of FGF-1. The data demonstrate that the exogenously added FGF-1 is constantly endocytosed and fractioned into the cytosol soluble compartment, whereas its nuclear localization is regulated at the nuclear translocation level and takes place preferably at late G1 phase of the cell cycle.