Nanoscale Dynamics of Protein Assembly Networks in Supersaturated Solutions.

Proteins in solution are conventionally considered macromolecules. Dynamic microscopic structures in supersaturated protein solutions have received increasing attention in the study of protein crystallisation and the formation of misfolded aggregates. Here, we present a method for observing rotational dynamic structures that can detect the interaction of nanoscale lysozyme protein networks via diffracted X-ray tracking (DXT). Our DXT analysis demonstrated that the rearrangement behaviours of lysozyme networks or clusters, which are driven by local density and concentration fluctuations, generate force fields on the femtonewton to attonewton (fN - aN) scale. This quantitative parameter was previously observed in our experiments on supersaturated inorganic solutions. This commonality provides a way to clarify the solution structures of a variety of supersaturated solutions as well as to control nucleation and crystallisation in supersaturated solutions.

Potentiation of anticancer agents by new synthetic isoprenoids. II. Inhibition of the growth of transplantable murine tumors.

The antitumor effects of combinations of synthetic isoprenoids-decaprenylamine.HCl, N-(p-methylbenzyl)decaprenylamine.HCl [N-(PMB)-decaprenylamine.HCl], and decaprenoic acid-with anticancer agents were studied in male ICR mice with ascites sarcoma 180 (S180) and in male BALB/c mice with fibrosarcoma Meth A. Decaprenylamine.HCl, N-(PMB)-decaprenylamine.HCl, and decaprenoic acid were tested in combination with bleomycin A2 (BLM) on S180. Decaprenoic acid was also examined in combination with BLM or 5-fluorouracil (FUra) for effects on fibrosarcoma Meth A. The dosages of synthetic isoprenoids used in the combination therapy were much below the median lethal dose. In the low-dosage schedule, decaprenylamine.HCl, N-(PMB)-decaprenylamine.HCl or decaprenoic acid considerably enhanced the antitumor effect of BLM on S180; e.g., the T/C value (mean survival time of treated mice/mean survival time of controls) for decaprenylamineHCl plus BLM, N-(PMB)-decaprenylamine.HCl plus BLM, or decaprenoic acid plus BLM was 294, 357, and 279% respectively, and the combinations increased life-span 2.4-fold, 2.3-fold, or 1.8-fold, respectively, as compared to the effects of BLM alone. The combination of BLM or FUra with decaprenoic acid, when administered by iv injection to mice with fibrosarcoma Meth A (solid tumor system), did not produce synergism. However, a preventive effect of decaprenoic acid monotherapy was observed: Decaprenoic acid at 3 mg/kg resulted in 38.9% suppression of tumor growth 21 days after inoculation.

Potentiation of anticancer agents by new synthetic isoprenoids. I. Inhibition of the growth of cultured mammalian cells.

In this study, 9 new synthetic isoprenoids with 9-10 isoprene chains were tested. One-fifth of the D10 value (concentration of drug that reduced colony-formation ability to 10% that of control) for each isoprenoid was calculated from dose-response curves of Chinese hamster V79 cells or human HeLa cells in culture. That dose was combined with various anticancer agent [1-(4-amino-2-methylpyrimidin-5-yl)methyl-3-(2-chloroethyl)-3-n itrosurea HCl, bleomycin A2 (BLM), cisplatin, doxorubicin (Dx), 5-fluorouracil, mitomycin, and 6-mercaptopurine] to test whether isoprenoids can potentiate the cytotoxic effect of anticancer agents against cultured mammalian cells. Among the 9 isoprenoids tested, decaprenoic acid, dacaprenylamine.HCl, and N-(p-methylbenzyl)decaprenylamine.HCl significantly potentiated BLM and Dx. Some isoprenoids increased intracellular levels of anticancer agents. Both enhanced uptake and reduced efflux of Dx were observed.

Differential effect of imidazole antibiotics on untransformed and virus-transformed rat cell lines.

The imidazole antimycotics clotrimazole and miconazole were tested on untransformed rat cell line 3Y1-B clone 1-6 (3Y1) and six transformed cell lines, which were independently isolated from 3Y1 or 3Y1-B clone 1 after infection with adenovirus 12 (AD-12) or with SV40, to determine their sensitivities to these drugs. The relative plating efficiency of three cell lines (T3, W4, and W5) transformed with AD-12 was reduced to 10(-1( of the initial value by clotrimazole (2 to 4 microgram/ml), whereas that of the parental cell line 3Y1 was reduced to 10(-1) of the initial value by clotrimazole (20 to 25 microgram/ml). By contrast, the differential effect of miconazole on 3Y1 and AD-12-transformed cell lines was found to be a factor of 2. The sensitivity of the SV40-transformed 3Y1 and the AD-12-transformed cell lines. The cellular sensitivity of untransformed 3Y1 cells to clotrimazole was significantly enhanced when exposed to various doses of the unsaturated fatty acid, linoleic acid. The untransformed and transformed cell lines showed sensitivities similar to the cytocidal activity of sterol-binding antimycotics, amphotericin B and filipin.

Imidazole-resistant phenotype and virus transformation in cultured rat cells.

Compared to an untransformed rat cell line, 3Y1, adenovirus 12-transformed cell lines of 3Y1 were highly sensitive to the cytotoxic action of an imidazole antibiotic, clotrimazole. In contrast, SV40-transformed rat cell lines derived from the 3Y1 cell line showed no appreciable difference in the response to clotrimazole when compared to 3Y1 cells. Clotrimazole-resistant clones, WCT-1 and WCT-2, which were spontaneously isolated from the adenovirus 12-transformed cell line W5, showed 5- to 10-fold higher resistance than did W5; the dose-response curves of clotrimazole-resistant clones were similar to those of the untransformed 3Y1 cells. The growth of 3Y1 cells was blocked in the presence of 1% serum, whereas those of W5, WCT-1, and WCT-2 cells were only slightly affected by the same dose of serum in the medium. The membrane fractions of 3Y1, W5, and WCT-1 cells were found to contain similar cholesterol:phospholipid ratios. The phospholipid composition in the membrane fraction of line WCT-1 was similar to that of line W5 but not to that of line 3Y1. By contrast, the fatty acid composition was specifically altered in clotrimazole-resistant clones; cellular contents and some species of fatty acid such as 16:1, 18:2, and 20:4 in WCT-1 and WCT-2 cells were similar to those of 3Y1 but not to those of W5 cells. Differential sensitivities of various cell lines to clotrimazole are discussed in relation to the lipid composition.

Expression of vascular endothelial growth factor and its possible relation with neovascularization in human brain tumors.

To examine which growth factors correlate with neovascularization in human brain tumors, the mRNA levels of transforming growth factor alpha, transforming growth factor beta, basic fibroblast growth factor, and vascular endothelial growth factor (VEGF) genes were determined by a Northern blot analysis in surgically obtained human gliomas and meningiomas. The vascular development was determined by counting the number of microvessels which were immunostained with von Willebrand factor. We normalized the growth factor mRNA levels versus the glyceraldehyde phosphate dehydrogenase mRNA level. In the 17 gliomas and 16 meningiomas examined, the mRNA of transforming growth factors alpha and beta, basic fibroblast growth factor, and VEGF were expressed at various levels. Among those 4 growth factors, the mRNA levels of VEGF, but not those of transforming growth factors alpha and beta and basic fibroblast growth factor, correlated significantly with vascularity in both gliomas (correlation coefficient r = 0.499; P < 0.05) and meningiomas (correlation coefficient r = 0.779; P < 0.001). These findings thus suggest that VEGF may be a positive factor in tumor angiogenesis in both human gliomas and meningiomas.

A programmable DNA origami nanospring that reveals force-induced adjacent binding of myosin VI heads.

Mechanosensitive biological nanomachines such as motor proteins and ion channels regulate diverse cellular behaviour. Combined optical trapping with single-molecule fluorescence imaging provides a powerful methodology to clearly characterize the mechanoresponse, structural dynamics and stability of such nanomachines. However, this system requires complicated experimental geometry, preparation and optics, and is limited by low data-acquisition efficiency. Here we develop a programmable DNA origami nanospring that overcomes these issues. We apply our nanospring to human myosin VI, a mechanosensory motor protein, and demonstrate nanometre-precision single-molecule fluorescence imaging of the individual motor domains (heads) under force. We observe force-induced transitions of myosin VI heads from non-adjacent to adjacent binding, which correspond to adapted roles for low-load and high-load transport, respectively. Our technique extends single-molecule studies under force and clarifies the effect of force on biological processes.

Macrophage infiltration and heme oxygenase-1 expression correlate with angiogenesis in human gliomas.

Macrophages are key participants in angiogenesis. In this study on human brain tumors, we first investigated whether macrophage infiltration is associated with angiogenesis and malignant histological appearance. Immunostaining of macrophages and small vessels in resected glioma specimens indicated that numbers of infiltrating macrophages and small vessel density were higher in glioblastomas than in astrocytomas or anaplastic astrocytomas. Macrophage infiltration was closely correlated with vascular density in human gliomas. Heme oxygenase-1 (HO-1), which is the rate-limiting enzyme in heme catabolism, was also associated with activated macrophages. Expression of mRNA encoding HO-1 was correlated with macrophage infiltration and vascular density in human glioma samples. Infiltrating macrophages were positively stained with anti-HO-1 antibody by immunohistochemical analysis, and in situ hybridization for HO-1 indicated that HO-1 was expressed in infiltrating macrophages in gliomas. HO-1 gene may be a useful marker for macrophage infiltration as well as neovascularization in human gliomas.

Time-resolved X-ray Tracking of Expansion and Compression Dynamics in Supersaturating Ion-Networks.

Supersaturation of a solution system is a metastable state containing more solute than can be normally solubilized. Moreover, this condition is thermodynamically important for a system undergoing a phase transition. This state plays critical roles in deposition morphology in inorganic, organic, polymer and protein solution systems. In particular, microscopic solution states under supersaturated conditions have recently received much attention. In this report, we observed the dynamic motion of individual ion-network domains (INDs) in a supersaturated sodium acetate trihydrate solution (6.4 M) by using microsecond time-resolved and high accuracy (picometre scale) X-ray observations (diffracted X-ray tracking; DXT). We found that there are femto-Newton (fN) anisotropic force fields in INDs that correspond to an Angstrom-scale relaxation process (continuous expansion and compression) of the INDs at 25 µs time scale. The observed anisotropic force-field (femto-Newton) from DXT can lead to new explanations of how material crystallization is triggered. This discovery could also influence the interpretation of supercooling, bio-polymer and protein aggregation processes, and supersaturated systems of many other materials.

Intracarotid infusion of leukotriene C4 selectively increases blood-brain barrier permeability after focal ischemia in rats.

Intracarotid infusions of leukotriene C4 (LTC4) were used to open selectively the blood-brain barrier (BBB) in ischemic tissue after middle cerebral artery (MCA) occlusion in rats. BBB permeability was determined by quantitative autoradiography using [14C]aminoisobutyric acid. Seventy-two hours after MCA occlusion, LTC4 (4 micrograms total dose) infused into the carotid artery ipsilateral to the MCA occlusion selectively increased the unidirectional transfer constant for permeability Ki approximately threefold within core ischemic tissue and tissue adjacent ot the ischemic core. No effect on BBB permeability was seen within nonischemic brain tissue or in ischemic tissue after only 24 h after MCA occlusion. gamma-Glutamyl transpeptidase (gamma-GTP) activity was decreased in capillaries in ischemic tissue at 48 and 72 h after infarction, compared to high gamma-GTP in normal brain capillaries and moderate gamma-GTP in capillaries in the ischemic tissue at 24 h after infarction. These findings suggest that normal brain capillaries resist the vasogenic effects of LTC4. In contrast, LTC4 increases permeability in capillaries of ischemic tissue, where gamma-GTP is decreased. gamma-Glutamyl transpeptidase, an enzyme that inactivates LTC4 to LTD4 and LTE4 to LTF4, may act as an "enzymatic barrier" in normal brain capillaries to leukotrienes.

Imaging peripheral benzodiazepine receptors in brain tumors in rats: in vitro binding characteristics.

Peripheral benzodiazepine binding constants for transplanted RG-2 gliomas and HD and LK Walker 256 tumors (metastatic breast carcinoma) were determined in Wistar rats using autoradiography. In addition, Kd and Bmax parameters for peripheral benzodiazepine receptors on RG-2 tumors were directly visualized using digital image analysis of autoradiograms. High specific binding of [3H]PK11195, a selective peripheral benzodiazepine ligand, had excellent topographical correlation to areas of histologically verified tumor. Scatchard analysis suggested a single class of peripheral binding sites with similar binding affinities in RG-2 and LK Walker 256 tumors and normal cortex. Bmax was 20-fold greater in glial tumors and 11.6- and 10.6-fold greater in LK and HK Walker 256 tumors, respectively, compared to normal cortex. The location of metastatic tumors, either intracerebrally or subcutaneously, did not effect their Kd or Bmax values. Kd and Bmax values for RG-2 tumors were similar whether determined densitometrically or by direct visualization with image analysis. Binding parameters within normal brain were difficult to visualize by image analysis due to the low level of specific binding. The ability to label specifically intracerebral tumor cells and to characterize the binding parameters shown in this study suggest that peripheral benzodiazepine receptor ligands could be utilized by PET to analyze directly a variety of tumors in humans.

A comparative study of apoptosis and proliferation in germinoma and glioblastoma.

Intracranial germinoma has a relatively good prognosis when treated with radiotherapy and chemotherapy, whereas glioblastoma has a poor prognosis irrespective of these treatments. Cell proliferation and cell death are opposing processes in tumor growth, with tumor progression reflecting the balance between proliferating and apoptotic cells. We investigated cell proliferation and cell death using MIB-1 staining and nick-end labeling in 13 germinomas in comparison with 11 glioblastomas. Expression of BAX and Bcl-2, which regulate apoptosis, were studied by immunohistochemistry. Although germinomas showed strong MIB-1 immunostaining similar to that seen in glioblastomas, germinomas included significantly more apoptotic cells. The ratio of apoptotic ratio to MIB-1 labeling index for germinomas was 72.9 +/- 36.9 (mean +/- SD), a higher, statistically significant ratio as compared with glioblastomas (14.5 +/- 11.2; P < 0.01). Furthermore, germinomas showed greater expression of BAX than did glioblastomas, while the expression of Bcl-2 was weak in both tumor types. A comparison of these apoptotic-related proteins showed that immunoreactivity for BAX was relatively higher in germinomas than in glioblastomas (P < 0.01), corresponding well to numerous apoptotic cells identified in germinoma tissues. These findings may account for the prognostic difference between germinoma and glioblastoma in the face of a similar proliferation potential according to MIB-1 immunostaining. The balance between cell proliferation and death should be considered when predicting outcomes in patients with intracranial tumors.

Stimulation of cell growth and DNA synthesis by peripheral benzodiazepine.

The effects of peripheral benzodiazepine receptor ligands on cell proliferation were evaluated. PK11195 increased the growth rate of C6 glioma cells by 20-30% in the nanomolar range in serum free medium. [3H]thymidine incorporation into C6 glioma cells also were increased 22% and 25% after treatment by PK11195 and Ro5-4864, respectively. The effect of PK11195 as a mitogenic agent was estimated by mitogenic agent was estimated by [3H]thymidine incorporation using Swiss 3T3 cells. PK11195 increased DNA synthesis 170% over control at 10 nM. Higher concentrations of benzodiazepines showed inhibition of the DNA synthesis. Peripheral benzodiazepine binding sites underwent downregulation after exposure to serum free medium or to 10 nM PK11195. These findings suggest that peripheral benzodiazepines may be involved in the regulation of cell proliferation as a growth factor in lower concentration and as a antiproliferative agent in higher concentration.

Photoconstructs of nerve guidance prosthesis using photoreactive gelatin as a scaffold.

We devised a novel nerve prosthesis composed of an elastomeric gelatinous tube and multifilament gelatinous fibers, both of which were prepared from styrene-derivatized gelatin, which allows in situ formation of a bioactive substance-incorporated gel. An in vitro study showed that the axonal regeneration potential of a photocured gelatin layer impregnated with laminin, fibronectin, and NGF was almost comparable with that of coated Matrigel. A nerve conduit and fibers prepared from photoreactive gelatin was subjected to visible-light irradiation with rotation in the presence of camphorquinone as a photoinitiator using a custom-designed apparatus. A sample of transparent gelatinous conduit with an inner diameter of 1.2 mm and a wall thickness of 0.6 mm and gelatin fibers ranging from 10 to 100 pm in diameter were produced. The photocured elastomeric gelatinous tube was flexible and had structural integrity that allowed mechanical handling without breaking. A novel nerve guidance prosthesis composed of tubes packed with fibers was assembled. This photofabrication technology may enable the design of a tailor-made shape and rapid morphogenesis and functional recovery of damaged nerve tissue.

Origin and evolution of the Department of Neurosurgery, Neurological Institute, Faculty of Medicine, Kyushu University.

The Department of Neurosurgery at Kyushu University had its origins within the First Department of Surgery and was established as a subspecialty at the Neurological Institute more than 30 years ago under the leadership of Katsutoshi Kitamura. Further development of the neurosurgical department has proceeded during the chairmanship of Masashi Fukui. These leaders and many other dedicated physicians and surgeons, nurses, investigators from other countries, and staff members have contributed to the creation of a research-oriented neurosurgical environment that interacts fruitfully with the other components of the Neurological Institute. This article describes the development of neurosurgery within Kyushu, which has been a highly cosmopolitan area throughout its long history. More specifically, this account outlines the origin and growth of the Department of Neurosurgery at Kyushu University.

Correlation of microanatomical localization with postoperative survival in posterior fossa ependymomas.

Twenty-two surgically treated infratentorial ependymomas were analyzed according to their anatomical origins and characteristics of extension in conjunction with the microsurgical anatomy of the fourth ventricle. The correlation between tumor origin and postoperative survival of the patients was also assessed. The tumors were classified into three types according to their origins and extensions: 1) midfloor-type: tumors originating from the caudal half of the fourth ventricular floor beneath the striae medullares. After occupying the fourth ventricular cavity, they extended downward through the foramen Magendie to the upper cervical level. 2) Lateral type: tumors arising from the vestibular area and/or the lateral recess. They grew not only inferiorly but also laterally to the cerebellomedullary cistern through the cerebellomedullary fissure and the foramen of Luschka. 3) Roof type: tumors originating from the roof of the ventricle. The overall cumulative survival rates at 2, 5, and 10 years were 84, 62, and 47%, respectively. Interestingly, the lateral-type tumors showed a significantly lower 5-year cumulative survival rate and mean survival time (21% and 40 months) when compared with midfloor-type tumors (73% and 170 months). Because the tumor originates near the vital neural structures and because each type has characteristics of extension, a clear knowledge of the microanatomical relationship between the tumor and the surrounding structures would be of great benefit for improving the operative outcome of posterior fossa ependymomas.

Three-dimensional comparison of peripheral benzodiazepine binding and histological findings in rat brain tumor.

Experiments were undertaken to determine the in vivo utility of the mixed benzodiazepine ligand [3H]flunitrazepam and the selective peripheral benzodiazepine ligand [3H]PK 11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide] to outline the borders of rat C6 glial tumors in three dimensions. Intravenous injection of [3H]flunitrazepam resulted in a tumor/cortex ratio of radioactive densities between 2.7 and 1.5 within the first 60 minutes after injection. [3H]PK 11195 demonstrated a higher tumor/cortex ratio (5.3) than [3H]flunitrazepam. For three-dimensional studies, images were generated from thionin-stained histological sections and autoradiograms. The mixed type benzodiazepine ligand [3H]flunitrazepam was superior in showing some of the normal anatomical structures surrounding the tumor, whereas [3H]PK 11195, a specific peripheral ligand, demonstrated higher tumor/brain contrast and superior topographical correlation between histological and autoradiographic images. Implications of peripheral benzodiazepine receptor ligands for positron emission tomography are discussed.

Patient survival and microsatellite instability in gliomas by high-resolution fluorescent analysis.

Deficient repair of nucleotide mismatches in the genome is considered a major factor in tumorigenesis. Such deficiency is evidenced by alterations in dinucleotide repeats of microsatellite sequences, specifically microsatellite instability (MSI) or replication errors. We investigated the frequency of MSI in human gliomas in terms of patient outcome. Frequency of MSI was estimated by examining five loci on chromosomes 2, 5, 10, 11, and 13 in 31 gliomas using high-resolution fluorescent microsatellite analysis. MSI was found at all loci in only 2 malignant gliomas (6.5%). MSI was detected at the D10S197 locus in 3 of 11 glioblastomas (27.2%) and 4 of 8 anaplastic astrocytomas (50%), while no MSI was detected in low-grade gliomas. Among patients with anaplastic astrocytoma, the 4 with MSI at D10S197 died from local recurrence less than 18 months after surgery, while 3 of the patients without MSI survived for more than 20 months. MSI at D10S197 may be a prognostic marker for patients with anaplastic astrocytomas.

Selective opening of the blood-tumor barrier by intracarotid infusion of leukotriene C4.

Intracarotid infusions of leukotriene C4 (LTC4) were used to selectively open the blood-tumor barrier in rats with RG-2 gliomas. Blood-brain and blood-tumor permeability was determined by quantitative autoradiography using 14C aminoisobutyric acid. Leukotriene C4 (4 micrograms total dose) infused into the carotid artery ipsilateral to the tumor increased twofold the unidirectional transfer constant for permeability within the tumor while no effect on permeability was seen in normal brain. No gamma glutamyl transpeptidase (gamma-GTP) activity was seen in tumor capillaries in contrast to high gamma-GTP in normal brain capillaries. These findings suggest that normal brain capillaries may resist the vasogenic effects of LTC4, while LTC4 will increase permeability in tumor capillaries. This could relate to the ability of gamma-GTP to act as an enzymatic barrier and inactivate leukotrienes in normal brain capillaries. Intracarotid LTC4 infusion may be a useful tool to selectively open the blood-tumor barrier for delivery of antineoplastic compounds.

Altered response to histamine in brain tumor vessels: the selective increase of regional cerebral blood flow in transplanted rat brain tumor.

The authors studied the effect of intracarotid administration of histamine on the regional cerebral blood flow (rCBF) in transplanted rat C6 glioma by the hydrogen clearance method. Histamine infusion at doses of 1 and 10 micrograms/kg/min produced an increase of rCBF in the tumor (24.6% +/- 16.4%, p < 0.002, and 37.6% +/- 18.2%, p < 0.0001, respectively) and also in brain surrounding the tumor (26.8% +/- 16.2%, p < 0.002, and 34.9% +/- 9.2%, p < 0.0001, respectively) without any significant changes in the ipsilateral hemisphere. Intravenous administration of pyrilamine (H1 antagonist) and cimetidine (H2 antagonist) reduced blood flow responses to histamine; cimetidine was a more effective blocking agent than pyrilamine. Intracarotid infusion of histamine (1 and 10 micrograms/kg/min) with intravenous injection of Evans blue dye disclosed the selective extravasation of dye in the tumor and the brain surrounding the tumor. These results indicated that brain tumor vessels could respond to histamine differently than normal brain capillaries. The mechanism of selective response to histamine could be explained either by increased permeability or by altered characteristics of histamine receptors in the tumor vessels.