Pseudorandom testing technique for the characterization of local hemodynamic control

Summary In order to investigate the low frequency properties of renal and femoral hemodynamic variables, pseudorandom testing techniques were used. The arterial flow of each bed, in separate experiments, was modulated by a low amplitude signal based on a pseudorandom binary sequence (PRBS) generated by digital computer.The cross-correlation functions between input flow and arterial pressure, venous pressure, and venous flow exhibit damped oscillations in all cases. These responses are parameterized in terms of a damping ratio () and an undamped natural frequency _n for a second order model. The parameters of the model are dependent upon the state of the bed as defined by mean arterial and venous pressures, mean flow through the bed, resistance, and oxygen consumption.The results of this study offer further insight into the dynamic low frequency autoregulation phenomenon for the renal and femoral beds of the dog.Supported by NIH Grant HE 11747.     

The molecular structures of 1,1-diphenylethyl methacrylate and triphenylmethyl methacrylate

The molecular structures of 1,1-diphenylethyl methacrylate (1,1-DPEMA) and triphenylmethyl methacrylate (TrMA) were determined by means of X-ray diffraction. 1,1-DPEMA: monoclinic, space group P2₁/a,a = 9,666(6), b = 19,94(2), c = 8,132(6) Å, β = 104,49(7)°, and Z = 4; TrMA: monoclinic, space group P2₁/n, a = 17,349(3), b = 9,487(2), c = 11,254(2) Å, β = 102,30(2)°, and Z = 4. Both structures were solved by the direct method and refined by the block-diagonal least-squares procedure to R = 0,175 and 0,056 for non-zero reflections, respectively. In both molecules, conformations about the C(1)? C(2) and C(1)? O(1) bonds are all synperiplanar and one of the two or three phenyl groups attached to the C(5) atom is in trans to the O(2).     

Difference in the expression of three tight junction proteins, barmotin, occludin, and ZO-1, in phenotypically different human colon cancer cell lines

Epithelioid disorganization is a hallmark of gastrointestinal cancers and is believed to be associated with malignant phenotypes such as invasiveness and the potentiality for metastasis. Although tight junctions (TJs) are known to be crucial for the maintenance of polarized organization of the gastrointestinal epithelium, changes in the TJ proteins in human cancers have not yet been fully elucidated. In this report, we investigated the expression and localization of three TJ proteins-barmotin (7H6 antigen), occludin, and ZO-1-in three phenotypically different human colon cancer cell lines exhibiting differnt grades of epithelioid organization. All three proteins were localized at the most apical part of the cell border corresponding to the site of TJs in T₈₄ cells, in which epithelioid organization was well preserved. In contrast, in COLO320DM cells, which showed no epithelioid phenotypes, occludin was not detectable at either the protein or mRNA level, although barmotin and ZO-1 were present in the cytoplasm. In the third cell line, DLD-1, which showed an epithelioid phenotype intermediate between T₈₄ and COLO320DM, aberrant expression of occludin was found in the basolateral cell membrane. On the other hand, barmotin was present in the cytoplasm, whereas ZO-1 was localized at the cell border. These observations showed that changes in the expression of TJ proteins occur in close correlation with epithelioid disorganization in human colon cancers.     

Antitumor immune response by CX3CL1 fractalkine gene transfer depends on both NK and T cells

The CX3C chemokine fractalkine (CX3CL1) exists as both a membrane-bound form promoting firm cell-cell adhesion and a soluble form chemoattracting leukocytes expressing its receptor CX3CR1. When adenoviral vector expressing mouse fractalkine (AdFKN) was transduced to the tumor cells, fractalkine was expressed as both membrane-bound form on the tumor cells and soluble form in the supernatant in vitro. Intratumoral injection of AdFKN (1 x 10(9)PFU/tumor) into C26 and B16F10 tumors resulted in marked reduction of tumor growth compared to control (C26: 86.5%, p<0.001; B16F10: 85.5%, p<0.001). Histological examination of tumor tissues revealed abundant infiltration of NK cells, dendritic cells, and CD8(+) T lymphocytes 3 and/or 6 days after treatment with AdFKN. Splenocytes from mice treated by AdFKN developed tumor-specific cytotoxic T cells, and thereby protected from rechallenging with parental tumor cells. Antitumor effects by AdFKN were completely abrogated in both NK cell-depleted mice and CD8(-/-) mice, and partially blocked in CD4(-/-) mice. These data indicated that fractalkine mediates antitumor effects by both NK cell-dependent and T cell-dependent mechanisms. This study suggests that fractalkine can be a suitable candidate for immunogene therapy of cancer because fractalkine induces both innate and adaptive immunity.     

Signalling during hypoxia in human T lymphocytes – critical role of the src protein tyrosine kinase p56Lck in the O2 sensitivity of Kv1.3 channels

T lymphocytes encounter hypoxia when they migrate to pathological sites such as tumours and wounds. The inability of T cells to provide an efficient defence at these sites can in part be explained by the hypoxic environment. Kv1.3 channels, important components of the T cell activation process are inhibited by hypoxia and their inhibition accounts for a hypoxia-induced decrease in T cell proliferation. Although Kv1.3 channels play a key role in T cell O₂ sensing, the signalling mechanisms mediating their response to hypoxia are still not understood. In this study, we show that the src-protein tyrosine kinase p56Lck (Lck) is required for Kv1.3 channel response to hypoxia. Pre-exposure to the src inhibitor PP2 abolished the hypoxia-induced inhibition of Kv1.3 channels in primary human T lymphocytes. Moreover, Kv1.3 channel sensitivity to hypoxia was lost in Lck-deficient Jurkat T cells. Further studies with recombinant Kv1.3 channels showed that Kv1.3 channels lack intrinsic O₂ sensitivity, but delivery of Lck into the cells and transfection of a constitutively active Lck (Y505FLck) restored their sensitivity to hypoxia. Although Lck is necessary for the Kv1.3 channel response to hypoxia, it does not directly inhibit Kv1.3 channels. Indeed, under normal oxygen tension, delivery of active Lck into L929 cells and overexpression of Y505FLck did not decrease recombinant Kv1.3 currents. On the contrary, activation of endogenous src kinases increased wild-type Kv1.3 currents in T lymphocytes. Our findings indicate that Lck is required for the acute response to hypoxia of human T lymphocytes as it is necessary to confer O₂ sensitivity on Kv1.3 channels.     

Increased production of lipocalin-type prostaglandin D synthase in leptomeningeal cells through contact with astrocytes

Lipocalin-type prostaglandin D synthase (L-PGDS) is dominantly expressed in the leptomeninges surrounding the brain and secreted into the cerebrospinal fluid as β-trace, a major cerebrospinal fluid protein. To examine the interaction between the leptomeninges and the brain parenchyma, we co-cultured rat leptomeningeal cells with cells dissociated from the neonatal rat cortex and found that the production of L-PGDS was remarkably increased after the co-cultivation. A similar increase in L-PGDS production was observed by the co-culturing of the leptomeningeal cells with cells dissociated from astrocyte-rich cultures or with 1321-N1 astrocytoma cells. When a crude membrane fraction prepared from 1321-N1 cells was added to leptomeningeal cell cultures, L-PGDS gene expression was slowly increased up to 48 h after the addition. These results indicate that leptomeningeal cells enhance their L-PGDS production by a slow activation of L-PGDS gene expression through their contact with astrocytes.     

Analgesic action of loperamide,an opioid agonist,and its blocking action on voltage-dependent Ca2+ channels

We investigated the relationship between the antinociceptive effect of the opiate agonist loperamide at the spinal level and its inhibitory effect on calcium influx. Intrathecal administration of loperamide showed a significant antinociceptive effect in the formalin test, which was not prevented by naloxone. On the other hand, no significant effects were observed by nicardipine, an L-type specific blocker, or by BAY K8644, an L-type specific agonist, suggesting no significant role of L-type calcium channels in nociceptive signal transduction. Loperamide suppressed the calcium influx in dorsal root ganglion neurons. As the antinociceptive effect of loperamide was not affected by naloxone or other calcium channel blocking toxins, and loperamide showed a direct inhibitory effect on calcium-influx, the analgesic effect of intrathecally injected loperamide might be due to its blockade of the voltage-dependent calcium channels at the terminals of the primary afferent fibers.     

A new pseudo-peptide of Arg-Gly-Asp (RGD) with inhibitory effect on tumor metastasis and enzymatic degradation of extracellular matrix

A series of pseudo-peptide analogs of the Arg-Gly-Asp (RGD) sequence of fibronectin have been synthe-sized, and their anti-metastatic effects in mice and inhibitory effects on tumor cell invasion in vitro have been examined. The partially modified retro pseudo-peptide of RGD, R_rev-COCH₂CO-D (FC-63), was more effective in inhibiting tumor metastasis than the original RGDS peptide. Replacement of the malonyl moiety of FC-63 with a carboxyethylene linkage (R_rev-COCH₂CH₂-D, FC-303 ) achieved more potent inhibition of lung metastasis of melanoma cells than FC-63. Among the analogs, FC-336, a p-xylylendiamine derivative having two FC-303 moieties, showed the most potent inhibitory effect on experimental lung metastasis produced by i.v. co-injection with B16-BL6 melanoma or colon 26 M3.1 cells in a dose-dependent manner. Multiple administrations of FC-336 after tumor inoculation also showed efficient therapeutic potency against spontaneous lung metastasis of B16-BL6 melanoma in mice. Furthermore, FC-336 effectively inhibited the invasion, migration and adhesion of tumor cells in vitro, but its inhibitory effects were not more than those of RGDS peptide. Zymography analysis revealed that FC-336 inhibited the degradation of gelatin substrate by matrix metalloproteinases (MMPs) produced by tumor cells, while the RGDS peptide did not affect the enzymatic degradation. These findings indicate that the pseudo-peptides of the RGD sequence, possessing the inhibitory property of the degradation by MMPs differently from original RGD-containing peptides, may be advantageous and useful in preventing tumor metastasis. © Rapid Science 1998     

Requirement of IL-5 for induction of autoimmune hemolytic anemia in anti-red blood cell autoantibody transgenic mice

IL-5, IL-10 and lipopolysaccharide (LPS) are known to activateB-1 cells in vivo in normal mice and anti-red blood cell autoantibodytransgenic mice (HL mice). To assess the exact role of IL-5in proliferation and activation of peritoneal B-1 cells, weanalyzed IL-5 receptor chain-deficient HL (IL-5R^–/–x HL) mice generated by the cross between IL-5R^–/–and HL mice. In IL-5R^–/– x HL mice, Ig-producingB-1 cells in the peritoneal cavity were negligible, althoughthe total number of B-1 cells in the peritoneal cavity wereas many as 30% of that in HL mice. Moreover, LPS- or IL-10-induceddifferentiation of B-1 cells into antibody-producing cells wasseverely impaired in IL-5R^–/– x HL mice. We alsoused in vivo 5-bromo-2'-deoxyuridine labeling to estimate theproliferation of B-1 cells in IL-5R^–/– mice. Theabsence of IL-5R did not affect spontaneous proliferation ofperitoneal B-1 cells. However, induced proliferation of peritorealB-1 cells by oral administration of LPS was markedly impairedin IL-5R^–/– mice. These results suggest that IL-5is required for activation-associated proliferation of B-1 cellsbut not for their spontaneous proliferation and support theidea that IL-5 plays an important role on the induction of autoantibodyproduction from B-1 cells.     

Detection of the Epstein-Barr virus in primary gastric lymphoma by in situ hybridization

The Epstein-Barr virus (EBV) has been shown to be associated with numerous human malignancies including Burktt's lymphoma and nasopharyngeal lymphoepithelioma. In addition, some typical gastric adenocarcinomas were also recently reported to demonstrate EBV relevance. The present study was designed to detect EBV in primary gastric lymphoma, using the in situ hybridization (ISH) method, in which oligo-nucleotide probes for the EBERl RNA and the EBV DNA W region have been used. Of the 49 cases of primary gastric lymphoma studied, which all showed B cell immunopheno-type, EBER1 sequences could only be found in four cases, including two low-grade cases and two high-grade cases of histological subtypes while the number of positive cells was less than 50% of the tumor cells. In one case of low-grade mucosa associated lymphoid tissue (MALT) lymphoma, the EBER1 -positive neoplastic cells were found in the regional lymph node, but the primary site of the stomach showed no positive signals. The EBV presence was further confirmed by the EBV DNA ISH. Using the ISH method, rare or occasional positive lymphoid cells (probably non-tumorous bystander cells) could be detected in 10 other cases including all histological subtypes. The present study shows that only a small proportion of primary gastric lymphoma is associated with EBV, and such positive cases could be found in both high- and low-grade histological subtypes. It is also suggested that the EBV presence in the neoplastic cells of some cases of primary gastric lymphoma is most likely a secondary phenomenon.