Membrane orientation of the precursor 74-kDa form of L-histidine decarboxylase

Objective We previously demonstrated that, when expressed in COS-7 cells, L-histidine decarboxylase (HDC), which has neither an amino terminal signal sequence nor a hydrophobic membrane anchor, was localized in the endoplasmic reticulum (ER), although its orientation in the membrane remains to be clarified. Methods & Results Protease digestion and immunofluorescence analyses of the cells, of which plasma membrane was selectively permeabilized, revealed that the amino terminal 50-kDa portion of HDC is hardly accessible to proteases and antibodies added exogenously from the cytosolic side. Green fluorescent protein fused with the carboxyl terminal 20-kDa region of HDC at its carboxyl terminus exhibited the same characteristics as native HDC. Conclusion These results indicate that HDC is tightly associated with the ER membrane with its carboxyl terminal region exposed on the cytosolic side. Received 22 November 2005; returned for revision 28 December 2005; accepted by A. Falus 22 January 2006     

Cell-type-specific excitatory and inhibitory circuits involving primary afferents in the substantia gelatinosa of the rat spinal dorsal horn in vitro

The substantia gelatinosa (SG) of the spinal dorsal horn shows significant morphological heterogeneity and receives primary afferent input predominantly from Aδ- and C-fibres. Despite numerous anatomical and physiological studies, correlation between morphology and functional connectivity, particularly in terms of inhibitory inputs, remains elusive. To compare excitatory and inhibitory synaptic inputs on individual SG neurones with morphology, we performed whole-cell recordings with Neurobiotin-filled-pipettes in horizontal slices from adult rat spinal cord with attached dorsal roots. Based on dendritic arborization patterns, four major cell types were confirmed: islet, central, radial and vertical cells. Dorsal root stimulation revealed that each class was associated with characteristic synaptic inputs. Islet and central cells had monosynaptic excitatory inputs exclusively from C-afferents. Islet cells received primary-afferent-evoked inhibitory inputs only from Aδ-fibres, while those of central cells were mediated by both Aδ- and C-fibres. In contrast, radial and vertical cells had monosynaptic excitatory inputs from both Aδ- and C-fibres and inhibitory inputs mediated by both fibre types. We further characterized the neurochemical nature of these inhibitory synaptic inputs. The majority of islet, central and vertical cells exhibited GABAergic inhibitory inputs, while almost all radial cells also possessed glycinergic inputs. The present study demonstrates that SG neurones have distinct patterns of excitatory and inhibitory inputs that are related to their morphology. The neurotransmitters responsible for inhibitory inputs to individual SG neurones are also characteristic for different morphological classes. These results make it possible to identify primary afferent circuits associated with particular types of SG neurone.     

TAC-101, a benzoic acid derivative, inhibits liver metastasis of human gastrointestinal cancer and prolongs the life-span

We examined the anti-tumor effect of a novel benzoic acid derivative, TAC-101 (4-[3,5-bis(trimethylsilyl) benzamide] benzoic acid) on models with liver metastasis. Oral administration of TAC-101 significantly inhibited spontaneous liver metastasis of AZ-521 (human gastric cancer ) by orthotopic implan-tation to athymic nude mice. It also inhibited both the liver metastasis of AZ-521 induced by intrasplenic injection and the secondary lung metastasis from the liver. In addition, TAC-101 inhibited the proliferation of Co-3 (human colon adenocarcinoma) that formed a single nodule in the liver of athymic nude mice by intrahepatic implantation. The growth inhibitory effect of TAC-101 on AZ-521 experimental liver metastasis was observed when treatment was started on day 7, 14, or 21 which may correspond to the progressive stage of liver metastasis in clinical settings. Multiple administration of TAC-101 (8 mg/kg/day) significantly prolonged survival time of the animals with liver met astasis by intrasplenic injection of AZ-521 (T/C = 230%) and A549 (human lung adenocarcinoma; T/C = 186%). These effects of TAC-101 were stronger than those of 5-FU, CDDP or ATRA. Furthermore, TAC-101 inhibited the binding of AP-1 to DNA on electrophoretic mobility shift assay using nuclear extract of AZ-521 cells, although ATRA did not inhibit. These findings suggested that TAC-101 may be a candidate for a new class of anti-cancer agents for liver metastasis. © Rapid Science Ltd.     

Thermodynamic studies on poly(a) · poly(u) duplex-dye systems

The interaction between poly(A) · poly(U) duplex and dyes (proflavine (PF) and 9-aminoacridine (9-AA)) was studied by means of the heat of mixing. From the results, thermodynamic quantities of poly(A) · poly(U) duplex-dye systems for the intercalation process were estimated. The free energy change for the poly(A) · poly(U) duplex-(PF) system is smaller than that for the poly(A) · poly(U) duplex-(9-AA) system, indicating that PF intercalated into poly(A) · poly(U) duplex is more stable than the intercalated 9-AA. This conclusion is based on the absolute value of the heat of interaction between PF and poly(A) · poly(U) duplex being larger than that between 9-AA and the poly(A) · poly(U) duplex.     

Effects of sex hormones on induction of intestinal metaplasia by X-irradiation in rats.

The influence of sex hormones on induction of intestinal rnetaplasia was examined in 5 week old Crj: CD (SD) rats of both sexes. At the age of 4 weeks, the animals were gonadectornized and given testosterone or dimethyl estradiol (DES). One week after operation, they were irradiated with two 10 Gy doses of X-rays to the gastric region at a 3 day interval for a total of 20 Gy. At the termination of the experiment, 6 months after the X-irradiation, the incidence of intestinal metaplasia with alkaline phosphatase (ALP) positive foci in males was significantly higher than in females, in orchidectomized males or orchidectomized plus DES treated rats (P <0.01). On the other hand, the incidence of intestinal metaplasia with ALPpositive foci in normal females appeared lower than in ovariectomized females (P < 0.01), and was increased in rats by treatment with testosterone or decreased by DES. Numbers of foci of intestinal metaplasias with Paneth cells and total numbers appeared to increase in males treated with DES. The results suggested a promoting role for testosterone in the development of ALP positive lesions and indicated considerable heterogeneity between intestinal metaplasia subtypes.     

Nature and significance of calcium oxalate crystals in normal human thyroid gland

To elucidate the significance and nature of calcium oxalate crystals in the thyroid, we studied these crystals clinicopathologically and immunohistochemically in 182 normal thyroids from patients autopsied within 5 h of death. Under polarized light, calcium oxalate crystals showed brilliant birefringence and were invariably found within the colloid. The crystals were found in 73.1% of all cases but were more prevalent and denser in older individuals, with the highest prevalence (85.2%) being observed in those over 70 years of age. No crystals were seen in those under 10 years of age. Although underlying diseases seemed to have little influence, post-mortem delay apparently affected the prevalence and density of occurrence since the crystals tended to disappear with hours after death. An immunohistochemical study using anti-thyroid hormone antibodies revealed that the crystals were within negatively or weakly stained colloid and were not common in strongly stained colloid. These findings support the hypothesis that the occurrence of calcium oxalate crystals in normal human thyroid is associated with a low functional state of the thyroid follicles.     

Effects of nerve growth factor and glucocorticoid on cultured human pheochromocytoma cells

Primary cell cultures of two human pheochromocytomas (PC) that were associated with high serum levels of adrenaline and noradrenaline were developed to study the effects of nerve growth factor (NGF) and dexamethasone on the morphology and function of PC cells in vitro. By phase-contrast microscopy, cultured cells were small and hyperchromatic on the first day of culture; neurite-like processes that extended to other cells developed several days later and were maintained for more than 3 months. NGF (100ng/ml), dexamethasone (10^–5M), or NGF + dexamethasone were added to the culture media 2 weeks after the cultured cells had stabilized. Catecholamine concentrations in the medium were maintained at higher levels after addition of NGF, dexamethasone, or NGF + dexamethasone as compared to control cells. In the presence of NGF, extension of neurite-like processes was clearly accelerated, while high levels of dexamethasone inhibited growth of processes. These in vitro studies showed that the addition of NGF or the removal of dexamethasone induces differentiation of adrenal neurons present in pheochromocytomas, suggesting that adrenocortical steroid hormones influence the morphological control of adrenal medullary cells.     

Heats of dilution of poly[styrene-ran-(butyl methacrylate)] solutions measured with an automatic flow microcalorimeter

In order to obtain information about the thermodynamic behaviour of copolymers in solution, we built an automatic flow microcalorimeter equipped with a computer that satisfies the requirements of accuracy, reliability, and rapidity for the measurement of heats of dilution of copolymer solutions. By using this apparatus, the heats of dilution of the random copolymer, poly[styrene-ran-(butyl methacrylate)] in ethyl methyl ketone solutions were measured at 298 K. Furthermore, light scattering of the copolymer and its constituent homopolymer solutions was measured at 298 K. From the combination of the heat of dilution and the light scattering results, thermodynamic and intramolecular interaction parameters of the copolymer chain were determined.     

Ultrastructure of human pheochromocytoma cells cultured for long periods

We conducted ultrastructural analysis of human pheochromocytoma (PC) cells maintained in primary culture for about 10 months. The cells were first isolated by the enzymatic treatment of a surgically resected tissue specimen obtained from a 37-year-old man with PC, a condition which is characterized by elevated blood levels of adrenaline and noradrenaline. It was found that noradrenaline production in the medium continued until the 90th day of culture (1330 pg/ml). The production level decreased to 20 pg/ml on the 180th day, and to 18 pg/ml on the 300th day. Examination under a transmission electron microscope (TEM) at 4 weeks of culture revealed electron-dense granules (about 200 nm in size and, presumably, rich in catecholamines), which were also observed in the tumor cells from the original PC tissue. Neurite-like processes grew at around 1 week of culture, and were still maintained at 6 months of culture. But, after 6 months of culture, the neurite-like processes contained a rosary-like elevated structure, which was suggestive of cell degeneration, as determined by a plasma polymerization replica method and observed with a scanning electron microscope. When cells were examined under the TEM, fewer electron-dense granules were observed in the cell bodies, with more numerous lipofuscin-like granules and filaments. Thus, electron-dense granules, which, presumably, contain catecholamines, were seen in a long-term culture of human PC cells. These granules decreased in number in parallel with the decrease in catecholamine levels in the culture.     

Evaluation of a latex agglutination method for the rapid identification of Campylobacter jejuni/coli

A latex agglutination assay was developed to identify Campylobacter jejuni and Campylobacter coli. We evaluated the specificity, reproducibility and utility of the assay for clinical use and the following results were obtained. 1) To prepare standardized antigen, bacterial cells must be suspended to a density of 1 to 5 McFarland unit, and heated at 121 degrees C for 10 to 30 min. 2) Bacterial cells may be suspended either in the solution provided with the kit, or in physiological saline, without affecting the results. 3) Of C. jejuni, 94 strains, 6 of C. coli, and 3 of "Campylobacter upsaliensis", all tested positive without exception. All other Campylobacter species, encompassing 13 species and 80 strains, were negative. An additional 9 species and 30 strains, of non-Campylobacter gram negative bacteria, isolated on the Campylobacter selection agar medium, also were uniformly negative. Based on these results, we conclude that bacteria testing positive with the kit can be identified as C. jejuni/coli. Interestingly, "C. upsaliensis", although isolated very rarely from the clinical specimens, also tested positive.