We recently developed a new dialysis culture system (termed LIFROC-device) for the cultivation of lymphokine-activated killer cells (Murata et al., 1990, 1991). In the present study, we applied the LIFROC-device (400 ml culture vessel) to the cultivation of mammalian cells for the production of biologically active substances. We cultured mouse-mouse hybridoma TP-709, secreting anti-tisse plasminogen activator (tPA) monoclonal antibody (mAb), recombinant CHO GT19, secreting hGH, and human melanoma Bowes cells, secreting tPA. With the LIFROC-device, TP-709 grew to a maximal cell density of 3.8 x 10(6) cells/ml and and produced 480 micrograms/ml (192 mg in total) of mAb. GT19 reached a cell density of 2.2 x 10(6) cells/ml and produced 302 micrograms/ml (120 mg in total) of hGH. Bowes cells expanded to 4.4 x 10(6) cells/ml and secreted 8.5 micrograms/ml (3.3 mg in total) of tPA. The protein concentration in the culture broths of the LIFROC-device became 7-200 times higher than that of batch culture. Thus, the LIFROC-device can be applied to protein production as well as cell growth with high efficiency.

Animals↗

PubMed1993

Unsuccessful attempt to detect genetic mutation in tuberous sclerosis utilizing the polymerase chain reaction.

Although tuberous sclerosis is supposed to be a phacomatosis inherited as an autosomal dominant trait, many cases develop without any affected parents or grandparents. In recent years, many vigorous investigations have been concentrated on finding the mutant gene, and possible candidate genes have been mapped on 9q34 and other chromosomes. In order to find a way of diagnosing asymptomatic carriers or patients, we tried to detect restriction fragment length polymorphisms (RFLPs) using the technique of polymerase chain reaction (PCR). We used a probe, MCOA12, which is located on 9q34 and has been known to show RFLPs in Caucasian tuberous sclerosis patients. However, we could not find a correlation between the phenotype and RFLP pattern in seven of eight families.

Base Sequence↗

PubMed1993

Isolation and characterization of a DNA fragment containing various kinds of repetitive sequences located on human chromosome 21.

In order to investigate the repetitive sequences located on human chromosome 21, we have isolated DNA fragments containing Alu sequences. One of the clones, p1, was chosen for further study, because it contained repetitive sequences different from the Alu sequence. Nucleotide sequence analysis of p1 indicates that p1 contains L1 and O-family sequences. Interestingly, when the L1 sequence was used as a probe, a discrete band of 5 kb was seen in HindIII-digested DNA from somatic cell hybrids containing human chromosome 21 as the sole human chromosome. The L1 sequence was rearranged and was interrupted by O-family sequence, which was flanked by 6 bp target site duplications. Since all three repetitive sequences are known to act as retroposons, these results imply that there is an integration hot spot on human chromosome 21. The sequence was mapped within 21q11-21.

Animals↗

PubMed1993

Proconvulsive effects of histamine H1-antagonists on electrically-induced seizure in developing mice.

The purpose of this study was to investigate the developmental differences in seizure susceptibility in mice and the roles of the histaminergic neuron system in inhibition of convulsions in development. First, we studied developmental differences in electrically-induced seizures. Since the 14-day-old mice showed a different seizure pattern from that of older mice, we evaluated the seizure susceptibility of mice older than 21 days. The durations of all the convulsive phases were significantly increased in 21- and 30-day-old mice, compared with older mice. Second, pyrilamine (or mepyramine), ketotifen, and d-chlorpheniramine, centrally-acting H1-antagonists, increased the durations of all the convulsive phases in the 21- and 30-day-old mice, but did not increase duration in 42-day-old mice. Terfenadine and astemizole, H1-antagonists that hardly enter the brain, did not affect the durations of all the convulsive phases in 21-, 30- and 42-day-old mice. The proconvulsant effect of centrally-acting H1-antagonists in 21- and 30-day-old mice were considered to be mediated via the central H1-receptors. Thus, the histaminergic neuron system may have an important physiological role in inhibition of seizures in 21- and 30-day-old mice which have higher seizure susceptibility. This would compensate for the immaturity of the other protective neuron systems such as NMDA receptor complexes and GABA receptors. In conclusion, the present findings support the view that the central histaminergic system plays a role in inhibition of convulsions.

Aging↗

PubMed1993

The behavioral and biochemical effects of thioperamide, a histamine H3-receptor antagonist, in a light/dark test measuring anxiety in mice.

We investigated the effects of thioperamide, a histamine H3-receptor antagonist, in a light/dark test measuring anxiety in mice. Thioperamide (20 mg/kg) slightly affected the locomotion and time spent in a light zone, and shuttle crossing. However, the decreases of these parameters were significant only when the animals were pretreated with zolantidine, a histamine H2-receptor antagonist. Moreover, the decreased parameters induced by the combination of thioperamide and zolantidine were reversed by pretreatment with pyrilamine, a histamine H1-receptor antagonist. These data suggest that thioperamide induces the release of neuronal histamine, which in turn stimulates both H1- and H2-receptors to produce the anxiogenic effect. The stimulation of histamine H1-receptors may mediate the anxiety, while H2-receptors may play a role in masking the anxiogenic effect. Thus, the present study suggests the involvement of endogenous neuronal brain histamine in anxiety. In the biochemical study, a previous report showed that thioperamide accelerated the release of neuronal histamine in the brains of mice [Sakai et al., Life Sciences, 48, 2397-2404(1991)]. This study also demonstrated that thioperamide did not affect the turnover rate of noradrenaline, dopamine, or serotonin in the brains of mice, which indicates that thioperamide is a good pharmacological tool for accelerating the release of neuronal histamine in the brain.

Animals↗

PubMed1993

Titanium lymph node pigmentation in the reconstruction plate system of a mandibular bone defect.

A case of titanium pigmentation that involved a submandibular lymph node is reported. A 41-year old man had been treated for ameloblastoma by partial resection of the mandible followed by reconstruction with the use of a titanium plate to bridge the mandibular defect. Titanium pigmentation occurred in the surrounding soft tissue adjacent to the plate and within a submandibular lymph node 2 years after reconstruction.

Adult↗

PubMed1993

Effects of (S)-alpha-fluoromethylhistidine and (R)-alpha-methylhistamine on locomotion of W/Wv mice.

We studied the effects of inactivators of the central histaminergic neuron system, (R)-alpha-methylhistamine, a histamine H3 receptor agonist, and (S)-alpha-fluoromethylhistidine, a histamine synthesis inhibitor, on locomotor activity and brain histamine content of mast cell-deficient W/Wv mice using a recently developed high-performance liquid chromatography system coupled with a fluorometric detector. IP injection of (R)-alpha-methylhistamine (6-50 mg/kg) increased brain histamine content after 1 h but caused no significant change in locomotor activity. IP injection of (S)-alpha-fluoromethylhistidine decreased brain histamine content at doses of 6-50 mg/kg and locomotor activity at doses of 12.5-50 mg/kg. However, locomotor activity was decreased significantly (in Student's t-test) by sequential administrations of (S)-alpha-fluoromethylhistidine (6 mg/kg) and (R)-alpha-methylhistamine (12.5 or 25 mg/kg), but not by (S)-alpha-fluoromethylhistidine (6 mg/kg) and other doses of (R)-alpha-methylhistamine (6 or 50 mg/kg). These results support the hypothesis that the central histaminergic neuron system is involved in the control of spontaneous locomotion or alertness.

Animals↗

PubMed1993

The spatial distribution of spindle microtubules in anaphase 3Y1 cells.

The spatial distribution of the microtubules (MT) in the rat 3Y1 cells in mitosis was investigated by immunoelectron microscopy and computer-graphic reconstruction of serial thin sections. In anaphase the interzone-MT increased in number gradually with advancing phase, while the kinetochore-MT in half-spindles decreased. The interzone-MT overlapped with each other at the equatorial region of the cell, and they formed a specific structure called the 'stem bodies'. The ends of the interzone-MT opposite to the stem bodies often attached to chromosomes but not to the poles. The stem bodies were not labeled with immunogold particles of anti-alpha tubulin. Some of the stem bodies or MT which originate from stem bodies were found just beneath the plasma membrane in the equatorial region where abundant actin filaments appear showing the formation of the contractile ring and subsequently the cleavage furrow begins. On the basis of these observations it is assumed that the interzone-MT is involved both in the separation of chromosomes in anaphase and in the formation of the cleavage furrow in telophase.

Anaphase↗

PubMed1993

Detection of Epstein-Barr virus DNA and EBV-determined nuclear antigen in angioimmunoblastic lymphadenopathy with dysproteinemia type T cell lymphoma.

Six cases of angioimmunoblastic lymphadenopathy with dysproteinemia (AILD)-like T cell lymphoma were analyzed by immunohistochemical staining, polymerase chain reaction (PCR) and Southern blot analysis. Five cases out of six showed gene rearrangements of the T cell receptor beta chain, indicating the existence of a clonal T cell proliferation. Epstein-Barr virus (EBV) DNA was detected in all six AILD type cases by PCR amplifying the sequence located in the internal repeat I, and confirmed by Southern blot hybridization, using a BamHI-W fragment as a probe. Detection of the EBV genome occurred more frequently as compared with other types of lymphoid disorders. Furthermore, EBV determined nuclear antigen (EBNA) was detected in UCHL-1 (CD45RO, pan-T cell marker) positive cells by immunostaining. These results suggest that a significant number of AILD type cases are T cell origin lymphomas and EBV infection may be relevant to the biological features of this type of lymphoma.

Adolescent↗

PubMed1993

Tissue factor production by cultured rat glomerular epithelial cells.

It is well known that fibrin deposition in Bowman's space in association with crescent formation may play an important role in progressive glomerular injury in crescentic glomerulonephritis. Recent reports describe the presence of a procoagulant activity (PCA) in the glomeruli and its increased expression in human and experimental nephritis. The cells that synthesize PCA have not yet been identified. We attempted to determine if glomerular epithelial cells (GEC), one of the prominent cell populations in the crescent, can produce PCA. The PCA of cultured rat GEC was measured by clotting and amidolytic assays. The cultured GEC yielded PCA with the characteristics of a tissue factor, and this PCA was stimulated by interleukin 1, tumour necrosis factor-alpha, and lipopolysaccharide. We concluded that GEC produce tissue-factor-like PCA and thereby may contribute to fibrin deposition, which, along with macrophage or monocyte infiltration, leads to crescent formation in crescentic glomerulonephritis.

Animals↗

PubMed1993

Evaluation of oxidized low-density lipoprotein and large molecular size low-density lipoproteins in atherosclerosis.

To study the roles of modified low-density lipoprotein (LDL) and the molecular size of LDL in atherogenesis, the following studies were carried out. Eight white male rabbits fed a standard oriental diet with 1% cholesterol were used to isolate LDL and to observe changes in the molecular size of LDL due to cholesterol feeding. The tissue LDLs in the aorta were analyzed to confirm the existence of modified LDL (namely, LDL with peroxidized cholesteryl ester) by thin-layer chromatography. In addition, plasma LDLs were isolated from 18 patients with myocardial infarction and 11 patients with angina pectoris to confirm the existence of LDL with peroxidized cholesteryl ester. Each LDL separated consisted of 3 fractions; namely, IDL (1.006-1.018), LDL1 (1.019-1.052) and LDL2 (1.053-1.063) by sequential ultracentrifugation. The molecular sizes of LDL were measured by a planimeter from electron microscopic photographs, with negative staining. The estimation of peroxidized cholesteryl linoleate in LDL was performed using our method. The modified LDLs with peroxidized cholesteryl ester were poorly estimated in the LDL separated from the plasma of cholesterol-fed rabbits and from the aorta extraction after 16 weeks of feeding. The peroxidized cholesteryl ester was clearly identified in the plasma LDLs of the patients with myocardial infarction and angina pectoris, and in whole extracts from human aortic atheroma, although it was not clearly identified in the tissue LDL fraction. The molecular sizes of LDL1 enlarged week by week with cholesterol feeding, but two fractions of IDL and LDL2 did not change in size. The infusion of cholesterol-rich LDL of large molecular size or LDL with peroxidized cholesteryl ester into the vessels led to fixation, on the surface of the arteries of many platelets, red cells, and white cells, and to marked irregularities in the endothelial folds. The evidence suggests that atheromas, formed in a short period in rabbits with cholesterol feeding, are caused mainly by the increase in LDL1 of large molecular size, and that foam cells, formed in human atheromas, are caused mainly by the production of modified LDL with peroxidized cholesteryl ester.

Animals↗

PubMed1993

[Effect of aging on sensitivity of blood pressure to salt].

We investigated the correlation between aging and sensitivity of blood pressure to salt. 88 non-treated essential hypertensives were divided into four groups: less than 40 years old (n = 20), 40-49 years old (n = 20), 50-59 years old (n = 39), and greater than 60 years old (n = 11). Changes of blood pressure, plasma renin activity (PRA), plasma aldosterone concentration (PAC), plasma norepinephrine (PNE), and plasma epinephrine (PE) due to salt load were compared among four groups. Salt sensitivity of blood pressure was increased with aging, and there was a positive correlation between them (r = 0.30, p < 0.01). Decrement of PRA due to salt load was decreased with aging, and there was a negative correlation between them (r = -0.35, p < 0.05). PCA and PNE were suppressed by salt load, and the decrement degrees did not change with age. PE did not change by salt load. We conclude that salt sensitivity is increased with age in essential hypertensives, and renin-angiotensin system might be involved in it.

Adult↗

PubMed1993

[Aging and serum protein].

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Adult↗

PubMed1993

Molecular cloning of cDNA encoding rat homologue of CD5 and CD8 alpha: evidence that R1-3B3 and R1-10B5 monoclonal antibodies detect rat CD5 and CD8 antigens, respectively.

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Amino Acid Sequence↗