Alternative splicing variants of IkappaB beta establish differential NF-kappaB signal responsiveness in human cells.

To release transcription factor NF-kappaB into the nucleus, the mammalian IkappaB molecules IkappaB alpha and IkappaB beta are inactivated by phosphorylation and proteolytic degradation. Both proteins contain conserved signal-responsive phosphorylation sites and have conserved ankyrin repeats. To confer specific physiological functions to members of the NF-kappaB/Rel family, the different IkappaB molecules could vary in their specific NF-kappaB/Rel factor binding activities and could respond differently to activation signals. We have demonstrated that both mechanisms apply to differential regulation of NF-kappaB function by IkappaB beta relative to IkappaB alpha. Via alternative RNA processing, human IkappaB beta gives rise to different protein isoforms. IkappaB beta1 and IkappaB beta2, the major forms in human cells, differ in their carboxy-terminal PEST sequences. IkappaB beta2 is the most abundant species in a number of human cell lines tested, whereas IkappaB beta1 is the only form detected in murine cells. These isoforms are indistinguishable in their binding preferences to cellular NF-kappaB/Rel homo- and heterodimers, which are distinct from those of IkappaB alpha, and both are constitutively phosphorylated. In unstimulated B cells, however, IkappaB beta1, but not IkappaB beta2, is found in the nucleus. Furthermore, the two forms differ markedly in their efficiency of proteolytic degradation after stimulation with several inducing agents tested. While IkappaB beta1 is nearly as responsive as IkappaB alpha, indicative of a shared activation mechanism, IkappaB beta2 is only weakly degraded and often not responsive at all. Alternative splicing of the IkappaB beta pre-mRNA may thus provide a means to selectively control the amount of IkappaB beta-bound NF-kappaB heteromers to be released under NF-kappaB stimulating conditions.

Functional interference of Sp1 and NF-kappaB through the same DNA binding site.

Gene activation by NF-kappaB/Rel transcription factors is modulated by synergistic or antagonistic interactions with other promoter-bound transcription factors. For example, Sp1 sites are often found in NF-kappaB-regulated genes, and Sp1 can activate certain promoters in synergism with NF-kappaB through nonoverlapping binding sites. Here we report that Sp1 acts directly through a subset of NF-kappaB binding sites. The DNA binding affinity of Sp1 to these NF-kappaB sites, as determined by their relative dissociation constants and their relative efficiencies as competitor DNAs or as binding site probes, is in the order of that for a consensus GC box Sp1 site. In contrast, NF-kappaB does not bind to a GC box Sp1 site. Sp1 can activate transcription through immunoglobulin kappa-chain enhancer or P-selectin promoter NF-kappaB sites. p50 homodimers replace Sp1 from the P-selectin promoter by binding site competition and thereby either inhibit basal Sp1-driven expression or, in concert with Bcl-3, stimulate expression. The interaction of Sp1 with NF-kappaB sites thus provides a means to keep an elevated basal expression of NF-kappaB-dependent genes in the absence of activated nuclear NF-kappaB/Rel.

The Bcl-3 oncoprotein acts as a bridging factor between NF-kappaB/Rel and nuclear co-regulators.

The proto-oncoprotein Bcl-3 is a member of the IkappaB family and is present predominantly in the nucleus. To gain insight into specific nuclear functions of Bcl-3 we have isolated proteins that interact with its ankyrin repeat domain. Using the yeast two-hybrid-system we identified four novel binding partners of Bcl-3 in addition to NF-kappaB p50 and p52, previously known to associate with Bcl-3. The novel Bcl-3 interactors Jab1, Pirin, Tip60 and Bard1 are nuclear proteins which also bind to other transcription factors including c-Jun, nuclear factor I (NFI), HIV-1 Tat or the tumor suppressor and PolII holoenzyme component Brca1, respectively. Bcl-3, p50, and either Bard1, Tip60 or Pirin are sequestered into quarternary complexes on NF-kappaB DNA binding sites, whereas Jab1 enhances p50-Bcl-3-DNA complex formation. Furthermore, the histone acetylase Tip60 enhances Bcl-3-p50 activated transcription through an NF-kappaB binding site, indicating that quarternary complexes containing Bcl-3 interactors modulate NF-kappaB driven gene expression. These data implicate Bcl-3 as an adaptor between NF-kappaB p50/p52 and other transcription regulators and suggest that its gene activation function may at least in part be due to recruitment of the Tip60 histone actetylase.

Purification and properties of a beta-galactoside-binding lectin from neonatal marmoset.

A beta-galactoside-binding lectin was extracted from whole neonatal marmoset homogenate with lactose solution and purified to homogeneity by ion-exchange chromatography on Q Sepharose Fast Flow and by affinity adsorption to trypsinized and glutaraldehyde-fixed ghosts of rabbit erythrocytes. The lectin has a dimeric structure composed of two 15K subunits. Its amino acid composition and partial amino acid sequences were quite similar to those of beta-galactoside-binding lectins from human placenta and lung.

Direct interaction between gingival fibroblasts and lymphoid cells induces inflammatory cytokine mRNA expression in gingival fibroblasts.

In inflamed periodontal lesions, dense infiltration of lymphocytes is usually observed in the extravascular periodontal connective tissue, adjacent to gingival fibroblasts. Our previous study revealed that activated lymphocytes can adhesively interact with gingival fibroblasts in vitro. In the present study, we investigated whether gingival fibroblasts are activated through direct interaction with lymphoid cells by monitoring the expression of inflammatory cytokine mRNA in human gingival fibroblasts (HGF). Co-culture with various human lymphoid cells in vitro resulted in a marked increase in the expression of IL-1alpha, IL-1beta, and IL-6 mRNA by the HGF. In addition, expression of the mRNA of the IL-1beta-converting enzyme (ICE), which is essential to produce the mature form of IL-1beta, was constitutively observed in the HGF, suggesting that mature IL-1beta is produced by these cells. When HGF were cultured with the culture supernatant of the lymphoid cells, the increase in the inflammatory cytokine mRNA expression was not observed. Similarly, when HGF and lymphoid cells were cultured in the same well but separated by a membrane which prevented direct contact between the cells, no increase in inflammatory cytokine mRNA expression was observed. These results strongly indicate that direct interaction between these heterotypic cell types transduces activation signals into HGF that induce an increase in inflammatory cytokine mRNA expression. Furthermore, IL-1beta mRNA expression in the HGF was synergistically increased when HGF directly interacted with lymphoid cells in the presence of exogeneous IL-1beta. The present study demonstrates that direct interaction between HGF and lymphoid cells stimulates HGF to increase inflammatory cytokine mRNA expression, and raises the possibility that heterotypic cell-cell interaction may facilitate local inflammatory reactions.

Effects of ursodeoxycholic acid and chenodeoxycholic acid on major histocompatibility complex class I gene expression.

We investigated the effect of ursodeoxycholic acid on major histocompatibility complex class I gene expression in cultured human hepatoma cells. Ursodeoxycholic acid, which is now being used for the treatment of various autoimmune liver diseases, paradoxically increased the mRNA level of major histocompatibility complex class I. However, endogenous bile acids, for example, chenodeoxycholic acid, increased major histocompatibility complex class I mRNA expression more strongly compared with ursodeoxycholic acid. Concerning the interplay between ursodeoxycholic and chenodeoxycholic acids, these bile acids additively induced major histocompatibility complex class I mRNA expression. In contrast, when the total concentration of ursodeoxycholic and chenodeoxycholic acids was kept constant, the expression of major histocompatibility complex class I mRNA appeared to decrease in a dose-dependent manner with an increasing ratio of ursodeoxycholic acid. These findings indicate that the beneficial action of ursodeoxycholic acid may be related to this relative decrease in major histocompatibility complex class I gene expression.

Natural course of diabetic peripheral neuropathy in spontaneous-onset diabetic Chinese hamsters.

We investigated metabolic and pathological changes in the peripheral nerve of the spontaneous-onset diabetic Chinese hamster. Electrophysiological examination revealed that the motor nerve conduction velocity was significantly decreased at 10 months and afterwards, however, the F-wave latency was significantly increased at 5 months and afterwards. Concerning sciatic nerve contents of sorbitol, myo- and scyllo-inositol, the content of sorbitol was not significantly increased at 5 months, but, myo- and scyllo-inositol were significantly decreased at 5 months and thereafter. At 10 and 15 months, however, sciatic nerve content of sorbitol was significantly increased. On morphological examination, loss of large myelinated fiber and reciprocal increase in degenerative fiber were also seen in sciatic nerve, but not in tibial nerve, at 5 months. At 15 months, these morphological changes were also found in the tibial as well as the sciatic nerve. Thus, we may hypothesize that F-wave latency is useful in the detection of initial diabetic neuropathy, and that the initial pathological changes in diabetic neuropathy of diabetic Chinese hamsters are predominantly found in the proximal site of peripheral nerves.

The micronucleus test with mouse spleen cells.

The results of this study show that the micronucleus test can be carried out with mouse spleen cells as well as with cells from bone marrow. Polychromatic erythrocytes occurred in the spleen at a frequency of about 9% of the whole spleen cells compared with about 13% in the bone marrow. 3 test compounds were used to compare the frequency of micronuclei in cells from the 2 tissues. Mitomycin C and cyclophosphamide induced micronucleated polychromatic erythrocytes in both spleen and bone marrow. Fosfomycin, an antibiotic having a broad spectrum of antimicrobial activities, did not induce micronucleated erythrocytes in either organ.

Innocuous mechanical stimulation of the neck and alterations in heart-rate variability in healthy young adults.

The present study examined the effects of cervical spinal manipulation, a widely applied form of physical therapy, which involves innocuous mechanical stimulation, on heart rate and heart-rate variability, in a cohort of healthy young adults. Using a cross-over treatment design, with a one-week washout period and, in contrast to a sham procedure, the authentic manipulation produced significant alterations in both heart rate and measures of heart-rate variability calculated from power spectrum analysis. In particular, there was an increase in the ratio of low-frequency (LF)-to-high-frequency (HF) components of the power spectrum of heart-rate variability, which may reflect a shift in balance between sympathetic and parasympathetic output to the heart.

Evaluation of the mutagenicity of aminoglycoside antibiotics in Salmonella typhimurium and Saccharomyces cerevisiae.

The mutagenicity of aminoglycoside antibiotics (KM, AKM, DKB, RSM, AMK, GM, TOB) has been studied in cells of the bacteria Salmonella typhimurium and in the yeast Saccharomyces cerevisiae. The bacterial strains (Ames') monitor reverse mutation (point mutation) and the yeast strain D5 monitors mitotic crossing-over, mitotic gene conversion and point mutation. None of these antibiotics demonstrated any mutagenic activities in either the bacteria or the yeast.

Evaluation of the mutagenicity of miokamycin (MOM), a new macrolide antibiotic in the Ames Salmonella test and the dominant lethal assay.

Miokamycin (MOM), a new macrolide antibiotic, was tested for mutagenicity in the Ames Salmonella test and the dominant lethal assay. MOM was not mutagenic against S. typhimurium strains TA1535-1538, TA100 and TA98 both with and without activation by S-9 mix. No increases in the frequencies of induced dominant lethal effects were found in MOM treated mice. MOM did not have mutagenic activities on either system.

Comparative in vitro and in vivo activity of cefminox (MT-141), cefotaxime and cefoperazone against gram-negative bacteria.

Against 17 Gram-negative bacteria, excluding Proteus species, the difference between cefminox and cefotaxime was significant in terms of MIC distribution, in favour of cefotaxime, but was not significant in terms of ED50 distribution. Against 22 Proteus species, the difference between cefminox and cefotaxime was not significant in terms of MIC distribution, but was significant for ED50 distribution, in favour of cefminox. The difference between cefminox and cefoperazone was not significant for either MIC or ED50 distribution against 17 Gram-negative bacteria, but was significant against 22 Proteus species, in favour of cefminox. The distribution of rank of the ED50/MIC ratios for cefminox was significantly lower than those for cefotaxime and cefoperazone, indicating that cefminox showed lower ED50 values than expected from the MIC values.

Successful treatment of refractory polymyositis with pulse intravenous cyclophosphamide and low-dose weekly oral methotrexate therapy.

A 36-year-old woman gradually developed dysphagia and muscle weakness of the lower extremities. Diagnosis of polymyositis was given from elevation of serum creatine kinase and pathological findings of a muscle biopsy. Despite oral prednisolone and intravenous pulse methylprednisolone therapy, her muscle weakness persisted, and then pulse intravenous cyclophosphamide (IVCY) therapy was initiated and repeated five times in total, which resulted in significant improvement in muscle strength. Thereafter, weekly administration of methotrexate at low dosage further normalized the serum creatine kinase level. We may conclude that IVCY and low-dose weekly methotrexate together could be an alternative in refractory polymyositis.

Human T cell leukemia virus type I-associated myelopathy in a patient with systemic lupus erythematosus.

A case of human T cell leukemia virus type I (HTLV-1) associated myelopathy (HAM)/tropical spastic paraparesis (TSP) with 14-year history of systemic lupus erythematosus (SLE) is reported. For 9 years, the numbness of the feet and sacral region progressed with occasional urinary incontinence and constipation. She was admitted to hospital due to gait disturbance and aggravation of SLE and the diagnosis of HAM/TSP was confirmed, indicating that HTLV-1 infection is associated with the development of not only HAM/TSP but also SLE.

Histo-chemical studies on the anti-ulcer effect of bamboo grass in rats.

Oral administration of a hot-water extract (Folin) of bamboo grass (Sasa albomarginata Makino & Shibata) significantly reduced the incidence of water-immersion and restraint stress-, ethanol-induced and indomethacin-induced gastric ulcers in rats. Histological examination of the Folin-treated gastric mucosa showed that microscopic blood clots overlaid the superficial epithelium, maintaining the cellular integrity of gastric mucosa, especially against stress ulcer. In addition, Folin suppressed the incidence of hyperaemia and a decline of acid mucopolysaccharides in the ethanol-induced ulcer. Folin suppressed a release of histamine from rat mast cells, and stabilized erythrocytes and accelerated their agglutination under acid conditions. These results suggest that a microscopic haemostatic effect of Folin reinforced by a membrane-stabilizing effect might be responsible for the prevention of the gastric lesions.

[A case of parkinsonism due to pontine and extrapontine myelinolysis].

A 43-year-old man who presented parkinsonism due to pontine and extrapontine myelinolysis was reported. Late in February, 1990, the patient presented suffered from a flu-like illness and was seen at a community hospital. Physical finding showed the pigmentation on the whole body and hypotension, and laboratory examination revealed severe electrolyte imbalance (serum sodium 100 mEq/l, serum potassium 6.9 mEq/l, serum chloride 68 mEq/l) and hypoglycemia (postprandial serum glucose 78 mg/dl). Given these results, adrenal failure was strongly suspected. Prompt correction of electrocyte imbalance was performed by the infusion of sodium chloride, and four days later the serum sodium level reached 131 mEq/l. On the other hand, the patient was noticed lethargic and showed parkinsonism i.e., rest tremor, cog-wheel rigidity, and hypokinesia. Fourteen days after the onset of neurological abnormalities, the patient was referred to our hospital for further evaluation of parkinsonism. Additionally, neurological examination revealed dysphagia, mutism and positive pyramidal tract sign. On admission brain computed tomography was unremarkable, but on the 14th hospital day it showed low density area in the pons. Brain magnetic resonance imaging also showed a striking increase in T2-weighted signal from the pons, the midbrain, and the bilateral thalamus. Based on these findings, a diagnosis of parkinsonism due to pontine and extrapontine myelinolysis was made, and levodopa therapy was started. After the initiation of levodopa therapy, improvement of tremor, rigidity, and hypokinesia ensued with marked functional benefit, and the patient was discharged on the 49th hospital day. Levodopa was stopped three weeks after discharge but, all neurological abnormalities were not recurrent.(ABSTRACT TRUNCATED AT 250 WORDS)

[The mutagenicity evaluation of MT-141, a new cephamycin].

Mutagenicity of MT-141, a new cephamycin, was evaluated by in vitro and in vivo assays. MT-141 did not induce mutations of the test strains, Escherichia coli WP2 (uvr A) and Salmonella typhimurium TA1535, TA1537, TA1538, TA100 and TA98, with and without metabolic activation in vitro. In bone marrow micronucleus assay with male mice, MT-141 showed no induction of micronucleated polychromatic erythrocyte at 6 hours and 30 hours after administration. In addition MT-141 was found not to cause any dominant lethal effects on male mice for 8 weeks after administration.

[Toxicological studies on dibekacin for intravenous injection use. II. Chronic toxicity in rats (author's transl)].

The toxic effects of dibekacin sulfate (DKB) in male and female rats were examined in chronic toxicity test (intraperitoneal injection), and the following results were obtained. 1) No death was noted in both male and female. 2) In general conditions, the excretion of soft or diarrheal stool was noted in groups of more than 20 mg/kg of either sex. The mean body weight was less than the control during a certain period in the male group of 40 mg/kg and in the female group of 20 mg/kg. But, in the food intakes, no particular change was noted in each group of either sex. 3) In the auricle reflex, no abnormality was noted in each group of either sex. 4) In the hematological test, the findings such as an increase of BUN, anemia, etc. were noted in the groups more than 10 mg/kg of male and more than 20 mg/kg of female. 5) In the histopathological study, evident degenerative changes of renal tubular epithelia were noted in the groups which were administered more than 20 mg/kg of DKB in both male and female, but no evident pathological findings due to the renal failure were noted in the groups of less than 10 mg/kg. Several slight changes of the thyroid gland noted in a few rats of DKB administration group of both male and female seemed to be artifact, and inflammatory changes owing to intraperitoneal injection were occasionally noted in the peritoneum of DKB injected animals. 6) Considering the above results, "the maximal non effective dose" was estimated to be 10 mg/kg in both male and female.

[Toxicological studies on dibekacin for intravenous injection use. I. Subacute toxicity in rats and rabbits (author's transl)].

Dibekacin sulfate (DKB) dissolved in physiological saline J.P. was administered to rats intraperitoneally and rabbits intravenously for subacute 35-day toxicity test. The results were as follows: I. Wistar-strain rats (1) All the animals of both male and female died in the group with 500 mg/kg. (2) In general conditions stretching physical positions, decrease in spontaneous movements, decrease in respiration rates, unsteady steps of walking and muscular relaxation developed in the groups of high doses of either sex. The effects through the administration of this drug were also noted on the progress of body weights and food intakes in the groups of high doses. (3) In the hematological and histopathological studies, degenerative and reparative changes of tubular epithelia were evidently noted in the groups which were administered more than 100 mg/kg of DKB in both male and female. No pathological findings were noted in administration groups less than 20 mg/kg. (4) Microscopically, slight inflammatory changes were noted in the bladder of the male groups of high doses and the direct stimulative effects on the peritoneum due to intraperitoneal administration were noted but slightly in the serous membrane of the liver, spleen and the gastrointestinal tracts. (5) Judging from the above-mentioned results, "the maximal non toxic dose" through the intraperitoneal administration to rats of this drug was assumed 20 mg/kg in either sex. II. Albino rabbits (1) Neither remarkable change in the general conditions nor death was noted in each administration group. (2) The increase in the mean body weight in each group was almost similar to the control value. They consumed the amounts of food given. (3) The specific abnormal finding was not noted in the hematology and biochemical tests of serum or urine. (4) Since no change was noted on ERG in each rabbits, we estimate there is no effect to the visual organs. (5) In histopathological study, several changes revealed in some organs through the macroscopic findings, organ weights and microscopic findings but they were no more the serious changes attributable to administration. (6) We estimate "the maximal non effective dose" in this test was 10 mg/kg.