Novel non‐alcoholic steatohepatitis model with histopathological and insulin‐resistant features

Although several non‐alcoholic steatohepatitis (NASH) models have been reported to date, few of these models fully reflect the histopathology and pathophysiology of human NASH. The aim of this study was to establish a novel NASH model by feeding a high‐fat (HF) diet and administering both carbon tetrachloride (CCl₄) and the Liver X receptor agonist T0901317. Male C57BL/6J mice were divided into four groups (each n = 5): HF, HF + CCl₄, HF + T0901317, and the novel NASH model (HF + CCl₄ + T0901317). CCl₄ (0.1 mL/kg) and T0901317 (2.5 mg/kg) were intraperitoneally administered four times and five times, respectively. The livers of the novel NASH model group presented a whitish colour. The serum levels of TNF‐α and IL‐6 were significantly increased in the novel NASH model group, and mice in this group exhibited histopathological features and insulin resistance reflective of NASH, i.e., macrovesicular hepatic steatosis, ballooning hepatocytes, Mallory‐Denk bodies, lobular inflammation and fibrosis. The novel NASH model group presented significantly upregulated expression levels of mRNAs related to lipogenesis, oxidative stress, fibrosis and steatosis and significantly downregulated expression levels of mRNAs related to triglyceride export. We successfully established a novel experimental NASH model that exhibits similar histopathology and pathophysiology to human NASH.     

Alpha1-adrenoceptor-mediated breakdown of phosphatidylinositol 4,5-bisphosphate inhibits pinacidil-activated ATP-sensitive K+ currents in rat ventricular myocytes

Phosphatidylinositol 4,5-bisphosphate (PIP2) stimulates ATP-sensitive K+ (K(ATP)) channel activity. Because phospholipase C (PLC) hydrolyzes membrane-bound PIP2, which in turn may potentially decrease K(ATP) channel activity, we investigated the effects of the alpha1-adrenoceptor-G(q)-PLC signal transduction axis on pinacidil-activated K(ATP) channel activity in adult rat and neonatal mouse ventricular myocytes. The alpha1-adrenoceptor agonist methoxamine (MTX) reversibly inhibited the pinacidil-activated K(ATP) current in a concentration-dependent manner (IC50 20.9+/-6.6 micromol/L). This inhibition did not occur when the specific alpha1-adrenoceptor antagonist, prazosin, was present. An involvement of G proteins is suggested by the ability of GDPbetaS to prevent this response. Blockade of PLC by U-73122 (2 micromol/L) or neomycin (2 mmol/L) attenuated the MTX-induced inhibition of K(ATP) channel activity. In contrast, the MTX response was unaffected by protein kinase C inhibition or stimulation by H-7 (100 micro mol/L) or phorbol 12,13-didecanoate. The MTX-induced inhibition became irreversible in the presence of wortmannin (20 micro mol/L), an inhibitor of phosphatidylinositol-4 kinase, which is expected to prevent membrane PIP2 replenishment. In excised inside-out patch membranes, pinacidil induced a significantly rightward shift of ATP sensitivity of the channel. This phenomenon was reversed by pretreatment of myocytes with MTX. Direct visualization of PIP2 subcellular distribution using a PLCdelta pleckstrin homology domain-green fluorescent protein fusion constructs revealed reversible translocation of green fluorescent protein fluorescence from the membrane to the cytosol after alpha1-adrenoceptor stimulation. Our data demonstrate that alpha1-adrenoceptor stimulation reduces the membrane PIP2 level, which in turn inhibits pinacidil-activated K(ATP) channels.     

Zinc Increases ABCA1 by Attenuating Its Clearance Through the Modulation of Calmodulin Activity

Aim: We previously revealed that Ca⁺⁺-activated calmodulin binds to ABCA1 by the region near the PEST sequence and retards its calpain-mediated degradation to increase HDL biogenesis. Calmodulin activity is reportedly modulated also by other nutritional divalent cations; thus, we attempted to determine whether Zn⁺⁺ is involved in the regulation of ABCA1 stability through the modulation of calmodulin activity.Methods: The effects of Zn⁺⁺ on ABCA1 expression was investigated in J774 mouse macrophage cell-line cells and HepG2 human hepatoma cell-line cells.Results: Zn⁺⁺ increased ABCA1 expression, not by increasing the mRNA but by attenuating its decay rate, more prominently in the presence of cAMP. Accordingly, it enhanced cell cholesterol release with extracellular apolipo-protein A-I. Calmodulin binding to ABCA1 was increased by Zn⁺⁺ and Ca⁺⁺. Zn⁺⁺ suppressed calpain-mediated hydrolysis of the peptide of ABCA1 cytosolic loop, including the PEST sequence and the calmodulin-binding site, in a calmodulin-dependent fashion, in the presence of the minimum amount of Ca⁺⁺ to activate calpain, but not calmodulin. Calpain activity was not directly inhibited by Zn⁺⁺ at the concentration for enhancing calmodulin binding to ABCA1.Conclusion: Nutritional divalent cation Zn⁺⁺ is involved in the regulation of ABCA1 activity and biogenesis of HDL through the modulation of calmodulin activity. The results were consistent with previous clinical findings that Zn⁺⁺ increased plasma HDL in the conditions of sympathetic activation, such as type 2 diabetes and chronic hemodialysis.     

Cimetidine inhibits salivary gland tumor cell adhesion to neural cells and induces apoptosis by blocking NCAM expression

Background  

Cimetidine, a histamine type-2 receptor antagonist, has been reported to inhibit the growth of glandular tumors such as colorectal cancer, however the mechanism of action underlying this effect is unknown. Adenoid cystic carcinoma is well known as a malignant salivary gland tumor which preferentially invades neural tissues. We demonstrated previously that human salivary gland tumor (HSG) cells spontaneously express neural cell adhesion molecule (NCAM), that HSG cell proliferation may be controlled via a homophilic (NCAM-NCAM) binding mechanism and that NCAM may be associated with perineural invasion by malignant salivary gland tumors. We further demonstrated that cimetidine inhibited NCAM expression and induced apoptosis in HSG cells. Here, we investigated the effects of cimetidine on growth and perineural/neural invasion of salivary gland tumor cells.     

Brain metastases from asymptomatic adenocarcinoma of the pancreas: an autopsy case report

BACKGROUND: Brain metastasis from pancreatic cancer is extremely rare. Because pancreatic cancer usually has a rapidly progressive nature, the majority of affected patients die from primary lesions before exhibiting clinical signs suggestive of brain metastases. CASE DESCRIPTION: The patient was a 62-year-old man who developed generalized convulsion followed by right hemiparesis accompanied by decreased consciousness level. Computed tomography (CT) scan revealed multiple brain tumors with ring-like contrast enhancement. Stereotactic biopsy disclosed mucinous adenocarcinoma. A marked increase in the serum CA19-9 level was noted, but the patient did not exhibit any other signs of pancreatic disease. Repeated whole body CT scan and ultrasonography demonstrated no primary lesions. The patient died of multi-organ failure during chemotherapy combined with radiation for metastatic brain tumors. Autopsy revealed well-differentiated papillary adenocarcinoma in the pancreatic head and systemic metastases associated with tumor emboli were widely distributed in various organs. CONCLUSION: This patient initially presented only with symptoms of neurologic disorder and no pancreatic symptoms. Moreover, repeated radiologic examinations did not reveal the primary lesion. We considered that the unusual clinical course in our patient may be partly explained by the autopsy findings: diffuse sclerotic changes of the pancreas without swelling. The present report suggests that undetected pancreatic cancer may have been the primary lesion classified as "unknown origin" in some cases of metastatic brain tumors.     

Variation in MT expression in early-stage depressed-type and polypoid-type colorectal tumours

Metallothionein (MT) expression is observed in various carcinomas, but its role is not fully understood. To clarify the clinicopathological significance of MT, 87 colorectal adenomas and 128 early-stage carcinomas were immunohistochemically analysed for MT expression. The degree of MT immunostaining of a specimen was graded according to the proportion of MT-positive cells; negative (<5%) and positive (focally 5-50%, diffusely >50%). MT expression significantly decreased with tumour development. For carcinomas, MT-positivity was significantly associated with depth of invasion (T1 60% versus T2 33%; P<0.01), vascular involvement (positive 35% versus negative 61%; P<0.01) and morphology (polypoid 62% versus depressed 26%; P<0.01). Regarding MT-positive distribution, the diffuse-positive rate in MT-positive polypoid lesions was 28%, while MT-positive depressed lesions were all diffusely stained (P<0.01). In conclusion, our results suggested that decreasing MT expression is an early event in colorectal carcinogenesis and may reflect local invasion. Furthermore, MT-positive distribution may reflect genetic differences between the polypoid and depressed-type.     

Early postnatal protein malnutrition affects learning and memory in the distal but not in the proximal cue version of the Morris water maze

Learning and memory of early postnatal protein malnourished rats were investigated in the Morris water maze. During the lactation period (21 days) each litter (mother plus six male and two female pups) was provided with 16% (well-nourished) or 6% (malnourished) protein diets. After weaning, rats remained on the same diet until 49 days of age. From day 50 on all animals were fed a commercial lab chow. Experiments started on day 70. In experiment I (proximal cue version) the animals were trained to escape from water to a visible platform (3 cm above the water level) in six trials daily for four consecutive days, completing 24 trials. In experiment II (distal cue version) the animals were trained to escape from water to a submerged platform using the same procedure as in experiment II. After the 24th trial, the platform was removed and the animals were submitted to a 60-s trial (probe trial). Seven and twenty-eight days after training, the retention test was conducted in one 180-s trial. The results showed no impairment of the learning or memory of malnourished animals tested in the proximal cue version but an increased latency and distance traveled to find the submerged platform in the distal cue version of the procedure. In the distal cue version the malnourished animals also showed increased latency to find the platform 7 and 28 days after the test training. No difference due to diet was found in the probe trial test indicating that, once the task is acquired, malnourished rats can manage extra-maze cues as easily as well-nourished rats. It is suggested that the present results can be due to alterations produced by protein malnutrition in the hippocampal formation or also to reflect the higher emotionality of rats following early malnutrition, specially considering the fact that postnatally malnourished animals are more reactive to unpleasant or aversive stimuli as cold water.     

Prevention of progression of interstitial lung lesions by early combination therapy with corticosteroids and cyclosporine/cyclophosphamide in two patients with amyopathic dermatomyositis

Two patients with amyopathic dermatomyositis complicated by interstitial lung lesions were effectively treated with a combination of corticosteroids and cyclosporine and/or cyclophosphamide. A 48-year-old female patient was treated with pulse methylprednisolone and cyclosporine 2 months after onset of dermal symptoms. A 45-year-old male patient was treated with oral prednisolone and pulse cyclophosphamide 2 1/2 months after onset of dermal symptoms. Early evaluation of interstitial lung lesions and early extensive therapy may improve prognosis of interstitial lung lesions in patients with amyopathic dermatomyositis.     

Early Diagnosis of Postoperative Infection: Assessment of Whole Blood Chemiluminescence

(Received for publication on Oct. 19, 1998; accepted on Nov. 11, 1999)