An enzyme-linked immunosorbent assay (ELISA) for the measurement of antibodies to different parts of the gram-negative lipopolysaccharide core region

Bovine serum albumin was complexed with the core antigens of either Escherichia coli J5 LPS, Salmonella minnesota R595 LPS or E. coli lipid A. These core-BSA complexes were used for solid-phase coating in ELISAs for anti-core antibodies. Antibodies, binding to various parts of the core region were easily quantified in a single experimental set-up, which was hitherto not possible. The ELISA has only 3 incubation steps and is not costly as only moderate amounts of the core antigens (i.e., 1 microgram per test) were needed for coating. The sensitivity proved to be excellent and the complexes were biologically fully active (compared to native, smooth LPS), which make them suitable for the screening (after fusion) of monoclonal anti-core antibodies. Another possible application is the large-scale screening of blood-bank sera in order to find samples with a high anti-core antibody content.     

硫酸软骨素酶ABC对大鼠脊髓损伤修复的影响

为了探讨硫酸软骨素酶ABC对脊髓损伤后损伤局部瘢痕形成和脊髓传导功能修复的影响,本研究首先制作大鼠脊髓全横断损伤动物模型,并将其分为脊髓损伤组(A组)和脊髓损伤治疗组(B组)。在观察期内对动物的行为学表现进行BBB评分;用免疫荧光组织化学方法观察损伤4周后硫酸软骨素酶ABC对损伤局部的硫酸软骨素蛋白聚糖(CSPGs)的裂解作用;用辣根过氧化物酶(HRP)示踪法观察损伤8周后神经纤维的再生情况。结果显示:A组与B组之间动物的行为学评分B组优于A组,有显著性差异(P<0.01);B组动物脊髓内硫酸软骨素蛋白聚糖阳性物质的表达明显低于A组,具有显著性差异(P<0.05),而硫酸软骨素核心蛋白的表达无显著性差异(P>0.05);HRP示踪法显示B组脊髓损伤头端可见少量HRP标记的神经元胞体和纤维。本研究结果提示硫酸软骨素酶ABC能够裂解CSPGs中的葡胺聚糖链,减少瘢痕,促进损伤的神经纤维再生。     

Matrix metalloproteinase expression in basal cell carcinoma: relationship between enzyme profile and collagen fragmentation pattern

Matrix metalloproteinases (MMPs) with collagenolytic and gelatinolytic activities are up-regulated in basal cell carcinoma. In the present study we demonstrate that the major collagenolytic enzyme detected is MMP-1 (interstitial collagenase) while gelatinolytic enzymes include both MMP-2 (72-kDa gelatinase A) and MMP-9 (92-kDa gelatinase B). Significant fractions of all three enzymes are present as active forms. In spite of the fact that high levels of gelatinolytic enzymes are present, the major fragmentation products resulting from digestion of intact type I collagen are the 1/4 and 3/4 fragments (products of MMP-1-mediated digestion). Thus, it appears that the gelatinolytic enzymes are not capable of degrading the collagen fragments as rapidly as they are produced. Since previous studies have demonstrated that interaction of interstitial fibroblasts with high molecular weight fragments of type I collagen leads to increased MMP production, the present results suggest a mechanism underlying altered function of stromal elements in the connective tissue adjacent to the growing neoplasm.     

Significance of beta-catenin and pRB pathway components in malignant ovarian germ cell tumours: INK4A promoter CpG island methylation is associated with cell proliferation

To clarify the mechanisms underlying cell cycle promotion in malignant germ cell tumours of the ovary (MGCTOs), beta-catenin and components of the pRB pathway, cyclin D1 and p16, were analysed in relation to cell proliferation. Immunohistochemically, p16 protein was not expressed in a number of MGCTOs (9 of 42 tumours: 21.4%) and was associated with p16 gene (INK4A) promoter 5'-CpG islands methylation. Amplification of the cyclin D1 gene (CCND1) was detected in a small number of MGCTOs (5 of 42 tumours: 13.5%). Reduced expression of p16 due to promoter methylation correlated significantly with increased cell proliferation as evidenced by Ki-67 labelling index (p < 0.001) and mitotic index (p < 0.01). In some tumour types, nuclear localization of beta-catenin has been reported to be associated with beta-catenin gene (CTNNB1) mutation, cyclin D1 overexpression, and increased cell proliferation. Nuclear localization of beta-catenin, which was observed in MGCTOs other than dysgerminoma, was not associated with cyclin D1 expression and increased cell proliferation, but appeared to be related to tumour differentiation. Furthermore, CTNNB1 mutations were not detected in any of the MGCTOs examined. Our results suggest that reduced expression of p16 due to INK4A promoter methylation is one of the principal factors that promote cell proliferation in MGCTOs. Thus, p16 may be a novel target for gene therapies to treat MGCTOs.     

Bicuculline-induced circling from the rat superior colliculus is blocked by GABA microinjection into the deep cerebellar nuclei

In a recent electrophysiological experiment, we showed the deep cerebellar nuclei to be a major source of excitatory input to the superior colliculus. Furthermore, target neurons in the colliculus were found, in every case, to receive convergent tonic inhibitory input from the substantia nigra pars reticulata. In the present study, we investigated these effects in the awake rat. We asked whether circling behaviour, induced by unilateral injection of a GABA antagonist into the lateral colliculus, could be suppressed by concurrent cerebellar inactivation. Rats were chronically implanted with bilateral guide cannulae located above the superior colliculus and deep cerebellar nuclei. Bicuculline methiodide (25 pmol) was microinjected unilaterally into intermediate layers of the colliculus at increasing depths until an optimal contralateral circling response was elicited. This behaviour was taken as the baseline response and was the first of three treatments. The second was an identical manipulation of the colliculus with a concurrent 200-nl microinjection of 1 M GABA into the contralateral deep cerebellar nuclei. The third was a repeat of BIC alone into the colliculus or, if rotation had been suppressed by more than 50% on test 2, the treatment was collicular BIC plus deep cerebellar saline. This latter treatment was used as a control for possible non-pharmacological injection effects. The effect of cerebellar GABA at 26 sites (17 within cerebellar nuclei and 9 outside) on BIC-induced rotation at 15 collicular sites was studied in ten animals. Only GABA injections at sites that fell within the cerebellar nuclei significantly reduced turning (P<0.0001). A full behavioural analysis showed that this was a specific suppression of turning, not the result of general motor impairment. These results provide clear behavioural evidence that opposing, convergent influences from the basal ganglia and cerebellum interact in the lateral superior colliculus to control head and body movements. They furthermore suggest that the tonic deep cerebellar excitation of the superior colliculus could be the driving force in the expression of rotation induced by manipulations of the basal ganglia.     

Evidence that non-NMDA receptors are involved in the excitatory pathway from the pedunculopontine region to nigrostriatal dopaminergic neurons

Summary Extracellular single-neuron recordings were obtained from electrophysiologically identified nigrostriatal neurons in chloral hydrate anesthetized rats, in order to test the hypothesis that excitatory amino acid receptors are involved in responses of these neurons to electrical stimulation of the pontine region where the pedunculopontine nucleus (PPN) is located. The effects of iontophoretic application of excitatory amino acids and their antagonists as well as of cholinergic antagonists were tested on the fast orthodromic excitation of nigrostriatal neurons evoked by stimulation of the PPN region. The N-methyl-D-aspartate (NMDA) receptor antagonist D-a-aminoadipic acid as well as the cholinergic receptor antagonists mecamylamine and atropine failed to suppress the synaptic excitation of nigral neurons. The NMDA receptor antagonist DL-2-amino-5-phosphonovalerate exerted a weak depressant action on the synaptic response in a few neurons only. On the contrary, the broad spectrum antagonists of excitatory amino acid receptors kynurenic acid and gamma-Dglutamyl-amino-methyl-sulphonate were found to block simultaneously both the synaptic excitation and the neuronal responses to iontophoretic pulses of glutamate while leaving unaffected the neuronal responses to local application of acetylcholine or carbachol. The competitive antagonist of non-NMDA receptors 6-cyano-2,3-dihy-droxy-7-nitro-quinoxaline suppressed the synaptic excitation at ejection currents which antagonized neuronal responses to quisqualate and kainate. These results suggest that PPN excitatory fibers synapsing onto pars compacta nigrostriatal neurons utilize an excitatory amino acid as a synaptic transmitter acting preferentially on non-NMDA receptors.     

An electron microscopic and immunohistochemical study of Merkel cell carcinoma

Two cases of Merkel cell carcinoma (MCC) were examined by electron microscopy and immunohistochemistry. Histologically, tumor cells, which extended from the dermis into the subcutis, showed anastomosing bands with partial trabecular pattern. The ultrastructural study showed tumor cells in case 1 with numerous neurosecretory granules. The number of granules in case 2, however, was smaller compared with that in case 1. Perinuclear bundles of filaments were present in case 2, but few bundles were observed in case 1. By immunohistochemistry, cytokeratin (CK)-8, -18, -19, and -20 and epithelial membrane antigen were stained positively within tumor cells in both cases. It was interesting that staining patterns of chromogranin A and of neuron-specific enolase were different in the two cases. These data indicated that CK-20 is a useful marker for diagnosing MCC and that ultrastructural and immunohistochemical differences in both cases were the result of phenotypic variation.     

The affinity of anti-HBc antibodies in acute and chronic hepatitis B infection.

Antibodies to hepatitis B core antigen (anti-HBc) are found in the sera of all individuals infected with hepatitis B virus. A role for these antibodies has been suggested in determining the outcome of infection. In this study, the affinity of anti-HBc antibodies in asymptomatic virus carriers was compared with that of antibodies present in the sera of patients with chronic liver disease. Persistently infected individuals with no evidence of clinical disease were found to have anti-HBc antibodies of greater affinity, compared with the chronic liver disease group. Sera from patients with chronic hepatitis contained high levels of low-affinity antibody whereas antibody levels in asymptomatic carriers were significantly lower. These findings are discussed in relation to the predicted role of anti-HBc antibodies in mediating hepatitis B virus-related hepatocellular injury.     

A role for the basal ganglia in nicotinic modulation of the blink reflex

Summary In humans and rats we found that nicotine transiently modifies the blink reflex. For blinks elicited by stimulation of the supraorbital branch of the trigeminal nerve, nicotine decreased the magnitude of the orbicularis oculi electromyogram (OOemg) and increased the latency of only the long-latency (R2) component. For blinks elicited by electrical stimulation of the cornea, nicotine decreased the magnitude and increased the latency of the single component of OOemg response. Since nicotine modified only one component of the supraorbitally elicited blink reflex, nicotine must act primarily on the central nervous system rather than at the muscle. The effects of nicotine could be caused by direct action on lower brainstem interneurons or indirectly by modulating descending systems impinging on blink interneurons. Since precollicular decerebration eliminated nicotine's effects on the blink reflex, nicotine must act through descending systems. Three lines of evidence suggest that nicotine affects the blink reflex through the basal ganglia by causing dopamine release in the striatum. First, stimulation of the substantia nigra mimicked the effects of nicotine on the blink reflex. Second, haloperidol, a dopamine (D₂) receptor antagonist, blocked the effect of nicotine on the blink reflex. Third, apomorphine, a D₂ receptor agonist, mimicked the effects of nicotine on the blink reflex.