Trivalent heat-labile- and heat-stable-enterotoxin probe conjugated with horseradish peroxidase for detection of enterotoxigenic Escherichia coli by hybridization.

A 1,268-bp polynucleotide probe for heat-labile and heat-stable enterotoxins (LTh, STIa, STIb) was conjugated with horseradish peroxidase (HRP). The HRP-conjugated trivalent probe was applied to the detection of enterotoxigenic Escherichia coli (ETEC) by colony and stool hybridizations. The binding of the probe to its targets was assayed by the addition of HRP substrates hydrogen peroxide and luminol in the presence of an enhancer, and the chemiluminescence was recorded by exposure to X-ray film. Slot blot hybridization demonstrated that the HRP-conjugated trivalent probe specifically hybridized with the DNA isolated from ETEC strains. The trivalent probe also specifically identified bacterial colonies of ETEC that produced LTh, STIa, STIb, LTh-STIa, or LTh-STIb. Treatment of targets with sodium dodecyl sulfate and proteinase K remarkably reduced nonspecific hybridization to DNAs of non-ETEC strains. Furthermore, this probe was able to detect stool specimens seeded with 10(2) original ETEC cells per 5 mg of feces. These results suggest that the HRP-conjugated trivalent probe is a candidate for use in the clinical laboratory to detect ETEC.     

Cardiomyopathy characterized by abnormal accumulation of desmin-type intermediate filaments in cardiac muscle fibers. A case report and review of the literature

A 42-year-old Japanese male, who had been suffering from congestive heart failure and electrocardiographic abnormalities (A-V block, intraventricular conduction disturbance, ventricular tachycardia), died after a clinical course of 2 years and 1 month. Macroscopic investigation revealed dilation of the left ventricle and thickening of the right ventricular wall. The unique finding in this case was a circumferential fibrous scar in the median circular layer and outer oblique layer of the left ventricular wall. Biopsy and autopsy materials revealed diffuse loss of myofibrils in the central zone of cardiac muscle fibers, and replacement with aniline blue-positive homogeneous material (17-35% of the area of one muscle fiber). Electron microscopy revealed abnormal accumulation of fine filamentous material (7.5-25 nm in diameter), which was immunohistochemically proved to be desmin-type intermediate filament. Moreover, sarcoplasmic reticulum-like material was detected in the degenerated area. At autopsy, degeneration was detected all over the heart. The ventricular muscle fibers were more severely affected than the atrial muscle fibers. The conduction system was also affected, in some parts more severely than the surrounding ordinary muscle fibers. The pathogenesis of this disorder remains to be clarified.     

Skeletal disorders associated with skin pigmentation: a role of melatonin?

Although frequently encountered, no available consensus about the association between skeletal abnormalities and skin pigmentation. Several syndromes are characterized by the presence of skin pigmentation in association with skeletal disorders like neurofibroamtosis 1, McCune-Albright Syndrome, Jaffe-Campanacci Syndrome and Jaffe-Lichtenstein Syndrome. Even in the absence of these syndromes, skeletal abnormalities were detected in all radiologically examined patients having patterned skin pigmentation. Although skin pigmentation is controlled by several factors, melatonin is the most reliable factor to have relation to development of skeletal abnormalities. Recent research works support that melatonin might play a role in bone development and several hypotheses link melatonin with some bone diseases associated with skin pigmentation. It seems that melatonin deficiency is a probable operating co-factor in a lot of clinical situations characterized by skin pigmentation and skeletal disorders. This would explain some of the un-explained observations related to these syndromes and research works along these lines might lead to the development of efficient treatment for these diseases.     

Phase-dependent filtering of sensory information in the oscillatory olfactory center of a terrestrial mollusk

With electrophysiological techniques, we found phase-dependent modification of the efficacy of signal transmission in the procerebrum (PC), the oscillatory olfactory center, of the terrestrial mollusk Limax marginatus and elucidated its neuronal mechanism. Previous studies have indicated that about 10(5) PC neurons can be classified into only two types: bursting (B) neurons and nonbursting (NB) neurons, and both types of neurons have ongoing and phase-locked periodic oscillation of their membrane potentials. On olfactory nerve stimulation, excitatory postsynaptic potentials (EPSPs) were evoked with a constant latency in NB neurons, while EPSPs with a variable latency were evoked in B neurons. These findings suggest a monosynaptic connection from the olfactory nerve to NB neurons, but a polysynaptic connection between the olfactory nerve and B neurons. This polysynaptic transmission is most likely mediated by NB neurons because the olfactory nerve makes synaptic connection only with NB neurons in the PC. The latency of the evoked EPSPs in B neurons depended on the phase of the PC oscillatory activity, presumably because of the oscillation of the intervening NB neurons. These results suggest that the efficacy of olfactory nerve-B neuron polysynaptic transmission is regulated by the activity level of the phasically oscillating NB neurons. Thus, the intrinsic oscillation in the PC can serve as a filter for olfactory information conveyed from the olfactory nerve as a train of neuronal spikes. This filtering system may also produce a phase-dependent modification by the olfactory input of the PC oscillation frequency.     

Observation of cavitation in a mechanical heart valve in a total artificial heart

Recently, cavitation on the surface of mechanical heart valves has been studied as a cause of fractures occurring in implanted mechanical heart valves. The cause of cavitation in mechanical heart valves was investigated using the 25 mm Medtronic Hall valve and the 23 mm Omnicarbon valve. Closing of these valves in the mitral position was simulated in an electrohydraulic totally artificial heart. Tests were conducted under physiologic pressures at heart rates from 60 to 100 beats per minute with cardiac outputs from 4.8 to 7.7 L/min. The disk closing motion was measured by a laser displacement sensor. A high-speed video camera was used to observe the cavitation bubbles in the mechanical heart valves. The maximum closing velocity of the Omnicarbon valve was faster than that of the Medtronic Hall valve. In both valves, the closing velocity of the leaflet, used as the cavitation threshold, was approximately 1.3-1.5 m/s. In the case of the Medtronic Hall valve, cavitation bubbles were generated by the squeeze flow and by the effects of the venturi and the water hammer. With the Omnicarbon valve, the cavitation bubbles were generated by the squeeze flow and the water hammer. The mechanism leading to the development of cavitation bubbles depended on the valve closing velocity and the valve stop geometry. Most of the cavitation bubbles were observed around the valve stop and were generated by the squeeze flow.     

A new method for assaying adhesion of cancer cells to the greater omentum and its application for evaluating anti-adhesion activities of chemically synthesized oligosaccharides

A new ex vivo method for assaying adhesion of cancer cells to the greater omentum has been developed using mouse greater omentum and [³H]labelled human gastric and mouse colorectal cancer cells. Since the adhesion rates were found to increase up to 18 h and labelled cells seemed to be stable during the period, the present method could be useful for investigating adhesion of cancer cells to the greater omentum, which must occur at the first step of the peritoneal dissemination. The adhesion of cancer cells to the greater omentum was inhibited by a series of chemically synthesized oligosaccharides and Galβ1,3[3OMeGalβ1,4GlcNAcβ1,6]αBn was found to be the best inhibitor. The anti-tumor effect of this novel tetrasaccharide in vivo was shown in preliminary experiments using Balb/c mice and colon26 cells.     

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.     

Positive regulator for the expression of Mba protein of the virulence plasmid, pKDSC50, of Salmonella choleraesuis.

A positive regulator was identified within a 2.3 kb fragment of the 6.4 kb mouse bacteremia region (mba region) of the virulence pKDSC50 plasmid of Salmonella choleraesuis. Sodium dodecyl sulphate polyacrylamide gel electrophoresis showed that Escherichia coli K-12 carrying the recombinant plasmids of the 2.3 kb fragment produced Mba1 protein with a molecular mass of 32 kDa. The recombinant plasmids carrying a 4.1 kb fragment, the other part of 6.4 kb region, produced Mba2 (32 kDa), Mba3 (70 kDa) and Mba4 (29 kDa) proteins. All three proteins were expressed by using the lacZ promoter under isopropyl thiogalactoside induction. In contrast to this, Mba3 protein was overexpressed independently of the lacZ promoter when the 2.3 kb fragment coexisted either in cis or trans. These results suggest that Mba1 is a trans-acting positive regulator for the expression of the Mba3 protein of mba region of pKDSC50.