A simple and sensitive method for detection of Bacillus anthracis by loop-mediated isothermal amplification.

AIMS: To develop a rapid and simple system for detection of Bacillus anthracis using a loop-mediated isothermal amplification (LAMP) method and determine the suitability of LAMP for rapid identification of B. anthracis infection. METHODS AND RESULTS: A specific LAMP assay targeting unique gene sequences in the bacterial chromosome and two virulence plasmids, pXO1 and pXO2, was designed. With this assay, it was possible to detect more than 10 fg of bacterial DNA per reaction and obtain results within 30-40 min under isothermal conditions at 63 degrees C. No cross-reactivity was observed among Bacillus cereus group and other Bacillus species. Furthermore, in tests using blood specimens from mice inoculated intranasally with B. anthracis spores, the sensitivity of the LAMP assay following DNA extraction methods using a Qiagen DNeasy kit or boiling protocol was examined. Samples prepared by both methods showed almost equivalent sensitivities in LAMP assay. The detection limit was 3.6 CFU per test. CONCLUSIONS: The LAMP assay is a simple, rapid and sensitive method for detecting B. anthracis. SIGNIFICANCE AND IMPACT OF THE STUDY: The LAMP assay combined with boiling extraction could be used as a simple diagnostic method for identification of B. anthracis infection.

Lysosomal enzyme activities are decreased in the retina and their circadian rhythms are different from those in the pineal gland of rats fed an alpha-linolenic acid-restricted diet.

The retinal rod outer segment (ROS) is shed and digested daily by phagosomes in retinal pigment epithelial (RPE) cells. We previously observed significantly fewer large phagosomes in rats fed an alpha-linolenic acid (ALNA)-deficient diet. Rats fed a safflower oil diet (ALNA-restricted) or a perilla oil diet (ALNA-sufficient) through two generations were adapted to a 24-h cycle with light from 0700 to 1900 h. They were killed at 0500, 0900, 1300 and 1700 h to determine the activities of four lysosomal enzymes in retina, including beta-glucosidase, beta-glucuronidase, hexosaminidase and acid phosphatase. The enzyme activities at 0500 h were the lowest and then increased gradually until 1700 h, exhibiting similar circadian rhythms in the two dietary groups. However, the activities at each time point were significantly lower in the safflower group. In the pineal gland, the activities were maximum at 1300 h, except for beta-glucosidase, and were not different between groups. These diets had qualitatively similar but quantitatively different effects on the fatty acid compositions of the retina and the pineal gland. These results indicate that decreased amplitudes in electroretinogram and altered size distribution of phagosomes, as induced by a restricted intake of ALNA, are associated with decreased lysosomal enzyme activities in the retina but not in the pineal gland.

Diurnal rhythms of retinal phospholipid synthetic enzymes are retained but their activities are decreased in rats under alpha-linolenic acid deficiency.

Rats fed a safflower oil (alpha-linolenic acid (ALNA)-deficient) diet over the course of two generations had significantly decreased docosahexaenoic acid (22:6n-3) and increased docosapentaenoic acid (22:5n-6) contents in the major retinal phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) when compared with those fed a perilla oil (ALNA-sufficient) diet, but the compositions of phosphatidylinositol acyl chains were relatively unaffected. The contents of individual phospholipids in the retina were essentially the same for the two dietary groups. The activities of the rate-limiting enzymes in the de novo synthesis of PC and PE, CTP:phosphocholine cytidylyltransferase (CT), and CTP:phosphoethanolamine cytidylyltransferase (ET), respectively, were measured in the retinas excised at 5:00, 9:00, 13:00, and 17:00 h from rats adapted to a 24-h cycle with lights on from 7:00 to 19:00 h. Both enzymes exhibited significant diurnal rhythms with the lowest activities at 5:00 h and gradually increasing activities following exposure of the rats to light; the maximum activities were at 13:00 h for CT and 17:00 h for ET. The diurnal rhythms were not significantly affected by the above-mentioned diets. However, both enzyme activities at each collection time point were significantly lower in the safflower oil group than in the perilla oil group. These results suggest that retinal phospholipid turnover associated with shedding, phagocytosis, and resynthesis of the rod outer segments is limited by ALNA deficiency.

Recurrent mixed tumor of the right lacrimal gland causes acute contralateral visual loss.

A 23-year-old woman underwent excisional biopsy of a right palpebral mass: the histopathologic study showed a benign mixed tumor of the lacrimal gland. Thereafter, the tumor recurred repeatedly, and multiple excisions were done. Subsequently, right orbital exenteration was performed, and some orbital and frontal bones were removed. When the patient was 52 years old, the orbital tumor recurred. A histopathologic study revealed malignant transformation. At the age of 54 years, the patient suffered acute loss of vision in her left eye. Computed tomography disclosed invasion of the tumor into the posterior paranasal sinuses and brain.

[Experimental study on the difference of ischemic threshold between cerebral cortex and thalamus with middle latency auditory evoked potentials].

Middle latency auditory evoked potentials (ML-AEPs) have not yet been established as a device of assessing neurological events because their origins have not been definitely identified. In this study, we assessed the neurological usefulness of MLA-EPs through experimental approach using canine models of acute ischemia localized within the cerebral cortex or thalamus. Two types of localized cerebral ischemia were produced in mongrel dogs by clipping of major cerebral arteries and inducing of hypotension; they were, unilateral cortical ischemia involving the right primary auditory area (group A) and unilateral thalamic ischemia involving the right medial geniculate body (group B). Using these two ischemia models, auditory evoked potentials (AEPs) were intracranially and extracranially recorded in addition to the measurement of local cerebral blood flow (1-CBF). Prior to the induction of ischemia, it was confirmed that positive waves with a latency of 20 ms (P20) were evoked in the bilateral primary auditory areas by sound stimulation (90 dB 5 Hz click) given to the ear contralateral to the planned ischemic side in both groups. The right P20 disappeared when the 1-CBF in the right primary auditory area decreased below the ischemic flow threshold of synaptic transmission failure, approximately 18 ml/100 g/min, in group A, and when the 1-CBF in the right medial geniculate body decreased below the threshold, 10 ml/100 g/min, in group B.(ABSTRACT TRUNCATED AT 250 WORDS)

[A comparison of the functional vulnerability of the cerebral cortex and thalamus to acute ischemia].

Using cerebral cortical and thalamic ischemia models produced in mongrel dogs, the reversibility of short-latency somatosensory-evoked potentials (SSEPs) and the effects of ischemic brain edema on reversibility were compared. The mean systemic blood pressure (MSBP) of animals was reduced by exsanguination until cortical SSEPs disappeared, and was held constant at that level. The MSBP was recovered by autogenous blood transfusion at 30 minutes (subgroup A), 60 minutes (subgroup B) and 90 minutes (subgroup C) after SSEP disappearance in the cortical ischemia group; and at 15 minutes (subgroup D) and 30 minutes (subgroup E) after SSEP disappearance in the thalamic ischemia group. Local cerebral blood flows (lCBF) were measured and SSEPs were monitored serially up to 2 hours after blood transfusion. At the end of measurement, the leakage of Evans blue was evaluated and brain tissue water content was measured. Cortical SSEPs disappeared when lCBF in the right cerebral cortex, measured by hydrogen clearance method decreased to 18.4 +/- 5.4 ml/100 g/min (mean +/- SD) and neuronal transmission failure in thalamus occurred when thalamic blood flow decreased to 10.0 +/- 3.3 ml/100 g/min. After blood transfusion. SSEP reappeared in all 12 animals in subgroup A, but did not appear in 2 of 9 animals in subgroup B and in all of 7 animals in subgroup C.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular pH regulation of normal rat brain: 31P-MRS study.

Intracellular pH changes in rat brain tissue were investigated during low or high extracellular pH induced by acetazolamide or sodium bicarbonate, respectively. Intracellular pH was measured by 31P-MRS in the brain of spontaneously breathing rats under intraperitoneal sodium pentobarbital anaesthesia. Extracellular pH was calculated from the results of blood gas analysis. After intravenous injection of sodium bicarbonate (280 mg/kg), the extracellular pH rose significantly (p less than 0.05) from 7.47 +/- 0.06 to 7.82 +/- 0.15 (mean +/- SE). After administration of acetazolamide (50 mg/kg), the extracellular pH dropped significantly (p less than 0.05) from 7.45 +/- 0.02 to 7.34 +/- 0.03. Despite the changes in extracellular pH, the intracellular pH of rat brain did not change significantly under either condition. The following four factors are thought to contribute to the maintenance of intracellular pH in the normal brain: 1) production and consumption of H+ by brain metabolism, 2) physicochemical buffering, 3) transmembrane transport of H+ and its equivalent, 4) compensatory adaptation of circulatory factors. These mechanisms are not disturbed in the brain of rats that are breathing spontaneously, because the cerebral circulation and energy metabolism are preserved in the normal range.

Cavernous angioma of the cerebellum--case report.

Computed tomography (CT) allows earlier diagnosis of intracranial cavernous angioma than was formerly possible. However, cerebellar lesions are uncommon. Cerebellar cavernous angioma with hemorrhage was diagnosed in a 54-year-old male who suddenly developed nausea and vomiting, then declined over the following week and developed ataxia and nystagmus. Blood pressure was normal, and he had no history of hypertension or hemorrhagic diathesis. Precontrast CT revealed an irregularly-shaped, hyperdense lesion in the left cerebellum and an associated hematoma. Injection of contrast medium did not significantly enhance the lesion. Vertebral angiography demonstrated only an avascular mass in the cerebellum. A semiliquid hematoma was evacuated and a vascular nodule was removed from the posterior wall of the hematoma cavity. The histopathological diagnosis was cavernous angioma. Nine sufficiently documented reports of cerebellar cavernous angioma are reviewed and the clinical and radiological features of these lesions are discussed.