The AMeX method: a multipurpose tissue-processing and paraffin-embedding method. III. Extraction and purification of RNA and application to slot-blot hybridization analysis.

RNA was extracted from tissues processed by a new fixation and paraffin-embedding method (the AMeX method) and examined by Northern blot analysis and slot-blot analysis. The RNA extraction method for AMeX-processed tissue sections after the deparaffinization step was the same as that for fresh materials. The total amount of cellular RNA extracted from AMeX-processed mouse liver tissue was slightly less than that extracted from fresh tissue. In tissues of malignant lymphoma, the total amount of cellular RNA extracted from 25 sections each 20 microns thick was about 1.6-1.8 micrograms/mm2, regardless of the histological subtype and period of storage. The extracted RNA was moderately degraded, and usually could not be used for Northern blot hybridization analysis. The intensity of ethidium bromide staining and the hybridization signals of RNA extracted from AMeX-processed tissues were usually reduced in comparison with RNA from fresh material, but specific signals could be detected by slot-blot hybridization analysis. We have demonstrated previously that the AMeX method preserves high-molecular-weight DNA and various antigens. Since the present study showed that information on mRNA can be obtained from AMeX-processed tissue, the versatility and usefulness of this method were further proven.     

Ventilatory response to hypercapnia in sprint and long-distance swimmers

Summary Ventilatory response lines to carbon dioxide at rest were determined by the rebreathing method in 10 untrained subjects, 17 sprint swimmers, and 11 long-distance swimmers. It was found that the mean slope of the ventilatory response line of the swimmer was lower than that of the untrained group, and the mean slope of the long distance swimmer was lower as compared with the sprint swimmer, though these differences were statistically not significant. The differences in the hypercapnic drive between untrained subjects and swimmers obtained here is discussed in connection with their maximum oxygen uptake.     

Influence of Various Immunomodulators on the Induction of Experimental Autoimmune Encephalomyelitis in Lewis Rats

The effect of various immunomodulators on the induction of experimental autoimmune encephalomyelitis (EAE) is evaluated in the Lewis rat. Bordetella pertussis (BP) is the optimal inductor of EAE in this rat strain. Treatment of the animals with BP either before or after or simultaneously with guinea-pig spinal cord preparation (GpSC) resulted in an EAE about two weeks thereafter. Additional injection of living BCG, of CFA, IFA (incomplete Freund's adjuvant) or Vibrio cholerae neuraminidase (VCN) did not augment or mitigate the effect induced by BP or GpSC. Living BCG, IFA, VCN or Corynebacterium parvum (CP) did not induce EAE when given in combination with GpSC but without BP. CFA combined with GpSC only occasionally induced EAE. However, EAE could be induced by the combination of CFA and GpSC or IFA and GpSC in a part of the animals tested if they had been pretreated or simultaneously been injected with living BCG by intravenous route. EAE could not be enhanced by the additional injection of VCN. Surprisingly, most of the animals peracutely died after injection of CFA and BP in combination with GpSC when they had been pretreated with CP. This effect was most pronounced when pretreatment was done on day -4. No acute effect could be seen when CP was given simultaneously to CFA, BP and GpSC. Animals which did not peracutely succumb developed EAE similarly as those in the positive control groups. CP treatment simultaneously with BP but without CFA resulted in a reduction of the EAE specific mortality. This reduction could not be seen if treatment with CP was done after injection of GpSC and BP.     

A method for simultaneous recording of electrical activities and spectrophotometric signals from brain slice preparations in vitro

Organ spectrophotometry has been applied to analyze cytochrome redox changes in brain slice preparations. An interface-chamber method for maintaining metabolism of brain slice tissues was devised to reduce noise on recording traces of spectrophotometric signals, and then used for continuous monitoring and simultaneous recording of electrical and optical signals from brain slices. With this method, the noise level during the recording of redox states of cytochromes was decreased to 0.0004 A unit.     

The effect of a series of fixatives on the AgNOR technique

With increasing interest being shown in nucleolar organizer regions (NORs) in pathology, it was considered of great importance to evaluate the effect of some of the more commonly used and more specialized fixatives on the demonstration of these moieties. NORs can be demonstrated in paraffin sections by a silver technique (AgNOR method) which was developed from a method used by cytogeneticists for the demonstration of NORs in chromosome spreads. The degree of staining is dependent on the fixation regime employed and results may vary greatly from one fixative to another. The fixative schedules and post-treatments used in this study were based on standard sequences from the literature. We have shown that, in general, alcohol-based fixatives give optimal results, Carnoy's fluid being especially recommended. Mercurial and dichromate-containing fixatives were found to have highly detrimental effects on NOR staining. 'Routine' 10 per cent formol saline fixation gave adequate results whereas 10 per cent neutral buffered formalin gave optimal staining, similar to alcohol-based fixation.     

Segmentation of Bacteria Image Based on Level Set Method

In biology ferment engineering, accurate statistics of the quantity of bacteria is one of the most important subjects. In this paper, the quantity of bacteria which was observed traditionally manuauy can be detected automatically. Image acquisition and processing system is designed to accomplish image preprocessing, image segmentation and statistics of the quantity of bacteria. Segmentation of bacteria images is successfully realized by means of a region-based level set method and then the quantity of bacteria is computed precisely, which plays an important role in optimizing the growth conditions of bacteria.     

Laminar distribution and morphology of NADPH-diaphorase containing neurons in the superior colliculus and underlying periaqueductal gray of the rat

Summary We have studied the laminar distribution of reduced nicotinamide dinucleotide phosphate diaphorase (NADPH-d) activity and the morphology of positive neurons in the superior colliculus (SC) and the underlying periaqueductal gray (PAG) of the rat. The morphology of NADPH-d-positive neurons has been compared to that of Golgi-impregnated cells. The highest activity occurs in the stratum zonale and stratum griseum superficiale, contrasting with the pale neuropil in the stratum opticum, where only a few positive neurons are found. In the stratum griseum intermedium positive neurons are grouped in patches separated by narrow, NADPH-d-negative bands. In the deeper layers, the neuropil is NADPH-d-negative, and few neurons show enzymatic activity. In contrast, numerous neurons in the dorsolateral part of the PAG are intensely positive. They are continuous with the positive neurons in the stratum album profundum, with no clear border between the two centers. In both SC and PAG, only small and medium sized neurons are NADPH-d-positive. In comparison with Golgi material, all types of small neurons in the superficial layers show NADPH-d activity; NADPH-d histochemistry, however, does not visualize the characteristic dendritic appendages of these neurons. The large neurons of the SC and PAG, probably representing the long-projecting neurons of these centers, do not contain the enzyme.     

The depolarizing action of 5-hydroxytryptamine on rabbit vagal primary afferent and sympathetic neurones and its selective blockade by MDL 72222

Summary MDL 72222 (1H,3,5H-tropan-3-yl-3,5-dichlorobenzoate) is a novel compound with potent and selective blocking actions at certain excitatory 5-hydroxytryptamine (5-HT) receptors on mammalian peripheral neurones. In the present study, the sucrose-gap technique has been used to record depolarizing responses to 5-HT from the cells of the rabbit nodose and superior cervical ganglia and to investigate the potency and selectivity of MDL 72222 as an antagonist of these responses.On nodose ganglia, responses to 5-HT were inhibited surmountably by MDL 72222 at concentrations up to 100 nmol/l. The threshold for antagonism was 2–10 nmol/l and the apparent pA₂ value (Schild 1947) was 7.7±0.2,n=10. Blockade was selective since responses to GABA and noradrenaline were unaffected by MDL 72222, 100 nmol/l. With concentrations of MDL 7222 higher than 100 nmol/l, antagonism was concentration-related but not in a manner consistent with simple competitive antagonism and even a concentration of 1 mol/l failed to abolish the response to 5-HT.The results from the superior cervical ganglion were essentially similar to those obtained from the nodose ganglion. The threshold concentration of MDL 72222 for inhibition of 5-HT was 1–10 nmol/l and blockade was selective in that depolarizing responses to dimethylphenylpiperazinium (DMPP) was unaffected by a concentration of MDL 72222 of 1 mol/l.The data provide direct evidence that MDL 72222 is a potent and selective antagonist of the receptors for 5-HT which mediate depolarizing responses in vagal primary afferent cell bodies and in sympathetic ganglion cells.     

Dendritic arbors and dendritic excrescences of abnormally positioned neurons in area CA3c of mice carrying the mutation "hippocampal lamination defect"

BALB/cJ and BALB/cByJ mice are homozygous for the autosomal gene "hippocampal lamination defect" (provisional gene symbol: Hld) which produces an abnormality in the lamination of the pyramidal cell layer of area CA3c of the hippocampus such that early-generated neurons are superficial and late-generated neurons are deep. Other inbred strains of mice are wild-type (+/+) at the Hld locus and do not have this inversion in cell position in area CA3c. The Golgi method was used to analyze the dendritic arbors of the abnormally positioned pyramidal cells and to compare the distribution of dendritic excrescences (i.e., the termination sites of the mossy fibers) in +/+ and Hld/Hld mice. It was found that in +/+ mice the late-generated pyramidal cells (whose cell bodies are positioned just below the suprapyramidal mossy fiber layer) have one set of dendritic excrescences on their apical dendrites as they extend through the suprapyramidal mossy fiber layer and a second set on their basal dendrites as they pass through the infrapyramidal mossy fiber layer. In contrast, in Hld/Hld mice the late-generated pyramidal cells (whose cell bodies are abnormally positioned just below the intrapyramidal mossy fiber layer) have two sets of dendritic excrescences on their apical dendrites, as they pass through the intrapyramidal and suprapyramidal mossy fiber layers, and none on their basal dendrites. In addition, in the vicinity of the apparent point of contact of the intrapyramidal mossy fibers, the apical dendrites of some of the abnormally positioned pyramidal cells have several fine-caliber branches.(ABSTRACT TRUNCATED AT 250 WORDS)     

非开胸食管内翻剥脱术

目的　研讨非开胸食管内翻剥脱的手术方法、优缺点及适应症。方法 :分析我院 1 992年至 2 0 0 0年 6月非开胸食管内翻剥脱胃重建食管术的 1 8例病历 ,其中下咽癌 5例、甲状腺鳞癌 1例、喉癌侵犯下咽者 4例、食管颈段癌 8例。结果 :本组病人食管床出血量均在 1 0 0ml以下 ,无吻合口漏等严重并发症 ,无手术死亡。结论 :本手术安全具有简便创伤小的优点 ,适用于颈段食管癌、下咽癌及甲状腺癌和喉癌侵及下咽者、部分早期食管癌和贲门癌。