Sandwich Embedding of Monolayers of Cells in Epoxy Resin for Ultrathin Sectioning

In this procedure for embedding monolayers of cells, the usual glass slides are replaced by plates of resin 1-1.5 mm thick. Unlike the open-face embedding technique, the present procedure uses only a few drops of unpolymerized resin, which are applied to the fixed and dehydrated cells. During polymerization this small amount of liquid resin spreads across a relative large area, leaves the cells covered by a very thin layer, and permits phase contrast observations through it. Ultrathin sections of a particular cell encircled by a rotary scriber can be obtained by sectioning the resin slide, which has been trimmed and mounted directly in the specimen holder of the ultramicrotome.     

Pattern of simulated snoring is different through mouth and nose

Cineradiography of the pharynx during simulated snoring was done in 6 healthy volunteers, and supraglottic pressure and flow rate were recorded in 12 others. We observed, immediately before snoring, a decrease in the sagittal diameter of the oropharynx followed, during snoring, by high-frequency oscillations of soft palate and pharyngeal walls. The pattern of soft palate oscillations was different while snoring through the nose or mouth. During inspiratory snoring through the nose, the soft palate remained in close contact with the back of the tongue and only the uvula presented high-frequency oscillations. Snoring through the mouth resulted in ample high-frequency oscillations of the whole soft palate. Frequency of airflow and supraglottic pressure oscillations was less (P less than 0.05) during mouth (28.2 +/- 7.5 Hz) than during nasal snoring (77.8 +/- 36.7 Hz). This difference may be related to the smaller oscillating mass (i.e., uvula) during nasal snoring. At variance with our previous data, which showed that snoring during sleep, in both heavy (nonapneic) snorers and obstructive sleep apnea patients, was systematically preceded by flow limitation, this was not true during simulated snoring.     

Estimation of regional cutaneous cold sensitivity by analysis of the gasping response.

Regional cutaneous sensitivity to cooling was assessed in males by separately immersing four discrete skin regions in cold water (15 degrees C) during head-out immersion. The response measured was gasping at the onset of immersion; the gasping response appears to be the result of a nonthermoregulatory neurogenic drive from cutaneous cold receptors. Subjects of similar body proportions wore a neoprene "dry" suit modified to allow exposure to the water of either the arms, upper torso, lower torso, or legs, while keeping the unexposed skin regions thermoneutral. Each subject was immersed to the sternal notch in all four conditions of partial exposure plus one condition of whole body exposure. The five cold water conditions were matched by control immersions in lukewarm (34 degrees C) water, and trials were randomized. The magnitude of the gasping response was determined by mouth occlusion pressure (P0.1). For each subject, P0.1 values for the 1st min of immersion were integrated, and control trial values, although minimal, were subtracted from their cold water counterpart to account for any gasping due to the experimental design. Results were averaged and showed that the highest P0.1 values were elicited from whole body exposure, followed in descending order by exposures of the upper torso, legs, lower torso, and arms. Correction of the P0.1 response for differences in exposed surface area (A) and cooling stimulus (delta T) between regions gave a cold sensitivity index [CSI, P0.1/(A.delta T)] for each region and showed that the index for the upper torso was significantly higher than that for the arms or legs; no significant difference was observed between the indexes for the upper and lower torso.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ploidy and morphology in osteosarcoma

In a cytometric DNA study of high-grade osteosarcoma, the relationship between DNA content and morphology was analyzed. The investigation, based on microspectrophotometry of tissue sections and flow cytometry (FCM), included both primary lesions and recurrences. FCM analysis, applied to a consecutive series of 47 primary osteosarcomas, disclosed that 2 were diploid and 45 were nondiploid, 8 of which were tetraploid. Multiple aneuploid peaks were detected in 13 tumors. Among the nondiploid tumors, there was no clear relationship between the peak DNA value(s) and the histologic subtype (osteoblastic, chondroblastic, fibroblastic) or grade (III-IV). The proliferative activity, as reflected by the percentage of S-phase cells, could be determined in 38 of the 47 tumors analyzed by FCM. The percentage was higher for aneuploid than for tetraploid lesions; however, the distribution of S-phase cells was not related to the histologic subtype or the grade of the tumors. To assess the reliability of a single sample for FCM, the DNA content of biopsy and surgical specimens was compared in 20 tumors; there was complete agreement in all cases with respect to the classification of the lesion as diploid, tetraploid or aneuploid. Analysis by FCM or microspectrophotometry of 12 local recurrences and 16 metastases and the corresponding 19 primary tumors showed that an aneuploid characteristic of the primary lesion was retained during progression of the disease. In 12 tumors analyzed by microspectrophotometry in tissue sections, comparison of chondroblastic and osteoblastic/fibroblastic areas within the same lesion consistently disclosed hyperploidy in both areas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution and metabolism of leukotriene C4 after cisternal injection in guinea pigs

Following cisternal injection of [3H8]LTC4 into guinea pigs, leukotriene metabolites were identified in the brain, cerebellum, perilymph, blood, liver and kidneys. LTC4 was metabolized into LTD4 and LTE4 in the cerebrospinal fluid and LTE4 was transported into the blood for general circulation and uptake into the liver and kidneys. The excretion of LTE4 from CNS to blood seemed to be the rate-limiting step in the elimination of leukotrienes from the body. Leukotrienes were also transported into the perilymph. The conversion of LTC4 into LTD4 and LTE4 was lower in perilymph as compared to the cerebrospinal fluid, suggesting a rate limiting function of the cochlear aqueduct that can be defined as a cerebrospinal fluid-labyrinth barrier.     

Human monocytes convert leukotriene B4 to two dihydro-leukotriene B4-metabolites

Human monocytes metabolize LTB4 by an additional pathway different from omega-oxidation. Reverse-phase high performance liquid chromatography showed four metabolites: 20-COOH-LTB4, 20-OH-LTB4 and two metabolites less polar than LTB4 with an UV maximum at 232 nm. Gas-chromatography mass-spectrometry showed nearly identical mass spectra for both metabolites. The main mass fragments of the two metabolites were increased by two mass units compared to LTB4. Our findings suggest that LTB4 had been reduced to a known and a new dihydro-metabolite of LTB4. Both metabolites together amounted to 85% of total metabolites. The remaining 15% were omega-oxidation products. Thus, the major pathway of LTB4 metabolism by human monocytes is reduction to dihydro-LTB4.     

The calcium-binding site of clathrin light chains

Clathrin light chains are calcium-binding proteins (Mooibroek, M. J., Michiel, D. F., and Wang, J. H. (1987) J. Biol. Chem. 262, 25-28) and clathrin assembly can be modulated by calcium in vitro. Thus, intracellular calcium may play a regulatory role in the function of clathrin-coated vesicles. The structural basis for calcium's influence on clathrin-mediated processes has been defined using recombinant deletion mutants and isolated fragments of the light chains. A single calcium-binding site, formed by residues 85-96, is present in both mammalian light chains (LCa and LCb) and in the single yeast light chain. This sequence has structural similarity to the calcium-binding EF-hand loops of calmodulin and related proteins. In mammalian light chains, the calcium-binding sequence is flanked by domains that regulate clathrin assembly and disassembly.     

Subunit selectivity and epitope characterization of mAbs directed against the GABAA/benzodiazepine receptor

mAbs bd 17, bd 24, and bd 28 raised against bovine cerebral gamma-aminobutyric acid (GABAA)/benzodiazepine receptors were analyzed for their ability to detect each of 12 GABAA receptor subunits expressed in cultured mammalian cells. Results showed that mAb bd 17 recognizes epitopes on both beta 2 and beta 3 subunits while mAb bd 24 is selective for the alpha 1 subunit of human and bovine, but not of rat origin. The latter antibody reacts with the rat alpha 1 subunit carrying an engineered Leu at position four, documenting the first epitope mapping of a GABAA receptor subunit-specific mAb. In contrast to mAbs bd 17 and bd 24, mAb bd 28 reacts with all GABAA receptor subunits tested but not with a glycine receptor subunit, suggesting the presence of shared epitopes on subunits of GABA-gated chloride channels.     

Resistance to H-2-restricted but not to allo-H2-specific graft and cytotoxic T lymphocyte responses in lymphoma mutant

The lymphoma mutant RMA-S escaped graft rejection after transplantation over a minor histocompatibility barrier, whereas it was rejected in H-2 allogeneic mice. The parental control line was rejected in both situations. The mutant, which had been selected against MHC class I molecules retained 5 to 10% of the wild-type H-2Db, Kb, and beta 2-microglobulin expression on the cell surface. It remained sensitive to allo-H-2b CTL in vitro, but was completely resistant to minor histocompatibility antigen-specific, H-2b-restricted CTL. It was equally resistant to other H-2b-restricted responses against internally derived Ag, such as tumor-specific CTL or a CTL clone specific for the influenza virus nucleoprotein. The results indicate a target cell defect that selectively abolishes the sensitivity to H-2-restricted CTL directed against internally processed Ag. This appears sufficient to shift the transplantation response over a minor histocompatibility Ag barrier from rejection to acceptance. There are two possible explanations for the results: 1) a block in the MHC class I-directed pathway for internal Ag processing, and 2) subthreshold H-2/Ag ligand density in relation to triggering requirements of restricted CTL. Regardless of the type of defect, the results demonstrate a difference between allo-H-2-specific and H-2-restricted CTL recognition at the level of the target cell.