ISOLATED NERVE ENDINGS (NEUROSECRETOSOMES) FROM THE POSTERIOR PITUITARY : Partial Separation of Vasopressin and Oxytocin and the Isolation of Microvesicles

Subcellular fractions of the bovine posterior pituitary, including one composed almost exclusively of pinched-off nerve endings (neurosecretosomes), were characterized electron microscopically, hormonally, and enzymically. 15% of the nerve terminals in the gland were isolated as neurosecretosomes, as estimated from determinations of lactic dehydrogenase, a soluble, cytoplasmic enzyme. Neurosecretosomes were subdivided into three fractions by density-gradient centrifugation. The three subfractions, each shown to be nearly homogeneous populations of neurosecretosomes by means of electron microscopic and enzymic criteria, differed from each other in their vasopressin/oxytocin (VP/OT) ratios. The VP/OT ratio increased from the lightest to the densest fraction, indicating that VP is localized to denser and OT to lighter neurosecretosomes; similar results have been obtained previously for subfractions of neurosecretory granules (NSG). No morphological differences were apparent in neurosecretosomes among the three subfractions. Although complete separation of VP and OT was not achieved, the findings suggest that VP and OT are each stored in a different species of nerve ending and support the hypothesis that a given neurosecretory cell synthesizes, stores, and secretes only one of the peptide hormones. Microvesicles, 40–80 mµ diameter and contained in typical neurosecretory cell terminals, are believed to be degradation products of membrane ghosts of depleted NSG; electron micrographs indicative of this transformation are presented. A fraction rich in microvesicles, but containing some NSG membranes, was prepared by density-gradient centrifugation of an osmolysate of neurosecretosomes. Smaller, apparently nonneurosecretory nerve endings, lacking NSG but filled with small vesicles, are occasionally seen in sections from whole gland. The vesicles in these atypical posterior pituitary nerve endings may be true neurohumor-containing, "synaptic" vesicles.     

Extraction, Characterization, and Cellular Localization of the Lipids of Staphylococcus aureus

Satisfactory extraction and assay procedures have been developed for the lipids of Staphylococcus aureus. The following lipids have been characterized in detail: the vitamin K(2), which is shown to exist as isoprenologues with side chains of 35, 40, and 45 carbon atoms; monoglucosyldiglyceride and diglucosyldiglyceride, which account for all the carbohydrate in the lipid extracts; the lysyl ester of phosphatidyl glycerol, phosphatidyl glycerol, and cardiolipin, which account for 98% of the phosphate in the lipid extract. The extraction procedure removes 98% of the total bacterial fatty acids. Acidification of the medium before harvest and refluxing in isopropanol are critical in the extraction procedure for the maximal recovery of lysyl-phosphatidyl glycerol and the glucolipids. The lipids have been shown to be a part of the same membrane as the respiratory pigments.     

Formation of Extracellular Sphingolipids by Microorganisms: IV. Pilot-Plant Production of Tetraacetylphytosphingosine by Hansenula ciferrii

Tetraacetylphytosphingosine (TAPS) formation by the F-60-10 mating type strain of the yeast Hansenula ciferrii, previously observed on agar plates, has been shown to take place in submerged cultures. The optimal conditions for TAPS formation, and the correlation of TAPS production and sugar utilization under aerobic conditions, were studied in 10-liter fermentors. For each gram of glucose consumed, 5 mg of TAPS were formed; for each gram of yeast solids produced, 15 mg of TAPS were synthesized. A 750-liter pilot-plant run yielded 175 g of crude TAPS, which were obtained by hexane extraction of centrifuged yeast cells.     

POLYESTER-METHACRYLATE EMBEDMENTS FOR ELECTRON MICROSCOPY

It has been found that tissues fixed for electron microscopy and dehydrated in acetone can be embedded in mixtures of n-butyl methacrylate and polyester resin. Activation with 1 per cent tert-butyl hydroperoxide followed by 12 to 48 hours at 60°C produces blocks that section well with glass knives. The ribbons are cleared of methacrylate by heat (200–250°C for 1 hour) and/or immersion in organic solvents (CCL₄, acetone-ether). After removal of the methacrylate the residual polyester matrix provides thermostable and insoluble support for the tissue. Its insolubility permits staining by immersion of cleared preparations in organic solvents carrying heavy metal compounds in solution. Clearing by heat stabilizes section-grid relationships. The removal of volatile materials by clearing substantially reduces contamination of both specimen and microscope. Tissue fine structure is well preserved in these preparations.     

Histoplasmosis in Montreal During the Fall of 1963, with Observations on Erythema Multiforme

Although Montreal is in an endemic area, significant clinical histoplasmosis with systemic manifestations has been, until recently, infrequently diagnosed. However, since the autumn of 1963, 31 cases of clinically significant histoplasmosis have been seen by the authors. These were divided into two groups: (1) patients in whom the diagnosis was established on the basis of histological and/or cultural demonstration of the fungus; (2) patients in whom the diagnosis was based on a positive histoplasmin skin test, a complement fixation antibody titre of 1:32 or greater and compatible clinical and radiological findings. An additional group of 11 patients who presented with erythema multiforme was investigated and a heretofore unrecognized relationship between histoplasmosis and erythema multiforme was established.