Interpretation of isolated agenesis of the pituitary.

Cholesterol synthesis inhibitors administered to rats caused more or less complete forms of the holoprosencephalic syndrome, consisting of severe abnormalities of the brain, sense organs and pituitary. The absence of the pituitary was also observed in fetuses without externally visible cephalic abnormalities. These observations suggest that the isolated absence of the pituitary is the lesser form of the holoprosencephalic syndrome. This interpretation is also valid for cases of isolated absence of the pituitary observed in humans.     

Glutathione conjugation by isolated lung cells and the isolated, perfused lung. Effect of extracellular glutathione

Cells isolated from rat lung by protease digestion were found to catalyze the reduced glutathione (GSH) conjugation of 1-chloro-2,4-dinitrobenzene. The rate of conjugation was stimulated severalfold in the presence of GSH, indicating uptake and utilization of extracellular GSH by the lung cells. The stimulation was dependent on the GSH concentration and not due to a spontaneous nonenzymatic reaction or to extracellular GSH-transferase activity. Conjugation of 1-chloro-2,4-dinitrobenzene was also measured using isolated perfused rat lung. The conjugation, which was linear for a longer time than with the isolated cells, was also stimulated in the presence of GSH in the perfusion medium. The results indicate the ability of rat lung to utilize extracellular GSH.     

Photoswelling and light-inactivation of isolated chloroplasts II. Functional and structural changes in isolated chloroplasts under the influence of lysolecithin

Lysolecithin was added to spinach chloroplasts in suspension,and its effect as a detergent on structure and electron transportactivities was examined. At low concentrations of lysolecithin,the activities of FeCN photoreduction and O₂-linked DCPIPH₂photo-oxidation were stimulated but the photoreduction of NADPwas not enhanced and decreased with an increase in concentration.Absorption bands over the whole visible region were intensifiedwithout any shifts. At high concentrations, the activities ofFeCN photoreduction and O₂-linked DCPIPH₂ photo-oxidation decreasedfrom a maximum to 10–50% of the control activity, andNADP photoreduction was completely inhibited. Absorption bandswere further intensified and the red band was slightly shifted.Results indicate that lysolecithin treatment is useful in chloroplastbiochemistry. Lysolecithin formation and the deterioration ofchloroplasts during light-aging is also discussed. (Received August 15, 1974; )     

Lactobacilli isolated from the stomach of conventional mice.

Twenty strains of lactobacilli isolated from the stomach of conventional mice were tested for their ability to ferment or hydrolyze substrates that may be present in the stomach habitat. The lactobacilli could be placed in four groups (A to D) depending on their ability to ferment N-acetylglucosamine, dextrin, cellobiose, gum arabic, and xylan. The majority of the isolates belonged to groups A and D. Group A strains did not resemble previously described Lactobacillus species, but group D strains were identified as L. leichmannii. A representative group A isolate colonized the surface of the nonsecretory epithelium of the stomach of gnotobiotic mice; a group D isolate did not.     

Ectoprotein kinase activity of the isolated rat adipocyte.

Intact adipocytes exhibit ectoprotein kinase activity as reflected by their ability to catalyze the transfer of the terminal phosphate of (γ-³²P) ATP to histone added to a cell suspension. This activity is substrate, time and cell number dependent. Lineweaver-Burk plots gave Km and Vmax values for ATP of 5 × 10^?5 M and 7.14 pmoles/min/1.5 × 10⁵ cells. Cyclic AMP but not cyclic GMP in μM concentrations stimulates ectoprotein kinase activity. The controlled tryptic digestion of intact cells results in reduction of ectoprotein kinase activity. This activity is not due to leakage of intracellular protein kinases during the preparative procedure nor to penetration of histone into the cells. Additional phosphoproteins not accessible to endogenous protein kinase activity are also localized on the external surface of the intact fat cell.     

Studies on isolated cell components. IV. The effect of various solutions on the isolated rat liver nucleus

1. The effects of morganic ions, electrolyte concentration, and pH on the appearance and volume of the isolated rat liver nucleus have been studied. Nuclei were isolated by differential centrifugation in a buffered salt-sucrose mixture at pH 7.1. Nuclear volumes were determined photographically. 2. In solutions of NaCl, of KCl, and in potassium phosphate buffers the nuclear volume decreased markedly with an increase in concentration from 0.001 M to 0.05 M but remained essentially constant with further increase in concentration to 1.0 M. The effects of CaCl(2) and MgCl(2) differed from those of NaCl and KCl in that a smaller volume was obtained in concentrations less than 0.15 M, and in the case of CaCl(2) an increase in volume was obtained in more concentrated solutions. The volume changes are considered to be due primarily to ionic effects on the nuclear colloids rather than to osmotic behavior. 3. Treatment of nuclei with DNAase prevented the characteristic volume changes resulting from ion effects, suggesting the importance of DNA in nuclear volume changes. 4. The optical changes in isolated nuclei in various concentrations of KCl, NaCl, CaCl(2), MgCl(2), and in potassium phosphate buffers as observed under phase contrast illumination are described. CaCl(2) gave the most marked nuclear changes from the conditions in the uninjured cell and caused shrinkage and granulation in 0.001 M concentration. The effects of CaCl(2) were also manifested in 0.88 M sucrose, in mixtures with monovalent salts, and in serum. Changes in nuclear volume and optical appearance which occurred in salt solutions and in 0.1 N HCl were readily reversible. 5. Nuclear volume remained constant between pH 8.91 and 5.12 and decreased in more acid solutions. 6. Sucrose had no appreciable osmotic effect, and in hyperosmotic solution. (0.88 M) nuclei showed swelling and rupture comparable to that in distilled water. 7. The results are considered in relation to the requirements of nuclear isolation media. 8. Rat liver nuclei isolated in a buffered salt-sucrose medium by differential centrifugation exhibited a pattern of size distribution similar to that of fixed nuclei but were of considerably larger volume. The ratio of the volumes of the peak frequencies of the two chief size groups was 1:1.9.     

Pleomorphism of fusobacteria isolated from the cockroach hindgut.

Fusobacteria are commonly isolated from the hindgut of the cockroach Eublaberus posticus . Eleven strains isolated from E. posticus by us were keyed to four species, Fusobacterium necrophorum, F. varium , F. gonidiaformans , and F. prausnitzii , using current taxonomic criteria. With the exception of F. gonidiaformis , all species showed rods with swollen centers and large bodies. The pleomorphism of F. varium was examined by phase microscopy and scanning and transmission electron microscopy. The pleomorphic process begins with a gradual swelling at the center of the rod until a large round body is formed. Some of these round bodies then fragment, giving rise to rod-shaped cells. When 10% yeast extract was added to growth media, pleomorphism was not induced. A dialyzable factor was found to account for this observation. Fermentation of [1-14C]glutamic acid gives rise to butyrate labeled in the carboxyl carbon, indicating that butyrate is formed by the hydroxyglutarate pathway which may be characteristic for the genus Fusobacterium.     

Permeability parameters of the toad isolated stratum corneum.

A technique for isolating the stratum corneum from the subjacent layers of the epithelium was developed which permits studying the stratum corneum as an isolated membrane mounted between half-chambers. The method basically consists of an osmotic shock induced by immersing a piece of skin in distilled water at 50 degrees C for 2 min. When the membrane is bathed on each surface by NaCl-Ringer's solution, its electrical resistance is 14.1 +/- 1.3 omega cm2 (n=10). This value is about 1/100 of the whole skin resistance in the presence of the same solution. The hydraulic filtration coefficient (Lp) measured by a hydrostatic pressure method, with identical solutions on each side of the membrane, is 8.8 X 10(-5) +/- 1.5 X 10(-5) cm sec-1 atm-1 (n=10) in distilled water and 9.2 X 10(-5) +/- 1.4 X 10(-5) cm sec-1 atm-1 (n=10) in NaCl-Ringer's solution. These values are not statistically different and are within the range of 1/80 to 1/120 of the whole skin Lp. The stratum corneum shows an amphoteric character when studied by KCl diffusion potentials at different pH'S. The membrane presents an isoelectric pH of 4.6 +/- 0.3 (n=10). Above the isoelectric pH the potassium transport number is higher than the chloride transport number; below it, the reverse situation is valid. Divalent cations (Ca++ or Cu++) reduce membrane ionic discrimination when the membrane is negatively charged and are ineffective when the membrane fixed charges are protonated at low pH.     

Conformation and elasticity of the isolated red blood cell membrane skeleton.

We studied the structure and elasticity of membrane skeletons from human red blood cells (RBCs) during and after extraction of RBC ghosts with nonionic detergent. Optical tweezers were used to suspend individual cells inside a flow chamber, away from all surfaces; this procedure allowed complete exchange of medium while the low-contrast protein network of the skeleton was observed by high resolution, video-enhanced differential interference-contrast (DIC) microscopy. Immediately following extraction in a 5 mM salt buffer, skeletons assumed expanded, nearly spherical shapes that were uncorrelated with the shapes of their parent RBCs. Judging by the extent of thermal undulations and by their deformability in small flow fields, the bending rigidity of skeletons was markedly lower than that of either RBCs or ghosts. No further changes were apparent in skeletons maintained in this buffer for up to 40 min at low temperatures (T less than 10 degrees C), but skeletons shrank when the ionic strength of the buffer was increased. When the salt concentration was raised to 1.5 M, shrinkage remained reversible for approximately 1 min but thereafter became irreversible. When maintained in 1.5 M salt buffer for longer periods, skeletons continued to shrink, lost flexibility, and assumed irregular shapes: this rigidification was irreversible. At this stage, skeletons closely resembled those isolated in standard bulk preparations. We propose that the transformation to the rigid, irreversibly shrunken state is a consequence of spectrin dimer-dimer reconnections and that these structural rearrangements are thermally activated. We also measured the salt-dependent size of fresh and bulk extracted skeletons. Our measurements suggest that, in situ, the spectrin tethers are flexible, with a persistence length of approximately 10 nm at 150 mM salt.     

Biosynthesis and excretion of gangliosides by the isolated perfused rat liver.

De novo synthesis and excretion into perfusate and bile fluid of hepatic gangliosides were studied in isolated perfused rat livers. Addition of N-acetyl-[6-3H(n)]D-mannosamine to the perfusate resulted in radioactive synthesis of at least eight gangliosides labeled in their sialic acid residues. About 10% of total de novo synthesized gangliosides were excreted into the perfusate, less than 1% into the bile fluid. Labeled gangliosides were tentatively identified by cochromatography with known standards. All of them are known to occur in rat liver and sera. The results indicate that most, if not all, normal serum gangliosides are synthesized in the liver; excretion with bile fluid is negligible. They explain previous observations, and indicate clinical implications, which are discussed.