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Preparation of cell walls and protoplasm of Neisseria with the Ribi cell fractionator.

The varied pressures required for disruption of Neisseria gonorrhoeae and other species of Neisseria when the Sorvall-Ribi refrigerated cell fractionator is used in the preparation of cell walls and cellular protoplasm are reported. Optimal disruption pressure for the gonococcus was considerably less than that required for other members of the genus Neisseria. Pressures varied from 8,000 psi for N. gonorrhoeae F62, colony type 4, to 22,000 psi for the nonpathogenic Neisseria-N. sicca, N. flava, and N. catarrhalis. Representative electron photomicrographs are shown.

Bacteriological Techniques↗

Preparation of apical plasma membranes from cells grown on coverslips. Electron microscopic investigations of the protoplasmic surface.

We introduce here a simple method which permits an efficient isolation of apical plasma membranes from tissue culture cells and the electron microscopic examination of their protoplasmic surfaces by use of the platinum/carbon replica technique. Different procedures were tested with regard to the efficiency of isolation and preservation of ultrastructure. Best results were obtained by prestabilization of cell surfaces with low concentrations of tannic acid prior to isolation. To demonstrate the possible applications and versatility of the method, studies were done on virus-infected cells in combination with immunocytochemical labeling. With this model system, we show that it is possible to correlate the structures seen on the cytoplasmic surface of the plasma membrane with the distribution of virus antigens at the cell surface labeled with immunogold markers prior to preparation.

Capsid↗

Plasmalemma invaginations, contraction and locomotion in normal and caffeine-treated protoplasmic drops of Physarum.

The de novo formation of plasmalemma invaginations and vacuoles of light microscopic dimension in protoplasmic drops at different age-stages was studied quantitatively by applying morphometric methods and marker techniques. The study includes: i) the normal morphogenetic development to a drop age of 1 hour, ii) the influence of caffeine treatment, and iii) the effects of removal of this drug. In untreated drops, formation of invaginations and vacuoles is accomplished within 10 to 15 min. By application of 5 mM caffeine, the formation of plasmalemma invaginations is inhibited for 20 to 30 min. The onset of oscillating contraction activity is delayed, but not hindered by the drug. Drug removal 20 min after drop generation leads to an immediate initiation of plasmalemma infolding. Although caffeine does not hinder initiation of normal contraction activity, the locomotory ability of the drop is blocked if the drug is not removed from the drop and the substrate. Thus, caffeine uncouples motive force generation from locomotion in all plasmodial stages of Physarum. The cellular sites of drug action are discussed.

Amoeba↗

Enzyme-linked immunosorbent assay for detection of bovine immunoglobulin G1 antibody to a protoplasmic antigen of Mycobacterium paratuberculosis.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of antibodies to Mycobacterium paratuberculosis in the sera of cattle. This assay was designed to minimize the nonspecific ELISA reactions caused by immunoglobulin (Ig)M by measuring only IgG1 antibodies against a protoplasmic antigen from the organism. The ELISA detected IgG1 antibodies in the sera of 58% of cattle with positive fecal cultures for M paratuberculosis compared with detection of 45% of culture-positive animals with an immunodiffusion test. In addition to its sensitivity, the ELISA apparently is highly specific because only 4% of the sera from fecal culture-negative animals gave a false-positive result.

Animals↗

Ultrastructural changes of basal laminae and protoplasmic astrocytes in craniostenosis with epilepsy.

Human brain biopsy specimens from twenty-four patients with epilepsy associated with craniostenosis and characterized by electroencephalography, were studied with an electron microscope. Characteristic ultrastructural changes were found in the basal lamina covering capillaries in the gray matter where the basal lamina was partially thickened, split and transformed into a heterogeneous network of strand enclosing electron-lucent areas. In the gray matter associated with epileptic convulsions, numerous filaments 60-100 nm in diameter appeared in protoplasmic astrocyte endfeet, apparently inserting in the plasma membrane adjacent to the basal lamina. Although the development of such filaments seemed to depend on precursor dense material appearing on the plasma membrane, their physiological function remains unknown. In cases with repeated epileptic convulsions, numerous fibrous astrocytes were seen in the affected portion of the gray matter, suggesting that they were the results of the repeated convulsions.

Astrocytes↗

Inhibition of pulmonary granuloma formation in mice by treatment with Mycobacterial protoplasm and immuno-suppressants and its relation to protection against aerosol infection with virulent Mycobacterium tuberculosis.

Because the pulmonary granuloma formation by mycobacterial cell walls (CW) is likely mediated by cellular immunity, the effect of specific antigens and various immunosuppressants on pulmonary granuloma formation and protection against virulent m. tuberculosis, strain H37Rv was investigated in mice immunized with oil-treated CW from BCG or M. Tuberculosis, strain Aoyama B. Results of desensitization experiments with specific antigens showed that multiple intravenous injections of BCG protoplasm inhibited the footpad delayed hypersensitivity, macrophage migration inhibitory activity of lung cells and pulmonary granuloma formation as well as the enhancement of resistance against aerosol challenge with M, tuberculosis, strain H37Rv. However, treatment with nonspecific immunosuppressants like con A, silica or carrageenan induced only abrogation of footpad delayed hypersensitivity but not prevention of pulmonary granuloma formation or resistance against aerosol challenge. These results suggest a close relationship between pulmonary granuloma formation and protection against H37Rv in mice immunized with oil-treated mycobacterial CW.

Aerosols↗

Spatio-temporal relationships between protoplasmic streaming and contraction activities in plasmodial veins of Physarum polycephalum.

Plasmodial veins of Physarum polycephalum were investigated by combining cinematographical, morphological, and tensiometrical techniques. Translucent and nontranslucent (thicker) strands remaining in their original position on filter paper were analyzed in respect to their radial contraction cycles and the resulting streaming activity of the endoplasm. A new technique for measuring the flow intensity by a microbalance method allowed testing of the influence of radial contraction on actual streaming processes within a strand. Stationary ectoplasm and flowing endoplasm are involved in a mutual transformation process: thereia an exchange of ectoplasm and endoplasm in the form of protoplasmic streamlets crossing the borderline between the endoplasmic stream and the ectoplasmic tube. Radial contraction cycles show the same time periodicity as the endoplasmic shuttle streaming. Relationships between radial contractions, streaming rates of endoplasm and streamlets between ectoplasm and endoplasm were analyzed. The participation of the ectoplasmic walls in motive force generation and their function in control of streaming throughput is discussed.

Cytoplasm↗

Cleft lip and palate etiology and its meaning in early 20th century England: Galton/Pearson vs. Bateson; polygenically poor protoplasm vs. Mendelism.

At the outset of the 20th century in England there arose a venomous dispute between Mendelian geneticists such as Bateson and anti-Mendelian biometricians such as Pearson over the genetic etiology of such "physical deformities" as cleft lip and palate. To Pearson et al., such traits were an expression of physical and racial degeneracy which could be traced to polygenically poor protoplasm. To Bateson et al., such traits were Mendelian unit characters whose segregation could be seen in carefully constructed family pedigrees. Bateson dismissed the work of the anti-Mendelians as "unsound in construction" and predicted such thinking would inevitably lead to "brutal" control of those the larger society deemed unfit. History proved Bateson astutely prescient.

Cleft Lip↗