Acquisition of adjectives and adverbs in sentences written by hearing impaired and aphasic children. The effect of an instructional package, which included modeling, reinforcement, and remedial feedback on the rate, accuracy, and topography of sentences composed by four hearing impaired and aphasic children, was examined. In a specially designed classroom, students wrote sentences describing a stimulus picture on acetate sheets placed on the stage of an overhead projector which was built into each student's desk. This arrangement provided the teacher and other students immediate and continuous visual access to each student's sentences. In a multiple baseline design across behaviors, model sentences were projected and token reinforcment and remedial feedback were made contingent upon writing correct sentences containing prenominal adjectives only, then adverbs only, then prenomial adjectives plus adverbs. During baseline all student displayed poor written language skills and seldom wrote sentences containing modifiers. When the instructional package was implemented, all students demonstrated significant increases in response rate, accuracy, and percentage of correct sentences including prenominal adjectives and adverbs.


alpha 2 Macroglobulin binding to the plasma membrane of cultured fibroblasts. Diffuse binding followed by clustering in coated regions. Using transmission electron microscopy, we have studied the interaction of alpha 2 macroglobulin (alpha 2 M) with the surface of cultured fibroblasts. When cells were incubated for 2 h at 4 degrees C with ferritin-conjugated alpha 2 M, approximately 90% of the alpha 2 M was diffusely distributed on the cell surface, and the other 10% was concentrated in "coated" pits. A pattern of diffuse labeling with some clustering in "coated" pits was also obtained when cells were incubated for 5 min at 4 degrees C with alpha 2 M, fixed with glutaraldehyde, and the alpha 2 M was localized with affinity-purified, peroxidase-labeled antibody to alpha 2 M. Experiments in which cells were fixed with 0.2% paraformaldehyde before incubation with alpha 2 M showed that the native distribution of alpha 2 M receptors was entirely diffuse without significant clustering in "coated" pits. This indicates that some redistribution of the alpha 2 M-receptor complexes into clusters occurred even at 4 degrees C. In experiments with concanavalin A(Con A), we found that some of the Con A clustered in coated regions of the membrane and was internalized in coated vesicles, but much of the Con A was directly internalized in uncoated vesicles or pinosomes. We conclude that unoccupied alpha 2 M receptors are diffusely distributed on the cell surface. When alpha 2 M-receptor complexes are formed, they rapidly cluster in coated regions or pits in the plasma membrane and subsequently are internalized in coated vesicles. Because insulin and epidermal growth factor are internalized in the same structures as alpha 2 M (Maxfield, F.R., J. Schlessinger, Y. Schechter, I. Pastan, and M.C. Willingham. 1978. Cell, 14: 805--810.), we suggest that all peptide hormones, as well as other proteins that enter the cell by receptor-mediated endocytosis, follow this same pathway.


Regulation of surface topography of mouse peritoneal cells. Formation of microvilli and vesiculated pits on omental mesothelial cells by serum and other proteins. The mesothelial cells of the mouse omentum provide an in vivo model for the study of the mobilization of labile microvilli on the cell surface. These mesothelial cells are sparsely covered with microvilli and large pits 150--400 nm in diameter, termed vesiculated pits. On the unstimulated cell, the microvilli average 44/100 microns2 and pits, 30/100 microns 2 of surface and they are rapidly induced to increase in number by the intraperitoneal injection of isologous mouse serum. After 2 min, microvilli increase threefold, continue to sevenfold at 30 min, and decrease to fourfold at 90 min. Vesiculated pits increased with similar kinetics. Bovine serum albumin and gamma globulin also stimulate the microvilli and pits to form, but the response is a slow, gradual rise to five- or sixfold the normal value at 90 min. Evidence indicates that multiple factors, possibly including insulin and immunoglobulins, are involved in the effect of serum. The close physical and temporal relationship between microvilli and pits suggests that a correlation exists in their mobilization by the cell and it is hypothesized that microvilli function in the regulation of the cortical microfilament network in effecting this mobilization.


Selective retrograde transsynaptic transfer of a protein, tetanus toxin, subsequent to its retrograde axonal transport. The fate of tetanus toxin (mol wt 150,000) subsequent to its retrograde axonal transport in peripheral sympathetic neurons of the rat was studied by both electron microscope autoradiography and cytochemistry using toxin-horseradish peroxidase (HRP) coupling products, and compared to that of nerve growth factor (NGF), cholera toxin, and the lectins wheat germ agglutinin (WGA), phytohaemagglutinin (PHA), and ricin. All these macromolecules are taken up by adrenergic nerve terminals and transported retrogradely in a selective, highly efficient manner. This selective uptake and transport is a consequence of the binding of these macromolecules to specific receptive sites on the nerve terminal membrane. All these ligands are transported in the axons within smooth vesicles, cisternae, and tubules. In the cell bodies these membrane compartments fuse and most of the transported macromolecules are finally incorporated into lysosomes. The cell nuclei, the parallel golgi cisternae, and the extracellular space always remain unlabeled. In case the tetanus toxin, however, a substantial fraction of the labeled material appears in presynaptic cholinergic nerve terminals which innervate the labeled ganglion cells. In these terminals tetanus toxin-HRP is localized in 500-1,000 A diam vesicles. In contrast, such a retrograde transsynaptic transfer is not at all or only very rarely detectable after retrograde transport of cholera toxin, NGF, WGA, PHA, or ricin. An atoxic fragment of the tetanus toxin, which contains the ganglioside-binding site, behaves like intact toxin. With all these macromolecules, the extracellular space and the glial cells in the ganglion remain unlabeled. We conclude that the selectivity of this transsynaptic transfer of tetanus toxin is due to a selective release of the toxin from the postsynaptic dendrites. This release is immediately followed by an uptake into the presynaptic terminals.


Autoradiographic identification of acetylcholine in the rabbit retina. Rabbit retinas were studied in vitro under conditions known to maintain their physiological function. Retinas incubated in the presence of [3H]choline synthesized substantial amounts of both [3H]phosphorylcholine and [3H]acetylcholine. With time, [3H]phosphorylcholine proceeded into phospholipids, primarily phosphatidylcholine. Retinas pulse-labeled by a 15-min exposure to 0.3 microM [3H]choline were incubated for a subsequent hour under chase conditions designed either to retain newly synthesized acetylcholine within synapses or to promote its release. At the end of this time the two groups of retinas were found to contain equal amounts of radioactivity in the phospholipid pathway, but only the retinas incubated under the acetylcholine-protecting conditions contained [3H]acetylcholine. Freeze-dried, vacuum-embedded tissue from each retina was autoradiographed on dry emulsion. All retinas showed silver grains over the photoreceptor cells and faint labeling of all ganglion cells. In the retinas that contained [3H]acetylcholine, silver grains also accumulated densely over a few cells with the position of amacrine cells, over a subset of the cells of the ganglion cell layer, and in two bands over the inner plexiform layer. Fixation of the retina with aqueous osmium tetroxide retained only the radioactive compounds located in the photoreceptor and ganglion cells. Sections from freeze-dried tissue lost their water-soluble choline metabolites when exposed to water, and autoradiography of such sections again revealed radioactivity primarily in the photoreceptor and ganglion cells. Radioactive compounds extracted from the sections were found to faithfully reflect those present in the tissue before processing; analysis of the compounds eluted from sections microdissected along the outer plexiform layer showed [3H]acetylcholine to have been synthesized only by cells of the inner retina. Taken together, these results indicate that the photoreceptor and ganglion cells are distinguished by a rapid synthesis of choline-containing phospholipids, while acetylcholine synthesis is restricted to a few cells at both margins of the inner plexiform layer. They imply that the only neurons to release acetylcholine within the rabbit retina are a small group of probable amacrine cells.


Estrogen and progestin regulation of the progesterone receptor concentration in human endometrium. The concentration of the progesterone receptor (PR), both cytosol and nuclear, has been measured in the endometrium of 31 normal menstruating women during the 2 phases of their menstrual cycle and compared with the plasma 17 beta-estradiol and progesterone concentrations. There was no relationship between PR concentrations and the plasma steroid levels when the 2 phases of the cycle were considered; however, a statistically significant correlation (r = 0.70;  P less than 0.005) was observed between PR concentration and plasma 17 beta-estradiol when only the follicular phase was considered. PR was then measured in the endometrium of 14 postmenopausal patients treated with ethinylestradiol at increasing doses with or without association of chlormadinone acetate. Ethinylestradiol was shown to increase PR concentration (P less than 0.05), and chlormadinone acetate was found to prevent this increase. These data suggest that in humans, as in other mammalian species, the endometrial PR concentration is under estrogen and progestin control.


Pulmonary carcinoid tumours: a comparative regional study. Twenty-two pulmonary carcinoid tumours (18 central, 4 peripheral) were seen in this department over an 11-year period. The majority of the tumours displayed a mixed pattern on microscopic examination, and cell-nest formation was a prominent feature in sixteen. The findings are at variance with the results of a similar series reported from Japan and suggest that there is regional variation in the tumour pattern of pulmonary carcinoids. Various aspects of the histopathology are discussed, and a causal relationship between ossification of bronchial cartilage in these tumours and locally produced calcitonin is postulated.


Early detection of Chlamydia trachomatis using fluorescent, DNA binding dyes. HeLa 229 cells were infected with genital tract strains of Chlamydia trachomatis. After incubation for varying times the infected cells were fixed and stained with the fluorescent DNA binding dyes Hoechst 33258 or DAPI for comparison with conventional Giemsa stain. Fluorochrome-treated preparations were examined by incident ultraviolet fluorescence microscopy and the Giemsa-stained preparations by dark-ground light microscopy. Chlamydial inclusion bodies could be identified unambiguously as early as 18 hours after infection of HeLa 229 cells using either Hoechst 33258 or DAPI but not until some 48 hours in Giemsa-stained preparations. The DNA rich chlamydial elementary bodies in infected egg yolk suspension were readily detected using Hoechst 33258. The fluorescent dye technique was simpler and more rapid than Giemsa staining. Using Hoechst 33258 it is possible to speed up the identification of chlamydial isolates growing in tissue culture.


Rapid diagnosis of beta-haemolytic streptococcus group B endocarditis by buffy-coat examination and gas-liquid chromatography. A semicomatose 34-year-old man had fever, heart murmur, and multiple gangrenous lesions. Several blood cultures were negative. Cultures of the heart valve grew beta-haemolytic streptococcus group B. The organism was seen in buffy-coat examination of the venous blood. The gas-liquid chromatogram of the serum was different from those of normal controls and of patients with invasive candida infection; it closely mimicked the one obtained from the organism itself. These studies promise to be valuable diagnostic adjuncts.


Distribution of T-lymphocytes in follicular lymphomas as revealed by acid alpha-naphthol acetate esterase. Twelve cases of follicular centroblastic/centrocytic malignant lymphoma were studied for acid non-specific esterase. The majority of the lymphocytes in interfollicular areas showed a pattern of enzymatic activity consistent with a T-cell nature. Variable numbers of lymphocytes with a similar enzymatic pattern were also seen among the negative centroblasts and centrocytes in neoplastic follicles.


Black and white human skin differences.

Black and white human skin differences. This review of black and white human skin differences emphasizes the alleged importance of factors other than the obvious, i.e., skin color. Physicochemical differences and differences in susceptibility to irritants and allergens suggest a more resistant black than white skin. Differences appear to exist in the frequency of which several skin diseases occur among blacks and whites. A striking feature in this literature is the disagreement between authors. Common for much of this information is difficulty of interpretation, because of socioeconomic influences and other environmental factors.


Distributional uptake of gamma-globulin in small intestine of neonatal calves. Newborn calves deprived of colostrum were used to determine distribution of uptake of gamma-globulin labeled with iodine-125 in small intestine. Ten calves less than 12.5 h of age (average 7 h) were anesthetized, and intestines were exteriorized through an abdominal incision. Intestine was ligated into 10-cm segments, 70 cm apart, beginning at the ileocecal junction and progressing anteriorally, then each segment injected with 100 mg (appoximately 1  microCi) labeled gamma-globulin in 5 ml electrolyte solution, and incubated for 1.5 h. One additional segment was formed adjacent to segments 1, 5, and 10 to assess uptake after .5 h incubation with [iodine-125] gamma-globulin. After prescribed gamma-globulin exposure, segments were excised; then volume of lumen contents, segment weight, and tissue activity were measured. The location of each segment was the percentage of distance from cecum to abomasum. Uptake was milligrams gamma-globulin per gram of segment tissue. Distribution of gamma-globulin uptake after 1.5 h incubation was a cubic function of segment position. Uptake was greatest in a region 15% of the cecum-abomasum distance and declined progressively toward the abomasum. After .5-h incubation with gamma-globulin, regression of uptake on segment position was a quadratic function with greatest uptake at 30% of cecum-abomasum distance. Uptake was greater in segments exposed to gamma-globulin for 1.5 h than .5 h.


Radiochemical determination of the metabolic activity of collagen in mature dentin. Tritiated proline was administered to domestic cats during the development of their permanent premolars. The metabolic activity of collagen in the mature premolar dentin was determined by quantitating the amount of tritiated hydroxyproline in the dentin as a function of time. It has been demonstrated that the metabolic activity of the dentinal collagen was extremely low and remained within the experimental error for a period of 45 weeks.


Study on the significance of bronchial hyperreactivity in the bronchus obstruction after inhalation of cat dander allergen. The role of bronchial hyperreactivity in the process that leads to bronchial obstruction after inhalation of an allergen was investigated. In 30 asthmatic children selected because of a positive skin test to cat dander allergen, we measured the histamine threshold, the reaction after allergen inhalation, the allergen-specific IgE concentration in serum, the lowest allergen concentration to which the intracutaneous skin test was positive (skin titer), and the histamine release of leukocytes after challenge with allergen. These variables were correlated with each other. The highest correlation was found between the inhalation reaction and the combination of the histamine threshold and either the allergen-specific IgE or the skin titer. Inhalation was only positive with a decreased histamine threshold (less than or equal to 8 mg/ml). With a low histamine threshold, a positive reaction to inhalation is likely to occur at an allergen-specific IgE concentration of > or = 2 U/ml or at a skin titer of < or = 2.5 x 10(-1) micrograms/ml.


Allergenic and biologic activity of commercial preparations of house dust extract. The endotoxin, protein nitrogen unit (PNU) content, and allergenic activity of seven different extracts of house dust were determined. The PNU content varied from 1,600 to 44,000 PNU/ml. The endotoxin content as measured by the limulus assay varied from 450 to 500,000 ng/ml. Pyrogenic activity was present by rabbit fever assay and correlated well with the limulus assay. There was no correlation between endotoxin and PNU content of the different dust preparations. Leukocytes from 15 patients with a clinical history of allergy to dust were used for histamine release studies. The allergenic activity did not correlate with the PNU or endotoxin content of the different house dust preparations. The dust extracts could be divided into three groups according to their activities: three extracts which were the most reactive (30% histamine release with 0.12 to 0.39 PNU/ml), two which were about 20 times less active (4.75 to 6.43 PNU/ml for 30% histamine release), and a final group of two which were about 600 times less active. The leukocytes of three out of 15 patients failed to release histamine with one of these least reactive extracts. The reactivity pattern of five of the dust preparations appeared to correlate, suggesting that the different preparations contained the same allergens. Reactivities of two of the other extracts correlated poorly, and the reactivity of the least reactive sample did not correlate with six out of the seven preparations. Therefore, some dust preparations contain significant endotoxin-like material. Wide variations in the endotoxin, PNU content, allergenic activity, and reactivity patterns of different dust preparations were observed.


Structure and activity of ragweed antigen E. II. Allergenic crossreactivity of the subunits. Recently we have reported the isolation of the two subunits (alpha and beta) of ragweed antigen E (AgE) in their active form. In the present work we have compared the allergenic activity of the two subunits with native AgE by the radioallergosorbent test (RAST) and by inhibition of the RAST. Also, the amounts of IgE antibodies in 30 ragweed-sensitive sera bound to alpha and beta subunits as well as native AgE were measured by RAST. In all individuals the percentage of RAST binding was greatest to native AgE; however, varying patterns of reactivity to alpha and beta subunits were noted. In RAST inhibition experiments, free beta subunit was equally effective in inhibiting the binding of IgE antibody (from a pooled ragweed-sensitive sera) to immoblized alpha or beta subunit. Similarly, fluid-phase alpha subunit inhibited in a comparable manner the binding of IgE antibody to solid-phase alpha or beta subunits. In further RAST inhibition experiments 7 x 10(-13) moles of fluid-phase native AgE was required for 50% inhibition of the binding of IgE antibody to solid-phase native AgE. Larger molar amounts of fluid phase beta (3.5 x 10(-11) and alpha (3 x 10(-10) were needed for 50% inhibition of the binding of IgE antibody to solid-phase native AgE. AgE allergenicity as measured by RAST could not be totally reconstituted by recombination of equimolar quantities of alpha and beta subunits. Less than 1 x 10(-12) moles of fluid-phase native AgE inhibited the binding of IgE antibody to solid-phase alpha or beta, greater than 50%. Finally, the isolated chains are bioligcally active as demonstrated by their ability to provoke wheal-and-flare skin reactions in subjects allergic to ragweed pollen, and the activities of the chains were approximately equivalent on a weight basis to native AgE.


An in vitro procedure for the estimation of thyroid hormone releasing factors in sera of thyrotoxic patients. LATS containing sera and a number of Graves' disease sera stimulated T4 release from mouse thyroids in vitro as determined by RIA, thus confirming the presence of a thyroid hormone releasing factor in sera of thyrotoxic patients. The pattern of stimulation was similar to that previously shown for TSH in terms of T4 release time sequence. cAMP increase and catecholamine inhibition via alpha-adrenergic receptors. In the same in vitro system, neutralization with a human thyroid homogenate showed presence of LATS-Protector (LPA) in LATS negative thyrotoxic sera. The present study describes a simpler procedure for estimating LATS or similar activity, as compared to the McKenzie assay, and suggests identical receptor sites for TSH and other thyroid stimulators.


Myoglobin as an ultrastructural probe for studying permeability of the nephron. Hemeproteins, like cytochrome c (12,500 M.W.; Karnovsky and Rice, 1969) and myoglobin (17,816 M.W.; Anderson, 1972; Simionescu et al., 1973) are advantageous over the true peroxidases with larger molecular weights (e.g. horseradish peroxidase, ca. 40,000 M.W) as ultrastructural probes in that they do not elicit vascular leakage in the inflammatory response (Cotran and Karnovsky, 1967) and are relatively nontoxic immunologically inert substances. The main disadvantage in using cytochrome c and myoglobin is that they have weak peroxidatic activity compared to the true peroxidases (Nakamura et al., 1960; Keilin, 1961; Kurozimi et al., 1961). These hemeproteins, however, offer the following advantages: 1) they retain sufficient peroxidatic activity after aldehyde-fixation to oxidize 3,3'-diaminobenzidine (DAB), 2) they may be localized by virtue of an insoluble reaction product (osmium black) deposited at the site of hemeprotein immobilization by fixation, and 3) they represent low molecular weight probes. This brief report emphasizes the advantages of myoglobin in the study of glomerular permeability, transport by endocytosis in proximal tubules and translocation of protein in the lower segments of the nephron.


Application of microspectrophotometry to biomedical research. During recent years, the utilization of microspectrophotometry in biomedical research has increased at a rapid pace. This growth has been facilitated by both the advent of commercially available instruments which are reliable and manageable as well as improvements in histochemical techniques. This paper discusses the fundamental principles of visible light microspectrophotometry and important design criteria as well as briefly reviewing a wide range of applications. Special emphasis will be given to the Vickers M85 scanning integrating microspectrophotometer which is being routinely employed for both clinical and laboratory studies by a sizeable number of investigators.


Nonspecific light loss and intrinsic DNA variation problems associated with feulgen DNA cytophotometry. Nonspecific light loss by the cell-wall-plus-cytoplasm (CWC) can cause a 50% increase in Feulgen absorption units in peanut root-tip nuclei as determined by scanning at 450 nm, whereas this phenomenon is not evident with chicken erythrocytes. A two wavelength scanning method of subtracting nonspecific 450 nm absorption from 550 nm Feulgen absorption values eliminated the nonspecific light loss in CWC, However, the two wavelength scanning method is time consuming and somewhat impractical with a regular scanning microdensitometer such as Vickers M85. Elimination of the problem of nonspecific light loss is suggested by careful determination of background setting with the spot position close to the nucleus in CWC. The accuracy of the CWC background setting method was further tested by comparison with subtraction method. The use of plant nucleis as an internal standard in plant DNA measurements was also evaluated. Significant variation among the replicate slides due to the variation in pine nuclear DNA amounts was observed and plant nuclei generally are not reliable internal standards. Mature chicken erythrocytes are recommended as an internal standard because the cell type and metabolic state is known.


Applications of microcomputer technology to cytophotometry. Lack of appropriate software support for microprocessor program development has previously limited the applications of such technology in the field of microspectrophotometry. This paper describes our use of a Vickers M86 integrating microdensitometer coupled through a custom-designed interface circuit to a Processor Technology SOL III microcomputer. W e have developed a series of interactive, user-oriented programs for DNA-Feulgen cytophotometry with this instrument to allow automatic storage of data in files on floppy disks and instant retrieval of sets of measurements for statistical processing. The same data files can also be used to generate graphic displays in the form of bar histograms or plots of linear regressions on a video monitor and to produce hardcopy output of data files and graphic displays through the use of a high speed DIABLO printer.


Immunoelectron microscopic localization of herpes simplex virus antigens in infected cells using the unlabeled antibody-enzyme method. Subcellular localization of viral antigens was demonstrated during viral morphogenesis using herpes simplex virus type 1 (HSV-1) infected monolayers of rabbit cornea cells. The localization was done by immunoelectron microscopy employing the peroxidase-antiperoxidase (PAP) immunocytochemical technique and the postembedding staining method. The localization of viral antigens was followed at time intervals during infection from 2 to 19 hr. After exposure of sections to either polyspecific antibodies against total HSV-1 antigens or monospecific antibodies against HSV-1 antigen No. 8, specific immunological reaction products were identified both in the cytoplasm and nucleus after 2 hr. The distribution and quantity of reaction products varied in the infected cells during the viral morphogenesis. The present results on the subcellular distribution of the HSV-1 antigens are related to current biochemical findings.


Cytochemistry of human catalase. The demonstration of hepatic and renal peroxisomes by a high temperature procedure. The cytochemical demonstration of marker enzymes for subcellular organelles permits light microscopic analysis of their structure and function in normal and diseased tissues. Currently available staining procedures for the peroxidatic activity of catalase in peroxisomes of plant and animal cells yield weak and inconsistent light microscopic staining when applied to human tissues. We have developed a simple and sensitive high temperature procedure that clearly and reproducibly stains these abundant, but poorly understood, organelles in biopsy specimens of human liver and kidney. This method utilizes formaldehyde fixation, a modified diaminobenzidine (DAB) medium, incubation at 45 degrees C and postosmication for both light and electron microscopy.


Evaluation of the validity of the histochemical lead nitrate technique for alkaline and acid deoxyribonuclease. The results of several tests and the characteristic morphological distribution of the enzymatic activity appeared to be in favor of the validity and specificity of the histochemical lead nitrate technique for alkaline and acid deoxyribonuclease (DNAse) detection. These tests included thermal inhibition, omission of substrate, use of different chemical inhibitors and the reproduction of histochemical staining on Coujard's slides. Most of these results were in conformity with the biochemical data gathered from literature. Topographically selective inhibition of alkaline or acid DNAse by different factors suggested that there might exist two kinds of alkaline or acid DNase--one cytoplasmic and the other one nuclear. The whole histochemical procedure produced relatively small loss of alkaline and acid DNAse activities as verified by biochemical methods.


Generation and enrichment of C4d in whole serum. A comparison of two methods of generation of C4d (the degradation product of complement component C4) and a method for the preparation of an enriched serum fraction of this fragment are presented.


An improved method for selecting T-cell hybridomas by the fluorescence-activated cell sorter. A two-step sorting procedure with the fluorescence-activated cell sorter (FACS) is described to select T cell hybridomas from cell populations obtained from the fusion of activated T cells and T- lymphoma cells. Cells are first sorted on the basis of differences in cell-surface antigens. After a period of growth in culture, the cells are then stained with the DNA-labeling dye dis-benzimidazole 33258 Hoechst and sorted on the basis of their DNA content. From 15 to 25% of the resulting cell clones, growing out of limiting dilutions, were hybrid lines as judged by chromosome analysis.


Sensitive radioimmunoassays using partially purified gamma globulins coupled to enzacryl (acrylamide polymer) solid support. Enzacryl polythiolactone is a cross-linked acrylamide polymer with an active thiolactone group, capable of coupling to lysine, serine and tyrosine residues. Gamma globulins from antisera specific to human chorionic gonadotropin, carcinoembryonic antigen, alpha-foetoprotein or casein were isolated using Protein A Sepharose and were subsequently coupled to enzacryl. The resulting coupled antibodies were found to provide greater sensitivity and convenience in radioimmunoassay studies than conventional double antibody precipitation methods using the same antisera.


An investigation of the optimal conditions for the in vivo production of immunologically sensitised rat mast cells. Experiments were designed to develop an optimal method for inducing in vivo production of sensitised peritoneal mast cells. Rats of different strains were sensitised with whole egg-white and killed at suitable intervals to harvest the peritoneal mast cells. Release of histamine was induced in vitro by both whole egg-white and its major protein constituents, and assayed by a standard spectrofluorometric method. Wistar rats showed higher levels of sensitisation than black-hooded Lister rats; it was more convenient to harvest erythrocyte-free peritoneal mast cells from males than females. Very young (less than 150 g) and very old (greater than 300 g) rats showed sub-optimal sensitisation. Optimal sensitisation was obtained by simultaneous administration of antigen (doses of 50 micrograms whole egg-white and above) and adjuvant (1.0 ml pertussis vaccine); mast cells harvested between 20 and 50 days after the sensitising dose exhibited maximal histamine release upon in vitro challenge with 'whole' egg-white (100 micrograms). Routine use of plastic ware, and ice-cold phosphate-buffered saline (pH 7.2) for handling cells and avoidance of heparin and excessive centrifugation ensured optimal preservation of histamine-releasing capacity of the harvested peritoneal mast cells.


Molecular heterogeneity of pregnancy specific beta 1 glycoprotein: the effect on measurement by radioimmunoassay and electroimmunoassay. A comparison of methods for the quantification of circulating pregnancy specific beta 1 glycoprotein in late pregnancy was performed to assess the influence of the presence of a high molecular weight glycoprotein with alpha 2 electrophoretic mobility (SP1 alpha) which is immunochemically identifiable with pregnancy-specific PBETA 1 glycoprotein (SP1 beta). Serum samples from 47 volunteers in the 3rd trimester of pregnancy were subjected to measurements of pregnancy specific beta 1 glycoprotein in rocket immunoelectrophoresis, quantitative crossed-immunoelectrophoresis and radioimmunoassay. Rocket immunoelectrophoresis gives a result which reflects the total SP1 content, i.e. SP1 alpha and SP1 beta while quantitative crossed-immunoelectrophoresis permits differentiation between the two molecules. Radioimmunoassay predominantly measures authenic SP1, i.e. SP1 beta in the presence of physiological amounts of SP1 alpha.


Intrathymic injection of antigen: a potent procedure for the induction of suppressor T cells. Immunological tolerance is induced in mice by intrathymic injection of HSA. The tolerance thus induced is mediated by suppressor T cells. Strong tolerance persists more than 56 days after the induction, and the high efficiency of the tolerance thus induced is accounted for in terms of the number or the potentiality of suppressor cells. Possible mechanisms of suppressor T cell induction by iT injection of the antigen are discussed briefly.


Microplate enzyme-linked immunosorbent assay in the study of the structural relationship between myosin light chains. A microplate enzyme-linked immunosorbent assay (microELISA) for the study of immunochemical relationships between rabbit myosin light chains is described. Purified individual fast-muscle myosin light chains (LC1F, LC2F and LC3F) and their respective antisera, obtained in chicken, were used. Optimal conditions for antigen concentration, antiserum dilution, substrate concentration, incubation time and reproducibility with time were established. The observed cross-reactivities between the different types of light chains associated with rabbit fast-muscle myosin confirm and extend previous results obtained by other authors using radioimmunoassay procedures. It was concluded that microELIAS may be successfully employed also to the study of macromolecule cross-reactivities.


Efficacy of purified influenza subunit vaccines and relation to the major antigenic determinants on the hemagglutinin molecule. Inactivated whole-virus vaccine of influenza A/Scotland/74 (H3N2) virus containing 700 or 1,400 chick cell-agglutinating (CCA) units, a purified subunit vaccine of equivalent dosage, or placebo were studied in 186 adult volunteers. Placebo was least reactogenic, 1,400-CCA unit whole-virus vaccine was most reactogenic, and others were intermediate. Vaccines were equally antigenic, and delineation of antibody specificities revealed antibody cross-reacting with A/Hong Kong/68 (H3N2) virus in all sera. Antibody specific for A/Hong Kong/68 virus was found in 82% of sera and for A/Scotland/74 virus in 46%. When compared with volunteers given placebo, volunteers given 700 CCA units of subunit or whole-virus vaccine exhibited significant protection against infection with live A/Scotland/74 virus. Infections in vaccinees occurred only in those with low titers of antibody to A/Scotland/74 virus, and this antibody was of the cross-reacting type. Persons with moderate and high levels of antibody resisted infection regardless of the absence or presence of antibody specific for A/Scotland/74 virus. Purified subunit vaccines provide an alternative to whole-virus preparations in primed individuals. Efficacy of vaccines may be dependent on the nature of the antibody response.


An electron microscopic India ink technique for demonstrating capsules on microorganisms: studies with Streptococcus pneumoniae, Staphylococcus aureus, and Neisseria gonorrhoeae. A technique using India ink in electron microscopic preparations was used to study bacterial capsules. Capsules were demonstrated on Streptococcus pneumoniae and Staphylococcus aureus (strain M and the Smith diffuse variant) from in vitro cultures. Two types of false capsules were observed on Neisseria gonorrhoeae grown in vitro or obtained from human urethral exudates. No true capsules were definitively identified on gonococci from these sources. The technique described does not require the use of specific anticapsular antibody or specific polysaccharide stains and permits the detection of artifacts that may, by the use of light microscopic methods, be misinterpreted as capsules.


Concealed quadrigeminy and quintageminy. Electrocardiographic rhythm strips were analyzed from three patients who had frequent unifocal extrasystoles. Parasystole was excluded in all three cases. Analysis of the distributions of the numbers (S) of conducted sinus beats between extrasystoles revealed that the distributions conformed to two variants of concealed quadrigeminy (namely, the "S = 4n" and "S = 4n - 2" variants) and to one variant of concealed quintageminy (namely, the "S = 5n - 2" variant). The binomial theorem was used to test the likelihoods that these patterns of distribution of extrasystoles could occur on the basis of chance alone; the probabilities were all extremely small.


Inhibition of H-Y cell-mediated cytolysis by H-2Db antiserum. Assays of H-Y-specific cell-mediated cytolysis (CMC) in vitro were carried out with B6 female effector cells and B6 male target cells. This lytic system depends on compatibility for H-2Db but not for H-2Kb. Antibody to H-2Db but not to H-2Kb blocked the CMC assay. However, maximal blocking required exposure to the target cells to H-2Db antibody for up to 2 h. This interval is far longer than would be required for blocking of a pre-existing H-Y:H-2Db complex, and seems more consonant with a membrane reorganization bringing H-Y and H-2Db together, as proposed from mapping studies. The question whether the postulated configurational change affecting H-Y and H-2Db is a necessary feature of CMC awaits further study.


Hapten-specific T-cell responses to 4-hydroxy-3-nitrophenyl acetyl. II. Demonstration of idiotypic determinants on suppressor T cells. The ability of NP-coupled syngeneic spleen cells to induce antigen-specific T-suppressor cells capable of binding to NP-BSA-coated Petri dishes and mediating transfer of specific suppressive activity to NP was demonstrated. Furthermore, in strains of mice bearing the Ig-1b allotype, including SJL, and in (non-Ig-1b x Ig-1b)F1 hybrids, the NP-specific suppressor cells also interferes with expression of immunity after priming with NIP-BGG. Anti-NPb anti-idiotype antiserum plus complement treatment effectively abrogated the ability to transfer suppression. Formal genetic mapping of the fine specificity of cross-reactivity with Ig-1 allotypic congenic mice implies that expression of this trait is linked to the Ig-1b heavy chain linkage group. The sensitivity of NP-suppressor cells of appropriate strains to anti-idiotype treatment was also consistent with the formal mapping data. These experiments suggest that there are shared V-region structures on antibody and T cells that are crucial in the suppression pathway for the same antigen.


Studies on the clonal origin of multiple myeloma. Use of individually specific (idiotype) antibodies to trace the oncogenic event to its earliest point of expression in B-cell differentiation. IgA myeloma proteins of kappa- and lambda-types were isolated from two patients. These were used to produce and purify anti-idiotype antibodies of both broad (myeloma-related) and narrow (individual myeloma) specificities. The anti-idiotype antibodies were conjugated with fluorochromes and used as immunofluorescent probes to trace in the patients clonal expansion at different levels of B-cell differentiation. Our results (a) confirm that B lymphocyte precursors in IgA plasma-cell myelomas are involved in the malignant process, (b) show that B lymphocytes of the malignant clone include those expressing each of the major heavy-chain isotypes, mu, delta, gamma, and alpha, and (c) provide strong circumstantial evidence that pre-B-cell members of the malignant clone are also increased in frequency. T cells expressing idiotypic determinants were not detected. These findings argue that the initial oncogenic event may occur in a B-stem cell and is not influenced through stimulation by antigen. An interesting association was the increased frequency of related clones of B lymphocytes as detected by their reactivity with anti-idiotype antibodies of broad specificity. Neither plasma cell nor pre-B-cell members of these related clones were increased in frequency. Anti-idiotype antibodies or helper T cells reactive with myeloma-related idiotypes could be responsible for this phenomenon. We discuss other implications of these findings and speculate that all of the various phenotypes of B-lineage malignancies may result from oncogenic processes affecting stem cell targets.


Suppression of hapten-specific delayed-type hypersensitivity responses in mice by idiotype-specific suppressor T cells after administration of anti-idiotypic antibodies. Delayed-type hypersensitivity (DTH) responses specific for the phosphorylcholine (PC) hapten were induced in BALB/c mice by immunization with syngeneic peritoneal exudate cells (PEC) coupled with diazotized phenyl-phosphoryl-choline. PC-specific DTH responses were elicited in such immunized mice after footpad challenge with PC-derivatized syngeneic spleen cells. Moreover, PC-immune lymph node cells could passively transfer PC-specific DTH responses to naive BALB/c mice and it was possible to demonstrate that the cells responsible for such passively transferred responses were T lymphocytes. Because the T-15 idiotypic determinant displayed on the TEPC-15 PC-binding myeloma protein is known to be a dominant idiotype associated with anti-PC antibody responses in BALB/c mice, an analysis was made of the effects of anti-T-15 idiotypic antibodies on the induction and expression of murine PC-specific DTH responses. Repeated injections of anti-T-15 idiotypic antiserum, raised in A/J mice by immunization with TEPC-15 myeloma protein, into recipient BALB/c mice both immediately before and after sensitization with PC-PEC virtually abolished the development of PC-specific DTH responses. Although administration of anti-T-15 antiserum effectively inhibited the induction phase of PC-specific DTH responses, these anti-idiotypic antibodies had no suppressive activity at the effector phase of these responses. The inhibition observed with anti-T-15 antibodies was highly specific for the PC hapten, and for PC-specific DTH responses of BALB/c but not A/J mice. Studies were conducted to address the possibility that anti-Id treatment induced suppressor T lymphocytes capable of specifically inhibiting the activity of PC-specific T cells participating in DTH responses. The results demonstrate that idiotype-specific suppressor T cells are, indeed, induced by treatment with anti-Id; moreover, such suppressor T cells, once induced, are highly effective in abrogating both the induction and the effector phases of PC-specific T cell-mediated DTH responses in BALB/c mice.


T-lymphocyte response to cytochrome c. I. Demonstration of a T-cell heteroclitic proliferative response and identification of a topographic antigenic determinant on pigeon cytochrome c whose immune recognition requires two complementing major histocompatibility complex-linked immune response genes. The T-lymphocyte proliferative response to pigeon cytochrome c was studied in the mouse. H-2a and H-2k strains were responders to this antigen whereas H-2b, H-2d, H-2f, H-2ja, H-2p, H-2q, H-2r, H-2s, and H-2u strains were low or nonresponders. Genetic mapping demonstrated that two major histocompatibility complex (MHC)-linked Ir genes control the response, one in I-A, the other in I-E/I-C. The major antigenic determinant recognized in this response was localized by cross-stimulations with species variants and cyanogen bromide cleavage fragments of cytochrome c. It was found to be a topographic surface determinant composed of an isoleucine for valine substitution at residue 3, a glutamine for lysine substitution at residue 100 and a lysine for glutamic acid substitution at residue 104. Tobacco hornworm moth cytochrome c, which contains a glutamine at residue 100 but a terminal lysine at residue 103 (one amino acid closer to the glutamine), stimulated pigeon cytochrome c immune T cells better than the immunogen. This result demonstrates for the first time a functional T-cell heteroclitic proliferative response in a system under Ir gene control. Immunization with the cyanogen bromide cleavage fragments revealed that only pigeon cytochrome c fragment 81-104 was immunogenic. This fragment primed for a T-cell proliferative response whose specificity was nearly identical to that of the T-cell response primed for by the whole molecule, suggesting that the glutamine at 100 and the lysine at 104 form the immunodominant portion of the antigenic site. Furthermore, mixing experiments using the two cross-reacting antigens, hippopotamus cytochrome c and Pekin duck or chicken cytochrome c fragment (81-104), each of which contains only one of the two immunodominant substitutions, demonstrated that the T lymphocytes responding to the major antigenic determinant comprise a single family of clones that recognize both amino acids as part of the same determinant. Thus, two complementing MHC-linked Ir genes can control the immune response to a single antigenic determinant.


Macrophage oxygen-dependent antimicrobial activity. I. Susceptibility of Toxoplasma gondii to oxygen intermediates. A sensitive method for evaluating extracellular parasite viability was used to determine the in vitro susceptibility of virulent Toxoplasma gondii to selected oxygen intermediates. By acridine orange fluorescent staining criteria, toxoplasmas were resistant to up to either 10(-3) M reagent H2O2 or H2O2 generated by glucose-glucose oxidase. In keeping with a lack of sensitivity to H2O2, toxoplasmas contained endogenous catalase (5.7 x 10(-4) Baudhuin units/10(6) organisms). The addition of a peroxidase and halide, however, markedly accelerated killing and lowered the H2O2 requirement by 1,000-fold. In contrast, toxoplasmas were promptly killed after exposure to products generated by xanthine (1.5 x 10(-4) M) and xanthine oxidase (50 micrograms). The inhibition of this system's microbicidal activity by scavengers of O2- (superoxide dismutase) and H2O2 (catalase) indicated that although neither O2- nor H2O2 were toxoplasmacidal, their interaction was required for parasite killing. Quenching OH. and 1O2, presumed products of O2--H2O2 interaction, by mannitol, benzoate, diazabicyclooctane, and histidine, also inhibited toxoplasma killing by xanthine-xanthine oxidase. These findings suggested that O2- and H2O2 functioned in precursor roles and that OH. and 1O2 were toxoplasmacidal. The capacity of normal peritoneal macrophages to pinocytose an oxygen intermediate scavenger, soluble catalase, was also demonstrated. Appreciable extraphagosomal concentrations of catalase were achieved by exposing macrophages to 1 mg/ml of the enzyme for 3 h. Maintenance of high intracellular levels required constant exposure because interiorized catalase was rapidly degraded.


Mice whose B cells cannot produce the T15 idiotype also lack an antigen-specific helper T cell required for T15 expression. The X-linked CBA/N defect in B cell function precludes an antibody response to phosphorylcholine (PC). Accordingly, (CBA/N X BALB/c)F1 male mice are unresponsive to PC and lack circulating immunoglobulin bearing the T15 idiotype characteristic of BALB/C anti-PC antibody. In contrast, (CBA/N X BALB/c)F1 female mice respond to PC and greater than 80% of the anti-PC antibody is T15+. No T-cell abnormalities are known to be associated with the CBA/N mutation. These experiments compared the ability of helper T cells from either (CBA/N X BALB/c)F1 male (T15-) or F1 female (T15+) mice to help F1 female B cells respond to PC and to influence the level of T15 expression. The results indicate that although F1 male T cells collaborated with F1 female B cells just as efficiently as F1 female T cells for the total anti-PC response, the percentage of T15 expression induced by F1 male T cells fell dramatically. The (CBA/N X BALB/c)F1 male thus appear to lack a helper T-cell subset required for dominant idiotype production. This helper T cell defect could be repaired by adding F1 female T cells primed to a second carrier to F1 male T cells and restimulating the cell mixture with PC coupled to the antigen used to prime the F1 male cells plus free second carrier. This result implies that conventional helper T cells derived from the F1 male donor can collaborate with a distinct helper T-cell subset from the F1 female donor which recognizes both carrier and idiotype to induce an anti-PC antibody response dominated by the T15 clonotype.


Ia determinants on human T-cell subsets defined by monoclonal antibody. Activation stimuli required for expression. The nature of Ia antigens which appear on human T cells after activation and the stimuli required for their expression was examined utilizing a monoclonal antibody reactive with the Ia antigen framework. T cells were purified using monoclonal antibodies directed either at the entire T-cell population (OKT3) or the T-cell inducer subset (OKT4). By indirect immunofluorescence, it was shown that the human T-cell population contains no detectable Ia+ cells in the resting state. In contrast, in excess of 60% of the T-cell population expresses Ia antigen after alloactivation in the mixed lymphocyte culture. Moreover, these Ia antigens are expressed within both the OKT4+ and OKT4- subsets. Similarly, phytohemagglutinin and concanavalin A induced approximately 20% of peripheral T cells to express Ia antigen and the expression of these antigens is not restricted to either OKT4 subset. In contrast, only the inducer T-cell population which proliferates maximally to soluble antigen expresses Ia antigens after activation by tetanus toxoid. Thus, the expression of human Ia antigens on unique T-cell subsets depends upon the activation stimuli utilized and ability of the individual subset to respond to a given stimulus. Additional studies indicated that Ia antigens appear on previously Ia- T cells after activation and do not result from clonal expansion of a small subset of Ia+ T cells.


Continuous proliferation of murine antigen-specific helper T lymphocytes in culture. Murine helper T cells activated to sheep or horse erythrocyte antigens in vivo have been established as continuous cell lines in culture. T cells require the presence of a T-cell growth factor (TCGF) for continuous proliferation. TCGF purified from murine, rat, or human sources all stimulate murine T-cell growth. The T-cell mitogens concanavalin A and phytohemagglutinin do not stimulate cell proliferation in continuous T-cell lines. All cells that grow in the presence of TCGF express Thy-1 antigens. Helper activity of T-cell lines is both antigen specific and effective for syngeneic or F1 B cells. Supernates from T-cell lines do not contain antigen-specific or nonspecific helper factors. Although several T-cell lines have shown stable helper activity for greater than 50 wk in culture, other cell lines have shown a gradual decline in effector function. The procedure used to establish and maintain proliferation of T cells in culture should be suitable for the selection and growth of antigen-specific effector T cells from each subclass.


Primary in vitro cytotoxic response of NZB spleen cells to Qa-1b-associated antigenic determinants. We have shown that cytotoxic lymphocytes generated in primary cultures of NZB spleen cells with H-2-identical BALB/c or B10.D2 stimulator cells exhibit specificity for Qa-1b-associated antigenic determinants. This unidirectional cytotoxicity constitutes the initial demonstration of a primary in vitro response to antigens of the Qa-Tla system. Such responses do not require H-2 homology between effector and target cells in the assay system. In fact, when H-2Dd homologous target cells were employed there was little, if any, evidence for development of primary H-2-restricted responses to minor locus histocompatibility antigens or viral antigens. In view of the recently defined role of Qa-1+, Ly-1,2,3+ cells as regulators of antibody responses, and of the deficiency of such cells in NZB mice, the observation of hyperreactivity for determinants of this system may be relevant to the development of autoimmunity in these animals.


Altered phospholipid composition in mutants of Escherichia coli sensitive or resistant to organic solvents. Mutants of Escherichia coli with altered resistance to low molecular weight organic solvents were isolated. Solvent-resistant mutants showed a decrease in the ratio of phosphatidylethanolamine to the anionic phospholipids (phosphatidylglycerol and cardiolipin) relative to the wild-type, whereas solvent-sensitive strains showed an increase. Reversion studies on representative mutants demonstrated that the phenotypic response to solvents and the changes in phospholipid composition were genetically associated. The fatty acid and lipopolysaccharide compositions of the various mutants showed no significant differences from the parental strain. The lesions in two of the solvent-sensitive mutants (DC7 and DC9) and one of the resistant mutants (DC11) were mapped by cotransduction with phage P1 and shown to lie very close to the pss locus at 56 min on the Escherichia coli map.


Acute experimental allergic encephalomyelitis. Myelin basic protein-reactive T cells in the circulation and in meningeal infiltrates. MBP-reactive early T cells were determined simultaneously in the circulation and in inflammatory cells from the meninges of guinea pigs with acute EAE. In addition, early and late T cells as well as B cells were estimated in both compartments. Within the circulation, early T cells were found to decrease at the onset of clinical signs, and MBP-reactive early T cells were significantly higher than in normals. Different changes were found in inflammatory cells from the meninges. In this cell population, early T cells were increased but did not show significant, further elevation after incubation with MBP. This may indicate that the majority of early T cells in meningeal infiltrates had been previously activated by MBP in vivo.


Stimulation of active E-rosette forming lymphocytes by myelin basic protein and specific antigens from multiple sclerosis brains. Peripheral blood lymphocytes from 30 patients with multiple sclerosis (MS) responded to low doses (i.e. 0.1--5 pg total protein) of crude MS myelin basic protein (BP) as assayed by the active E-rosette test (AER). Of the MS patients studied 20 (65%) responded to control BP. The optimum response of MS lymphocytes to MS BP was obtained at a lower concentration than their response to control BP. Thirty percent of other neurological patients (OND) were stimulated by both MS and control BP. Lymphocytes of all MS patients but none of the OND responded to partially purified protein fraction of MS brain ("peak 2"). Crossed immunoelectrophoresis revealed the presence of one common specific antigen in crude MS BP and MS "peak 2" antigens. The nature of these antigens is discussed.


Correlation between the capacity of bacterial lipopolysaccharide to supress experimental allergic encephalomyelitis and its mitogenic activity for lymph node cells in guinea pigs. Protection of guinea pigs from experimental allergic encephalomyelitis (EAE) was attempted using bacterial lipopolysaccharide (LPS) from 4 sources. The ability of these LPS to induce DNA synthesis in guinea pig lymph node (LN) cells in vitro was also investigated. It was found that there existed a good correlation between the capacity of LPS to suppress EAE and their degree of mitogenic activities for LN cells. LPS from Escherichia coli 0111:B4 (Ec-LPS), which was most effective in suppressing EAE and also best inducer of DNA synthesis in LN cells, enhanced the proliferation of cells forming antibody to myelin basic protein (BP) in the regional LN. These results, in addition to the previous report, suggested that at the inductive phase the proliferation of B lymphocytes or their products, antibodies to BP, could inhibit formation of T lymphocytes sensitized to BP, resulting in suppression of EAE. Lipid A but not PS fraction of Ec-LPS showed a protective activity against EAE and a mitogenic activity for LN cells although less so than whole LPS. In addition, Lipid A appeared to exert its mitogenic effect mainly on B rather than on T lymphocytes.


Further studies on cell-mediated immunity to myelin basic protein in schizophrenic patients. Cell-mediated immunity (CMI) toward myelin human basic protein (HBP) was studied in 19 chronic drug-free schizophrenic patients and in 30 patients with primary affective disorders receiving psychoactive drugs, 74% of the schizophrenics showed an inhibition of macrophage migration, whereas only one patient from the affective group showed such a response. These results indicate that drugs are not responsible for the appearance of CMI to HBP in schizophrenics. One out of 9 ECT-treated patients developed a CMI to HBP.


Hypersomnia with simultaneous waking and sleep patterns in the electroencephalogram. A case report with neurotransmitter studies. A mildly dyslexic boy of 11 years, with no neurological deficit or history of epileptic seizures, had marked hypersomnia for 2 years, which was most pronounced in the morning hours. Repeated EEG studies and power spectral analysis revealed simultaneous posterior alpha rhythm and sleep patterns (spindles, vertex waves, K complexes) over vertex and frontocentral regions, while the patient was behaviorally awake. Bilateral synchronous anterior spikes were frequently noted in association with sleep patterns. A polysomnogram over 24 h confirmed excessive sleep, night and day (especially morning hours) and there was evidence of a large REM sleep percentage (on EMG and EOG basis) while the EEG had predominantly non-REM sleep patterns. Special neurotransmitter studies were performed in view of a presumed disturbance affecting the neurobiochemical sleep regulation. These studies were based on CSF metabolite levels and provided evidence for a high serotonin metabolite (5HIAA) level. It is tempting to hypothesize that the biochemical disturbance has led to encroachment of non-REM sleep patterns on both wakefulness and REM sleep. Further discussion deals with the bilateral-synchronous spike activity and its relationship to arousal patterns in sleep.


Right/left differences of median nerve evoked scalp potentials in multiple sclerosis. Scalp potentials evoked by electrical stimulation of the median nerve at the wrist were examined in multiple sclerosis patients and healthy controls. The latencies of the first negative peak (about 18 ms latency) of the response to right and to left-sided stimulation were compared. Forty-eight of 60 measured latency differences in 15 suspected or certain MS patients were more than 3 standard deviations beyond the average difference in controls (arbitrary norm limit), whereas none of the 56 results of the 14 controls was in that range. Fifteen of 24 latency differences in 6 patients without anamnestic or clinical sensory disturbances in the arms were above the limit. On the other hand, conduction velocity between wrist, neck and scalp did not differentiate controls from patients. We suggest using latency differences of the early components of right and left median nerve-evoked scalp potentials as a mean for the early detection of functional disturbances in multiple sclerosis.


Progressive extrinsic ophthalmoplegia with peripheral neuropathy and storage of muscle glycogen. A case of progressive extrinsic ophthalmoplegia associated with peripheral neuropathy and hypertriglyceridemia type IV is described. Motor and sensory conduction velocities of the spinal nerves were severely decreased, while the EMG of the facial muscles was more suggested of a myopathic disorder. Electron microscopic study of biopsies of biceps and peroneus brevis muscles disclosed many ragged red fibers, mainly type I, which contained typical abnormal mitochondria. Other fibers, all type II, contained increased amounts of glycogen between myofibrils or beneath the sarcolemmal membrane, but the mitochondria were normal. These fibers were more abundant in the peroneus brevis than in the biceps muscle. Nerve biopsy revealed marked loss of myelinated fibers, but neither mitochondrial changes nor glycogen storages were evident in Schwann's cells. Biochemical investigations confirmed the increased amount of glycogen in both muscle biopsies and revealed a decrease of guanilcyclase. Phosphorylase, phosphorylase b kinase, adenilcyclase, and carnitine concentrations were all normal. The pathogenesis of this syndrome is discussed and the relationship between mitochondrial abnormalities and glycogen accumulation in muscle tissue are considered.


Isoelectric focusing and crossed immunoelectrofocusing of CSF immunoglobulins in MS. The 3 main Ig classes and the presence of free light chains were studied by isoelectric focusing and crossed immunoelectrofocusing in 100 CSF samples from patients with clinically definitie or probable MS. Minute quantities of IgM and free light-chain (mostly lambda) components were found in 2 out of 11 and 5 out of 14 samples respectively. IgG and IgA were detected in all samples examined for these proteins and were found in the pI-ranges of 5.3-9.8 and 4.6-6.4 pH-units respectively. The gammaglobulin abnormalities found on isoelectric focusing were identified as microheterogeneous, oligoclonal IgG with predominantly kappa light-chain determinants. The IgG immunoprecipitates differed from those of normal subjects and the major abnormal components most frequently exhibited pI-values greater than 8 pH-units. The IgA immunoprecipitates had 2-4 main components with some tendency to discontinuous subfractionation. This Ig class, however, did not exhibit the marked tendency to oligoclonal distribution found for IgG.


Erythrocyte unsaturated fatty acid test (E-UFA test): a biological test to detect optic neuritis as initial feature of multiple sclerosis. Retrobulbar optic neuritis, though suspicious is not always considered as a possible onset of MS. For this reason we submitted to the E-UFA (erythrocyte unsaturated fatty acid) test, 41 subjects who suffered from one or several episodes of retrobulbar optic neuritis with no other neurological signs. The test was positive in 14 subjects (34, 1%).


Plasma exchange in motor neuron disease. A controlled study. In vitro studies seem to indicate that a serum factor may be involved in the pathogenesis of motor neuron disease. If so, plasmaphoresis might influence the course of amyotrophic lateral sclerosis (ALS) favorably. In the present study, therefore, ALS patients were subjected to weekly 21 plasma exchanges, using a Haemonetics blood separator. Seven other ALS patients, matched as closely as possible with the treatment group regarding age, sex, duration of symptoms as well as degree of involvement, served as control group. The progression of the disease was followed by an arbitrary grading system, assessment of muscular power by Zadig's dynamometer, and by tests for motor speed, coordination and for pulmonary function. Duration of treatment was from 6 to 15 months. Monthly evaluations indicated that the rate of deterioration was approximately the same in treatment and control groups. Plasmaphoresis carried out in this way does thus not alter the downhill course of ALS.


Primidone metabolism in renal insufficiency and acute intoxication. Primidone (PRIM) is metabolized into phenobarbital (PB) and phenylethylmalonamide (PEMA). During anticonvulsant therapy with PRIM under normal conditions PB represents by fat the largest portion of the total concentration of all three components (PRIM + PB + PEMA). In combined therapy with diphenylhydantoin (DPH), and during chronic PRIM overdosage, the relative concentration of PB is even higher. A case of renal insufficiency while on PRIM therapy and a case of acute PRIM intoxication are presented. In both cases PRIM and PEMA are elevated while PB is relatively low. The mechanisms involved in this phenomenon are discussed. Excluding young children with chronic PRIM overdosage, and the endogenous and exogenous intoxication described here, a relative PB concentration below 40% indicates a lack of patient compliance if a steady treatment schedule has been maintained for at least 3 weeks.


Roving eye movements with bilateral symmetrical lesions of the thalamus. Typical roving eye movements (pendular deviations of the bulbi) were observed in a 41-year-old patient with bilateral symmetrical softenings in the anterior thalamic region. They constantly appeared after lid closure with a mean latency of 7.3 sec. The mean frequency was 0.25 cps, the mean amplitude 30 degrees. The phenomenon of roving eye movement is discussed with regard to the supranuclear structures regulating binocular eye movements.


Hyperacute experimental allergic encephalomyelitis in rhesus monkeys as a model of acute necrotizing hemorrhagic encephalomyelitis. A comparative study of clinical and morphological findings in three fatal cases of acute necrotizing hemorrhagic encephalomyelitis (ANHE) and hyperacute experimental allergic encephalomyelitis (HEAE) in rhesus monkeys is reported. In all cases ANHE was characterized clinically by definite prodromal respiratory infection. The course was rapidly progressive with fatal termination. The salient histopathological changes were necrosis of blood vessels with plasma exudation and fibrin impregnation, hemorrhages and inflammatory reaction in the damaged cerebral tissue. Perivascular lymphoid histiocytic infiltration with glial proliferation was also noted in all cases. Numerous compound granular cells were found in one case. HEAE was detected in five rhesus monkeys immunized with homological spinal cord emulsion with complete Freund adjuvant. The illness was acute or subacute and the course was rapidly progressive with a fatal end. There was multiple necrosis of small blood vessels with plasma exudation, fibrin impregnation and massive neutrophila infiltration of the damaged brain tissue in all rhesus monkeys with HEAE. There was also widespread glial proliferation and numerous compound granular cells alongside with necrosis of blood vessels in the brain. These findings suggest that HEAE in rhesus monkeys can be viewed as an adequate model of ANHE.


Recurrent transient global amnesia in a case with cerebrovascular lesions and livedo reticularis (Sneddon Syndrome). Eight attacks of transient global amnesia were observed in a female patient who suffered from livedo reticularis and a series of other neurological symptoms, which were transient in most stances. The neurological deficits include focal epileptic attacks, unilateral loss of vision, paresis of left arm and/or leg and dysarthria. The first amnestic attack was seen at the age of 19. The episodes lasted from a few to 3 days. The intervals between the amnestic episodes varied between a few days and 11 years. The livedo reticularis became more obvious during each neurological episode and was less pronounced during the time of remission. A benign type of essential hypertension and parproteinemia (gamma-M) was found. The investigations failed to show any evidence of essential thrombocythemia, polyarteriitis nodosa, lupus erythematodes and other immune complex diseases. The underlaying disease remained unclear.


Familial meralgia paresthetica with an autosomal dominant trait. Familial occurrence of Meralgia paresthetica is uncommon. Only few familial case studies have been reported up to now. The author presents a family with Meralgia in four generations, suggesting a distinctly autosomal dominant trait.


An unusual case of paroxysmal facial pain. Paroxysmal facial pain is a rare sequel to brainstem infarction. This is a report of a patient who developed paroxysmal facial pain with the exceptional features of a trigger zone and relative refractory pain-free periods in an area previously anesthetic after infarction of the brainstem. The characteristic of this patient's pain is compared with the pain is compared with the pain of trigeminal neuralgia and possible similar pathogenetic mechanisms.


Varieties of the locked-in syndrome.

Varieties of the locked-in syndrome. The locked-in syndrome (LiS) was broken down on the basis of neurological symptoms in 12 patients. The criteria of classical LiS are total immobility except for vertical eye movements and blinking. If any other movements are present one should consider the condition as incomplete LiS. Total immobility, including all eye movements, combined with signs of undisturbed cortical function in the EEG led to the concept of total LiS. The anatomical basis for this condition consists of lesions in both cerebral peduncles which interrupt the pyramidal and corticobulbar tracts, the supranuclear fibers for horizontal gaze and the postnuclear oculomotor fibers. As to the course, chronic and transient LiS have been described.


The value of CT and EEG in cases of posttraumatic epilepsy. The results of the clinical neurological investigation were compared with those of electroencephalography (EEG) and computed tomography (CT) in 64 patients suffering from verified posttraumatic epilepsy. Only 18 patients (28%) showed central neurological features with corresponding focal disorders on CT (15 cases) and EEG (11 cases). The combined application of both methods led to positive results in 94% on the part of at least one accessory examination. The clinical neurological investigation as well as the EEG and CT were normal in only 3 cases, although the traumatic etiology of epilepsy was beyond doubt. In addition to the clinical neurological investigation, EEG and CT are most important accessory examinations for the diagnosis and followup studies in cases of posttraumatic epilepsy.


Intracranial giant cell arteritis.

Intracranial giant cell arteritis. A case of giant cell arteritis of intracranial vessels diagnosed by autopsy is described. Giant cell arteritis of the proximal basal brain arteries was exceptionally marked in a man of 60 years. The clinical course, laboratory findings and the pathological alterations of the brain and intracranial blood vessels are described. The case is discussed on the basis of the literature on giant cell arteritis with cerebral symptoms as well as on granulomatous giant cells arteritis of the brain. A separation of these two entities does not seem justified.


Isotachophoresis of CSF proteins in gel tubes especially gammaglobulins. An analytical and preparative technique for high-separation of CSF proteins. An isotachophoretic method using polyacrylamide gel (PAG-ITP) in a simple disc electrophoretic equipment with plastic tubes containing the gels, was elaborated and especially designed for studying the gammaglobulins in CSF and serum from control subjects and patients with neurological disorders, especially known or probable MS. The device and the ITP system used, including leading and terminating electrolytes and spacer substances, dividing the gammaglobulins in a reproducible way, are described. No cooling of the gel tubes was needed. The sample volumes varied between 5--500 microliters, and the separation time was 1.5--3.0 h. CSF from patients with verified or probable MS revealed characteristic, increased low-mobility gammaglobulin fractions. Using other ITP systems, such as other spacer compositions, the anodic proteins can also be studied in more detail. PAG-ITP in gel tubes is a simple and inexpensive technique which can be used for both analytical and preparative procedures for biological material such as CSF, serum and extractions from nervous tissues.


Isotachophoresis in capillary tubes of CSF proteins -- especially gammaglobulins. Isotachophoresis in polyacrylamide gel tubes (PAG-ITP) and in capillary tubes (Tachophor, LKB) have previously been found by the authors, to be very promising high-separation methods for CSF and serum proteins, especially regarding the diagnosis of MS. PAG-ITP methods for analytical and preparative use have been described by the authors elsewhere, while in this paper proper cationic systems for ITP in capillary tubes for studying gammaglobulins in microliter amounts of CSF and serum are described, i.e. the albumin injection-clog problem is avoided and the preparation time can be forced. By using microdialysis of the CSF samples for desalting, with a technique easy to perform and with high reproducibility, microliter amounts of native CSF can be performed in less than half an hour. The method seems to be even more applicable for clinical and scientific use if the capillary isotachophoretic apparatus is connected to a synchronized equipment (LKB Tachophrac) with a cellulosa acetate strip onto which the separated fractions are ejected for further analysis by immunological tests. The analytical systems used have been especially directed to gammaglobulins in CSF and serum regarding further studies on demyelinating and infectious disorders of the nervous system.


Intrathecal IgG synthesis in multiple sclerosis and other neurological diseases: a comparative evaluation by IgG-index and isoelectric focusing. Intrathecal IgG synthesis has been investigated by determining the IgG index and by isoelectric focusing in 30 cases of definite multiple sclerosis, in 15 cases of probable multiple sclerosis and in 128 patients affected by other neurological diseases. The blood-brain barrier function was evaluated at the same time by serum albumin/CSF albumin quotient and isoelectric focusing. The IgG index was found elevated in 73.3% of definite multiple sclerosis patients, while oligoclonal IgG bands occurred in 90%. In the other neurological diseases the IgG index was abnormally increased in 35.1% but IgG bands were present only in cerebrospinal fluid (CSF) in 1.5% and both in the CSF and serum in 7% of patients. The high capacity of isoelectric focusing to detect IgG oligoclonal bands in the CSF is pointed out as an extremely useful diagnostic tool in multiple sclerosis.


The discriminating role of EEG power spectra in dyslexic children. The aim of this investigation is to compare EEG power-density spectra between dyslexic and normal children. The children have been selected on the basis of specific reading and writing tasks; those with severe organic diseases and mental disorders were excluded. Groups aged 8, 9 and 10 years have been compared. In the 8-year-old group the power in the alpha band is higher in normals, in the 9-year-old group there was a higher power of the mu rhythm in normals and in the 10-year-old group the power in the temporal theta band is lower in normals. A possible relation between these findings and psychomotor development is discussed.


EEG spectra in children aged 8, 9 and 10 years. Reference values. In a group of normal children aged 8, 9 and 10 years power density spectra of a 100-second EEG period were made over four different brain regions. All children had to undergo medical and psychological examinations prior to this EEG and the criteria for normality were based on these examinations. Reference values of the power density distribution for children between 8 and 10.5 years are given.


Unusual features in a case diagnosed as subacute sclerosing panencephalitis (SSPE). SSPE is characterised by progressive mental deterioration, myoclonic and similar motor disorders and final severe comatose states, increase of immunoglobuline G in the CSF, strongly elevated antibody titers to measles virus in serum and CSF and typical periodic K-complexes in the EEG. The disease appears commonly in childhood and has a fatal course. Cases with atypical signs have occasionally been reported. The case described in this paper shows a number of uncommon features: late onset, partial remission and stationary course, increased antibody titers to measles virus but relatively low in comparison to others, dissociation of cytoplasmic and nuclear fluorescent antibodies against SSPE brain tissue and an initial increase of antibodies against rubella virus. The patient was treated with isoprinosine. Improvement was observed before the start of this therapy and stabilized while treatment was being continued.


A case of priapism with ruptured intracranial aneurysm. A man of 35 years, who had had three attacks of subarachnoid hemorrhage in the previous 3 years, was admitted to hospital with complaints of headache and priapism. There had been intermittent priapism with abnormal acceleration of sexual desire since the first attack, and erection of the penis had persisted with intolerable pain after the last attack of subarachnoid hemorrhage. A carotid angiogram revealed an aneurysm at the junction of the left internal carotid and posterior communicating arteries. Clipping of the aneurysmal neck was successfully performed. However, priapism continued for 22 days after the operation and resulted in sexual impotence. The neurological problems of priapism are discussed with special reference to a hypothalamic lesion caused by the ruptured intracranial aneurysm in this report.


Quantitative and qualitative changes in phospholipid in the intestine of the gerbil and the development of lipodystrophy. Female gerbils fed a diet containing 20% coconut oil develop an intestinal lipodystrophy that is not seen in animals fed a diet containing 20% safflower oil or a diet of 20% coconut oil supplemented with 0.1% inositol. The coconut oil diets contained 1.5% safflower oil to prevent essential fatty acid deficiency. The level of inositol in the intestinal tissue of animals fed the coconut oil diet not supplemented with inositol has been shown to be decreased. Phospholipid analyses of the intestinal tissue were undertaken to determine if this decrease in total inositol was reflected in a decrease in phosphatidylinositol or resulted in an altered phospholipid pattern. No difference in the phosphatidylinositol level was seen between animals fed 20% coconut oil with and without inositol supplementation (microgram P/gut section), although animals fed coconut oil diets had lower levels of phosphatidylinositol than animals fed safflower oil diets. Fatty acid analyses of total phospholipid and phosphatidylinositol in gut tissue revealed that animals which deveoped the lesion had an ultered phosphatidylinositol fraction with a depressed level of arachidonic acid and an elevated level of oleic acid. This suggests that the development of the intestinal lipodystrophy may be correlated with qualitative rather than quantitative changes in phosphatidylinositol.


Histogenesis of the so-called "intravascular bronchioloalveolar tumour". Three cases of Dial and Liebow's intravascular bronchioloalveolar tumour are described, with emphasis on ultrastructure and histogenesis. The tumour cells are quite different from adjacent reactive alveolar cells and electron microscopy does not support the suggested alveolar cell origin. The tumour cells contain abundant microfilaments, moderate amounts of rough endoplasmic reticulum and varying numbers of Weibel-Palade bodies. With features of smooth muscle, myofibroblast and endothelial cell differentiation, a vascular origin from precursor mesenchymal cells such as the vasoformative reserve cells is suggested.


Isolation and characterization of surface antigens from Schistosoma mansoni. II. Antigenicity of radiolabeled proteins from adult worms. Adult Schistosoma mansoni were radiolabeled in vitro with 125I Bolton-Hunter reagent. Surface membrane antigens were solubilized with non-ionic detergent, then reacted with infection or normal serum. The antigen-antibody complexes were then precipitated with staphylococcal protein A immunoadsorbent, eluted with urea and SDS, and fractionated by SDS-PAGE. The results indicated the presence of 6 to 8 tegument antigens, depending on the type of antisera used. Human antisera to S. japonicum and S. haematobium reacted with some but not all of the antigens identified with human S. mansoni infection serum; this implies the presence of species-specific tegument antigens. The molecular weights of the radiolabeled antigens ranged from 10,000 to 100,000. A large (greater than 100,000) molecular weight glycoprotein and an uncharacterized lipid fraction appeared to be precipitated nonspecifically. Immunoprecipitation methods with anti-mouse IgG and anti-mouse whole serum failed to detect the presence of hostlike antigens in the labeled extracts. Several of the labeled proteins from S. mansoni were found to react with serum from patients infected with either S. haematobium or with S. japonicum.


Antigenic make-up of Trypanosoma cruzi culture forms: identification of a specific component. A comparison of Trypanosoma cruzi water soluble antigens with those of stercorarian and salivarian trypanosomes, and Leishmania using immunoprecipitation in gels and immunoelectrophoresis, with the aid of hyperimmune rabbit serum and heterologous adsorptions showed the following. 1) There is a high complexity of soluble antigens of T. cruzi and T. rangeli. 2) At the intraspecific level our results demonstrated the antigenic stability of T. cruzi when maintained in vitro, and that there was quantitative antigenic consistency of the culture forms of different strains of T. cruzi from diverse geographic and parasite sources. At the interspecific level, the antigenic relationships between T. cruzi and the other Trypanosomatidae were established, as follows: 6/10ths of the antigens are shared by stercorarian species (T. dionisii, T. rangeli); 4/10ths by a salivarian trypanosome (T. brucei); and 3/10ths by Leishmania (L. donovani, L. mexicana). 3) Among the 4/10ths of antigenic components specific to T. cruzi, one component was characterized by its antigenicity and immunogenicity in natural and experimental infections, and in immunization experiments; this component was specific to T. cruzi when compared to the other Trypanosomatidae antigens.


Evidence for reappearance of Trypanosoma brucei variable antigen types in relapse populations. Seven out of 11 bovines infected with different clones of Trypanosoma brucei showed 2 peaks of antibody activity against the infecting clone within 7 weeks, as measured by immunofluorescence, radioimmunoassay, and neutralization of infectivity tests. Using other clones from an unrelated Stock, antibodies to these clones were not detectable, indicating that the antibodies produced were specific to the infecting organisms. These results suggest that there was a reappearance or increase in numbers of the infecting organisms or of organisms with variable surface antigens similar to those of the infecting clones. The reappearance of variable antigen types in the presence of specific antibodies would imply that antibody plays a selective rather than an inductive role in the process of antigenic variation in African trypanosomes.


Endodermal sinus (yolk sac) tumors in infants and children. Endodermal sinus tumors, as a specific entity, were first proposed and described by Teilum on the basis of morphological and histogenetic features. Recent work utilizing tumor markers (alpha-fetoprotein), immunofluorescent and electron microscopic studies have supported Teilum's original concept as to the origin of these tumors. Five typical presentations of endodermal sinus tumors seen in the pediatric age group were reviewed with reference to the original presentation, the utilization of tumor markers (alpha-fetoprotein) and the prognostic implications of this neoplasm.


Comparative pharmacokinetics of coumarin anticoagulants. XLII: Effect of phenobarbital on systemic availability of orally administered dicumarol in rats with ligated bile ducts. The purpose of this investigation was to determine if the previously demonstrated inhibitory effect of phenobarbital treatment on the systemic availability of orally administered dicumarol in rats is related to the known effect of phenobarbital on bile output. It was found that phenobarbital had no apparent effect on the systemic availability of an aqueous dicumarol suspension in rats with ligated bile ducts. Compared to results obtained previously on normal rats, bile duct-ligated rats absorbed and eliminated dicumarol much more slowly and absorbed much less of the anticoagulant. On the other hand, the relative inductive effect of phenobarbital treatment on dicumarol elimination was similar in normal and in bile duct-ligated animals. The latter exhibited substantial serum transaminase elevations, indicative of liver damage presumably secondary to cholestasis. These results demonstrate that a drug-drug interaction can depend markedly on the pathophysiological status of the animals.


Benzalkonium chloride: selective inhibitor of histamine release induced by compound 48/80 and other polyamines. Benzalkonium chloride (BAC) is a mixture of quaternary benzyldimethylalkylammonium chlorides which was found to inhibit histamine release induced by polyamines (48/80, ATP, bradykinin, curare, guanethidine, polylysine, polymyxin B, poly-THIQ, protamine, stilbamidine or substance P), but not that caused by antigens, concanavalin A, dextran, lonophores (A23187 or X-537A), enzymes (chymotrypsin or phospholipase C), monoamines (dextromethorphan, meperidine or chlorpromazine) or detergents (decylamine, Triton X-100 or a fire ant venom alkylpiperidine). Inhibition by 1.5 and 3 microgram of BAC per ml caused parallel shifts of the 48/80 dose-response curves to the right with no loss of efficacy, indicating that the antagonism was surmountable. Phospholipase C was partially inhibited by BAC, but Triton X-100 also inhibited phospholipase C (but not 48/80), indicating that the inhibition of phospholipase C by BAC was probably a nonspecific, detergent effect. BAC caused histamine release by itself at concentrations over 5 microgram/ml. Heat inactivation (50 degrees C for 15 min) of the mast cells did not prevent this release, suggesting a lytic mechanism for this action. Structure-activity relations studies on various members of the BAC family for their ability to inhibit 48/80-induced histamine release indicated that benzyldimethyltridecylammonium chloride was the most potent.


Development, utilization, and evaluation of educational programs for patients with maxillary and mandibular defects. Prior to the development of these two rehabilitation programs, there was no informational material available for patients having undergone debilitative maxillary or mandibular surgery. Realizing that what little information the patients and their families did receive during the rehabilitative process was often fragmented, misunderstood, or simply forgotten, two audiovisual programs were designed to fulfill a long-neglected need. Experience with this project indicates that if patient rehabilitation programs are to be effective, it is essential that the patients contribute to the program's development. Only in this way can members of the health-care team be assured that the transmission of pertinent information through variatious educational materials is totally effective.


Distribution of alpha 1-fetoprotein in fetal plasma, allantoic fluid, amniotic fluid and maternal plasma of cows. Maximal concentrations of AFP, measured by RIA, were obtained in fetal plasma and amniotic and allantoic fluid between the 3rd and 4th month of gestation, with levels declining thereafter until term. AFP values in maternal plasma were unchanged. Throughout gestation, AFP values were higher in allantoic than in amniotic fluid and the ratio of allantoic fluid/amniotic fluid AFP was significantly correlated with gestational age.


Biologically active polycycloalkanes. 6. Antiviral 1-tricyclo[4.3.1.1 2,5]undecyl derivatives. Functionalization reactions via cationic intermediates of tricyclo[4.3.1.1 2,5]undecane (2) were investigated to prepare derivatives with potential antiviral activities. Bromination of 2 took place regiospecifically at C-1, and the resulted bromide 5 was converted into the hydroxide 9, the carboxylic acid 12, and the amine 22, from which were synthesized a variety of secondary derivatives, including homologous esters 10 and 20, amides 14 and 19, carbamates 24, and ureas 17 and 25. The hydroxide 9, the acid 12, and the acetamide 21 were also obtainable directly from tricyclo[5.2.1.0 2,6]dec-endo-2-ylcarbinol (1), the precursor for the synthesis of the hydrocarbon 2. Success in these functionalization-rearrangements was attributed to the inability of the intermediate 2-1-yl cation (2+) for further skeletal isomerizations. Among the 1-substituted derivatives of 2 prepared, the amine hydrochlorides (16 and 23), a few esters (20b and 20d), and some N-alkylamides (19c, 19d, and 19e) exhibited marked antiviral activities as compared to amantadine hydrochloride, when tested in vitro on a monolayer culture of chick embryo fibroblasts against Newcastle disease virus.


Synthesis and antitumor activity of 5-azacytosine arabinoside. 5-Azacytosine arabinoside (ara-AC) can be considered a combination of structural elements derived from the antitumor nucleosides cytosine arabinoside (ara-C) and 5-azacytidine (5-AC). The synthesis of ara-AC, for which standard methods were inadequate, was accomplished using the stable dihydro derivative as a synthetic intermediate. A novel dehydrogenation of the latter through the application of a trimethylsilylation-oxidation procedure gave ara-AC in good yield. Using murine L1210 leukemia as a test system, ara-AC was evaluated for antitumor properties in parallel determinations with 5-AC and ara-C. Although higher dose levels were necessary, ara-AC demonstrated a reproducibly greater efficacy in the L1210 system (% ILS = 144-148) than that shown by 5-AC (% ILS = 126-124) or ara-C (% ILS=127-121 ). Moreover, initial data suggest that ara-AC exhibits less host toxicity than either 5-AC or ARA-C. Although ara-AC can equally be considered an analogue of either 5-AC or ara-C, preliminary results indicate that ara-AC is chemically similar to 5-AC but biologically more closely related to ara-C.


Formation of ionic channels in black lipid membranes by succinic derivatives of gramicidin A. Different succinyl derivatives of Gramicidin A were synthesized and their activity was investigated with different methods on lipid bilayer membranes. The succinyl derivatives of Gramicidin A can be classified as three different types, the O-succinyl derivative, the N-succinyl derivative and the N-O-succinyl derivative of Gramicidin A. An O-pyromellityl-N-succinyl gramicidin was synthesized which can be attributed to the latter class. It was found that O-succinyl gramicidin behaves like the unmodified Gramicidin A despite a charge effect on single-channel conductance, arising from the negative charge of the succinic residue at the mouth of the channel. The activity of N-succinyl and N-O-succinyl gramacidin and of O-pyromellityl-N-succinyl-gramicidin depends strongly on the pH of the electrolyte solution. It is demonstrated that at low pH (less than or equal to 5) the N-succinyl derivatives show high activity, whereas at high pH (greater than or equal to 7) the activity is sharply reduced or disappears totally. From these experiments it can be concluded that, for the formation of a dimeric gramicidin channel, the hydrogen of the formyl group can be replaced by a protonated carboxylic group of a succinic residue. Further results, obtained by measurement of the single-channel conductance and of the reaction rate constants for the channel formation, are discussed in terms of the structural basis of the single stranded model for the gramicidin channel. On this basis the double stranded helix can be excluded and an interesting head-to-head single stranded beta(pi L.D.) helical channel is described which contains carboxyl groups at the head-to-head junction.


Dicarboxylic acid analogs of gramicidin A: dimerization kinetics and single channel properties. According to the model of Urry, the cation-permeable gramicidin channel is a dimeric helix formed by association of two peptide monomers linked at their amino ends. In this paper the channel properties of gramicidin analogs are described which have been obtained by chemical modification at the coupling site of the two half-channels. In these analogs the amino terminal -CHO group is replaced by -CO(CH2)nCCOH (n = 2, 3, 4, 5, 6). All analogs form conducting channels in black lipid membranes with the same general properties as found for gramicidin A. The observation that the channel-forming activity decreases with increasing pH is consistent with the notion that the half-channels are linked at the amino terminus. The channel lifetime of the different analogs varies between 2 msec and greater than of equal to 50 sec, the longest lifetime being found for the compound with n = 3. The single-channel conductance : formula : (see text) is always smaller than that of gramicidin A, but the reduction of : formula : (see text) depends on the nature of the permeable ion. Ion specificity was studied at 1 M electrolyte by measuring the conductance : formula : (see text) for different permeable ions (Na+, K+, Cs+). The conductance ration : formula : (see text) (Cs+)/ : formula : (see text) (Na+) was found to vary between 2 and 10.5 for the different analogs.


Mutant of B-tropic murine leukemia virus synthesizing an altered polymerase molecule. A nonconditional mutant of B-tropic murine leukemia virus (MuLV), defective in polymerase, has been isolated by cloning chronically infected cells. The cell clone containing the mutant produced virus particles which were noninfectious. However, superinfection of the cells by replication-competent XC-negative viruses resulted in the rescue of virus capable of forming plaques in a modified XC test, termed the "complementation plaque assay" (A. Rein and R. H. Bassin, J. Virol. 28:656-660, 1978). Analysis of the noninfectious virions produced without superinfection demonstrated that they contained only 2 to 5% of the wild-type level of reverse transcriptase activity. Purification of this activity indicated that it was associated with a smaller molecule than that produced by wild-type virus. Cells producing the mutant virions did not contain the gag-pol precursor, Pr180gag-pol; however the cells contained proteins of 147K and 114K daltons precipitable with anti-pol serum. All of the normal structural proteins as well as 70S genomic RNA could be detected in the mutant particles. An interference test indicated that a functional ecotropic glycoprotein was synthesized by the mutant. These results indicate that the mutant has a unique defect in the pol gene.


Preparation of syngeneic tumor regressor serum reactive with the unique determinants of the Abelson murine leukemia virus-encoded P120 protein at the cell surface. Antisera reactive with the Abelson murine leukemia virus (A-MuLV)-specified P120 (anti-AbT sera) were produced in C57L/J mice. Of many strains tested, only C57L/J reproducibly rejected syngenic A-MuLV-induced tumor cells; after multiple immunizations their sera would immunoprecipitate both P120 and Moloney-MuLV (M-MuLV) proteins. Using labeled A-MuLV-induced nonproducer cells, only P120 could be detected by anti-AbT sera, suggesting that it may be the only A-MuLV-specified protein. Reactivity of anti-AbT sera with P120 was not blocked by M-MuLV virion proteins, implying that the sera recognize a portion of P120 that is not homologous to any M-MuLV product. Anti-AbT sera stained the surface of live, A-MuLV-transformed nonproducer cells in a two-stage immunofluorescence assay, and such staining was not blocked by M-MuLV protein. Also, intact A-MuLV-transformed cells absorbed much of the reactivity of certain anti-AbT sera for P120. Thus a portion of P120 appears to be exposed on the surface of transformed cells. P120 lacks detectable carbohydrate, is not affected by endoglycosidase H, and cannot be labeled by lactoperoxidase-catalyzed iodination. Thus P120 is an unusual surface protein.


Establishment of a lymphoblastoid cell line and isolation of an Epstein-Barr-related virus of gorilla origin. A B-lymphoid cell line was established from a normal gorilla. The cells contained Epstein-Barr virus-related antigens, and herpesvirus particles were demonstrated by electron microscopy. DNA-DNA reassociation kinétics revealed 30 to 40% hybridization to Epstein-Barr virus with 50 genomes per cell. Examination of the viral nuclear antigen with gorilla sera showed this to be a unique isolate termed Herpesvirus gorilla. H. gorilla transformed gibbon B-lymphocytes in vitro.


Characterization of intracellular precursor polyproteins of Moloney murine leukemia virus. Intracellular Moloney murine leukemia viral precursor polyproteins were compared with mature viral proteins by immunoprecipitation and tryptic peptide mapping experiments. The results were consistent with precursor roles for Pr65gag, Pr200gag-pol, Pr135pol, and gPr83env. The glycosylated gag gene product gPr85gag, although containing sequences characteristic of all four core proteins plus additional sequences not found in Pr65gag, lacked a major tyrosine-containing p30 tryptic peptide, suggesting that gPr85gag is not processed to p30.


Polypeptides of hepatitis B virus surface antigen produced by a hepatoma cell line. The PLC/PRF/5 cell line derived from a human hepatoma produces hepatitis B surface antigen (HBsAg) in 22-nm particles of the same buoyant density as those found in the serum of infected patients. The HBsAg particles from this cell line were labeled with [35S]methionine and purified, and the polypeptides were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with those of serum-derived particles. The two major polypeptides of serum-derived HBsAg particles (p20 and p23) were found in the same relative amounts in the particles from the cell line. The three smallest of the five minor components observed in HBsAg particles from serum were present in particles from the cell line. These polypeptides (p31, p36, and p43), as well as p20 and p23, were precipitated with anti-HBs-containing serum. The two largest polypeptides of serum particles (p49 and p66) were not detected in particles from these cells. When the PLC/PRF/5 HBsAg particles were radiolabeled with tritiated sugars, p23, and not p20, was found to contain radioactivity, indicating that the pattern of polypeptide glycosylation is similar to that of serum HBsAg. None of the other possible gene products of hepatitis B virus was detected in the PLC/PRF/5-derived HBsAg particles, in the cells, or in the cell supernatants.