A new optical multichannel microspectrofluorometer. A new microspectrofluorometer has been developed that combines a photometric fluorescence microscope with an optical multichannel analyzer. This instrument provides fluorescence emission spectra of biological materials by detecting the entire spectrum simultaneously in real time. These spectra are subsequently recorded and corrected so as to identify the fluorescent reaction products or to test whether fluorescent cytochemical probes bind to the expected substrate within cells. The procedures and advantages of optical multichannel analysis are described, and an application of microspectrofluorometry to acriflavine-Feulgen cytochemistry is given.


Microfluorimetric quantitation of catecholamine fluorescence in rat median eminence. I. Aspects on the distribution of dopamine and noradrenaline nerve terminals. Using the fluorescence histochemical technique of Falck and Hillarp, a quantitative microfluorimetric study of the catecholamine fluorescence in the median eminence has been performed. On the basis of morphologic criteria, the median eminence was subdivided into various areas from which the microfluorimetric measurements were made; the subependymal layer, the medial and lateral palisade zone of the rostral and the central and caudal region of the median eminence (for definitions of the various areas and regions, see Anatomical Subdivision). The highest fluorescence intensities were recorded from the lateral palisade zone, indicating that this area has the most dense catecholamine innervation, whereas the lowest fluorescence intensities were recorded from the subependymal layer. Dopamine-beta-hydroxylase inhibition produced by FLA-63, fusaric acid or diethyldithiocarbamate resulted in all cases in a 50-70% reduction of the catecholamine fluorescence in the subependymal layer, whereas only minute effects were observed in the lateral palisade zone. In the medial palisade zone, these treatments generally led to a substantial reduction (30-50%) of the catecholamine fluorescence. Basal hypothalamic deafferentation according to Halasz, or lesioning of the ventral catecholamine bundle, produced an almost complete disappearance of the fluorescence in the subependymal layer, while both procedures were largely ineffective in affecting the catecholamine fluorescence in the lateral palisade zone. On basal hypothalamic deafferentation the catecholamine fluorescence in the medial palisade zone was markedly reduced (40-60%), while the ventral bundle lesions were less efficient in this respect. From the present results it is suggested that the subependymal layer is mainly innervated by noradrenaline nerve terminals and the lateral palisade zone is mainly innervated by dopamine nerve terminals, whereas the medial palisade zone receives a mixed innervation of dopamine and noradrenaline terminals, the dopamine proportion being in the order of 50-75% of total catecholamine content.


Microfluorimetric quantitation of catecholamine fluorescence in rat median eminence. II. Turnover changes in hormonal states. Catecholamine nerve terminals in the rat median eminence have been studied using the fluorescence histochemical technique of Falck and Hillarp in combination with quantitative microfluorimetry. The catecholamine fluorescence intensities recorded from various parts of the median eminence were all found to be within the linear part of the dopamine or noradrenaline concentration-fluorescence relationship as studied in an agar-albumin model system. The catecholamine fluorescence was also found to disappear with time in an exponential manner following tyrosine hydroxylase inhibition produced by alpha-methyl-p-tyrosine methylester (H44/68). Similar results were obtained when measuring the dopamine decline by mass fragmentography in the median eminence after H44/68 treatment. These results and analysis of fluorescence frequency histograms strongly indicate that the catecholamine fluorescence values recorded are proportional to the catecholamine concentration. It is concluded that the microfluorimetric technique used is a reliable method for catecholamine quantitation in discrete nerve terminal areas of the median eminence. The main advantages of the technique are that a high sensitivity and quantitative data on the transmitter content can be obtained in strict relation to the neuroanatomy. Measurement of the catecholamine fluorescence disappearance after H44/68 was used to evaluate catecholamine turnover during various endocrine states. The results showed that two dopamine systems with different transmitter turnover may be distinguished. Tuberinfundibular dopamine neurons projecting to the lateral palisade zone were thus shown to have a slower turnover than those projecting medially to the capillary loops. No definite changes in catecholamine turnover were observed after adrenalectomy and castration in the male, although there was a tendency toward increased noradrenaline turnover in both states. During pregnancy an increase in noradrenaline as well as dopamine turnover was noted. The present results therefore give further evidence for the view that catecholamine nerve terminals in the median eminence may participate in the regulation of gonadotrophin secretion.


Simultaneous visualization by light microscopy of two pituitary hormones in a single tissue section using a combination of indirect immunohistochemical methods. Two indirect methods involving enzyme-labeled antibodies were used to demonstrate simultaneously two distinct tissue antigens in the same histologic section without a need for antigen-antibody dissociative procedures. Sections of rat pituitary gland were incubated with rabbit anti-rat luteinizing hormone followed by goat anti-rabbit gamma-globulin conjugated to horseradish peroxidase. The same sections were then further incubated with monkey anti-rat growth hormone followed by goat anti-monkey gamma-globulin conjugated to glucose oxidase. Antigenic luteinizing hormone was subsequently localized with hydrogen peroxide-3,3'-diaminobenzidine as substrate for peroxidase, and growth hormone was localized with a glucose-phenazine methosulfate-nitroblue tetrazolium mixture as a substrate for glucose oxidase. The method relies on the availability of specific primary antibodies raised in different animal species in addition to corresponding specific secondary antibodies linked covalently to separate enzymes.


Aurothiomalate as an ultrastructural marker. Electron microscopy and x-ray microanalysis of various tissues after in vivo gold injections. Rabbits and rats were given single injections of aurothiomalate by different routes. The animals were killed at progressive intervals, and sections from various organs were examined by electron microscopic x-ray microanalysis. Ultrastructurally, characteristic material was regularly found in vacuoles, dense and heterogeneous bodies of macrophages, hepatocytes and renal epithelial cells. Occasionally, other mesenchymal cells also contained gold. Histochemical and analytical tests showed that the gold-containing organelles were devoid of acid phosphatase activity. The generalized rapid spread, retention and selectivity of localization after a single small dose make aurothiomalate a useful marker substance for ultrastructural studies.


Transformation of human peripheral lymphocytes by galactose oxidase. Human peripheral lymphocytes can be transformed by treatment with galactose oxidase alone. Prior treatment with neuraminidase enhances this effect. The aldehyde blocking agents thiocarbohydrazide, hydroxylamine, dimedone, and sodium borohydride block transformation when they follow, but not when they precede, galactose oxidase treatment. Thus, as is the case for periodate-induced lymphocyte transformation, the formation of free aldehyde at the cell surface would seem to be a critical event in the triggering of transformation by this agent. The degree of transformation is highly variable from individual to individual, and also for the same donor at different times. However, the lymphocytes of some people give a consistently poor response to galactose oxidase. Similar results have been obtained for periodate-induced transformation of human lymphocytes, but to this date this is unexplained.


Experimental allergic orchitis and aspermatogenesis. VI. Transfer of allergic orchitis with immune cells. Typical experimental allergic orchitis (EAO) and aspermatogenesis were successfully transferred to strain 13 guinea pigs with peritoneal exudate and lymph node cells from male and female donor guinea pigs (lacking detectable antibody) previously sensitized with 9 mug of highly purified GP1 glucoprotein isolated from the sperm acrosome. Attempts to transfer the disease with circulating antibody from hyperimmunized animals were not successful. These studies support a cell-mediated basis for the immunopathologic events in EAO.


Resistance to murine leukemia in mice rejecting syngeneic somatic hybrid cells. (A/J X C3H/HeJ) F1 mice reject somatic cell hybrids of ASL-1 cells (A origin) and LM(TK)- cells (C3H origin), but die from leukemia within 10 days after the inoculation of approximately 10(6) viable ASL-1 cells. Mice rejecting hybrid cells survive for prolonged periods after challenge with otherwise lethal numbers of ASL-1 cells. The hybrid cells, rejected by syngeneic F1 recipients, retained their oncogenic potential as determined by the appearance and progressive growth of tumors in immunologically deficient nu/nu mice injected with the cells. Similar results were obtained using hybrids of a radiation-induced cell line (RADA-1), maintained by serial transfer in strain A mice and LM(TK)- cells. Syngeneic mice injected with RADA-1 X LM(TK)- cells failed to form tumors. Mice rejecting RADA-1 X LM(TK)- hybrid cells survived for prolonged periods after challenge with otherwise lethal numbers of RADA-1 cells.


Production of large amounts of antibodies in individual mice. A method is described for the elicitation of substantial amounts of anti-hapten and anti-protein antibodies in ascitic fluids of individual mice of various strains. The method utilizes repeated i.p. inoculations of u.5-mg quantities of antigen in complete Freund's adjuvant. A high volume ratio of adjuvant to antigen solution, and the volume used per inoculation, were shown to be critical factors. Ascitic fluids developed after three to five inoculations and were subsequently tapped repeatedly over a period of several weeks. All strains tested produced substantial amounts of anti-KLH antibodies, but marked differences were noted with respect to response to a hapten. Titers were enhanced in low responders by priming before the induction of ascites. The method is also useful for the production of "nonspecific" IgG and other serum components.


Degranulation of basophilic leukocytes in allergic contact dermatitis reactions in man. Basophilic leukocytes participating in allergic contact dermatitis in man underwent progressive degranulation over the course of several days after skin test. Less than 10% of basophils were degranulated in 24-hr reactions, but more than 60% were degranulated by 72hr. In electron microscopic individual basophil granules developed looser packing of particles by finely granular material, and eventual loss of all visible granule contents, changes that were progressive between 2- and 3-day reactions.


Tolerance and contact sensitivity to DNFB in mice. V. Induction of tolerance with DNP compounds and with free and membrane-associated DNFB. Immunologic unresponsiveness (tolerance) was induced in a mouse model of contact sensitization to DNFB. The ability to induce tolerance varied with the chemical reactivity of the tolerogen; DNFB was highly tolerogenic, DNBSO3 was moderately tolerogenic, and DNP-lysine was not tolerogenic. Although DNFB is considered a highly reactive compound, tracer studies of injected DNFB showed that it was rapidly excreted. Further studies were therefore done with DNFB attached to mouse erythrocytes. Tolerance to DNFB-RBC was highly specific in vivo; mice tolerant to DNFB showed normal reactivity to TNCB (picryl chloride.) Cells of mice tolerant to DNFB-RBC were also unresponsive to DNBSO3 in vitro. Tolerance to DNFB, DNBSO3, and DNFB-RBC all required time to develop, suggesting that an active process was involved.


Tumor-associated blocking factors: isolation from sera of tumor-bearing mice. An isolation and partial purifications of tumor-associated blocking factors from the sera of tumor-bearing mice is described. Columns for affinity chromatography were prepared by coupling syngeneic tumor-immune antibodies to Sepharose 4B. Passage of serum through such immunoadsorbent columns removed all blocking activity from tumor-bearers' sera; subsequent elution of the absorbent with 3 M NaSCN recovered the activity. The blocking material was further purified on Sephadex G-200. The data provide evidence for the presence of antigen in tumor-associated blocking factors and are compatible with the hypothesis that blocking factors often consist of antigen and antibodies in the form of immune complexes.


Genetic control of immune response against random copolymers of glutamic acid and alanine (GA) and tyrosine (GT) in inbred mice. An immune response (lr) gene(s) controlling responses to synthetic random copolymers of two amino acids of glutamic acid and alanine (GA) in mice has been well documented. This specific lr gene is linked to the major histocompatibility complex of the species. Mice of the H-2a,b,d,f,k,r and s haplotypes produce high titer antibodies and thus are high responders; whereas mice of the H-2 haplotypes j, ja, p and q produce no detectable antibody and are non responders. The response is "all or none" and high response is dominant. Attempts to detect an Ir-GT gene (glutamic acid and tyrosine) after immunization with two GT random copolymers were unsuccessful. However, GT-antibody could be produced after immunization with GT-methylated bovine serum albumin aggregates.


Development of rat mast cells in vitro. I. Differentiation of mast cells from thymus cells. Mast cells were differentiated by long-term culture of rat thymus cells on rat embryonic fibroblasts monolayers. Mature mast cells obtained in the culture were morphologically similar to normal peritoneal and thoracic mast cells and possessed specific receptors for IgE on their surface. In culture, blast cells appeared on the monolayer several days after seeding of thymus cells. These cells developed into young mast cells in the monolayer and became free in the culture medium with maturation. Receptors for IgE were detected on the surface of mastoblasts which contained a small amount of metachromatic granules. Evidence was obtained which suggested that the number and/or affinity of the receptors for IgE increases with maturation of mast cells. It was found that some mast cells differentiated from monolayers of embryo cells without seeding thymus cells. The present experiments, however, clearly showed that mast cells can be differentiated from thymus cell culture without monolayer. It appears that both thymus and embryo tissues contain precursors of mast cells.


Visual detection of carcinoembryonic antigen on surfaces. An attempt is made to detect carcinoembryonic antigen (CEA) at clinically interesting concentrations by using a simple immunologic surface test. Antibodies to CEA are detected in a direct test at concentrations below 1 ng/ml. The sensitivity of this assay is mainly limited by diffusion to the reacting surface. CEA is detected in an inhibition test at concentrations down to 20 ng/ml. The sensitivity of this inhibition test is limited by the average equilibrium constant in solution of the antibody-antigen reaction.


Regulation of the immune response to tumor antigens. II. The nature of immunosuppressor cells in tumor-bearing hosts. Immunosuppressor (IS) cells were found to be generated in tumor-bearing animals (TBA) within 24 hr after inoculation of tumor cells of the methylcholanthrene-induced Sarcoma 1509a and appeared to persist in the hosts as long as the tumor was progressing. However, IS cells disappeared with 5 days after extirpation of the tumor. Increasing doses of thymus cells of TBA increased the degree of suppression of tumor rejection in immune syngeneic animals. Ten million thymus cells of TBA were capable of suppressing significantly the tumor rejection. The IS cells were detected in the thymus, spleens, and draining lymph nodes, as well as in bone marrow of TBA, but could not be detected in the peripheral circulating blood. Since immunosuppressive activity of bone marrow cells from TBA was entirely abolished by the in vitro treatment of the cells with anti-theta serum and complement, the observed immunosuppression appears to be mediated by the T cell population.


Analysis of the humoral immune response to influenza virus in vitro. The induction of in vitro primary and secondary humoral immune responses to influenza virus in murine splenic explant culture is described. Anti-influenza antibody synthesized in vitro was detected and quantitated by a radioimmunoassay which utilized influenza coupled to bromoacetylcellulose. Both in vitro primary and secondary responses could be stimulated over a large range of virus doses. In vitro secondary responses were maximal when spleen donors had been immunized by the parenteral route although secondary type responses could be demonstrated as well after primary immunization by pulmonary infection. In vitro stimulation with influenza virus was relatively insensitive to inhibition at high antigen doses. The results are discussed in terms of the response of other antigens in spleen fragment culture.


Immune serum-mediated cytotoxicity against Trypanosoma rhodesiense. The metabolic integrity of Trypanosoma rhodesiense can be assessed in vitro by the extent of incorporation of radiolabeled leucine into trichloroacetic acid precipitable material or into material retained after filtration on a glass fiber filter. Incorporation is an approximately linear function of time, and the rate of incorporation is linearly dependent on cell concentration in the presence of normal rat serum. Incorporation is completely prevented if the organisms are reacted wiith fresh serum from animals immunized with gamma-irradiated parasites; the degree of inhibition is a function of the dose of immune serum used. This serum-mediated cytotoxic activity is abrogated by heating the serum, but can be fully restored by addition of fresh rat or guinea pig serum to the heated immune serum. The serum activity arises promptly after one to four immunizing doses of irradiated parasites, falls to lower levels by 1 month, but persists for at least 2(1/2) months, and is unaffected by challenge with viable trypanosomes.


Preferential expression of anti-azobenzenearsonate antibodies of heterozygous a1a3 rabbits in the a1 allotype. The preferential expression of anti-As antibodies in the allotype a1 of heterozygous a1a3 rabbits immunized with As-TMA-BSA has been investigated by means of quantitative methods. The average content of the anti-As antibodies in a1 and a3 allotypes was 84 and 11%, respectively; the analogous values for anti-TMA antibodies were 41 and 56%, and for anti-BSA antibodies they were 54 and 41%. The molar anti-As/anti-TMA ratios in the heterozygous a1a1 rabbits sensitized with As-TMA-BSA. The very low yields of anti-As-antibodies of allotype a3 cannot be caused by a lack of genes for the production of anti-As antibodies of allotype a3 because a3a3 homozygotes produce considerable amounts of anti-As antibody of allotpye a3. Competition between lymphoid cells having anti-As receptors of different allotype and different affinity for the antigenic p-azobenzenearsonate determinant is discussed as a possible cause for the preferential expression in the a1 allotype.


Synthesis of Gm allotypes by human tonsillar lymphocytes in culture: concordance with serum phenotype. Isolated human tonsillar lymphocytes were cultured with pokeweed mitogen, phytohemagglutinin, and without mitogen for 9 to 28 days. IgK, Gm(a) and Gm(f) were then quantitated in the cell suspensions. In cultures of cells derived from persons whose blood was heterozygous for IgGl allotype antigens Gm(a+f+), approximately equal amounts of Gm(a) and Gm(f) were found. In cultures of cells of Gma or Gmf homozygotes, there was complete concordance between the Gm allotype antigens produced by the cultures and the donor's serum phenotype-with no instance, either at zero time or at culture termination in which a Gm antigen was detected which was absent from the donor's serum. It was concluded that in vitro genetic allotype synthesis in tonsillar lymphocytes during short-term culture mirrored accurately in vivo Gm expression. IgK and Gm antigen synthesis was highest in the flasks containing pokeweed mitogen although both phytohemagglutinin and no-mitogen control flasks showed, in certain experiments, proliferation and an increase in the Ig per viable cell. It was observed that no-mitogen flasks contained twice as much allotype antigen as did phytohemagglutinin flasks suggesting an inhibition of Ig synthesis associated with the mitogen. The tonsillar lymphocytes, under the experimental conditions employed, were shown by a radio-incorporation and immunoprecipitation technique to be synthesizing polyclonal Ig de novo, at the termination of the cultures.


Radioimmunossay for detection of antigen and antibodies to Newcastle disease virus. When Newcastle disease virus (NDV) is treated with NP-40 and ether a membrane fraction of 150,000 m.w. is obtained. This fraction which is composed of two polypeptides with m.w. of 56,000 and 76,000 was used in a radioimmunoassay (RIA). The assay was developed for both antigen and antibody and was found to be reproducible, specific, and highly sensitive. Titers of 1:51,200 were determined by RIA as compared to 1:4 by agar gel diffusion and 1:200 by hemaglutination inhibition (HI). As little as 5 ng of viral protein were detected by RIA inhibition technique. Labeled antigen could be stored in the presence of serum, KCI and Triton X-100 at -20 degrees C for as long as 6 weeks and retained similar reactivity as fresh reagent.


The host antigen phenomenon in experimental murine schistosomiasis. II. Failure to demonstrate destruction of parasites transferred from hamsters to mice. Adult Schistosoma monsoni grown in hamsters and transferred directly to the mesenteric vessels of C57BL10J mice previously immunized with hamster cells survive normally despite evidence of cytoxic and hemagglutinating antibodies directed against hamster cells. This finding indicates that species differences may play an important part in the manifestions of the host antigen phenomenon in experimental schistosomiasis.


Gentic control of the immune response to the Ea-2 alloantigen system of the mouse. I. The humoral antibody response. The immunization of selected congenic strains and hybrids against the Ea-2.1 cellular alloantigen of the mouse demonstrated that the hammagglutinating antibody response to Ea-2.1 is regulated by a gene or genes associated with the H-2 gene complex. H-2r and H-2b are, respectively, responder and non-responder haplotypes.


A phosphorylcholine idiotype related to TEPC 15 in mice infected with Ascaris suum. A serum component, binding antigens having phosphorylcholine (PC) determinants were induced in several strains of mice by infection with Ascaris suum. This component was isolated and demonstrated to be an IgM (K) anti-PC antibody having idiotypic determinants in common with the IgA PC-binding myeloma protein TEPC 15. Rabbit anti-idiotypic antisera prepared with this component had idiotypic specificity for TEPC 15 and cross-idiotypic recognition of MOPC 167 and McPC 603, all IgA PC-binding myeloma proteins. The antisera also recognized determinants not present on TEPC 15. IgM and idiotype levels were quantitated by radial immunodiffusion and PC-specific antibody measured by hemagglutination (HA) with sheep erythrocytes coated with pneumococcal-C-polysaccharide. Mean IgM levels ranged from 2.5 to 8.7 mg/ml, idiotype from 0.54 to 5.3 mg/ml; and HA titers from 1:512 to 1:130,000 in different mouse strains. The high PC-specific antibody response was not duplicated by immunization with dead ascaris larvae or by infection with two other nematode species.


Amyloid fibril protein AA: purification and properties of the antigenically related serum component as determined by solid phase radioimmunoassay. The isolation by gel filtration of a serum component (SAA), antigenically related to the major filbrillar amyloid protein (AA), associated with "secondary" amyloidosis, has been monitored by a solid phase radioimmunoassay for the AA protein to detect cross-reacting serum fractions. Evidence is presented that not all cross-reacting antigenic determinants are accessible in native SAA, since additional determinants are revealed during the isolation procedure. The native structure of SAA appears to be quite labile. SAA from freshly collected serum has a m.w. of 180,000 and co-chromatographs with IgG. However, species of higher m.w. are observed after storage of serum at 4 degrees C or upon chromatography of serum in ammonium bicarbonate. Denatured SAA has a tendency to aggregate under strong dissociating conditions. A 12.500 m.w. antigenic species (SAAL) was detected upon guanidine-HCl denaturation of SAA, by earlier studies employing double immunodiffusion. However, evidence is presented here that the major part of the antigenic acitivity after guanidine-HCl treatment was of m.w. greater than 12,500, but was unreactive in double immunodiffusion. Formic acid treatment of cross-reacting serum fractions does result in virtually complete dissociation of SAA to SAAL, however. Furthermore, Formic acid-dissociated SAAL is of comparable immunoreactivity with AA, on a molar basis, unlike SAAL obtained from SAA by guanidine-HCl denaturation.


The indiction by complement of a change in KSCN-dissociable red cell membrane lipids. During complement lysis of antibody-sensitized sheep erythrocytes (EA) there was a larger loss of membrane phospholipids than during lysis elicited by hypotonic buffer. In addition, membranes prepared from complement-lysed EA had a marked reduction in KSCN (2.4 M)-dissociable membrane cholesterol and phospholipids, as compared to membranes from EA lysed hypotonically. Complement lysis caused a mild reduction in the amount of KSCN-dissociable membrane hexose but no change in the amount of dissociable protein. The impairment in dissociation of membrane lipids was related to the action of C8; it did not occur with membranes from EA that were treated with heat-inactivated (56 degrees C for 30 min) human serum, C4-deficient guinea pig serum, C6-deficient rabbit serum, or the first seven human complement components. EA lysed with limited amounts of complement exhibited a partial impairment in KSCN-dissociable lipids. Membranes from erythrocytes lysed with melittin showed a large increase in dissociation by KSCN of lipids, proteins,and hexoses. Membranes from erythocytes lysed with lysolecithin or phospholipase C showed, in addition to a reduction in dissociable lipid, a much larger reduction in dissociable hexose than a membranes from complement-lysed cells. These profiles of reactivity with 2.4 M KSCN inidcate that the membrane pertubations caused caused by complement may be specific. We conclude that complement-lysis is accompanied by a major rearrangement of membrane cholesterol and phospholipid which could be demonstrated in membranes from cells lysed by only one or very few complement lesions. Therefore, it appears that the lesions induce a propragated change in the lipid organization which extends throughout large areas of the membrane. This change might be responsible for the impairment of membrane permeability that follows the action of complement and results in cell destruction.


Temporary inhibition of antibody-dependent, cell-mediated cytotoxicity by pretreatment of human attacking cells with ammonium chloride. Treatment of lymphoid cell preparations with ammonium chloride buffers to remove contaminating erythroid cells markedly reduced the ability of these cells to function as attacking cells in antibody dependent cell mediated cytotoxicity tests (ADCC). The lymphoid cells, however, retained their ability to adhere to antibody-coated target cells. Recovery of function 20 hr after exposure to ammonium chloride suggests a metabolic rather than receptor effect of these salts on the ADCC function of lymphocytes.


Cell membrane IgD: demonstration of IgD on human lymphocytes by enzyme-catalyzed iodination and comparison with cell surface Ig of mouse, guinea pig, and rabbit. A substantial fraction of human cord blood and peripheral blood lymphocytes have recently been shown to bear IgD. Although IgD has not been identified in mice, it has been suggested that it is also a major surface immunoglobulin of murine lymphocytes. Thus, lactoperoxidase-catalyzed iodination of surface immunoglobulin of mouse spleen and lymph node cells reveals the existence of an IgH chain differing from mu, gamma, and alpha-chain both antigenically and by mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This new H chain class has been previously proposed to be the mouse homologue of delta-chain. In this paper, we analyzed human, mouse, guinea pig, and rabbit lymphoid cell membrane Ig by lactoperoxidase-catalyzed iodination, extraction with non-ionic detergent precipitation with a variety of specific anti-Ig sera, and electrophoresis of dissolved reduced precipitates on sodium dodecyl sulfate-polyacrylamide gels. Our studies confirm the previous reports of a new mouse cell membrane H chain with a mobility more rapid than that of mu-chain. However, we fail to detect a molecule with this electrophoretic mobility on the surface of guinea pig or rabbit lymph node and spleen cells. Moreover, neither anti-kappa nor anti-delta antibody precipitates a molecule with an H chain of this mobility from labeled extracts of human cord blood or peripheral blood lymphocytes. Cell surface delta was identified on both human cord blood and peripheral blood lymphocytes, but it proved to have mobility similar to human and mouse mu-chain. This result indicates either that mouse delta-chain has an electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels which differs appreciably from that of human membrane delta-chain or that the newly described mouse H chain is not the homologue of human delta-chain.


Antibody-dependent cell-mediated cytotoxicity in humans. III. Effect of protease inhibitors and substrates. Different classes of protease inhibitors and substrates were tested for their effect on the ability of human lymphocytes to mediate antibody-dependent cytotoxicity (Ab CMC). All the inhibitors tested (serine esterase inhibitors, chloromethyl ketone derivatives of tosyl-amino acids, synthetic protease substrates), except for the naturally occurring protease inhibitors (derived from soybean, lima bean, and porcine pancreas), were able to suppress, or to reduce insignificantly, the cytotoxicity. In the absence of a direct demonstration of an esterase activity, sensitive to the action of the inhibitors in the effector lymphocytes, careful controls were used to restrict the possibility that some nonspecific effect of the drugs was being interpreted. Particularly, the dependence of the inhibition of cytotoxicity as an effect of drugs on membrane transport mechanisms or on energy metabolism was excluded. The similarity between results obtained with compounds of different chemical characteristics and different molecular mechanisms of action supports a specific effect of the inhibitor on cellular esterase(s) or possibly protease(s). The fully reversible inhibition obtained with serine esterase inhibitors suggests that the relevant enzymes are activated only after effector-target cell interaction; the irreversible effect of chloromethyl ketone derivatives, however, does not allow the participation of already activated enzymes to be excluded. The results presented in this study on the probable role of cellular esterases, on cation requirement and on the sequence of biochemical steps in Ab CMC add  a new element to the analogy between this  cellular phenomenon and different types of cytotoxicity or other immunologically induced cellular reactions, suggesting that the biochemical mechanisms of cytotoxicity may partly reflect a common pattern of cellular response to external stimuli.


Neutrophil receptors for IgG and complement: their roles in the attachment and ingestion phases of phagocytosis. The functional roles of IgG and C3b in phagocytosis by human peripheral neutrophils were investigated. Phagocytosis of Staphylococcus aureus in the presence of human serum was severely depressed by heat inactivation of serum at 56 degrees C for 30 min. Experiments with varying particle: leukocyte ratios in the presence of complement-inactivated sera showed that particle-bound C3b can mediate a 10-fold enhancement of the overall phagocytic rate. When sheep erythrocytes were sensitized with either IgG or IgM, only the former were bound to and readily internalized by neutrophils. Erythrocytes  sensitized with both IgM and C3b were bound but not internalized. Furthermore, the presence of Fc fragments during incubation of S. aureus or latex beads with neutrophils in the presence of IgG or fresh serum affected a total inhibition of internalization but did not significantly alter adherence. Quantitative data regarding IgG sensitization indicated that bound C3b results in at least a 3-fold decrease in the amount of sensitizing IgG required for 50% maximal phagocytic response by neutrophils. On the basis of the above results, it is argued that particle-bound C3b functions primarily in the adherence phase and that bound IgG serves as a trigger for the internalization phase of phagocytosis.


Separate Fc-receptors for immunoglogulins IgG2a and IgG2b on an established cell line of mouse macrophages. The specificity of Fe-receptors on IC-21 cells, an established line of mouse peritoneal macrophages with antibody-dependent effector cell activity has been examined. Only IgG2a and IgG2b myeloma proteins bound readily to IC-21 Fc-receptors, the former in nonaggregated as well as aggregated form, the latter only as aggregated complexes. Thus, IgG2a bound in a manner characteristic of classically defined cytophilic antibody, whereas the binding of IgG2b appeared to be mediated by Fc-receptors for antigen-antibody complexes. Evidence is presented in support of the view that IC-21 macrophages possess separate and distinct Fc-receptor sites for these two immunoglobulins.


In vitro hemolysis of autologous erythrocytes caused by immune murine spleen cells and spherules of the fungus Coccidioides immitis. This communication describes an in vitro system wherein mouse erythrocytes are lysed in the presence of spherules of the fungus Coccidioides immitis and spleen cells from syngeneic mice immunized with a variety of antigens. The antigens include: tobacco mosaic virus in complete Freund's adjuvant (CFA), CFA alone, separate components of CFA, sheep erythrocytes, and allogeneic tumor. Spleen cells from mice sublethally infected with C. immitis are also capable of participating in this response. The lytic phenomenon, which does not require complement, is dependent upon the number of spleen cells per culture, the number of spherules per culture, the time of culture incubation, the amount of antigen injected into the animal and the time after immunization at which spleen cells are recovered. Live spherules or spherules killed with heat, with dimethylsulfoxide, or with formalin were effective participants, together with immune spleen cells, in the lytic reaction.


Age-associated involution of cellular immune function. I. Accelerated decline of mitogen reactivity of spleen cells in adult thymectomized mice. Autoimmune-susceptible (A/J and C57BL/6J) and nonautoimmune-susceptible (DBA/1J) strains of mice were adult thymectomized (aTx). Groups of mice treated by aTx, aTx and 330 rads (R), aTx and 650 R, and 330 R OR 650 R only, were compared to normal age-matched control (normal and sham Tx) mice. T cell functions were then followed as measured by in vitro spleen cell responsiveness to the T cell mitogens phytohemagglutinin (PHA) and staphylococcal enterotoxin B (SEB) in a long-term study. A decline in spleen cell responsiveness with natural aging up to 18 months of age was seen to PHA in all three strains and to SEB in A/J and C57BL/J mice. Adult Tx alone was followed by an accelerated decline in spleen cell responsiveness to PHA and SEB in all three strains of mice. Irradiation combined with aTx-reduced spleen cell responsiveness to SEB in A/J and DBA/1J mice. Irradiation with or without aTx diminished the bone marrow stem cell pool as measured by exogenous colony-forming units, and the hematocrit of all three strains of mice. Thus, the natural decline in cellular immunity with aging was accelerated by aTx in both autoimmune-susceptible and non-autoimmune-susceptible strains of mice. These results emphasize that physiologic thymic function(s) must continue throughout life in order to maintain T cell function.


The chromic chloride method of coupling antigens to erythrocytes: definition of some important parameters. Factors affecting the efficiency of the chronic cloride method of coupling antigen to erythrocytes were studied using a radioactive tracer. The amount of chromic chloride required for optimal coupling was related to the concentration of protein in the reaction mixture. Coupling was considerably more efficient when the chromic chloride solution was allowed to 'age' prior to use. In the system studied, the addition of piperazine buffer made little difference to the degree of coupling. The reaction was shown to proceed very rapidly. 50% of the final uptake of antigen took place within seconds, and the reaction was essentially complete 5 min after initiation. The importance of adequate mixing during the addition of the chromic chloride solution was emphasised.


Reliability of the enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Trichinella spiralis infections in conventionally raised pigs. An enzyme immunoassay with horse radish peroxidase as marker enzyme for detection of antibodies to Trichinella spiralis in pigs is described. In the enzyme-linked immunosorbent assay (ELISA) quantitation of specific antibodies is obtained by means of peroxidase labeled anti-species-immunoglobulin in antigen-coated tubes. The enzyme remaining in the tube after washing provides a measure of the amount of specific antibodies in the serum. A crude saline extract of T. spiralis muscle larvae served as antigen, 5 mug protein/ml being a satisfactory concentration. Lyophilization of antigen had no adverse effect on sensitivity. To decrease background staining the use of an optimal conjugate dilution was important. Adding bovine serum albumen to the conjugate was essential to decrease background reactions. Suitable substrate incubation times were studied. Washing was performed with tap water and Tween 20. In experiments with conventionally raised slaughter pigs infected with different numbers of T. spiralis larvae a positive correlation was found between initial dose of larvae and amount of antibodies detected by ELISA. Compared with immunofluorescence (IF) ELISA was more sensitive. IF yielded positive results in 11 out of 34 infected animals, whereas ELISA results were positive in 27. To evaluate ELISA under practical conditions extinction values of sera from infected and non-infected conventional pigs were compared with the highest extinction value in a group of 74 negative conventional pig sera. The relatively high background reaction of some of these negative sera decreased the number of positive practical ELISA results from 27 to 19 out of 34. In 1 out of 10 non-infected animals a false positive practical ELISA result was obtained.


Lymphocyte activation. VIII. The application of a whole blood test to the quantitative analysis of PHA responsive T cells. In diluted blood predominantly T cells responded to purified PHA (pure leucoagglutinin Wellcome). Red cells and non-T mononuclear cells increased PHA responsiveness of T cells at concentrations present in the peripheral blood. Addition of polymorphonuclear leucocytes was a very slight further advantage. The incorporation of [3H] thymidine reflected the numbers of T cells in culture, although not perfectly linearly, since counts increased when more T cells were added to the cultures. It was therefore concluded that in cultures of diluted whole blood non-specific amplification mechanisms were provided and responses reflected the performance of stimulated T lymphocytes. Since the method is simple and the number of technical variables is relatively small, it is possible to establish a "normal" range of PHA responsiveness expressed in absolute counts per minute, and thus directly compare results from different laboratories.


A new screening method for adenosine deaminase and nucleoside phosphorylase. An improved screening test for both adenosine deaminase and nucleoside phosphorylase is described.


Persistence of neuroadapted mumps virus in brains of newborn hamsters after intraperitoneal inoculation. Neuroadapted mumps virus produces systemic infection in newborn hamsters after intraperitoneal inoculation. Virus is disseminated via a low-level viremia and appears to enter the central nervous system by passage through the choroid plexus. At such sites, choroidal and ependymal epithelial cells are productively infected and become a source for further viral spread throughout the brain parenchyma. The development of neutralizing and hemagglutination-inhibiting antibodies in serum correlates with the clearance of virus from most systemic sites. However, peristence of virus in both brain and kidney is demonstrated late in this infection.


Activity of combinations of antimicrobial agents against Bacteroides fragilis. Thirty-two clinical isolates of Bacteroides fragilis were tested against nine pairs of antimicrobial agents by means of an agar dilution technique. A synergistic effect was observed with the combination of clindamycin and metronidazole. End points were achieved with 17 strains. Of these strains, 13 (76%) were inhibited by the combination of clindamycin and metronidazole; each drug was present at a concentration of less than or equal to 25% of its minimal inhibitory concentration when tested alone. This combination also showed synergistic bactericidal activity against three of six strains examined by a tube dilution technique. No antagonism was noted with any strain. The other eight combinations tested failed to show a consistent synergistic effect, although no antagonism was observed. These in vitro data indicate that antagonism is not likely to be encountered when combination therapy is used for B. fragilis infections. For selected B. fragilis infections, the combination of clindamycin and metronidazole may be useful.


Relation of infection with Neisseria gonorrhoeae to ABO blood groups. The relation of infection with Neisseria gonorrhoeae to the blood groups A, B, AB, and O was examined in 584 women attending a prenatal clinic. The occurrence of gonorrhea was significantly higher in black patients with blood group B than in those with blood groups A, AB, or O.


Equal susceptibility of males and females on Santa Cruz Island to the carrier state of hepatitis B surface antigen. Various studies have shown that hepatitis B surface antigen is detected more frequently in the blood of asymptomatic males than in that of asymptomatic females. The reason for this difference might be that males are more frequently infected than females or that males more frequently become asymptomatic carriers when they are infected. The sex distribution of hepatitis B surface antigen and antibody, as measured by reversed passive hemagglutination and passive hemagglutination, was determined in a Melanesian population. The frequency of the antigen was higher among males, but the ratio of antigen to antibody was the same in both sexes. This finding indicated that the higher frequency in males was due to a higher rate of infection rather than to a greater susceptibility of the males to the chronic carrier state.


Biological properties of the M-44 strain of Coxiella burneti. The attenuated M-44 strain of Coxiella burneti was investigated for biological characteristics affecting its use as a live vaccine. After 25 serial passages in guinea pigs, the strain did not develop phase I properties detectable by the microagglutination test. Infectious organisms were not detected in untreated guinea pigs later than 12 weeks after inoculation; however, infection could be reactivated at later times by the production of stress (by pregnancy or drug treatment) in guinea pigs that had recovered. Evidence of reactivated infection was observed in recovered pregnant animals just before parturition at 46 weeks after infection, in methyl prednisolone-treated animals at 56 weeks, and in cyclophosphamide-treated guinea pigs at 96 weeks. A low rate of intercage and intracage infection was recorded.


Chemical characterization of the structure of cholera toxin and its natural toxoid. Acrylamide gel electrophoresis demonstrated that the toxin of Vibrio cholerae is comprised of three polypeptide chains, alpha, beta, and gamma, of molecular weights 24,000, 9,700, and 9,700 daltons, respectively. Amino acid sequence analysis of intact toxin indicated a molecular composition of alpha gamma beta4. Acrylamide gel electrophoresis and sequence analysis confirmed that the natural toxoid (choleragenoid) is identical to the toxin beta-chain. The alpha- and gamma-chains of the toxin are disulfide-linked (fragment A) but are noncovalently bound to the beta-chains. About 50% of the primary structure of the N-terminal portion of the beta-chain has been identified and a small segment of the D-terminus has also been characterized. Twenty residues of the N-terminal portions of the alpha- and gamma-chains have been tentativly identified. The amino acid composition of the beta-chain was determined and compared to that of the natural toxoid.


Aspects of the interaction of Vibrio cholerae toxin with the pigeon red cell membrane. The ganglioside galactosy-N-acetylgalactosaminyl [sialosyl] lactosyl ceramide (GM1) is readily accumulated in pigeon red cell membranes soaked with [3H]GM1 (1-100 mug/ml) at 37 C for 30 min. This treatment enhances the activation of adenyl cyclase by the toxin of Vibrio cholerae. An attempt was made at correlation of the amount of incorporated GM1 with the increased binding of toxin and activation of adenyl cyclase. Cells with less than 2 mug of incorporated GM1 per 4 X 10(9) cells bind 5-10 mug more toxin than do untreated cells, which bind 0.25 mug per 4 X 10(9) cells. Cells with more than 2 mug of GM1 bound (per 4 X 10(9) CELls) which has been incorporated from micellar solutions of GM1 (greater than 20 mug/ml), do not bind any more extra toxin. In untreated cells, 0.1 mug of toxin is involved in the activation of adenyl cyclase. In GM1-treated cells 0.25-0.5 mug of toxin is involved, although at least 5 mug of toxin is bound. It is concluded that 90% of the extra toxin-binding sites on the GM1-treated cell are nonproductive.


Stimulation of adenylate cyclase by Vibrio cholerae toxin and its active subunit. The mechanism by which Vibrio cholerae toxin and its active subunit A stimulate adenylate cyclase of rat liver plasma membrane after in vivo or in vitro exposure was investigated. Stimulation of adenylate cyclase was more efficient with cholera toxin than with subunit A in whole animals and in "soluble" preparations of adenylate cyclase. Toxin-stimulated plasma membrane adenylate cyclase activity persisted after solubilization from the membrane by detergent, while subunit A stimulation was destroyed by detergent treatment. Detergent-free "soluble" enzyme could be stimulated in vitro by cholera toxin, but such stimulation by subunit A was very weak. Binding studies with iodotoxin or iodosubunit A confirmed that the interaction of toxin with adenylate cyclase in "soluble" preparations could be the result of specific binding to the enzyme. The uncertainty as to whether G M1 ganglioside (galactosyl-N-acetylgalactosaminyl [sialosyl] lactosyl ceramide; GGnSLC) is present in these fractions limited the interpretation of the data on binding. A biological modification of subunit A prior to the activation of adenylate cyclase is inferred from these data.


Aspiration biopsy. An aid in the diagnosis of para-nasal tumours. Three cases are reported to demonstrate (1) the difficulty in diagnosing the nature of nasopharyngeal tumours (2) the assistance given by the fine-needle aspiration biopsy technique in cases of paranasal tumours. Reliance should not be placed on this technique alone. It should be performed at the initial examination together with conventional techniques. In this way, the patient may be spared the need for multiple anaesthetics and a diagnosis may be arrived at more promptly so that treatment may be instituted more quickly.


Massive acquired cholesteatoma of the temporal bone. Nine patients with massive acquired cholesteatoma of the temporal bone were treated by radical mastoidectomy, facial nerve decompression, and excision of the inner ear. The commonest presenting symptoms were otorrhea and deafness, but otalgia, vertigo, external swelling and facial nerve palsies were also seen. Polytomography was helpful in assessing the degree of bone erosion preoperatively. The large cavity present following cholesteatoma excision was partially obliterated by a superiorly based temporalis muscle pedicle which helped to seal the internal auditory meatus and prevented the development of a cerebral hernia. Those patients with preoperative facial nerve palsies showed evidence of nerve degeneration and had incomplete recovery at a one year follow-up.


Acid-base equilibrium in Menière's disease. 57 patients suffering from Meiere's disease have been examined: metabolic alkalosis has been found in 39 and metabolic acidosis in 4. Electrolytes (Na, K and Cl) were normal. Among the 31 subjects in the control series a slight metabolic alkalosis qas detected only twice. Follow-up of several patients, from one to three years, resulted in a realization of a parallel between metabolic disturbance and Meniere symptoms. A provocation test, by sodium bicarbonate oral administration, has beem attempted twice: in both instances alkalosis did increase strongly but, only once, vertigo was elicited. Meniere patients found in alkalosis were given acidifying drugs to normalize their acid-base equilibrium. Such treatment proved effective in preventing acute cochleo-vestibular crises and in improving tinnitus and deafness (in the fluctuating stage). On the interruption of treatment symptoms reappeared. The physiological-pathological significance of metabolic alkalosis in Meniere's disease still remains unclear and, consequently, this can be considered merely a predisposing factor in endolymphatic hydrops.


Benign cartilagenous tumour of the temporomandibular joint. An unusual case of a benign cartilagenous tumour of the temporomandibular joint is presented. The difficulties encountered in histological diagnosis, the benign behaviour of the tumour and the need for restraint in treatment are emphasized.


Carotid body tumour. Exicsion with resection of carotid arteries. A case of carotid body tumour is reported and the salient features of its histopathology have been described. Its excision with resection of the carotid arteries can be safely undertaken if the tumour is of long duration, the angiography shows marked displacement of the carotid arteries and if the blood pressure is maintained during surgery by regulating the blood transfusion and lowering the head end of the patient, thereby preventing fatal brain hypoxia. The fact that it is a potentially malignant and radioresistant tumour, makes its excision with or without ligation of the carotid arteries almost imperative.


Unilateral blindness following vidian neurectomy. A case with unilateral blindness is being reported, to arouse awareness in surgeons about the possibility of such a serious complication after Vidian neurectomy by the trans-septal route.


11, 15, 19-trihydroxy-9-ketoprost-13-enoic acid and 11, 15, 19-trihydroxy-9-ketoprosta-5, 13-dienoic acid in human seminal fluid. Two novel prostaglandins (PG) have been found in human seminal fluid which had been frozen immediately after collection. They were characterized by combined gas-liquid chromatography-mass spectrometry of various derivatives as 19-hydroxy prostaglandin E1 (11, 15, 19-trihydroxy-9-ketoprost-13-enoic acid) and 19-hydroxy prostaglandin E2 (11, 15, 19-trihydroxy-9-ketoprosta-5, 13-dienoic acid). They were present in three to five times the quantity of prostaglandins E1 and E2. Incubation of seminal fluid for 3 hr at 25 degrees C reduced levels of 190H-PGEs2.5-fold and PGE22-fold, while increasing levels of PGAs and PGBs 2-fold. No 190H PGA or 190H PGB was detected in extracts of unincubated fluid. The PGAs, PGBs and their 19-hydroxy analogs are probably artifacts arising metabolically or as a result of classical isolation techniques.


Bile acids. XLIX. Allocholic acid, the major bile acid of Uromastix hardwickii. Tauroallocholate is the major bile salt of the lizard, Uromastix hardwickii. Alkaline hydrolysis of bile from 25 gallbladders provided 1.21 g of acidic material, about 90% of which was allocholic acid. Analyses by gas-liquid chromatography, and mass spectrometry verified the presence of almost 10% of deoxycholic acid and smaller amounts of other 5alpha and 5beta-bile acids.


Measurement of human high density lipoprotein apolipoprotein A-1 in serum by radioimmunoassay. A sensitive and specific double antibody radioimmunoassay for the major apolipoprotein (apo A-I) of human serum high density lipoprotein (HDL) was developed. Initial studies indicated that direct measurements of apo A-I concentration in whole untreated sera or isolated high density lipoprotein fractions yielded variable results, which were lower than those obtained in the corresponding samples which had been subjected to delipidation. Subsequently, it was observed that heating diluted sera or HDL for 3 hr at 52 degrees C prior to assay resulted in maximal increases in apo A-I immunoreactivity to levels comparable to those found in the delipidated specimens. This simple procedure permitted multiple sera to be assayed efficiently with full recovery of apo A-I.


Measurement of squalene in human tissues and plasma: validation and application. A method is described for accurate and reproducible measurement of squalene in plasma, feces, urine, bile, and tissue that depends on isolation by alumina column chromatography after mild saponification and on measurement by gas-liquid chromatography. Recoveries from all tissues exceeded 80% and from plasma 96%; losses were accurately corrected by appropriate additions of squalene as an overall recovery standard.


Effect of p-chlorophenoxyisobutyrate on the antilipolytic action of insulin and insulin binding in isolated adipocytes. The present study was undertaken to investigate the potentiation by p-chlorophenoxyisobutyrate (CPIB) of the antilipolytic effect of insulin in isolated adipocytes from rats fed a (1) sucrose diet, (2) glycerol-lard diet, or (3) chow diet. CPIB supplementation in the diet consistently resulted in decreased serum triglyceride levels in rats from the three dietary groups. The catecholamine-stimulated glycerol release was significantly depressed to a greater extent by insulin when the fat cells were obtained from rats given CPIB compared to those without drug treatment. The enhanced insulin sensitivity was, however, not accompanied by any changes in insulin binding to adipocytes. These two observations were found in cell preparations from rats fed any one of the diets, although differences among dietary groups could be detected. In an in vitro experiment, epinephrine-stimulated glycerol release was progressively inhibited by increasing concentrations of CPIB in the incubation medium. However, the antilipolytic response to an optimal concentration of insulin (100 muU/ml) was augmented in the presence of CPIB. Thus, it seems that CPIB can potentiate the action of insulin in inhibiting mobilization of free fatty acid from the adipose tissue, and the coordinated effect of both antilipolytic agents is important in lowering serum triglyceride concentration. The mechanism by which CPIB facilitates the effect of insulin is discussed.


Duvatrienediols in cuticular wax of Burley tobacco leaves. 4, 8, 13-Duvatriene-1, 3-diol diastereoisomers have been identified in the cuticular wax of fresh Burley tobacco leaves. Their structures were determined by gas-liquid chromatography, mass spectrometry, and infrared and ultraviolet spectroscopy. Butylboronic acid derivatives of the alpha, beta-isomers were separated by gas-liquid chromatography and identified by mass spectrometry. The quantitative determination by gas-liquid chromatography revealed that the duvatrienediols are major components in the cuticular wax of Nicotiana tabacum. The duvatrienediol content in young leaves is higher than in old leaves, and in young leaves this compound may account for half of the cuticular wax.


Synthesis of 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol and 5beta-cholestane-3alpha, 7alpha, 245, 25-pentol. This paper describes syntheses of 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol and 5beta-cholestane-3alpha, 7alpha, 12alpha, 24xi, 25-pentol which give higher yields than previously published methods. In addition, 5beta-cholestane-3alpha, 7alpha, 12alpha, 24xi, 25-pentol was synthesized by a different procedure, namely via performic acid oxidation of the correspinding unsaturated triol, which gave a lower yield but avoided the formation of 5beta-cholestane-3alpha, 7alpha, 12alpha, 25, 26-pentol, which normally tends to contaminate the final product. Structures were confirmed by gas-liquid chromatography, infrared-, proton magnetic resonance- and mass spectrometry, 5beta-Cholestane-3alpha, 7alpha, 12alpha, 25-tetrol and 5beta-cholestane-3alpha, 7alpha, 12alpha, 24xi, 25-pentol were required for in vivo and in vitro studies of the (hypothetical) 25-hydroxylation pathway of cholic acid biosynthesis.


A sterospecific synthesis of 7alpha-hydroxycholesterol. The five step synthesis of 7alpha-hydroxycholesterol utilizes the solvolysis of 7alpha-bromocholesterol benzoate with potassium acetate in acetic acid as the key step in controlling the stereospecificity of the reaction sequence. This reaction yields 7alpha-acetoxycholesterol benzoate with retention of configuration at position seven. The diester is readily reduced with lithium aluminum to 7alpha-hydroxycholesterol.


Measurement of cortisol, cortisone, 11-deoxycortisol and corticosterone in foetal sheep plasma during the perinatal period. A method is described for the resolution and individual quantitation of cortisol, cortisone, 11-deoxycortisol and corticosterone in foetal sheep plasma. The steroids were extracted by solvent partition and separated by LH-20 Sephadex column chromatography. Radioimmunoassay was used for the measurement of 11-deoxycortisol and cortisone and competitive protein-binding for corticosterone and cortisol. The relative levels of these steroids in the plasma of chronically catheterized sheep foetuses from 12 days before birth to term and then in the newborn lamb until 2 days of age are recorded. Cortisol gradually increased from a basal concentration of between 0 - 5 and 3 - 0 mug/100 ml plasma between days 12 and 5 pre partum, and then rose rapidly to 10 mug/100 ml plasma during the last 5 days of pregnancy to reach a maximum during or just after birth. Two days post partum the levels had fallen to approximately 3 mug/100 ml plasma. The mean value for 11-deoxycortisol between days 8 and 3 pre partum was  0 - 4 mug/100 ml plasma and increased in the final days before delivery to  1 - 0 mug/100 ml. Corticosterone initially showed slightly higher levels (approximately 1 - 5 mug/100 ml) in the earlier period of investigation but then fell during the immediate pre-partum period to  0 - 8 mug/100 ml. Cortisone was not detected at any stage of the investigations. The relationship between levels of cortisol and 11-deoxycortisol in foetal plasma and myometrial contractility is shown. An increase in uterine activity was seen to occur at the time that cortisol levels were at their maximum. The 11-deoxycortisol values throughout this particular study remained low. The results are discussed in relation to recorded levels in the adult and to previous studies in vitro with regard to changing steroid biosynthetic enzyme activity.


Hormone storage in individual neurosecretory granules of the pituitary gland: A quantitative ultrastructural approach to hormone storage in the neural lobe. A quantitative ultrastructural study of the neurosecretory granules in the neural lobe of the rat was performed in order to calculate the number of such granules in the neural lobe and thus, provided all hormone is intragranular, to derive the hormone content of individual granules. The results suggest that the gland contains 1 - 44 X 10(10) granules and that an individual granule (mean diameter 160 nm) contains about 84000 hormone molecules. The errors involved in the many measurements made are analysed, and the calculated value for the amount of space available for each hormone molecule within the granule is shown to agree well with independently reported data for the expected size of a molecule of protein-hormone complex, and for the size of subunits visualized in freeze-etched material. These results are compatible with exclusive intragranular hormone storage as a 'solid' core, and the physical form that this core might take is discussed. The data are used to express what is known of hormone turnover and release in terms of the numbers of granules involved. Comparison of different parts of the neural lobe suggests that, apart from the most anterior part of the gland, there is little regional variation in quantitative aspects of granule storage in the neural lobe.


Distribution of neurosecretory granules among the anatomical compartments of the neurosecretory processes of the pituitary gland: a quantitative ultrastructural approach to hormone storage in the neural lobe. The distribution of neurosecretory granules in various anatomical compartments of neurosecretory axons of the neural lobe of the rat pituitary has been studied. Apart from the most anterior tip of the gland, where granules are largely restricted to undilated axons and a few 'swellings', the proportional compartmental storage of granules is essentially homogeneous for the rest of the gland: 13% of granules are found in undilated axons, 31% in axonal 'endings' (which contain microvesicles and abut the basement membrane) and 55% in axonal 'swellings' (which are devoid of significant numbers of microvesicles). These values indicate that the 'endings' contain a much greater proportion of the total number of granules stored in the neural lobe than would be predicted if the readily releasable pool of hormone were composed of all the granules in the 'endings'. Some further constraint on granule release either physiological or anatomical (e.g. the position of the granule in relation to the plasmalemma of the 'ending') must be operative.


Circadian rhythm of corticosterone levels in rat brain. In the present study the circadian changes which occur in the levels of corticosterone in the brain and plasma in Sprague-Dawley rats are reported. The levels of corticosterone in the brain were found to have a daily trough and crest with timing similar to that observed for the plasma steroid. In addition, the effect of histamine stress on the corticosterone content of the particulate and the soluble fractions at the trough and crest was examined. The levels of both brain fractions were significantly higher 20 min after histamine injection. The time of day at which the stress was applied was not a significant factor in the magnitude of the stress response.


Effect of cortisol on utilization and hepatic release of glucose in the marsupial brush-tailed opossum, Trichosurus vulpecula. Brush-tailed opossums were prepared surgically with indwelling heatic and jugular venous catheters for blood sampling without disturbance in the conscious state.  Hepatic extraction of Rose Bengal was 21 +3 (s.d.) % and hepatic clearance, used as a measure of hepatic blood flow, was 42.5 +7 ml/kg/min.  Hepatic relaease of new glucose, calculated from the thorias vena caval-hepatic venous difference in glucose specific activity at equilibrium during i.v. infusion of [14C]glucose and hepatic blood flow, was 3.5 + o.0 mg/kg/mim.  This was not changed by i.v. infusions of 10% ethanolic aline or cortisol in ethanolic saline, at 1mg/kg/h for 90 min, although the cortisol infusion caused the peripheral blood glucose concentration to rise form 56.5 + 7.3 to 83.2 + 10.3 mg/100ml.  The rate of metabolic clearance of glucose fell from 6.1 +1.1 to 4.2 +0.9 ml/kg/min during i.v. cortisol infusion.  Daily i.m. injection of 1 mg cortisol accetate/kg for 5 days caused an increase in hepatic new glucose release to 8.0 + 1.6 mg/kg/min.  The findings support the propostion that, in the marsupial, the short-term effect of cortisol on plasma glucose concentration is due to inhibition of peripheral glucose utilization, whereas the long-term effect is due to increased hepatic glucose production.


Solvolysable deoxycorticosterone conjugates in human urine. The nature of the urinary conjugate converted by solvolysis, to free unconjugated deoxycorticosterone (DOC) was studied. A comparison of 11 solvolysis techniques has shown that the method employed in this study yielded 86% of the highest yield by any of the techniques tried. Three successive chromatographic systems on paper showed that no appreciable amounts of contaminants were present in the free DOC eluates, following solvolysis. By preparing authentic [3H]DOC sulphate and subjecting it to solvolysis it was shown that more than 90% of the tritiated DOC was recovered, after chromatography of the free DOC extract. This suggests that much of the solvolysable DOC in human urine is present in the form of the sulphate conjugate. The levels of DOC, excreted as the solvolysable conjugate in a variety of urine specimens, were shown to be much higher than those of free DOC, the former being 4-8 to 127 times higher than the amount of the latter. This highly variable ratio suggests that the site of production of solvolysable DOC is different from that for free DOC. The only correlation between free and solvolysable DOC was shown in dexamethasone-suppressed patients, in whom the mean percentage remaining after suppression was 30-6% for free DOC, 24-1% for solvolysable DOC and 22-2% for cortisol. As solvolysable DOC is present in much larger amounts in urine, care is necessary in the storage of urine samples in which free DOC estimates are to be made, as we found that urine specimens left at room temperature for 1 week could show rises of as much as 400% of their starting free DOC levels.


Metabolic clearance rate and production rate of oestradiol in conscious rabbits. A continuous isotope infusion technique was used to measure the metabolic clearance rate (MCRB) and production rate (PRB) of oestradiol-17beta in a group of six non-pregnant conscious Chinchilla rabbits. The mean MCRB of oestradiol was 162 ml/min (0 X 079 1/min/weight0 X 75), and at equilibrium a significant proportion of the radioactivity in the circulation was present as oestrone (conversion ratio oestradiol to oestrone, 27 X 6%). The mean PRB of oestradiol was 4 X 3 ng/min, which is equivalent to about 5 mug/day. Some day-to-day variation was seen in the endogenous concentrations of oestradiol, oestrone and progesterone although in general the levels of the two oestrogens were less than 30-40 pg/ml, and progesterone levels were below 300 pg/ml apart from short peaks up to 600 pg/ml. These values were compared with previous measurements of the ovarian secretion rate of oestradiol measured directly in anaesthetized animals.


Egg transport in ovariectomized rabbits as affected by different combinations of oestrogen and progesterone. A total of 1548 eggs was transferred to the oviducts of rabbits ovariectomized 45 days to 7 months earlier. The pattern of egg transport was disrupted, and the majority of eggs were in the vagina 60-72 h after transfer. To determine the role of the ovarian hormones on egg transport, the effects of various combinations of oestrogen and progesterone were studied. None of the hormonal treatments produced the normal transport pattern and large proportions of transferred eggs were retained in the oviducts. It is concluded that as long as progesterone is the dominant hormone, eggs move very slowly through the isthmus, and that a surge of oestrogen is required to modify the action of progesterone and to speed the movement of eggs to the uterus.


Stimulation of carp interrenal function by adrenocorticotrophin. The effect of ACTH on cortisol concentration in the plasma of the carp has been studied; cortisol was determined using a competitive protein-binding radioassay. Blood was sampled at 12.00 h (the diurnal minimum) by cardiac puncture. The plasma cortisol level of undisturbed carp (blood obtained 1--3 min after netting) was 16 +/- 14 (S.E.M.) ng/ml. One hour after injection of ACTH (1-0 i.u./100 g body wt., i.m.) the level of cortisol increased to 415 +/- 62 ng/ml and reached a maximum of 656 +/- 62 ng/ml after 2 h. The level of cortisol in saline-injected control carp after 2 h was 72 +/- 33 ng/ml. A transient rise of cortisol was noted in both groups 30 min after injection (315 +/- 51, 315 +/- 62 ng/ml). This was attributed to a surge of endogenous ACTH, presumably due to handling. Carp head kidneys were superfused in vitro. They released cortisol spontaneously, but the releasing rate declined exponentially and reached a minimum after 135 min. Addition of ACTH to the medium was followed by a prompt increase in cortisol release. The rate of cortisol release was maximal 30--45 min after addition of ACTH and was identical to the rate at the beginning of the superfusion.


Inability of actinomycin D to act upon the uterine refractory state resulting from nidatory oestrogen action in rats. After priming with oestradiol, ovariectomized rats were given 6 days of progesterone treatment in which two doses of 50 ng oestradiol were superimposed on days 3 and 6. This basic regimen allows the oestradiol-induced (1st injection) inhibition of uterine sensitivity to decidual stimuli to appear. Actinomycin D given instead of, or together with, the first dose of oestradiol could not mimic, or prevent oestrogen action in the occurrence of the uterine refractory state. This drug was unable to interfere with the minimal 48 h progesterone treatment which is requisite to uterine sensitization. Our results together with other data from the literature, suggest that luteal hormones do not need to act at the transcriptional level to sensitize and desensitize the uterus to implantation.


Placental lactogen in the cow.

Placental lactogen in the cow. Placental lactogen secretion has been demonstrated in cows on days 36, 178, 182 and 270 of pregnancy by co-culture of cotyledonary tissue with mouse mammary gland explants. Bovine placental lactogen probably originated from the foetal cotyledon and showed no detectable cross-reaction in immunoassays for bovine prolactin or growth hormone. Peripheral plasma samples collected from seven primiparous heifers at 2-week intervals throughout pregnancy showed a seasonal rhythm in prolactin concentration, measured by radioimmunoassay, with high levels in the summer. Monthly samples were bioassayed for total lactogenic activity by a rabbit mammary gland organ culture method. Postive responses were obtained only when prolactin levels were high (greater than 70 ng/ml), indicating that levels of placental lactogen in the circulation are much lower in the cow than in sheep, goats or women.


The influence of oestradiol-17beta and progesterone on the contractility of the oviduct of the turtle, Chrysemys picta, in vitro. The contractility of the oviduct of Chrysemys picta (the fresh-water turtle) was studied in vitro. Significant regional differences in contractile activity were observed. Contractions increased in amplitude and duration and decreased in frequency from the proximal to the distal end of the oviduct. Injections of oestradiol-17beta in vivo increased the amplitude of contractions and decreased the duration of the rest period in segments taken from the diatal end of the oviduct. In contrast, progesterone injections significantly reduced the duration of both contractile and resting periods.


Uptake and metabolism in vivo of tritiated oestradiol-17beta in tissues of ageing female mice. The uptake in vivo of tritiated oestradiol-17beta has been compared in young and aged ovariectomized CBA/H-T6 mice by examining the levels of radioactivity in tissues 1 h after injection. The specificity of oestradiol uptake was demonstrated by previous treatment of some animals with either diethylstilboestrol or progesterone. The levels of radioactivity in whole tissue extracts were similar in both age groups for the uterus, hypothalamus, cerebrum and serum, but the uptake in the pituitary gland was significantly lower in old mice. There was no evidence of altered hormone metabolism with age.


Absorption of 125I-labelled sheep growth hormone in single proximal tubules of the rat kidney. Techniques of kidney micropuncture and electron microscope autoradiography have been used to study the uptake of 125I-labelled sheep growth hormone (GH) in rat renal proximal tubules. After microperfusion of a proximal tubule with 125I-labelled GH, the transport of label by the tubular epithelium was studied autoradiographically at selected times up to 1 h. The sequential transfer of labelled material from tubule to microvilli, then to small and large apical vacuoles and finally to lysosomes followed the pattern of absorption that has been described for other proteins. Evidence of lysosomal degradation of the transported protein was obtained from studies in vitro; lysosomes isolated from the renal cortex rapidly converted 125I-labelled GH to products of lower molecular weight. In addition to the absorptive pathway through the intracellular vacuolar apparatus is appeared that there was also an alternative pathway, less well defined, whereby GH could be absorbed without being degraded.


Corticosteroid binding by plasma proteins and the effects of oestrogens in the marsupial brush-tailed opossum, Trichosurus vulpecula (Kerr). Using the techniques of equilibrium dialysis at 36 degrees C and gel filtration at 4 degrees C, a high-affinity, transcortin-like, corticosteroid binding system has been demonstrated in the blood plasma of the marsupial Trichosurus vulpecula. The affinity constant for non-albumin binding was 4-018 +/- 1-032 X 10(7) (S.C.) 1/mol for males and 4-046 +/- 0.981 X 10(7) for females. The concentration of non-albumin binding sites was 1-82 +/- 0.76 X 10(-7) mol/1 in males and 1-86 +/- 0-57 X 10(-7) mol/1 for females. Oestrogen administration, sufficient to cause marked hypertrophy of the genital tract in the femlaes, had no effect on the affinity constant or the concentration of the non-albumin binding sites in either males or females. The general condition of the animals deteriorated during oestrogen administration and there were significant falls in the concentrations of albumin and cortisol in the blood plasma. In one animal which died during oestrogen treatment, the adrenal glands were significantly hypertrophied.


A possible role for oxytocin in sperm transport in the male rabbit. Administration of methallibure (50 mg/kg body weight, daily) to male rabbits resulted in a 45% reduction in sperm number in ejaculates obtained during the treatment period. Recovery occurred within 48 h after the last dose of methallibure. This decrease in sperm number did not occur when oxytocin (0-2 i.u./kg body weight) was administered simultaneously with methallibure. This suggests that methallibure prevents the release of oxytocin during ejaculation.


Behavioural responses to sex steroids of gonadectomized and sexually regressed quail. The purpose of these experiments was to compare the behavioural and morphological effects of exogenous sex hormones in gonadectomized quail (Coturnix coturnix japonica) with those in quail having regressed gonads as a result of exposure to short days. In Expt 1, male quail were assigned to one of three treatment groups: (1) intact, exposed to 16 h light: 8 h darkness and injected with oil (group 16L); (2) gonadectomized, exposed to 16 h light: 8 h darkness and injected with 2-5 mg testosterone propionate (TP)/day (group 16L-castrated); and (3) intact, exposed to 8 h light: 16 h darkness, and injected with 2-5 mg TP/day (group 8L). Groups 16L-castrated and 8L responded similarly to testosterone, copulating with equal frequency and rapidity after the same number of days of treatment, and also developing proctodeal (foam) glands of a similar size. Only on day 7 of testosterone treatment did the results for these two groups differ. By day 14, the behaviour of both groups resembled that of the 16L birds. In Expt 2 female quail were assigned to the same three treatment groups, except that the hormone treatment was 25 mug oestradiol benzoate/day. Group 8L became sexually receptive sooner than the 16L-ovariectomized quail, but by day 13 both groups had oviducts of similar size, were equally receptive, and were as receptive as the 16L females. The results suggest that the effects of photoperiod on sexual behaviour in this species are mediated largley, if not wholly , by the gonads.  They also suggest exposure to short days and surgical gonadectomy are rather similar experimental procedures in the quail.


Immunoreactive gastrin in differential and density gradient fractions of rat gastric mucosa. Immunoreactive gastrin was measured in subcellular fractions of rat gastric mucosa. The sedimentational properties of subcellular gastrin-containing structures were distinct from those of mitochondria. After centrifugation in sucrose density gradients using a zonal rotor, the peak of immunoreactive gastrin was found in 1-17--1-18 g cm(-3) density sucrose (1-35 M; 39-5%, W/W). A thermolabile component with 125I-labelled gastrin-binding activity present in gastric mucosal homogenates and fractions was not associated with the gastrin storage vesicles sedimenting in density gradients.


Incorporation of radioactive precursors into the membrane and contents of the neurosecretory granules of the rat neurohypophysis as a method of studying their fate. Rat neural lobes have been separated into subcellular fractions by differential centrifugation at various times after an intracisternal injection of [35S]cysteine or [3H]choline. Both isotopes led to a rise and fall in the radioactivity of neurosecretory granules (NSG) which paralleled that found previously for the neurohypophysial hormones and the neurophysins. While the radioactivity of the NSGs resulting from [35S]cysteine injection was predominantly associated with granular contents, [3H]choline injections led to a preferential labelling of the granular membrane. There was no indication of a sequential movement of radioactivity from the NSG-membrane fraction into the microsomal fraction (containing the so-called small vesicles) which might be expected if granular membrane were recaptured as small vesicles after release of secretory product by exocytosis. When release was stimulated in injected animals by giving them 2% NaCl solution to drink, 35S diappeared from the gland as expected, but 3H was retained and, moreover, appeared in the NSG-membrane fraction; results compatible with membrane conservation occurring by recapture of large vesicles. There was an indication that some of the neurophysin in the NSG was membrane-bound and that this too was retained after release of the granular contents.


Prolactin inhibition test with L-dopa: decrease and restoration of plasma prolactin levels in the rat by a peripheral process. L-DOPA, within 30 min after administration, induced a highly significant decrease of plasma prolactin levels (phase 1) in a number of groups of rats, differing in age and/or endocrine status, apparently by direct inhibition of prolactin release from the pituitary. Three hours after administration of L-DOPA these low plasma prolactin concentrations in treated animals had increased (phase 2) and did not differ significantly from levels in control animals, indicating that the effect of L-DOPA on plasma prolactin levels is only of short duration. During this process some interesting phenomena were observed, especially in the animals treated with oestrone. The elimination rate of prolactin from plasma was very high (t 1/2 = 2.8 min), as indicated by decreasing concentrations of the hormone during phase 1. Pituitary prolactin content did not change during phase 1, suggesting that prolactin synthesis was also stopped. Notwithstanding the high elimination rate, plasma prolactin regained initial concentrations in phase 2, suggesting release of a substantial part of the pituitary prolactin content. The latter,however, remained constant during the whole experiment (i.e. before L-DOPA administration and during phase 1 as well as phase 2). The results suggested another working mechanism of L-DOPA in decreasing plasma prolactin levels, namely by stimulating the uptake of this hormone in the periphery. After the effect of L-DOPA had ceased, most of the prolactin from the periphery returned into the bloodstream, causing a rapid restoration of plasma prolactin levels without substantial release from the pituitary. The nature of the processes responsible for the peripheral uptake of prolactin is discussed.


Parturition in the goat: plasma concentrations of prostaglandin F and steroid hormones and uterine activity during late pregnancy and parturition. Changes in plasma concentrations of hormones and uterine activity associated with spontaneous parturition in the goat were examined. No change in oestradiol-17beta concentration was detected during the experimental period, but oestradiol-17alpha consistently increased in concentration 3-4 days before parturition. This was followed by an increase in prostaglandin F two days later, while a pre-partum decline in progesterone concentration occurred 18-20 h after the significant increase in prostaglandin F. The onset of uterine contractions coincided with a significant decline in progesterone concentration and a period of continually rising prostaglandin F and oestradiol-17alpha concentrations. Uterine activity expressed in Montevideo units did not change until 10-8 h before foetal expulsion. These results support the hypothesis that in the goat the elevated prepartum concentration of  prostaglandin F brings about luteolysis and a subsequent decline in progesterone concentration.


Oestrogens and prolactin as possible regulators of puberty. At day 15 after birth, high serum oestradiol levels, high FSH levels and occasionally high LH levels were observed in control female rats. Injections of potent and specific antisera for oestrogen between days 8-15 prevented LH peaks and decreased basal LH levels. Serum FSH levels were increased after treatment. High oestradiol levels at this time appear to exert a positive feedback action on phasic as well as tonic LH release. Injections of ovine prolactin (0.5 mug/g body wt twice daily) between days 8-25 of life significantly advanced the day of vaginal opening in immature rats and initiated regular oestrous cycles. Such treatment completely prevented high LH levels at day 15 but serum FSH levels remained high. Prolactin and progesterone levels were higher at day 25 than at day 15 in control rats and at that time LH and FSH levels were low. Prolactin treatment had a depressant effect on endogenous prolactin and progesterone levels. It is proposed that after day 20 the positive feedback threshold of oestradiol on LH release is at a mature, highly sensitive level. The steady increment in serum prolactin and/or progesterone levels between day 20 and puberty, however, inhibited phasic LH release thus preventing early puberty. The possible mechanisms of action of prolactin are discussed.


Inhibition of human chorionic gonadotrophin-induced hypertrophy of the ovaries and uterus in immature mice by some pineal indoles, 6-hydroxymelatonin and arginine vasotocin. Administration of the indoles 5-methoxytryptophol, 6-hydroxymelatonin, melatonin, N-acetylserotonin or 5-methoxyindole-3-acetic acid (six 100-mug injections given at intervals of 12 h) inhibited both the absolute and relative ovarian and uterine weights of immature female mice pre-treated with HCG. Administration of 5-hydroxytryptophol or 5-methoxytryptamine at the same dosage inhibited ovarian but not uterine hypertrophy in mice pre-treated with HCG.


Role of maternal and foetal adrenal activity in the development of the rat mammary gland near term. The essential role of adrenal corticosteroids in the initiation of mammary gland secretory activity near term in the rat has been investigated. Mammary gland DNA and RNA were estimated as a quantitative indication of the extent of mammary gland development. Maternal adrenalectomy or hypophysectomy resulted in a decrease in the mammary gland RNA:DNA ratio. Treatment of adrenalectomized animals with cortisol acetate completely restored the RNA:DNA ratio to the control level. Treatment of hypophysectomized animals with cortisol acetate resulted in an increase in the RNA:DNA ratio but did not restore the ratio to the control level. Removal of the pups had no effect on the RNA:DNA ratio. The findings are discussed in relation to the foetal and maternal contribution to the level of glucocorticoids in the circulation and with respect to changes in mammary gland sensitivity to lactogenic stimulation near term.


Tamoxifen as an anti-tumour agent: oestrogen binding as a predictive test for tumour response. Rats with growing 7,12-dimethylbenz(alpha)anthracene (DMBA)-induced rat mammary carcinomata were biopsied and oestrogen-binding capacity was measured using a Sephadex LH-20 chromatography method. Tumours were measured with calipers and animals were treated for 3 weeks with tamoxifen (50 mug/day, S.C.). Tumour response was determined by the size (cm2) before and after therapy. An increase in tumour regression (ten tumours) was seen with increasing oestrogen-binding sites determined by Scatchard analysis (P less than 0.01). Thirty tumours were used to determine oestrogen binding with a single dose of [3H]-oestradiol. The percentage tumour regression was linearly correlated with oestrogen-binding capacity (P less than 0.01), although some tumours with high oestrogen-binding capacities only partially regressed in response to tamoxifen therapy. The time of the oestrous cycle when biopsy occurred was not a critical factor in determining oestrogen binding for prediction of response. Oestrogen binding was reduced during tamoxifen therapy.


Effect of luteinizing hormone releasing hormone on plasma levels of luteinizing hormone, oestradiol and testosterone in the male dog. An injection of luteinizing hormone releasing hormone (LH-RH) increased plasma LH and testosterone concentrations in the male dog, but no significant increase in plasma oestradiol-17beta levels was observed. Repeated injections of LH-RH produced an increase in plasma LH levels but there was a progressive decline in the response with each injection. The concentration of plasma testosterone reached a maximum within 40 min of the first injection of LH-RH and remained constant thereafter while plasma oestradiol concentration gradually increased with successive injections of LH-RH.


The effects of antigen on the migration of recirculating lymphocytes through single lymph nodes. The increased input of RL into LN starts within 3 h after antigenic stimulation and is due to an increase in the number of RL passing through a LN. It is not associated with an altered transit time through a LN of the majority of RL and it cannot occur in the absence of high endothelial PCV. The immediate decrease in the output of RL from an antigen-stimulated LN is due to a delay in the exit from that LN of RL which had entered the LN before antigen was given. The decrease in cell output from a LN after antigen administration affects blast cells produced within the LN as well as small lymphocytes and is not specific for RL. There are at least two distinct mechanisms controlling the migration of RL through an antigen-stimulated LN. The first controls the increased input of RL which occurs only through high endothelial PCV. The second controls the immediate decrease in cell output, which although it does not occur at the level of the high endothelial PCV, can only occur in organized lymphoid tissue. The increased input of RL into an antigen-stimulated LN and the decreased cell output from the LN can occur independently and are possibly controlled by different mechanisms.


Inclusion group systems and cis-trans effects in responses controlled by the two complementing Ir-GLphi genes. The immune responses to the random linear terpolymers of L-amino acids, poly-(glu57, lys38, tyr5), poly-(glu51, lys34, tyr15,), and poly-(glu53, lys36, phe11) are each controlled by two dominant H-linked Ir genes. The immune responses to these three related terpolymers demonstrate different H-2 distributions, however, the H-2 patterns are part of a single inclusion group system. The alpha- and beta-genes are dominant; however, most effective gene interactions occur when the two genes are in the cis configuration. The potential significance of this cis-trans effect is discussed.


Differences between pig tissues in the expression of major transplantation antigens: possible relevance for organ allografts. Evidence is presented that there are differences between pig liver and kidney parenchymal cells in the expression of major histocompatibility complex antigens. Analogues of I-region antigens of the mouse are detectable on kidney cells and some peripheral blood leukocytes (PBL) but not on liver, platelets, and other PBL. Such differences between liver and kidney may explain (a) the fate of these organs as allografts and (b) the donor-specific protection of kidney by liver. Other antigens peculiar to skin and kidney have been detected but require further characterization.


Effects on adult newt limb regeneration of partial and complete skin flaps over the amputation surface. The influence of the wound epithelium on the cellular events preceding blastema formation was examined by comparing dedifferentiation, DNA labeling indices, and mitotic indices of the distal mesodermal tissues in control regenerating newt forelimbs and in amputated forelimbs covered with a flap of full thickness skin. Three kinds of results were seen following the skin-flap graft operations. Epidermal migration across the amputation surface was completely inhibited in 22% (8) of the cases and these limbs repaired the amputation wound but did not form regeneration blastemas. In 11% (4) of the experimental limbs, essentially normal wound epithelia displaced the skin flaps and the limb stumps formed blastemas and regenerated. The majority of the skin grafts (67%) exhibited epidermal migration restricted to the free edges of the flaps. These limbs formed eccentric blastemas on the ventral side of the limb next to the dermis-free epidermis and regenerated laterally in that direction.


Evidence for the role of a trypsin-like enzyme in the hamster sperm acrosome reaction. Acrosome reactions occurring in vitro in hamster sperm capacitated by bovine follicular fluid were severly inhibited by four synthetic trypsin inhibitors and by Zn2+. Three polypeptide trypsin inhibitors and a synthetic chymotrypsin inhibitor did not inhibit the acrosome reaction, and Ca2+ overcame the inhibition by Zn2+. These results suggest that a trypsin-like enzyme (possibly acrosin) plays a role in the acrosome reaction.


Spontaneous maturation of oocytes isolated from ovaries of immature hypophysectomized rats. Oocytes were collected from preantral follicles (200-300 mu in diameter) from 30-day-old immature rats 7 days after hypophysectomy. The ova were cultured in vitro for 17 hrs in a chemically defined medium and scored cytologically for meiotic maturation. Of 534 oocytes that were cultured 89% resumed meiosis; however, 98% of these oocytes arrested in either metaphase or anaphase I. In contrast, 82% of the oocytes isolated from preovulatory follicles (approximately 600 mu in diameter) of adult proestrus rats progressed to metaphase II. These results are discussed in terms of functional FSH and LH receptors on the granulosa cells.


Neurohemal organ extracts effect the ventilation oscillator in crustaceans. Extracts of the pericardial organs of crabs injected into intact animals cause an increase in the frequency of scaphognathite beating. The active factor is not dopamine of 5-hydroxytryptamine, which are present in pericardial organs; it is probably a peptide. The factor may represent a hormonal mechanism of coordinating heart rate and ventilation.


Changes in volume of the rhabdom in the compound eye of Aedes aegypti L. The volume of the rhabdom in compound eyes of mosquitoes decreases upon illumination. This decrease is probably mediated by a bleaching of the visual pigment, since blue light is most effective in producing the change and red light is least effective. The reduction in rhabdom volume appears to be a result of rhabdomal membrane loss to coated vesicles and multivesicular bodies. These organelles were seen most frequently in blue adapted eyes, markedly less frequently in red adapted eyes, and only rarely in dark adapted eyes.


Biosynthesis of lipovitellin by the crustacean ovary. II. Characterization of and in vitro incorporation of amino acids into the purified subunits. The amino acid composition has been determined for two of the five subunits of the crayfish lipovitellin. In addition the molecular weight of each subunit has also been ascertained. Most importantly, it is demonstrated that following in vitro incubation radioactive amino acids are incorporated into each of three purified subunits. These data strongly support the suggestion that the vitellogenic ovary of the crayfish is capable of in situ synthesis of lipovitellin.


Cleavage initiation activities of microtubules and in vitro reassembled tubulins of sperm flagella. Cleavage of fish (Oryzias latipes) eggs was induced by injection of heterologous (sea urchin Hemicentrotus pulcherrimus and oyster Crassostrea gigas) sperm microtubules. Cleavage initiating CI activity of microtubules was higher in 3% PVP suspension than in 6% BSA, and not affected significantly by the concentration of microtubules themselves. The CI activity of microtubules suspended in 3% PVP was comparatively stable in the frozen state. Heat-treatment at more than 55 degrees C resulted in the loss of most or all of their CI activity. Such activity was observed in side-by-side aggregates of tubulin linear polymers of sea urchin (Hemicentrotus pulcherrimus) spermatozoa but not in dispersed linear polymers or tubulin dimers. Microtubules with CI activity seem to participate in initiating cleavage as astral centers, or a "seed" for polymerization of ooplasmic tubulins in activated eggs.


Time-course of potential spread along a skeletal muscle fiber under voltage clamp. The equations describing the time-course of potential spread into a terminated segment of muscle fiber are given for the condition that a step of voltage is applied at x - 2l. Measurements of V(2l) - V(l) were made at 16.7-19.5 degrees C, using a three-microelectrode voltage clamp, to compare with the theory. Best least squares fits of calculated curves to data obtained in Ringer's solution (5 mM K) gave GL = 10 mumho/cm and Cm' = 1.6 muF/cm2. Similar measurements in 100 mM K solution, with the inward rectifier shut off by a positive prepulse, gave GL = 20 mumho/cm and Cm' = 2.0 muF/cm2. The time-course of V(2l) - V(l), measured when the inward rectifier was fully activated by a negative prepulse, was in good agreement with the curve calculated assuming no change in GL and Cm' and that the only effect of the negative prepulse was to increase the conductance of surface and tubular membranes.


Sensitivity of the sodium and potassium channels of Myxicola giant axons to changes in external pH. Myxicola giant axons were studied using standard voltage-clamp techniques in solutions whose pH values ranged from 3.9 to 10.2. Buffer concentrations of 50 mM or greater were necessary to demonstrate the full effect of pH. In acidic solutions the axon underwent a variable depolarization, and both the sodium and potassium conductances were reversibly depressed with approximate pKa's of 4.8 and 4.4, respectively. The voltage dependence of GNa was only slightly altered by acidic conditions, whereas there occurred large shifts in GK along the voltage axis consistent with a substantial decrease in net negative surface charge in the vicinity of the K+ channels. The sodium and potassium activation rate constants were decreased by acidic conditions, but the results could not be described as a simple translation along the voltage axis.


Signal transformation with pairing of sensory stimuli. Rotation of the isolated nervous system of Hermissenda in a caudal orientation causes a synaptic hyperpolarization accompanied by elimination of impulse activity during the steady-state phase of type A but not type B photoreceptors' responses to light. Rotation of the isolated nervous system in a cephalic orientation causes a synaptic depolarization with increase of impulse activity during the steady-state phase of both type A and type B photoreceptors' responses to light. These effects of rotation on photorecptors are explained by known synaptic interactions. Sufficient redundancy is found to be provided by the neural organization of the visual system and its interaction with the statocyst to preserve much of the visual information in spite of signal transformation in specific photorecptors resulting from pairing of rotation with light.