Acid mucopolysaccharides in mammary tumors of dogs. The predominant acid mucopolysaccharides found in selected epithelial mammary tumors of dogs stained with alcian blue and were labile to hyaluronidase digestion. These histochemical characteristics identified them as hyaluronic acid, chondroitin-4- and chondroitin-6-sulfate. The intensity of the staining of these acid mucopolysaccharides varied in a transitionary process from a precartilaginous to a pseudocartilaginous intercellular matrix to mature hyaline cartilage. The tumor acid mucopolysaccharides were indistinguishable from those associated with formation of cartilage in developing mammals; such cartilage is reported to be produced only by cells of mesodermal origin. There was no evidence to suggest transitional changes in myoepithelial cells, neoplastic epithelial cells or their components that could contribute to the formation of the acid mucopolysaccharides. It was concluded that the heterotopic tissues (cartilage, bone and fibrous connective tissue) in the epithelial mammary tumors were derived from cells of mesodermal origin and formed the adjacent stroma in areas of neoplasia.


A comparison of the antibacterial properties of some udder creams and ointments. The antibacterial activity of three udder creams and two ointments was assayed using three different methods. Seven different bacterial genera were used as test organisms providing a total of 17 strains. Only a single strain of corynebacterium was inhibited by all the preparations. Certain strains of staphylococci and streptococci were inhibited by four of the preparations. The gram-negative organisms showed greatest resistance to the antibacterial agents tested. The in vitro assays showed only one of the five preparations to have even slight bactericidal activity. These preparations when tested on the teats of dry and lactating cows showed similar results to the in vitro experiments.


Epithelioid cell granulomata in the mucosa of the small intestine in Whipple's disease. This report is the first description of sarcoid-like epithelioid cell granulomata in the mucosa of the small intestine in a case of Whipple's disease. The epithelioid cells do not contain PAS-positive material or products of bacterial degradation. Their ultrastructural features characterize them as histiocytes which have become secretory rather than phagocytic. These sarcoid-like lesions are considered to be a morphological manifestation of an immunological process in Whipple's disease and not to represent evidence of sarcoidosis as a concomitant or associated disorder.


[Immunochemical study of the alpha-globulins in human kidney tumor tissue]. Four individual tissue proteins were studied both in normal and tumoral tissue of kidney (hypernephroma, Wilms tumor) using immunochemical analysis. Occurrence of an antigen, which was absent in definitive tissue of homologous origin, as well as a decrease in biosynthesis of some homologous antigens were found in malignant tissue.


Differentiation of herpes simplex virus type 1 and type 2 by immunofluorescence: discriminative staining by labelled IgG preparations. While evaluating herpes simplex virus (HSV) typing by indirect immunofluorescence staining, an undesired specific staining pattern turned out to be a reliable marker for herpes simplex type 1. Cells infected with herpes simplex type 1 displayed clear staining with a FITC-conjugated antiglobulin preparation, also in the absence of herpes simplex-specific antibodies. Using the same conjugate, herpes simplex type-2-infected cells exhibited no fluorescence. The particular type of staining observed was influenced by neither the anatomical site of origin of the virus isolate nor the cell type used for virus preparation. Herpes simplex type 1-specific fluorescence was only obtained with the use of FITC-conjugates possessing anti-IgG activity. Both reliability and specificity of this discriminating procedure as a diagnostic tool has been established by typing 282 virus isolates over a period of 4 years.


[Uniform method for determining the alpha 1-antitrypsin and alpha 2-macroglobulin activity in human blood serum (plasma)]. A modified spectrophotometric method is developed for simultaneous estimation of alpha 1-antitrypsin and alpha 2-macroglobulin in human blood serum (plasma); the method is based on dissimilar interaction of these inhibitors with trypsin in the systems with a low molecular substrate N-alpha-benzoyl-l-arginine ethyl ester. alpha 1-Antitrypsin was estimated by inhibition of the arginine esterase activity of trypsin in a mixture containing human blood serum diluted 50-fold. alpha 2-Macroglobulin was estimated by maintained arginine esterase activity of the trypsin-alpha 2-macroglobulin complex, formed after interaction of an excess of trypsin with blood serum, diluted 10-fold and after subsequent inactivation of free, unbound with alpha 2-macroglobulin, trypsin by treatment with the soy bean inhibitor of trypsin. alpha 1-Antitrypsin and alpha 2-macrog-obulin were estimated by means of the method described in blood serum of healthy persons and in patients with burns or with carcinoma of pancreas. The method enables to estimate two main inhibitors of blood plasma proteinases in a small volume of blood serum (0.1 ml) very rapidly and specifically using commercially available substrate; the method might be recommended for routine clinical analysis.


Strain-specific antigens in Rhizobium leguminosarum. Fifty strains of Rhizobium leguminosarum, isolated from five species of host plant (Pisum sativum, P. arvense, Vicia sativa, V. faba, and a Lathyrus sp.) were examined for the presence of strain-specific somatic antigens by immune-diffusions against 13 antisera. Thirty eight strains (76 per cent) were found to belong to the same sero-group and were serologically indistinguishable from each other, but four of these strains also exhibited non-reciprocal cross reactivity with other antisera. In contrast to this, five Australian strains, isolated from P. sativum, showed a high degree of strain specificity.


[In vitro study of the sensitizing activity of an antirabies immunoglobulin and its fractions]. The dynamics of the blood in guinea pigs sensitized with heterogenous antirabic immunoglobulin was studied. The damage of neutrophils in the blood was found to be an index which allowed to detect changed sensitivity to heterogenous protein already at an early period (on day 6) after immunization, whereas the indirect mast cell degranulation test detected it only at a later period (on day 21). The above methods revealed that beta 1 and beta 2 globulin fractions possessed high sensitizing activity.


[Submicroscopic organization of M. pneumoniae studied by means of negative contrasting, ultrathin sections and scanning electron microscopy]. The ultrastructure of M. pneumoniae, grown on a solid culture medium and in a liquid one, was studied by a number of methods. Two types of cells were shown to prevail in the culture: spherical cells (0.5--1 micrometer) forming chains of different configurations and filamentous cells (5 micrometer long and greater) with spherical enlargements along their whole length. The absence of microcapsules made M. pneumoniae different from other species of mycoplasms, and the organism proliferated by division into 2 daughter cells, equal or unequal in size, by the segmentation of the cytoplasm and the formation of elementary bodies inside the cell and on its surface.


Effect of some insecticides on the periodic activity of the fresh-water mussel (Anodonta cygnea L.). The effect of the insecticides Hungaria L-7, Dimecron-50, Thimet-10 G and Satox 20 WSC containing lindane, phosphamidon, phorate and trichlorfon, respectively, as active ingredient was investigated on the survival and pumping behaviour of the fresh-water mussel Anodonta cygnea L. The shortening of active periods was used as indicator of the sublethal effects. Hungaria L-7, Thimet-10 G and Satox 20 WSC caused significant inhibition of the filtering activity, while Dimecron-50 was less effective. Fifty per cent shortening of active periods required 6 X 10-3 g/l Hungaria L-7, 8 X 10-3 g/l Thimet-10 G and 2 X 10-2 ml/l Satox 20 WSC. Thimet and Satox were lethal at 10-1 g/l and 1 m/l concentrations, respectively. Although the mechanism of the effect can be different with various substances, the results have shown, that monitoring of the activity of mussels can be a suitable test for the biological indications of water pollution.


Purification of RNA-directed DNA polymerase from mouse spleen infected with Rauscher leukemia virus. RNA-directed DNA polymerase was purified from spleens of Balb/c and NMRI mice infected with Rauscher murine leukemia virus. The method includes cell fractionation and lysis of microsomal fraction, chromtography on Sephadex G-200 and phosphocellulose. Estimation of molecular weight from the sedimentation rate of the purified enzyme in a glycerol gradient was consistent with a structure containing one polypeptide with a molecular weight of 70,000. Purified RLV DNA polymerase from spleen could transcribe purified DNA polymerase from purified virions. This simple preparation method offers a procedure for large scale preparation of the RNA-directed DNA polymerase which can be used for synthesis of DNA complementary to mRNA.


[Antigenic composition of adenovirus hexons]. Quantitative relations between the group-specific and the type-specific components of the hexons of adenovirus type 2 and 5 were studied by means of FITC-conjugated Fab-fragments of antibodies directed against type 2 and type 5 hexons. From the sedimentation constant of the complexes of hexons and Fab in the region of excess of Fab we conclude that there are at least 20 determinants on the hexon. Half of these are type-specific and the others are group-specific. Both components of the type 2 hexon consist of equal parts of carbodiimide sensitive and carbodiimide resistent determinants.


A technique for demonstrating of dehydrogenase activities in Epon-embedded, semithin sections of the nervous tissue. Experiments were performed aiming at establishing optimal conditions for demonstrating the activity of dehydrogenases in Epon-embedded, semithin sections. Small fragments of the nervous tissue were incubated 1, 4, 10 and 24 h 37, 20 and 4 degrees C in the presence of NBT or TC NBT. The incubated fragments were embedded in Epon and semithin sections prepared thereof. The activities of succinate dehydrogenase and of alpha-glycerol dehydrogenase was evaluated. The experiments have shown that semithin, Epon-embedded sections may be used for demonstration and cytological localization of respiratory enzymes. Various experimental conditions were tried. Optimal results obtained when unfixed tissue fragments were incubated for 24 h at 4 degrees C.


Effect of mast cell chymase of rat skin on intercellular matrix: a histochemical study. The effects of purified mast cell chymase of rat skin on intercellular matrix were studied histochemically using cartilage as a model. The enzyme effectively releases proteoglycans from neonatal human costal cartilage. An analysis of the primary site of proteolytic attack by chymase in the matrix was attempted. The results suggest that components containing the more acidic radicals (e.g. sulphates) are hydrolyzed first. After a prolonged attack, the matrix loses most of its staining with Toluidine Blue and Alcian Blue, and staining of collagen is increased. The hydrolyzed regions of cartilage in Alcian Blue-stained sections exhibited a purple interference colour in polarized light. A similar interference colour appeared after incubating sections of rat skin with chymase. The mast cell chymase presumably promotes diffusion of plasma to the site of injury by the direct breakdown of connective tissue ground substance.


Histochemical and morphological evaluation of cerebral cortex of newborn rat in the course of joint ethanol and pyrazole administration. The object of the study was the cerebral cortex of newborn rat. Tissue material was collected from the 2-nd to the 8-th hour after birth both from control animals and newborns whose mothers had been given both ethanol and pyrazole throughout gestation period, and also either ethanol alone or pyrazole. The drug were administered by gastric tube, in doses: ethanol 8.0 g/kg body weight, pyrazole 36 mg/kg body weight. Histochemical studies revealed variation in the intensity of reaction of the respiratory enzymes during examination of the cortex of newborns whose mothers had been fed with ethanol alone, or pyrazole alone. A strong inhibition of enzymatic reaction was observed in the examined cerebral cortex of newborns after a joint ethanol and pyrazole administration. Morphological studies showed an inhibition of maturing process of the cerebral cortex cells of animals treated with ethanol alone, whereas the cerebral cortex of animals whose mothers had been given both ethanol and pyrazole, presented symptoms allowing to diagnose encephalitis congenita symptomatica.


Timolol transitory manifestations of dry eyes in long term treatment. In 64 patients treated with timolol eye drops (0.25% and 0.50%) seven patients developed transitory sensation of dry eyes. Two of these subjects also had xerostomia. Conjunctival and corneal defects were disclosed simultaneously with rose bengal staining. Morphologically, some of these lesions might have the same appearance as of the early stages of kerato-conjunctivitis sicca. A reduction of Schirmer test and break-up time was noted. The duration of symptoms ranged from 3 to 13 days. The mean time of treatment at the début of symptoms was 30 weeks (range 23--43 weeks). A pathogenesis of these seemingly harmless findings is at the present obscure. In none of our patients was the treatment discontinued and the symptoms did not reappear.


Fibrinolytic factors in aqueous humour and serum from patients with Fuchs' dystrophy and patients with cataract. The concentration of alpha 2-macroglobulin, alpha 1-antitrypsin, plasminogen, C3-complement, fibrinogen degradation products (FDP) and fibrinolytic activity, were studied in the aqueous humour and serum from nine patients with Fuchs' endothelial dystrophy, 17 patients with uncomplicated senile cataract and in the secondary aqueous from six cataract patients. Finally, the aqueous humour and serum from two patients anterior uveitis were studied. An increased concentration of alpha 2-macroglobulin, alpha 1-antitrypsin, plasminogen and C3-complement was found in both the aqueous and the serum from patients with Fuchs' dystrophy when compared with the primary aqueous and serum from patients with cataract but this was only significant for alpha 1-antitrypsin in aqueous humour. A significant increase in the amount of FDP was found in the serum of the Fuchs' patients compared with the cataract patients. Fibrinolytic activity could not be demonstrated in the serum in any of the patient groups. The concentrations of the various factors found in the secondary aqueous of the cataract patients differed only slightly from the content of the primary aqueous of the Fuchs' patients.


Comparative study of the influence of aminoglycoside antibiotics on the activity of the horizontal semicircular canal in the frog. This work is an electrophysiological study made in the frog. The technique allows one to test and to compare the actions of a number of aminoglycoside antibiotics, directly introduced into the labyrinthic cavity, on the spontaneous activity of a vestibular receptor--the horizontal semicircular canal. The effects of aminoglycoside solutions have been compared with those of physiological solutions (NaCl 7 g/l, Ringer) and of penicillin (not ototoxic). The results obtained show: (1) After the introduction of a physiological solution the activity disappears only very briefly (electrical artefact, probably); after a few minutes the activity returns to its initial value. A similar phenomenon is obtained with penicillin. (2) When used at a dose of 10 microgram, all the aminoglycosides studied generally induced an important and lasting decrease in semicircular canal activity. (3) These aminoglycosides have been classified according to their vestibular local toxicity. Their descending order of influence is as follows: streptomycin, dihydrostreptomycin, amikacin (BBK 8), neomycin, sisomycin, gentamicin and lividomycin, tobramycin, kanendomycin. (4) A parallel can be drawn between local vestibular toxicity and clinical ototoxicity. The role and importance of the hemolabyrinthic barrier are noted and the notion of ototoxicity is discussed.


Tumorigenicity and karyotype of rat embryo cell lines transformed by BK virus. A rat embryo cell line transformed by BK virus was used to induce tumors in rats. Cell lines were established from these tumors. Other sublines were obtained by in vitro cloning of the parental line. Growth characteristics and karyotypes were compared to the tumorigenicity of these cell lines. The in vitro cloned sublines had a low tumorigenicity. Tumorigenicity of the tumor cell lines varied from high to undetectable. The tumor cell line with the highest tumorigenicity also had the highest saturation density in vitro, but otherwise there was little correlation between tumorigenicity and the in vitro characteristics of the cells. Karyotype analysis was done for two cell lines with high or low tumorigenicity which both had a near-diploid complement of chromosomes. The findings were in agreement with the expression-supression model of Rabinowitz and Sachs (1970). The suppression chromosomes seemed to be confined in group A, the expression chromosomes in group B.


Histological diagnosis of myocardial injury. Comparison of hematoxylin-basic fuchsin-picric acid (HBFP)-stained sections obtained during autopsy with isolated viable rat cardiac myocytes exposed to anoxia. The light microscopic appearances in hematoxylin-basic fuchsin-picric acid (HBFP)-stained histological sections from cardiac and skeletal muscle tissue were put in relation to the reactions of isolated viable rat cardiac myocytes exposed to anoxia in suspension and their morphology in paraffin-embedded sections. Special attention was paid to prenecrotic phases of myocytic injury which were followed, in viable rat cardiac myocytes, by light microscopy, and confirmed with biochemical assays indicating increased plasma membrane permeability. In cases of sudden death, and traumatic injury to the heart and skeletal muscle, there was good agreement between alterations demonstrated with the HBFP technique and alterations of viable rat cardiac myocytes exposed to anoxia. In isolated myocytes, these alterations were associated with irregular contractility which, when occurring in situ, might have influenced the cardiac function prior to death. Moreover, the changes develop at a much faster rate than the inflammatory reaction following tissue injury and may therefore be regarded as an early vital phenomenon of significance in clinico-pathological and medico-legal considerations.


Immunochemical analysis of the teichoic acid from Staphylococcus hyicus. The wall teichoic acid of Staphylococcus hyicus has been isolated and characterized. The teichoic acid is a glycerol phosphate polymer with glycosidically linked N-acetylglucosamine. Interaction with concanavalin A and susceptibility to alpha- but not to beta-N-acetylglucosaminidase showed that the sugar is in the alpha-configuration.


Characterization of fractions containing the thermostable agglutinogens of Neisseria gonorrhoeae. Extract was prepared by heat treatment (100 degrees C. 2 h) of bacteria of N. gonorrhoeae strain 8551. The acid precipitate (pH 2.5) of the extract was resolved by gel chromatography (Sepharose CL-6B) into material eluted with the void volume (Fr. I) and material retarded by the column (Fr. II). Fr. I contained at least three precipitinogens, the antigenic determinant a of LPS and the protein determinant b. The antigens demonstrable in Fr. I were also detected in an outer membrane preparation obtained by extraction of the bacteria with a lithium acetate buffer. Fr. II contained LPS and several other precipitinogens. The antigens of Fr. I included the heat stable agglutinogens of the gonococcus, and antibodies of these could be raised by immunization of rabbits with the Fr. I material.


Microvascular permeability to macromolecules in thermal injury. Thermal injury of the skin causes an inflammatory reaction with formation of edema, mainly due to vasodilatation, increased extravascular osmotic activity and increased microvascular permeability to macromolecules. These reactions are due to local and systemic effects . At the site of injury heat itself causes microvascular damage. Vascular changes may also be mediated by vasoactive compounds, e.g. liberation of histamine and increased synthesis of prostaglandins. Reflex mechanisms elicited via the nervous system must also be considered. Morphological studies revealed increased number of vacuoles, a large number of open endothelial intercellular junctions and changes of the interstitial tissue at the site of thermal injury. The vascular reactions described can be modified by drugs, especially indomethacin which inhibits the synthesis of prostaglandins and drugs which competitively antagonize the effects of histamine at H2-receptors.


Identification of the small pore in muscle capillaries. Electron microscope studies have been carried out in order to identify the structural counterpart of the small pore of muscle capillaries. Experiments with horseradish peroxidase (40,000 MW) and microperoxidase (1,900 MW) indicate that the clefts between capillary endothelial cells, rather than endothelial vesicles or transendothelial channels, are the counterpart of the pore. The structural properties of the clefts that enable them to serve as the small pore are presumably related to the nature of the attachment between plasma membranes of neighboring endothelial cells.


Capillary permeability to interstitial microinjections of macromolecules and influence of capillary hydrostatic pressure on endothelial ultrastructure. The transcapillary flux of horseradish peroxidase (HRP, mol. diam. 50--60 A) and ferritin (mol. diam. 110--120 A), microinjected interstitially into the biceps femoris muscle of rats, was analyzed in the electron microscope. From 1 min postinjection HRP was observed to occupy endothelial plasmalemmal vesicles in all positions to be expected if a vesicular transendothelial transport of material occurs in the interstitium-to-lumen direction. Permeation of HRP through the intercellular clefts of the endothelium could not be demonstrated unequivocally. The periendothelial basement membrane appeared to constitute a significant permeability barrier against ferritin. The endothelial ultrastructure in muscle capillaries was analysed morphometrically after artificial perfusions of rat hindquarters at venous outflow pressures of -4 cm H2O and +10 cm H2O. In the "high pressure" material the endothelium was thinner and the frequency of vesicles open towards the interstitium was lower than after perfusion at low outflow pressure. The findings may indicate that net transendothelial transport via vesicles varies with hemodynamics.


Ultrastructure of rat renal tubular basement membrane--meshwork structure demonstration by negative staining. The tubular basement membrane (TBM) (i.e. tubular basal lamina) of rat kidney was shown to be a fine meshwork by electron microscopy after negative staining. Strands of the meshwork formed a regular three dimensional lattice work. The pores of the meshwork were polygonal. There were two main pore sizes: one approximately 30 A in diameter, the other 42--60 A. In view of our previous observation that glomerular and alveolar basement membranes were made up fine meshwork, it is quite possible that the basement membranes of other organs are also made up such fine meshwork.


Observations concerning the staining properties of the macroconidia of certain dermatophyton species. Staining of the macroconidia of several Dermatophyton species by lactophenol-cotton blue was investigated. Young macroconidia stain variably; their cytoplasm may appear homogeneous or inhomogeneous. The possible explanation seems to be variation in the composition of the cytoplasm. In some macroconidia dark-staining filament was seen along the longitudinal axis. Mature macroconidia showed basal or basal+apical homogeneous deep staining. The intensive apical staining suggests that the apical structure differs from the structure of the side wall.


Liberation of histamine by compound 48/80, tolazoline, betahistine and burimamide from isolated spontaneously beating guinea pig and rabbit atrial pairs. Tolazoline, betahistine, burimamide and compound 48/80 release histamine from isolated guinea pig atria resulting in histamine concentrations that stimulate H2-receptors. Tolazoline, burimamide and 48/80 also release histamine from rabbit atria but do not result in histamine concentrations that will stimulate either H1- or H2-receptors. However, betahistine (which does not release histamine from rabbit atria) and tolazoline stimulate the rabbit atrial chronotropic response by releasing catecholamines.


Histamine-releasing effect of a corticotrophin derivative. II. Mechanism of action of histamine release by C 44 680-Ba, compared with that of Cpd. 48/80, dextran and triton. The mechanism of the histamine-liberating action of the synthetic polypeptide C 44 680-Ba, an alkyl-prolyl derivative of beta 1-19 corticotrophin, was investigated and compared with those of Compound 48/80, dextran, Melittin and Triton X-100. It was found that the release of histamine from rat peritoneal cells induced by the polypeptide is dependent on temperature, pH, calcium ions and energy-providing processes. In regard to these criteria, the mode of action of this histamine liberator resembles that of Compound 48/80 but is quite distinct from that of the unspecific substance Triton X-100.


[Situation and trends of hyposensitisation treatment (author's transl)]. Since the introduction of the hyposensitization treatment by Noon and Freeman in 1911, an essential change of the hyposensibilisation technique, despite numerous recently published results, has not been able to be demonstrated up to now. In most cases, treatment is still done with and allergen mixture consisting of a variety of unknown compounds with changing composition according to own experiences concerning initial and final dosis of the allergen or the application intervals. The linkage of the allergens to chemical substances, the modification into so-called allergoids as well as the investigation of the hyposensitisation success, such as determination of blocking antibodies, the course of the titre of specific antibodies by the RAST technique, the histamin liberation from leucocytes of allergic persons after antigen contact and the lymphocyte stimulation by allergens are starting points for developing a scientific base to the hyposensitisation treatment and for introducing new results mainly from experiments on animals into the clinical practice. Standardisation of allergens, optimisation of the dosis and the application intervals as well as the further decrease of the allergenicity and the optimal choice of the adjuvants have to be regarded as the problems to be solved in the next future. On the other hand, the use of allergen determinants linked to non-immune carriers or the induction of a tolerance by another way seem to be not yet a subject with clinical relevance during the next years.


A route for direct retinal input to the preoptic hypothalamus: dendritic projections into the optic chiasm. With the use of Golgi, horseradish peroxidase, and electron microscopic techniques, neurons within a broad region of the preoptic hypothalamus of the mouse were shown to have dendrites that projected well into the depths of the optic chiasm. Further experimental and ultrastructural investigation demonstrated synapses between these dendrites and retinal axonal boutons within the chiasm. All synapses located in the chiasm were classified as Gray's type I. The possible function of these dendritic projections is discussed.


alpha 2-Macroglobulin in vitamin A-deficient children. Serum samples were obtained from 43 children 14 years old or younger in Malaysia and Guatemala. The levels of the serum glycoprotein alpha 2-macroglobulin (alpha 2-M) were assayed by two methods: the trypsin-binding assay of Ganrot (Clin. Chim. Acta 14:493, 1960) and a radial immunodiffusion assay against alpha 2-M antiserum. The two methods gave the same results. When serum alpha 2-M levels were plotted against serum vitamin A concentrations, they were significantly correlated (r = 0.505, P less than 0.001); children with serum vitamin A levels greater than 40 micrograms/100 ml had alpha 2-M levels of 3.71 +/- 0.79 mg/ml (mean +/- SD, n = 13), while those with level less than 40 micrograms/100 ml had alpha 2-M levels of 2.78 +/- 0.51 mg/ml (n = 30); the difference was significant (P less than 0.001). Normal, apparently healthy children had alpha 2-M levels of 3.90 +/- 0.39 mg/ml. Most of the children sampled suffered from a variety of infections; of these, measles appeared to counteract the effect of vitamin A deficiency by elevating alpha 2-M levels. Vitamin A-deficient children with measles had alpha 2-M levels not significantly lower than those of normal children. The difference between deficient and normal values of alpha 2-M was still significant (P less than 0.05) when expressed per milligram of serum protein, showing that the effect was not caused by lowered serum protein concentrations associated with protein-calorie malnutrition, from which most of the deficiency children suffered.


Comparative study of ascorbic acid and disodium cromoglycate in some models of experimental anaphylaxis. The pharmacological activity of vitamin C and the antiallergic drug disodium cromoglycate are compared in models of rat mast cell degranulation and histamine release, and in protracted anaphylactic shock in the guinea pig. Our results demonstrate that ascorbic acid has a very weak inhibitory effect on degranulation and histamine release from mast cells, and therefore cannot be considered a prophylactic antiallergic drug such as disodium cromoglycate.


Immunologic localization of elastin by electron microscopy. Research on the pathogenesis of experimental emphysema has involved studies of the distribution of and destruction of elastin in the alveolar interstitium. The ill-defined organization of elastin in the alveolar interstitium makes it difficult to identify the elastin specifically by staining procedures ordinarily used for electron microscopy. This problem becomes more significant when the elastic tissue is fragmented during emphysema development and localization of the elastin fragments is essential. Therefore, a specific technique using high-titer antibodies against purified canine lung elastin was developed. The primary antibody was used on preembedded or etched postembedded sections. Localization of the antielastin IgG was accomplished with ferritin-labeled rabbit antisheep IgG as the secondary antibody. Treatment with the preimmune serum gave negligible ferritin background staining. The antielastin antibody did not react with lung connective tissue proteins such as the microfibrillar component of elastin or collagen or proteoglycan. The antielastin antibody appeared to be species specific. The method may be useful for studies of experimental emphysema.


Bleomycin-induced injury and metaplasia of alveolar type 2 cells. Relationship of cellular responses to drug presence in the lung. Metaplastic epithelial cells are often observed lining alveoli in bleomycin-induced pulmonary fibrosis. The hypothesis that these cellular changes are induced by the direct action of the drug on differentiating Type 2 cells is now examined in a sequential study to correlate the presence of 3H bleomycin in the lung with the pattern of injury and repair of the alveolar epithelium. A single intravenous dose or multiple small intraperitoneal doses induce focal necrosis of Type 1 epithelial cells followed by Type 2 cell regeneration. At the time of maximal deoxyribonucleic acid (DNA) synthesis in these cells, significant amounts of 3H bleomycin are demonstrable in the lung by scintillation counting; and in autoradiographs, the drug appears to concentrate in epithelial cells. Subsequently many abnormal Type 2 cells are seen. Some are binucleate, and others show nuclear disruption. The usual process of differentiation to Type 1 cells does not occur; instead, a variety of epithelial forms are found, including fetal-like tubular structures and ciliated and squamous metaplastic cells. The correlation of epithelial injury and repair with the direct demonstration of bleomycin in the lung indicates that Type 2 cells are susceptible to injury in the division and differentiation phases of the cell cycle and may then produce a variety of inappropriate alveolar lining cells.


Effect of encephlitogenic protein. PPD and tetanus toxoid on leukocyte migration in agarose. A study of "cross-reactivity". The reactivity to three antigens: bovine encephalitogenic protein (EP), PPD, and tetanus toxoid, was studied with blood leukocytes from healthy humans using Clausen's (5) leukocyte migration in agarose technique. There is an obvious correlation between the reaction of EP (all concentrations studied) and to low concentrations of PPD; and between the reactivity to low concentrations of EP and low concentrations of tetanus toxoid. After vaccination with tetanus vaccine, a marked increase in reactivity to the toxoid sometimes occurred; at the same time, a marked reactivity to EP appeared. Various explanations are discussed: a true immunological cross-reactivity, that the correlations are due to a variability in individual response with respect to lymphokine production, and that BCG and tetanus vaccinations produce an adjuvant effect increasing a pre-existing low reactivity to EP.


The effect of isoproterenol on hypoxia-mediated histamine release. In 13 guinea pigs the pulmonary circulation was isolated from the systemic circulation and perfused with Tris buffer alone or with isoproterenol. The lungs were initially ventilated with 100% O2 and then switched to 100% N2 to induce alveolar hypoxia. The effluent was collected from the aorta and assayed for histamine. In the control group (n = 7) the histamine levels rose markedly with hypoxia. In the group perfused with Tris buffer and isoproterenol (n = 6) the rise in histamine levels was markedly diminished. Therefore, the authors conclude that isoproterenol, in their in vivo model, had a dramatic effect on hypoxia (non-immunologic) mediated histamine release.


Motor unit irritability in Beagles Before and after exposure to cholinesterase inhibitors. Electromyographic (EMG) examinations were performed on Beagles before and for 7 days after oral administration of one of the following organophosphate (OP) compounds; ronnel (55.0 or 110.0 mg/kg), dichlorvos (29.7, 59.4, or 148.5 mg/kg), or cythioate (24.8 or 33.0 mg/kg). The EMG values determined were evoked potentials, after-discharge activity, F-wave activity, nerve conduction velocity, and motor unit potential activity associated with interosseous and pectineal reflexes. Erythrocyte cholinesterase (ChE) activities were measured in some dogs. Ronnel did not have an effect on ChE activity, whereas dichlorvos and cythioate, at all dosage levels, had an inhibitory effect. Some dogs had minor signs of OP toxicosis. The EMG changes for individual OP compounds were not statistically significant (P greater than 0.05), but pooled results revealed an increased duration of evoked potentials, increased after-discharge activity, and decreased F-wave activity; however, only the effect on duration was significant (P less than 0.05). Reflex motor unit potential activity and nerve conduction velocities were not affected. Effects of neostigmine (0.1 to 0.4 mg/kg) given IV to anesthetized, atropinized Beagles were similar to those effects shown by pooled data for the OP compounds, but considerably more muscle fasciculation was produced. Results of this study indicate that even when erythrocyte ChE activity is reduced by OP compounds at dosage levels that produce no or minimal visible signs of toxicosis, EMG reveals little evidence for increased motor unit irritability.


Leukogenic properties of purified BLV and possible routes of its transmission with blood cells present in milk. The results of five years studies on the possibility of experimental induction of bovine leukemia with purified BLV and different BLV-containing materials demonstrated that inoculation of calves with purified BLV (structures of 1.14-1.17 g/ml density in 20-60% sucrose gradient) can induce oncornavirus infection and bovine leukemia. From 41 experimentally infected calves 9 showed evidence of bovine leukemia. 5 of these 9 animals were from a group of 22 calves inoculated with purified BLV. Diagnosis of bovine leukemia in these 9 animals was established by hematologic (3 animals) and histologic method (6 animals). BLV was isolated from leukocytes of all of them. It was shown also that before the development of bovine leukemia the development of oncornavirus infection takes place. But only a small part of the animals with oncornavirus infection and presence of antibodies became ill with typical forms of hemoblastosis. The investigation of the role of milk and blood cells in milk in bovine leukemia transmission was also carried out. The presence was demonstrated of oncornavirus structures antigenically identical with BLV in milk and reproduction of these structures in blood cells being present in milk. These results, and also the induction of bovine leukemia, in cattle by rearing with raw milk from cattle with leukemia, allow us to suppose the possible routes of bovine leukemia transmission in nature.


Studies on cell lines derived from calf, thymic and skin forms of bovine lymphosarcoma. The etiology of sporadic bovine leukosis (SBL) is not known. Long-term monolayer cultures were made from calf (CLS), thymic (TLS) and skin (SLS) forms, and serological tests, electron microscopic observations and reverse transcriptase assays were employed for the detection of an etiological agent. Bovine leukosis virus (BLV) antigen and reverse transcriptase activities remained negative in cultures from SBL cases. Treatment of a culture from CLS 3178 with 5'-iodo-2'-deoxyuridine and dexamethasone resulted in production of BLV which may have been acquired from the BLV-infected dam of CLS 3178, and in an alteration of cell morphology. Focus formation in monolayer cultures and colony formation in soft agar cultures were observed in this treated cell line. Human fetal lung fibroblast cells cocultivated with the cultures from SBL resulted in rapid proliferation of cells with an increased focus formation.


Innervation of the feline eustachian tube. The distribution of adrenergic, cholinergic and peptidergic nerves in the feline eustachian tube was studied using histochemical techniques. Adrenergic, acetylcholinesterase-positive and vasoactive intestinal polypeptide immunoreactive nerves were numerous in the tubal wall. All three types of nerve fibers occurred in the subepithelial layer, around small blood vessels and around the acini of seromucous glands. No nerves displaying substance P or enkephalin immunoreactivity were observed.


Does a defect of energy metabolism in the nerve fiber underlie axonal degeneration in polyneuropathies? A number of chemically unrelated neurotoxic compounds and several types of metabolic abnormalities cause strikingly similar patterns of distal symmetrical polyneuropathy in humans and animals. Experimental studies with laboratory species have demonstrated that many toxic polyneuropathies are associated with distal and retrograde axonal degeneration occurring in vulnerable nerve fiber tracts in the central as well as the peripheral nervous system. This has been termed central-peripheral distal axonopathy. Recent observations from the authors' laboratories regarding (1) the spatial-temporal evolution of nerve fiber degeneration in experimental toxic neuropathies and (2) the inhibition of glycolytic enzymes by chemically unrelated neurotoxic compounds point to a common metabolic basis for many distal axonopathies. It is postulated that neurotoxic compounds deplete energy supplies in the axon by inhibiting nerve fiber enzymes required for the maintenance of energy synthesis. Resupply of enzymes from the neuronal soma fails to meet the increased demand for enzyme replacement in the axon, causing the concentration of enzymes to drop in distal regions. This leads to a local blockade of energy-dependent axonal transport, which produces a series of pathological changes culminating in distal nerve fiber degeneration. The idea provides a working hypothesis with which to study the cause of inherited and acquired human and animal polyneuropathies.


Chronic lead intoxication: effects on developing optic nerve. The effects of chronic lead intoxication were studied by examining the optic nerve of mice given lead-containing mother's milk from day 1 to day 21 of life. Biochemical assays for myelin basic protein, 2',3'-cyclic nucleotide phosphodieterase, and cerebroside sulfotransferase showed that the total amount of myelin produced by the lead-exposed animals was decreased. Lead exposure did not alter the number of oligodendroglia or the relationship between axons and myelin sheaths. The hypomyelination was paralleled by a reduction in size of optic axons in the lead-exposed animals. The data suggest that chronic exposure to lead in developing mice results in a primary effect on neurons and that hypomyelination is secondary to reduction in axon size.


[Induction of reaginic hypersensitivity to tartrazine in the rabbit immunization by ingestion of the covalent conjugate tartrazine-human serum albumin (author's transl)]. Sensitization to simple chemical substances acting as haptens is not well known in food allergy. For that purpose, a study was made in rabbit using tartrazine alone with adjuvant, or mixed with human serum albumin (HSA), or conjugated by covalent BOP bonds with HSA. Hypersensitivity was evaluated by rabbit basophil degranulation test which would offer more specificity through cell membrane IgE-antibody reactions, and thus, more accuracy than classical passive cutaneous anaphylaxis. The data showed that sensitization could not be induced by ingestion of tartrazine with adjuvant or mixed with a protein. But covalent conjugate T-HSA mixed with adjuvant sensitized 8 of the 9 rabbits studied. It seems that preceding irritation of intestinal mucous membrane by acetylsalicylic acid enables one to obtain a more long lasting sensitization.


Giant exomphalos--conservative or operative treatment? The rate of survival for infants with intact giant exomphatos has much improved during the last 20 years; this is partly due to better respiratory and nutritional support. The use of a staged operative closure using a sialon prosthesis has been advocated for 12 years, but our data do not show this to be superior to nonoperative management.


Dynamic redistribution of concanavalin A binding sites on isolated guinea pig keratinocytes. Modifications in the distribution of the binding sites for concanavalin A (Con A) were studied on trypsin isolated living guinea pig keratinocytes. Fluorescein-labelled Con A was used and the in vitro procedure has included short-term cultures, experiments at 37 degrees C and 4 degrees C, the study of colchicine and vincaleucoblastine effects. It was possible to induce different patterns of staining corresponding to distinct redistribution of Con A binding sites; the distinct redistribution was correlated to the effects of Con A, colchicine and vincaleucoblastine. These findings demonstrated that the system used was appropriate to the study of some dynamic events on the keratinocytes membranes.


Esophagomyotomy versus forceful dilation for achalasia of the esophagus: results in 899 patients. Between 1949 and 1976, 899 patients underwent treatment for achalasia of the esophagus at the Mayo Clinic, 431 by forceful hydrostatic or pneumatic dilation and 468 by a standardized transthoracic esophagomyotomy. Esophageal leak and mediastinal sepsis was an uncommon but major complication of both types of therapy, occurring four times more often with dilation (4%) than with myotomy (1%), although no deaths resulted from this in either group. The 30-day mortality was 0.2% after myotomy and 0.5% after forceful dilation. Although there was minimal morbidity and mortality with either modality, the late results were significantly superior after myotomy. Excellent to good results were obtained by 85% of the group treated with myotomy but only by 65% of those treated with hydrostatic dilation. Late poor results were encountered three times more frequently after dilation (19%) than after myotomy (6%). Analysis of poor results after myotomy indicates that late serious complications of gastroesophageal reflux developed in only 3% of patients operated on.


Esophageal carcinoma: an aggressive approach. During a 3-year period, 45 patients with esophageal carcinoma (18 of the lower and 27 of the middle third) underwent esophagectomy and esophagogastrostomy. All patients were considered to be in Stage I and Stage II preoperatively, but at the time of operation, 66% were found to have Stage III disease. All patients underwent mobilization of the stomach through a laparotomy, and resection of the esophagus and reconstruction of the continuity of the gastrointestinal tract through a separate right lateral thoracotomy incision. Following operation, 22 patients (10, Stage I and II and 12, Stage III) received no radiotherapy and 23 patients (5, Stage I and II and 18, Stage III) received radiotherapy. Two patients died in the immediate postoperative period, for a surgical mortality of 4.8%. Because of the acceptable survival and enhancement of quality of life, esophagectomy and esophagogastrostomy should be offered not only to Stage I and II patients with esophageal carcinoma but also to selected patients with Stage III disease and those with lower and middle third esophageal lesions.


Recurrent idiopathic anaphylaxis.

Recurrent idiopathic anaphylaxis. Eighteen patients with recurrent episodes of life-threatening anaphylaxis of unknown cause were studied. Each patient had repeated detailed histories taken and had repeated physical examinations, hypersensitivity skin tests to foods, and complete blood cell counts. In addition, each patient underwent stringent dietary manipulation. Seventeen of the 18 patients underwent a series of studies. Three patients were hospitalized for study. Results of all tests were essentially normal except for an elevated plasma histamine level during attacks in two patients. Episodes could not be prevented with antihistamine therapy. Attacks were treated successfully by instructing the patients in the self-administration of epinephrine. It thus appears that recurrent episodes of anaphylaxis due to nonimmunologic histamine release of anaphylaxis due to nonimmunologic histamine release can occur without discernible cause and can be lifethreatening.


Immunochemical analysis of cartilage proteoglycans. Antigenic determinants of substructures. Antibodies were raised in rabbits by injection of cartilage proteoglycan monomers, isolated hyaluronic acid-binding region, polysaccharide-peptides prepared by trypsin digestion of proteoglycans and link-protein. The rabbits injected with the proteoglycan monomers made antibodies reacting with the intact proteoglycan. The antiserum contained antibodies specific for, and also reacting with, the isolated hyaluronic acid-binding region and the keratan sulphate-rich region. In addition there were probably antibodies reacting with other structures of the proteoglycan monomer. When isolated hyaluronic acid-binding region was used for immunization the antibodies obtained reacted specifically with the hyaluronic acid-binding region. The antibodies obtained from rabbits immunized with the polysaccharide-peptides reacted with the proteoglycan monomers and showed a reaction identical with that of the chondroitin sulphate-peptides isolated after trypsin digestion of proteoglycans. The antibodies prepared with the link-protein as the antigen reacted only with the link-protein and not with any preparation from the proteoglycan monomer. Neither did any of the antisera raised against the proteoglycan monomer or its substructures react with the link-protein. Separately it was shown that the peptide 'maps' prepared from trypsin digests of the link-protein and the hyaluronic acid-binding region were different. Therefore it appears that the link-protein is not structurally related to the proteoglycan or the hyaluronic acid-binding region. Digestion of proteoglycan monomers or isolated hyaluronic acid-binding region with trypsin did not destroy the antigenic sites of the hyaluronic acid-binding region. In contrast trypsin digests of previously reduced and alkylated preparations did not react with the anti-(hyaluronic acid-binding region). The trypsin digests, however, reacted with both the antibodies directed against the chondroitin sulphate-peptides and those against the keratan sulphate-peptides. Trypsin digestion of the link-proteins destroyed the antigenic site and the reactivity with the antibodies. By combining immunoassay of proteoglycan preparations before and after trypsin digestion it is feasible to quantitatively determine its substructures by using the antisera described above.


Mutant analysis of glyceraldehyde 3-phosphate dehydrogenase in Escherichia coli. NAD(+)-specific glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from Escherichia coli was purified to homogeneity by a relatively simple procedure involving affinity chromatography on agarose-hexane-NAD(+) and repeated crystallization. Rabbit antiserum directed against this protein produced one precipitin line in double-diffusion studies against the pure enzyme, and two lines against crude extracts of wild-type E. coli strains. Both precipitin lines represent the interaction of antibody with determinants specific for glyceraldehyde 3-phosphate dehydrogenase. Nine independent mutants of E. coli lacking glyceraldehyde 3-phosphate dehydrogenase activity all possessed some antigenic cross-reacting material to the wild-type enzyme. The mutants could be divided into three groups on the basis of the types and amounts of precipitin lines observed in double-diffusion experiments; one group formed little cross-reacting material. The cross-reacting material in crude cell-free extracts of several of the mutant strains were also tested for alterations in their affinity for NAD(+) and their phosphorylative activity. The cumulative data indicate that the protein in several of the mutant strains is severely altered, and thus that glyceraldehyde 3-phosphate dehydrogenase is unlikely to have an essential, non-catalytic function such as buffering nicotinamide nucleotide or glycolytic-intermediate concentrations. Others of the mutants tested have cross-reacting material which behaved like the wild-type enzyme for the several parameters studied; the proteins from these strains, once purified, might serve as useful analogues of the wild-type enzyme.


Structure of antigenic determinants in the N-terminal region of dermatosparactic sheep procollagen type I. About half of the rabbit antisera raised against type-I procollagen, p alpha 1(I) chain or nonreduced procollagen peptides reacted in a radioimmunoassay with the reduced form of peptide Col 1, which  comprises the whole non-collagenous region at the N-terminus of procollagen. Proteolytic fragments prepared from reduced peptide Col 1 were still effective inhibitors of the antibodies and allowed the localization of two antigenic determinants. The antigenically active regions have the sequences less than Glu-Glu-Glu-Gly-Gln-Gln-Glu and Gly-Asp-Thr-Gly-Pro-Arg, and are located at the N- and C-termini of the peptide respectively. Antibodies raised against reduced peptide Col 1 bind to a determinant localized in a different region of the peptide.


[Ultrastructure of Mycoplasma sp. demonstrated by phosphotungstic acid-chromic acid pH 0,4]. Ultrastructural observation of Mycoplasma sp. revealed by the phosphotungstic acid-cromic acid, pH 0,4. Arq. Inst. Biol., São Paulo, 45(3) :169-176, 1978. Cells of IB-RS-2 (clone C-26-3) swine line were examined under electron microscopy, employing the differential coloration with PTA-ACr 5 %, pH 0,4, during 30 minutes, at 37 degrees C. Through this method we could observe the intracitoplasmatic forms, electronically dense, rather pleomorfic, with sizes varying between 60nm and 640nm and a unitary membrane of 90A similar to the extracellular forms described as Mycoplasma sp.


Demonstration of cross-reacting material in Tay-Sachs disease. Antibodies against placental hexosaminidase A and kidney alpha-subunits were raised in rabbits after cross-linking the antigens with glutaraldehyde. Anti-(alpha(n)-subunit) antiserum (anti-alpha(n)) precipitated hexosaminidase A but not hexosaminidase B, whereas anti-(hexosaminidase A) antiserum precipitated both hexosaminidases A and B. Specific anti-(hexosaminidase A) antiserum was prepared by absorbing antiserum with hexosaminidase B. Both anti-alpha(n) and anti-(hexosaminidase A) antisera precipitated the CR (cross-reacting) material from eight unrelated patients with Tay-Sachs disease. Immunotitration, immunoelectrophoresis, double-immunodiffusion and radial-immunodiffusion techniques were used to demonstrate the presence of CR material. The CR-material-antibody complex was enzymically inactive. Antiserum raised against kidney or placental hexosaminidase A, without cross-linking with glutaraldehyde, failed to precipitate the CR material, implying that treatment of the protein with glutaraldehyde exposes antigenic determinants that are hidden in the native protein. Since anti-(hexosaminidase B) antiserum did not precipitate the CR material during the immunoelectrophoresis of Tay-Sachs liver extracts, it is suggested that altered alpha-subunits do not combine with beta-subunits. By using immunotitration we have demonstrated the competition between the hexosaminidase B-free Tay-Sachs liver extract and hexosaminidase A for the common binding sites on monospecific anti-(cross-linked hexosaminidase A) antiserum. The amount of CR material in the liver samples from seven cases of Tay-Sachs desease was found to be in the same range as theoretically calculated alpha-subunits in normal liver samples. Similar results were obtained by the radial-immunodiffusion studies. The present studies therefore suggest that Tay-Sachs disease is caused by a structural-gene mutation.


Interactions of rat alpha-foetoprotein with bilirubin. The binding of bilirubin to rat-alpha-foetoprotein has been measured by changes in the absorption spectrum and by the appearance of two intense circular-dichroism bands. Furthermore binding of this bile pigment has been demonstrated by a decrease of its enzymic oxidation rate and by competition experiments with bovine serum albumin-agarose. The binding parameters have been determined as follows: n = 1.0 mol bound/mol of protein and Ka = 2.9 X 10(6) M-1. Competition of oestradiol for the bilirubin-binding site has not been established.


[Alterations in pharmacokinetics during toxicity tests (author's transl)]. Some possible reasons are discussed which may lead to changes in the pharmacokinetics of a drug during long-term toxicity tests. Since most of these alterations have toxicological consequences, their evaluation can be helpful in the interpretation of toxicity studies. Furthermore, and altered pharmacokinetic profile may be a diagnostic tool for the detection of organ damage (e.g., eliminatory organs). In this paper three examples of the "radioactive test dose method" are shown. This method allows a direct comparison of the pharmacokinetics between pretreated animals and controls by administration of a single radioactive test dose to both groups.


Mechanism of action studies of lidamidine hydrochloride (WHR-1142A), a novel antidiarrheal agent. 1-(2,6-Dimethylphenyl)-3-methylamidinourea hydrochloride (WHR-1142A), lidamidine hydrochloride), a novel antidiarrheal agent, inhibited contractile activity in isolated guinea pig ileum stimulated by acetylcholine, histamine, serotonin, dimethylphenylpiperazinium, prostaglandin E2, BaCl2 and KCl. WHR-1142A also blocked spontaneous and stimulated contractile activity measured with extraluminal strain gauges in the duodenum, ileum and colon of dogs. Studies on the autonomic effects of WHR-1142A indicated little, if any, peripheral adrenergic stimulatory or cholinergic blocking activity. Inhibition of intestinal motility by WHR-1142A was not antagonized by naloxone. WHR-1142A also showed no morphine-like analgesic effects and was devoid of any H1-antihistamine activity. WHR-1142A appears to be a pharmacologically unique antidiarrheal agent.


Preliminary studies on nasal decongestant activity from the seed of the shea butter tree, Butyrospermum parkii. 1 The seed of Butyrospermum parkii yields shea butter which according to local traditional healers relieves inflammation of the nostrils. 2 Since there is as yet no absolutely satisfactory nasal decongestant in clinical use, it was decided to investigate the effects of shea butter in nasal congestion. The substance was prepared in the laboratory. 3 The human subjects used were those suffering from rhinitis with moderate to severe nasal congestion. They were divided into the test group which received shea butter, the control group which was treated with xylometazoline and the 'placebo' group which received white petroleum jelly B.P. 4 The results showed that nasal congestion was relieved more satisfactorily in the test group than in the other two groups. 5 It is concluded that shea butter may prove more efficacious in nasal congestion than conventional nasal drops.


Changes in the molecular composition of circulating hydroxyethyl starch following consecutive daily infusions in man. 1 Changes in the circulating molecular composition of hydroxyethyl starch (HES) were determined in four normal men following three consecutive daily 500 ml infusions (total 1,500 ml), by passage of trichloroacetic acid filtrates of plasma through a Sepharose CL4B gel filtration column. 2 The HES recovered from the intravascular space 10 min following the injection on Days 1, 2 and 3, was of a narrower molecular size distribution than the injected material, with a noticeable shift to molecules of a low molecular weight (LMW) size. 3 The HES in the sampled plasma 24 h post-injection on Days 1, 2 and 3 consisted of molecules possessing a LMW distribution, concomitantly with a slight shift to molecules of a larger molecule size. 4 The HES recovered from the bloodstream 480 h after the third and final injection consisted of molecules possessing an intermediate size distribution, between LMW and high molecular weight (HMW) size material. 5 The results indicate that large HES molecules contained in the injected material are eliminated from the bloodstream; the HMW fraction at least partially by a alpha-amylase mediated catabolism, and the resulting LMW fraction by excretion.


An antigen associated with mesenchyme in human tumours that cross-reacts with brain glycoprotein. Anti-NSA3 antiserum was found to react with many kinds of benign and malignant tumours, as well as foetal skin and intestinal extracts. The corresponding antigens isolated from nervous tissue, benign breast adenoma, and a fibrosarcoma were compared. Immunoprecipitation cannot distinguish between these antigens, and their amino-acid contents were comparable. However, immuno-absorption identified an antigenic determinant that was confined to nervous tissue. Indirect immunofluorescence further confirmed the validity of the concept of a nervous form vs a mesenchymal form of the antigen. Furthermore, immunofluorescence enabled the localization of the antigen found in non-nervous tissue to mesenchyme (mesenchyme-associated antigen: MAA), whether the mesenchymal tissue be normal (foetal organs), tumoral (fibrosarcoma) or reactional (connective-tissue stroma of epithelial tumours).


Antibody-dependent cellular cytotoxicity to human colon-tumour cells. II. Analysis of the antigens involved. The relationship between carcinoembryonic antigen (CEA) and A antigenic determinants on the cell surface of colon-tumour cells was studied by the ADCC assay. Antiserum prepared in 2 rabbits to an undecapeptide analogous to the amino terminal of CEA(1-11) was found by us either to participate in (Rabbit 2) or specifically inhibit (Rabbit 1) ADCC. The binding spectra of these two antisera and of antiserum to the whole CEA molecule were similar. All of them react with A and non-A colon-tumour cells as well as red blood cells of Type A (RBC-A) and their activity was completely absorbed on RBC-A but not on B or O. O-type, ADCC-reactive human sera always react with A-type colon-tumour cells and RBC-A, and some of them with non-A colon-tumour cells also. The degree of inhibition of their reactivity by anti-CEA(1-11) R1 varied between sera, from none to almost a complete inhibition, and is not related to whether the serum is of cancer or non-cancer origin. Non-reactive O-type sera contain anti-A activity demonstrable by haemagglutination and immunofluorescence. However, they cannot participate in ADCC reaction nor inhibit it. The sera, which contain lymphocyte-dependent antibody to A-type colon-tumour cells, lysed RBC-A, without the addition of lymphocytes or complement, in an immunologically specific way. It is concluded that the reactivity seen in our ADCC system is related to a determinant common to A and CEA (and maybe to other normal cross-reacting antigens) which most probably resides in the amino terminal part of these molecules. This determinant elicits the production of lymphocyte-dependent antibodies in about 50% of people with blood group O. Thus, the amino terminal part of CEA is not a tumour-specific part of the CEA molecule. No specific anti-tumour activity was found in patients' serum by this method, and claims for its demonstration by other methods may well be related to the non-specific activity observed here.


Induction of foci of altered, gamma-glutamyltranspeptidase-positive hepatocytes in carcinogen-treated rats fed a choline-deficient diet. A series of experiments was performed to investigate whether, after exposure of rats to a chemical hepatocarcinogen, feeding a choline-deficient (CD) diet would promote the proliferation of initiated liver cells, and their evolution to foci of altered γ-glutamyltranspeptidase (GGT)-positive hepatocytes, without subjecting the animals to further experimental manipulations.Diethylnitrosamine (DEN), in single doses of 15-150 mg/kg body weight, was injected into male, Sprague-Dawley rats, either intact or 18 h after a partial hepatectomy (PH). The animals were then fed either a CD or a choline-supplemented (CS) diet for 2-8 weeks. Emergence in the liver of foci of altered, GGT+ hepatocytes was studied by histological and histochemical techniques. Foci, in varying numbers, developed in the liver of all rats fed the CD diet. The number of foci induced was larger when DEN was administered after PH rather than to intact rats. Foci developed in none of the livers of rats fed the CS diet, except in one experiment in which 30 mg DEN/kg body weight was injected after a PH. In all cases, foci of altered, GGT+ hepatocytes were shown to be α-foetoprotein after immunofluorescence staining of liver sections.It is concluded that feeding a CD diet exerts a strong promoting action on the proliferation and further evolution of liver cells initiated by a chemical carcinogen, providing the basis for a new and efficient procedure for the induction of foci of altered hepatocytes in rat liver.


Morphological Abnormalities in cultured erythroid colonies (BFU-E) from the blood of two patients with HEMPAS. The results of cytological and ultrastructural analysis of erythroid burst colonies derived from the peripheral blood of two patients with HEMPAS have been compared to those obtained in normal controls. Using the plasma clot technique, in studies on 10 subjects we confirmed that most of the colonies consisted or erythroblasts with a synchronous and normal maturation involving a wave of nuclear extrusion at day 13. In contrast, the majority of well-haemoglobinized colonies from HEMPAS consisted of numerous bi- or multinucleated erythroblasts displaying the supplementary double membrane beneath their plasma membrane. This excessive membrane may be present as a continuous or fragmented structure in different erythroblasts from the same colony. These findings suggest that the progeny derived from one BFU-E may vary considerably in their morphological defects. Furthermore, one third of the packed colonies appeared to be formed by non-haemoglobinized cells which were clearly identified by electron microscopy as very early erythroblasts. These cells were unable to mature and subsequently lysed. Thus dyserythropoiesis occurred in culture both at early and late stages of maturation. These studies clearly demonstrate that HEMPAS is a disorder resulting from defective erythroid committed cells.


Cut-off levels for maternal plasma alpha-fetoprotein in the diagnosis of neural tube defects: validation of the use of multiples of the normal median. A retrospective study was made of measurement of maternal plasma alpha-feto-protein (AFP) by a standard radioimmunoassay technique, as a method of screening for neural tube defects (NTDs) between 15 and 20 weeks gestation. There was a good fit of a log-normal distribution to the data at each week and multiples of the normal median were equivalent to constant centiles over weeks 15 to 20 and possibly at later weeks also. There were 700 plasma samples from women with normal prenancies and 60 from women with pregnancies complicated by fetal NTDs (30 with anencephaly and 30 with spina bifida). At 15 to 20 weeks of pregnancy, 90 per cent of women with fetuses affected by anencephaly, 70 per cent of those with fetuses affected by open spina bifida and 3 per cent ofthose with normal pregnancies had plasma AFP levels at or above 2.4 times the normal median for each gestational week. It was estimated that 2.1, 2.4 and 3.1 times the normal median maternal plasma AFP levels were equivalent to the 95th, 97th and 99th centiles respectively. The interassay coefficients of variation estimated on control sera throughout the entire study were 4 to 6 per cent.


The relation between maternal serum alpha-fetoprotein levels and fetomaternal haemorrhage. Spontaneous fetomaternal haemorrhage at 14 to 20 weeks gestation resulted in raised serum alpha-fetoprotein (AFP) levels in 13 of 150 patients attending a genetic counselling clinic. In all 13 patients, the placenta was anterior or fundal in position. By allowing for a rise in serum AFP levels of 4 microgram/l for each fetal cell seen in 30 high power fields (Kleihauer test), a 62.5 per cent reduction in the number of patients selected for amniocentesis because of raised serum AFP levels would have been achieved. The occurrence of fetomaternal haemorrhages at the time of amniocentesis can be detected by either the Kleihauer technique or the measurement of maternal serum AFP levels.


Amino acid sequence of California quail lysozyme. Effect of evolutionary substitutions on the antigenic structure of lysozyme. To examine the effect of amino acid substitutions in lysozyme on the binding of antibodies to lysozyme, we purified lysozyme from the egg whites of California quail and Gambel quail. Tryptic peptides were isolated from digests of the reduced and carboxymethylated lysozymes and subjected to quantitative analysis of their amino acid compositions. The two proteins were identical by this criterion. Each peptide from the California quail lysozyme was then sequenced by quantitative Edman degradation, and the peptides were ordered by homology with other bird lysozymes. California quail lysozyme is most similar in amino acid sequence to bobwhite quail lysozyme, from which it differs by two substitutions: arginine for lysine at position 68 and histidine for glutamine at position 121. California and bobwhite quail lysozymes were antigenically distinct from each other in quantitative microcomplement fixation tests, indicating that substitutions at one or both of these positions can alter the antigenic structure of lysozyme. Yet neither of these positions is among those claimed to account for the precise and entire antigenic structure of lysozyme [Atassi, M. Z., & Lee, C.-L. (1978) Biochem. J. 171, 429--434]. Two possible explanations for this discrepancy are discussed.


Comparison of fluorescence energy transfer and quenching methods to establish the position and orientation of components within the transverse plane of the lipid bilayer. Application to the gramicidin A--bilayer interaction. Fluorescence quenching and resonance energy transfer methods have been used to investigate the position of fluorophores in the lateral and transverse planes of the lipid bilayer. A series of n-(9-anthroyloxy) fatty acids (n = 2, 6, 9, and 12) have been used as energy-transfer acceptors so that apparent transfer distances from a membrane-bound donor (N-stearoyltryptophan) have a transverse as well as a lateral component. Both theory and experiment show that the energy-transfer method is not precise enough to discriminate between the positions of the fluorophores in the transverse plane of the bilayer. The n-(9-anthroyloxy) fatty acids are also susceptible to quenching by the indole moiety of tryptophan. The relative quenching efficiency can provide a semiquantitative measure of the position of quenching molecules in the lipid bilayer. The quenching techniques are applied to the determination of the orientation of gramicidin A in lipid bilayers. The tryptophan residues of gramicidin appear to be located near the membrane surface in agreement with the head-to-head dimeric structure proposed by D. W. Urry et al. [(1971) Proc. Natl. Acad. Sci. U.S.A. 68, 672--676].


Biosynthesis of streptomycin. Enzymic oxidation of dihydrostreptomycin (6-phosphate) to streptomycin (6-phosphate) with a particulate fraction of Streptomyces griseus. Resting cells and to a greater extent permeabilized cells of Streptomyces griseus can oxidize dihydrostreptomycin to streptomycin. The dihydrostreptomycin oxidoreductase activity was localized in the 100,000 X g particulate fraction. Sucrose density gradient centrifugation of the particulate suspension gave a band at a density of 1.09 which consisted mainly of membrane vesicles. This fraction had high dihydrostreptomycin oxidoreductase activity. S. griseus protoplasts also contain high oxidoreductase activity. These data are consistent with localization of the enzyme in the cell membrane. Dihydrostreptomycin and dihydrostreptomycin 6-phosphate can both serve as substrates for the oxidoreducatase, but the phosphate was the better substrate in the cell free system. Addition of cofactors was not required for the bound dihydrostreptomycin oxidoreductase. The electron acceptor for the oxidation is unknown. Oxidation of dihydrostreptomycin 6-phosphate to streptomycin 6-phosphate very probably represents the penultimate step in the biosynthesis of streptomycin.


Effects of hexachlorocyclohexane isomers on concanavalin A 'capping' in bovine lymphocytes. The effects of alpha, beta, gamma, and delta isomers of hexachlorocyclohexane on concanavalin A 'capping' in bovine lymphocytes were evaluated, gamma and delta hexochlorocyclohexane inhibited 'capping' whereas the alpha and beta isomers were without effect. In addition, gamma-hexachlorocyclohexane has been shown to antagonize the maintenance of preformed 'caps' and cause the rapid dispersal of the concanavalin A-receptor complexes over the surface of cells by a temperature-dependent mechanism. The possible role of a gamma-hexachlorocyclohexane-sensitive process in the organization of microflow patterns in the lectin-activated lymphocyte membrane is discussed.


Maternal plasma alpha-feto-protein in missed abortion. Maternal plasma alpha-fetoprotein (AFP) levels in 8 pregnancies, which several weeks later ended as missed abortions, were evaluated. In 7 patients, blood sampling before or after the death of their fetuses revealed almost normal maternal AFP values. The relatively viable placenta of missed abortion probably hampers the eventual leakage/transudation of fetal AFP into maternal circulation.


Specific precipitation of mouse and rat alpha 2-macroglobulin by immune sera directed against murine leukemia viruses. Leukemia viruses preparations obtained from plasma of leukemic mice or rats were used to immunize rabbits. By immunoelectrophoresis, these non-absorbed antisera reacted specifically with one single mouse serum alpha 2 globulin which was further identified as alpha 2-macroglobulin by several criteria, including gel chromatography and nitracentrifugation. When mouse plasma derived virions were used as antigen, they gave rise to antibodies cross-reacting completely with the corresponding mouse and guinea pig protein and partially with rat and calf globulin. Immunization of rabbits with rat plasma derived virions induced antibodies directed preferentially against the rat alpha 2-macroglobulin.


Effects of toyocamycin on the biological activity of a murine oncornavirus produced by a chronically infected cell line. Production of Friend virus by chronically infected cells (Friend-Eveline cell line) is depressed by Toyocamycin, an adenosine analogue, at doses which stop cellular growth. The infectivity and the endogenous reverse transcriptase activity of the virions are diminished. These effects are likely to be due to the incorporation of Toyocamycin into the 70S RNA.


Hageman-factor-dependent fibrinolysis: generation of fibrinolytic activity by the interaction of human activated factor XI and plasminogen. Human coagulation factor XI has been purified, and upon activation with Hageman factor fragments, was found to convert the fibrinolytic proenzyme plasminogen to plasmin. This proactivator activity was shown to be functionally and antigenically distinct from prekallikrein. When the gamma-globulin fractions of plasma deficient in Hageman factor, prekallikrein and factor XI were isolated, factor-XI-deficient plasma possessed two-thirds of the plasminogen proactivator activity of the Hageman-factor-deficient plasma, while prekallikrein deficient plasma had only one-third of the plasminogen proactivator activity. Thus, the Hageman-factor-dependent plasminogen proactivator previously reported to be present in the gamma-globulin fraction of normal human plasma is a function of prekallikrein and factor XI, while the activity observed in prekallikrein-deficient plasma is attributable to factor XI. When compared utilizing digestion of iodinated fibrin, prekallikrein and factor XIa had similar potency per active site; they were, however, far less active than urokinase.


A radioimmunoassay for erythropoietin.

A radioimmunoassay for erythropoietin. We describe the development of a radioimmunoassay (RIA) for erythropoietin. Antisera were raised in rabbits with an impure human urinary erythropoietin preparation used as immunogen, but with pure human erytropoietin serving as the labeled antigen in the RIA and as a primary standard. The immunoreactivity of erythropoietin is not altered significantly by the mode of labeling with radioiodine, even though the biologic activity is lost. With this method, it is possible to detect 2--3 mU of erythropoietin in a volume of 0.1--0.3 ml. Therefore, the method can be used for detection of normal and subnormal serum titers as well as elevated titers. RIA for erythropoietin does not distinguish between the native (active in vivo) and the asialo form (inactive in vivo) and cannot yet be used for routine monitoring of crude fractions during purification.


The implementation of a large-scale self-instructional course in medical information resources. The implementation of library orientation and bibliographic instruction in health sciences centers presents some interesting as well as perplexing problems. The Rowland Medical Library at The University of Mississippi Medical Center had to confront and reexamine these problems when faced with the requirement to teach 298 freshman and sophomore medical students in one ten-week quarter. This paper outlines the development and implementation of a large-scale self-instructional approach to library instruction. The package consisted of an audiotape, a videotape, a written program, self-teaching quizzes, a performance test, and a student evaluation. Performance test results and student evaluation data are presented which indicate that this format can successfully be employed to meet course objectives and to be accepted by students.


Self-report reliability of skid-row alcoholics. The stability of self-report of 72 skid-row alcoholics over a one to six month interval was explored. Relatively high response agreement on reinterview was indicated for demographic items, but less reliability was observed on items assessing social functioning and drinking patterns. Implications of the findings for the evaluation of treatment programs are discussed.


Serum alphafetoprotein (AFP) in bilharziasis. Two hundred and eighty-four patients of various ages from an endemic area, diagnosed as having bilharziasis by the presence of living Schistosoma haematobium's ova in the mid-stream urine sample, have been investigated for the presence of alphafetoprotein (AFP) in the serum. One hundred and forty-one (49.6%), were positive; no false positives have occurred. Radioimmunoassay is capable of increasing the positivity rate still further. The detection of AFP will be of limited value as a screening test in countries where bilharziasis is endemic.


Cytological changes due to urinary calculi: a consideration of the relationship between calculi and the development of urothelial carcinoma. Urinary calculi can induce urothelial cellular abnormalities comparable with those of malignancy; this was found in 11 out of 62 lithiasis cases. Severe cellular changes, comparable with those of carcinoma in situ, may be seen in the epithelium adjacent to a calculus. Squamous metaplasia was frequently observed in cases with staghorn stones in the renal pelvis. The abrasive effect of the calculus may result in many multinucleated cells in the sediment. When the calculi are removed the cytological atypia and the observed multinucleation disappeared, and none of these 62 patients developed urothelial carcinoma. A possible relationship was found between a long-term clinical history of lithiasis and the development of cancer of the upper urinary tract in a study of 92 cases of carcinoma of the ureter and renal pelvis.


Non-operative arterial embolisation in primary liver tumours. Three patients with primary hepatic tumours were treated by selective arterial embolisation with gelatin-foam fragments to induce necrosis. In the two with histologically proved hepatocellular carcinoma ultrasonography suggested that necrosis had been induced, as did the rapid initial falls in serum alpha-fetoprotein concentration by 95 and 81% of the original values respectively. Treatment was continued with a course of adriamycin, and both patients remained well and symptom free at 10 and 12 months. In the third patient, who had an expanding and highly vascular benign hepatic adenoma associated with use of a contraceptive pill, embolisation obliterated the tumour mass. Tumour embolisation should be regarded as only the first step in managing hepatocellular carcinoma and as a means of reducing appreciably the viable tumour mass before chemotherapy. It may be used as the primary and definitive treatment in patients with benign liver tumours.


Axonal transport of actin: slow component b is the principal source of actin for the axon. Axonally transported proteins were studied in guinea pig retinal ganglion cells using the standard radioisotopic labeling procedure. Two slowly moving groups of proteins were identified in guinea pig retinal ganglion cells. The more slowly moving group of proteins, designated slow component a (SCa) was transported at 0.2-0.5 mm/day. Five polypeptides contained greater than 75% of the total radioactivity transported in SCa. Two of these polypeptides correspond to the subunits of tubulin, while the other three correspond to the slow component triplet. The other slowly moving group of proteins, which is designated slow component b (SCb), was transported at approximately 2 mm/day. Twenty labeled polypeptides were identified in SCb. The major labeled polypeptides transported in SCb differ from those transported in SCa. One of the polypeptides transported in SCb co-migrates with skeletal muscle actin in SDS-polyacrylamide slab gels. This polypeptide behaved identically to skeletal muscle actin on DNaseI affinity columns. Since DNaseI is a highly specific affinity ligand for actin, we conclude that the labeled SCb polypeptide which comigrates with actin in SDS-gels is actin. Between 1.4 and 5.7% of the total radioactivity transported in SCb is attributable to action. Detailed comparison of the distribution of total radioactivity in the optic axons with the distribution of radioactive actin in the optic axons at post-injection times between 6 and 77 days showed that actin was transported specifically in SCb, and not in SCa. Furthermore, analyses of the proteins transported in the fast component of guinea pig retinal ganglion cells by DNaseI affinity chromatography failed to reveal an actin-like moiety. Slow component a, SCb and the fast component are the major components of axonal transport in guinea pig retinal ganglion cells. Thus, in these neurons, actin is transported principally and possibly only in SCb. Guinea pig retinal ganglion cell axons project principally to the lateral geniculate nucleus and superior colliculus. The fate of actin axonally transported to the region of the axon terminals was studied by determining the kinetics by which radioactivity associated with actin accumulates and then decays in the superior colliculus. The results of these studies indicate that labeled actin has a half-life in the superior colliculus of approximately 28 days.


The role of axonal transport in the regulation of enzyme activity in sympathetic ganglia of adult rats. The relationship of perikaryal and presynaptic enzyme activity to axonal transport was studied in adult sympathetic neurons in the rat superior cervical ganglion (SCG). Surgical axotomy or local colchicine application to the postganglionic nerves resulted in a significant decrease in ganglionic tyrosine hydroxylase (T-OH) activity without a significant alteration in choline acetyltransferase activity. Colchicine did not appear to block axonal impulse conduction since pupillary and eyelid function remained normal. Consequently, the reduced T-OH activity resulted from alteration of other axonal functions. Axotomy or colchicine application decreased T-OH activity in decentralized ganglia, suggesting that the depression of perikaryal T-OH was not secondary to altered orthograde transsynaptic interactions. Colchicine did not prevent transsynaptic induction of T-OH by reserpine, suggesting that axonal transport is not necessary for enzyme induction. Nerve growth factor (NGF) treatment partially prevented the effects of colchicine application. It is concluded that in adult sympathetic neurons both orthograde transsynaptic mechanisms and the retrograde transport of NGF normally govern perikaryal T-OH activity.


The effects of drug which destroy the sympathetic nervous system on the retrograde transport of nerve growth factor. It has been proposed that the drugs (6-hydroxydopamine, guanethidine, vinblastine) which are known to destroy sympathetic neurons in neonatal animals do so by preventing the accumulation of retrogradely transported nerve growth factor (NGF). It was found, consistent with the proposal, that administration of 6-hydroxydopamine (100 mg/kg s.c.) or vinblastine (0.4 mg/kg s.c.) 16 h prior to the administration of [125I]NGF complete prevented the accumulation of retrogradely transported [125I]NGF in superior cervical ganglia of neonatal rats. Administration of 6-hydroxydopamine or vinblastine to adult rats (where it does not cause sympathetic neuron cell death) did not completely prevent the retrograde transport of NGF, although 6-hydroxydopamine produced an alteration of the time course of accumulation (early times unaffected, later times depressed). The administration of guanethidine to adult rats (50 mg/kg/day) produced a modest decrease in the accumulation of NGF (40-60%). It would appear, however, that this decrease cannot account for the cytotoxic effects of guanethidine since: (1) sub-cytotoxic doses of guanethidine and non-cytotoxic guanidinium blocking agents also produce modest decreases in the retrograde transport in NGF; and (2) the retrograde transport of [125I]NGF is not affected in neonatal animals until after the neurons are clearly damaged. Hence, the data are entirely consistent with the hypothesis that NGF deprivation caused by 6-hydroxydopamine and vinblastine is the mechanism of the cytotoxic effects of these drugs on sympathetic neurons in neonatal animals. Guanethidine destroys sympathetic neurons by some other mechanism.


Influence of cysteamine on differential staining of BUdR-substituted human chromosomes. In 5-bromodeoxyuridine (BUdR)-substituted human chromsomes stained with 4'-6-diamidino-2-phenylindole (DAPI) differential staining is suppressed totally by the H+-donor cysteamine (concentration 0.08 M). We propose that differential staining appears because the double BUdR-substituted chromatid will be disintegrated via a photosensitive dye-visible light system. It is suggested that cysteamine prevents the production of strand breaks in DNA and, consequently, differential staining in BUdR-substituted chromosomes. Furthermore it is shown that differential staining with DAPI causes irreversible changes in the double BUdR-substituted chromatid. This finding can be explained with the above mentioned mechanism.


Experimental studies of the pathogenesis of infections due to Pseudomonas aeruginosa: extracellular protease and elastase as in vivo virulence factors. The effects on mortality of supplemental injections of protease and elastase were determined in burned mice infected with non-lethal inocula of a toxin-producing but non-proteolytic-enzyme-producing strain of Pseudomonas aeruginosa. When a variety of solutions containing proteolytic enzyme were injected under these conditions, the mortality increased significantly. This did not occur when organisms other than P. aeruginosa were used. Injections of the enzyme solutions alone were non-lethal. Injection of a solution of alpha 2-macroglobulin, which was shown to inhibit proteolytic activity, together with a proteolytic enzyme--toxin producing strain of P. aeruginosa caused a significant delay in mortality when compared with controls. It was concluded that protease, elastase, and toxin production were necessary for P. aeruginosa to express full virulence in the burned mouse model.


Relationship of hepatitis B surface antigen to serum alpha-fetoprotein in nonmalignant diseases of the liver. Serum alpha-fetoprotein (AFP) levels were measured by radioimmunoassay in 89 healthy adult Chinese, 170 patients with histologically verified non-malignant liver diseases, and 14 hepatitis B surface antigen (HBsAg) carriers with normal liver histology. In 97% of the healthy adults, AFP levels were under 20 ng/ml, which is then regarded as the normal upper limit. Cases with supranormally elevated AFP levels ranged from 15-51% in chronic hepatic disorders and were 33% in acute hepatitis. None of the healthy HBsAg carriers had abnormal AFP level. HBs antigenemia was found to be related to AFP elevation in chronic active hepatitis, cirrhosis, and acute hepatitis but not in chronic persistent hepatitis and healthy HBsAg carriers. The correlation could be demonstrated only when the sensitive third generation test was employed to define seropositivity of HBsAg. Events after hepatic injury induced by hepatitis B virus, rather than the HBs antigenemia itself, are probably responsible for the association. Whether the association of HBsAg and elevated serum AFP in these nonmalignant hepatic disorders contributes to the higher risk of subsequent development of hepatocarcinoma in Taiwan is unknown and requires further long-term longitudinal study.


Use of PM-2 DNA degradation as a pharmacokinetic assay for bleomycin. The PM-2 DNA fluorescence assay has been shown to be a rapid, sensitive, and reproducible assay for bleomycin biochemical activity. The assay can detect bleomycin in human serum in the nmol range. The method measures DNA degradative activity of bleomycin and could be used to determine activity of bleomycin analogs and metabolites. The usefulness of the assay to perform bleomycin pharmacokinetic studies in cancer patients has been demonstrated. Linear regression analyses of parallel bleomycin assays with the radioimmunoassay gave a coefficient of correlation of 0.98 to 0.78 with trichloroacetic acid-treated serum. These results indicate excellent agreement between the two assays.


Ultrastructural and autoradiographic study of the effects of bleomycin on the interphase nucleus of cultured normal cells. Primary cultures of hepatocytes and epithelial endometrial cells were treated with bleomycin (10 to 200 microgram/ml) for 30 to 300 min. Structural changes were studied with a staining method which contrasts ribonucleoproteins. The earliest visible alteration was the accumulation of perichromatin granules in association with the nucleolus. This disturbance was frequently accompanied by modifications in the nucleolar architecture. After larger treatments, the most striking changes were nucleolar segregation and the appearance of spherical clear bodies in the nucleolus. In the extranucleolar area, a remarkable diminution of ribonucleoprotein fibrils and clustering of interchromatin granules were observed. Functional disturbances in the synthesis and transporting of RNA to the cytoplasm were studied by high-resolution quantitative autoradiography after labeling with tritiated uridine. Bleomycin produces a strong inhibition of RNA synthesis in nucleolar and extranucleolar areas. Important decreases of [3H]uridine incorporation were observed as early as 30 min after the administration of drug. Alterations of processing and/or transporting of RNA to the cytoplasm were found after treatments with bleomycin (100 microgram/ml) for 30 to 300 min. It is suggested that the diminution of ribonucleoprotein fibrils is related to the inhibition of RNA synthesis while the accumulation of perichromatin granules is connected to alteration of the transporting and/or processing.


Electrofocusing patterns of fucosyltransferases in plasma of patients with neoplastic disease. Electrofocusing patterns of plasma fucosyltransferases provide information concerning marrow status of patients with myeloproliferative disorders. Three enzymes were detected in normal plasmas using an acceptor terminating in the sequence N-acetylglucosamine-galactose. The enzyme which focused at pH 4.7 was elevated during rapid proliferation of myeloid cells, e.g., acute myelogenous leukemias and certain infectious diseases. Activity at pI = 5.1 was decreased in acute myelogenous leukemia patients, and from other observations, appears related to the level of erythropoietic activity. Acceptor studies show this enzyme to be specified by the H gene. A third enzyme focused at pH 5.5 and appeared to be correlated with a later step in granulocytes maturation. Two other plasma fucosyltransferases (pl = 5.6 and 8.3) were detected with a high-molecular-weight acceptor terminating in N-acetylglucosamine. This activity was markedly elevated during regeneration of a normal marrow population during drug-induced remission of acute myelogenous leukemia. Additional isoenzymes were detected, using this acceptor, in plasma of patients with certain solid tumors and multiple myeloma. However, the new isoelectric points observed (pH 6.0, 6.9, and 7.8) suggest these enzymes are probably not derived from hematopoietic tissues.


alpha-Fetoprotein levels and hepatic alterations during chemical carcinogenesis in C57BL/6N mice. Hepatocellular carcinomas were induced by administration of acetylaminofluorene or chlordane to C57BL/6N mice. Lesions which closely resembled the neoplastic nodule described as a putative premalignant lesion in rats were evident. alpha-Fetoprotein elevations were noted only in the presence of the malignant lesions for both carcinogens. In this regard, the responses of these mice were similar to those seen during spontaneous hepatocarcinogenesis.


Differential expression of alpha-fetoprotein and gamma-glutamyltranspeptidase in chemical and spontaneous hepatocarcinogenesis. The expression of two markers of fetal liver, alpha-fetoprotein (AFP) and gamma-glutamyltranspeptidase (GGT), was studied in chemical and spontaneous hepatocarcinogenesis in mice. Serum AFP concentration increased within 3 weeks 3 weeks from the commencement of feeding of o-aminoazotuluene. This early elevation subsided about 3 months after the beginning of the administration of the carcinogen. A new, sustained elevation of the serum AFP level followed at 5 to 6 months accompanied by the appearance of liver tumors. In immunofluorescence, some small oval cells and scattered adult-type hepatocytes contained AFP during the early stage of chemical carcinogenesis. During the later phase, AFP was detected in a few of the nodular areas, in solitary hepatocytes, and in groups of carcinoma cells. GGT activity in the liver increased within 1 week after the carcinogen regimen was started, preceding the early increase of AFP production. At the final stage, the chemically induced hepatomas contained about 80 times more GGT than did normal liver. In histochemical staining, proliferating oval cells and small areas of hepatocytes stained for GGT during the early weeks, and later most nodules, small areas of nonnodular parenchyma, and carcinomas contained GGT. During spontaneous carcinogenesis in male C3HeB/FeJ mice, premalignant lesions, accompanied  by a slight increase of serum AFP, precede the appearance of liver tumors. No cells staining for AFP were detected during this early stage. Once overt liver cancers had developed, AFP was readily detectable in the tumors and was localized to some but not all carcinoma cells. The corresponding serum AFP levels were highly elevated. In contrast to the high levels of GGT found during chemical carcinogenesis, no elevation of GGT was found in livers at various stages of spontaneous carcinogenesis, including cancers in eight individual mice. These results indicate that the production of AFP and GGT is not turned on as a single "genetic package," and that these two markers differ in their behaviour in liver carcinogenesis.


Modulation of cellular interactions between C3H/10T1/2 cells and their transformed counterparts by phosphodiesterase inhibitors. It has been demonstrated previously that nontransformed C3H/10T1/2CL8 mouse embryo fibroblasts (10T1/2) can induce a state of reversible growth inhibition in cocultured malignantly transformed mouse fibroblasts and that this inhibition is modulated by serum concentration. The present study suggests that cyclic nucleotides may be implicated in this intercellular communication. The phosphodiesterase inhibitors theophylline, caffeine, and 3-isobutyl-1-methylxanthine (IBX) at concentrations of 10(-3) M, maintained continuously, were all found to inhibit the expression of 3-methylcholanthrene-induced malignant transformation when added 7 days after removal of carcinogen. IBX was the most potent, causing 100% inhibition at 10(-4) M and 70% inhibition at 10(-5) M. This inhibition was partially reversible in the former case and completely reversible in the latter case by removal of drug. Complete inhibition by 10(-4) M IBX was still observed when treatment was delayed 21 days postcarcinogen. In reconstruction experiments, utilizing confluent monolayers of 10T1/2 cells overlaid with transformed cells, IBX caused a dose-dependent inhibition of colony size of the transformed cells. Adenosine cyclic 2':3'-monophosphoric acid (cAMP) and N6,O2'-dibutyryladenosine cyclic 3':5'-monophophoric acid potentiated this response. The presence of non-transformed 10T1/2 cells was required for this effect, since a concentration of IBX (10(-4) M) inhibitory for the growth of transformed cells in mixed cultures was without effect on the growth rate, plating efficiency, or saturation density of pure cultures of 10T1/2 cells or of their transformed counterparts. Conditioned medium removed from IBX-treated 10T1/2 cells was not growth inhibitory for transformed cells, indicating a requirement for cell-cell contact. IBX caused a dose-dependent increase in intracellular cAMP in confluent 10T1/2 cells and a more pronounced increase in cAMP concentration in the culture medium of these cells. The dose-response effects of IBX on growth inhibition of malignant cells in mixed cultures appear to correlate well with its ability to elevate cAMP levels. Thus, IBX increased the capacity of 10T1/2 cells to cause reversible growth arrest of transformed cells and appears to act in a manner analogous to the previously reported effects of serum.


alpha-Fetoprotein as a carrier protein in plasma and its bilirubin-binding ability. The bilirubin-binding ability of human alpha-fetoproteins, which were purified from fetal cord serum and from ascites fluid of a hepatoma-bearing patient, was examined by the difference spectrum and the Jacobsen peroxidase methods. The difference spectrum observed as a result of the specific binding of bilirubin to alpha-fetoprotein had a maximum at 482 nm, and this pattern was quite similar to that observed for serum albumin. The result obtained by the difference spectrum method showed that 1 mol of each alpha-fetoprotein bound 1 mol of bilirubin at pH 8.3 and that the dissociation constants of the complexes of bilirubin with fetal alpha-fetoprotein and hepatoma-derived alpha-fetoprotein were 2.6 x 10(-7) and 5.0 x 10(-7) M, respectively. The Jacobsen enzymatic method using horseradish peroxidase gave the same values for molar binding ratios and similar dissociation constants, 7.1 x 10(-7) M for fetal alpha-fetoprotein and 7.4 x 10(-7) M for hepatoma-derived alpha-fetoprotein. These results indicate that alpha-fetoprotein may function as a carrier protein for bilirubin as has been shown for serum albumin.