Histochemistry of neuronal degenerative changes.

Classical forms of degenerative changes of neurocytes, defined by Nissl and Spielmeyer as neuronal diseases were produced experimentally in rats. The individual neuronal changes were induced with the aid of X-ray irradiation, TET administration, ligation of the carotid artery followed by oxygen deficient breathing (anoxia), and by sectioning of the sciatic nerve. The so produced experimental models of neuronal diseases were the subject of morphological and cytochemical studies with special attention payed to enzymic and autoradiographic reactivity. Basing on the results of performed investigations, the author inferred that from a pathogenetic point of view, not all of the respective forms of neuronal diseases postulated by the classification of Nissl and Spielmeyer can be regarded as separate, distinct forms of degenerative changes. From the results obtained it would appear that pathogenetically distinct are only the following diseases: acute nerve cell swelling, shrinkage of perikaryons, homogenizing changes of Purkinje cells and axonal degeneration. The so called severe disease as well as the ischemic disease should be regarded as transitory forms of neuronal changes developing from shrunken neurocytes. It has also been suggested that the pathogenesis of homogenizing changes in Purkinje cells is distinct from that responsible for the development of ischemic changes in the course of cerebral anoxia and ischemia. This suggestion is substantiated by the observed differences in cytoenzymic reactivity between homogenized Purkinje cells and ischemically changed neurocytes.


Does Freund's adjuvant denature protein antigens? EPR studies of emulsified hemoglobin.

Use of complete Freund's adjuvant for production of antibodies to study protein conformation is valid only if emulsification in adjuvant does not denature protein antigens. Using electron paramagnetic resonance to observe directly the protein in situ in the opaque emulsions, we demonstrate that hemoglobin is not denatured by emulsification or storage in adjuvant for 24 hr at 4 degrees C, conditions comparable to the usual handling of antigens before immunization.


A microtiter solid-phase radioimmunoassay for hepatitis A antigen and antibody.

A microtiter solid phase radioimmunoassy for hepatitis A antigen (HA Ag) and antibody (anti-HA) was developed. The test was more sensitive than immune adherence hemagglutination for detecting HA Ag and almost as sensitive for detecting anti-HA. The specificity and sensitivity of reagents were examined and optimum conditions for the test were determined. Radioimmunoassay, immune adherence hemagglutination, and immune electron microscopy were compared for detecting anti-HA. A serologic response to HA Ag was detected in paired sera from patients with type A hepatitis but not from patients with type B or non-A, non-B hepatitis by all three techniques.


[Histoenzymologic modifications of placental tissue in experimental fetal hypotrophy in the rat].

Chronic placental vascular insufficiency in the rat was achieved by ligature of the uterine and utero-placental arteries. The fetal and placental hypotrophy observed confirmed the previous observation. However, the placental enzymatic changes showed an increasing gradient of cellular hypoxy from decidua basalis to yolk sac placenta; this gradient is unfavourable to fetuses. The strong depletion of yolk sac enzyme activities demonstrates the reduction of fetomaternal exchanges which are usually predominant in this placental area. The other parts of rat placenta appear relatively less involved in fetal nutrition. Further studies are necessary to understand the changes observed in these activity stainings, such as: oxidoreductases II, acid phosphatase and 5-nucleotidase.


Neuraminidase reversal of resistance to lysis of herpes simplex virus-infected cells by antibody and complement.

The susceptibility to lysis by antibody and complement was examined in four human cell lines. The cells were infected with herpes simplex virus type 1 and lysis was assessed by the 51Cr release test by using antibodies to herpes simplex virus and guinea pig serum as a source of complement. The four cell lines were found to differ in their susceptibility to lysis, although virus replication was readily demonstrated in the different cell lines. By indirect immunofluorescence, no differences in the expression of virus antigens at the surface of the cells could be found between the different cell lines. Treatment of cells with neuraminidase markedly enhanced the sensitivity of the cells which were relatively insensitive to lysis. The enhancement of susceptibiltiy to lysis by neuraminidase occurred if cells were treated before reaction of the cells with antibody and if the cells were reacted with antibody before treatment with the enzyme. No enhancement was observed when cells were reacted with antibody and complement before neuraminidase treatment. Neuraminidase treatment did not seem to enhance appreciably the quantity of antibody which reacted at the cell surface. The observations suggest that surface properties of certain cells render the cells resistant to lysis by antibody and complement and that the resistance to lysis can be abrogated by treating the cells with neuraminidase.


Regulation of antibody response in vitro. X. Biphasic effect of cyclic AMP on the secondary anti-hapten antibody response to anti-immunoglobulin and enhancing soluble factor.

The effect of elevation of an intracellular cyclic AMP level on in vitro anti-hapten antibody response was studied, by using mesenteric lymph node cells of rabbits which were primed with dinitrophenylated Ascaris antigen (DNP-Asc) or DNP-ragweed antigen (DNP-Rag). The anti-hapten antibody response was induced by stimulation of the primed B cells by either DNP-heterologous carrier conjugate or anti-immunoglobulin (anti-Ig) for 24 hr (first stage), followed by 6-day culture of the activated cells in the presence of nonspecific enhancing factor (second stage). The stimulation with anti-Ig induced IgG anti-hapten antibody response and enhanced the formation of total IgG. Addition of dibutyryl cyclic AMP or aminophylline with anti-Ig or DNP-heterologous carrier during the first stage enhanced IgG anti-hapten antibody response. The optimal concentration of these reagents for the enhancement was 5 x 10(-4) M to 10(-3) M. The presence of 5 x 10(-6) M prostaglandin E1 during the first stage also enhanced the antibody response. Similarly, the presence of dibutyryl cyclic AMP or aminophylline during the stimulation of DNP-Rag-primed cells with homologous antigen (first stage) enhanced the antibody response. If the same concentration of dibutyryl cyclic AMP or aminophylline was added together with enhancing soluble factor during the second stage after the stimulation of the primed cells with anti-Ig, both the antibody response and the formation of IgG were suppressed. The antibody response of DNP-Rag-primed cells stimulated with homologous antigen was also suppressed if dibutyryl cyclic AMP or aminophylline was added during the subsequent culture (second stage). Evidence was obtained that suppression of antibody response by cyclic AMP during the second stage is probably due to inhibition of the proliferation of B cells. Neither of these drugs suppressed the formation of enhancing soluble factor from the carrier-specific cells stimulated with the homologous carrier. The results obtained in the present experiments suggested that stimulation of hapten-primed B cells with anti-gamma chain in the presence of an optimal concentration of dibutyryl cyclic AMP resulted in the formation of a significant amount of IgG anti-DNP antibody without participation of T cells.


Cell mediated immunity to herpes simplex in man. IV. A correlation of lymphocyte stimulation and inhibition of leukocyte migration.

The cell mediated immune response to herpes simplex virus was assessed by a modification of the leukocyte migration inhibition test, developed to increase its sensitivity and correlated with the in vitro stimulation of lymphocytes. The introduction of a preincubation step allowed us to demonstrate significant inhibition of leukocyte migration in 15 out of 24 subjects susceptible to recurrent herpes labialis. This was not present in any of the 10 subjects without such infections. However lymphocytes from all 24 subjects demonstrated stimulation in response to herpes virus antigen. The degrees of lymphocyte stimulation and migration inhibition in the susceptible subjects were well correlated and do not support the suggestion that there is a focal defect in this aspect of the cell mediated immune response to herpes simplex virus in those patients.


Turnover of xanthoma cholesterol in hyperlipoproteinemia patients.

The turnover of xanthoma cholesterol was measured in 9 hyperlipidemic and one normocholesterolemic patients. Sequential biopsies of the xanthomas were obtained 13 to 364 days after the administration of isotopic cholesterol and were then analyzed for cholesterol specific activity. A total of 34 xanthomas of 3 different types - 10 tendon xanthomas, 3 tuberous xanthomas, and 21 xanthelasmas - comprised the material for analysis. The cholesterol specific activity ratio of tendron xanthomas to that of the plasma varied from 11 per cent at 21 days to a maximum of 543 per cent at 122 days after the intravenous administration of isotopic cholesterol. This ratio declined to 426 per cent at 182 days and was still 131 per cent at 364 days. Similarly, the cholesterol specific activity of xanthelasmas increased gradually. In most instances, the xanthelasma cholesterol attained isotopic equilibration with plasma cholesterol by about 50 days but varied from patient to patient (minimum time, 46 days and maximum time, 91 days). The cholesterol content of xanthomas ranged from 10.7 to 197.0 mg per gram of dry weight of the tissue. Sixty-one to 87 per cent of the total xanthoma cholesterol was esterified. No other sterols were identified in these xanthomas. Thus, the cholesterol of 3 types of xanthoma readily attained isotopic equilibration with the plasma cholesterol which suggested total exchangeability of cholesterol between plasma and xanthomas. The uptake of cholesterol by the xanthomas from plasma was rapid considering the large mass of cholesterol in the lesions. The turnover of xanthoma cholesterol was intermediate between that of the rapidly exchangeable pool and of the slowly exchangeable pool of body cholesterol. Comparison of these results with those obtained in human advanced atheroma suggest that the turnover of xanthoma cholesterol and atheroma cholesterol are quite different.


A study on the reactions of tubercidin triphosphate with myosin and actomyosin.

The triphosphate ester of tubercidin (tubercidin triphosphate, TuTP) was synthesized. This is an analog of ATP in which a CH group replaces the N-7 of the adenine ring. The rate of TuTP hydrolysis by myosin in the presence of Mg2+ was the same as that of ATP in the 10(-7)-10(-3) M range, whereas the increment in the optical density  of myosin ihe 290mmu region caused by TuTP was twice that caused by ATP. TuTP is hydrolyzed by actomyosin faster than ATP, the value of Vmax being about 4 times larger while the Km values were of the same order of magnitude. The rate of superprecipitation induced by TuTP was 50% of that caused by ATP at nucleotide concentrations of 3-60 muM. A similar difference was observed with respect to the rate of tension development by glycerol-extracted rabbit psoas fibers upon addition of these two substances. Substitution of ADP by tubercidin diphosphate (TuDP) in F-actin did not affect the rate of superprecipitation or enzymic activity of actomyosin.


In vitro study of chorionic and ectoplacental trophoblast differentiation in the mouse.

Mouse chorioallantoic pre-placental structures alone or in association with the embryo were explanted during the 9th day of gestation (7-somite stage) and cultured in a static medium for 24 to 48 h. From the subsequent morphological study of trophoblast differentiation, using both light and electron microscopy, we draw the following conclusions. 1. The allantoic mesoderm cells migrate inside the trophoblastic population but they do not differentiate a capillary network and trophoblast cells phagocytose the existing foetal erythrocytes. 2. In the absence of allantoic mesoderm, chorionic trophoblast cells remain undifferentiated. 3. The development of the chorionic trophoblast is modified in that chorionic trophoblast cells fail to establish close junctions with ectoplacental trophoblast, and some chorionic cells initiate the formation of multinucleated syncytia. The genesis of these syncytia is discussed.


Regulation of the T-cell response to ectromelia virus infection. I. Feedback suppression by effector T cells.

Spleen cells and serum from mice immunized with ectromelia virus suppressed the immune response to infectious virus when transferred intravenously into recipient mice given an immunizing virus dose. The suppression was reflected in decreased cytotoxic T-cell activity directed against H-2 compatible virus-infected target cells in the spleens of recipients. Suppression was observed when immune cells or serum were transferred 1-2 h or 1 day after immunization of recipients, but not 2 days after, and was maximal when 6-day immune spleen cells were used as suppressor cells. H-2 compatibility between donor and recipient mice was necessary for suppression to be expressed. Use of recombinant mice showed that I-region compatibility was neither sufficient nor necessary, and that D-region compatibility was sufficient. Specificity of suppression was suggested by the finding that cells and serum from mice immunized with Listeria monocytogenes, a bacterium, had no suppressive activity on the antiviral response. Anti-theta treatment eliminated the ability of immune cells to suppress, and the suppressive effect was not markedly dose-dependent with respect to both cell dose and virus dose under the conditions employed. Virus levels in the spleens of recipients were significantly reduced after injection of immune cells. Adult thymectomy had no effect on the primary immune response to ectromelia virus infection, thus indicating no role for T1 cells in the suppressive mechanism. The results obtained therefore suggested that suppression in this system was due to effector T cells which triggered clearance of virus (and thus, of virus-induced antigens) necessary for the induction of precursors of effector T cells, and that this simple feed-back mechanism normally plays an important role in the regulation of the primary immune response to ectromelia infection at the level of precursor induction. The existence of other postinduction regulatory mechanisms, however, is unknown and under investigation.


Macrophages genetically resistant to mouse hepatitis virus converted in vitro to susceptible macrophages.

Genetic resistance to mouse hepatitis, which resides largely in the macrophages of resistant C3H mice, may be altered by exposing the cells in vitro to fluid from allogeneic mixed lymphocytes. A 1,000-fold increase in susceptibility was produced in these genetically resistant cells by exposure to this fluid. This presumed lymphokine was effective without producing any change in host adaption of the virus.


Influence of auxiliary enzymes on the spectrophotometric measurement of alanine aminotransferase and aspartate aminotransferase activities.

We investigated the enzyme activity of the blank in the spectrophotometric determination of the aminotransferase activities and aspartate aminotransferase activity. 6 lactate dehydrogenase and 3 malate dehydrogenase preparations from different manufactures and from different organs showed additional and contaminating activity. The additional activity depends upon the 2-oxoglutarate concentration. The contaminating activity is caused by alanine aminotransferase and aspartate aminotransferase in the auxiliary enzymes. We propose that exact definitions must be given for the auxiliary enzymes in the recommendations of standard determinations for enzyme activities.


[Specificity of 3 alpha, 20 beta-hydroxysteroid: nad+ -oxidoreductase (author's transl)].

The enzyme 3 alpha, 20 beta-hydroxysteroid:NAD+ oxidoreductase (EC 1.1.1.53) catalyses the stoichiometric reduction of 20-oxo groups of C-21 steroids by NADH, and the non-stoichiometric oxidation of 3 alpha-hydroxy groups of 5 alpha-androstane derivatives by NAD. The activity of the enzyme towards the most important C-18, C-19 and C-21 steroids of human urine was tested in the presence of NAD and NADH. The rate of reduction of 20-oxo steroids is influenced by substituents at various positions of the steroid skeleton. These studies are intended as the basis of an eventual clinical chemical application.


[Phakomatoses. Ophthalmological and genetic aspects].

Detailed ophthalmological and genetic studies on the four classical types of phacomatoses and more succint studies of twelve syndromes which can be considered as atypical forms of phacomatoses. Discussion of the association between the different forms of phacomatoses. Establishment of genetic prognoses.


Non-hypertrophic familial neuropathy associated with intention tremor. A variety of Charcot-Marie-Tooth disease?

A family with an association of sensorimotor neuropathy and intention tremor is reported. Clinical examination of 3 affected family members showed in varying degrees areflexia, muscle wasting, impairment of deep sensation with an ataxic gait, pes cavus and disabling intention tremor. Motor nerve conduction velocities were moderately slowed. A superficial peroneal nerve biopsy showed axonal degeneration without segmental demyelination or onion bulb formation. Our observation seems to indicate an association of intention tremor with the non-hypertrophic variety of Charcot-Marie-Tooth disease. It can therefore be suggested that the two classical types of Charcot-Marie-Tooth syndrome possess variants which are associated with intention tremor. This association is well-known for the hypertrophic type; our report gives an example of the non-hypertrophic type.


Congenital oculoskeletal myopathy with abnormal muscle and liver mitochondria.

We report a 22-year-old very thin man, who showed various symptoms and signs of oculoskeletal muscle weakness, episodes of myalgia, prolonged diarrhea, severe myopia, perceptive deafness, electrocardiographic and electroencephalographic abnormalities, and endocrinological abnormality. The onset was at about 3 months of age and a positive family history was strongly suggested. The light-microscopic, histochemical and electron-microscopic findings showed abnormal mitochondria not only in the skeletal muscle, but also in liver cells. From the clinical as well as morphological points of view, this case was suspected to be one of congenital oculoskeletal myopathy probably due to an unidentified systemic metabolic disorder.


Characteristics of myosin in nemaline myopathy.

Electron-microscopic, morphometric, histochemical and biochemical studies were carried out on muscle biopsies from a patient with the characteristic clinical and pathological findings of nemaline myopathy. The mean fiber diameter was decreased, and the vastus lateralis muscle biopsy consisted exclusively of slow twitch (Type I) fibers. Quantitative biochemical investigations revealed significantly low calcium uptake and ATPase activity of the fragmented sarcoplasmic reticulum and decreased myosin ATPase activity. The electrophoretogram of myosin showed an abnormality in the light chain pattern which could not be explained by a disproportion of normal fiber types.


The effects of dbcAMP on adreanl chromaffin cells in organotypic culture.

Chromaffin cells in organotypic cultures of adrenal glands from 19 day rat embryos developed a marked increase in the volume of their rough endoplasmic reticulum (RER) and enlargement of nucleoli in response to 1 mM dibutyryl cyclic AMP (dbcAMP) treatment for 17 days in vitro. 0.1 mM dbcAMP had a similar but less dramatic effect. When dbcAMP was removed from the medium after 11 days in vitro, cells became more like control explants treated with no additives, suggesting the effects of dbcAMP were reversible. ACTH which selectively stimulates adrenal cortical cells had no effect on RER morphology of chromaffin cells. The 'neuronal-like' configuration of dbcAMP treated chromaffin cells is discussed.


Ventral horn synaptology in the rat.

The synaptology of the normal ventral horn of the rat was studied. Presynaptic boutons were classified as S (Spherical vesicles), F (flattened vesicles), and G (predominance of 700-1200 A granular vesicles). In addition, Cf, Cs, M, and T synaptic complexes were defined and quantitated. Synaptology was studied on alpha-motoneuron somata, alpha-motoneuron primary dendrites, peripheral dendrites and interneuron somata. In addition, organelles were quantified for the pre- and postsynaptic members of the synaptic complex. All counts were made on coded material and these data were analyzed statistically. Motoneuron somata had significantly more (P less than 0.01) F (58%) than S (33%) boutons. This was also the case for the motoneuron primary dendrite (P less than 0.01; F, 61%; S, 37%). The small dendrites had more (P less than 0.05) S (56%) than F (44%) boutons. More Cf bulbs (P less than 0.01) were found on motoneuron somata (9%) than on motoneuron primary dendrites (2%) or interneruon somata (3%). The C complex presynaptic bouton contained spherical (Cs) or flattened (Cf) synaptic vesicles which were attributed to the fixation employed. Cf bulbs were not observed on small dendrites. G bulbs were observed (less than 1%) only on small dendrites M bulbs were not observed on any postsynaptic structure. The boutons of the motoneuron primary dendrites (15% of total afferents) and peripheral dendrites (14% of total afferents) were frequently branched whereas there was significantly (P less than 0.01) less branching of boutons on motoneuron and interneuron somata. Small postsynaptic subsurface cisterns were associated with boutons of both the S and F type on all structures. In addition, these cisterns were observed in motoneuron somata (4%) and interneuron somata (2%) without an accompanying bouton. C postsynaptic organelles were observed in motoneuron somata (3%) and primary dendrites (1%) with an overlying neuroglial cell process and no presynaptic bouton. The synaptology of the rat ventral horn is comparable to that in the cat and monkey. However, M (R) and P bulbs were not observed in the rat. This could be due to the sampling method which indicated that synapses with less than 1% occurrence fall at the level of statistical resolution in quantitative electron microscopy. The presence of postsynaptic specialization usually associated with presynaptic boutons with no presynaptic component may be a reflection of the dynamics of normal bouton renewal in the rat ventral horn.


Chemistry of 99mTc tracers. II. In vitro conversion of tagged HEDP and pyrophosphate (bone-seekers) into gluconate (renal agent). Effects of Ca and Fe (ii) on in vivo distribution.

Sodium gluconate transforms the bone-seekers 99mTc-HEDP and 99mTc-pyrophosphate into the renal agent 99mTc-gluconate. In these in vitro processes, pyrophosphate is displaced faster than HEDP, while the HEDP reaction is accelerated by calcium ions. The in vivo distributions of these bone and kidney agents are altered by the prior local injection of calcium or iron(II). These transformation and translocation phenomena are explained in terms of the mechanistic behavior of Tc(IV) complexes.


Studies on myoinositol. Effects of caffeine and theophylline on incorporation of myoinositol into phosphatidylinositol in rat liver homogenate.

Both caffeine and theophylline, which were known to be potent inhibitors of cyclic-AMP phosphodiesterase, stimulated the incorporation of myoinositol into phosphatidylinositol in rat liver homogenate. However, cyclic-AMP had no effect. The effect of dibutyryl-cyclic-AMP differed with different concentrations. These results suggest that the stimulation cannot be explained by the increase in the amount of cyclic-AMP. This view was supported by the fact that papaverine, cyclic-AMP phosphodiesterase inhibitor, did not stimulate the incorporation and imidazole, the phosphodiesterase stimulator, did not inhibit the incorporation, and that adenylcyclase stimulators, epinephrine and glucagon, did not stimulate the incorporation.


Collagenase studies in bones of guinea pigs.

A specific collagenase has been demonstrated in culture fluids of guinea pig bones. Based on the acrylamide gel electrophoretic pattern, the mechanism of action of guinea pig bone collagenase appears similar to other known vertebrate collagenases. 2 peaks of enzymatic activity were found upon assay of fractions from gel chromatography, corresponding to molecular weights in the range of 137,000 daltons and 29,000 daltons. The enzyme is inhibited by cysteine and EDTA. Alpha1-macroglobulin is a much stronger inhibitor of the collagenase than alpha-antitrypsin. Activation studies suggested the presence of a proenzyme to guinea pig bone collagenase that was converted to an active enzyme by proteases present in the culture fluid.


Atypical hidradenoma arising in a dermoid cyst of the spinal canal.

A spinal dermoid cyst with an invasive sweat-gland tumour in its wall is reported. This appears to be the first of this type to be reported in the nervous system.


Chronic cytomegalovirus infection, immunodeficiency, and monoclonal gammopathy-antigen-driven malignancy?

An infant with severe combined immunodeficiency had normal numbers of lymphocytes which bore E rosette and surface Ig markers in an appropriate distribution. However, only minimal responsivity to in vitro stimulation by mitogens and allogenic cells, and none to antigens could be elicited; functional antibody responses were also nil, except to cytomegalovirus. Intrauterine-acquired cytomegalovirus may have caused his immune dysfunction, although the possibility of a postnatal infection cannot be excluded. Therapy with transfer factor and thymus transplantation was unsucessful in restoring immunity and may have aggravated a pre-existing monoclonal gammopathy. It is possible that the monoclonal protein was derived from B-cells transplacentally received from the patients mother.


Parathyroid function tests with EDTA infusions in infancy and childhood.

To determine the functional capabilities of the parathyroid glands, 17 EDTA infusions were given to 11 children (ages 1 month to 12 years) and to two mothers of four of the children. Serum ionized Ca fell from 4.1 mg/dl to 3.4 mg/dl. Excessive parathyroid hormone responses were elicited during seven of nine EDTA infusions in five children and in one adult with hypophosphatemic rickets, during the active phase of rickets. In four of five subjects with problems related to hypercalcemia, borderline low or undetectable PTH responses were elicited. Three relatively normal PTH responses were obtained, two in an infant after phosphate-induced hypocalcemic tetany was corrected, and one in a child with a malabsorption syndrome. The renal tubular reabsorption of phosphate was inversely related and the urinary cyclic AMP excretion was positively related to the PTH response. Thus EDTA infusions in infants and children might be useful in the identification of hyper-, normo-, or hypoparathyroid states and would be of value in defining the functional condition of the parathyroid glands in children with deranged Ca or P metabolism.


Eye movements during REM sleep: the influence of personality and presleep conditions.

Male subjects scoring high (n=17) or low (n=14) on the Maudsley Neuroticism scale were randomly assigned to a positive (n=17) or negative (ego threat) (n=14) presleep condition and then slept a single night in the laboratory. The negative condition elicited significantly more rapid eye movement activity (REM density) than did the positive condition (pless than.03). There was a significant interaction for Groups X Conditions (pless than.007) indicating that low neuroticism subjects had greater REM density in the negative condition, and less REM density in the positive condition, compared to high neuroticism subjects under either condition. The low neuroticism subjects in the negative condition showed a discrepancy between greater REM density but less admission of stress on a presleep mood questionnaire compared to high neuroticism subjects. These results support the hypothesis that REM density is a correlate of stress-induced arousal that is subject to the modifying effects of individual differences.


Effect of bradykinin on transepithelial transfer of sodium and water in vitro.

Mucosal sodium and water transfer were measured in everted sacs of rat jejunum. 2. Bradykinin (7.86 X 10(-12)M), when present in both mucosal and serosal solutions, produced a biphasic effect on mucosal sodium and water transfer. When basal transfer was low a stimulation was observed whereas an inhibition of transfer was observed when basal transfer was high. Bradykinin at concentrations of 7-86 X 10(-11) and 7-86 X 10(-13) M produced qualitatively similar effects. 3. Inhibition of transfer was observed whether bradykinin was present in the mucosal, serosal or both solutions. Stimulation of transfer was observed only when bradykinin was present in the serosal solution. 4. Theophylline (1 mM), alone inhibited water transfer at high and intermediate levels of basal transfer, and significantly potentiated the inhibitory effect of bradykinin (7-86 X 10(-12)M) on water transfer at intermediate levels of control transfer. 5. Cyclic AMP (1 mM) inhibited water transfer when basal transfer was high. Dibutyryl cyclic AMP (1 mM) inhibited water transfer at all levels of basal transfer. Dibutyryl cyclic AMP (1 mM) and bradykinin (7-86 X 10(-12)M) together produced a significantly greater inhibition of water transfer than either agent alone, at intermediate basal transfer. 6. It was observed that the action of bradykinin upon sodium and water transfer consists of two different and opposing effects. It is possible that the inhibitory effect of bradykinin upon water transfer is related to increased cyclic AMP activity.


Effects of divalent cations and of catecholamines on the late response of the superior cervical ganglion.

The characteristics of the late response of the superior cervical ganglion of dogs were studied by close-arterial injection of catecholamines and divalent cations to the ganglion. 2. Dopamine, noradrenaline and adrenaline inhibit the late response as well as ganglionic activity induced by other means. The effect of dopamine is brief but that of adrenaline is prolonged. 3. Cd2+, Co2+, Ni2+, Zn2+, Hg2+ and Fe2+ markedly potentiate the late response, whereas Mn2+, Ca2+ and Mg2+ inhibit it. 4. may potentiate ganglionic activity triggered by other ganglionic stimulants. The Cd-augmented activity may be blocked by a ganglion-blocker which is specific to the stimulant. 5. CdCl2 may exhibit a direct ganglion-stimulating action on a ganglion which shows prominent late responses and has been conditioned by tetanic preganglionic stimulation. 6. CdCl2 and MnCl2 may inhibit ganglionic transmission by suppressing acetylcholine release from presynaptic nerve terminals. 7. It is concluded that the late response represents the late discharges of ganglion cells, which are very sensitive to inhibition by CaCl2 and MnCl2 and may be potentiated by CdCl2.


Effects of physiologic alterations on binomial transmitter release at magnesium-depressed neuromuscular junctions.

1. Transmitter release from Mg2+-treated frog neuromuscular junctions can be described by binomial statistics. Good agreement between the observed amplitude-frequency distribution of e.p.p.s. and that predicted by binomial statistics is observed with relatively low concentrations of Mg2+. Conversely, good agreement is found with Poisson predictions when higher concentrations of Mg2+ are used to depress transmission. 2. Binomial analysis at these junctions shows that Mg2+ reduces quantal content (m), the probability of release (p) and to a lesser extent the available stores of transmitter (n). Raising Ca2+ causes an increase in n and p and a small but significant increase in n. K+ increases m and p but not n. 3. During 'frequency-facilitation' (1-6 Hz), e.p.p.s., m and n are increased but p is unaffected. 4. It is concluded that binomial statistics can be used to estimate the quantal parameters of transmitter release and that these parameters can be identified as discrete entities.


An electrophysiological study of chemical and electrical synapses on neurones in the parasympathetic cardiac ganglion of the mudpuppy, Necturus maculosus: evidence for intrinsic ganglionic innervation.

1. The cardiac ganglion of the mudpuppy is situated on a thin sheet of tissue. Two nerve cell types can be distinguished readily in the living preparation - principal cells and smaller interneurones which synapse with the principal cells. The purpose of this study was to investigate synaptic transmission and the functional organization of neuronal connections of ganglion cells with intracellular micro-electrodes. 2. Stimulation of the preganglionic, vagus, nerves evoked a large excitatory response in principal cells. About three quarters of these neurones were innervated by a single vagal axon. The remaining cells received two or more preganglionic nerve fibres. 3. The quantum content of vagal excitatory post-synaptic potentials (e.p.s.p.s) was measured. Normally, the e.p.s.p. was suprathreshold and consisted of about twenty-two quanta, whereas only about nine quanta were required to reach threshold and initiate an action potential. 4. Intracellular stimulation of principal cells evoked e.p.s.p.s in neighbouring principal cells. The responses were blocked by cholinergic antagonists. These potentials were caused by excitation of principal cell axon collateral synapses. 5. Principal cells also formed electrical junctions with each other. These electrical junctions were very weak. Although they transmitted slow potential changes, only a small response was recorded in one cell when an electrically coupled neighbouring cell fired an impulse. The resistance of the electrical junction between principal cells was calculated to be about 5-8 X 10(8) omega. 6. Stable penetrations of interneurones were only rarely achieved, making it difficult to study their functional relationship to principal cells. Action potentials were recorded from interneurones in a few instances. 7. These data demonstrate that parasympathetic ganglion cells in the heart of the mudpuppy receive innervation from more than one source involving both chemical and electrical synapses, and that some of the synapses are intrinsic to the ganglion.


The acetylcholine sensitivity of the surface membrane of multiply-innervated parasympathetic ganglion cells in the mudpuppy before and after partial denervation.

1. The surface chemosensitivity to iontophoretically applied acetylcholine (ACh) of single nerve cells in the cardiac ganglion of the mudpuppy was examined. 2. Some synapses on the neurones can be recognized in the living preparation with differential interference contrast optics. Identified synaptic regions of the ganglion cells were more sensitive to ACh than were other areas. The mean sensitivity of synaptic areas was 509 mV/nC, but that of random spots on the cell surface (which were mainly non-synaptic) was only 190 mV/nC. The mean rise time of ACh responses at synapses was 23 msec and at random spots was 36 msec. These data suggest that the density of ACh receptors is highest under the synapses on the post-synaptic membrane. 3. When some, but not all, of the presynaptic terminals on the ganglion cells are destroyed by cutting the vagus nerve, the sensitivity of the entire surface membrane to applied ACh increases. This increase in sensitivity reaches a maximum about 4-6 weeks after the operation. 4. Synaptic transmission at excitatory collateral synapses which remain after vagal degeneration is not altered by this hypersensitivity. 5. Neurones from ganglia which have been isolated and maintained in organ culture also become hypersensitive to applied ACh. this heightened chemosensitivity deveoops much faster in vitro; hypersensitivity in cultured ganglia becomes manifest within 4-5 days, in contrast with 4-6 weeks after vagus degeneration in vivo.


How to be a helping person.

This paper is an attempt to summarize the essential ingredients of helping someone in need. The main element needed is the helpers interest in the person, be he student, employee, Army draftee, or patient. Eleven points of interviewing are outlined along with possible uses in training situations.


Membranes of Tetrahymena. IV. Isolation and characterization of temperature-responsive smooth and rough microsomal subfractions.

Temperature-responsive microsomes of the ciliate protozoan Tetrahymena have been originally fractionated by step centrifugation on two-layered, Mg2+-containing sucrose gradients. Three fractions have been obtained, which are termed smooth I, smooth II and rough according to the appearance of the membrane vesicles upon electron-microscopy. Smooth I, smooth II, and rough microsomes exhibit RNA/protein ratios of 0.09, 0.20, and 0.34; their phospholipid/protein ratios and their neutral lipid/phospholipid ratios were 0.52, 0.43 and 0.25, and 0.17, 0.18 and 0.13, respectively. All three fractions contain equivalent, low succinic dehydrogenase and 5'-nucleotidase activities. Glucose-6-phosphatase and acid phosphatase are more concentrated in smooth I membranes than in rough membranes. The reverse is true for ATPase. The smooth II membranes occupy an intermediate position except that their ATPase activity is the lowest of the three fractions. The specific activities of these enzymes of the three microsomal fractions are compared to those of homogenates of whole cells. Thin-layer chromatography reveals a very similar polar and nonpolar lipid pattern of the three microsomal fractions. The major phospholipid compounds are phosphatidlethanolamine, glycerideaminoethylphosphonate and phosphatidylcholine, while diglycerides, an unknown NL-compound, and triglycerides are the major apolar lipids. Gas liquid chromatography shows that the fatty acids are mainly even-numbered ranging between C12 and C18. The smooth I, smooth II and rough membranes contain 65.2, 69.3 and 72.7% unsaturated fatty acids in their polar lipids, whereas only 52.7, 49.7 and 48.3% unsaturated acids are found in their apolar lipids, respectively. The fatty acids are more unevenly distributed among the individual polar lipids than in the apolar ones.


Measure of enzymatic activity coincident with 2450 MHz microwave exposure.

Enzyme preparations were exposed to microwave radiation at 2450 MHz and enzymatic activity was simultaneously monitored spectrophotometrically with a crossed-beam exposure detection system. Enzymes studied were glucose 6-phosphate dehydrogenase from human red blood cells and yeast, adenylate kinase from rat liver mitochondria and rabbit muscle, and rat liver microsomal NADPH cytochrome c reductase. No difference was found between the specific activity at 25 degrees C of unirradiated controls and enzyme preparations irradiated at an absorbed dose rate of 42 W/kg.


A review of adenoviruses in the etiology of acute hemorrhagic cystitis.

In a comparison of children with acute hemorrhagic cystitis it was noted that 51 per cent of those in Sendai, Japan and 23 per cent of those in Chicago, Illinois had adenoviruria during their illness. These 2 populations are the only ones in which the disease has been studied and reported. In both populations male outnumbered female patients, with a ratio of 3.4 to 1 in Japanese children and 2.3 to 1 in American children. Adenovirus type 11 was isolated exclusively in both studies, with the exception of 2 American children who had adenovirus type 21 in the urine. While the Japanese children experienced their illnesses for approximately 2 weeks American children had symptoms for only 4 to 5 days. Virus shedding continued throughout the illness in both populations. The adenovirus 11 strains from American children were antigenically similar to the prototype adenovirus type 11 strain as measured by cross hemagglutination-inhibition procedures. Adenovirus antigen was identified by immunofluorescence in exfoliated bladder epithelial cells from patients with acute hemorrhagic cystitis. Studies to date indicate that adenovirus type 11 is the etiologic agent in some but not all pediatric cases of acute hemorrhagic cystitis.


Vidarabine therapy for severe herpesvirus infections. An unusual syndrome of chronic varicella and transient immunologic deficiency.

Six patients with severe herpesvirus infections were successfully treated with vidarabine. One patient had a previously undescribed syndrome of chronic cutaneous varicella infection of eight months' duration, associated with transient but complete duppression of lymphocyte response to conconavalin A. Other diagnoses were severe varicella pneumonia, progressive cytomegalovirus pneumonia associated with acute lymphocytic leukemia, herpes simplex encephalitis, severe zoster associated with stage IV lymphoma, and disseminated herpes simplex in a patient receiving high doses of steroids. All patients showed cessation of new lesions or abrupt clinical improvement between days 2 and 4 after initiation of therapy, and all were cured of their clinical infection. Dramatic improvement in all of our patients and the minimal toxicity observed make vidarabine suitable for use in severe herpesvirus infections.


Hepatic changes in young women ingesting contraceptive steroids. Hepatic hemorrhage and primary hepatic tumors.

Primary hepatic tumors developed in 13 young women who had been ingesting contraceptive steroids. Nine tumors were benign, and four malignant. Six tumors ruptured spontaneously and caused life-threatening hemorrhage. Benign tumors were successfully resected in eight women. One woman died during hepatic resection for malignant hepatoma; in two other cases of hepatocellular carcinoma, tumors were nonresectable. In two patients, the tumor was left in situ after neoplasm had been excluded by biopsy. The increase of such tumors in women of childbearing age warrants the suspicion that these contraceptive steroid hormones play an etiologic role.


Clostridia in soil of the Antarctica.

From the soil in the area around the Syowa Station, the East Ongul Island, the Antarctica, a total of 193 strains of clostridia were isolated and identified. It was surprising that the soil samples taken from the places which were considered to be scarcely contaminated by human beings and animals contained many clostridia. One hundred and fifty-five strains were assigned to 11 species, including C. perfringens, C. bifermentans, C. sordellii, C. sporogenes, C. plagarum, C. paraperfringens, C. septicum, C. tertium, C. cadaveris, C. butyricum and C. felsineum, but 38 strains remained unidentified. C. perfringens, C. bifermentans and C. sordellii were isolated very frequently and C. sporogenes less frequently. All the strains of C. sordellii were nonpathogenic and had almost the same characteristics as those of C. bifermentans except for the attitude in the urease test. The peculiar distribution and characteristics of the clostridia in the Antarctic soil were discussed in comparison with those found in the soil in Japan.


Monolayer cell culture of human pancreatic beta cell tumor: effect of glucose and somatostatin on insulin release.

A human pancreatic beta cell tumor was maintained in monolayer cell culture for 80 days. The culture was terminated because of bacterial infection. Probably because extensive trypsin-collagenase dissociation was unnecessary, the dissociated cells attached much more quickly to the surface of the culture flask than do rat pancreatic cells obtained by enzymatic dissociation. Insulin release not only oscillated widely during the first 40 days of culture but also showed a decline from 380 mU the first week to about 50 mU/week the seventh week. For some unknown reason fibroblast overgrowth was not a major problem. Reduction of the medium glucose concentration from 16.5 mM to 5.5 mM did not alter insulin release rate. At glucose concentration of 16.5 mM, somatostatin 1.0 mug/ml reduced insulin release by 40%. From our previously reported studies on the effect of somatostatin on insulin release by monolayer cell cultures of rat endocrine pancreas, we conclude that the constant release of insulin by the tumor cells is relatively nonstimulated. We have confirmed that monolayer cultures of human pancreatic beta cell tumor do not represent a good model for normal human beta cell function because of the major shortcoming of an apparent inability to recognize glucose as a secretogogue.


Dysuria in women.

Only 46% of 84 women with dysuria were found to have significant bacteriuria while 8% had gonorrhea, 1% had herpes progenitalis and 17% had monilia and Trichomonas vaginitis. The remainder were considered to have the urethral syndrome. Neither pyuria, microscopic bacteriuria, nor any single subjective or objective datum definitively predicted cystitis at the initial visit. Therefore, we advise formulating the problem as "dysuria" at the initial evaluation even though one treats with an antimicrobial. The initial workup should include a routine urinalysis, gram stain of the urinary sediment, urine culture, an examination of the urethra and labia as well as a urethral culture. Complete pelvic examination is useful in documenting gonorrhea, herpes progenitalis or vaginitis. If the pelvic examination is deferred and the initial urine culture is negative, reevaluation of the dysuria should include a pelvic examination.


[Clinical features and pathology of Resochin - implantation in the cornea (author's transl)].

The long-term therapy with Resochin leads in a proportion of cases to reversible changes in the cornea. There is no direct relationship between the total dose used and the keratopathy. Point-like "Infiltrations" can be identified histologically in the cytoplasm of the epithelium and also vacuolised cells and cell necrosis, mainly in the surface epithelial layers. Under the electron microscope one can see, alongside intact cells, cells with multiple lyosomal inclusions, surrounded by a membrane, and myelin figures. The changes described are probably to be seen as after-effects of the influence of the drug.


[Influence of somatastatin on oral glucose tolerance in autonomous hypersecretion of growth hormone, prolactin or insulin (author's transl)].

Oral glucose tolerance tests (OGTT) were performed for two subsequent days in 4 patients with active acromegaly, 2 patients with prolactin-producing pituitary adenomas and one insulinoma patient. Thirty minutes before the second OGTT 250 mug of somatostatin were injected intravenously as a bolus followed by a somatostatin infusion (500 mug) over 21/2 hours. The OGTTs were pathologic due to the hGH- and hPRL-induced insulin antagonism; they could not be normalized or improved by somatostatin. Only the peak of the blood sugar curve was shifted from one to two and a half hours after glucose administration; insulin and hGH levels were regularly suppressed after somatostatin whereas hPRL remained unchanged in most instances. Gastrin levels increased in all patients during the OGTT, the increase was suppressed in 4 patients. These findings show that the pathologic glucose tolerance due to insulin antagonism could not be improved by somatostatin in contrast to the deteriorated glucose tolerance in insulinopenic states.


[Diarrhea and islet cell tumor. Clinical and morphological report on 2 observations].

The combination of diarrhea with an islet cell tumor (Verner-Morrison Syndrome) is a clinical picture presenting many diagnostic problems. It is probable that larger numbers of cases will be encountered with more widespread knowledge of this disease. The clinical features and the pathologic-anatomical findings of two characteristic cases are reported and discussed.


[The Verner-Morrison syndrome. The clinical picture and pathologic anatomy].

The Verner-Morrison Syndrome is a clinically defined entity caused by an islet cell tumor of the pancreas. More than 60 cases have been described so long. The syndrome is characterized by diarrhea, hypokalemia and hypochlorhydria. In addition to a diabetic disposition, raised calcium levels and skin alterations may be present. The diagnosis is a clinical one. A pancreatic tumor should be searched for and removed. Morphologically a benign and a maligne islet cell tumor or a diffuse hyperplasia of the islets of Langerhans can be found. Until now identification of the tumor cells has not been possible. There seems no doubt that the tumor cells produce a peptide hormone. Secretin, gastric inhibitory polypeptide, vasoactive intestinal polypeptide and combinations of hormones are discussed. The results are contradictory. Theories concerning the formal and causal pathogenesis are only incomplete and unproved up to now.


An ultrastructural study of nuclear and centriolar configurations in multinucleated giant cells.

Multinucleated giant cells were examined with electron microscopy from (1) four peripheral giant cell granulomas of the jaws, (2) a central giant cell tumor of the maxilla, (3) five giant cell tumors of tendon sheath, (4) experimentally induced foreign body granulomas in rats, (5) a virus-induced sarcoma, and (6) osteoclasts from the mandibles of rats and (7) the femurs of hamsters. The purpose of the study was (1) to examine the location and arrangement of the nuclei and the centrioles of the multinuclueated cells and compare them with the osteoclast which has previously been thought to have a unique arrangement and (2) to observe whether the physical location of the centrioles and the nuclei were such that the nuclei could undergo mitosis within the cytoplasm. All of the multinucleated cells were found to have a similar arragnement of nuclear concentration areas and nucleus-free areas. A giant centrosphere containing multiple pairs of centrioles was found in the nucleus-free areas, unassociated with any particular nucleus. In the case of the foreign body giant cell and the osteoclast, this giant centrosphere was located very close to the foreign material or bone. When occasional single pairs of centrioles were found, they were located in the area of nuclear concentration, closely associated with a particular nucleus at the periphery. These findings have shown that a common centrosphere containing multiple centrioles is not an exclusive feature of the osteoclast as was previously thought. The findings suggest that a mononuclear cell containing a centriole pair fuses to the larger cell and maintains its centriole pair in close proximity to its nucleus for a short period of time, during which it may undergo mitotic activity. Eventually, the centrioles proceed to a common centrosphere whereas the nuclei aggregate in another area making further mitotic activity an unlikely possibility.


Zonal changes in renal structure and phospholipid metabolism in potassium-deficient rats.

Morphologic alterations and membrane metabolism were studied in the kidneys of rats fed a low potassium diet. Transmission and scanning electron microscopy following perfusion-fixation of kidneys revealed that the earliest morphologic change occurs in cells of the papillary tip in which multivesicular bodies, a specific type to lysosome, appear after 1 day. Increased depletion leads to extension of the lesion to all cells of the papilla. After 1 week, a narrow band of hyperplasia in the inner red medulla appears; this band is characterized by adenomatous proliferation of intercalated and light cells and partial obstruction of collecting tubules. These alterations and cortical growth in the normal pattern result in increased renal weight. New membrane formation for lysosomes and growing cells was studied by measuring the rate of [14C]choline incorporation into phospholipid in slices from five zones of the kidney. In the papilla the rate increased 39 per cent after 18 hours, the earliest change detected. After 36 hours the rate increased in inner red medulla by 28 per cent, inner cortex by 25 per cent and outer cortex by 40 per cent. [14C]choline was a specific precursor of the three renal phospholipids, phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin. The relative distribution of the label did not change with growth induced by potassium depletion. The results indicate that potassium depletion induces early increases in the formation of cell membrane phospholipid which correlate with specific morphologic changes in different zones within the kidney.


Proliferation of endothelial cells in estrogen-stimulated rat liver. A light and electron microscopic cytochemical study.

The mononuclear phagocytes (Kupffer cells) in the normal rat liver can be distinguished from the endothelial cells on the basis of their endogenous peroxidase activity in the endoplasmic reticulum and their exclusive ability to phagocytose large (0.81 mum.) latex particles. Using these cellular markers we have investigated the effects of an estrogen upon the mitotic activity and the ultrastructure of individual types of littoral cells in the rat liver. Adult female rats received a single 10-mg. injection of diethylstilbestrol, and at daily intervals up to 6 days their livers were fixed by perfusion and processed for localization of peroxidase. Mitotic figures were rare in untreated control animals, but dividing littoral cells with both positive and negative peroxidase reaction could be identified. The exclusive localization of injected latex particles in dividing peroxidase-positive cells indicated that peroxidase reaction identified the Kupffer cells not only in the interphase but also during the mitotic division. In estrogen-treated animals there was a sharp rise in the mitotic activity of littoral cells; the activity reached its peak on the 3rd day and returned to normal levels on the 6th day after the initial injection. A breakdown of the dividing cells on the basis of their peroxidase reactivity revealed that nearly the entire population of dividing cells consisted of peroxidase-negative endothelial cells. In addition, numerous hyperactive Kupffer cells containing large phagolysosomes with phagocytosed peripheral blood cells and latex particles were seen. Intermediate cell-types with cytochemical features between Kupffer cells and endothelial cells or between monocytes and Kupffer cells were not encountered. Because of the limited phagocytic capacity of hepatic endothelial cells, our observations would provide morphologic evidence in support of previous physiologic studies, indicating that estrogen treatment has little or no effect upon the particle clearing function of the reticuloendothelial system in rats. The rare but clear demonstration of dividing Kupffer cells in liver sinusoids would indicate that these cells are capable of self-replication in situ. Finally, our observations suggest that estrogens may play an important role in the pathophysiology of endothelial cells.


Abnormally large lamellar bodies in type II pneumocytes in Chediak-Higashi syndrome in beige mice.

Lamellar bodies in type II pneumocytes are markedly enlarged in beige mice. The irregular shapes of some of these enlarged specialized lysosomes suggest fusion of smaller ellipsoidal bodies to form the giant organelles, consistent with the proposal of others for the mechanism of formation of abnormal lysosomes in Chediak-Higashi syndrome. Examination of the structure of lamellar bodies by the freeze-fracture technique reveals highly ordered stacking or concentric wrapping of the constituent lamellae. The corrugated patterns evident on the surfaces of normal lamellae are also present in Chediak-Higashi syndrome lamellae. Both normal and Chediak-Higashi syndrome lamellae appear free of typical intramembranous particles. The corrugations seen in the lamellae closely resemble those seen on freeze-fracture of dispersions of lecithin in water.


[Pathophysiology of prolonged hypokinesia].

Hypokinesia is an important problem in modern medicine. In the pathogenetic effect of prolonged hypokinesia the main etiological factor is diminished motor activity; of major importance are disorders in the energy and plastic metabolism which affect the muscle system; the contributing factors are cardiovascular deconditioning and orthostatic intolerance. This is attributed to a decreased oxygen supply and eliminated hydrostatic influences during a prolonged recumbency. Blood redistribution in the vascular bed is related to the Gauer-Henry reflex and subsequent changes in the fluid-electrolyte balance. Decreased load on the bone system induces changes in the protein-phosphate-calcium metabolism, diminished bone density and increased calcium content in the blood and urine. Changes in the calcium metabolism are systemic. The activity of the higher nervous system and reflex functions is lowered. Changes in the function of the autonomic nervous system which include a noticeable decline of its adaptive-trophic role as a result of the decrease of afferent and efferent impulsation are of great importance. Changes in the hormonal function involve a peculiar stress-reaction which develops at an early stage of hypokinesia as a response to an unusual situation. Prolonged hypokinesia may result in a disturbed function of the pituitary-adrenal system. It is assumed that prolonged hypokinesia may induce a specific disease of hypokinesia during which man cannot lead a normal mode of life and work.


Human cord blood lymphocytes. Ultrastructural and immunologic surface marker characteristics; a comparison with B- and T-cell lymphomas.

The ultrastructural and surface marker characteristics of human cord blood lymphocytes were studied. These properties were compared with those in cells of patients in the leukemic phase of both malignant lymphoma, poorly differentiated lymphocytic type, and mycosis fungoides. Nuclear folding in cord blood lymphocytes was similar to that seen in lymphocytes of patients with malignant lymphoma, poorly differentiated lymphocytic type and mycosis fungoides. Surface marker characteristics of cord blood lymphocytes included increased percentages of surface IgD on cells bearing surface immunoglobulins and decreased percentages of E-rosette-forming cells. The hypothesis that both malignant lymphoma, poorly differentiated lymphocytic type and mycosis fungoides represent an arrest in the normal lymphocyte maturation sequence is discussed.


The effects of long chain free fatty acids on human neutrophil function and structure.

Neutrophils from healthy volunteers were isolated and incubated with varying concentrations of albumin-bound long chain free fatty acids. Standard in vitro function tests including phagocytosis, bactericidal activity, and chemotaxis were performed after the incubation. It was found that unsaturated fatty acid (oleic acid) caused no changes in bactericidal activity and only moderate decreases in phagocytosis and chemotaxis at very high concentrations. Saturated fatty acid (palmitic acid) produced, at high concentrations, virtually complete inhibition of chemotaxis and moderate depression of phagocytosis and bactericidal ability. Most significantly, lower concentrations of saturated free fatty acids, within the range reported clinically in various diseases, caused a marked inhibition of chemotaxis. These functional disturbances were associated with ultrastructural alterations. Neutrophils treated with oleic acid contained numerous cytoplasmic neutral lipid droplets. Neutrophils incubated with palmitic acid showed elongated cleftlike dilations of the endoplasmic reticulum and degenerative degranulated cytoplasmic areas. It is postulated that these represent crystallization of excess saturated free fatty acids or triglyceride which interfere with chemotaxis, either mechanically or by causing cell injury.


Desmoid tumor associated with cystosarcoma phyllodes of the breast.

A case of desmoid tumor of 17 years' duration associated with cystosarcoma phyllodes of 17 months' duration is presented. Good initial response to radiation therapy of the desmoid tumor was shown. Poor response of cytosarcoma phyllodes to radiation therapy was noted. A comparison between the similarities and differences of the two tumors is presented.


[Ultrastructural and cytological study of atypical fibroxanthoma].

A case of atypical fibroxanthoma of the skin is related. Ulstrastructural findings include: fibroblast-like cells, histiocytic-like cells, xanthomatous cells, and multinucleated cells. Strikingly similar findings were found by Fu and cols. (1975) in four cases of malignant fibrous histiocytoma. The authors postulated that both tumours could represent different aspects of a single proliferating process.


[Histiocytofibroma. Spontaneous regression, leaving skin atrophy].

Three cases of histiocytoma with atypical course are described by the authors. Two of them dissappeared completely or almost completely leaving a scar where some remnants of the original tumor were present. In the third patient the tumor grew deeply in the dermis leaving the same atrophic scaring on the surface. By palpation the original tumor was also detectable.


Diagnostic importance of lipoprotein-X (LP-X) in the diagnosis of cholestasis.

In 317 patients with diseases of the liver and bile tract the authors determined LP-X in the serum and the obtained results were compared with the morphological findings in liver tissue or intraoperative examination of the extrahepatic bile ducts. Int he same patients serum bilirubin, total cholesterol, alkaline phosphatase activity, gamma-glutamyltranspeptidase and leucylaminopeptidase activity were determined. In the light of these results the authors believe that detection of LP-X in the serum is a sensitive and specific method in the diagnosis of cholestasis and that it is of greater usefulness than determination of alkaline phosphatase activity. Determinations of other biochemical parameters is of small diagnostic importance in these cases.


[Xeroradiography of the neck and of the salivary glands (author's transl)].

This paper describes the possibilities of xeroradiography in the neck area. 6 clinical samples demonstrate the usefulness of this method in the diagnosis of different disorders of the neck organs. The advantage of xeroradiography is that in contrast to conventional x-ray technique all tissue densities are represented in a single image. The problems of the different radiation exposure are discussed.


[Clinical diagnosis of the auditory pathway using different acoustical evoked responses (author's transl)].

Evoked responses originating from cochlea, brain stem, and cortex are clinically used for differential diagnosis of hearing losses. The diagnostic range and the reliability of the different ERA methods are discussed. In our clinic brain stem potentials recorded by a nonsurgical method have been used as a routine audiometric test in more than 900 cases. The procedure has proved to be easier than electrocochleography and gives nearly the same information about cochlear and middle ear function if there is no VIIIth nerve or lower brain stem damage. For topical diagnosis of the lower auditory pathway additional recording of ECoG is necessary. In the examination of a cortical deafness the recording of brain stem potentials yields the same result as the electrocochleography according to Aran (Fig. 2). In a case of an Apallic syndrome (Fig. 3) brain stem potentials are found only for high intensity clicks, and latencies are abnormally increased. The cochlear potentials simultaneously recorded from the promontory are quite normal. So damage of the brain stem is confirmed.


Specific formation of arachidonoyl phosphatidylinositol from 1-acyl-sn-glycero-3-phosphorylinositol in rat liver.

The conversion of 1-acyl-sn-glycero-3-phosphorylinositol-3H into phosphatidylinositol-3H was studied using rat liver microsomal and homogenate preparations. The nature of the molecular species of phosphatidyl inositol so formed in the absence of added acyl moieties was determined after fractionating the radioactive product by means of argentation thin layer chromatography. In other experiments, the possible specificity of the microsomal acyl-CoA:1-acyl-sn-glycero-3-phosphorylinositol acyltransferase towards different acyl-CoA derivatives was investigated. Maximum conversion of 1-acyl GPI to the diacyl analogue was dependent on the addition of adenosine triphosphate and CoA when exogensou acyl groups were omitted from the incubation medium. Under these latter conditions, the tetraenoic species comprised 56-74% of the total molecular species of newly-formed phosphatidylinositol. The microsomal acyl-CoA:1-acyl-sn-glycero-3-phosphorylinositol acyltransferase showed a marked preference for arachidonoyl-CoA. The present results suggest that the enrichment of rat liver phosphatidyl inositol in arachidonic acid may arise when 1-acyl-sn-glycero-3-phosphorylinositol is acylated to form phosphatidylinositol.


Effect of dietary polyunsaturated pork on plasma lipids and sterol excretion in man.

Pork, enriched in linoleic acid content, was compared iwth conventional pork in the diet of three human subjects with respect to the plasma cholesterol concentration and the excretion in feces of neutral sterols and bile acids. Since the fatty acids in pork glyceride have an unusual positional distribution, the redistribution that might occur during the absorption and disposition of a fat meal was also studied. The plasma cholesterol was lower with polyunsaturated pork, the difference, 14 mg/100 ml plasma, being of the order expected from the change in polyunsaturated to saturated fatty acid ratio. On average, the excretion of neutral sterols was 57% greater with polyunsaturated than with conventional pork in all three subjects, and in this respect the results resembled the findings with polyunsaturated ruminant fats. During the absorption of pork fat, the high proportion of palmitate in the 2 position of lard triglyceride served as a useful marker, since human triglyceride carries mainly unsaturated fatty acids in that position. There were stepwise changes in the fatty acid composition at the 2 position of triglyceride as the fat was absorbed, transported through, and cleared from plasma, the palmitate being gradually replaced by oleate and linoleate. By contrast, the total fatty acid profile in the triglyceride changed relatively little, implying selective reacylation with palmitate at the 1 and/or 3 position. During the clearing of dietary triglyceride, the porcine triglyceride was thus converted to the form occuring in humans.


Lung lamellar bodies lack certain key enzymes of phospholipid metabolism.

Palmitoyl CoA-glycerol-3-phosphate acyltransferase, phosphatidate phosphohydrolase, and phospholipase A were assayed in subcellular fractions of rat lung, including lamellar bodies, the putative site of storage and secretion of lung surfactant. The specific activity of each of these enzymes in lamellar bodies was relatively low and could be entirely accounted for by a small contamination of the lamellar bodies fraction by microsomes, as quantitated by the presence of the microsomal marker reduced triphosphopyridine nucleotide cytochrome c reductase. These data indicate that lamellar bodies are not the site of synthesis of the lipid component of pulmonary surfactant by pathways involving these enzymes.


Cholesteryl ester metabolism in tissue culture cells: II. Source of accumulated esterified cholesterol in Fu5AH rat hepatoma cells.

Previous investigations had demonstrated that Fu5AH rat hepatoma cells accumulated large quantities of esterified cholesterol when grown in hyperlipemic rabbit serum. The present investigation has determined the sources of the cellular esterified cholesterol when the cells were grown in hyperlipemic serum. Cellular esterification of endogenous and exogenous free cholesterol contributed 10% and 30%, respectively. The remaining 60% of the accumulated cellular esterfied cholesterol was derived from exogenous (serum) cholesteryl esters. Evidence for the hydrolysis of a portion of the incorporated esterified cholesterol is presented. A stimulation of free cholesterol incorporation and cellular esterification is elicited by hyperlipemic serum and serum lipoproteins when compared to normolipemic serum present at equivalent exogenous cholesterol concentrations. The effect of hyperlipemic serum is reduced by Tween-80 and Triton WR-1339. Comparative data on esterified cholesterol accumulation, free cholesterol incorporation, and cellular cholesterol esterification in Fu5-5 rat hepatoma cells, L-cells, and rabbit aortic medial cells are presented.


Interaction of glucocorticoid hormones with rat skeletal muscle: catabolic effects and hormone binding.

The mechanism of action of glucocorticoid hormones on rat skeletal muscle was studied by following their effect on muscle weight, free amino acid content, activity of amino acid-metabolizing enzymes, and binding to cytoplasmic receptor proteins. A significant reduction of gastrocnemius muscle and body weight occurred following administration of cortisol, triamcinolone diacetate, and triamcinolone acetonide to adrenalectomized rats. Treatment with triamcinolone diacetate also reduced the level of several free amino acids and enhanced the activity of a myofibrillar protease in skeletal muscle. The hormone had, however, no effect on the activity of various enzymes involved in amino acid catabolism in muscle. In nephrosis, another condition of muscle wasting, the level of several muscle amino acids were also reduced to a lesser extent. Cortisol and triamcinolone acetonide, both of which induce muscle wasting, were found to bind to two distinct cytoplasmic proteins in muscle. Binding of the labeled hormones was followed at 0 C and could be observed in presence of a 1000-fold excess of the catabolically inactive steroid epicortisol. Binding of 3H-triamcinolone acetonide. In vitro competition experiments further suggest a correlation between steroid binding to the 3H-dexamethasone or 3H-triamcinolone acetonide site and their potency to induce muscle catabolism. It is concluded that skeletal muscle is a direct target organ for glucocorticoids, and that muscle responsiveness involves binding of the active hormones to cytoplasmic receptor sites.


Steroidogenic responses to varying parenteral routes of administered (alpha 1-18) ACTH.

In healthy human subjects, the steroidogenic responses to a single injection of alpha 1-18 ACTH by the subcutaneous, intramuscular, and intravenous (IV) routes were assessed by measurement of plasma cortisol levels, 17-hydroxycorticosteroid excretion, and cortisol secretion rates. Results showed that regardless of the parenteral route used, alpha 1-18 ACTH at doses of 1.0 and 0.5 mg was an effective adrenocorticotropic agent; however, with the IV route, the effect was considerably prolonged. The patterns of disappearance within the plasma of alpha 1-18 ACTH and alpha 1-24 ACTH were compared following a single IV injection of these compounds labeled with 125I. ALPHa 1-18 acth produced only a slight initial rise and a slower decline in plasma radioactivity compared to alpha 1-24 ACTH, suggesting slower degradation of alpha 1-18 ACTH. Administered as a single IV injection, alpha 1-18 ACTH has a prolonged effect on steroidogenesis and has potential for being a useful diagnostic and therapeutic ACTH preparation.


On the lipolytic action of parathyroid hormone in man.

An investigation was carried out to determine whether bovine PTH stimulates lipolysis in human fat tissue, whether this action is mediated by cyclic adenosine 3', 5'-monophosphate and whether the N-terminal 1-34 peptide of bovine PTH is responsible for the lipolytic effect. Studies were also performed to determine if parathyroid extract (PTE) produces lipolysis in normal subjects and in patients with pseudohypoparathyroidism in whom there is a defect in the adenylate system in response to PTH in the renal cortex and presumably in the skeletal system as well. It was found that highly purified bovine PTH in the concentration range between 10(-9) M and 10(-5) M stimulated lipolysis in vitro by human fat in a dose-dependent manner. Significant increases in glycerol production were observed at concentrations of PTH as low as 10(-9) M and maximal increases were seen at 10(-6) M. The hormone significantly increased the concentration of cyclic adenosine 3' ,5'-monophosphate in fat tissue. The synthetic N-terminal 1-34 peptide of bovine PTH was as effective as the native hormone in stimulating glycerol production at a concentration of 10(-9) M-10(-6) M. PTE, 100 mU per kg per min for 30 min given intravenously, produced transient increases in the concentration of plasma free fatty acid in each of eight normal subjects, three patients with hypoparathyroidism and eight patients with pseudohypoparathyroidism. Purified bovine PTH also increased plasma free fatty acid in each of two normal subjects. It is concluded that PTH stimulates lipolysis in human subcutaneous fat, that this action of the hormone is mediated through cyclic adenosine 3', 5'-monophosphate and that the N-terminal 1-34 peptide portion of the hormone is responsible for this lipolytic action. Further, PTE stimulates lipolysis in vivo in man. There appears to be no defect in the adenylate cyclase system in the fat cell in response to PTH in patients with pseudohypoparathyroidism.


Alterations in levels and interrelations of plasma apolipoproteins induced by diet.

Diets high in carbohydrates increase the rates of hepatic secretion of VLDL in man. VLDL particles are secreted in greater numbers; they also tend to contain more triglycerides and to be larger in size. Comparable changes in intestinal secretion of chylomicrons and VLDL accompany fat absorption. We studied the metabolism of the ApoC apoproteins under these circumstances because in vitro studies suggest that these apoproteins may play important roles in the catabolism of VLDL. ApoA-I and ApoB were also examined because of their importance in HDL and VLDL structure, respectively. The fasting plasmas of 16 normolipemic young adults were examined before and after they had been fed high carbohydrate diets for 4-5 days. ApoA-I and ApoB were measured by radioimmunoassay. The relative proportions of ApoC-II and ApoC-III in VLDL were determined by disc gel electrophoresis. Lipids in plasma and in individual lipoproteins were determined chemically. Lipoproteins were isolated by ultracentrifugal and precipitation methods. VLDL-TG and VLDL-chol and VLDL-protein rose by factors of 2.4, 1.67, and 1.88, respectively. (Final value divided by initial value.) Since TG rose more than the others, VLDL became enriched by TG. LDL-chol fell by a factor of 0.78 while LDL-TG and LDL-ApoB remained constant, i.e., LDL became relatively enriched in TG and ApoB. HDL-TG rose by 1.42 and HDL-chol fell by 0.74, i.e., HDL too became TG enriched. Plasma Apo-A-I fell by 0.84. Thus, there were alterations in both the levels and compositions of all lipoproteins. The proportion of ApoC-II relative to ApoC-III in VLDL increased in each subject on the carbohydrate diet (mean rise was 1.55-fold). The increases in ApoC were detectable after as little as 48 hr of diet, and the same results were obtained in two people who were each tested twice on two separate occasions several months apart. Thus, the changes in ApoC were reproducible. Proportions of ApoC-II were greater in SF greater than 400 than in SF 20-400 subfractions of VLDL on ad lib diets. Proportions of ApoC-II rose in both density subfractions on carbohydrate diets. The changes in both subfractions suggest that rises of ApoC-II in total VLDL (SF greater than 20) fraction were not due solely to the accumulation of SF greater than 400 particles of unaltered ApoC composition in postdiet plasma. The changes following carbohydrate could have been due to secretion of altered lipoproteins or due to diet induced alterations in apoprotein catabolism. Three subjects were given 150 gm of corn oil to drink. Lipoproteins were analyzed, as above, at 0 and 3-12 hr after the drink. In spite of several-fold rises in the SF greater than 20 TG and proteins, the relative proportions of ApoC-II and ApoC-III remained constant. Thus, an acute dietary fat load and 2-4 days of carbohydrate diets provoked different responses. The fluctuations in the levels and relative proportions of ApoC support the notion that the ApoC group may have importance in vivo in the metabolism of VLDL.


Inhibitory effect of somatostatin on dibutyryl cyclic AMP-induced insulin and growth hormone release in human subjects.

The effect of somatostatin on the responses of blood glucose, plasma immunoreactive insulin (IRI), growth hormone (GH), and free fatty acids (FFA) to the injection of dibutyryl cyclic AMP (DBC) was studied in six normal volunteers. DBC, when injected alone, induced a rapid increase in blood glucose and plasma IRI levels, while GH concentrations showed a less marked and more delayed increase and plasma FFA showed a clear downtrend. Somatostatin infusion suppressed the GH and IRI release induced by DBC, potentiated its hyperglycemic effect and changed the pattern of FFA. These results suggest that somatostatin inhibits hormone secretion distal to the generation of cyclic AMP.


Pituitary regulation of human growth hormone binding sites in rat liver membranes.

We have studied the binding of 125I-human growth hormone (hGH) to crude 100,000 X g membrane preparations from rat liver, and have studied factors which might regulate the capacity and affinity of hGH binding sites. Membrane preparations have livers of pregnant rats bound between 8% and 18% of the 125I-hGH initially added, and 70%-80% of that bound was displaced by 1 mug of unlabeled hGH. Humans prolactin (hPrl) displaced 125 I-hGH in a manner parallel to hGH itself but with about one-third the potency. Ovine, porcine, and rat Prl, and rat and bovine GH were much less effective. Scatchard analysis of specific hGH binding by a variety of different rat liver membrane preparations revealed a single order of binding site in each case with a  binding affinity of 0.93-1.62 X 10(-9) M-1. Membranes from pregnant rats had twice the binding capacity of membranes from nonpregnant female rats, and about six times the capacity of sites present in preparations from normal adult male rats and hypophysectomized (Hx) male or female rats. Female or male rats with extremely high circulating GH an Prl levels, due to the presence of transplantable GH/Prl secreting pituitary tumors showed a significantly greater binding capacity than did the pregnant rats. Estradiol (E2) treatment (25 mug/day for 10-12 days) of normal male rats led to an increase in specific hGH binding. Treatment of hypophysectomized male rats with bovine GH (100 or 500 mug/day) +/- E2 (25 mug/day) for 5-10 days stimulated both body weight gain and the incorporation of sulfate by cartilage from the treated rats, but no significant increase was observed in the characteristics of 125I-hGH binding. These results indicate that high levels of E2, GH, and/or Prl play an important role in the regulation of hGH binding sites in rat liver membranes. The restoration of binding sites in liver from hypophysectomized rats, however, apparently requires additional factors which are as yet unidentified. The role of the hGH binding sites in the physiologic actions of GH also remains to be determined.


[Effect of endogenous respiration of Candida mycoderma on the operation of the cytochrome system and the oxidation of glucose, ethanol and acids of the Krebs cycle].

The kinetics of oxidation of glucose, ethanol, and acids of the Krebs cycle was studied in resting cells of Candida mycoderma which differed by the level of endogenous respiration (ER). The cells with ER close to zero oxidized glucose and ethanol almost at once and at a high rate. During oxidation of succinic or other acids of the Krebs cycle, QO2 was maximal only after a long period of adaptation (tad); a latent period (tlat) was observed sometimes during which assimilation of oxygen was either insignificant or not detected at all. The values of tad and tlat were much lower in the cells with high ER or upon preliminary introduction of small amounts of easily oxidized substrates (e. g. glucose or ethanol). During oxidation of succinate (citrate, malate or alpha-ketoglutarate) by the cells with ER close to zero, reduction of cytochrome c takes place only some time after the beginning of oxygen uptake. Such a delay suggests that oxidation of aforementioned substrates is initiated by some other electron carriers rather than by the cytochrome system.


Specificity of the stimulation of in vitro ribonucleic acid synthesis by guanosine 5'-diphosphate 3'-diphosphate.

The in vitro synthesis of ribonucleic acid (RNA) by S-30 extracts of Escherichia coli K-12 is stimulated from two-to fourfold by 0.16 mM to 0.32 mM guanosine 5'-diphosphate 3'-diphosphate (ppGpp) when either gammacI857St68h80 deoxyribonucleic acid (gammah80 DNA), gammah80dilv DNA or gammah80dlac DNA are employed as templates. Hybridization analysis of the 3H-RNA product transcribed from gammah80dilv DNA in the presence of ppGpp indicates that both bacteriophage- and bacterial-specific transcription is stimulated to an equivalent degree. In the absence of cyclic 3'-5'-adenosine monophosphate (cyclic AMP), correct lac-specifci RNA synthesis from gammah80dlac DNA is not stimulated by 0.32 mM ppGpp although total RNA synthesis is increased nearly twofold. In the presence of 0.5 mM cyclic AMP, correct lacspecific RNA synthesis is stimulated preferentially by ppGpp. These data suggest that ppGpp is capable of stimulating in vitro transcription in both a general and selective manner.


DNA-cellulose column chromatography of phosphorylated nucleolar nonhistone proteins.

A salt-extraction procedure was used to isolate a nucleolar nonhistone protein fraction, containing [32P]phosphoserine, from the nucleoli of Novikoff hepatoma ascites cells. These proteins are similar in amino-acid composition to whole nuclear (chromosomal) nonhistone proteins. DNA-cellulose column chromatography showed that this fraction contains DNA-binding phosphoproteins, some of which will bind only to homologous (Novikoff) nucleolar or nuclear DNA.


Cucumber mosaic virus RNA contains 7-methyl guanosine at the 5'-terminus of all four RNA species.

Purified cucumber mosaic virus (CMV) RNA was reacted with NaIO4 and then [3H]KBH4 to label ends containing exposed 2',3'-hydroxyls. The four major RNA species were then fractionated by polyacrylamide gel electrophoresis. Analysis of digests of these RNAs by ribonucleases T1 plus T2, alkaline phosphatease, snake venom diesterase and nucleotide pyrophosphatase by means of paper electrophoresis and ion-exchange chromatography indicated that the sequence m7G(5')ppp(5')N was present at the 5'-end of 75-92% of all RNA molecules. In addition, about 90% of the 3'-ends terminated in either adenosine or cytidine.


Paget's disease of the breast.

Criteria of the morphological diagnosis as well as the histogenesis of the lesions in Paget's disease are presented; the authors claim that the initial lesion occurs in the lactiferous ducts, the skin being secondarily involved. Likewise, the dyskeratotic lesions are considered as secondary to the neoplastic process, either by neoplastic induction or by mobilization and proliferation of cancerous cells from lactiferous ducts. The utility of biopsies in all cases of nipple eczema, and of the histologic investigation of the profound tissues is pointed out. In this way the canalicular starting point in the vicinity of the nipple can be noticed.


[Study of the conformational changes of serum albumin molecules within a pre-denaturation temperature range by a spin probe technique].

To study conformational changes of protein molecules in a pre-denaturation temperature range, nitroxyl radicals adsorbed by a protein are suggested to be used. A spin probe technique is specially developed to be implied for this aim by using a probe specifically bound to the bovine serum albumin molecule, it was possible to reveal a dependence of the rotation correlation time of the adsorbed radical and the environment polarity on the temperature. The data obtained testify in favour of a change occurring in the intramolecular structure of the protein under study due to a temperature change within the pre-denaturation temperature range.


[Selected carboxymethylation of ferri-hemoglobin from insect larvae Chironomus thummi thummi].

Specific modification of the monomeric fraction III of ferri-hemoglobin from insect larvae Chironomus thummi thummi (Hb CTT) was studied on histidyl residues His-G19 (pK 4,8), His-E5 (pK 7,3) and Met-H22 at different pH using iodacetamide and spin label 2,2,6,6-tetramethyl-4-bromacethyl-piperidin-1-oxyl, an analogue of bromacetate. The analysis of the products of carboxymethylation (CM) showed that at pH 5,0 two products of modification CM-(His-G19)-Hb CTT, and CM-(Met-H22)-Hb CTT were obtained. In the case of modification at pH 7,2 with a spin label dicarboxymethylatid product CM-(His-G19)-CM (His-E5)-Hb CTT is obtained. In all products the degree of modification was one spin label per mole protein. Based on the data on the primery and tertiary structures Hb CTT and the results of the investigation, different reactivity of His-G19 and His-E5, as well as the cause of the absence of the product of carboxymethylation on His-G2 have been discussed. By analizing the absorption spectra of carboxymethylated derivatives of hemoglobin in the ultraviolet and visible region, as well as from the pH dependence curves of the absorption at Soret band in the interval pH 5,5-11,5 it has been shown that carboxymethylation of His-G19 and His E5 is not accompanied by any substantial disturbance of the structures of aquous-complexes Hb CTT. Modification of Met-H22 leads to strong changes in the absorption spectrum and to the absence of pH dependence of the absorption at Soret band, which indicates a change in the aquous-complexes Hb CTT structure.


Comparative mutagenic effects of ethyl methane-sulfonate, n-methyl-n'-nitro-n-nitrosoguanidine, ultraviolet radiation and caffeine on Dictyostelium discoideum.

A high frequency of morphogenetic mutants of Dictyostelium discoideum can be induced by treatment with MNNG under conditions which result in relatively low cell killing. Six temperature-sensitive growth mutants induced by this treatment were isolated by replica plating. Among these, five showed spontaneous reversion rates of 10(-4) to 10(-5). The mutagenic activity of ems, measured for the induction of both morphogenetic and temperature-sensitive mutants, was weaker than that of MNNG and UV radiation. High frequencies of morphogenetic mutants were obtained only with doses of UV irradiation that resulted in high killing of cells or spores. Caffeine, at concentrations that slightly decreased the growth rate of amoebae in axenic medium, induced morphogenetic defects and also enhanced the mutagenic effect of UV irradiation. However, all the aggregateless clones derived from caffeine treatment that were studied reverted to the wild-type phenotype after a variable number of clonal re-isolations.


[Specific modification of phenylalanine:tRNA-ligases of E. coli MRE-600 with N-chlorambucilyl-14c-phenylalanyl-tRNA].

N-Chlorambucilyl-[14C]phenylalanyl-tRNA was used for the affinity modification of phenylalanine : tRNA-ligase from E. coli MRE-600. It has been found that N-chlorambucilyl-[14C]phenylalanyl-tRNA selectively inactivates phenylalanine : tRNA-lagase that results in formation of a covalent bond between the tRNA derivative and the enzyme at pH 5.8, 25 degrees C. The rate fall of the aminoacylation of tRNA with [14C]phenylalanine was observed after the enzyme incubation with N-chlorambucilyl-[14C]phenylalanyl-tRNA at pH 7.5, 25 degrees C. It has been shown that this modification results in a similar rate decrease of tRNA aminoacylation with [14C]phenylalanine, ATP-[32P]pyrophosphate exchange and reaction of the enzymatic deacylation of [14C]phenylalanyl-tRNA. This fact evidences in favour of the possibility of the alkylation to proceed in the proximity of the active centre of the enzyme. The covalent complex obtained seems to be an interesting model for the studies of the mechanisms involved in tRNA aminoacylation as well as for elucidation of the tertiary structure of tRNA bound with the enzyme.


Studies on chemically induced dominant lethality. I. The cytogenetic basis of MMS-induced dominant lethality in post-meiotic male germ cells.

Young adult male mice were injected intravenously with doses of methyl methanesulfonate(MMS) ranging from 25 to 100 mg/kg body weight. These males were serially mated to superovulated females from day 1 post injection to day 23 post injection. The morning after mating (about 4-6 h post-copulation) the females were sacrificed and ova flushed from the ampulla. The ova were cultured, in the presence of colchicine, for 26 h and metaphase preparations made of the first cleavage division. Chromosome analysis was done and the types, and extent, of chromosome aberrations correlated to previously published dominant lethal data at the same MMS doses and time intervals. The types of aberrations seen were predominantly double fragments (presumably isochromatid deletions), chromatid interchanges, and some chromatid deletions, as well as shattering effect on the male complement at the highest dose and the time of peak sensitivity to dominant lethal induction. When the frequency of cells containing a cytologically visible aberration is compared to the total dominant lethal data an excellent correlation is obtained. Furthermore, the frequency of highly damaged cells, agrees very well with estimated frequencies of preimplantation loss. These data strongly suggest that chromosome aberrations seen at the first cleavage stage are the basis of MMS-induced dominant lethality.


[MS2 RNA hydrolysis by snake venom phosphodiesterase and template properties of RNA produced by limited exonucleolytic action].

The action of snake venom phosphodiesterase on bacteriophage MS2 RNA under conditions of limited hydrolysis has been studied. The content of 5'-mononucleotides released during hydrolysis does not correspond to the calculated one based on the 3'-terminal nucleotide sequence of MS2 RNA. This finding suggests the occurrence of endonucleolytic splits in MS2 RNA with concomitant exonucleolytic digestion of some newly formed fragments. A high molecular weight RNA fraction, comprising unfragmented MS2 RNA with about 5-63'-terminal nucleotides removed is separated by sucrose gradient centrifugation and shown not to differ from the native MS2 RNA in its template function in a cell-free protein synthesizing system. It is demonstrated that template activity, polarity of translation, and nascent peptide chain termination on the MS2 replicase cistron are not affected by the removal of 3'terminal nucleotides of MS2 RNA. Consequently, these nucleotides are unessential in translation of native MS2 RNA.


Chemical induction of presumed dominant-lethal mutations in postcopulation germ cells and zygotes of mice. II. Sensitivity of different postcopulation-precleavage stages to three alkylating chemicals.

The relative sensitivities of various postcopulation-precleabage and pronuclear stages to dominant-lethal effects of isopropyl methanesulfonate (IMS), ethyl methanesulfonate (EMS), and triethylenemelamine (TEM) were investigated. The pattern of sensitivity differed with the chemical. IMS was most effective when pronuclear formation was already completed and the majority of the zygotes were presumably undergoing DNA synthesis. EMS, on the other hand, induced its most pronounced effects when eggs in the course of second meiotic division and zygotes in early pronuclear stages were treated. The greatest effect of TEM was observed when zygotes were treated at the early pronuclear stage. EMS and TEM, in contrast to IMS, are similar to radiations in that zygotes undergoing DNA synthesis are more resistant to them than are the early pronuclear stages. In the case of IMS, effects induced in the most sensitive postcopulation-precleavage stage were 6 to 9 times greater than in the most sensitive precopulatory dictyate oocytes or male germ cells. On the other hand, in the case of EMS and TEM, the most sensitive precopulatory male germ cells, but not the dictyate oocytes, were more sensitive than the most sensitive postcopulation stages.


Inadequate parathyroid response in acute pancreatitis.

We studied nine consecutive hypocalcemic patients with acute pancreatitis to elucidate the mechanism of hypocalcemia. Mean serum ionized calcium, 0.97 mM, was below the normal mean of 1.16 mM (P less than 0.001). Seven of eight patients tested had normal parathyroid hormone levels. All responded to parenteral parathyroid extract by increasing serum ionized calcium and urinary cyclic AMP, indicating parathyroid-hormone-responsive target organs. Calcitonin and glucagon concentrations were increased above normal in some patients, but there was no relation with serum ionized calcium. Parenteral glucagon had no significant effect on serum ionized calcium or calcitonin concentrations. These findings suggest that neither glucagon nor calcitonin was primarily responsible for the hypocalcemia, which did not produce expected increases in serum parathyroid hormone concentrations. Relative parathyroid insufficiency may account for the persistent hypocalcemia frequently observed in patients with acute pancreatitis.


Transiently reduced activity of carbamyl phosphate synthetase and ornithine transcarbamylase in liver of children with Reye's syndrome.

Since Reye's syndrome is associated with hyperammonemia, we measured the urea-cycle enzymes in hepatic tissue of 13 patients. Expressed as nanomoles of citrulline per milligram of hepatic protein per minute, mean activity of carbamyl phosphate synthetase (6.27 +/- 2.45 S.D.) and ornithine transcarbamylase (136.19 +/- 41.83) in Reye's syndrome was reduced significantly (P less than 0.005) when compared with that of 25 "normal" controls (11.54 +/- 4.24 and 307.49 +/- 94.15, respectively). Activity was maximally reduced during the first days of clinical symptoms; it returned toward normal during the following week regardless of whether the disease ended in death or recovery. The activity of the two enzymes was normal in patients with salicylate intoxication or heritable argininosuccinic acid synthetase deficiency. The apparent Km of hepatic ornithine transcarbamylase for ornithine was in the normal range in patients with Reye's syndrome (mean 0.24 mM). These observations indicate that Reye's syndrome is associated with acquired and transient dysfunction of hepatic mitochondrial urea-cycle enzymes.


Receptor-binding region of insulin.

X-ray analysis, circular dichroism, receptor binding and biological potencies of chemically modified insulins suggest that the conformation of the insulin molecule is critical to the formation of both the zinc insulin hexamer and the insulin-receptor complex. Results are consistent with an insulin receptor-binding region including many of the hydrophobic residues important to dimerisation in addition to more polar surface residues. There is a further possibility of formation of an antiparallel sheet structure between the insulin and receptor molecules in the complex similar to that between monomers in the insulin dimer.


[Topochemistry of hormone action].

The site of hormone action is the cells of target tissues. Now that ultrastructural research has revealed the complexity of eukaryotic cells, we must ask which cellular structures are involved. Some hormones act at the cell membrane, mainly via adenylate cyclase and the "second messenger" cAMP. The cytosol and the mitochondria, the main sites of intermediate metabolism and enzyme activity, are apparently not involved, although in the past interaction of hormones with enzymes has been postulated. Most of the steroid hormones act at the level of the cell nucleus, regulating transcription of specific genes and inducing enzymes or other proteins. The mechanisms by means of which this regulation is achieved are not fully understood, but it is likely that derepression is involved. The active agent seems to be a complex of the hormone with a receptor protein.


Neoplastic transformation of mouse embryo cells by virus released from tumorigenic mouse cells transformed by avian sarcoma virus B77.

Mouse C3H embryo cells were transformed in vitro by avian sarcoma virus Bratislava 77 (B77) released scantily from a mouse cell line transformed earlier by the same virus. B77 virus transformed C3H embryo cells contained B77 viral genome and were transplantable into syngeneic as well as allogeneic DBA/2J young mice in which autochthonous sarcomas were induced. Tumors in both strains of mice were virogenic. The probable reasons for an increased transformation capacity of B77 virus in mammals are discussed.


Viruses harboured in blasts of the rat myelogenous leukemia (RBA-Le), induced by chicken sarcoma virus.

It was shown that the rat myelogenous leukemia RBA-Le harbours three kinds of viruses. 1. Rat leukemogenic virus, which can be recovered from leukemic cells, medium of cultured leukemic cells and the plasma of leukemic rats. About 12% of rats inoculated when newborn with the recovered virus developed leukemia. 2. Chicken sarcoma virus, which seems to have a lowered infectivity or altered host range as the B77(RBI) virus by which this leukemia had been induced. 3. A cytopathic, hemagglutinating virus, very pathogenic for baby rats, which was shown to be closely related, if not identical in biological as well as immunological properties with Kilham rat virus. The RBA-Le leukemic cells were adapted for growth in tissue culture.


Virus infections and acute renal transplant rejection.

The date of onset of 360 acute renal transplant rejection episodes from 1969 to 1973 have been compared with the prevalence of various common viral infections and infections due to Mycoplasma pnuemoniae. A positive correlation was found for influenza B infections (r=0.43, p less than 0.01) up to 5 months before transplantation and for adenovirus infections (r=0.32, p less than 0.05) at 1 month before kidney grafting.