Link-proteins and non-collagenous proteins from normal and chondrodysplastic cartilages.

Baboon and human articular and growth cartilage was extracted with 4M guanidinium chloride in the presence of proteolysis inhibitors. After dialysis against 8M urea pH 6.8 the proteins were separated from proteoglycans by ion-exchange chromatography. The concentrated and reduced protein fractions was analyzed by SDS-PAGE. Bands corresponding to collagen and to 6 major non-collegenous proteins were found. Two of the latter were identified with the link-proteins. By using small columns and microconcentration procedures, a gel-electrophoretic analysis of link-proteins extracted from small pieces of cartilage was performed and ten cases of osteochondrodysplasias were studied. No abnormalities were detected in the following syndromes: achondroplasia, diastrophic dwarfism, thanatophoric dwarfism, Jeune disease, spondyloepiphyseal dysplasia congenita, Kozlowski syndrome, osteogenesis imperfecta, polyepiphyseal dysplasia with diabetes mellitus.


Cardiovascular and adrenergic effects of cigarette smoking during immediate non-selective and selective beta adrenoceptor blockade in humans.

The cardiovascular and adrenergic responses to cigarette smoking during acute selective and non-selective beta adrenoceptor blockade were studied in seven young healthy volunteers in a double blind cross-over fashion. Heart rate, arterial blood pressure, forearm blood flow and plasma levels of adrenaline and noradrenaline were determined before and during the terminal 5 min period of 15 min smoking test. During smoking, plasma concentrations of adrenaline increased markedly and evenly by approximately 0.3 ng/ml in all three experimental sessions. Plasma concentrations of noradrenaline remained unchanged. Propranolol, a non-selective beta blocker, caused a marked rise in diastolic and mean blood pressure and forearm vascular resistance during smoking. This response was not seen in the control series or after selective beta-1 blockage with atenolol. This difference is attributable to propranolol's blockade of adrenaline's vasodilating effect mediated by beta-2 receptors in the resistance vessels. Furthermore, atenolol attenuated the systolic blood pressure and tachycardiac responses induced by cigarette smoking by comparison with placebo. This study suggests that selective beta-1 blockers are preferable in the management of patients who are habitual smokers.


Biliary lipid composition during treatment with different hypolipidaemic drugs.

In an attempt to clarify the possible lithogenic effects of commonly used hypolipidaemic drugs, gallbladder bile was obtained from patients with primary hyperlipoproteinaemia before and during treatment with nicotinic acid (n = 13), cholestyramine (n = 19), clofibrate (n = 11), and a combination of cholestyramine and clofibrate (n = 11). Each treatment period was minimum 6 weeks, and standardized dietary conditions were obtained. Both nicotinic acid and clofibrate treatment caused an increase in biliary cholesterol concentration relative to biliary total lipids (bile acids, phospholipids, and cholesterol). During cholestyramine medication the relative cholesterol concentration fell. A combination of cholestyramine with clofibrate medication led to a decrease of bile saturation to pretreatment levels in nine of the eleven subjects. In the other two a further increase in the cholesterol saturation of the bile occurred. Treatment with nicotinic acid and clofibrate but not with cholestyramine is thus probably associated with an increased risk for development of cholesterol gallstones. It is suggested that addition of cholestyramine may be a possible way to prevent the lithogenic effect of clofibrate in patients with hyperlipoproteinaemia when not only hypocholesterolaemic but also hypotriglyceridaemic effects are wanted.


The significance of gross elevations of the erythrocyte sedimentation rate in a general medical unit.

One hundred consecutive patients with an ESR of 100 mm or more in the first hour admitted to a general medical unit were studied. Their mean age was 67 years and forty-seven were male. Three patients recovered without a satisfactory diagnosis. In thirty-three of the remainder a single diagnosis was considered responsible for the elevation of the ESR, and in the others multiple diagnoses were found. Infection was found in 60% of patients, malignancy in 28% (including 7% with myelomatosis), rheumatoid disease in 20% and renal disease in 11%. 34% of patients died within 6 months of entry into the study. In the absence of rheumatoid disease or a paraproteinaemia, elevation of the ESR in excess of 60 mm in the first hour at 1 month or longer was associated with a particularly poor prognosis. This study has shown the diagnostic implications of an ESR of 100 mm or more in the first hour and the prognostic significance of a persistent elevation of the ESR.


Hereditary deficiency of triosephosphate isomerase in four unrelated families.

Triosephosphate isomerase deficiencies in erythrocytes and leucocytes were discovered in three unrelated families by a heterozygote screening of 3000 blood samples. In addition, a family found by Schroter et al. [not published] was studied. In these four families, only heterozygote carriers were found. In the family described by Freycon et al. with hetero- and homozygote carriers of triosephosphate isomerase deficiency, the heterozygotes were reinvestigated. There was 51% of normal enzyme activity in three of the families. In the other two families the enzyme activity was 64% and 71% of normal. Two of the eleven heterozygotes, both children, were diseased, but it seems unlikely that the disorders resulted from the deficiencies. The activities of thirteen enzymes, the Km of triosephosphate isomerase for glyceraldehyde phosphate and the concentrations of metabolites were normal. Antibody titration showed normal specific activities in four families and 50% of normal in one family. No electrophoretic variant was detected. From the proved heredity, a heterozygous frequency of at least 1/1000 is indicated. A maximal frequency of 5/1000 is estimated by using further instances of triosephosphate isomerase deficiency where heredity has not yet been investigated. An explanation for the small number of known cases is that this enzyme is not routinely assayed.


Studies on cortisol metabolism during haemodialysis in man.

Plasma clearance rates and dialysance of labelled cortisol were measured in patients on chronic intermittent dialysis during and after dialysis. Plasma clearance rates during dialysis were 30--63% higher than after dialysis; dialysance was 0.3--0.7 1/h and accounted only for 20--35% of the increase of plasma clearance rates during dialysis. After bolus injection of labelled cortisol, disappearance curves were obtained in two normal subjects and two uraemic subjects on and off dialysis. Biological halftimes of the different segments of the disappearance curves and distribution volumes were calculated. The size of the inner compartment (V1) is greater in uraemia. The disappearance curves flatten after the end of dialysis, indicating an increase of biological halftime after dialysis. Plasma 17-OHCS-glucuronides dropped from 2.13 +/- 0.62 micromol/l (n = 20) at the beginning to 1.05 +/- 0.65 micromol/l at the end of dialysis and increased to 1.51 +/- 0.43 micromol/l (n = 12) within 3 h thereafter. During dialysis cortisol metabolites are removed from the blood. This may decrease inhibition of cortisol metabolism by the end products of cortisol metabolism which might explain the more rapid plasma clearance and the shortened cortisol half-life during haemodialysis.


Metabolic activity of human polymorphonuclear leucocytes: relation to ingestion rate.

The ingestion rate and oxygen-dependent metabolic activities of normal human polymorphonuclear leucocytes were measured with heat-killed Klebsiella as the particle. Since the experimental conditions were similar for each measurement, it was possible to make direct correlations between each oxygen-dependent reaction and (1) ingestion rate and (2) the other oxygen-dependent reactions. In the controls, oxygen-uptake was more reliably correlated (r = 0.960) with ingestion rates than with (in order of reliability) hydrogen peroxide produced (r = 0.860) and iodination (r = 0.858 and 0.813 for 100 and 20 micromol/l iodide respectively). Hydrogen peroxide production (r = 0.988), nitroblue tetrazolium reduction (r = 0.969) and cytochrome c reduction (r = 0.862) were more reliably correlated to oxygen-uptake than to ingestion rate, and iodination was better related to hydrogen peroxide production (r = 0.90 and 0.819 for 100 and 20 micromol/l iodide respectively) than to ingestion rate. From these findings it was possible to locate primary defects in abnormal polymorphonuclear leucocytes from individual patients with pyogenic infections, idiopathic refractory anaemia or idiopathic oesteomyelofibrosis with splenomegaly, even when several deficiencies existed.


The behavioral effects of cocaine: rate dependency or rate constancy.

The behavioral effects of cocaine were studied in squirrel monkeys trained to press a response key under an 8-min fixed-interval (FI) schedule of electric shock presentation. Overall mean rate of responding increased at 0.03--0.3 mg/kg (i.m.) and decreased at 1.0--3.0 mg/kg. Increased responding during the initial and middle periods of the fixed-interval accounted for the increase in overall mean rate; response rate during the final two min of the interval did not increase at any dose. An analysis based on response rate during individual 1-min segments of the 8-min interval showed that the rate during the interval became more uniform, and the pattern of positively accelerated responding became more linear, as dose increased. At 0.3--1.0 mg/kg, response rate was relatively constant and independent of the control, pre-drug rate of responding.


Effect of meseclazone and other non-steroidal anti-inflammatory drugs on isolated tracheal chain tone.

Meseclazone, 5-CSA and several representative NSAIDs caused concentration-dependent relaxation of the tracheal ring preparation and are listed in order of descending potency: isoproterenol greater than naproxen greater than ibuprofen greater than diflunisal greater than tolmetin approximately equal to fenoprofen approximately equal to indomethacin greater than phenylbutazone greater than meseclazone greater than 5-CSA greater than aspirin. This relaxation may be related to inhibition of prostaglandin synthetase, but relative potencies of NSAIDs in this test do not necessarily correspond to their potency in inhibiting PG synthethase in other tissue. Thus other factors may play a role.


[Photographic methods for observation of the eye-fundus of rhesus monkeys (author's transl)].

The method for observing the eye-fundus of rhesus monkeys was devised by the use of Kowa RC-2 Hand-held Fundus Camera. 1. For monochrome photography, Fuji Neopan F (ASA 32) and Fuji Neopan SS (ASA 100) films were used. The best result was obtained under the following conditions : the Strong Power Pack "50W" X 6 for Neopan F and "50W" X 3 for Neopan SS. 2. For color photography, Fuji Color (ASA 100 : Negative, Positive) and Sakura Color (ASA 100 : Negative) films were used. The best result were obtained under the following conditions : the Strong Power Pack "50W" X 2 for Fuji Color (ASA 100 : Negative), "50W" X 3 for Fuji Color (ASA 100 : Positive) and "50W" X 3 for Sakura Color. 3. Four fluorescein angiography, 0.3ml/kg of 10% sodium fluorescein was injected into the vein, and then photographs were taken under the condition of "100W" X 6. For monochrome photography (Kodak Tri-X ASA 400 : Negative), a combination of Kodak Wratten Filter 47 (excitor) X K. W. 15 (barrier) brought about the best photographic result. FOR COLOR PHOTOGRAPHY (Kodak Ektachrome High Speed ASA 160 : Positive), the filters (K. W. 32 X K. W. 56, K. W. 47 A X K. W. 12 . 15, K. W. 47 X K. W. 12 . 15 and Fuji Filter BPB 45 X BPB 55) were used and the films were developed by a double sensitivity method to obtain the best photograph.


The retino-geniculo-cortical pathway in Callithrix. II. The geniculo-cortical projection.

After monocular injections of tritiated tracer precursors and transneuronal transport of tritiated compounds, a continuous band of radioactivity in layer IV of striate cortex of Callithrf the crossed and uncrossed retino-cortical pathways. Comparison with the organization of these pathways in other primates suggests that a segregation of the retino-cortical pathways of opposite ocularities into alternating ocular dominance columns in striate cortex has developed independently in the different primate lines. The significance is discussed of the weak labeling of the region of area 17 representing the central retina as compared to the representation of the peripheral retina.


Short-latency peripheral inputs to thalamic neurones projecting to the motor cortex in the monkey.

One hundred seventy-five neurones in the n.ventroposterior lateralis (VPL) and n.ventralis lateralis (VL) in the thalamus of anaesthetised monkeys have been tested antidromically for projection to the cortex and for somatosensory input from the contralateral arm. Using bipolar stimulation of the cortical surface, 113 thalamic neurones were successfully identified as antidromically driven from the hand area of the postcentral gyrus (48 neurones) or from the hand area of the precentral gyrus (65 neurones). All but one of these 113 neurones could only be antidromically discharged from the postcentral cortex or from the precentral cortex, and not from both. Most had antidromic latencies between 0.5 and 1.5 ms. Twenty-five/sixty-five precentrally projecting neurones and 45/48 postcentrally projecting neurones were activated by stimulation of the contralateral median or radial nerves. Both groups responded at short latency (4--8 ms) and many were activated by low-threshold shocks (0.8--1.3 T) and had restricted receptive fields on the hand. Precentrally projecting neurones responded most powerfully to joint movement or deep pressure, and some of these neurones were also responsive to cutaneous stimuli. Precentrally projecting neurones with peripheral inputs were all found in the oral subdivision of the VPL (the VPLO). The properties of these neurones suggest that they may be partly responsible for rapid somatosensory input to the motor cortex.


An HRP and autoradiographic study of cerebellar corticonuclear-nucleocortical reciprocity in the monkey.

Combined injections of 3H-leucine and HRP were made into the monkey cerebellar cortex in order to identify any reciprocal connections between the corticonuclear and the nucleocortical pathways. These combined intraaxonal labeling experiments have demonstrated a considerable overlap of orthogradely labeled Purkinje cell axons and terminals with retrogradely labeled HRP-positive neurons in the ventrolateral region of the dentate nucleus following combined injections into the lateral hemisphere, and in the dorsal area of the dentate following combined injections into medial cortical areas of the anterior lobe. There were also areas within the deep cerebellar nuclei where orthogradely labeled corticonuclear terminals did not overlap with retrogradely labeled nucleocortical neurons.


Transplantability of chemically induced skin tumors in syngeneic strains of mice, rats, and guinea pigs.

Syngeneic tumour transplants to inbred female animal strains were carried out with basaliomas, squamous cell carcinomas and fibrosarcomas of the skin, induced by DMBA (9,10-dimethyl-1,2-benzanthracene) in the mouse, rat and guinea pig. A completely successful transplantation of all tumors was only achieved in the group of fibrosarcomas of the rat when a myocardial suspension of a young rat was used as a "supporting agent". This may be a new immuno-biological aspect in tumor transplantation. In the other groups, five tumors developed in the host animals after a period of latency of 10 months or more. Therefore, it is discussed whether two of these tumors grown in the host rats should be considered spontaneous. In addition I have found no earlier reports in the literature with successful DMBA-induced epidermal tumors transplantations in homologous or syngeneic animal strains. Tumors were grafted on to altogether 100 animals.


Endothelium-associated IgG in the post-capillary venules of the lymphatic tissues in mice bearing experimental neoplasia.

Post-capillary venules (PVC) of the lymph nodes and Peyer's patches were studied in frozen sections stained with an indirect immunoperoxidase technique to demonstrate their endothelium-associated IgG in DBA/2 mice bearing mastocytoma. The previously established three types of IgG-distribution (luminal, intraendothelial and basement membrane site), each confined to one of the three PCV grades (graded on the basis of the endothelial cell height), were found in these tumor-bearing mice, too. This finding shows that the tool applied in T-lymphocyte deprivation, be it a tumor or anti-theta-globulin, does not influence the behavior of this IgG-distribution profile. This fact lends further support to the thesis that the endothelium-associated IgG could be involved in the regulation of the T-cell passage through the wall of the post-capillary venules.


Adenosine 5'-triphosphate sulphurylase from Spirulina platensis.

ATP-sulphurylase from an unicellular blue-green alga, Spirulina platensis was localized in the soluble fractions of cell-free homogenate, and it was stable for over 3 weeks at -6 degrees C.


Effect of phytic acid on diamine oxidase activity in germinating pea seeds.

Diamine oxidase present in the cotyledons of germinating pea seeds is induced by phytic acid but the embryo enzyme is not affected. Polyamines have no effect on phytase activity of the cotyledon or embryo.


Effects of calcium-EGTA buffers on active calcium transport in inside-out red cell membrane vesicles.

In inside-out red cell membrane vesicles, the free calcium concentration half-maximally stimulating active calcium uptake is about 2 orders of magnitude smaller in a calcium-EGTA buffer than in media containing unbuffered calcium. In calcium-EGTA buffer, the maximum rate of calcium uptake is determined by the total calcium concentration present. A possible model for explaining these findings is presented.


Alteration in thermal stability of ribosomes from Drosophila melanogaster with age.

Thermal analysis of high salt (0.5 M) washed ribosomal monomers from young and old male Drosophila melanogaster revealed an 8 degrees C downshift in the mean temperature of denaturation (Tm). Moreover, there was observed a marked loss in the ability of ribosomes extracted from older flies to reassociate upon cooling. These observations suggest that age-dependent alterations in the structural integrity of the rRNA-r protein complex could, at least in part, be responsible for the diminished capacity for protein synthesis in this species with advancing age.


Changes in free amino acid level due to physical and chemical agents in ageing Drosophila.

The effects of gamma rays and caffeine on total free amino acids were assessed in unirradiated and irradiated ageing Drosophila. As age advances, there is a rise in the quantity of free amino acids in untreated males and females. Gamma rays and caffeine strongly reduce the level of free amino acids in both the sexes.


Effect of the polychlorinated biphenyl preparation, Aroclor 1242, on the quantity of neurosecretory material in the medulla terminalis X-organ of the fiddler crab, Uca pugilator.

Exposure of the fiddler crab, Uca pugilator, to the PCB preparation, Aroclor 1242, produces an increase in the quantity of neurosecretory material in the medulla terminalis X-organ. This Aroclor apparently inhibits release, but not synthesis, of one or more neurohormones.


The effect of dietary fat on the anticoagulant activity of aflatoxin B.

A single i.p. dose of aflatoxin B1 had no significant effect on the thrombotest clotting times of monkeys subsisting on low-fat and high-fat dietary regimens, respectively. There was a significant interaction between aflatoxin and dietary fat level.


The localization of mutarotase in rat kidney.

The localization of mutarotase in rat kidney was investigated by fluorescein-labelled and peroxidase-labelled antibody techniques, and by method of isolation of the nuclei and cytoplasm in non-aqueous solvents. In these immunohistochemical studies, mutarotase was almost exclusively recognized in the nuclei of epithelial cells of renal tubules and glomeruli in rat. The specific activity of mutarotase was found to be 1.5 times higher in the nuclei (122 units/g dry wt) than that in the cytoplasm (80 units/g dry wt) isolated with non-aqueous solvents. These results suggest that mutarotase may be involved in the metabolism of D-glucose in nuclei.


[Light and electron microscopic study of hepatic lesions in the course of hyperlactatemia in diabetic patients (author's transl)].

Histopathological study of the liver has been undertaken on twenty-one diabetics with hyperlactatemia exceeding 5 mEq/1 of whom seven were treated with phenformin, six with metformin and eight not biguanide-treated. Hyperlactatemia occurred during the course or during resolution of severe ketoacidosis or of hyperosmolar coma. Hepatic lesions were invariably present. By light microscopy, massive steatosis, steatonecrosis or necrosis of variable extent were observed. Ultrastructural study showed constant mitochondrial abnormalities. These results support the hypothesis of a major role for mitochondrial changes in hepatic cells in provoking pathological hyperlactatemia. In diabetic patients, these mitochondrial lesions could be induced either by an anoxic process resulting from a variety of metabolic insults or by some as yet undefined toxic action of biguanides or by the combination of both of these factors.


Combined therapy of T3, and antidepressants in depression.

Since there are some patient groups whos symptoms do not improve despite the fact that the use of the antidepressants will alleviate the symptoms to some extent, we have conducted a TRH test for depression and found that there are not a few cases who show a low TSH response. We therefore used a small amount of T3 together with the antidepressants in these cases and have found that the therapy is useful for improvement of tye symptoms. When the TRH test is made at the time of improvement of the symptoms due to the combined use of these two drugs and compared with the state at pre-treatment, it has been confirmed that the TSH response will go back to normal. Hence, we would like to present here two markedly improved cases due to the combined use of imipramine and clomipramine plus T, and to refer to the result of the TRH test.


Olfactory and visual differentiation of synthetically scented surrogates by infant squirrel monkeys.

Twelve infant squirrel monkeys were reared with distinctively colored and scented inanimate surrogates (Green/Floral or Black/Clove) and tested monthly from 1 to 6 months of age in 3 paired-comparison conditions that assessed the efficacy of synthetic olfactory cues and color cues as attractive properties of the surrogates. The infants developed specific attractions to both the rearing odor and color of the surrogates by approximately the end of Month 4, although substantial differences existed between the 2 rearing groups, particularly with respect to color responsiveness. When compared with previous findings on infant squirrel monkeys reared with naturally scented surrogates, odor preferences developed relatively late, suggesting that natural, species-typical odors may have inherently stronger attractive properties for Saimiri than arbitrary synthetic odors.


Absorption of elemental and complex nutritional solutions during a continuous jejunal perfusion in man.

The jejunal absorption of either an elemental solution (amino acids, glucose and glucose oligosaccharides), or of nonelemental diet (chicken meat, egg-yolk power, soya flour, glucose, saccharose, maltose and dextrin maltose, corn and wheat oils) were compared in 25 healthy subjects by the technique of intestinal perfusion with a three-lumen tube. The test solutions were perfused just beyond the ligament of Treitz. The samples were collected 35, 70 and 105 cm below the infusion point. In a small segment of jejunum, the absorption increased the nutritional imbalance of the element diet 1 m below the infusion point, the flow rate in the lumen was similar for the two solutions and higher than the initial flow rate; the absorption, in terms of calories, is similar for the two solutions.


A physical map of the genome of temperate phage phi 3T.

A physical map of the genome of Bacillus subtilis bacteriophage phi 3T was constructed by ordering the fragments produced by cleavage of phi 3T DNA with restriction endonucleases AvaII (2 fragments), BglI (2 fragments), SmaI (3 fragments), BamHI (6 fragments), SalI (7 fragments), AvaI (7 fragments), SacI (12 fragments), PstI (14 fragments), and BglII (26 fragments). Two techniques were used to order the fragments: (1) Sets of previously ordered restriction fragments were isolated and redigested with the endonuclease whose cleavage sites were to be mapped. (2) Fragments located near the ends of the genome or near the ends of other restriction fragments were ordered by treating the DNA with lambda exonuclease prior to restriction endonuclease cleavage. The susceptibility of phi 3T DNA to 15 other restriction endonucleases is also reported.


Luteotrphic capacity of TRH in the baboon.

An attempt was made to determine the synergistic effect of TRH stimulated prolactin with LH on the luteinization of corpus luteum in baboons. Four normally cycling female baboons were used in this study. Synthetic LHRH (100 micrograms) was given during the early luteal phase and blood samples were collected sequentially for assay of progestin. In additional experiments, 40 micrograms of synthetic TRH was given at 30 and 60 minutes after LHRH injection and blood samples were assayed for progestin. Injection of LHRH elevated the plasma level of progestin. Subsequent treatment with TRH in LHRH treated baboons enhanced the elevation of plasma progestin in three of four baboons. The evidence illustrates the nature of synergistic effect of prolactin with LH on the luteinization of corpus luteum in baboons.


Pituitary-thyroid function of fetuses of hypothyroid and growth hormone treated hypothyroid rats.

Maternal hypothyroidism induced by surgical thyroidectomy (Tx) of the rat resulted in significantly higher fetal serum levels of thyroid stimulating hormone (TSH) and thyroxine (T4) on day 22 of gestation. Surprisingly, administration of growth hormone (GH) to hypothyroid mothers increased further the fetal serum T4 and TSH. The in vitro uptake of 131I-T4 by erythrocytes was elevated significantly when incubated with serum from fetuses of both hypothyroid and hypothyroid GH-treated mothers. Although the plasma protein levels of hypothyroid mothers and their fetuses are decreased significantly as compared to controls this is not true of hypothyroid GH-treated mothers and their fetuses. The T4 levels of both groups of Tx mothers were significantly below that of controls. However, as in the case of their fetuses, the serum T4 of GH-treated hypothyroid mothers was elevated from that of Tx only animals. It is concluded that the pituitary-thyroid system of fetuses of hypothyroid mothers is activated excessively during late gestation, that considerable T4 can be transported from the fetus to the mother during this period and that these fetuses are in fact born in a hyperthyroid state which is aggravated by maternal treatment with GH.


Assessing medical efficacy:a neglected administrative necessity.

Administrators must balance time, cost, benefits, and safety against technological innovations when assessing medical efficacy, a task requiring assertiveness, well-chosen priorities, and familiarity with patient care.


Associations between atopic diseases and the polymorphic systems ABO, Kidd, Inv and red cell acid phosphatase.

In 239 German patients with atopic conditions (atopic dermatitis, hay fever, allergic rhinitis, bronchial asthma, and acute urticaria) the phenotype and gene distribution of 15 genetic blood polymorphisms (ABO, MNSs, rhesus, P, Kell, Duffy, Kidd, Hp, Gc, Gm, Inv, aP, PGM1, EsD, and 6-PGD) were analyzed and compared with those in 151 selected controls (individuals clinically free of allergic conditions and without allergy in the family history). The incidence of blood group antigens A and B was somewhat higher in patients than in controls. These observations are in accordance with the results of previous studies in other populations. In addition, our observations favor the hypothesis that there are also associations between the phenotypes Jk (a-b+), Inv(1) and red cell acid phosphatase aP A and aP AP on the one hand and atopic disposition on the other. The possible reasons for these associations are discussed.


Long-term in vitro cell culture of Sinclair swine melanoma.

The melanoma of Sinclair swine exhibits several characteristics similar to human melanoma but demonstrates an unusually high incidence of spontaneous regression. A total of 66 finite cell lines derived from 21 swine melanotic lesions, both cutaneous and visceral, were studied in vitro over their life spans of up to 14 months. The growth characteristics of the cultures varied with the age of the swine from which the tumors were obtained. Cell cultures of tumors obtained from swine aged less than 2 months grew steadily in cluture with a population-doubling time of 120 to 180 hr until growth and division ceased after a maximum of 25 to 35 population doublings (6 to 8 passages). Cell cultures of tumors obtained from swine aged 3 months or older showed a biphasic growth pattern with an early slow growth rate (population-doubling time 120 to 160 hr), which shifted after 3 to 6 passages to a faster rate (80 to 110 hr population-doubling time) until termination of growth and division after a maximum of 75 to 85 population doublings (18 to 20 passages). The cultures were morphologically heterogeneous including cuboidal, spindle and dendritic cell types. Electron microscopy showed classic melanosomes only in the primary and passage 1 cultures although vesicular inclusions were numerous in later-passage cells. However, continued melanin synthesis was indicated by the spectroscopic characteristics of material obtained from medium of passage 8 cultures and by DOPA staining of cultures as advanced as passage 18.


Interaction of Mycoplasma pneumoniae with alveolar macrophages: viability of adherent and ingested mycoplasmas.

Guinea pig peritoneal or alveolar macrophages were inoculated with Mycoplasma pneumoniae cells. Extracellular mycoplasms were killed by complement treatment, and the effect of macrophage action on the number of the remaining viable mycoplasmas was observed. The complement killing was to some extent inhibited by the presence of the macrophages, but the mechanism of this protection remains unknown. Opsonized mycoplasmas were ingested, and approximately 98% were killed within 4 h. The killing rate was somewhat lower than comparable data for bacteria, but lack of cell wall and high lipid content of the membrane apparently do not cause a significant delay in intracellular destruction.


Induction of Listeria monocytogenes infection by the consumption of ponderosa pine needles.

An infectious microorganism, identified as Listeria monocytogenes, has been isolated from the bloodstream of pregnant mice fed a diet containing Pinus ponderosa needles. When the isolate was injected into pregnant mice, reproductive dysfunction and other changes, including speckled livers, spleen atrophy, and hemorrhagic intestines, appeared to mimic the signs of the disease in pregnant mice fed pine needles. Moreover, these pathological changes are similar to those observed in cattle and other mammals experiencing abortions or toxemia, or both, attributed to the ingestion of P. ponderosa needles, suggesting that L. monocytogenes may be a part of the etiology of "pine needle abortion."


Immunization against Schistosoma mansoni in rhesus monkeys and the requirement of activation of both cell-mediated and humoral mechanisms.

When groups of rhesus monkeys were pretreated with BCG plus hyperimmune serum from monkeys with chronic schistosomiasis or with dialyzable transfer factor from uninfected monkeys plus hyperimmune serum and were challenged with 1,500 cercariae of Schistosoma mansoni, the mean worm burdens were significantly lower than that of untreated controls. Pretreatment with neither BCG alone nor Corynebacterium parvum plus a membrane antigen of adult worms of S. mansoni affected susceptibility. Neither lymphocyte proliferation in the presence of mitogens or schistosome antigen nor serological responsiveness (as measured by gel diffusion, Cercarienhüllenreaktion, circumoval precipitation, or enzyme-linked immunoabsorbent assay) correlated with the degree of resistance of the animals to S. mansoni. The pretreatment procedures used did not cause any abnormal histopathological responses and did not alter the characteristic host response to schistosome eggs in the lungs, liver, mesenteric lymph nodes, and colon.


Immune responses to Schistosoma mansoni in rhesus monkeys with multiple chronic and early primary infections.

Immunological reactivity in 10 rhesus monkeys was monitored over a 22-week period. Cellular and humoral responses of three animals were studied after primary infection with Schistosoma mansoni. Two uninfected animals served as controls. Increased lymphocyte proliferative responsiveness to mitogens and adult worm antigen was evident during the prepatent period of the infection. Marked suppression of these responses occurred during the acute phase of the disease, but by weeks 9 and 11 the animals were again responsive to mitogens and antigen, respectively, and remained so throughout the remainder of the observation period. No antibody response to various cercarial, adult worm, and egg antigens could be detected until weeks 5 to 7, after which these responses also persisted. Comparison of the immunological reactivities of these animals with primary infection and those of five chronically infected immune animals indicated possible correlations between protective immunity and (i) strong Cercarienhüllenreaktion reactivity, and (ii) lymphocyte proliferative responsiveness to adult worm antigen.


Enhancement of host resistance against Trypanosoma cruzi infection by the immunoregulatory agent muramyl dipeptide.

N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide [MDP]) enhanced resistance against Trypanosoma cruzi infection in mice. This effect was evidenced by significant reductions in both parasitemias and mortality rates and increased survival time in MDP-treated animals compared with untreated infected mice. MDP effectively augmented host resistance when administered in any one of the following ways: (i) continuous subcutaneous release from an osmotic minipump for a 7-day period starting 2 days before infection; (ii) as a single dose of 0.5 mg injected intraperitoneally 48 h before infection; or (iii) injected intraperitoneally at 48-h intervals during the first 16 days after infection. CBA/J mice, which exhibit very low, insignificant augmentation of reticuloendothelial activity by MDP but are susceptible to its adjuvant effect, failed to manifest enhancement of resistance to T. cruzi infection when treated with MDP under regimens that cause increased resistance in other mouse strains. These results suggest that MDP enhances resistance against T. cruzi infection by stimulating the activity of the phagocytic cells of the host. Adjuvant effect appears to play either a less significant role or no relevant role, except when MDP is administered repeatedly after infection.


Antibody-dependent cellular cytotoxicity of Trypanosoma cruzi: characterization of the effector cell from normal human blood.

The antibody-dependent cellular cytotoxicity activity of normal human blood cells against epimastigotes of Trypanosoma cruzi was measured by the release of incorporated [3H]uridine. Sera from patients with chronic Chagas' disease were used to sensitize the parasites to the lytic activity of the effector cells. Different steps of peripheral blood cell purification were employed, and different cell subpopulations were tested as effectors in the system. The main cytotoxic activity was detected in the granulocyte-rich fraction.


Phenotypic expression of genetically controlled host resistance to Listeria monocytogenes.

Several inbred mouse strains, all of them derived from the C57BL background, have genetically determined increased resistance to infection with Listeria monocytogenes, whereas a variety of other strains are relatively sensitive to this infection. Comparison of the host response to L. monocytogenes in the sensitive A strain and the resistant C57BL/6 (B6) strain revealed that the B6 mice were superior to A mice both in the T-cell-independent and in the T-cell-dependent phase of the response. Although animals of both strains had equal ability to clear their circulation of intravenously administered Listeria and to take up comparable amounts of bacteria in their livers and spleens, already 24 to 48 h after infection the genetic advantage of B6 strain mice to suppress bacterial proliferation was apparent. Both the primary (early and late) and the secondary responses as well as the ability to inactivate the bacterial load after adoptive protection by syngeneic immune lymphocytes were more efficient in the B6 animals, suggesting that the common effector macrophage arm of the antilisterial resistance rather than the lymphocyte arm (mediating the T-cell-dependent phase of response) is genetically controlled.


Extraction and partial characterization of a leukotoxin from a plaque-derived Gram-negative microorganism.

The plaque-derived gram-negative microorganism Y4 identified as a member of the genus Actinobacillus, was tested for a soluble cytotoxic factor(s). Sonication or incubation of viable Y4 microorganisms in distilled water or normal human serum resulted in liberation of a soluble material which was cytotoxic in vitro for human polymorphonuclear leukocytes (PMNs). The Y4 soluble sonic extract was also cytotoxic to human peripheral blood monocytes. However, human lymphocytes, platelets, and fibroblasts, as well as rabbit, rat, and mouse leukocytes and chicken embryo fibroblasts, were not killed by exposure to the Y4 sonic extract. No hemolytic activity was detected in the Y4 sonic extract. No hemolytic activity was detected in the Y4 sonic extract. Consequently, the factor(s) in the Y4 sonic extract was referred to as Y4 leukotoxin. The Y4 leukotoxin was inactive at 4 degrees C, heat sensitive (56 degrees C, 30 min), and inactivated by proteases. The cytotoxic effect of Y4 leukotoxin on PMNs was dose, time, and temperature dependent. The leukotoxin did not bind to viable PMNs at 4 degrees C but did bind to dead PMN membrane components at both 4 and 37 degrees C. The addition of bovine serum albumin (51 mg/ml) to PMN-Y4 leukotoxin cultures inhibited the release of lactate dehydrogenase from the PMNs, but did not prevent the death of the cells as indicated by electron microscopy. Lysosomal markers were released in parallel to the cytoplasmic enzyme lactate dehydrogenase from Y4 leukotoxin-treated PMNs. The addition of 0.02 M ethylenedinitrilotetraacetic acid to these cultures inhibited release of lysosomal markers but enhanced the release of lactate dehydrogenase. These results suggested that a soluble leukotoxin with specificity for only human PMNs and monocytes can be liberated from viable Y4. What role this leukotoxin plays in the pathogenicity of the Y4 microorganism is not yet known. However, this leukotoxin is one of the first materials from a plaque-derived microorganism with a potential role in the pathogenesis of juvenile periodontitis.


Interaction of Mycoplasma pneumoniae with human lung fibroblasts: characterization of the in vitro model.

The interaction of pathogenic Mycoplasma pneumoniae and host cells was studied in cell cultures of MRC-5 human lung fibroblasts. A comparison of results obtained with fibroblasts in a monolayer format and with hamster tracheal explant cultures indicated that the former can bind significantly larger numbers of mycoplasmas. In addition, the attachment was 96% specific, that is, mediated through a neuraminidase-sensitive receptor on the host cell. Uptake of mycoplasmas was directly related to the number of mycoplasma cells present in the inoculum, and attachment was virtually complete within a 30-min period at 37 degrees C. High doses of M. pneumoniae induced a marked cytopathic effect, whereas doses of less than or equal to 10(6) colony-forming units per ml produced grossly observable cell damage that was moderate and variable. Transmission electron microscopy studies indicated that attachment of M. pneumoniae to the surface of lung fibroblasts occurred with the specialized terminal structure or binding site oriented closest to the epithelial cell surface. The filamentous mycoplasma cells were spatially arranged in several configurations and were not limited to a vertical orientation. The advantages and disadvantages of human lung fibroblast monolayer cultures, in reference to other in vitro models are discussed. A new mycoplasma agar medium (G-200 agar) with a defined tissue culture base and 10% horse serum is also described.


Interaction of Mycoplasma pneumoniae with human lung fibroblasts: role of receptor sites.

The biochemical nature of the neuraminidase-sensitive Mycoplasma pneumoniae receptor site on human lung fibroblast cells was studied. Purified, mixed sialoglycolipid (ganglioside) preparations from human and bovine tissues did not bind to M. pneumoniae organisms and block their subsequent attachment to fibroblasts. Fibroblasts incubated for 24 h in sialoglycolipid solutions to increase the ganglioside content of their membranes did not show increased pathogen attachment when later incubated with mycoplasmas. HeLa cells grown in the presence of sodium butyrate to increase GM3 ganglioside levels likewise did not have significantly increased uptake of M. pneumoniae organisms. Treatment of fibroblasts with enzymes indicated that the mycoplasma receptor site is trypsin and papain resistant but Pronase sensitive. Pronase digests of fibroblast membranes contained a product(s) which combined with M. pneumoniae cellls and cosedimented with them during centrifugation. Glycoproteins, purified from fibroblast membranes by a lithium diiodosalicylate solubilization technique, similarly bound to M. pneumoniae organisms. Collectively, these data suggest that the major component of the M. pneumoniae receptor site is a sialoglycoprotein with little or no lipid.


Herpesvirus papio: state and properties of intracellular viral DNA in baboon lymphoblastoid cell lines.

Herpesvirus papio (HVP) is an indigenous B-lymphotropic virus of baboons (Papio sp.) present in latent form in baboon lymphoblastoid cell lines. It shares cross-reacting viral capsid and early antigens with the Epstein-Barr virus (EBV), and HVP DNA and EBV DNA show partial sequence homology. EBV-specific complementary RNA was employed here as a probe to investigate the physical state of the HVP DNA component in baboon lymphoblastoid cells after fractionation of cellular DNA by density gradient centrifugation. Five virus-producing cultures contained both free and integrated HVP DNA sequences while one non-producing cell line had two or three viral genome equivalents per cell in an apparently integrated form. Further analysis of one virus-producing line showed that the free HVP DNA fraction was composed of both linear and circular viral DNA. Contour length measurements of HVP circular DNA molecules by electron microscopy revealed that they were similar in length to the EBV circular DNA present in human lymphoblastoid cells.


Sites of breakdown of the blood-aqueous barrier after paracentesis of the rhesus monkey eye.

Sites of breakdown of the blood-aqueous barrier following rapid paracentesis have been investigated in rhesus monkeys with two independent procedures. (1) The entrance of fluorescein into the anterior and posterior chambers following paracentesis was studied in vivo in normal eyes, in eyes with laser-induced experimental glaucoma, and in totally iridectomized eyes. (2) Scanning electron microscopy was used to study the ciliary body and trabecular meshwork regions in normal eyes and paracentetic eyes. These investigations show that the ciliary body, particularly the anterior pars plicata region, is a source of secondary aqueous humor protein. In addition, the study shows reflux of blood into Schlemm's canal and plasma movement across the inner wall into the anterior chamber.


Response of the vestibulo-ocular reflex to differing programs of acceleration.

The vestibulo-ocular reflex of normal monkeys was tested by a modified Bárány spinning test (B-test) and by sinosoidal rotation. The two tests gave concordant estimates of the time constant and corner frequency. Deviation from concordance was caused largely by modification of the reflex during the testing procedures. As a means of estimating both time constant and corner frequency in the vestibulo-ocular reflex, the B-test is to be preferred to sinusoidal rotation because although both tests provide similar information, the B-test is the more rapidly performed and analyzed and the B-test modifies the reflex less.


Echothiophate-induced structural alterations in the anterior chamber angle of the cynomolgus monkey.

Four cynomolgus monkeys were treated topically twice daily in one eye with echothiophate iodide (PI) doses of 63, 75, or 250 micrograms per treatment for 7.7 weeks to 7 months. The opposite eyes of two monkeys received a control solution (diluent). The anterior ocular segments of all six eyes were studied by light and transmission electron microscopy during treatment. In the PI-treated eyes, the cribriform and outer corneoscleral meshwork were unusually dense. The trabecular meshwork was collapsed and the lamellae showed thickened basement membranes and thickened sheaths of elastic-like material. Most endothelial cells were enlarged and activated. Some contained many glycogen particles; others showed evidence of degeneration. The cribriform meshwork contained much more extracellular fine fibrillar material than normal, and the endothelium of the inner wall of Schlemm's canal was damaged. The PI-contracted ciliary muscle had a more rectangular shape than normally contracted muscles, and the inner edge extended so far anteriorly that in some areas it overlapped and occluded the trabecular meshwork. The muscle cells appeared damaged, and their basement membranes were thickened. The nonpigmented epithelial cells of the ciliary processes showed signs of degeneration, and within the pars plana some contained large, weakly osmiophilic inclusions. The basement membrane of the ciliary epithelium was thickened everywhere. The stromal vessels of the pars plana were dilated, and signs of inflammation were present. The sphincter iridis was damaged and there were iridocorneal adhesions. The 5 month diluent-treated eye demonstrated mild structural abnormalities in the meshwork.


Polychlorinated biphenyls poisoning in monkey eye.

Poisoning by polychlorinated biphenyl(s) (PCB) in humans leads to cutaneous and ocular findings. A white, cheeselike secretion issuing from the orifice of the Meibomian gland duct when the eyelid is squeezed is one sign of this intoxiation. In the rhesus monkey, abnormal hyperkeratosis of the ductal epithelium was observed histopathologically.


Optic nerve sheath imaging with metrizamide.

A technique is described for opacifying the optic nerve sheaths with intrathecally injected metrizamide. The clinical and investigational applications are discussed.


Radiographic study of gastric hyperplasia induced by polychlorinated biphenyls in the rhesus monkey.

Polychlorinated biphenyls (PCBs) are a group of compounds which have many industrial uses and have become an environmental pollutant. In the rhesus monkey, an unusual type of gastric hyperplasia has been produced by feeding the animals small amounts of these chemicals. By using a double contrast gastrogram with simultaneous pneumoperitoneum, we studied the development of the lesions radiographically. Over a period of several months the gastric abnormality progressed from minimal thickening of the gastric wall, with normal appearing mucosa, to extensive nodularity and irregularity in the area of involvement. This technique provides a method for studying the characteristics of the gastric hyperplasia over an extended time, and therefore has several advantages over autopsy studies or surgical biopsies. Although no similar lesions related to PCB exposure have been seen in humans, no survey of possible gastric changes has been done among persons exposed to these materials.


Virus-cell interactions as seen by scanning electron microscopy.

Scanning electron microscopy (SEM) has been used during the past few years to determine surface alterations of cells infected with a variety of RNA and DNA viruses. Infection of different cultured cells by RNA and DNA viruses usually revealed dramatic shape changes with surface alterations. The most frequent changes seen were "rounding up" of cells and the development of surface microprojections such as ruffles and microvilli. In the light of available knowledge, suggestions are made in this review for future SEM studies in this field with more sensitive methodologies, which will enable even better characterization of virus-induced cell surface changes.


Pharmacokinetics of iopanoic acid in the rhesus monkey: biliary excretion, plasma protein binding and biotransformation.

A dynamic infusion method, originally developed for the pharmacokinetic studies of Iodoxamic acid, was applied to the kinetic studies of the biliary excretion of another cholecystographic agent, iopanoic acid. This dynamic method has an important advantage in that the pharmacokinetic parameters involved in the hepatic uptake or biliary excretion can be evaluated from a single infusion experiment. Using the equilibrium dialysis technique, iopanoic acid was found to be highly bound to the plasma proteins. A linear relationship was found when the logarithm of unbound plasma concentration of iopanoic acid was plotted vs. the logarithm of its blood concentration. When the biliary excretion rates of iopanoic acid were fitted by a computer to the Michaelis-Menten equation against its unbound plasma concentration, the average Vm value was found to be 0.85 micron/kg/min and the average Km value was found to be 0.253 micron. Iopanoic acid was found to exist in monkey blood as unchanged species and in the bile mainly as the ester glucuronide.


[Effect of a corticosteroid additive on the result of local therapy of bacterial superinfection in tinea pedis intertriginosa].

Sixty cases of tinea pedum intertriginosa were topically treated in a double-blind trial. In 34 cases a cream was used, which contained 5-chloro-8-hydroxyquinoline, gentamycin and fluprednyliden-21-acetate. In 26 cases a cream was used, which contained 5-chloro-8-hydroxyquinoline and gentamycin alone. Treatment was continued over three weeks. In weekly intervalls mycological, bacteriological and clinical controls were performed, which showed that the steroid-component had no disadvantageous effect on the course of infection. In contrast the steroid containing preparation proved to be slightly superior.


Radioiodination of biologically active compounds: a simplified solid-state enzymatic procedure.

A simplified solid-state enzymatic iodination procedure for routine labeling of unstable pure protein or complex amino acid-containing molecules is presented. The procedure was designed using agarose-bound lactoperoxidase to iodinate human IgG with iodine-125. This method consistently resulted in a labeling efficiency greater than 90% with high stability and undetectable gross structural alterations of the substrate as evaluated by immunodiffusion and electrophoresis. The technique presented is simple, efficient, and may be employed to yield a sterile, pyrogen-free labeled species.


The biochemical and histochemical demonstration of lactase induction in fetal rat intestine by intra-amniotic injection of lactose.

A single dose of 20 mg beta-D-lactose injected into the amniotic sac of rats on day 17 of pregnancy induced an increase in lactase activity in fetal jejunum. This effect was first noted two days after injection and lasted for at least two additional days. Histoenzymatic investigation indicated that this enzyme was located on the surface of the absorptive cells lining the villi and thus corresponds to the "dietary" form of beta-galactosidase. A much smaller increase, based presumably on progressive increase in fetal size (age) was found in control fetuses which had received glucose or no injections. Peak lactase values in fetuses receiving lactose were substantially higher than peak values in control fetuses. In both lactose-injected and non-injected rats which were allowed to deliver, there was a sharp drop in lactase values coincident with birth.


Insertional inactivation of trpC in cloned Bacillus trp segments: evidence for a polar effect on trpF.

Plasmid pUB110 was previously used as a vector to clone fragments of deoxyribonucleic acid that complement the trpC2 mutation in Bacillus subtilis from endonuclease EcoRI digested B. licheniformis, B. pumilus, and B. subtilis cellular deoxyribonucleic acid. Each of several such trp plasmids was subsequently shown to contain a segment of the trp gene cluster on the basis of genetic complementing activity. In the present study, analysis of the Trp enzyme levels in B. subtilis harboring the constructed trp plasmids confirms the genetic constitution of the plasmids. Thus, plasmids that complement mutations in specific trp genes specify the corresponding enzyme activities. The levels of the plasmid-specified Trp enzymes in B. subtilis were generally above the repressed level of the chromosomally specified Trp enzymes and equal to or below the derepressed levels of the chromosomally specified Trp enzymes. Certain cloned trp segments contain a single HindIII-sensitive site. Insertion of HindIII-generated deoxyribonucleic acid fragments into these trp plasmids resulted in inactivation of trpC complementing activity, loss of the trpC-specified enzyme activity, and a 10-fold reduction in the specific activity of the plasmid-specified trpF product. The HindIII insertions had no detectable effect on the level of the trpD product, nor did the insertions detectably alter plasmid-specified complementing activity other than to abolish trpC complementation. Removal of the HindIII insertions was accompanied by recovery of trpC complementing activity and restoration of the trpC-and trpF-determined enzymes to the levels specified by the parent plasmids.


Positive control of ilvC expression in Escherichia coli K-12; identification and mapping of regulatory gene ilvY.

The construction of a plasmid carrying the ilvC::lacZ fusion is described. This plasmid provides a convenient source of template deoxyribonucleic acid for use in an in vitro protein-synthesizing system. We screened strains deleted in regions of the ilv cluster for their ability to support ilvC-dependent beta-galactosidase synthesis. The fact that two deletions prevented beta-galactosidase production indicated that ilv-C expression is under positive control. By use of plasmids carrying the positive-control factor structural gene ilvY, we were able to restore protein-synthesizing ability to these strains. These plasmids also enabled us to map ilvY between ilvA and ilvC.


Alterations in peptidoglycan chemical composition associated with rod-to-sphere transition in a conditional mutant of Klebsiella pneumoniae.

Klebsiella pneumoniae Mir M7 is a spontaneous parentless morphology mutant which grows as cocci at pH 7 and as rods at pH 5.8. This strain has been characterized as defective in lateral wall formation (at pH7). Data suggest that the cell wall is mainly made up of poles of the rods (G. Satta, R. Fontana, P. Canepari, and G. Botta, J. Bacteriol. 137:727--734, 1979). In this work the isolation and the biochemical properties of the peptidoglycan of both Mir M7 rods and cocci and a nonconditional rod-shaped Mir M7 revertant (strain Mir A12) are described. The peptidoglycan of Mir M7 (both rods and cocci) and Mir A12 strains carried covalently bound proteins which could be easily removed by pronase treatment in Mir M7 rods and Mir A12 cells, but not in Mir M7 round cells. However, when the sodium dodecyl sulfate-insoluble residues of Mir M7 cocci were pretreated with ethylenediaminetetraacetic acid (EDTA), pronase digestion removed the covalently bound proteins, and pure peptidoglycan was obtained. EDTA treatment of the rigid layer of Mir M7 cocci removed amounts of Mg2+ and Ca2+, which were 10- and 50-fold higher, respectively, than the amount liberated from the rigid layer of Mir  M7 rods and Mir A12 cells. Amino acid composition was qualitatively similar in both strains, but Mir M7 cocci contained a higher amount of alanine and glucosamine. Mir M7 cocci contained approximately 50% less peptidoglycan than rods. Under electron microscopy, the rigid layer of the Mir M7 rods and Mir A12 cells appeared to be rod-shaped and their shape remained unchanged after EDTA and pronase treatment. On the contrary, the Mir M7 cocci rigid layer appeared to be round, and after EDTA treatment it collapsed and lost any definite morphology. In spite of these alterations, the peptidoglycan of Mir M7 cocci still appeared able to determine the shape of the cell and protect it from osmotic shock and mechanical damages. The accumluation of divalent cations appeared necessary for the peptidoglycan to acquire sufficient rigidity for shape determination and cell protection. We concluded that the coccal shape in Mir M7 cells is not due to loss of cell wall rigidity but is a consequence of the formation of a round peptidoglycan molecule. The possibility that the alterations found in the Mir M7 cocci rigid layer may reflect natural differences in the biochemical composition of the septa and lateral wall of normally shaped bacteria is discussed.


Transport systems for branched-chain amino acids in Pseudomonas aeruginosa.

The cells of Pseudomonas aeruginosa showed high activity for leucine transport in the absence of Na+, giving a Km value of 0.34 microM. In the presence of Na+, however, two Km values, 0.37 microM (LIV-I system) and 7.6 microM (LIV-II system), were obtained. The former system seemed to serve not only for the entry of leucine, isoleucine, and valine, but also for that of alanine and threonine, although less effectively. However, the LIV-II system served for the entry of branched-chain amino acids only. The LIV-II system alone was operative in membrane vesicles, for the transport of branched-chain amino acids in membrane vesicles required Na+ and gave single Km values for the respective amino acids. When cells were osmotically shocked, the activity of the LIV-I system decreased, whereas the LIV-II system remained unaffected. The shock fluid from P. aeruginosa cells showed leucine-binding activity with a dissociation constant of 0.25 microM. The specificity of the activity was very similar to that of the LIV-I system. These results suggest that a leucine-binding protein(s) in the periplasmic space may be required for the transport process via the LIV-I system of P. aeruginosa.


Genetic and physiological characterization of Pseudomonas aeruginosa mutants affected in the catabolic ornithine carbamoyltransferase.

In Pseudomonas aeruginosa arginine can be degraded by the arginine "dihydrolase" system, consisting of arginine deiminase, catabolic ornithine carbamoyltransferase, and carbamate kinase. Mutants of P. aeruginosa strain PAO affected in the structural gene (arcB) of the catabolic ornithine carbamoyltransferase were isolated. Firt, and argF mutation (i.e., a block in the anabolic ornithine carbamoyltransferase) was suppressed specifically by a mutationally altered catabolic ornithine carbamoyltransferase capable of functioning in the anabolic direction. The suppressor locus arcB (Su) was mapped by transduction between hisII and argA. Second, mutants having lost suppressor activity were obtained. The Su- mutations were very closely linked to arcB (Su) and caused strongly reduced ornithine carbamoyltransferase activities in vitro. Under aerobic conditions, a mutant (PA0630) which had less than 1% of the wild-type catabolic ornithine carbamoyltransferase activity grew on arginine as the only carbon and nitrogen source, at the wild-type growth rate. When oxygen was limiting, strain PA0630 grown on arginine excreted citrulline in the stationary growth phase. These observations suggest that during aerobic growth arginine is not degraded exclusively via the dihydrolase pathway.


Regulation of expression of the flagellin gene (hag) in Escherichia coli K-12: analysis of hag-lac gene fusions.

Previous studies have defined 28 genes necessary for the synthesis of the flagellar apparatus of Escherichia coli K-12. This study analyzed the influence of the flagellar genes on the expression of the hag gene (structural gene for flagellin). To this end, a hag::Mu d(Apr lac) mutant which had the lac genes fused to the promoter of the hag gene was constructed. This allowed the measurement of hag gene expression by detection of beta-galactosidase activity. The following observations were made. (i) The hag gene was expressed constitutively in Fla+ cells. (ii) hag gene expression was positively regulated by flaA, FLAB, flaC, flaD, flaE, flaG, flaH, flaI, flaK, flaL, flaM, flaN, flaO, flaP, flaQ, flaR, flaV, flaW, flaX, flaY, flaZ, flbA, and flbB genes.hag-lac expression was not observed in strains with these fla mutations. (iii) The hag gene was expressed in mutants with flaS, flaT, flaU, and flbC defects. Therefore, these genes were not involved in regulation of hag gene transcription.


recE4-Independent recombination between homologous deoxyribonucleic acid segments of Bacillus subtilis plasmids.

A plasmid (pLS104) carrying a tandem repetition of the leu region of the Bacillus subtilis chromosome arose spontaneously from pLS103, which carried a single copy of the leu region. Plasmid preparations from strains harboring pLS104 also contained the original plasmid, pLS103, and, in some preparations, plasmids carrying three or four repetitions of the leu region. These plasmids were shown to be generated by recombination between homologous deoxyribonucleic acid (DNA) segments in the tandemly repeated DNA regions on the plasmids, but not by recombinations between specific DNA sites. These phenomena were observed in a recE4-Independent background, showing that recombination of the homologous DNA sequences does not require the recE-Independent gene product(s).


Effect of protein synthesis on plasmid maintenance in Streptococcus faecalis.

Plasmid-to-chromosome ratios in Enterobacteriaceae, upon interruption of protein synthesis by chloramphenicol, are either conserved or increased when measured by dye buoyant density centrifugation. We have found, on the other hand, that the effect of inhibition of protein synthesis on the amount of covalently closed circular deoxyribonucleic acid visualized by this method in two strains of Streptococcus faecalis appears to differ from these established systems. A three- to sixfold decrease in covalently closed circular deoxyribonucleic acid was observed when lysates of chloramphenicol-treated cultures were submitted to dye buoyant density centrifugation. A loss of covalently closed circular deoxyribonucleic acid was also evident from electrophoretic profiles of these lysates. Several conditions which could account for the apparent loss of covalently closed circular deoxyribonucleic acid upon inhibition of protein synthesis are discussed.


Transposable plasmid deoxyribonucleic acid sequence in Pseudomonas aeruginosa which mediates resistance to gentamicin and four other antimicrobial agents.

A 9.1 x 10(6)-dalton transposable deoxyribonucleic acid sequence resides within Pseudomonas aeruginosa plasmid R1033 and mediates resistance to gentamicin, streptomycin, sulfamethoxazole, chloramphenicol, and mercuric chloride. Transposability was demonstrated in Escherichia coli when this sequence, designated Tn1696, excised from R1033 and integrated into plasmid pMB8. Excision and insertion of Tn1696 occurred independently of the host Rec phenotype and may involve the 140-base pair, inverted deoxyribonucleic acid repeated region that flanks this sequence. Occurrence of a multiresistance transposon on a transferrable plasmid that has a broad host range may have serious epidemiological and therapeutic consequences.


Membrane enrichment of genetic markers close to the origin and terminus during the deoxyribonucleic acid replication cycle in Bacillus subtilis.

A temperature-sensitive Bacillus subtilis initiation mutant was used to achieve one cycle of synchronized deoxyribonucleic acid (DNA) replication. Markers near the origin of replication and the terminus were assayed for association with the cell membrane at intervals during the DNA replication cycle. DNA near the origin and terminus was found to be enriched in the membrane fraction throughout the DNA replication cycle. The magnitude of membrane enrichment or origin and terminus markers varied coincidentally, possibly as a consequence of incubating the cells at 45 degrees C.


Synthesis of 1,3-beta-glucanases in Saccharomyces cerevisiae during the mitotic cycle, mating, and sporulation.

Upon fractionating Saccharomyces cerevisiae asynchronous cultures by sucrose density gradient centrifugation in a zonal rotor and examining the exo-1,3-beta-glucanase and deoxyribonucleic acid content of the cells, a periodic step increase in the activity of this enzyme was observed, indicating a discontinuous pattern of synthesis or activation of exo-1,3-beta-glucanase during the mitotic cycle at the transition from the S to the G(2) phase. Similar results were obtained for endo-1,3-beta-glucanase by assaying activity against oxidized laminarin in permeabilized cells, suggesting that the synthesis of endo-1,3-beta-glucanase is controlled in the same way. When a and alpha strains were mated, the specific activity of cell extracts against laminarin, oxidized laminarin, and pustulan remained constant while zygote formation was taking place. However, when growth resumed, active synthesis of 1,3-beta-glucanases took place as shown by the occurrence of a significant increase in the specific activity against the three substrates. Specific changes in the level of glucan degradative enzymes, not observed in a haploid parental strain, occurred when the diploid S. cerevisiae AP-1 was induced to sporulate. The sporulation process triggered the activation of first the pustulan degradative capacity and then the capacity to hydrolyze oxidized laminarin. The specific activity against this substrate was 10 times higher than that against pustulan.


Use of gene fusions to determine a partial signal sequence of alkaline phosphatase.

We have isolated strains of Escherichia coli in which an amino-terminal portion of the cytoplasmic enzyme beta-galactosidase is replaced by an amino-terminal portion of the periplasmic enzyme alkaline phosphatase. The synthesis of these hybrid proteins is regulated by inorganic phosphate and they are located in the cytoplasm. One of these proteins was purified, and 14 amino acids of the amino-terminal sequence were determined. The first five amino acids, Met-Lys-Gln-Ser-Thr, appear to represent a portion of the signal sequence of the precursor of alkaline phosphatase, and the remaining sequence corresponds to that of beta-galactosidase, beginning at amino acid residue 20. The approach described here could be used for the analysis of signal sequences of exported proteins and for partial amino acid sequence determination of certain of certain other proteins.


Effect of repair deficiency and R plasmids on spontaneous and radiation-induced mutability in Pseudomonas aeruginosa.

The effect of R plasmids on spontaneous and radiation (ultraviolet and gamma)-induced mutability in Pseudomonas aeruginosa was studied in strains containing the radiation-sensitive markers polA3 or rec-2 and the revertable auxotrophic markers hisO27 and trpB1. In the absence of an R plasmid, the radiation-induced mutability was dependent on the recA+ genotype and independent of the polA+ genotype, whereas spontaneous mutability was similar in all genetic backgrounds. R plasmids pPL1, R2, and pMG15 increased the ultraviolet radiation survival and ultraviolet-induced mutability of wild-type and polA host cells but did not alter either effect in a recA mutant. These R plasmids also increased the gamma radiation survival and gamma-induced mutability of wild-type host cells bud pMG15 also enhanced the level of spontaneous mutagenesis in wild-type host cells but not in a polA or recA mutant. These data suggested that a common plasmid gene product(s) may participate in various recA-dependent, error-prone deoxyribonucleic acid repair pathways of P. aeruginosa. The properties of a mutant R plasmid, pPL2, originally selected because it lacked enhanced ultraviolet-induced mutability, supported this conclusion.


Fatty acid omega and (omega-1)-Hydroxylation in rabbit intestinal mucosa microsomes.

The microsomes from rabbit intestinal mucosa which had been washed quickly and thoroughly with phenylmethylsulfonyl fluoride were found to catalyze the hydroxylation of fatty acids in the presence of NADPH and molecular oxygen. Myristic and palmitic acids were converted to the corresponding omega-and (omega-1)-hydroxy fatty acids, whereas lauric acid was converted only to 12-hydroxylauric acid, and capric acid, to 9-and 10-hydroxycapric acids together with an unknown polar acid. Among these fatty acids, both myristic and lauric acids appeared to be the most efficient substrates. The inhibition of the hydroxylation by SKF 525-A and carbon monoxide suggested that the activity depended upon cytochrome P-450. The specific activity of the fatty acid hydroxylation was almost constant along the small intestine, while the aminopyrine N-demethylation activity and the cytochrome P-450 content were highest at the proximal end of the intestine and progressively declined toward the caudal end. The cytochrome P-450 was solubilized from the intestinal microsomes and purified by 6-amino-n-hexyl Sepharose 4B chromatography. The partially purified cytochrome P-450 was active in fatty acid hydroxylation in combination with intestinal NADPH-cytochrome c reductase and phosphatidylcholine.


Turnover of pegeon breast muscle pyruvate dehydrogenase complex.

The pigeon breast muscle pyruvate dehydrogenase complex was resolved into three component enzymes: lipoate acetyltransferase, pyruvate dehydrogenase, and lipoamide dehydrogenase. The antibodies against each component enzyme were prepared. All of the antibodies against component enzymes precipitated the pyruvate dehydrogenase complex. The enzyme complex was recovered as the immunoprecipitate from the extract of breast muscle of a pigeon that had received a single injection of L-[4,5-3H]leucine. The immunoprecipitate was separated into each component enzyme by SDS-polyacrylamide gel electrophoresis. The relative isotopic leucine incorporations per mg of protein into each component enzyme 4 h after the injection were 1.0 : 0.9 : 1.4 : 2.7 for lipoate acetyltransferase, alpha- and beta-subunit of pyruvate dehydrogenase, and lipoamide dehydrogenase, respectively. The half-lives of lipoate acetyltransferase, alpha- and beta-subunit of pyruvate dehydrogenase, and lipoamide dehydrogenase were 7.7, 2.5, 2.6, and 1.8 days, respectively. These results indicate that the component enzymes of the pyruvate dehydrogenase complex were synthesized and degraded at different rates.


Conformational stability of ribonuclease T1. II. Salt-induced renaturation.

In the presence of high concentrations of the monovalent salts, sodium chloride and potassium fluoride, disulfide-reduced RNase T1 having four cysteinyl residues intact regenerates the spectral properties characteristic of native RNase T1, e.e., the fluorescence spectrum of the aromatic side chains and the ultraviolet circular dichroism spectrum. The folding of the polypeptide chain proceeded without formation of disulfide bonds to yield an enzymatically active species having an activity toward RNA equivalent to 25% of that of the native enzyme at the same salt concentration of 2 m. Unfolding of RNase T1 by a denaturant, urea, was suppressed in the presence of salts, and the salt-induced chain folding was observed spectroscopically even in 6.9 m urea solution. The salts also induced the chain folding of disulfide reduced and modified (carboxymethylated or carboxamidomethylated) RNase T1 into the native conformation, as indicated by its spectroscopic properties, but did not restore the enzymatic activity.


Structural components of sphingophosphonolipids from the ciliated protozoan, Tetrahymena pyriformis WH-14.

1. Two sphingophosphonolipids were isolated from the lipids of the ciliated protozoan, Tetrahymena pyriformis WH-14. They were ceramide N-methyl-2-aminoethylphosphonate (CMAEP) and ceramide 2-aminoethylphosphonate (CAEP), in yields of 0.05 mg/g and 1.74 mg/g dry cells, respectively. 2. Two chromatographically distinguishable CAEP species were found, a slow-moving major component and a minor component which moved faster; the slow-moving one contained primarily hydroxy fatty acids, while in the other one nonhydroxy fatty acids were predominant. However, their long-chain base constituents were similar. 3. The major fatty acids of CAEP were 2-hydroxy acids with carbon numbers of 16 to 19, which were almost exclusively iso-types. The fatty acids of CMAEP consisted mainly of palmitic, iso-octadecanoic, and 2-hydroxy iso-heptadecanoic acids. 4. The long-chain bases were dominated by C16, C17, and C19 iso-4-sphingenine homologs.


Studies on the glycosphingolipids of the starfish, Asterina pectinifera. II. Isolation and characterization of a novel ganglioside with an internal sialic acid residue.

1. Three gangliosides, provisionally named Gangliosides 1, 2, and 3, were obtained from the lipid extract of the starfish, Asterina pectinifera by silicic acid, DEAE-Sephadex, and Iatrobeads column chromatography. The most abundant, Ganglioside 3 (37.7 microgram/g wet weight of starfish) was isolated in the pure state and its chemical structure was studied. 2. The sugar composition of Ganglioside 3 consisted of arabinose, glucose, galactose, and sialic acid (as N-glycolylneuraminic acid) in a molar ratio of 1:1:3:1. Three sialic acid-containing oligosaccharide fragments were isolated from partial acid hydrolysates of the ganglioside by Dowex 1 X 8 (acetate form) column chromatography and preparative paper chromatography, and identified as Gal leads to NeuGc, Gal (1 leads to 4)[Gal(1 leads to 8)]NeuGc and Ara-(1 leads to 6)Gal(1 leads to 4)[Gal(1 leads to 8)]NeuGc. 3. The structure of Ganglioside 3 was postulated to be: Araf,p(1 leads to 6)Galpbeta(1 leads to 4)[Galpbeta(1 leads to 8)]NeuGc(2 leads to 3)Galpbeta(1 leads to 4)Glcpbeta(1 leads to 1)-ceramide. This is a unique structure with the sialic acid residue internally located in the sugar chain. 4. The ganglioside contained saturated 2-hydroxy fatty acids ranging in length from C16 to C24, among which C22, C23, and C24 acids were predominant.  The long-chain bases consisted exclusively of C16, C17, and C18 phytosphingosines of iso and anteiso types.


Calcium regulation in chicken gizzard muscle and inosine triphosphate-induced superprecipitation of skeletal acto-gizzard myosin.

Inosine triphosphate (ITP) does not serve as a substrate for myosin light-chain kinase from gizzard muscle. That is to say, myosin light-chain is not phosphorylated in ITP media. Nevertheless, at pH 6.8, 1 mM or 5 mM ITP induces superprecipitation of skeletal acto-gizzard myosin. The ITP-induced superprecipitation occurs in the absence or presence of calcium ions, and regardless of whether gizzard myosin is phosphorylated or not. On the other hand, at pH 8, 5 MM ITP induces practically no superprecipitation of skeletal acto-gizzard unphosphorylated myosin, whereas it does induce a strong superprecipitation of skeletal acto-gizzard phosphorylated myosin. Superprecipitation is also independent of the presence or absence of calcium ions.


Effect of deglycosylation on the binding and immunoreactivity of human thyroxine-binding globulin.

Thyroxine-binding globulin (TBG), prepared from human serum by an improved purification method, was treated with a mixture of neuraminidase, beta-galactosidase, alpha-mannosidase, and beta-N-aectylglucosaminidase, which resulted in the removal of approximately 86% of saccharides. Purification by thyroxine-Sepharose affinity chromatography gave a homogeneous protein as shown by equilibrium sedimentation and sodium dodecylsulfate-polyacrylamide gel electrophoresis. Amino acid and NH2-terminal sequence analysis indicated that the protein moiety was intact. Deglycosylation had no effect on the stoichiometry of the binding of L-thyroxine as shown by tryptophanyl fluorescence quenching and equilibrium dialysis at pH 8.6 and 25 degrees C. However, the affinity constant for L-thyroxine was reduced from 1.6 X 10(9) M-1 to 0.58 X 10(9) M-1. Analysis of radioimmunoassay data revealed that deglycosylation resulted in a slight decrease of the affinity constant for anti-TBG antibody from 3.9 X 10(10) M-1 to 1.8 X 10(10) M-1. These results suggest that the polypeptide moiety, rather than the heterosaccharides, contains the antigenic determinants. Removal  of the majority of the heterosaccharides of TBG has only a minor effect on its immunoreactivity and on the binding of thyroid hormone.


Primary structure of a human IgA1 immunoglobulin. V. Amino acid sequence of a human IgA lambda light chain (Bur).

The sequence of the lambda light chain of the Bur IgA1 molecule has been determined. It comprises 214 amino acid residues with a blocked NH2 terminus and lacks carbohydrate. The V-region sequence is of the VlambdaII subgroup and contains the coupled interchanges Arg-7 and Cys-87. The Lv3 region is comparatively short and hydrophobic in nature and lends support for the designation of this area as a hypervariable deletion region. The C-region exhibits the Mcg+ Kren+ Oz- isotypes. These appear coupled with substitution at position 100 (in the V-region). The pattern of nonrandom association of V- and C-regions and H and L chains is discussed in terms of the generation of antibody diversity. With the companion papers in this series, the complete primary structure of a human IgA1 molecule is established.


Conformation-specific antibodies to the alpha chain COOH terminus of hemoglobin A0.

An anti-hemoglobin antiserum obtained from a sheep immunized with human carboxyhemoglobin A0 demonstrated little difference in its reactivity with deoxy- or carboxyhemoglobin A0. However, a subpopulation of this antiserum isolated by synthetic peptide affinity chromatography clearly distinguished between these two hemoglobin species. This subpopulation, designated alpha(129-141) anti-hemoglobin antibodies, represents less than 1% of the total anti-hemoglobin antibodies. They are nonprecipitating by Ouchterlony analysis, and fluorescence-quenching studies demonstrate the interaction of a single antibody binding site per hemoglobin dimer. These antibodies bind preferentially to carboxyhemoglobin with a median affinity constant of 5 X 10(8) M-1 compared to binding to deoxyhemoglobin with a binding affinity of less than 1 X 10(8) M-1. Furthermore, the presence of these antibodies in stoichiometric amounts increases the oxygen affinity of hemoglobin, and thus antibody and oxygen binding to hemoglobin can be considered as a linked function.


Purification of Bacillus subtilis RNA polymerase with heparin-agarose. In vitro transcription of phi 29 DNA.

We have devised a new procedure for the purification of highly active preparations of Bacillus subtilis RNA polymerase holoenzyme. A column of heparin-agarose A-15m is used to rapidly and quantitatively adsorb RNA polymerase from the initial crude extract fraction. This affinity procedure obviates the necessity of including nucleic acid precipitation or partitioning steps and allows for rapid separation of RNA polymerase from proteolytic activity. The enzyme is further purified by preparative glycerol gradient centrifugation resulting in an overall purification in 200-fold in 24 h with near quantitative recovery of polymerase protein and activity. RNA polymerase holoenzyme is obtained by chromatography on single-stranded DNA-agarose. The in vitro transcription products made by purified preparations of B. subtilis and Escherichia coli RNA polymerase holoenzymes in response to B. subtilis phage phi 29 DNA have been analyzed, and an in vitro transcription map is presented. The E. coli RNA polymerase holoenzyme initiates transcription from three promoter sites not efficiently utilized by the B. subtilis holoenzyme under optimal conditions for RNA synthesis.


Precursor sequence of MOPC-315 mouse immunoglobulin heavy and light chains.

Partially purified mRNA coding for the MOPC-315 heavy (alpha) or light (lambda 2) immunoglobulin chain was translated in a nuclease-treated reticulocyte lysate containing 20 labeled amino acids. Radiolabeled precursor heavy and light chains, purified by immunoprecipitation and preparative gel electrophoresis, were subjected to Edman degradation. The labeled phenylthiohydantoin derivatives obtained in each degradative cycle were identified and quantitated by high pressure liquid chromatography. Both heavy and light chain precursor segments were hydrophobic in nature; however, they were not homolgous in sequence. To establish whether COOH-terminal proteolytic processing of the heavy chain might also be occurring during secretion, the cyanogen bromide peptides of the heavy chain precursor were compared to those of the mature secreted heavy chain. The results indicated that the COOH termini of the two chains were identical.


Fibrodysplasia ossificans progressiva. A survey of forty-two cases.

Fibrodysplasia ossificans progressiva in children can often be recognized at birth because of the presence of shortening of the great toe, characteristic hallux valgus, and short thumbs. The majority of patients have the onset of symptoms by the age of four years, but there is often a delay of many months before the diagnosis is made. Early recognition will allow protection of the child from injuries in which the damaged tissue will serve as a focus of calcification. Most patients with this condition are physically disabled. The site of onset and of greatest involvement is axial. Biopsy and surgery are best avoided. Use of EHDP (disodium ethane 1-hydroxy, 1-diphosphonate, or disodium etidronate) is not as yet of proved value in most patients. Data from this study support the concept that most cases of this lesion arise as dominant mutations.


The stress-tenogram in the diagnosis of ruptures of the lateral ligament of the ankle.

The stress-tenogram is a radiological technique for the investigation of injuries to the lateral ligament of the ankle, and combines the information previously provided by inversion and anterior stress radiographs, and the peroneal tenogram. It is designed to differentiate between stable and unstable ankles, and between isolated ruptures of the anterior talofibular ligament and combined tears of the anterior talofibular and calcaneofibular ligaments. A high degree of diagnostic accuracy has been confirmed at operative repair in a group of thirty-two patients.


Reconstruction of the anterior talofibular ligament for the treatment of the unstable ankle.

Chronic instability of the ankle is frequently due to disruption of the fibres of the anterior talofibular ligament and the anterolateral capsule. This allows momentary subluxations at the ankle joint; the talus tilts into an abnormal varus position and rotates forwards and inwards about a vertical axis which passes through the medial malleolus. The use of a free tendon graft to reconstruct the anterior talofibular ligament restores the stability of the ankle without restricting movement at the subtalar level.


Cytostatic effect of macrophages from non-immunised mice on mastocytoma P-815 cells in vitro.

Peritoneal macrophages from non-immunised C57/bl, C3H, and DBA/2 mice were examined for their capability of inhibiting the growth of mastocytoma P-815 cells in vitro. The rate of tumour cell growth was measured by the uptake of 125IUDR into the DNA of the tumor cells. Sodium thioglycollate-induced macrophages as well as resident macrophages inhibited the growth of allogeneic tumor cells distinctly, when incubated for 48 h. The effect depended on the effector to target cell ratio. Tumor cell growth was still reduced at a ratio of one macrophage to one tumor cell. A decrease in the proliferative activity of mastocytoma cells was observed in the presence of non-stimulated and non-immune macrophages from DBA/2 mice. This decrease in target cell proliferation, however, occurred more slowly.


Multiple actins in Drosophila melanogaster.

The tissue and developmental specificities of the three Drosophila isoactins, originally identified in primary myogenic cultures and in the permanent Schneider L-2 cell line, have been investigated. Of these three isoactins (I, II, and III), actins I and II are stable and actin III is unstable. Two-dimensional polyacrylamide gel electrophoretic analyses of total cellular extracts after 1-h [(35)S]methionine pulses were performed on a large variety of embryonic, larval, and adult muscle and nonmuscle tissues. The results suggest that isoactins II and III are generalized cellular actins found in all drosophila cell types. Actin I, on the other hand, is muscle-associated and is found exclusively in supercontractile muscle (such as larval body wall and larval and adult viscera) including primary myogenic cell cultures. Although actin I synthesis is not detectable during very early embryogenesis, it is detectable by 25 h and actin I is a major stable actin in all larval muscle tissues. Actin I is synthesized in reduced amounts relative to the other actins in late third instar larvae but is again a major product of actin synthesis in the adult abdomen. A stable actin species with the same pI as actin III has been identified in the adult thorax and appears to be unique to flight muscle tissue. This new stable form of thoracic actin may be the result of a stabilization of the actin III found in other tissues or may be an entirely separate gene product.


Increase of gap junctions between pancreatic B-cells during stimulation of insulin secretion.

The development of gap junctions between pancreatic B-cells was quantitatively assessed in freeze-fracture replicas of isolated rat islets under different conditions of insulin secretion. The results show that in resting B-cells, gap junctions are small and scarce but that these junctions increase when insulin secretion is stimulated. Both a short (90 min) stimulation by glucose in vitro and a prolonged (2.5 d) stimulation by glibenclamide in vivo raise the number of gap junctions; in addition, the glibenclamide stimulation causes an increase in the size of individual gap junctions. As a consequence, the total area occupied by gap junctions on the B-cell membrane and the ratio of this area to the cell volume were found significantly increased in the latter condition. The slight increase of these values observed after the glucose stimulation did not reach significance. These data indicate a change of gap junctions during the secretory activity of the pancreatic B-cells. The possibility that the coupling of the cells is affected by the treatment is discussed.


Analysis of the time of utilization of proteins for the initiation and completion of division in synchronized Tetrahymena pyriformis.

We exposed division-synchronized Tetrahymena pyriformis to pulse treatments with the structural amino acid analogue p-fluorophenylalanine. We related the timing of the treatments to the delays in onset or completion of division, or both. The results enable us to distinguish between early and late phases in cell division in which the cells bring into action proteins which contribute to initiate and complete division, respectively. Normally, these events are separated by 25-30 min.


A ligand-receptor model for the cohesive behaviour of Dictyostelium discoideum axenic cells.

Axenically grown cells of D. discoideum Ax-2 harvested in the log phase of growth, cohere rapidly when shaken in phosphate buffer. After 3.5 days in the stationary phase of growth, cells become completely non-cohesive. Although they do not stick to each other, stationary phase cells do stick to both log phase cells and aggregation-competent cells. The cohesion of stationary phase cells with these other 2 cell types is inhibited by both EDTA and the low-molecular-weight factor which we have previously demonstrated in stationary-phase growth medium. There is a decline in the sensitivity of slime mould cell cohesion to the low-molecular-weight inhibitory factor as the cells become aggregation-competent. This effect parallels the developmentally-regulated decline in sensitivity to EDTA. The low-molecular-weight inhibitor is not a chelating agent, however. The effect of the inhibitor seems to be specifically against contact sites-B mediated cohesion. We suggest that the simplest cohesive mechanism which can explain our results, is that the EDTA-sensitive cohesion of log phase cells could be dependent on a ligand-receptor system.


Antibody staining of 10-nm (100-A) filaments in cultured smooth, cardiac and skeletal muscle cells.

Antibodies were prepared against the SDS-denatured 10-nm filament protein 'skeletin' extracted from chicken gizzard. The specificity of the antibody to the 10-nm filament protein was shown by immunodiffusion before and after purification of the protein on SDS gels by the enzyme-linked immunoabsorbent assay (ELISA) and by its specific absorption with purified skeletin. In immunofluorescence (where preimmune sera and antigen-absorbed antisera gave negative results), cultured cardiac, skeletal and smooth muscle cells and endothelial cells stained intensely. No staining was observed in fibroblasts present in these cultures, nor was there staining in glial cells or nerve cell bodies and fibres from sympathetic ganglion and Auerbach's plexus cultures. Smooth muscle cells (regardless of their source and phenotypic state) and endothelial cells stained intensely in the perinuclear region and in a fine filamentous network that existed throughout the cytoplasm. In both chick and rat skeletal and cardiac muscle (cultures and frozen sections) filamentous network staining was observed, while in rat muscle the antibody was additionally localized in a regular pattern in the region of the Z-disk, and in the case of cardiac muscle associated with the intercalated disk. The addition of 10(-6) M colchicine to the culture medium of smooth and striated muscle and endothelial cells resulted in an aggregation of the filaments in the nuclear region. Cultured smooth and striated muscle and endothelial cells and freshly isolated smooth muscle cells extracted of actomyosin and tubulin by high and low ionic strength solutions gave a staining pattern similar to non-extracted cells and in the electron microscope, exhibited filaments of predominantly 10 nm diameter.


[Malignant plasma cell lymphoma of the stomach. Two cases and review of the literature (author's transl)].

Malignant plasma cell lymphomas have rarely been studied in the literature. We report two personal cases, the first survived 9 years, in the second case, histo-immunofluorescence revealed the presence of IgA kappa immunoglobulin. 59 cases have until now been reported to the best of our knowledge. They were reported under the term plasmocytoma. Analysis of data in the literature shows that plamocytomas resemble clinically and in prognosis, non-Hodgkin malignant lymphomas, of which Lukes and Lennert have recently given a new classification. Histo-immunofluorescence will permit in the future better understanding of the immunoglobulins secreted. Treatment is not yet standard. Three methods are used, depending on the spread of the tumour (surgery, radiotherapy and chemotherapy).


Comparative effect of calcium and of the adrenergic system on calcitonin secretion in man.

This study evaluated the effects of adrenergic agents on immunoreactive calcitonin (iCT) secretion in normal man, and compared the time course and magnitude of these adrenergic effects with those caused by modifying calcium (Ca) ion concentration. Ca infusion (15 mg Ca++/kg iv in 4 h) significantly increased plasma iCT within 1 h, reaching 140 +/- 8% of baseline at 4 h. EDTA (50 mg/kg iv in 2 h) significantly decreased plasma iCT within 15 min, with nadir value of 53 +/- 4.9% of baseline at 2 h. The beta-adrenergic agonist, isoproterenol, significantly increased plasma iCT with 5 min, reaching 136 +/- 5.9% of baseline at 30 min. The alpha-adrenergic antagonist, phentolamine, significantly increased iCT within 15 min, reaching 132 +/- 8.6% of baseline at 45 min. The beta-adrenergic antagonist, propranolol, significantly suppressed iCT with 15 min, reaching 51.8 +/-6.3% of baseline at 2 h. Therefore, 1) the adrenergic system (without induced change in serum Ca) can modify CT secretion to as great a degree as can change in Ca ion concentration induced by standard Ca and EDTA infusion tests and 2) even basal secretion of CT can be modified by adrenergic influences. These data strongly suggest 1) that the adrenergic system is an effective modifier of CT secretion and 2) that the adrenergic system, as well as Ca ion concentration, may play an improtant physiological role in control of CT secretion in man.


Evidence for a thyrotropin inhibitory effect of histamine in man.

Because of certain side effects of cimetidine therapy which may be hormonally mediated (e.g. gynecomastia), there has been recent interest in the possible endocrine effects of this H2 histamine receptor-blocking agent used in the treatment of peptic ulcer disease. Accordingly, the effect of chronic cimetidine therapy on anterior pituitary function was examined in 12 adult men with mild peptic ulcer disease. TRH and insulin-hypolycemic stimulation tests were performed by standard methods. Serum for TSH and PRL RIA was obtained after TRH; serum for GH, cortisol, and PRL RIA was obtained after insulin-induced hypoglycemia. In addition, serum for LH, FSH, testosterone, and PRL was obtained every 4 h for 24 h. After these baseline studies, 300 mg cimetidine were administered orally 4 times a day for 4--8 weeks and the studies were repeated as before. Chronic treatment with cimetidine caused a significant increase in the peak TSH response to TRH at 30 min (mean peak TSH value before cimetidine, 7.0 microU/ml; after cimetidine, 10.2 microU/ml; P less than 0.05) as well as a significant increase in the TSH area under the curve. There was no statistically significant effect of cimetidine on basal TSH or basal or stimulated PRL secretion. Cimetidine had no effect on the GH, PRL, or cortisol response to insulin-induced hypolycemia or the 24-h secretion of LH, FSH, testosterone, or PRL.


Rickettsial hemolysis: rapid method for enumeration of metabolically active typhus rickettsiae.

A new assay is described for enumerating biologically active typhus rickettsiae (Madrid E strain), based on adsorption of rickettsiae to erythrocytes in the presence of NaF (which allows adsorption but not lysis) and lysis in the presence of anti-Rickettsia prowazeki immune serum (which allows only a single round of lysis). The number of lysed erythrocytes is then used to estimate the number of active rickettsiae.


Coordinate secretion of acid hydrolases in rat bile.

Three lysosomal glycosidases, beta-glucuronidase (EC 3.2.1.31), beta-galactosidase (EC 3.2.1.23), and N-acetyl-beta-glucosaminidase (EC 3.2.1.30) have been investigated in bile that was freshly collected from rats through a complete bile fistula. Assay conditions have been established on the basis of appropriate kinetic studies. The biliary excretion patterns for these enzymes were found to vary considerably from rat to rat during the 24-h collection period. In a given animal, however, the three hydrolases were excreted in parallel and showed a gradual increase in activity with time, most marked after 10- 12 h of collection. 24-h biliary outputs of the three hydrolases averaged congruent with3% of their respective contents in total liver, and bile diversion had no effect on hepatic glycosidase activity or total protein content. Other enzymes known to be associated primarily with mitochondria, endoplasmic reticulum, and cell sap were also detected in bile, generally in smaller amounts. The biliary excretion of the plasma membrane markers, alkaline phosphodiesterase I and 5'-nucleotidase, however, was comparable to that of the lysosomal hydrolases. Biliary excretion of total protein was relatively constant and corresponded to 3.0% of the total hepatic protein content per day, whereas biliary bile acid secretion decreased during the first 12 h and then remained constant. Exocytic bulk discharge of hepatocyte lysosomes is proposed as the most likely mechanism for the biliary excretion of lysosomal enzymes. These results call attention to the possible pathophysiologic significance of biliary excretion of hepatic lysosomal contents as a means of residue disposal.


Inferior temporal lesions do not impair discrimination of rotated patterns in monkeys.

Ablation of inferior temporal cortex in the rhesus monkey produces a visual discrimination learning deficit. The severity of this deficit has often been found to be a function of task difficulty. This report concerns a type of visual discrimination problem that, although difficult, is not sensitive to inferior temporal lesions. Monkeys with anterior, posterior, and complete inferior temporal lesions were repeatedly unimpaired or only slightly impaired in learning to discriminate a pattern from the same pattern rotated 90 degrees or 180 degrees; yet they were very severely impaired in learning equally or more difficult discriminations of two different patterns. This demonstration that discrimination of orientation of patterns is relatively spared after inferior temporal lesions helps specify the pattern-recognition processes that require inferior temporal cortex.