Surface localization of virus production on a glucocorticoid-stimulated oncornavirus-producing mouse mammary tumor cell line by scanning electron microscopy. A chronically infected continuous mouse mammary tumor cell line containing virus particles of type B morphology, free of contaminating type C virions, has been grown in tissue culture. These cells were treated with dexamethasone, a synthetic glucocorticoid, a potent stimulator of mouse mammary tumor virus expression. Surfaces of untreated and dexamethasone-treated cells were investigated by scanning electron microscopy. Untreated cells demonstrated a moderate expression of mouse mammary tumor virus (80 particles/cell) distributed diffusely over the cell surface. However, virions on dexamethasone-treated cells were localized in clusters of 100 to greater than 2000 virus particles, often with more than one cluster per cell. Dexamethasone-treated cells typically showed a 10-fold increase in cell-associated virus over untreated cells. Concentrated extracellular fluids from untreated and dexamethasone-treated cultures were quantitated for free virus. Dexamethasone-treated culture fluids demonstrated a similar 10-fold increase of extracellular particles, in contrast to untreated cultures. This increase in virus particles on the cell surfaces as well as in the extracellular fluids supports the theory that dexamethasone has a stimulatory effect on viral replication, not just on the release of budding particles. The ultrastructure of budding mouse mammary tumor virus during dexamethasone stimulation, determined by scanning and transmission electron microscopy, and the significance of such an in vitro system for viral immunodiagnosis are discussed.


Carcinogenesis by carbamic acid esters and their binding to DNA. The tumor-initiating potency of three simple alkyl carbamates and mono-N-substituted ethyl carbamates was examined in Hall strain mice. The binding of 14C-labeled carbamates of DNA was measured in Crackenbush mice. Ethyl carbamate was the most potent carcinogen for the epidermis, liver, and lung, followed by its N-alkyl derivatives. Methyl carbamate was without effect but n-propyl and n-butyl were possible carcinogens. The ethyl esters bound to a greater extent to DNA in liver and skin than the methyl, n-propyl, and n-butyl esters and only this binding persisted. A preliminary application of croton oil increased the yield of skin tumors but not of liver or lung tumors. It also increased the binding of the alkyl carbamates to DNA in skin, the increase being greatest with ethyl carbamate. The binding persisted longer in treated than in non-croton oil-treated mice.


Receptor characteristics of specific estrogen binding in the renal adenocarcinoma of the golden hamster. Linear sucrose gradient analyses reveal that all estrogen-induced and -dependent primary renal tumor cytosols examined contain an 8 S and variable amounts of 4 S receptor in low ionic buffer concentrations. Similar results were obtained with extracts of primary metastases of these tumors. Sucrose gradients containing high salt (0.4 M KCl) convert the 8 S receptor in both the hamster renal tumor and uterus to a 4 to 5 S complex. Scatchard plot analysis reveals that the renal tumor cytosol estradiol-receptor complex has a Ka of 1.7 X 10(9) M-1 and 9.2 X 10(-10) M binding sites. Competition for the tritiated 17beta-estradiol binding sites in the renal tumor was similar to that in the uterus with respect to estrogenic compounds. Nonestrogenic steroids exhibited minimal competition at the same concentrations or higher. Substitution in the ring structure, particularly in position 3 of the phenolic A-ring, resulted in a considerable loss in the ability of such compounds to compete for these receptors. Aniestrogens were effective competitors for these estrogen receptors only at higher concentrations relative to the tritiated estradiol.


A quantitative study of the subcellular localization of 67Ga. This paper describes a quantitative procedure for the isolation of 67 Ga-binding granules (GBG) from normal rat liver and Morris 5123C hepatoma homogenates by a combination of rate and isopyknic density gradient zonal centrifugation. Another class of GBG has been found that is much smaller than the lysosomal GBG we have previously described. These smaller particles, or microvesicles, bind the largest portion of the 67Ga found in the hepatoma whereas, in the liver, the GBG lysosomes are the major binding component. Previously, we had shown that considerably more 67Ga is taken up in hepatoma than in liver (as percentage of administered dose per g of tissue). The preferential association of 67Ga with these microvesicles in the 5123C hepatoma may be indicative of a basic difference between normal and malignant tissue.


Some ultrastructural observations on the midgut epithelium of Isohypsibius augusti (Murray, 1907) (Eutardigrada). The midgut epithelial cells of the eutardigrade Isophypsibius augusti are organized into a convoluted monolayer. Only a single cell type could be distinguished although this cell type displayed considerable morphological variation. The midgut begins with crescent-shaped cells. More distally the cells are of variable height depending on the stored amount of nutritional material. No regenerative cells are present. Adjoining cells are held together by apical zonulae continuae. All the cells are characterized by a striated border, some basal infoldings, cytosis vesicles, numerous mitochondria, and abundant rough endoplasmic reticulum. Dictyosomes occur in small numbers. Ovoid or spherical inclusions (spherites), often concentrically laminated, are common. The cells, especially those along the middle part of the gut, are filled with large amounts of nutritional storage which includes polysaccharide material. The possible functions of the midgut in Tardigrada are discussed.


Immunohistochemical study on adenohypophysial primordia in organ culture. Rathke's pouches isolated from rat fetuses on day 12 were maintained in organ culture for 9 days and investigated immunohistochemically to test whether or not the hypothalamus is involved in the cytodifferentiation of the adenohypophysis. The unlabeled antibody enzyme method demonstrated that the cultured tissue contains different types of glandular cells, i.e., adrenocorticotropin (ACTH)-, growth hormone (GH)-, luteinizing hormone (LH)-, thyrotropin (TSH)-, and prolactin-producing cells. Indirect evidence was also obtained to indicate the presence of melanocyte stimulating hormone (MSH)-cells. These findings suggest that adenohypophysial primordial cells of rats start to synthesize their respective hormones without stimuli from the neurosecretory substances of the brain which are known to be essential for the maintenance of the secretory activity of the adult gland.


The fine structural localization of testicular phosphatases in man: the control testis. Electron microscopic cytochemistry was used to determine the localization of five phosphatase enzymes-glucose-6-phosphatase, inosine diphosphatase, thiamine pyrophosphatase, acid phosphatase, and adenosine triphosphatase-in control human testes. Glucose-6-phosphatase occurred in the endoplasmic reticulum and nuclear envelope of Sertoli cells, Leydig cells and primitive spermatogonia, but was not observed in more advanced spermatogenic cells. The presence of glucose-6-phosphatase activity paralleled the presence of glycogen in spermatogenic cells, i.e., both occurred in type AL and AD spermatogonia but not in type AP or B spermatogonia or in more advanced spermatogenic cells. Inosine diphosphatase activity was found in the endoplasmic reticulum, nuclear envelope, and Golgi complex of Sertoli cells and all spermatogenic cells except late spermatids. Additionally, inosine diphosphatase activity was localized at the junctions between Sertoli cells and late spermatids, but was not associated with any other plasma membrane. Thiamine pyrophosphatase reaction product was found in the Golgi bodies of Sertoli cells and in spermatogenic cells through immature spermatids. Neither inosine diphosphatase nor thiamine pyrophosphatase was observed in the Golgi bodies of spermatids during acrosomal formation. Acid phosphatase activity was found in lysosomes of spermatogonia, spermatocytes, and spermatids, in lysosomes of Leydig cells, and in lysosomes, lipofuscin bodies, and Golgi cisternae of Sertoli cells. It is thought that Sertoli lysosomes play a role in the phagocytosis of degenerating germ cells; however, the role of spermatogenic or Leydig lysosomes is unknown. Adenosine triphosphatase activity occurred at the interfaces between two spermatogonia, and between Sertoli cells and spermatogonia, but was not observed in the spaces between two Sertoli cells, two spermatocytes, two spermatids, or between Sertoli cells and spermatocytes, or between Sertoli cells and spermatids.


The formation and repair of single-strand breaks in DNA of cultured mammalian cells treated with UV-light, methylating agents or 4-nitroquinoline-1-oxide. The technique of sedimentation in alkaline sucrose was used to examine the formation and repair of single-strand (SS) breaks in cultured mammalian cells that were treated with methyl methanesulfonate (MMS), methyl nitrosourea (MNUA), 4-nitroquinoline-1-oxide (4NQO) or UV-light. The SS breaks induced by MMS and 4NQO were largely repaired by HeLa cells during a 5-h post-treatment incubation. The SS breaks induced by MNUA and UV-light were not repaired by HeLa cells. L-cells were not able to repair the SS breaks induced by any of the agents, which correlates with the deficiency of these cells for repair synthesis of DNA. The following conclusions are discussed. MNUA and UV-light produce modifications in DNA which are not repaired but are translated into SS breaks in alkali. MMS produces SS breaks intracellularly but these are not derived from a simple depurination of methylated purines. 4NQO produces a modification in DNA which is translated into an SS break in alkali but which can be removed by an intracellular process.


Alkylation of meseenger RNA by dimethylnitrosamine. Rats were treated with radioactive dimethylnitrosamine or methyl methanesulphonate and the alkylation of various RNA fractions was determined 4 h later. It was found that after administration of dimethylnitrosamine, hepatic messenger RNA (isolated by binding to oligo(dT)-cellulose) and nuclear RNA were alkylated slightly, but significantly less than ribosomal RNA. After treatment with methyl methanesulphonate nuclear RNA was alkylated to a greater extent than either messenger or ribosomal RNA. The implications of these findings with respect to the mechanism by which dimethylnitrosamine inhibits liver protein synthesis are discussed.


Mechanism of the autocatalytic formation of ferrihemoglobin by N,N-dimethylaniline-N-oxide. Structure and ferrihemoglobin forming activity of the purple dye. The structure of the leuco compound of the purple dye which is formed in mixtrues of N,N-dimethylaniline-N-oxide (DANO) and ferrihemoglobin or ferricytochrome c was elucidated. IR, NMR, mass spectroscopy, and synthesis by oxidation of mixtures of N-methylaniline and 2-dimethylaminophenol showed that the leuco compound is produced by condensation of these two compounds. But only X-ray analysis proved the structure: 2-dimethylamino-4-(N-methylanilino)-phenol. The purple dye was produced from the leuco compound by withdrawal of two electrons and may be considered as resonance hybrid of the p-quinonimine and the o-quinonimine. When DANO was incubated with ferrihemoglobin or ferricytochrome c the oxygen of DANO was used for the production of the dye by oxidation of N-methylaniline and 2-dimethylaminophenol. The amount of N,N-dimethylaniline found in the incubation mixtures corresponded with the amount of purple dye produced. In the absence of molecular oxygen from incubation mixtures of DANO with cytochrome c the purple dye was formed at the same rate as under air. In blood in vitro the purple dye catalytically transferred electrons from ferrohemoglobin to molecuar oxygen. Its ferrihemoglobin-forming activity was lower than that of 4-dimethylaminophenol but higher than that of 2-dimethylaminophenol. The chemical mechanism of the autocatalytic formation of ferrihemoglobin by DANO is described.


[Selection of cell lines resistant to cyclic AMP and theophylline from murine plasmocytoma MOPC 173. Cross resistance to ouabain and concanavalin A]. Using cAMP or theophyllin as selective agents, we obtained from contact inhibited and non contact inhibited cell lines, derived from the same plasmocytoma, different resistant cell lines. All of them were cAMP and theophyllin resistant and also ouabain and con A resistant as judged by the growth curves in presence of the different drugs.


[Estradiol and progesterone receptors in human endometrium during the menstrual cycle]. The concentrations of estradiol and progesterone receptors are measured in the cytoplasmic and nuclear fractions of endometrial samples obtained by biopsy throughout the menstrual cycle. The standardized micromethod accounts for the sites occupied by endogenous hormones and for interference by androgen receptors and plasma binding proteins. In normal women, the cellular concentration of estradiol receptor increases early in the cycle and decreases steadily after ovulation. The progesterone receptor is maximum between the 10th and 15th day of the cycle and decreases during the luteal phase. The nuclear/cytoplasmic ratios of both receptors follow the variations of circulating hormones. These data constitute a new basis for physiopathological and pharmacological investigations on the hormonal control of the human uterus.


[Early stimulation of protein kinase activity during hormonal meiosis reinitiation in starfish ovocytes]. Total endogenous protein kinase activity has been compared in homogenates prepared from Marthasterias glacialis and Asterias rubens oocytes treated or not by 1-methyladenine, the hormone which triggers meiosis reinitiation. A 5 mn treatment at the threshold 10(-7) M concentration causes a significant increase (+ 20%) of these activities. This stimulation is maintained or even enhanced when homogenates are complemented with exogenous histones.


[Short and long term depression of neuromuscular transmission in crayfish]. Following single  excitation of the abdominal giant motoneuron of crayfish, an important depression of neuro-muscular transmission is observed for about 15 mn. The time course of recovery is not modified even after low frequency (1/30 Hz) repetitive stimulation. Recovery curves obtained for different Mg++ concentration in physiological solutions show two phases of depression: short-term depression which is largely reduced in high Mg++ concentrations - thus probably depending on transmitter depletion in motoneuron terminals - long-term depression which is not modified by changing Mg++ concentration and seems to be independent of depletion.


[Isolation of murine sarcoma virus not associated with XC plague forming leukemia virus]. The N-type MLg cells transformed by B-tropic MuSV (WN1802B) produced defective MuSV without detectable among of MuLV; the focus formation by the culture fluid required the co-infection of exogenous MuLV. After one passage of the virus in MLg cells, we obtained a virus preparation which was capable of inducing foci without the exogenous MuLV, but which remained negative in the XC test.


Radionuclide methods in the evaluation of myocardial ischemia and infarction. Radionuclide techniques that assess regional myocardial perfusion and detect acute myocardial infarction promise to provide critical information in the detection and evaluation of coronary artery disease and in the assessment of therapies aimed at limiting the degree of ischemia and the extent of tissue necrosis. Radioindicators such as 99mTc-tetracycline and 99mTc-pyrophosphate which are sequestered by acutely infarcted myocardium provide a direct method to detect an infarct and to determine its size. Regional alterations in myocardial perfusion can be assessed by myocardial scintigraphy performed after the injection of radiopotassium or one of its analogues with the patient either at rest or at exercise. A somewhat more accurate evaluation of the extent of altered perfusion can be obtained after the intracoronary injection of macro-aggregated particles. A quantitative index of altered perfusion can be obtained after the intracoronary injection of an inert gas such as 133Xe.


Changes in electrophoretic mobilities of alpha-and beta-lipoproteins as a result of plasma delipidation. A two-phase system containing the ternary mixture butanol/disopropyl ether/plasma in different proportions yields ordered delipidation of alpha-lipoproteins, pre-beta-lipoproteins-  and beta-lipoproteins in plasma, as quantitated by densitometry after electrophoresis. As a consequence of delipidation the electrophoretic mobilities of pre-beta-lipoproteins and beta-lipoprotein increased, that of alpha-lipoprotein decreased.


Determination of apolipoprotein A and its constitutive A-I and A-II polypeptides by separate electroimmunoassays. Electroimmunoassays ("rocket" electrophoresis) are described for human serum apolipoprotein A and its constitutive A-I and A-II polypeptides. Purified lipoprotein A, A-I, and A-II were used to prepare monospecific antisera and to standardize assays. These specific, rapid (5-8 h), precise (the within-and between-assay coefficients of variations are 5 and 7%, respectively), and accurate (by gravimetry) assays are applicable to measurement of these polypeptides in whole serum and in various density classes of lipoproteins. Comparable results are obtained with intact and delipidized lipoproteins. Results correlated well with those obtained by radial immunodiffusion or radioimmunoassay. However, the present procedure is more rapid than the former and simpler than the latter immunoassay. Concentrations of A-I and A-II in the serum of normal men and women were similar (143 +/- 24 and 146 +/- 78 mg/dl, respectively, for A-I and 78 +/- 17 and 83 +/- 25 mg/dl for A-II). Subjects with type lla, llb, and IV hyperlipoproteinemias had similar concentrations of both polypeptides, while patients with type I disease, lecithin:cholesterol acyltransferase deficiency and LP-A deficiency had lowest concentrations of A-I (0.3-30 mg/dl) and A-II (11-20 mg/dl). The molar ratio of A-I/A-II in the serum and high-density lipoproteins was close to unity.


Statistical characterization of the random errors in the radioimmunoassay dose--response variable. We have developed practical methods for evaluating the magnitude of the random errors in radioimmunoassay dose--response variables, and the relationship between this error and position on the dose--response curve. This is important: to obtain appropriate weights for each point on the dose--response curve when utilizing least-squares curve-fitting methods; to evaluate whether the standards and the unknowns are subject to error of the same magnitude; for quality-control purposes; and to study the sources of errors in radioimmunoassay. Both standards and unknowns in radioimmunoassays for cAMP and cGMP were analyzed in triplicate. The same mean (Y), sample standard deviation, sy, and variance (2-y) of the response variable were calculated for each dose level. The relationship between s 2-y and y was calculated utilizing several models. Results for standards and unknowns from several assays were pooled, and a curve smoothing procedure was used to minimize random sampling errors. This pooling increased the reliability of the analysis, and confirmed the presence of the theoretically predicted nonuniformity of variance. Thus, the calculation of results from these radioimmunoassays should utilize a weighted least-squares curve-fitting program. These analyses have been computerized, and can be used as a "pre-processor" for programs for routine analysis of results of radioimmunoassay.


Stability of fatty acids in hyperlipoproteinemic plasma during long-term storage. Aliquots of hyperlipoproteinemic plasma were stored at -20 degrees C in a nitrogen atmosphere with added disodium ethylenediaminetetraacetate for up to one year, to determine the stability of fatty acids in the four major lipid classes under these conditions. Assays were performed at zero time and at 2, 5, and 12 months. No uniform fatty acid change was found. Minor statistically significant changes were found in total triglyceride fatty acids and in some of the individual fatty acids in cholesterol esters, phospholipid, and free fatty acids. These minor changes could not be accounted for by plasma lipid concentrations, lipolysis, or fatty acid peroxidation. Evidently, plasma can be stored in this manner for as long as a year without substantial change in fatty acids in any of the four major lipid classes.


Direct radioimmunoassay for serum estriol during pregnancy. I describe an assay for serum estriol during pregnancy, which does not require prior extraction or chromatography of the serum. Fifty microliters of 20-fold diluted serum can be assayed to detect as little as 20 pg of estriol. For a 20 mug/liter serum estriol concentration the CV was 16%; for 100 mug/liter concentration it was 5%. There was no significant interference by serum "binding" proteins. The assay measures unconjugated estriol, estriol conjugated with either sulfate or glucuronide at the 3' position, and a portion of the estriol 16 alpha (beta-D-glucuronide). Serum estriol concentrations increase during gestation from a low at 20 weeks of less than 10 mug/liter to a high of 100-150 mug/liter at term. Serum estriol values correlate well with those for urinary total estrogens and with values for human placental lactogen in serum at various gestational ages.


Erythrocyte adenosine kinase activity in gout. Erythrocyte adenosine kinase (AK) (EC 2.7.1.20) and guanosine monophosphate (GMP) reductase (EC 1.6.6.8) were measured in healthy male controls and primary gout subjects. Adenosine kinase activity in 19 controls and 26 gouty subjects was 0.717 +/- 0.176 and 0.615 +/- 0.128 nmol/mg protein/h, respectively. The difference was statistically significant (p less than 0.05). GMP reductase activity in 39 controls and 46 gouty subjects was 30.90 +/- 6.28 and 33.43 +/- 7.97 mumol/mg protein/h, respectively, without statistically significant difference.


Changes in the immunoelectrophoretic characteristics of serum alpha-I-lipoprotein induced by various dyes. The effect of 8 dyes on the electrophoretic mobility of normal human serum alpha-1 lipoprotein has been examined. The two characteristics of increase in anodal mobility, and conformational changes of the immunoelectrophoretic precipitin arc suggesting polymorphism, were found to vary independently among the dyes tested. The results suggest that dye induced electrophoretic mobility alteration may represent a useful tool for examination of heterogeneity of alpha-1 lipoprotein.


Chemotherapy of herpesvirus infections.

Chemotherapy of herpesvirus infections. Herpesviruses commonly produce lesions that come to the attention of physicians. Many different chemicals are known to suppress the growth of herpesviruses in vitro, but only a few of these have found application in clinical practice. A critical assessment of the place of some of these forms of chemotherapy was briefly presented.


Head pain.

Head pain. Head pain can be a challenging, difficult, and interesting diagnostic problem. A detailed history and carefully performed and directed physical and laboratory examination will aid in the complex differential diagnosis--which includes structural disease of the eye, ear, nose, throat, teeth; trauma; central and peripheral nervous system infections and tumors; and various neuropathies of unknown cause. Systemic abnormalities such as hypertension are a frequent cause, as are emotional upset and tension. Other isolated and extremely rare causes, such as the anatomic abnormality of an elongated styloid process, cannot be discussed in a presentation of this length, but are included in the bibliography.


Diffusion of bone morphogenetic activity from the residue of collagenase digested bone matrix gelatin through interstitial fluid. Bone morphogenetic activity is transmitted from the residue of a collagenase digest of bone matrix gelatin not only across cellulose acetate membranes but also through an interstitial fluid filled duplex diffusion chamber (a distance 300 + 2,000 mum). Collagenolysis enhances dissociation of the bone morphogenetic property of bone matrix and dissemination among mesenchymal cells proliferating in the host bed surrounding the diffusion chamber. The bone morphogenetic response is associated with secretion of interstitial fluid, enzymes, and fibrin as well as formation of new collagen fibrils beaded with coarse ruthenium red granules in the pores of the cellulose acetate membrane. Membranes with pore sizes too small to accommodate either new collagen fibrils or mesenchymal cell microvilli do not transmit the morphogenetic response.


Histological studies of subcutaneous and intraperitoneal implants of trypsin-prepared dermal collagen allografts in the rat. The possibility of using allograft collagen for the permanent replacement of lost or damaged connective tissues has been examined in the rat. The cellular components of skin, which are known to be of major importance in allograft rejection, were removed by treating skin with a solution of crystalline trypsin at 15 degrees C. Non-collagenous structures were largely removed by 7 days, but the purification process continued up to 28 days without damage to the collagen fibrils. Dermal collagen allografts, which were implanted intraperitoneally or subcutaneously and biopsied 3-83 days after operation, became recellularized and revascularized without being being resorbed. In contrast to skin allografts, there was no evidence of cellular rejection of the collagen grafts, even when recipient animals had been sensitized to allogeneic skin from the same donor. Densensitization of collagen to collagenase, by treating dermal collagen with solutions of glutaraldehyde at concentrations ranging from 0.001-1.0 per cent, was also investigated in vitro and by implantation. The best results, in terms of preservation of the collagen bundle architecture and graft recellularization without persisting inflammation, were achieved with collaged pre-treated with a solution of 0.01% glutaraldehyde.


Insoluble collagen I. The effect of substrate weight on the rate of solubilisation of polymeric collagen fibrils by bacterial collagenase. The enzymic solubilisation of insoluble collagen fibrils by bacterial collagenase has been examined employing unlabelled polymeric collagen fibrils (PC), rhodamine labelled fibrils (RB-PC), and fluorescein labelled fibrils (F-PC) as substrates. The presence of the label did not affect the kinetics of enzyme digestion but enabled the solubilized products to be rapidly estimated. The quantity of substrate used was found to play a major role in determining the quantity of peptides solubilised by a given enzyme concentration. It is suggested that in the PC fibrils only a limited number of TC molecules were suitably located to form an enzyme substrate complex with bacterial collagenase. Conditions were found in which the F-PC and RB-PC could be used as substrates in the assay of this enzyme.


Heparan sulfate of skin fibroblasts grown in culture. Primary cultures of normal human skin fibroblasts were examined for glycosaminoglycan content. Heparan sulfate was found in the growth medium of these cells, in fractions obtained by sequential collagenase and trypsin treatments, and in the remaining intact cells. Heparan sulfate was found to be the major sulfated glycosaminoglycan of the trypsin fraction but appeared as a smaller proportion of the collagenase fraction. The heparan sulfate of the growth medium, the collagenase fraction, and the trypsin fraction appeared to be proteoglycan while intracellular material appeared to be mainly free polysaccharide. The collagenase fraction is thought to be representative of "matrix" material produced by the cells, while the trypsin fraction may represent external cell surface material. The trypsin fraction heparan sulfate polysaccharide was relatively homogeneous in size with an average molecular weight of approximately 40,000 relative to a chondroitin sulfate standard. It was also relatively homogeneous in sulfate content, containing an average of 0.8 sulfate groups per disaccharide repeating unit. Approximately 50% of this was N-sulfate.


Studies on hyaluronic acid. V. Relationship between the protein content and viscosity of rooster comb dermis hyaluronic acid. Protein accounted for an average of 8.7% w/w of the hyaluronic acid obtained from rooster comb dermis extracts and three types of peptide constituents appeared to be present. A few collagen-like fibers were closely associated with the hyaluronic acid when samples were examined in the electron microscope and collagenase treatment decreased the intrinsic viscosity from 7000-5000 ml/g to 3900-2700 ml/g. The quantities of collagen present, however, were too small to detect chemically with the methods employed. The major peptide consituent was readily separated from the hyaluronic acid by fractionation in a cesium chloride gradient or by treatment with pronase. The viscosity was decreased by the density gradient procedure but not by the pronase digestion. Repeated fractionation in a cesium chloride gradient decreased the intrinsic viscosity still further and a small peptide constituent with a high glycine and serine content remained associated with a hyaluronic acid. The data suggest that an interaction or entanglement with collagen fibers is responsible for the high viscosity of hyaluronic acid in this tissue extract and that the viscosity of purified hyaluronic acid preparations is dependent upon interactions between adjacent polysaccharide chains. Interactions between the major peptide constituent and polysaccharide chains or the small residual peptide component remaining with hyaluronic acid after extensive purification procedures, however, appear to be involved in some organized structure because the presence of the major peptide constituent minimized the decrease in viscosity that occurred when hyaluronic acid samples were lyophilized.


Indoluble collagen II. The use of fluorescein labelled polymeric collagen fibrils in a very sensitive assay procedure for enzymes degrading insoluble collagen. 98% of the collagen in mature connective tissue is in the form of insoluble collagen fibers, consisting of bundles of polymeric collagen (PC) fibrils. The enzymes concerned in connective tissue remodeling degrade PC rather than tropocollagen (TC). TC is the most usual substrate for collagenase assays, and we believe it is essential to employ PC in any study of the activity of collagenolytic enzymes. In order to facilitate the study of enzymic degradation of PC we have labelled PC with fluorescein iso-thiocyanate to produce F-PC fibrils, containing 5 fluorescein labelled epilson-NH2 groups of lysine per TC molecule within the PC. The fluorescent F-PC is degraded at the same rate as PC with the release of hydroxyprolyl peptides but has the great advantage that the solubilised F-peptides can be quantitated by their fluorescent emission. The technique is described in detail employing bacterial collagenase and mammalian collagenase preparations to illustrate the methodology. The advantages of the fluorescent technique over the collagenolytic assay methods currently in use are outlined.


Mammary neoplasia in the guinea pig (Cavia porcellus). Mammary tumors are reported in four guinea pigs and the literature is reviewed. It is concluded that mammary adenocarcinomas are second only to pulmonary adenomas as the most common epithelial tumors in the guinea pig. The sex incidence of mammary tumors is almost equal and both young and old animals are susceptible. Mammary tumors are generally locally invasive, rarely metastasize, are often quite large, and very vascular. Surgical excision of mammary tumors is possible but recurrence should be expected if the primary tumor is incompletely removed.


Current status and usefulness of operative cholangiography. This article is a review of the recent literature on operative cholangiography. The objective is to summarize concisely the important contemporary viewpoints as an aid to students of radiology. To the practicing radiologist, it may offer a concise review of the subject and facilitate upgrading operative cholangiography in his hospital. The introduction covers statistical evidence of the growing dependence of biliary tract surgeons on operative cholangiography. The rudiments of diagnoses of the various diseases that can be detected by operative cholangiography are presented. Pertinent clinical facts along with cholangiographic diagrams are presented under arbitrary sections: I. Stones; II. Anomalies; III. Other Diseases; IV. Techniques of Operative Visualization of the Biliary System.


Ultrastructural surface alterations of serratia marcescens after exposure to polymyxin B and/or fresh human serum. Exposure of Serratia marcescens cells to 10, 5, and 2.5 mug/ml of polymyxin B resulted in outer cell surface alterations that consisted of coarse, pleomorphic projections which revealed a double-contoured membrane structure. In contrast, fresh, but not heat-inactivated human serum caused the deposition of very fine, thread-like aggregates on the outer cell surface of exposed cells. The combination of polymyxin B and fresh human serum caused clearly discernible ultrastructural changes of the polymyxin and the fresh serum type, respectively.


Antiviral 1,3,4-thiadiazoles. II. Effects on cellular and viral RNA synthesis in Mengo-virus-infected FL cells. Three antiviral 1,3,4-thiadiazoles caused, when incubated together with 3H-uridine, a reduction of the incorporation rate into acid-insoluble material of FL cells. Using the method of prelabelling cellular RNA synthesis was not remarkably influenced. However, the substances tested act as specific inhibitors of the virus-induced RNA synthesis.


Pneumatosis cystoides intestinalis.

Pneumatosis cystoides intestinalis. Pneumatosis cystoides intestinalis (PCI) is an uncommon disease of previously unknown etiology, characterized by the presence of gas-filled cysts in the walls of some portion of the gastrointestinal tract. This investigation showed that PCI was readily produced in the germfree rat by inoculating its peritoneal cavity with a pure culture of either C. perfringens or C. tertium. Similar inoculation of the germfree animal with any one of eight other clostridia species did not result in the formation of PCI. The bacterial theory for the formation of at least some cases of PCI is now established. This work suggests that part of the treatment of those fulminating diseases known to be frequently associated with PCI should be directed at controlling a possible clostridial infection.


Anal infections caused by herpes simplex virus. Herpes simplex infections of the perianal skin and anal canal are not uncommon, as evidenced by the present series of 16 cases, but have rarely been reported in the literature of the past. They are caused by the HSV-2 virus, which is also associated with genital lesions. There is abundant evidence that anogenital herpes is a venereal disease. Anal herpes is most commonly transmitted by anal intercourse. When the infection is present inside the anal canal, especially in a primary attack, the pain can be quite dibilitating. The diagnosis can usually be made on clinical grounds by recognizing the typical vesicles or aphthous ulcers, together with inguinal lymphadenopathy. The disease is self-limiting, but may be recurrent. Asymptomatic homosexuals may represent a large reservoir of the disease, which appears to be on the increase in our society. Cytology, immunofluorescence, viral cultures and serologic tests can be used for laboratory confirmation of the diagnosis. Newer methods of treatment with vaccine containing heat-inactivated virus, and use of photoinactivation following topical application of heterotricyclic dyes, show promise.


[Congenital cytomegalovirus infection after maternal renal transplantation (author's transl)]. A 34-year-old female gave birth to a 32 to 35 week-old girl one year after she had undergone renal transplantation. The child was dwarfed and microcephalic and also had congenital platelet deficiency and purpura. In addition it was anaemic and suffered from severe neonatal icterus. An initial hypogammaglobulinaemia disappeared except for low IgA levels. The immunoglobulin deficiency is probably correlated with the virologically and serologically proven cytomegalovirus infection. The incidence of this disease seems to be high in children of mothers with renal transplantations.


Phase transitions in the membrane of a marine bacterium, Pseudomonas BAL-31. An unsaturated fatty acid auxotroph, strain UFA, isolated from the marine pseudomonad Pseudomonas BAL-31, host cell of the lipid-containing bacteriophage PM2, was grown in media supplemented with different unsaturated fatty acids. Under these conditions the fatty acid composition of the cell could be altered drastically. The phase transition in the native membrane and in the extracted lipids was analyzed by electron spin resonance using a nitroxide spin probe. Membranes prepared from strain UFA grown in cis16:1 or trans16:1 showed one transition at 9.4 degrees C and 12.4 degrees C respectively. Extracted lipids in both cases had almost the same transition temperature as that of the intact membrane. Membranes prepared from Pseudomonas BAL-31 had one transition at approximately 12 degrees C, on the other hand there was no clear cut phase transition using extracted lipids. Replication of bacteriophage PM2 took place below the transition temperature of the membrane lipids in the case where strain UFA was grown in tran16:1. Other cases were not studied.


Norepinephrine-induced shift in levels of adenosine 3':5'-monophosphate and ATP parallel to increased respiratory rate and lipolysis in isolated hamster brown-fat cells. 1. Isolated brown fat cells from hamster respond to added catecholamines with a temporary increase in respiratory rate and an extended lipolysis. 2. From experiments with catecholamines and alpha and beta-blockers, the receptors of these cells are classified as beta according to classical definition. 3. Norepinephrine induces a rapid increase in adenosine 3':5'-monophosphate levels which parallels in time the stimulated respiration. Maximal adenosine 3':5'-monophosphate levels are reached within 1-3 min and are followed by a continuous decline. 4. Parallel to the catecholamine-induced respiration and lipolysis there is a pronounced drop in ATP levels. This energy depletion could be reversed by addition within 5 min after norepinephrine of the beta-blocker propranolol. 5. The nucleotide pattern in isolated hamster brown fat cells after norepinephrine addition was mimicked in experiments with isolated hamster brown fat mitochondria. From these experiments it is concluded that a high ratio of AMP and ADP over ATP decreases the respiratory rate when endogenous free fatty acids are oxidized.


Modifications in the release of rat growth hormone in vitro and the morphology of rat anterior pituitaries incubated in various ionophores. The ionophore A23187 increased the release of rat growth hormone in the presence of a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine; a second ionophore X537A inhibited growth hormone release induced by the methylxanthine. A23187 did not alter rat growth hormone release in the absence of 3-isobutyl-1-methylxanthine, but X537A enhanced hormone release in the absence of calcium or in the presence or somatostatin. These findings provide further evidence that both calcium and cyclic AMP are important in the regulation of growth hormone release. Tissue incubated in X537A combined electronlucent vesicles apparently derived from the Golgi apparatus, swollen granules and mitochondria with dense matrices. Tissue incubated in the presence of valinomycin or A23187 did not show altered morphology of either secretory granules or the Golgi complex. Possible mechanisms of these changes are discussed.


Human adipose tissue in culture. IV. Evidence for the formation of a hormone antagonist by catecholamines. Explants of human adipose tissue were cultured for several days with noradrenaline (NA). After the culture period the explants were carefully washed, preincubated and then incubated for 2 h by a technique analogous to those generally used to study adipose tissue metabolism. The results show that explants cultured with NA were considerably less responsive to the acute lipolytic effect of catecholamines than explants not previously exposed to NA. The diminished responsiveness could not be reversed by the addition of phentolamine or by preincubating the explants with a prostaglandin antagonist and it was dependent upon the concentration of noradrenaline used in the culture medium as well as upon the period of time that the explants were exposed to the hormone. The cyclic AMP (cAMP) levels did not increase in response to NA in these explants. Addition of theophylline or dibutyryl cAMP elicited a lipolytic response. However, the phosphodiesterase levels were not increased in the explants cultured with noradrenaline. When freshly excised tissue specimens were incubated in the culture medium which had previously contained NA the acute lipolytic effect of catecholamines was blunted. The results indicate that an antagonist is formed during the incubation with NA which inhibits the responsiveness to catecholamines.  The effect of the antagonist seems to be exerted at a step prior to the formation of cAMP.


Effect on plasma aldosterone, renin activity and cortisol of acute volume depletion induced by ethacrynic acid under constant infusion of angiotensin II and dexamethasone in man. The influence on plasma aldosterone of acute volume depletion induced by ethacrynic acid was studied in man. The experiments were performed during the morning in supine healthy males receiving a control infusion of 5% glucose or an infusion of angiotensin II (AII) to suppress endogenous renin production or an infusion of dexamethasone to suppress endogenous ACTH. Ethacrynic acid induced in all circumstances a similar diuresis and volume depletion. The rise of plasma renin activity (PRA) was effectively suppressed by AII and the rise of plasma cortisol by dexamethasone. Plasma aldosterone (PA) rose markedly even when the elevation of PRA or cortisol were suppressed. Yet when both endogenous renin and ACTH secretion were blocked, PA rose much less after ethacrynic acid. This residual increase could be attributed mainly to a decrease of the metabolic clearance rate (MCR) of aldosterone which had been measured before and after ethacrynic acid administration. The data presented indicate that multiple factors influencing PA after acute volume depletion could be dissected out and that renin, ACTH and a decrease of the MCR each contribute to the elevation of PA.


The pharmacological actions of some polymethylene-bis-(hydroxyethyl)-dimethyl-ammonium compounds on cholinergic transmission. Some polymethylene-bis-(hydroxyethyl)-dimethyl-ammonium (dicholine) compounds containing from 7 to 10 methylene groups between the quaternary nitrogen atoms, have been studied for activity at various sites of cholinergic transmission. The post-junctional activity of the compounds was investigated on the frog rectus abdominis muscle. Small doses of C8-, C9-, and C10-dicholine potentiated ACh contractions, however larger doses of all the dicholine compounds blocked the nicotinic receptor sites of the frog rectus muscle. The dicholine compounds blocked neuromuscular transmission in the rat phrenic nerve diaphragm. These actions at the rat neuromuscular junction are compared with the purely post-junctional actions on the frog rectus muscle. Although the compounds exert anticholinesterase activity in vitro it is suggested that this effect plays little part in the action of the drugs at the neuromuscular junction. It is concluded that the dicholine compounds have both pre- and post-junctional activity at the neuromuscular junction. The dicholine compounds are acetylated at varying rates by partially purified choline acetyltransferase (ChAc) although all are acetylated less readily than choline. The rate of acetylation of the dicholine compounds by ChAc parallels their activity in blocking neuromuscular transmission and it is suggested that other quaternary ammonium compounds containing hydroxyl or hydroxyethyl groupings may be acetylated by ChAc and this may affect their blocking action at the neuromuscular junction.


Effects of methylxanthines and imidazole on the contractions of guinea-pig ileum induced by transmural stimulation. Methylxanthines (10(-5) to 10(-3)M) were found to increase the amplitude of contractions of guinea-pig ileum induced by transmural stimulation but to inhibit those induced by acetylcholine or histamine. The order of the abilities of methylxanthines to augment the contractile responses was theobromine greater than caffeine greater than theophylline. When the contractions were completely suppressed by reduction of the calcium content in the medium or by addition of cyclic AMP, methylxanthines restored the responses effectively, just as does addition of calcium. Methylxanthines also accelerated the release of acetylcholine from the ileum associated with stimulation. Imidazole (3 X 10(-5) to 10(-3) M) had an essentially similar effect to methylxanthines in potentiating the contractile responses and in augmenting the release of acetylcholine. The present results indicate that the potentiating effects of methylxanthines and imidazole are due to an action on the nerve terminals, not on the postsynaptic membranes or contractile elements. Therefore, it si concluded that theit potentiating actions are due to facilitation of the movement of calcium in the nerve terminals on excitation, resulting in increased release of acetylcholine, and are not due to the effect of cyclic AMP formed as a result of their inhibitory actions on phosphodiesterase.


[Toxicologic and pharmacologic study of tributyltin oxide (TBTO)]. Toxicological study on bis-(tributyl-tin) oxyde shows a delayed toxicity of this compound in acute experiments. The same result is obtained in sub-chronic study. Most of the animals die after a great loss of weight. In pharmacological study, T. B. T. O. depresses central nervous system, but his action on ortho and parasympathetic system is slight and non specific. In rat, rabbit and dog, the effect is a fall in blood pressure that result from a depression of the vascular smooth muscle. The death of the animal is due to respiratory arrest by a central mechanism.


[The irritant and allergenic action of two isocyanates: toluene diisocyanate (TDI) and diphenylmethane diisocyanate (MDI)]. This is a study on the irritant capacity of two isocyanates (T. D. I. Toluene diisocyanate and M. D. I. diphenyl methane diisocyanate). The experiment was carried out on the hide and ocular mucous membrane of albino rabbits, using Draize method as well as the method for appraising cosmetics. An histopathological examination of the evolution of the lesions was then achieved together with a study on the delayed allergenic capacity. This study was carried out on albino guinea-pig hides using Kligman and Magnusson technique. In these experimental conditions, the conclusions were the following:--on rabbit hide, T. D. I. is a medium irritant, more caustic than M. D. I.,--on ocular mucous membrane, M. D. I. is a mild irritant; T. D. I. a strong one,--on guinea-pig hide, T. D. I. and M. D. I. may induce cutaneous sensitivity similar to contact allergy. Their allergenic capacity is long lasting but varies according to the allergen concentration.


[Binding of delta-8-THC to human serum proteins: binding as a function of lipoprotein composition]. In accordance with the function of the human lipoprotein composition, using serum of different lipid profiles, the distribution of delta8tetrahydrocannabinol, as isomer of delta9THC, was studied through the use of ultracentrifugation and fraction isolation, followed by biochemical analysis of the VLDL, LDL, and HDL fractions, as well as that of the non-lipoprotein protein fraction. The results indicate the existence of a linear correlation between the distribution of delta8THC and the phospholipids of the lipoprotein cenapse. No correlation between the quantity of bound delta8THC and the other lipoprotein constituents cholesterol, triglycerides, total lipids or protein could be observed.


Electrophysiological properties of nucleus reticularis tegmenti pontis cells: antidromic and synaptic activation. Antidromically and synaptically activated spike and synaptic potentials in the nucleus reticularis tegmenti pontis (NRTP) of the cat were recorded intracellularly. The antidromic firing of the NRTP neurone is composed of IS-SD spikes with short duration and short spike-after-hyperpolarization. Membrane resistance and firing patterns were studied by applying depolarizing and hyperpolarizing current through the recording electrode. Findings indicate that the NRTP neurone has a relatively high membrane resistance and is capable of firing at a high frequency. Single shock stimulation of the interpositus (IP) and lateral nucleus (LN) of the cerebellum, brachium conjunctivum (BC), red nucleus (RN) and cerebral peduncle (CP) induced monosynaptic EPSPs in the NRTP neurones. Superior vestibular nucleus (SVN) stimulation induced monosynaptic IPSPs. Collision tests showed that (1) NRTP neurones are activated by the axons of IP and LN neurones which travel through BC, (2) these axons also send collaterals to RN, and (3) there is convergence of cerebellar, cerebral and brain stem inputs to a single NRTP neurone.


Changes in excitability of the theta activity generating substrate by ACTH 4-10 in the rat. The present experiments tested the sensitivity of theta activity to ACTH 4-10 a peptide known to facilitate the maintenance of conditioned behaviors. Hippocampal theta activity was induced by electrical stimulation of the reticular formation in freely moving rats. The administration of ACTH 4-10 produced a typical 0.5 c/s shift indominant frequency and an increase in 7.5-9.0 c/s components of induced theta activity. The effects were maximal between 60-120 min after subcutaneous injection and lasted several hours. The fact, that a similar effect could be obtained by an increase in stimulus intensity suggests an elevated excitability in the theta generating system in the presence of the peptide. The possible significance of this finding for the maintenance of learned behavior is discussed.


The lateral reticular nucleus in the cat. IV. Activation from dorsal funiculus and trigeminal afferents. 1. The activation of neurones in the mLRN (major portion of lateral reticular nucleus comprising its parvi- and magnocellular parts) be a spinal path ascending in the dorsal funiculus (DF) and by trigeminal afferents has been studied. 2. Stimulation of the DF at C3 activated about one half of the mLRN neurones. The latencies were 2-28 ms. In experiments with the spinal cord interrupted at C3 except for the DF it was shown that cutaneous and high threshold muscle afferents in mainly forelimb nerves were effective. The latencies of the responses to nerve stimulation were 8-27 ms. 3. Stimulation of trigeminal afferents evoked a response in about one third of the mLRN neurones. The latencies were 2-27 ms. 4. Activation from the DF- and trigeminal paths occurred often in the same mLRN neurones and the neurones activated from the two paths had a similar location in the nucleus and a similar termination in the cerebellar cortex. 5. The DF- and trigeminal paths had similar properties. Activation was evoked from both ipsilateral and contralateral nerves. Fast adapting hair receptors were commonly effective. 6. Evidence is presented indicating that the DF- and trigeminal paths share a common final path to the mLRN neurones which is formed by brain stem interneurones intercalated between the DF- and trigeminal nuclei and the mLRN. It is suggested that these interneurones represent a supraspinal motor centre. 7. Activation from the DF- and trigeminal paths occurred with unequal frequency among groups of mLRN neurones activated from different spinal paths ascending in the ipsilateral lateral funiculus (cf. Clendenin et al., 1974a).


The effect of morphine on the activity evoked in ventrolateral tract axons of the cat spinal cord. The effect of morphine on the activity in ventrolateral tract axons was studied in intercollicularly decerebrate cats with and without spinal section. Activity was elicited by electrical stimulation of Adelta- and C-fibres in the sural nerves. In spinal animals, morphine injected intravenously in a dose as low as 0.5 mg/kg reduced the post-stimulus discharge of impulses recorded in ventrolateral tract axons below the site of transection. The depression was not only abolished but reversed by levallorphan and naloxone. Pretreatment with reserpine did not diminish the effect of morphine. The effect of morphine was considerably weaker in decerebrate cats. Reversible block of the spinal cord produced by cold revealed that morphine reduced inhibition from the brain stem controlling the impulse transmission to ventrolateral tract axons. It is concluded that a spinal effect contributes to the analgesic action of morphine.


Does an inhibitor of mitochondrial adenylate kinase also affect oxidative phosphorylation? Adenylate kinase activity of intact mitochondria is strongly inhibited by Ap5A, i.e. p1,p5-Di (adenosine-5') pentaphosphate, whereas oxidative phosphorylation is not affected. Therefore, Ap5A is a useful tool to distinguish between oxidative and non oxidative ATP generating reactions.


Effect of cyclic AMP and theophylline on phagocytotic activity of Tetrahymena pyriformis. Dibutyril cAMP and the cPDE-inhibitor theophylline both enhance the phagocytotic activity of Tetrahymena. Theophylline and cAMP-activating histamine are synergistic. It follows that the cAMP-adenylcyclase system functions in the unicellular animal Tetrahymena.


Preparation of isolated single cardiac cells from adult frog atrial tissue. Isolated cardiac cells from bullfrog atrial tissue can be readily prepared by digestion of intact fragments of atrial tissue with trypsin and collagenase. These isolated cells have dimensions of about 5 mum in width and range in length from 300 mum to over 500 mum. Such isolated cells may prove useful for the investigation of contractile activity of cardiac muscle at the single cell level and at the sarcomere level within the single cell.


[Influence of lithium chloride on lipid metabolism]. Lithium chloride was injected subcutaneously to mice (in amounts of 500 and 300 mg/kg) once a day for 10 and 15 days, and to rabbits in single doses via a tube into the stomach (in amounts of 400 and 850 mg/kg). After different time intervals following a single or course-wise administration of the compound the level of total lipids was determined in the muscles and liver of the mice, and of the total lipids, beta-lipoproteins, phospholipids, cholesterol, fatty acids and 11-oxycorticosteroids levels in the blood serum of rabbits and of the bile acids content in the vesical bile of these animals. Hyperlipemia that developed under the effect of lithium was caused by mobilization of fat from fat depots and by deranged interstitial lipids metabolism.


[Biochemical correlates of functional features of visual cortex neurons]. Changes of protein concentration and of different metabolic ferments activity were studied in the rabbit visual cortex after visual deprivation. Two groups of neurons with different sensitivity to visual deprivation were found. The less sensitive are supposed to have an additional source of activation. These findings corroborate the microelectrode data showing that the activity of about 40% of the visual cortex neurons are modulated by a different nonvisual stimulus.


[Antidromic identification of hypothalamic neurosecretory cells which participate in the anterior pituitary control (author's transl)]. Attempts were made to identify hypothalamic neurosecretory cells which participate in the anterior pituitary control with a special reference to the effects of ovarian hormones. Wave forms of the antidromically activated action potentials in the hypothalamus were investigated in the female rats of Wistar strain. The animals of different endocrinological environments, that is, proestrus and diestrus-I of 4-day cyclers; ovariectomized, ovariectomized and estrogen primed, ovariectomized and progesterone primed, were used. The operational procedures were performed under light urethane anesthesia. Different two types of craniotomy were adopted for the preparation. For recordings from the medial basal hypothalamus, the ventral surface of the brain was exposed by parapharyngeal approach, and a bipolar silver ball electrode for the application of electrical stimuli was placed on the surface of the median eminence (ME). Another method was used for recordings of the medical preoptic area (MPO) and the periventricular area of the MPO and the anterior hypothalamus (PVA). In the latter, the parietal surface of the brain was exposed and a side-by-side bipolar electrode for the stimulation was inserted to the arcuate (ARC-ME region. All the recordings were performed by using glass pipettes which were filled with 0.5 M sodium acetate solution. The solution was added with pontamine sky blue 6B to allow marking recording sites. Stimulatory pulses of 5 msec duration of variable intensity were generated by an electronic stimulator and applied by the above mentioned bipolar electrodes. The antidromic responses and the intensity of the stimuli were observed and photographed on a synchroscope. ...


[Further purification and immunohistological localization of human placental lactogen in syncytiotrophoblasts (author's transl)]. In 1936, Ehrhadt reported a prolactin-like substance which has been identified as a second protein hormone exists in extracted substance from the placenta. To date various reports have been published, but ther are still unsolved problems concerning the specifity of the substance, and its clinical significance is unclear. The purpose of this project was to purify and investigate the biochemical, immunological properties of HPL, and try to localize HPL secretion immunohistologically. The biological properties examined by intramuscular and intradermal pigeon crop sac reactions, both showed positive results. Biochemical properties were tested by 7.5% polyacrylamide disc electrophoresis and Free Fatty Acid mobilization. By these methods, the former showed a single band by electrophoresis with purified HPL 200 mug, and the latter showed a strong positive reaction both in vivo and in vitro. Immunological propert iese were examined by immunoelectrophoresis and the Ouchterlony method. In immunoelectrophoresis, the reaction of purified HPL 80 mug and anti-HPL serum resulted in a single strong zone. In constrast, anti-serum and HGH resulted in a weak zone of reaction. We confirmed that HGH cross reacts with our anti-HGH serum to HPL. The cross reaction of HGH with anti-HPL serum was reported by Josimovich et al, and other workers. In the Ouchterlony method, no precipitation line was detected between anti-HPL serum and 50-300 iu of HCG (PREGNYL). The results of investigating the localization of secritions of HPL immunohistologically, show that the specific fluorescence was limited to the strong reacting syncytial layer in the both the early chorionic villi of normal pregnancy and in the late stages of pregnancy. And the results of examining the localization with enzyme reactions with peroxdase from horseradish Type II, the reaction of benzidine brown was limited to the syncytial trophoblastic layer as in the case of FITC. These results suggests that the localization of the secretion of HPL exists in the syncytial layer of the chorionic villi.


Virus-induced diabetes mellitus. IV. Genetic and environmental factors influencing the development of diabetes after infection with the M variant of encephalomyocarditis virus. The M variant of encephalomyocarditis virus (EMC) infects pancreatic beta cells and causes the development of a diabetic syndrome in susceptible strains of mice. By examining four F1 crosses of susceptible and resistant strains of mice, we found that the development of diabetes after infection was inherited as a recessive trait. Analysis of the data from the F2 generation indicated that more than one gene was involved in the development of EMC-diabetes. The severity and frequency of abnormal glucose levels in EMC-infected animals was markedly influenced by environmental factors.


Influence of cell size on the effects of insulin and noradrenaline on human adipose tissue. In the present study dose-response relationships of the effects of noradrenaline and insulin on fat cells of different sizes were performed. Adipose cells larger than 100 mum were more responsive (expressed as absolute effects) to the lipolytic action of noradrenaline as well as to the antilipolytic effect of insulin. This suggests that in the larger cells the capacity, i.e. the sum of factors contributing to the ability to stimulate or inhibit the metabolic rates, was greater than in the smaller ones. In contrast the sensitivity to these agents, i.e. the readiness to respond, was not different between small and large cells. It is shown that the concentrations of insulin needed to obtain an antilipolytic effect is far below that needed to stimulate glucose incorporation. This discrepancy in insulin concentrations required may be due to binding of insulin to receptors with different affinity.


Axyl hydrocarbon hydroxylase in Morris hepatoma 5123D. Aryl hydrocarbon hydroxylase activity in Morris hepatoma 5123D is very low, but apparently increases by the administration of 3-methylcholanthrene, as demonstrated in the host liver, small intestine, and lung from the rats bearing this tumor. The peak level of the increased activity in tumor appears 48 hr after the initial administration when the repeated treatment is given intragastrically every 24 hr. 7,8-Benzoflavone increases the enzyme activity in tumor and lung, but not in liver and small intestine. Among the subcellular fractions of the tumor cells, induction of the enzyme by 3-methylcholanthrene was observed in microsomes and nuclei, and the increased level of the activity was higher in microsomes than in nuclei.


Inhibition of multinucleation of cells treated with cytochalasin-B by cyclic dibutyryl-AMP. Multinucleation of SV40-transformed mouse cells was induced by Cytochalasin-B as a result of inhibition of cytokinesis. Multinucleated cells with nuclei, whose number is other than that that can be obtained by raising the power of 2, were frequently observed. By simultaneous addition of Cytochalasin-B and cylic dibutyryl-AMP to SV40-transformad mouse cellls, multinucleation was fairly inhibited and the predominance of the cells with 2 or 4 nuclei was characteristic. In case of normal mouse cells (BALB/3T3), addition of both Cytochalasin-B and cyclic dibutyryl-AMP lessened the number of nucleic in a cell compared with treatment of the cells with Cytochalasin-B alone. These results suggested that cyclic dibutyryl-AMP inhibited multinucleation of cells treated with Cytochalasin-B and that the chemical regulated the division of nuclei in a cell to divide simultaneously.


Bicarbonate and cyclic AMP content of pure human pancreatic juice in response to graded doses of synthetic secretin. In seven healthy volunteers pure pancreatic juice was obtained by endoscopic cannulation of the papilla of Vater. Synthetic secretin was intravenously infused in doses doubled every 20 min. The volume of pancreatic juice was proportional to the log of the secretin dose. A significant rise (P less than 0.05) in pancreatic juice flow was elicited at a dose as low as 8.05 ng per kg per hr. Maximum flow rate approximating 250 mul per 5 min per kg of body weight was attained during infusion of 129 ng per kg per hr. At the same dose maximal bicarbonate outputs averaging 383 muEq per hr per kg of body weight were obtained, whereas bicarbonate ion concentration approached peak values (135 +/- 9 muEq per ml) at 32.2 ng per kg per hr. Bicarbonate concentrations showed a tendency to fall at supramaximal doses. The effect of increasing secretin doses on bicarbonate and cyclic AMP concentrations was remarkably similar (rs = 0.635, P less than 0.001) suggesting the participation of cyclic AMP in human pancreatic bicarbonate secretion.


Cyclic AMP in secretin choleresis. Evidence for a regulatory role in man and baboons but not in dogs. Cyclic AMP output in the bile in response to intravenous secretin was measured in 11 patients, 12 baboons, and 15 dogs. Secretin was given to patients with bile drainage tubes as an intravenous bolus (1 U per kg). In baboons and dogs both secretin infusion (4 U per kg per hr) and bolus injection (1 U per kg) were used. In baboons cyclic AMP was also determined in liver, extrahepatic duct tissue, and in perfusate from isolated segments of extrahepatic bile ducts. Secretin induced a marked choleresis in all three species. In humans, biliary cyclic AMP concentration increased an average (+/- 1 SE) of 68% +/- 12% and in baboons 4-fold, but no increase occurred in dogs. In baboons, cyclic AMP concentration increased in both bile duct tissue and perfusate from isolated bile ducts concomitant with secretin choleresis, but not in liver. In humans the choleretic effects of sodium dehydrocholate, aminophylline, and glucagon were compared to dibutyryl cyclic AMP (DBcyclic AMP). All agents increased bile flow 2- to 3-fold. Cyclic AMP concentration in bile markedly increased after glucagon and DBcyclic AMP but not after sodium dehydrocholate and aminophylline. We conclude that cyclic AMP is implicated in secretin choleresis in both humans and baboons, but not in dogs. The bile duct appears to be the site of cyclic AMP elaboration induced by secretin in baboons and probably is also in man.


Effects of cholera enterotoxin, glucagon, and dibutyryl cyclic AMP on rat liver alkaline phosphatase, bile flow, and bile composition. Cholera enterotoxin, 45 mug per 250 g body weight, administered intravenously to rats, caused a 6-fold rise in the activity of liver alkaline phosphatase in 12 hr. There was no change in bile volume or in the concentration or total bile content of Na+, K+, HCO3-, or Cl- for 36 hr after the administration of cholera toxin. However, bile phospholipid output fell markedly from a control level of 15.0 +/- 1.0 mumol per 6 hr to a low level of 4.0 +/- 1.2 mumol per 6 hr in the 12- to 18-hr collection, P less than 0.001. There was a similar fall in bile acid secretion, from a control value of 9.8 +/- 0.4 mumol per 6 hr to 4.1 +/- 0.9 mumol in the 12- to 18-hr period, P less than 0.01. The cholera effect was prolonged. Bile acid and phospholipid secretion rates did not return to control values until 30 to 36 hr after the administration of cholera enterotoxin. The cholera toxin-induced reductions in bile acid and phospholipid secretion into bile did not appear to be mediated by adenyl cyclase or cyclic AMP because neither glucagon, a known stimulator of liver adenyl cyclase, nor dibutyryl cyclic AMP had any effect on the secretion into bile of bile acids or phospholipid. The administration of cholera toxin was not associated with any increase in the secretion of free choline into bile. Glucagon and dibutyryl cyclic AMP, two other substances known to increase the activity of rat liver alkaline phosphatase, also had no stimulatory effect on the secretion of free choline into bile. The results do not support the hypothesis that the main function of rat liver alkaline phosphatase is to facilitate the excretion of free choline into bile.


[Present problems of somatic cell genetics]. A review of recent advances in genetics of somatic cells is given in the article. It contains three sections: 1) hybridization of somatic cells and its application to the mapping of genes and to the study of gene action; 2) relation of somatic cell genetics to genetic engineerings; 3) progress and perspectives of higher plant somatic cell genetics.


[A case of endogenous familial hereditary malformation of both hands]. A case of congenital malformation of both hands in a 22 year old television-technician, who otherwise showed no abnormalities, is reported. In his case, a familiar incidence was observed constantly in all males for a period of nearly 200 years. The morphological picture of this malformation is as follows: left hand: postaxial oligodactyly with a cutaneous syndactyly of the third and fourth fingers accompanied by a V-shaped os metacarpale and a synostosis of the os triquetrum, os lunatum and possibly of the os pisiforme right hand: postaxial oligodactyly with an accessory hypoplastic third finger on the ulnar side with a common base of the proximal phalanx which was enlarged at the end and shorter than the others (symbrachybasophalanx). Os multangulum minus and os capitatum as well as os triquetrum and possibly also the os pisiforme showed a synostosis. Subjective symptomatology and further clinical investigations (Phonocardiogramm) revealed an associated heart disease. This kind of malformation of both hands is transmitted inclompletely only in males.


[Preliminary report on the follow-up examination of 101 surgical cases of syndactylia]. A review of 48 patients in whom 101 syndactyly separations were performed between 1963 and 1972 is reported with the peaks of frequency of the inital operation (being in early infancy, and just before entering school). Different surgical techniques were used for restoration of the web space, with the two-flap-technique of ZELLER most often used. About 60% of the fingers were separated by longitudinal incisions with the remaining 40% with zig-zag incisions. Of the skin grafts, about two-thirds showed excellent cosmetic appearance with the remaining third showing evidence of hyperpigmentation. Digital abduction was measured and approximated the spread seen in normal children.


[A comparison of xero-tomography of the lung hilum with conventional tomography (author's transl)]. Because of its particular photoelectric properties, xero-tomography is particularly suited for the investigation of abnormalities in the hilar regions. It is superior to conventional tomography because of the improved edge contrast, which emphasises details of relatively non-opaque structures, and because of its high exposure latitude. This justifies its use despite the higher radiation dose that is necessary. The demonstration of relatively dense and of more transradient tissues on the same picture by xero-tomography, and the ability to read it by ordinary light, makes it less tiring to interpret than the translucencies of conventional radiographs.


Bone scanning with 99m-Tc-phosphates: a comparison and problems in the detection of tumor metastasis. A comparative study on 99m-Tc-phosphate compounds (TcPP) in detecting tumor metastasis to bone and problems accompanying it are reported. TcPP revealed metastatic foci which are unrecognized by conventional bone survey. To recognize these foci, exclusion of following problems is necessary: Accumulation at front of neck, asymmetrical image of joint, increased bone density of the aged, Tc-photon absorption and radiotherapy effect. The mechanism of TcPP accumulation is discussed.


[Angiographic findings of a mixed angiodysplasia (Klippel-Trenaunay-Weber syndrome) associated with choanal atresia (author's transl)]. The angiographic, phlebographic and lymphographic findings in a 3-year old girl with mixed angiodysplasia (Klippel-Trenaunay-Weber syndrome) combined with unilateral congenital choanal atresia are described.


[Percutaneous recanalisation of chronic arterial occlusions (Dotter principle) with a new double lumen dilatation catheter (author's transl)]. A modification of Dotter's transluminal recanalisation of stenoses and occlusions of the superficial femoral artery, and of stenoses of the iliac artery, is described. In this procedure the occluding material is compressed not by successive catheters, but by a catheter with a dilatable cover. This catheter consisted originally of a single lumen, but now has double lumen; one lumen accomodates contrast, or a guide wire or a stiff cannula, whereas the second lumen is used for filling the cover and the expandable segment. The procedure is described in detail. Results so far appear satisfactory; they are compared with the results which have been obtained during the last year with the single lumen catheter.


[Mode of fatty acid synthesis in rat liver mitochondria (author's transl)]. In order to clarify the mode of fatty acid synthesis in rat liver mitochondria, metabolism of labeled fatty acid was examined after portal and intraperitoneal administration of several labeled fatty acids. 1) After portal administration of palmitic acid-1-14C, alpha, beta-mono-unsaturated fatty acid (C18:1) was synthesized as a result of chain elongation by addition of endogenous C2 compound 20 min. after injection. 2) Similarly, after lauric acid-1-14C injection, myristic acid (C14) and palmitic acid (C16) were found 20 min. after injection and even C18:2 was detected 30 min. after injection. 3) Following intraperitoneal administration of lauric acid-1-14C, the appearance of palmitic acid (C16) was confirmed in inner mitochondrial membranes 60 min. after injection. From the analysis of intramolecular distribution of labeled carbon, it is surmised that lauric acid is first decomposed into C2 fragments which then are used for the chain elongation.


[Fractionation of inner and outer mitochondrial membranes in rat liver (author's transl)]. Mitochondrial membranes of rat liver were fractionated into inner and outer membranes by osmotic shock. Purity of the inner and outer membrane subfractions was examined by estimating marker enzyme activities of succinate-cytochrome c reductase and monoamine oxidase, respectively. Monoamine oxidase activity in the outer membrane subfraction was found to be 21.90 mmumoles of benzaldehyde produced/min./mg. protein and 1.57 mmumoles in the inner membranes, whereas succinate-cytochrome c reductase activity was 13.4 and 33.0 mmumoles/min./mg. protein, respectively. These results indicate higher purity of each membrane fraction obtained.


Investigations on isolated islets of Langerhans in vitro. Influence of temperature changes during preparation on some parameters of insulin metabolism. Pancreatic islets of Wistar rats were prepared by digestion with collagenase and then washed and isolated at three different temperatures (4, 22 and 37 degrees C). The efficiency of washing with regard to proteolytic and collagenolytic activities in the wash buffer was not affected by the temperatures used. The islet thiol:protein-disulphide oxidoreductase activity (EC 1.8.4.2) was apparently unchanged, whereas washing temperatures lower than 37 degrees C resulted in a diminished insulin content. The insulin secretion of islets, isolated at 4 degrees C, is reduced in response to glucose without changing the sigmoidal shape of dose-response curve.


Long-term follow-up studies on herpes simplex antibodies in the course of cervical cancer. II. Antibodies to surface antigen of Herpes simplex virus infected cells. In a serological follow-up of 88 women with active invasive cervical carcinoma, antibodies to membrane antigen of Herpes simplex virus (HSV)-infected cells were measured by the use of the mixed hemadsorption method. Sera were collected at the time of initial treatment and then at regular intervals. As controls, 85 healthy age-matched women and 70 patients suffering from other types of cancer were tested similarly. During the 6-to 60-month observation period 16 patients in the cervix carcinoma group died. The reactivity with membrane antigen of HSV-infected cells was positive in 71% of the cervical carcinoma patients, whereas the figure for the other cancer group was 27% and for the age-matched control group 27%. In the group of 16 patients who died, only 3 demonstrated antibodies against surface antigens of HSV-infected cells. In most cases radiation treatment of the tumor did not significantly alter the mixed hemadsorption titer but in 10 surviving patients there was a significant increase in reactivity as the tumor was treated; 1 patient who had recurrence of her cancer lost reactivity in later sera. Among the 11 cervix carcinoma patients in stage I (by clinical definition carcinoma strictly confined to the cervix) all but one showed positive reaction against the surface antigen of HSV-infected cells; the patient lacking reactivity was the only one out of the 11 patients in stage I who had a recurrence of her cancer. The results confirm that low or missing antibody titers to membrane antigen of HSV-infected cells are of prognostic significance, and decreasing antibody titers to membrane antigens run parallel to the severity of the lesions. There was poor correlation between antibody titers against membrane antigens and neutralizing antibody titers.


Transformation-enhancing factor(s) produced by virus-transformed and established cells. Chick embryo fibroblasts (CEF) and hamster BHK21 cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) release into the culture medium a factor or factors which enhance 2- to 7-fold the formation of transformed foci by chich embryo fibroblasts infected with the Bryan strain of RSV (B-RSV). The factor(s) also increase the number of foci failing to revert to normal phenotype at restrictive temperature (41 degrees C) in cultures infected with a temperature-sensitive mutant (FU-19) of SR-RSV which is defective for transformation. The factor(s) is produced also by BHK21 cells transformed by other tumor viruses and by BHK21 cells passaged for a long time, but not by normal CEF, CEF transformed by B-RSV, CEF infected by FU-19 at 41 degrees C, normal hamster embryo fibroblasts, established but density-inhibited mouse fibroblasts, or BHK21 cells of early passages. The relative enhancement of the number of B-RSV foci can be more than 100-fold when the medium contains fetal calf serum which suppresses focus formation in controls. The focus-enhacing factor(s) appears to act after infection and has been termed, operationally, transformation-enhancing factor(s) or TEF. The factor produced by RS2/3 cells which enhances the formation of B-RSV foci is non-dialyzable and thermolabile, and is presumably a protein. Its molecular weight is between 10(5) and 2 X 10(5) daltons.


Kinetics of serum factors mediating blocking, unblocking and antibody-dependent cellular cytotoxicity in hamsters given isografts of para-7 tumor cells. Sera collected at intervals after PARA-7 tumor-cell inoculation were monitored for blocking, unblocking, and antibody-dependent cellular cytotoxicity (ADCC) using the microcytotoxicity test. Within 7 days after isografting, significant levels of blocking were demonstrated. This activity increased in parallel with tumor growth and reached a maximum at day 21. In contrast, the capacity of the sera to mediate ADCC was maximal at day 7, and no longer detectable at 3 weeks. Unblocking was not demonstrable in tumor-bearer sera. Further studies showed that, when the tumor was exicised, blocking activity fell rapidly while ADCC was elevated. Sera collected 7 days after surgery could neutralize the blocking activity of tumor-bearer sera. Significant levels of unblocking were still evident 10 weeks after surgery while ADCC had generally disappeared after 5 weeks. These findings illustrate the complexity of the hamster's humoral immune response to tumor cells, and suggest that (1) the sequestering of antibody by circulating tumor antigen accounts for the demise of ADCC in tumor-bearer sera and the concomitant appearance of blocking activity; and (2) the serum factors mediating ADCC and unblocking differ.


Cell-killing by Epstein-Barr virus: analysis by colony inhibition procedure. The P3HR-1 and B95-8 strains of Epstein-Barr virus (EBV) were cytocidal for EBV-carrier human cell lines, as revealed by a colony inhibition procedure. The cytocidal activity was proportional to virus dose added. The cell killing was neutralized by anti-EBV antibody-positive but not -negative human sera. When the relative sensitivity to ultraviolet light of EBV activities was examined, the cytocidal actitivy was much more resistant than the viral infectivity as assayed by early antigen-forming activity (P3HR-1 virus) or leukocyte-transforming activity (B95-8 virus), but it closely paralleled the ability to adsorb to cells.


Characteristics of three strains of feline fibrosarcoma virus grown in cat and marmoset monkey cells. Two strains of feline fibrosarcoma virus (ST-FeSV and GA-FeSV) were found to induce tumors in cats and marmosets, and to transform feline and marmoset cells in vitro after primary inoculation. A third strain (SM-FeSV) failed to induce tumors or transform marmoset cells after primary inoculation; however, when SM-FeSV-injected marmoset cultures were passed 26 times in vitro, the cell cultures released infectious virus which transformed marmoset fibroblasts but still failed to induce tumors in marmosets. ST-FeSV induced mainly round-cell type transformation (r foci), GA-FeSV induced predominantly mixed round-fusiform cell type transformation (fr foci), and SM-FeSV induced r and fr type foci with a higher proportion of fusiform cells in the fr foci than seen with GA-FeSV. Transforming virus was obtained from r or mixed r/fr foci of ST-FeSV but not from fr foci; heat treatment changed the virus from producing almost exclusively r type foci to inducing an increased number of fr foci. Passage of FeSV in cat cells yielded viruses with a higher ratio of infectivity for feline vs marmoset cells, while passage of FeSV in marmoset cells yielded virus with a relatively higher infectivity ratio for marmoset cells; the three strains differed in the degree of change in the infectivity ratio. Despite the alteration of host range of SM-FeSV propagated in marmoset fibroblasts, the virus retained feline P-30 antigen by CF and FA assays. Neutralization tests did not indicate but also did not exclude an alteration of the surface antigens of ST-FeSV or SM-FeSV propagated in marmoset fibroblasts. The alteration of the relative infectivity of FeSV during passage in marmoset cells may be due to: (1) the selection of a variant present in the original heterogenous uncloned population; (2) mutation; or (3) recombination with some marmoset genetic material, possibly an as yet unidentified endogenous marmoset virus.


A radiometric method for predicting effectiveness of chemotherapeutic agents in murine leprosy. A simple radiometric method has been developed for evaluating the effect of drugs on the metabolism of M. lepraemurium. The method is based on the measurement of the 14CO2 produced through bacterial metabolism of acetate-U-14C. Seventeen drugs were tested: bacitracin, cephaloridine, chloramphenicol, cycloserine, dactinomycin, DDS, ethionamide, INH, kanamycin, methenamine mandelate, nitrofurantoin, oxacillin, polymyxin B, rifampicin, streptomycin, sulfadimethoxine and vancomycin. The drugs which caused most marked inhibition were chloramphenicol, INH, ethionamide and nitrofurantoin in order of increasing effectiveness. The radiometric study which is completed in 15 days permits direct study of the drug effect on the metabolism of M. lepraemurium and a more rapid screening of antileprosy drugs than has previously been possible. Currently, these observations are being extended to studies of the structure-activity relationships of antileprosy drugs and the metabolism and drug susceptibility of M. leprae in vitro.


Immune host response to corneal grafts sensitized to herpes simplex virus. Experiments were performed to study the fate of corneal grafts sensitized to herpes antigens when grafted to HSV-sensitized hosts. Nonsensitized grafts in systemically sensitized hosts remained clear, whereas all locally sensitized grafts in systemically sensitized hosts became opaque within ten days. The stroma showed severe lymphocytic and plasma cell infiltration and neovascularization. One-half of the corneas from systemically sensitized hosts grafted to eyes previously infected opacified in an accelerated manner. Round cells were found in the stroma and endothelium. The histological reaction in both groups was similar to that found in the immune graft reaction even though the clinical picture was not typical (peripheral and progressive edema with a rejection line). The accelerated reaction was probably related to the presence of virus antigens in graft stroma and subepithelial areas of the graft. The significance of HSV-sensitized donor tissue grafted onto HSV-sensitized hosts is discussed because of its possible implication in tissue transplantation.


Immunofluorescent detection of adenovirus antigen in epidemic keratoconjunctivitis. An immunofluorescent technique was used to demonstrate soluble adenoviral antigens in epithelial cells on conjunctival scrapings of patients with epidemic keratoconjunctivitis (EKC). Conjunctival scrapings for immunofluorescence and viral isolation studies were performed on 79 patients suspected of having EKC or other acute follicular or papillary conjunctivitides. Of 41 patients with clinical findings consistent with a diagnosis of EKC and three patients with pharyngoconjunctional fever, 43 were positive by immunofluorescence. All 39 patients with adenovirus isolation had positive immunofluorescence studies. The five remaining cases had other documented evidence of EKC. No false-positive responses were encountered in relation to the clinical diagnoses. The fluorescent staining was predominantly found in the cytoplasm. Speckled nuclear fluorescence was also noted. The cytoplasmic fluorescence is compatible with the adenovirus cellular replication cycle. The immunofluorescent technique was found to be a reliable, sensitive, specific, and rapid diagnostic technique for detection of group-reaction adenoviral antigens in conjunctival scrapings.


High-pressure liquid chromatographic determination of vitamin D3 in resins, oils, dry concentrates, and dry concentrates containing vitamin A. A high-pressur liquid chromatographic (HPLC) procedure has been developed for the determination of vitamin D3 in resins, oils, dry concentrates, and dry concentrates containing vitamin A. Specificity of the method for vitamin D3 in the presence of isomers and other related constituents was shown by ultimate recovery of the vitamin D3 and the individual constituents and their characterization by other methods such as ultraviolet spectrophotometry. Precision and accuracy are within 2%, and as many as 20 determinations may be completed in a working day. Excellent agreement with the official AOAC biological assay was found. A comparison of the response of isomers of vitamin D3 by the HPLC method vs. other instrumental and chemical procedures shows HPLC to be the most specific method for determining the bioactive isomers.


Cyclic guanosine 3',5'-monophosphate in the dimorphic fungus Mucor racemosus. The dimorphic fungus Mucor racemosus was found to contain the cyclic nucleotide guanosine 3',5'-monophosphate (cGMP). Approximately equivalent amounts of the compound were found in ungerminated spores, yeastlike cells, and mycelia. Germinating spores contained severalfold higher amounts of cGMP than the other cell forms. cGMP levels did not change significantly during the morphogenetic conversion of yeast to mycelia. Added exogenous cGMP or the dibutyryl derivative did not influence cell morphology in any way and did not alter the effect that cyclic adenosine 3',5'-monophosphate has upon cell morphology.


Control of transformation competence in Azotobacter vinelandii by nitrogen catabolite derepression. Azotobacter vinelandii (ATCC 12837) became competent to be transformed by exogenous deoxyribonucleic acid towards the end of exponential growth. Competence in wild-type and nitrogenase auxotrophic (nif-) strains was repressed by the addition of ammonium salts or urea to the transformation medium. Transformation of wild-type cells and nif- strains was optimal on nitrogen-free or nitrogen-limiting medium, respectively. Transformation of wild-type cells also was enhanced when the transformation medium had low molydbate content. During the development of competence, nitrogen was growth limiting, whereas carbon (glucose) was in excess. Carbon source shift-down was not effective in inducing competence. Shifting glucose-grown wild-type cells to medium containing 0.2% beta-hydroxybutyrate initiated encystment and also induced competence. The addition of glucose to this medium blocked encystment and early competence induction and reduced the transformation frequency to the basal level. Cyclic adenosine 3',5'-monophosphate induced competence in wild-type nitrogen-fixing cells and increased the transformation frequency 1,000-fold over the basal level. Exogenous cyclic adenosine 3',5'-monophosphate however, did not reverse nitrogen repression of competence in ammonia-grown wild-type or nif- strains.


Transcriptional and post-transcriptional control of beta-galactosidase synthesis. lac messenger ribonucleic acid synthesis is dissociated from its translation in certain lac operon mutants. More beta-galactosidase messenger ribonucleic acid (as determined by hybridization) is made in fully induced lacOc mutants and in trp-lac fusions than is eventually translated into enzyme. Consequently, beta-galactosidase synthesis may be post-transcriptionally controlled under these conditions.


Cytochrome spectrum of an obligate anaerobe, Eubacterium lentum. An obligately anaerobic bacterium, Eubacterium lentum, was shown to contain cytochromes a, b, and c and a carbon monoxide-binding pigment. Extracts of cells grown with hemin gave a typical absorption spectrum for cytochrome c with maxima at 424, 525, and 553 nm. Extracts from cells grown in the absence of hemin also had an absorption peak corresponding to cytochrome b (562 nm) in their reduced versus oxidized spectrum. Extraction of hemes and formation of pyridine hemochromes allowed quantitation of protoheme IX and heme c. Large amounts of cytochrome c masked the presence of cytochrome b in cells grown in medium containing hemin. When cells were grown in the presence of 50 mM nitrate, cytochrome A (606 nm) was detected. In anaerobic extracts of cells grown either with or without nitrate, cytochromes b and c were reduced by formate and oxidized by NO3. Cytochrome a appeared to be partially oxidized by NO3 and completely oxidized by air.


Binding of (125I)iodohydroxybenzylpindolol to putative beta-adrenergic receptors of rat glioma cells and other cell clones. [125I]Iodohydroxybenzylpindolol, an extremely potent beta-adrenergic antagonist, has been purified to theoretical specific activity (2200 Ci/mmol) and used as a ligand to characterize the beta-adrenergic receptors of cultured rat glioma cells and other cultured cell clones. Appropriate receptor sites were identified by stereoselective competition for binding by a number of adrenergic agonists and antagonists, by correlation between the potency of these compounds to inhibit binding and to affect adenylate cyclase activity, and by correlation of binding with the presence or absence of response to catecholamines (stimulation of adenylate cyclase) in various cell clones. In equilibrium experiments, the dissociation constant for binding of the iodinated ligand to the beta-adrenergic receptor of a clone of rat glioma cells (C6TG1A) was 250 pM; the corresponding value for a clone of human fibroblasts (VA2) was 15 pM. For C6TG1A, KD was verified by analysis of the kinetics of binding: k1 = 10(8) 1/mol/min: k-1 = 0.017/min. This rate of dissociation of ligand from the receptor was also established by study of the rate of activation of adenylate cyclase by isoproterenol after prior equilibration with iodohydroxybenzylpindolol. For VA2 cells, where affinity was higher, the rate of reversal of binding was only 0.0035/min. C6TG1A contained approximately 4000 receptor sites/cell (75 fmol/mg of protein), and these sites appeared to be coupled to adenylate cyclase in a stoichiometric manner. A second site with equal affinity for iodohydroxybenzylpindolol (KD = 250 pM) was also identified in C6TG1A by both kinetic analysis and equilibrium binding studies. While most compounds that interacted with the beta-adrenergic receptor also influenced binding to the second site, the latter did not distinguish between stereoisomers of propranolol, and its affinity for the other compounds tested was poorer.


Activation of C55-isoprenoid alcohol phosphokinase from Staphylococcus aureus. I. Activation by phospholipids and fatty acids. The activation of C55-isoprenoid alcohol phosphokinase by a variety of lipids has been investigated. A number of amphipathic lipids can serve as effective kinase activators. Both the nature of the polar and nonpolar groups are important, but kinase activation does not depend on any particular chemical structure or charge on the lipid. The structure of those lipids which are most effective, as well as an analysis of their temperature profiles, suggests that bulk physical properties are significant. Lipids which provide a hydrated, loosely packed, highly fluid environment are often effective activators.


Activation of C55-isoprenoid alcohol phosphokinase from Staphylococcus aureus. II. Biophysical studies. Physicochemical techniques were used to investigate the activation of C55-isoprenoid alcohol phosphokinase by synthetic lecithins. Complexes of the enzyme with phospholipids were prepared using a method employing sodium dodecyl sulfate as a protein-solubilizing agent. Circular dichorism and the intrinsic fluorescence of the kinase were used as optical probes of protein conformation with these complexes. No evidence for a major lipid-dependent conformational change in the protein was observed when these complexes were studied under conditions where the lipid mesomorphic transitions occurred. EPR studies of mixtures of synthetic lecithins and the C55-isoprenoid alcohol indicated a correlation between kinase activity and the rotational diffusion rate within the hydrophobic phase. It is concluded that the lipid physical state probably does not affect the enzyme activity by altering the protein conformation but more likely does so by affecting the motion of the molecular participants in the reaction.


Activation of C55-isoprenoid alcohol phosphokinase from Staphylococcus aureus. III. Activation by detergents. The activation of kinase by neutral detergents has been examined. Of those detergents tested, only those with short chain and unsaturated chain hydrophobes were successful activators at 25 degrees. In addition to these structural requirements, a dependence upon hydrophilic-lipophilic balance number was observed when mixing hydrophobic and hydrophilic detergents. Most pairs tested showed an optimal hydrophilic-lipophilic balance number for kinase activation in the hydrophobic range of 6 to 9. Thus, there appears to be both a structural and a relative hydrophobic requirement for activation, and hydrophilic-lipophilic balance number may be a measure of the physical properties of the lipid required for activation.


Protein asymmetry in the inner membrane of rat liver mitochondria. At least three groups of polypeptides of the inner membrane of rat liver mitochondria have been shown to be exposed to the exterior surface by several techniques: lactoperoxidase-catalyzed iodination of mitochondria and inner membrane/matrix vesicles (mitoplasts), reaction of mitochondria and mitoplasts with the membrane-impermeable diazonium salt of sulfanilic acid, and controlled proteolysis of mitoplasts. These classes of proteins, separated by dodecyl sulfate gel electrophoresis, have polypeptide molecular weights of 73,000, 31,000, and 26,000. In addition, four other groups have been shown to be exposed to the exterior surface by at least one of these techniques: these components have polypeptide molecular weights of 130,000, 87,000, 16,000, and 10,500. A class of proteins, which makes up 50 to 60% of the total mitochondrial protein and which can be easily extracted from mitoplasts by freeze-thaw fractionation or other procedures designed to separate "matrix" protein from "membrane" protein, is shown not to be exposed to the outer surface of the inner membrane by these techniques. This class of proteins contains polypeptides of various molecular weights and includes the major 165,000 molecular weight polypeptide, identified with carbamyl phosphate synthetase.