Mechanisms of inactivation of noradrenaline in the iris sphincter, tracheal muscle and facial artery of cattle: implications for beta-adrenoceptor-mediated responses.

1 The role of neuronal and extraneuronal pathways of amine inactivation in regulating the inhibitory actions of noradrenaline was investigated in three bovine smooth muscle preparations in which the primary adrenoceptor is of the beta-type.2 The extraneuronal uptake inhibitor, 17beta-oestradiol, sensitized the inhibitory responses to noradrenaline in the facial artery, the iris sphincter and in tracheal muscle preparations, indicating a major role for non-neuronal processes in agonist-inactivation in all three preparations. Cocaine also increased responses to noradrenaline, pointing to a role for neuronal uptake either as a terminating mechanism or as a process limiting access of exogenous agonist molecules to their site of action.3 Cocaine did not enhance significantly responses to isoprenaline, a potent beta-adrenoceptor agonist which is not taken up neuronally. Further, relaxations to metaraminol, a sympathomimetic amine which is taken up extraneuronally, but much less so than noradrenaline, were also less enhanced by 17beta-oestradiol in the three preparations tested. These findings support the specificity of action of cocaine and 17beta-oestradiol as neuronal and extraneuronal uptake inhibitors in the present experiments.4 Studies of the uptake of [(3)H]-noradrenaline revealed that 17beta-oestradiol reduced the uptake of amine in the presence of cocaine, confirming a cocaine-resistant site of action for the steroid in all three preparations.5 It is concluded that extraneuronal uptake sites are located sufficiently close to the beta-adrenoceptors to modulate the concentration and duration of action of noradrenaline at these sites of action. It is proposed that in smooth muscles which contain a preponderance of beta-receptors, extraneuronal metabolism is a key event in terminating the inhibitory effects produced.


Comparison of the receptor binding characteristics of opiate agonists interacting with mu- or kappa-receptors.

1 The receptor binding characteristics of various morphine-like and ketazocine-like opiate agonists were measured by inhibition of [3H]-naloxone binding in homogenates of brain and of ileal myenteric plexus-longitudinal muscle of the guinea-pig. No differences were found for the two tissues. 2 The depressant effect of Na+ on the inhibition of [3H]-naloxone binding by opiate agonists varies widely, giving sodium shifts between 5 and 140. The relationship between Na+ concentration and inhibition of binding is non-linear, the magnitude of the sodium shift varying directly with the slope of the regression of log IC50 on log [NaCl]. 3 The sodium shift of ketazocine-like agonists is lower than that of morphine-like agonists but higher than that of opiates with dual agonist and antagonist action. A working hypothesis is proposed which suggests that the kappa-receptors for the ketazocine-like drugs are less susceptible to the Na+ effect than the mu-receptors for the morphine-like drugs. 4 For most of the morphine-like but not the ketazocine-like agonists, a good correlation has been found for the pharmacological activity in the myenteric plexus-longitudinal muscle preparation and the inhibition of binding of [3H]-naloxone at 12 mM Na+. An exception is fentanyl which has a much greater pharmacological potency than may be expected from its potency in inhibiting [3H]-naloxone binding.


Stimulation of non-neuronal cell proliferation in vitro by mitogenic factors present in highly purified sympathetic neurons.

Sympathetic neurons have been demonstrated to contain one or more mitogens which are active on highly purified non-neuronal cells cultured in medium containing an optimal concentration of fetal calf serum. Neurons and homologous non-neuronal cells were separated by a method recently developed in this laboratory. The highly purified neurons were either sonicated or homogenized prior to addition to nonneuronal cultures. The presence of neuronal sonicate (1) greatly stimulated [3H]thymidine incorporation into acid-precipitable macromolecules without altering the soluble [3H]thymidine pool, (2) increased both the fraction of non-neuronal cells which took up [3H]thymidine and the density of labeling as observed by autoradiography, and (3) increased the number of cells present in treated cultures after 40 h. The enhancement of [3H]thymidine incorporation was dose-dependent and did not involve cyclic AMP. Addition of neuronal sonicate also caused marked non-neuronal cell elongation which resulted in the elaboration of very long cell processes. The active factor(s) in the neuronal sonicate were partially heat-labile. Norepinephrine was ruled out as a possible mitogenic factor.


Inhibitory and excitatory binocular convergence to visual cortical neurons of the cat.

Responses of 182 visual cortical (VC) neurons to electrical stimulation of both optic nerves (ON) were recorded intra- and extracellularly, and their eye dominance determined with visual stimuli. Many VC neurons could not be excited by ON stimulation without simultaneous activation by visual stimulation of the eye of the other side. The ON-excited units (25 simple and 71 complex cells) had essentially the same response latency from both ONs. In almost all VC neurons including those not driven from the ON, ON stimulation elicited inhibition which was shown to be post-synaptic in all neurons recorded intra- or quasi-intracellularly. The onset latency of IPSPs not preceded by an action potential or EPSP was 4.2+/-1.0 msec, suggesting that intracortical inhibition was initiated by afferent impulses mediated through fast conducting fibers. In visually monocular neurons, ON stimulation of the non-driving side also elicited primary inhibition. Visually binocular, but monocular-dominant neurons responded more reliably to ON stimulation of the dominant side than to the other. In most binocular neurons with equal visual responsiveness to both eyes, inhibition and excitation evoked from both ONs had about the same latency and magnitude.


The neuronal pathway subserving the pupillary light reflex.

The pupillary light reflex was investigated using electrical stimulation along the pathway and recording in the short ciliary nerves. The discharge of single units in the ciliary ganglion was compared during diffuse light stimuli and electrical stimuli. It was concluded that the early reflex discharge in the short ciliary nerves following electrical stimulation on the optic tract is due to excitation of fibres active during the pupillary light reflex. The light reflex is conveyed by slow (less than 10 m/sec) optic tract fibres which synapase in the medial part of the pretectal area. In turn, pretectal neurones with conduction velocities of about 6 m/sec pass to the Edinger-Westphal complex from which the preganglionic 'pupilloconstrictor' neurones originate. Latency measurements show that there are synapses in the pretectal region and the Edinger-Westphal nucleus. The amplitude of the 'pupilloconstrictor' responses in the short ciliary nerves can be used as a measure of the excitability in the pathway of the pupillary light reflex under various conditions, e.g. following conditioning stimuli of other interacting pathways. In addition to the 'pupilloconstrictor' response there is also another short-latency discharge in the short ciliary nerves following stimulation of the posterior commissure and the Edinger-Westphal nucleus. That discharge is presumably due to activation of fibres which cause accommodation of the lens.


Cerebellar influence on parasympathetic neurones innervating intra-ocular muscles.

Previous work has suggested that cerebellum may control the size of the pupil and transmission in the pathway mediating the pupillary light reflex. In this work we found that electrical stimulation of the cerebellar nuclei (nucleus fastigius and nucleus interpositus bilaterally) evoked a discharge in the short ciliary nerves which innervate the intraocular muscles. The latency was 5.7 msec, which is short enough for a direct excitatory connexion from cerebellar efferents onto the preganglionic neurones in the Edinger-Westphal nucleus. These neurones are controlling the ciliary muscle of the lens system as well as the constrictor muscle of the pupil and it was therefore important to elucidate whether both of these groups participate in the discharge. Studies on interaction between the response to cerebellar stimulation and 'pupilloconstrictor' responses from optic tract fibres as well as recording from individual cells in the ciliary ganglion (identified with diffuse light stimuli) led to the conclusion that the short latency excitation is more or less confined to units controlling accommodation. Cerebellar inhibition (or disfacilitation) of longer latency affects both pupilloconstrictor and accommodation units.


Burst generation by electrically coupled network in the snail helisoma: analysis using computer simulation.

The effectiveness of electrical coupling between neurons as a mechanism for mediating single and repetitive bursts is investigated here using computer simulation. The cyberchron network in the snail Helisoma generates repetitive bursts controlling the animal's feeding behavior and served as the basic model for the simulation studies described in this paper. The action potential properties of individual neurons were modeled by the Rall equations describing generalized action potentials. Several properties of electrical coupling and its role in burst generation were demonstrated, including: (1) A neuron in an electrically coupled network can generate action potentials at a higher frequency than an isolated neuron with similar membrane properties due to the loading through the electrical junctions. However, the ability of electrically coupled neurons to generate high-frequency bursts of action potentials requires a concomitantly greater amount of driving current to overcome the junctional loading. (2) Temporal and spatial summation of synaptic input onto a neuron is maintained at its most effective level because the postsynaptic current is integrated across the long postsynaptic membrane time constant. (3) Initial simulations concentrated on a pair of electrically coupled neurons which were below threshold. Stimulation of one of the two neurons with a short pulse resulted in a reverberation or regenerative excitation between the two neurons. The reverberation terminated after a number of action potentials dependent on the specific model parameters. Similar results were obtained with a network containing a greater number (20) of model neurons if approximately one-half of the neurons were stimulated simultaneously. However, none of the cases studied produced more than a single discrete burst. (4) Simulations were also conducted on 20-neuron networks containing two subpopulations of model neurons differing in their values of coupling resistance and excitability. Some networks of this type required stimulation of only one cell to make the two subpopulations of model neurons reverberate with one another. Such simulations suggest the possibility that 'preferred' input pathways involving a small number of neurons would be capable of 'turning on' the activity of the entire network.


Pathways and polarities of synaptic interactions in the inner retina of the mudpuppy: I. Synaptic blocking studies.

Intracellular recording techniques were applied to the perfused retina-eyecup of the mudpuppy (Necturus maculosus). The use of input resistance measurements and the application of cobalt as a synaptic blocking agent provided evidence about the mode of synaptic operation for depolarizing and hyperpolarizing bipolar cell input to cells of the inner retina. In addition, cobalt produced a more rapid block of hyperpolarizing bipolars compared to depolarizing bipolars; in some postbipolar neurons, a temporary simplification of neuronal response waveform was observed and such cells resembled the depolarizing bipolar. These experiments suggest that bipolar cell input is excitatory and that response characteristics of third-order neurons is partially dependent on the relative input from the two types of bipolar cells.


Pathways and polarities of synaptic interactions in the inner retina of the mudpuppy: II. Insight revealed by an analysis of latency and threshold.

Intracellular recording experiments in the mudpuppy (Necturus maculosus) have demonstrated that depolarizing bipolars show a one-half to one log unit higher threshold to light stimuli and a longer latency when compared to hyperpolarizing bipolars. This threshold difference cannot be ascribed to differences in rod and cone connections. Experimentally it was possible to use these differences to evaluate postbipolar cell connections; such experiments support the idea that some neurons are connected to one or the other bipolar cell type, while on-off cells receive input from both.


Excitability changes of dorsal inspiratory neurons during lung inflations as studied by measurement of antidromic invasion latencies.

(1) Glass microelectrodes were used to record extracellularly the activity of single dorsal (in the region of nucleus tractus solitarius (NTS)) inspiratory neurons in either chloralose- or pentobarbitone-anaesthetized, paralyzed cats. (2) In the lateral and ventral part of NTS, 89 cells were localized which responded antidromically to stimulation applied to the contralateral spinal cord (C3). Seventy were not excited by lung inflation and were classified as Ralpha, while 19 excited by that stimulus were classified as Rbeta. Usually Rbeta neurons fired both during each pump-induced lung inflation and during the central inspiratory excitation (CIE). (3) A technique similar to Merrill's recording small variations in the antidromic latency measured to the SD spike was used to ascertain excitability of Ralpha and Rbeta cells during the CIE time and during lung inflation in expiration. The technique was extended by averaging antidromic latency patterns within many respiratory cycles. (4) In the majority of Ralpha cells the averaging revealed a shortening of the antidromic latency during lung inflation in expiration, which indicated a subthreshold depolarization. Lengthening of the antidromic latency by this stimulus was never observed. These results are in disagreement with the concept of a direct inhibitory input from Rbeta to Ralpha neurons. (5) It is concluded that the dorsal Ralpha and Rbeta neurons differ less in their properties than was previously assumed on the basis of classical extracellular techniques. The inhibitory influence of pulmonary stretch receptor input on inspiratory discharge of Ralpha cells seems to be exerted through an indirect action on the source of the CIE.


Kinetic and pharmacologic characterization of gamma-aminobutyric acid receptive sites from mammalian brain.

Sodium-dependent (+Na) and sodium-independent (-Na) receptive sites for gamma-aminobutyric acid (GABA) have been characterized using synaptic plasma membranes from bovine and rat brain. Synaptic plasma membranes were prepared from either rat cerebellar cortex or calf cerebral cortex by discontinuous sucrose gradient flotation centrifugation of crude mitochondrial pellets, and assayed using equilibrium ligand binding assays to obtain the maximum binding capacity (Bmax) and the thermodynamic constant (KD). Values for KD from equilibrium studies were subsequently confirmed by kinetic analyses of association and dissociation reactions. The KD for +Na GABA binding (5.0 +/- 0.2 micron) corresponds to the apparent Michaelis constant for neuronal GABA transport (3.8 +/- 0.1 micron)22, while the KD for -Na binding (0.17 +/- 0.04 micron) agrees with that determined by Enna and Snyder for the putative postsynaptic receptor. Maximal binding activities of about 5 and 55 pmole/mg protein were obtained for -Na and +Na binding respectively. The pharmacologic specificities of the two sites were determined using competition binding studies. Nipecotic acid and diaminobutyric acid inhibit both synaptosomal GABA uptake (Ki approximately 25 micron and 120 micron respectively) and +Na binding of GABA to synaptic plasma membrane (IC50 approximately 40 micron and 350 micron respectively) but do not inhibit -Na binding. Bicuculline inhibits -Na [3H]GABA binding at low concentrations (IC50 approximately 15 micron), while affecting the uptake and +Na binding of [3H]GABA only at high concentrations (IC50 approximately 520 micron and 300 micron respectively). beta-Alanine inhibits the -Na binding site (IC50 approximately 100 micron), but is ineffective at the +Na binding site and does not interfere with synaptosomal uptake of GABA. Finally, chlorpromazine and N-ethylmaleimide inhibit the +Na binding, albeit at high concentrations (IC50 approximately 600 micron and 5 mM respectively) but are ineffective at the -Na binding site. From these results the -Na binding site is tentatively identified as a postsynaptic receptor and the +Na binding site is identified as the neuronal uptake receptive site.


Interspecific variations in proteins synthesized by mammalian mitochondria.

The products of mitochondrial protein synthesis in established cell lines of various mammalian species were labelled with [35S]methionine and their number and apparent molecular weights determined by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis and fluorography. Proteins synthesized by isolated rat liver mitochondria were labelled with [3H]valine and similarly characterized. Each species had a distinctive pattern of from 10 to 13 mitochondrially synthesized proteins with apparent molecular weights between 10,000 and 50,000. No differences were detected in the number or electrophoretic mobility of the mitochondrially synthesized proteins of SV-40-transformed and nontransformed WI-38 cells.


Selective degradation with phospholipases D and C of radioactive isomeric spin-labelled lipids bound to guinea pig liver microsomal membranes.

Membrane-bound lipids of isolated guinea pig liver microsomal membranes were selectively enzymatically labelled with isomeric (5-, 12-, and 16-)doxyl stearic acid. After reisolation, the membranes were degraded with phospholipases D and C under conditions not requiring detergents or organic solvent activators. The degradation of membrane-bound lipids occurred according to the recognized specificity of phospholipases D and C. Temperature-induced changes of degraded membranes containing radioactive spin-labelled isomeric lipids were followed by the electron spin resonance and spectral changes correlated with the lipid composition of membranes. Discontinuities in plots of experimental spectral parameters versus temperature detected in the case of microsomal membranes before and after degradation with phospholipases D and C were attributed to lipid-protein and lipid-lipid interaction(s). On the basis of these and control experiments, discontinuity at around 10-12 degrees C was attributed to the microsomal membrane phosphatidylcholine intrinsic microsomal membrane protein interaction(s), while discontinuities detected at 19-21 degrees C approximately and at 20-30 degrees C approximately were attributed to the phase separation of Ca or Zn salts of membranous phosphatidic acid and to the similar phenomenon involving membrane-bound diglycerides respectively.


Low density lipoprotein turnover in swine.

The catabolism of intravenously injected 125I-labelled low density lipoproteins (LDL) was followed in normal miniature swine for 2 weeks. When compared with the two-exponential model, the decay curve of the plasma radioactivity associated with the LDL fraction was best described by a three-exponential model. In this system, the half-lives were 4.5 +/- 3.7, 19.7 +/- 6.6, and 127 +/- 70 h (mean of four studies). Assuming a kinetic model with metabolism of LDL in the rapidly equilibrating compartment and two slower equilibrating compartments (a model requiring three exponentials), the mean fractional catabolic rate for apo-LDL was calculated to be 0.015 h-1. Therefore, if at steady state, the synthetic rate for apo-LDL in the same pigs would be 5.6 +/- 4.1 mg/h. Different kinetic models using two or three exponentials would provide different values for the synthetic rate of apo-LDL. However, in view of the known existence of at least three major equilibrating pools for LDL in plasma, liver, and lymph, and in view of the present results, the kinetic model for LDL metabolism should be better represented by a three-exponential system.


Oxytocin binding sites in lactating rabbit mammary gland.

Material which specifically binds oxytocin was prepared from a crude preparation of lactating rabbit mammary gland by purification on a sucrose density gradient. On examination of activities of enzyme markers and the molar ratio of cholesterol to phospholipid, this material was considered to be a highly purified plasma membrane fraction. For the determination of specificity and time course of oxytocin binding, a Scatchard plot analysis was carried out for the crude and purified fractions. Dissociation constant (Kd) and binding capacity values were found to be as follows: crude, Kd equals 1.83 X 10(-9) M, capacity equals 670 fmol/mg protein; purified, Kd equals 2.8 X 10(-9) M, capacity equals 1700 fmol/mg protein. Treatment of the purified material with different detergents resulted in loss of all [3H]oxytocin binding capacity. However, preincubation of this material with [3H]oxytocin prior to detergent treatment resulted in solubilization of a receptor-hormone complex. This complex remained in the supernatant even after centrifugation at 210 000 X g for 30 min. Using oxytocin analogs, we have shown this solubilized complex to be oxytocin specific.


Heterogeneity of human plasma low density lipoprotein.

Low density lipoprotein (LDL) was fractionated into subspecies by the use of DEAE-agarose column chromatography and the peptide compositions of the LDL subspecies which eluted at different NaCl concentrations were determined. LDL which elutes at low NaCl concentration has relatively less non-B apoprotein than does LDL which elutes at high salt concentration. The LDL subspecies which elute at high NaCl concentration contain more apo A-1 than do those which elute at the lower NaCl molarity. These results indicate that LDL consists of subfractions which differ in their peptide compositions.


Determination of the rates of synthesis and degradation of adenosine 3',5'-cyclic monophosphate in Escherichia coli CRP- and CRP+ strains.

We have developed a method for estimating the rates of synthesis and degradation of adenosine 3',5'-cyclic monophosphate (cAMP) in Escherichia coli during balanced growth. Applying this method, we have found that an E. coli CRP- mutant 5333 (deficient for cAMP receptor protein) synthesizes cAMP about 25 times faster than does its CRP+ parent 1100. This accounts for the abnormally high intracellular and extracellular cAMP accumulation in 5333.


Metabolic responses of Sertoli cells in culture to various concentrations of follicle stimulating hormone and cholera toxin.

The concentration of cholera toxin required for half-maximal stimulation of cAMP production by Sertoli cell enriched cultures (4.48 X 10(2) microgram/ml) is greater than that required for half-maximal stimulation of 17beta-estradiol synthesis from testosterone (2.34 X 10(-4) microgram/ml), [3H]thymidine incorporation into DNA (1.48 X 10(-5) microgram/ml), or androgen binding protein production (2.43 X 10(-6) microgram/ml). The same relative dose response hierarchy was obtained with respect to stimulation of Sertoli cells with follicle stimulating hormone (FSH) preparations. Again, highest concentrations were required to elicit maximal cAMP production. The data are discussed in relation to an apparent paradox: If cAMP is the mediating 'second messenger' following stimulation by FSH or cholera toxin, why should highest concentrations of these agents be required to elicit 50% of maximal cAMP levels?


Sphingomyelinases in human tissues. IV. Purification of sphingomyelinase from human placenta and effect of Triton X-100.

Sphingomyelinase was purified about 1700-fold from human placenta. The major steps in the procedure included chromatography on Concanavalin A-Sepharose, Sepharose 6B, and carboxymethyl-Sepharose (CM-Sepharose). The final preparation was stable for at least 3 months when stored at 4 degrees C. The enzyme was found to be heterogeneous on CM-Sepharose and isoelectric focusing. Triton X-100 which was present in most buffers used during the purification appears to be partially responsible for the heterogeneity. When Triton X-100 is removed by treatment with Bio Beads, heterogeneity was reduced. However, removal of the detergent also leads to loss of enzyme activity which could not be restored by readdition of Triton X-100. The data suggest that sphingomyelinase has a high hydrophobic character and that both its stability and electrofocusing behaviour are influenced by interaction with the nonionic detergent.


Spin-label study of the relation between enzymatic activity and lipid-protein organization in reconstituted cytochrome c oxidase.

Lipid-depleted cytochrome c oxidase (EC 1.9.3.1) containing less than 20 microgram lipids per milligram protein was reconstituted with pure phospholipids of well-defined chemical structure and fatty acid composition without using detergents and (or) sonication. For the maximal restoration of electron transport activity, lipid-depleted cytochrome c oxidase required acidic phospholipds such as phosphatidylglycerol or phosphatidylserine or lysophospholipids such as lysophosphatidylcholine or lysophosphatidic acid, but no specific phospholipid fatty acid composition was necessary. The organization of the lipid environment of the reconstituted cytochrome c oxidase, having a well-defined lipid composition, morphology, and a high specific activity, was examined by electron spin resonance spectroscopy using 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl (16-doxyl stearic acid) and 16-doxyl stearic acid - containing phosphatidylglycerol. The presence of boundary lipid was established in both lamellar and micellar organizations of reconstituted cytochrome c oxidase and was not necessarily related to the enzymatic activity of the complex. Our results have established that aside from structural considerations, the boundary lipid, at least in the reconstituted cytochrome c oxidase, is a necessary but not sufficient condition for the enzymatic expression of cytochrome c oxidase.


N-Terminal amino acid sequence of rat tonin: homology with serine proteases.

In this paper, we present the amino-terminal sequence of rat tonin, an endopeptidase responsible for the conversion of angiotensinogen, the tetradecapeptide renin substrate, or angiotensin I to angiotensin II. It is shown that isoleucine and proline occupy the amino- and carboxy-terminal residues respectively. The N-terminal sequence analysis permitted the identification of 34 out of the first 40 residues of the single polypeptide chain composed of 272 amino acids. These results showed an extensive homology with the sequence of many serine proteases of the trypsin-chymotrypsin family. This information, coupled with the slow inhibition of tonin by diisopropylfluorophosphate, classified this enzyme as a selective endopeptidase of the active serine protease family.


Relationship of herpes simplex virus type-2 antibodies and squamous dysplasia to cervical carcinoma in situ.

Serum specimens from 75 women with cervical carcinoma in situ, 84 with squamous dysplasia, and 132 controls, who had previously been interviewed and tested for complement fixing antibodies against a number of organisms, were analyzed for HSV-2 antibodies. Carcinoma in situ and severe dysplasia were associated with HSV-2 antibodies. Mild dysplasia was related to evidence of prior infection by Trichomonas vaginalis, adenoviruses, and Mycoplasma pneumoniae, plus a history of vaginal discharge. Severe dysplasia was less strongly related to these variables. The relative risk of dysplasia increased with the number of different pathogens by which a woman had been infected. It is concluded that HSV-2 may be a cause of carcinoma in situ; that much dysplasia is a nonspecific reaction of the cervical epithelium to chronic inflammation; and that dysplastic lesions that are caused by HSV-2, and hence may be a precursor to carcinoma in situ, tend to be distinguished by their severity.


Silica reaction simulating fibrous histiocytoma.

An unusual foreign body reaction to silica (quartz), resembling fibrous histiocytoma, is described in 7 patients (age range 22 to 71 years). These lesions occurred in the inguinal region (4 cases) or the abdominal wall (3 cases), and in 5 instances were associated with a history of injection at the same site 10 to 41 years previously. Most commonly the injection consisted of the administration of a sclerosing agent for the repair of a hernia. Microscopically these lesions contained broad sheets of histiocytes separated by bands or concentrically lamellated nodules of collagen. Intra- and extra-cellular birefringent crystals, identified by x-ray diffraction as silica (quartz), were present in all cases and served to distinguish this process from a true neoplasm. Although the injection therapy for hernia is now obsolete, it was formerly practiced in the United States with a variety of sclerosing agents including silica (quartz). Since these lesions are often confused with a benign or malignant fibrous histiocytoma it is recommended that fibro-histiocytic lesions associated with a previous hernia or injection at the same site be examined for the presence of silica.


Bronchiolo-alveolar carcinoma: a correlative clinical and cytologic study.

From 1970 to 1977, 101 patients with bronchiolo-alveolar carcinoma were admitted to the Toronto General Hospital. Cytology preparations from 97 patients were reviewed and analyzed in correlation with biologic behavior of the tumours. The value of cytologic diagnosis was reassessed. It appears that routine cytology methods were of limited value in the investigation of patients with a peripheral solitary tumor and therefore, percutaneous fine needle aspiration with positive results in 92% of cases examined, was the only useful cytologic examination for this type of lesion. For multicentric tumors, routine cytology methods achieved positive results in 87.9% of cases and fine needle aspiration 100%. Based on cytomorphologic features, bronchioloalveolar carcinoma can be subclassified into three types: secretory, nonsecretory and poorly differentiated. In this series, 84% of solitary tumors were secretory or nonsecretory type with favorable prognosis, and 16% of solitary tumors were poorly differentiated type with poor prognosis. 55.2% of multicentric tumors were poorly differentiated type and 77.3% of multicentric tumors showed positive lymph nodes at surgery. Our results demonstrate that patients with multicentric or poorly differentiated tumors had poor prognosis.


Mixed mesodermal tumor and clear cell carcinoma arising in ovarian endometriosis.

A case is reported of clear cell carcinoma, arising as several papillary masses from the lining of an endometrial cyst within the ovary, associated with a mixed mesodermal tumor arising in the stalk of the largest papillary nodule. The origin of this tumor from within a focus of endometriosis lends support to the theory that both the epithelial and mesodermal components are of Müllerian (Paramesonephric) origin.


Spinal subarachnoid metastasis from primary intracranial glioblastoma multiforme.

Twenty-five patients with glioblastoma multiforme were autopsied at our institution in 7 years. Spinal cords were examined in 20 and 5 were found to have spinal leptomeningeal metastases. Clinical and neuropathological findings of these 5 patients are presented and factors possibility influencing such spread are analyzed. Review of previous studies of intracranial glioblastomas discloses only 14 reported cases with spinal leptomeningeal metastases confirmed at autopsy since 1931. We conclude that spinal leptomeningeal metastases in glioblastoma multiforme are a common occurrence. These findings are of little significance at present with our poor success at control of the primary lesion. Frequency of involvement of the spinal subarachnoid space will be significant, when we are able to better treat the primary tumor. Knowledge of the significant possibility of this phenomenon will allow earlier and more frequent clinical diagnosis.


Growth rates of small cell bronchogenic carcinomas.

In reviewing a series of 144 patients with small cell bronchogenic carcinoma, 12 were found to have serially measurable roentgenographic lesions prior to therapy. Although caliper-based measurements and a silhouette cutout method gave comparable sets of tumor doubling time data, inter-observer variability was less with the silhouette cutout method. Tumor doubling times in small cell bronchogenic carcinoma ranged between 25 and 160 days, with a median of 77 days, a log mean of 81 days, and an arithmetic mean of 91 days. There was no apparent relation between tumor doubling time and tumor location, histologic subtype, response to therapy, or patient survival. The data indicate that small cell bronchogenic carcinoma of the lung is a relatively slowly growing tumor. Assuming that late subclinical disease exhibits growth characteristics that are similar to those seen in the clinical stages of growth, it can be estimated that residual body tumor burdens of 1 X 10(6) cells may be followed by tumor recurrence times of 2 years or longer; the likelihood of "cure" should not be entertained in patients with disease-free intervals shorter than 4-5 years.


Lung cancer as a second primary.

An unusually high association of other primary cancers (9.7%) was found during the analysis of 403 consecutive cases of carcinoma of the lung diagnosed at DGMC between 1960 and 1975. Incidence by stage included 17.3% for Stage I (75 cases) and 16.9% for Stage II (59 cases). Median survival by stage was not adversely affected by the associated malignancy. Incidence by histologic type was 15.6% for adenocarcinoma (132 cases), 7.7% for epidermoid (130 cases), 1.5% for oat (small cell) (67 cases), 12.5% for large cell (40 cases) and 11.8% for undifferentiated anaplastic type (34 cases). Of 31 cases of Stage I adenocarcinoma, 9 (29%) had second malignancies. Both adenocarcinoma and epidermoid carcinoma exhibited decreasing association of second malignances with increasing stage of lung cancer. The head and neck region was the location of the nonlung malignancy in 22 cases and the GU system in 11 cases. Two cases each of colon carcinoma and basal cell skin carcinoma were found and there was one case each of carcinoma of the pancreas, lymphoma and melanoma. The diagnosis of lung cancer was made first in only 3 instances. The appearance of solitary nodules in patients with known malignancy should receive strong consideration for vigorous diagnostic and therapeutic procedures. Future studies should consider carcinogenic stimuli that may be common etiologic factors in both malignancies.


Coexistence of bilateral pheochromocytoma and pancreatic islet cell tumor: report of a case and review of the literature.

A 14-year-old Japanese male with a previously undescribed combination of bilateral pheochromocytoma and an islet cell tumor of the pancreas is presented. The combination of endocrine neoplasms in this patient overlaps multiple endocrine neoplasia (MEN) Type 1 and Type 2. A total of 14 reported cases of MEN overlapping Type 1 and Type 2 is reviewed. Of the 14, 7 patients with acromegaly developed a paraganglioma(s), 2 patients with Sipple syndrome had a pituitary adenoma, and in the other 5 patients, an intestinal carcinoid or a pancreatic islet-cell tumor occurred in association with either a thyroid medullary carcinoma or a paraganglioma(s). We believe that the occurrence of MEN overlapping Type 1 and Type 2 is more than a fortuitous association, and can be explained on the basis of the neuroectodermal origin.


Damage of proliferating lymphoid cell deoxyribonucleic acid by methyl methanesulfonate and N-acetoxy-2-acetylaminofluorene.

Replicative synthesis of deoxyribonucleic acid by splenic lymphoid cells cultured from C57BL/6J strain mice and stimulated to proliferate with concanavalin A (Con A) was strongly suppressed by short exposure (10-60 min) of the cells to the direct-acting carcinogens methyl methanesulfonate or N-acetoxy-2-acetylaminofluorene. Carcinogen-treated lymphoid cells failed to recover stimulated levels of replicative synthesis after incubation for several hours in fresh medium without carcinogen. In contrast, spleen cells treated with hydroxyurea (a metabolic inhibitor of deoxyribonucleic acid replication) fully recovered stimulated rates of replicative synthesis upon incubation in medium without hydroxyurea. Furthermore, the inhibition of replicative synthesis by the carcinogens was accompanied by marked stimulation of deoxyribonucleic acid repair synthesis. Concomitant measurement of replicative and repair synthesis appears to be an effective approach for the in vitro study of carcinogen-induced damage in the deoxyribonucleic acid of proliferating lymphoid cells.


Heterotransplantation into nude mice of pancreatic carcinoma induced by N-bis(2-hydroxypropyl)nitrosamine in hamsters.

Heterotransplantation into nude mice by the subcutaneous (s.c.) and intraperitoneal (i.p.) routes of hamster pancreatic ductal cell carcinomas induced by N-bis(2-hydroxypropyl)nitrosamine (DHPN) was studied. By s.c. transplantation, tumors occurred in 1 of 4 mice at the first trial, and the tumor incidence was 100% for shorter periods by serial transfer. By i.p. transplantation, the malignant potential of hamster pancreatic ductal cell carcinoma was expressed as intra-abdominal invasive tumors with bloody ascites (carcinomatous peritonitis). Tumors in nude mice basically retained the histology of the original hamster pancreatic ductal cell carcinoma.


Preliminary results of combined surgery and adjuvant Bacillus Calmette-Guérin plus levamisole treatment of resectable lung cancer.

The administration of intrapleural bacillus Calmette-Guérin (BCG) alone and intrapleural BCG plus levamisole was compared to placebo in patients with resectable, non-small cell cancer of the lung. This report is based on an interim analysis of 100 eligible patients with a median followup of 245 days. No significant treatment effects are apparent at the present time, although current trends are consistent with an approximate 30% reduction in recurrence rate in patients receiving intrapleural BCG. There is little evidence to suggest that the addition of levamisole will contribute to this effect. A relationship between purified protein derivative conversion and tumor recurrence is apparent. Failure to manifest purified protein derivative conversion following administration of intrapleural BCG is associated with a significantly greater risk of tumor recurrence.


Overview of thymosin activity.

Our current research program centers around the biologic and chemical characterization of the family of polypeptides present in thymosin fraction 5. A system of nomenclature has been developed and the peptides are being systematically isolated and chemically characterized. Thymosin fraction 5 and its component parts influence a variety of lymphocyte properties including cyclic nucleotide levels, migration inhibitory factor production, T-dependent antibody production, and expression of certain surface markers. Thymosin is being used in clinical trials to investigate its effects on immunodeficiency diseases, malignant diseases, and autoimmune diseases.


Experiences with thymosin in primary immunodeficiency disease.

Thymosin has no effect in vitro on cyclic AMP levels in thymocytes. However, when thymosin was injected into patients lacking "serum factor" (SF) activity, it induced the appearance of SF or SF-like activity and the disappearance of target cells for SF among the peripheral blood lymphocytes of the treated patients. On the other hand, when thymosin was injected into one patient with normal SF activity, it induced a marked decrease of SF activity and the appearance of target cells for SF among the peripheral blood lymphocytes of this particular patient.


Thymosin in cancer patients: in vitro effects and correlations with clinical response to thymosin immunotherapy.

Studies on the effect of thymosin on T-cell levels in vitro among normal persons and cancer patients show that, in general, T-cell levels increase after incubation with thymosin in populations with low initial T-cell levels while the levels decrease in populations with high initial T-cell levels. In patients with small cell carcinoma of the lung receiving intensive chemotherapy also randomized to receive thymosin at a dose of 60 mg/m2, thymosin at a dose of 20 mg/m2, or placebo twice weekly, increased survival occurred in patients receiving the thymosin dose of 60 mg/m2. The increase in survival was greatest in patients with low pretreatment T-cell and alpha2HS-glycoprotein levels. These observations suggest that the cancer patients most likely to benefit therapeutically from adjuvant treatment with thymosin are those with relatively low initial T-cell levels and other parameters of cellular immunity.


Wilms' tumor--model of a curable pediatric malignant solid tumor.

Wilms' tumor is the model of the treatment of a pediatric solid tumor. Initially it appeared that multi-modality therapy, consisting of transabodominal nephrectomy, post-operative radiation therapy to the tumor bed and adjuvant, single agent chemotherapy provided the highest likelihood of disease-free survival. The identification of important prognostic factors, such as histology, tumor weight, lymph node involvement and age at diagnosis has led to a re-examination of the treatment of Wilms' tumor. Future therapeutic developments will include the administration of less therapy to some well defined groups of patients, and the exploration of new programs for patients who have been demonstrated to have a poor prognosis using currently accepted treatment techniques.


Immunologic localization of alpha- and beta-endorphins and beta-lipotropin in corticotropic cells of the normal and anencephalic fetal pituitaries.

The use of antibodies to alpha-(17--39) ACTH, beta-LPH, and alpha- and beta-endorphins has enabled the detection of immunoreactive cells as early as the eight week of fetal life. Immunoreactive cells are located in the anterior and intermediate lobes of the pituitary gland in normal human fetuses. The same cells reacted with different antisera used in the present study. No immunoreactive cells were observed in the posterior lobe. The specificity of the immunocytological reaction has been controlled. In seven anencephalic fetuses, the presence of immunoreactive cells suggests that ACTH and beta-LPH can originate from a common precursor and are synthesized independent of the hypothalamus.


Intranuclear inclusions in the developing neurons of the rat cuneate nuclei.

The ultrastructure of intranuclear rodlets, microtubules, fibrillar lattices and membranous inclusions found in the developing cuneate nuclei of rats is described. Rodlets, ranging in diameter from 96--312 nm and in length from 1--2 micrometer, are made up of tightly packed straight filaments measuring 5--8 nm in diameter. Microtubules with a diameter of 26 nm are clustered together. Fibrillar lattices are made up of fibrils with a diameter of 9 nm arranged in layers or sets. Two to nine sets make up a lattice, with a maximum width of 68 nm, in which the adjacent sets are arranged at an angle to each other. Rodlets and fibrillar lattices occur in 6.8% of the neurons. Membranous inclusions, reported here for the first time in normal neurons, are of 2 types: small vesicles of 0.1--0.6 micrometer and large vacuoles measuring 1--2 micrometer. Both types are bounded by either a single or a double membrane and generally have an electron lucent content. Membranous inclusions occur in 25.3% of the neurons. Changes in the frequency of occurrence of the various intranuclear inclusions in the course of postnatal development are also reported.


Intranuclear rodlets in woodchuck thyroid follicular cells.

Intranuclear rodlets are seen in thyroid follicular cells of woodchucks fixed during summer and fall. They are not observed in winter and spring animals. A functional significance of these structures is suggested.


An immunohistochemical study of epithelial cells in the posterior lobe and pars tuberalis of the human adult pituitary gland.

Pituitary glands were examined using reference staining (hematoxylin and eosin, periodic acid-Schiff and alcian blue) and the peroxidase-labeled antibody method, for 1) "invading" anterior cells in the posterior lobe, 2) intermediate colloid forming follicles, and 3) pars tuberalis cells. The results showed: 1) that the majority of cases possessed "invading" anterior cells of various amount. Most of these cells were positive for ACTH1-18, ACTH17-39 and betaMSH. However, on a few occasions, scattered GH, PRL, FSHalpha, FSHbeta, LHbeta and even TSHbeta cells were also present. 2) Colloid forming follicular cells were mostly ACTH cells, but also contained occasional other hormone-secreting cells. Hormone negative cells were correlated with salivary type epithelium. Well established acinic type salivary glands and ciliated epithelium were negative for any hormones immunohistochemically. 3) Pars tuberalis cells were predominantly gonadotrophs but also included TSHbeta and ACTH cells. Some cells appeared to contain both FSHbeta and LHbeta. When these cells underwent squamous metaplasia, they seemed to lose their hormone secreting activity.


Schwann cell growth factors.

Purified rat Schwann cells were found to proliferate very slowly in normal growth medium containing 10% fetal calf serum (FCS). Crude extracts of bovine pituitary or brain markedly enhanced Schwann cell growth, while similar extracts of nerve roots, liver and kidney did not. Pituitary extracts were more potent than brain extracts, and extracts from both anterior and posterior pituitary were active. The mitogenic activity of pituitary extracts was reduced by treatment with trypsin, and abolished by pronase and by boiling. A variety of known anterior and posterior pituitary hormones, as well as fibroblast, epidermal and nerve growth factors, were not mitogenic. FCS (greater than 1%) was required for Schwann cell proliferation, but even high concentrations of FCS did not substitute for pituitary or brain extracts, and serum from various other species did not support Schwann cell growth. Although various agents that increase cyclic AMP levels (such as cholera toxin) had been shown to be Schwann cell mitogens, extracts of pituitary or brain did not increase cyclic AMP levels. Extracts of various bovine tissues, including pituitary, brain, liver and kidney, acted synergistically with cholera toxin in stimulating Schwann cell proliferation, although the increase in cyclic AMP induced by the mixture was not greater than that seen with cholera toxin alone. We conclude that there are at least two separate pathways for stimulating Schwann cell division, only one of which involves an increase in intracellular cyclic AMP.


Immunocytochemical visualization of coated pits and vesicles in human fibroblasts: relation to low density lipoprotein receptor distribution.

The coated pit-coated vesicle system has a key role in the uptake of plasma low density lipoprotein (LDL) and other receptor-bound proteins in human fibroblasts. To study the distribution of coated pits and coated vesicles in fibroblasts by immunochemical techniques at both the light and electron microscopic levels, we immunized rabbits with coat protein extracted from bovine brain-coated vesicles. The resulting anti-coat protein antibody was directed predominantly against clathrin, the 180,ooo dalton protein that constitutes the major component of coat protein. By indirect immunoperoxidase electron microscopy, the anti-coat protein antibody was observed to bind specifically to coated pits on the surface of human fibroblasts and to coated vesicles within the cell. Indirect immunofluorescence and immunoperoxidase staining techniques at the light microscopic level revealed that the coat protein was distributed in fibroblasts in two distinctive patterns: as discrete foci on or near the cell surface that were linearly aligned in association with phase-dense cellular fibers (first pattern), and as intracellular foci that were randomly arranged around the cell nucleus (second pattern). The distribution of coat protein in fibroblasts was compared with the distribution of ferritin-labeled LDL, which was studied with the use of similar electron microscopic and immunofluorescence techniques. As previously reported, electron microscopic studies revealed that the LDL-ferritin binding sites at 4 degrees C were clustered in coated pits. By immunofluorescence microscopy, the LDL-ferritin that was bound to receptors within coated pits was shown to be arranged linearly over the cell surface in a pattern that was similar to the linear arrangement of coat protein (first pattern). Considered together, the current data indicate that coated pits in human fibroblasts contain a protein analogous to clathrin, and that those coated pits which contain receptors for LDL are located over intracellular fibers most likely corresponding to stress fibers. These observationa may have relevance to the mechanisms by which the coated pit-coated vesicle system efficiently delivers recptor-bound ligands to lysosomes.


The entry into host cells of Sindbis virus, vesicular stomatitis virus and Sendai virus.

We have compared the mechanisms of entry into host cells of three enveloped viruses: Sendai virus, vesicular stomatitis virus (VSV) and Sindbis virus. Virus entry by membrane fusion should antigenically modify the surface of a newly infected cell in such a way that it will be killed by anti-viral antibody and complement. On the other hand, virus entry by a mechanism involving uptake by the cell of the whole virion should not make cells sensitive to antibody and complement. As expected, cells newly infected with Sendai virus were readily and completely lysed by anti-Sendai antibody and complement. In marked contrast, however, cells newly infected with either Sindbis virus or VSV were killed by anti-viral antibody and complement only when infected at an extremely high multiplicity of infection, in excess of 1000 plaque-forming units per cell. We favor the following explanation for these results with Sindbis virus and VSV: a very large majority of the Sindbis and VSV virions entered the infected cells by some means other than membrane fusion, presumably engulfment of the whole particle. Efficient entry by way of membrane fusion may therefore not be a general characteristic of enveloped viruses.


Transformation of hamster embryo fibroblasts by a specific fragment of the herpes simplex virus genome.

Isolated restriction endonuclease fragments of the herpes simplex virus type 1 (HSV-1) genome were introduced into hamster embryo cells to identify DNA sequences capable to transforming the cells with respect to acquisition of properties correlated with tumorigenicity. One of the fragments generated by cleavage of HSV-1 DNA with the restriction endonuclease Xba I was found to induce transformation at a frequency of about 10 colonies per quantity of fragment recovered from 1 microgram of uncut DNA; fractions containing the other Xba I fragments failed to induce transformation reproducibly, although occasional colonies were detected. The fragment with transforming activity (Xba I-F) is 15.5 X 10(6) daltons in molecular weight and is located between 0.30 and 0.45 map units on the HSV-1 genome. The Xba I-F transformants obtained were selected for their ability to replicate in low concentrations of serum; in addition, they were found to attain high saturation densities in the presence of 10% serum and to form colonies in semisolid medium. Moreover, the transformed cells produced at least one of the viral gene products (a membrane glycoprotein) encoded in the fragment used for transformation, indicating not only that viral DNA was incorporated into the cells, but also that viral genes were expressed.


Progesterone-induced meiosis in Xenopus laevis oocytes: a role for cAMP at the "maturation-promoting factor" level.

Cholera toxin inhibition of progesterone-induced meiosis of Xenopus laevis oocytes in vitro has been correlated with increased cAMP levels. Inhibition of germinal vesicle breakdown (Gvbd) and cAMP increase occurred after a lag period of 2 hr, when cholera toxin was injected, or 4--5 hr, when applied externally. The ability of the maturation-promoting factor (Mpf) to provoke Gvbd when injected into recipient oocytes was found to be dependent upon whether the oocytes had been exposed to cholera toxin alone or to toxin and progesterone. With the former, cAMP levels were elevated and Mpf activity was abolished, whereas with the latter, the increase in cAMP was less pronounced and Mpf activity was observed. Injection of cAMP or its 8-thio derivatives shortly before the appearance of progesterone-induced Mpf abolished Gvbd. If injected earlier or later, no inhibition was observed. In contrast, cholera toxin inhibited maturation even when added several hours before progesterone, suggesting a sustained accumulation of cAMP. No Gvbd occurred when 8-thio-methyl-cAMP was injected together with Mpf. These data suggest that cAMP is involved in the control of the formation/amplification and/or activity of Mpf-a result which may be of general significance in cell division mechanisms.


Metabolism of acetylcholine receptor in chick embryo muscle cells: effects of RSV and PMA.

We have investigated some aspects of the metabolism of the integral membrane protein acetylcholine receptor (AChR) in normal and transformed cultures of chick embryo muscle cells. Turnover of AChR in control muscle cell cultures was compared with turnover in cultures infected and transformed by a temperature-sensitive mutant of Rous sarcoma virus (RSV) and with cultures treated with the tumor promoter phorbol myristate acetate (PMA). The parameters of AChR metabolism were estimated using 125I-alpha-bungarotoxin as a stoichiometric high affinity ligand for the AChR. We found that both RSV transformation and PMA increased the rate of degradation and decreased the rate of synthesis of AChR. The consequent reduction in steady state receptor levels suggests that oncogenic transformation and tumor promoter significantly alter the metabolism of cell surface membranes. We also observed that parameters of AChR metabolism in control cultures changed systematically in a pattern which depended upon the age of the culture as well as the use of embryo extract or fetal bovine serum as medium supplements. The muscle cell system allows quantitative measurement of an integral membrane protein and its metabolism, and may serve as a more general model for alterations in membrane and surface receptor metabolism associated with the transformed state.


Plasminogen activator in chick embryo muscle cells: induction of enzyme by RSV, PMA and retinoic acid.

To explore the generality of the effects of sarcoma viruses, tumor-promoting phorbol esters and retinoic acid, we have studied plasminogen activator production in differentiating chick myogenic cultures. Although slightly higher than in chick fibroblast cultures, the level of spontaneously synthesized enzyme is low; it reaches a peak shortly after maximum cell fusion has been completed and then declines. Rous sarcoma virus (RSV) transformation of differentiating myotubes was accomplished by infecting myoblasts with a temperature-sensitive mutant, maintaining cultures at the nonpermissive temperature until completion of fusion and shifting to permissive temperatures at selected times thereafter. RSV transformation, phorbol myristate acetate (PMA) and retinoic acid all induced high levels of plasminogen activator production by differentiating myotubes in the absence of DNA synthesis. In comparison with fibroblasts, virus-induced enzyme synthesis by myogenic cultures proceeded more slowly but ultimately reached comparably high levels. Whereas cAMP strongly repressed RSV- and PMA-induced plasminogen activator production by chick fibroblasts, it weakly stimulated enzyme synthesis by myotubes. This suggests that enzyme induction by RSV and PMA is not mediated primarily through effects on cAMP metabolism.


Kinase-negative mutants of S49 mouse lymphoma cells carry a trans-dominant mutation affecting expression of cAMP-dependent protein kinase.

Kinase-negative mutants of S49 mouse lymphoma cells are pleiotropically negative for all known cAMP-mediated responses of S49 cells and yield cell extracts which are deficient in cAMP binding activity and devoid of cAMP-dependent protein kinase activity. In hybrids between kinase-negative and wild-type cells, the mutant phenotype is dominant: the tetraploid hybrids have reduced cAMP-binding activity and undetectable cAMP-dependent kinase activity. The mutant phenotype is attributable to neither a soluble inhibitor of kinase catalytic subunit, nor a defective kinase regulatory subunit acting as an inhibitor, nor a defective catalytic subunit which sequesters regulatory subunits in inactive complexes. We propose that these mutants carry trans-dominant lesions in a regulatory locus responsible for setting intracellular levels of kinase expression.


Mapping unintegrated avian sarcoma virus DNA: termini of linear DNA bear 300 nucleotides present once or twice in two species of circular DNA.

Three major species of viral DNA have been observed in cells infected by retroviruses: a linear, double-stranded copy of a subunit of viral RNA; closed circular DNA; and proviral DNA inserted covalently into the genome of the host cell. We have studied the structures of the unintegrated forms of avian sarcoma virus (ASA) DNA using agarose gel electrophoresis in conjunction with restriction endonucleases and molecular hybridization techniques. The linear duplex DNA is approximately the same length as a subunit of viral RNA (approximately 10 kb) and it bears natural repeats of approximately 300 nucleotides at its termini. The repeats are composed of sequences derived from both the 3' and 5' termini of viral RNA in a manner suggesting that the viral DNA polymerase is transferred twice between templates. Thus the first end begins with a sequence from the 5' terminus of viral RNA and is permuted by about 100 nucleotides with respect to the 3' terminus of viral RNA; the linear DNA terminates with a sequence of about 200 nucleotides derived from the 3' end of viral RNA. We represent this structure, synthesized from right to left, as 3'5'-----3'5'. Two closed circular species of approximately monomeric size have been identified. The less abundant species contain all the sequences identified in linear DNA, including two copies in tandem of the 300 nucleotide 3'5' repeat. The major species lacks about 300 base pairs (bp) mapped to the region of the repeated sequence; thus it presumably contains only a single copy of that sequence. The strategies used to determine these structures involved the assignment of over 20 cleavage sites for restriction endonucleases on the physical maps of ASV DNA. Several strains of ASV were compared with respect to these sites, and the sites have been located in relation to deletions frequently observed in the env and src genes of ASV.


Proviruses of avian sarcoma virus are terminally redundant, co-extensive with unintegrated linear DNA and integrated at many sites.

We have analyzed the DNA from 15 clones of avian sarcoma virus (ASV)-transformed rat cells with restriction endonucleases and molecular hybridization techniques to determine the location and structure of proviral DNA. All twenty units of proviral DNA identified in these 15 clones appear to be inserted at different sites in host DNA. In each of the ten cases that could be sufficiently well mapped, entirely different regions of cellular DNA were involved. Thus ASV DNA can be accommodated at many positions in cellular DNA, but the existence of preferred sites has not been excluded. Six of the 15 clones carry only one normal provirus, two contain two normal proviruses, and seven harbor either one or two proviruses that appear anomalous in physical mapping tests. Both ends of at least 18 proviruses, however, were found to contain sequences specific to both the 3' and 5' termini of viral RNA. The organization of these terminally redundant sequences appeared identical to that of the 300 base pair (bp) repeats found at the ends of unintegrated linear DNA (Shank et al., 1978). Proviral DNA is therefore co-extensive, or nearly co-extensive, with unintegrated linear DNA and has a structure we denote as CELL DNA-3'5'----------3'5'-CELL DNA. Three of the four anomalous proviruses which were fully analyzed were deletion mutants lacking 25--65% of the genetic content of ASV; the fourth provirus had a novel site for cleavage by Eco RI but was otherwise normal. Tests for the biological competence of proviral DNA, based upon rescue of transforming virus after fusion with chicken cells, were generally consistent with the physical mapping studies.


Tandem integration of complete and defective SV40 genomes in mouse-human somatic cell hybrids.

We have analyzed the arrangement of SV40 DNA sequences integrated in human chromosome 7 in two lines of mouse-human somatic cell hybrids: one containing only one human chromosome 7 per cell and the other containing an average of about three. We found that the integration site differs in both the viral and host sequences in the two clones. However, the sites of integration into the several copies of human chromosome 7 of one clone are identical. Each chromosome 7 in both clones carries approximately six viral genomes tandemly linked. Some of these genomes lack about 20% of the DNA from the late region, including the Eco RI site.


Three species of polyoma virus tumor antigens share common peptides probably near the amino termini of the proteins.

Detergent extracts of polyoma virus-infected mouse cells contain three major proteins of approximately 100,000--108,000 (100K), 55,000 (55K) and 21,500 (22K) daltons, which react with sera obtained from rats carrying tumors induced by the virus. A comparison of the 35S-methionine-, 3H-leucine- and 3H-proline-labeled tryptic peptides of each of these proteins by cation-exchange chromatography followed by descending paper chromatography has shown that: at least five peptides are shared by all three T-reactive proteins; at least three peptides are shared by the 55K and 22K proteins, but not by the 100K protein; at least three peptides are found only in the 22K protein; at least six peptides are found only in the 55K protein; and at least sixteen peptides are found only in the 100K protein. The results are consistent with the hypothesis that the polypeptide chains of the 100K, 55K and 22K dalton tumor antigens of polyoma virus share a common virus-coded amino terminal region. The data also suggest that there is a portion of the polypeptide chains (probably immediately adjacent to the common amino terminal region of the molecules) that is shared by the 55K and 22K proteins, but not by the 100K protein (perhaps because this portion of the genetic information is spliced out of the messenger RNA coding for the 100K protein). The facts that all the peptides common to the 100K and 55K proteins are also found in the 22K protein and are thus assigned to the common amino terminal region of the molecules, and that there are several peptides unique to the 100K protein, as well as several peptides unique to the 55K protein, suggest that the presumed carboxy terminal portion of the polypeptide chain of the 100K protein is considerably, if not entirely, different from that of the 55K protein.


An enzymatic activity in uninfected cells that cleaves the linkage between poliovirion RNA and the 5' terminal protein.

The 5' terminal protein (VPg) on poliovirion RNA can be removed by cell-free extracts from a variety of uninfected cells. This soluble enzymatic activity is found in both nuclear and cytoplasmic extracts of heLa cells and is activated by Mg++. The enzyme activity cleaves the tyrosine-phosphate bond that links the protein to the RNA. In a partially purified form it has insufficient nonspecific protease or nuclease activity to account for its action. The existence of this enzyme implies that poliovirus RNA is translated in cell-free extracts in a form that lacks the 5' terminal protein. The role of this enzyme in the uninfected cell is not known.


The matrix (M) protein of vesicular stomatitis virus regulates transcription.

Temperature-sensitive mutants of vesicular stomatitis virus (VSV) belonging to complementation group III contain a lesion in the matrix (M) protein. This results in a 2--5 fold increase in transcription at the nonpermissive temperature. Co-infection of cells with one of these mutants and wild-type virus reverses this mutant phenotype. Separation of the transcriptional and translational products from mutant-infected cells reveals an overall increase in each of the viral mRNA species concomitant with degradation of the M protein at the nonpermissive temperature. The increase in mRNA, however, does not lead to increased synthesis of viral proteins. Quantitation of individual mRNA species indicates that M protein acts as a direct inhibitor of transcription as well as an attenuator of sequential transcription.


The International Reference Preparations of Clostridium welchii (C. perfringens) beta and epsilon toxoids.

The Central Veterinary Laboratory, Weybridge, England was requested by the WHO Expert Committee on Biological Standardization to obtain suitable materials for international standards for Clostridium welchii (C. perfringens) beta and epsilon toxoids and to arrange collaborative assays. Preparations were obtained and dispensed as freeze-dried toxoids in ampoules. The toxoids were assayed by nine laboratories in eight countries. On the basis of the results obtained, the materials have been established as the International Reference Preparations of Clostridium welchii (C. perfringens) Beta and Epsilon Toxoids.


Use of combined diet and colestipol in long-term (7--7 1/2 years) treatment of patients with type II hyperlipoproteinemia.

Long-term effects of diet and colestipol (a bile acid sequestrant) were studied in 25 patients with familial type II hyperlipoproteinemia. Serum lipids and body weights of an initial group of 30 patients were stabilized by low cholesterol-saturated fat-refined carbohydrate diet and the patients were then randomized into placebo and drug-treatment groups. After explaining that the drug is nontoxic and effective in lowering serum lipids, total cholesterol (C) and low-density lipoprotein cholesterol (LDL-C), colestipol (30 g/day) and diet were given to the 25 patients who remained in the long-term follow-up program. The treatment resulted in highly significant lowering of serum lipids (mg/dl, mean +/- SEM): C and LDL-C from 412.7 +/- 24.4 and 331.1 +/- 22.8 to 270 +/- 11.0 and 188.1 +/- 13.8, respectively (p less than 0.001 in each instance) over 7--7 1/2 years. Although we observed no absolute increase in high density lipoprotein (HDL), the HDL/LDL ratio was elevated. Long-term colestipol and diet treatment reduced the xanthoma size and stabilized serially angiographically visualized atherosclerotic lesions in 21 of the 25 patients who showed a satisfactory hypolipemic response. It did not cause nutritional or metabolic disturbances.


Effect of beta-adrenergic suppression by propranolol on coronary collateral development in response to chronic coronary ischemia in dogs.

Acute left circumflex coronary artery (LC) occlusion in conscious dogs caused marked ischemia in the myocardium supplied by the occluded artery, as judged by the radioactive microsphere technique for determining blood flow distribution. With the chest open, LC pressure distal to the occlusion fell to 21 +/- 1.9% of aortic pressure. By 8 weeks after gradual LC occlusion with an ameroid constrictor, collateral development had restored coronary blood flow distribution to near-normal under basal conditions and during pacing, at a heart rate of 200 beats/min. The only evidence for ischemia was in the subepicardium within the distribution of the unoccluded left anterior descending artery, which provided the extra collateral blood flow. Distal LC pressure was 70 +/- 1.7% of aortic pressure. Propranolol 160 mg orally every 6 hours for 8 weeks had no detectable effect on coronary collateral development, as judged by blood flow distribution or distal LC pressure. The only significant difference for the propranolol dogs was a slight transmural shift away from the subendocardium in the left anterior descending region.


Ventricular aneurysms complicating coxsackievirus group B, types 1 and 4 murine myocarditis.

Suckling Swiss Webster mice were inoculated with 10(4)TCD50 of coxsackieviruses, group B types 1 or 4. Virulent necrotizing myocarditis resulted in 185 infected mice. Of the latter group, three (14.3%) nurslings on the 17th and 23rd day after inoculations had left ventricular aneurysms postmortem. None of 61 concurrently matched control mice developed aneurysms. Ventricular aneurysm is a suggested but previously undocumented complication of murine, and possibly human necrotizing transmural coxsackievirus myocarditis.


New saccharogenic determination of alpha-amylase in serum and urine.

I describe a new kinetic enzymatic saccharogenic method for assaying alpha-amylase in human serum and urine. alpha-Amylase liberates maltose from starch. This is successively acted on by alpha-glucosidase, mutarotase, and glucose dehydrogenase. The resulting conversion of NAD+ to NADH, measured at 340 nm, during a 20-min incubation reflects amylase activity. Endogenous glucose is destroyed before measurement of amylase activity is begun.


Nephelometry of apolipoprotein B in human serum.

We studied the development of light scattering in the reaction between anti-apolipoprotein B and apolipoprotein B in intact very-low-density lipoproteins (I) and low-density lipoproteins (II) as well as in lipoproteins treated with lipases, and found considerable differences in the kinetics of the immunoreaction for the two lipoprotein classes. Pre-incubation with triglyceride lipase and cholesterol esterase caused a decrease of final light scattering in I but only minimal changes in the reaction with II. Non-ionic detergent not only decreased the original light scattering in hyperlipemic serum samples, but also accelerated the immunoreaction. Under standardized conditions, results of quantitative nephelometry correlated highly significantly with quantitative determination of apolipoprotein B by radial immunodiffusion, both for normolipemic and hyperlipoproteinemic serum samples. The nonspecific light scattering caused by neutral lipids in intact lipoproteins could be minimized when samples were pre-incubated with lipolytic enzymes.


Rapid fluorometry of estrogens in nonpregnancy urine, with use of chloroform extraction and purification by anion-exchange chromatography.

We describe a new method of extraction and purification of estrogens in low concentration in urine, involving a rapid enzymic hydrolysis of a 10-mL sample, automatic extraction (in a tube) with CHCl3/ethyl acetate, purification by chromatography on a disposable "mini-column" of AG 1-X2, and fluorometry by continuous flow according to Itrich's procedure [Acta Endocrinol. (Copenhagen) 35, 34 (1960)]. Conditions of hydrolysis, extraction, and purification were studied. Within-day precision (CV) was 7.5%, the mean between-day precision 8.5%. The sensitivity was 6 microgram of total estrogens per liter of urine. The specificity was assessed particularly by comparison with the results obtained by Schöller et al. [Acta Endocrinol. (Copenhagen) 57, suppl. 107 (1966)] and by gas-chromatography-mass-spectrometry. The normal limits as calculated by this technique were identical to values reported by others. The technique is rapid; results are obtained in 3 h. It is quite suitable for routine determinations: 100 assays can be done by a team of three technicians in one working day.


Reversed-phase liquid chromatographic separation of 3',5'-cyclic ribonucleotides.

A rapid, reversed-phase "high-performance" liquid-chromatographic separation of the five naturally occurring cyclic ribonucleotides is described. The separation, optimized for the measurement of these compounds in biological samples, is short (25 min), sensitive (50-100 pmol), and requires no sample pre-concentration steps. We also report an alternative isocratic elution mode, optimized for a rapid and selective analysis for adenosine 3',5'-cyclic phosphate. The identity of chromatographic peaks in biological extracts is confirmed by several methods: retention times, co-chromatography with the reference compounds, absorbance ratios, enzymatic peak-shift with cyclic nucleotide phosphodiesterase, and stopped-flow ultraviolet-scanning techniques.


Efficacy of a simplified primary screening procedure for detection of hyperlipoproteinemias in a pediatric population.

We examined the efficacy of a simple primary screening procedure for detecting beta- and pre-beta-lipoprotein abnormalities in 3183 children, ages 5-14, residing in Bogalusa, Louisiana. This procedure is based on the ability of beta- and pre-beta-lipoproteins to form insoluble complexes with heparin in the presence of Ca2+; the turbidity produced by the reaction was considered as an index of the concentration of these two classes of lipoproteins. Our results indicate a close relationship (r = 0.88) between the beta- + pre-beta-lipoprotein index (turbidity) and the concentrations of these lipoproteins. Comparison of serum lipid and beta- + pre-beta-lipoprotein values of 5% of the children whose results fell outside the normal limits (upper and lower 5%) indicated that serum total cholesterol was not reflecting the beta- + pre-beta-lipoprotein concentration of a given child. The variability of alpha-lipoprotein concentration in these children accounted for this discrepancy. Measuring the serum beta- + pre-beta-lipoprotein index may be more useful for large-scale screening and for detecting subtle abnormalities than are determinations of either cholesterol or triglycerides.


Re-evaluation of turbidimetry of proteins by use of aromatic sulfonic acids and chloroacetic acids.

From studies on 11 different proteins (including native albumin and albumin with reduced disulfide-bridges) treated with sulfosalicylic, 2-naphthalenesulfonic, toluenesulfonic, dichloroacetic, or trichloroacetic acids, we elucidate the interactions determining the resulting turbidities and other factors affecting turbidities, and we discuss the clinical utility of such turbidimetry. At least three interactions are important in determining turbidity: reduction of positive charges on the protein, hydrogen bonding of the non-ionized chloroacetic acids with the protein, and hydrophobic interaction of the aromatic sulfonic acids with albumin. Turbidity varies appreciably with the species of acid and protein, concentrations of acid, temperature, and standing time after acid is added. We conclude that this technique should be restricted to confirming proteinuria.


Lactate-to-pyruvate or pyruvate-to-lactate assay for lactate dehydrogenase: a re-examination.

The pyruvate-to-lactate assay for determining lactate dehydrogenase (EC 1.1.1.27) can now yield linearity equal to or better than that obtained by the lactate-to-pyruvate assay. In addition, there are significant advantages to the pyruvate-to-lactate reaction: (a) a greater change in absorbance per unit time, which allows more accurate spectrophotometric readout; (b) lower reactant concentrations are required, which substantially reduces the cost per assay; (c) solid reagents are used to prepare the assay solution; and (d) reagent solutions are more stable. However, impurities present in commercial NADH preparations may substantially affect measured lactated dehydrogenase activities; therefore, a Standard Reference Material for NADH is being developed for issuance by the National Bureau of Standards.


Gel isoelectric focusing method for specific diagnosis of familial hyperlipoproteinemia type 3.

We describe a gel isoelectric focusing procedure for resolving into at least five bands the arginine-rich protein of very-low-density lipoproteins, and use the method in diagnosis of hyperlipoproteinemia type 3. We find that deficiency of one band, designated E3, relative to E2, expressed as an E3/E2 ratio less than or equal to 0.3, is specifically associated with hyperlipoproteinemia type 3 as diagnosed by traditional criteria in a large family study. In addition, we used the procedure to test 47 referral samples with demonstrable beta-migrating very-low-density liproprotein grouped by very-low-density lipoprotein cholesterol/total triglyceride ratios of greater than or equal to 0.3, from 0.25 to 0.3, and less than 0.25. All 24 of the first group, three of six in the second, and only one of 17 in the third had E3/E2 ratios of less than or equal to 0.3. Also, two normolipidemic children, without detectable lipoprotein abnormalities but related to subjects with hyperlipoproteinemia type 3, had E3/E2 ratios of less than or equal to 0.3. Use of our procedure improves the specificity of diagnosis and allows sensitive detection of asymptomatic subjects who may be at risk of developing the disorder.


An enzymic and centrifugal method for estimating high-density lipoprotein cholesterol.

Enzymic measurement of high-density lipoprotein cholesterol with a centrifugal analyzer is described. We used polyethylene glycol (Mr 6000), final concentration 100 g/L, to precipitate low-density and very-low-density lipoproteins, thereby eliminating the difficulties of the commonly used heparin/Mn2+ precipitation method and facilitating the use of ethylenediaminetetraacetate-stabilized plasma. As measured by rocket immunoelectrophoresis, this final concentration of polyethylene glycol completely precipitates beta-lipoproteins, leaving the alpha-lipoproteins in solution. Between-run reproducibility (CV) was 3.6%, within-run reproducibility (CV) 0.8%. Reagent costs currently are $US 0.13 per test and large numbers of samples can be handled conveniently. Normal ranges were compiled for 539 men and 444 women. The high-density lipoprotein cholesterol for men was 1.20 +/- 0.31 (SD) mmol/L and for women 1.52 +/- 0.38 (SD) mmol/L.


A radiochemical method for the determination of transketolase activity in erythrocyte hemolysates.

A radiochemical method for the determination of transketolase activity is reported. It is based on incubation of erythrocyte hemolysates with radioactive ribose 5-phosphate followed by isolation of sedoheptulose 7-phosphate on anion-exchange columns. The experimental conditions are discussed and the presence of phosphatase activity in the incubation mixture is demonstrated. The proposed method is compared with a current colorimetric method. Reference values are given for the transketolase activity and the thiamine pyrophosphate effect.


Concentration and composition of human serum lipoproteins at birth.

By ultracentrifugation of sera from 69 cord bloods very low (VLDL), low (LDL) and high density lipoproteins (HDL) were obtained and analysed for triglycerides (TG) and cholesterol (CHOL). Electrophoresis in agarose gel was performed on serum, VLDL and LDL + HDL. VLDL lipids were present in all newborns. VLDL contained 30 and 5% of serum TG and CHOL. The range of TG in VLDL was wide (0.02-0.62 mmol/l), but there was a constant ratio between TG and CHOL. The electrophoretic mobility of VLDL was slower than in adults. LDL contained 50% of both serum TG and CHOL, and HDL contained 15 and 50% of these serum lipids. A published formula for calculating CHOL in VLDL and LDL from serum CHOL and TG and CHOL in HDL gave values for CHOL in LDL which corresponded well with the determined values. For VLDL, however, the calculated values were too high.


[Lipoprotein electrophoresis on acrylamide-agarose plates, with discontinuous acrylamide gradient].

We describe a modified lipoprotein electrophoresis on acrylamide gel. The lipoproteins, prestained with Sudan Black, are separated by running on acrylamide-agarose gels, with two different concentrations of acrylamide and a constant concentration of agarose. The sera are deposited in the first gel (2% acrylamide). Chylomicrons remain at origin and other lipoproteins run through the first gel to the second one (3% acrylamide gel). VLDL stays at the junction of both gels, LDL and HDL are separated in the 3% acrylamide gel. By this technique, we were able to detect an increase of Lp(a) and to identify the different types of hyperlipoproteinemia. After running, the plates can be treated for storage.


Hormonal control of oocyte meiosis, ovulation and luteinization in mammals.

The ovulatory process can be regarded as a series of biochemical and morphological changes ultimately leading to the release of a mature oocyte and the transformation of the Graafian follicle into the corpus luteum. This process involves acute changes in steroidogenesis, resumption of oocyte meiosis, and finally rupture of the follicular wall and luteinization of the granulosa cells. Normally, all of these changes are induced synchronously by the pre-ovulatory LH surge. Experimentally, however, these changes in steroidogenesis, oocyte maturation and follicular rupture can be dissociated from each other showing that the LH effect is mediated via different cellular messengers. The gonadotrophins act in an orderly sequence to induce follicular maturation. The granulosa cells increase their number of LH receptors and respond to LH with increased stimulation of cyclic AMP accumulation and progesterone secretion. Concomitantly, they decrease in their FSH receptors and their response to FSH diminishes in terms of ability to stimulate cyclic AMP accumulation. The ovulatory process is associated with increased uptake of LH by the follicle; when granulosa cells are obtained from pre-ovulatory follicles and cultured they luteinize spontaneously. Steroid hormones modulate the actions of gonadotrophins on follicular maturation. In addition, there are non-steroidal factors in follicular fluid which regulate follicular maturation: an oocyte maturation inhibitor keeps the oocyte in meiotic arrest; a luteinizing inhibitor prevents the granulosa cells from luteinizing prior to follicular rupture; a folliculostatin inhibits FSH release from the pituitary gland. The functional activity and the lifespan of the corpus luteum depend on adequate pre-ovulatory as well as post-ovulatory gonadotrophic stimulation. Its lifespan may also be regulated by an LH binding inhibitor.


Polymorphism of apolipoprotein E. I. Methodological aspects and diagnosis of hyperlipoproteinemia type III without ultracentrifugation.

Two methods for phenotyping apolipoprotein E are compared. One is based on preparation of VLDL by conventional ultracentrifugation, whereas the other uses heparin/Mg precipitation of VLDG. In principle, the same results were obtained by both methods. However, the group limits for the three different phenotypes Apo E-N, Apo E-ND and Apo E-D were slightly different by the two methods. Phenotype Apo E-D - the phenotype characterizing type III dyslipoproteinemia - is clearly definable by both methods. Hence the precipitation-I.E.F. method for Apo E phenotyping provides a simple tool for genetic and population genetic studies and also for the routine diagnosis of hyperlipoproteinemia type III, based on the only specific marker known for this disease.


Use of microtechniques for the detection of lysosomal enzyme disorders: Tay-Sachs disease, Gm1-gangliosidosis and Fabry disease.

A preliminary report on the use of microtechniques for the detection of three lysosomal storage diseases (Tay-Sachs, GM1-gangliosidosis and Fabry disease) is presented. This microassay method uses from 100 to 300 cultured amniotic fluid cells or skin fibroblasts. A comparison between values for total activity and heat inactivated forms of hexosaminidase (in Tay-Sachs disease) is presented. The feasibility of the use of this microtechnique in prenatal diagnosis is discussed.


Polymorphism of apolipoprotein E. II. Genetics of hyperlipoproteinemia type III.

Apolipoprotein E from human serum shows a genetic polymorphism determined by two autosomal codominant alleles, Apo En and Apo Ed. Homozygosity for the gene Apo Ed (phenotype Apo E-D) results in primary dysbetalipoproteinemia, but only some individuals with this phenotype develop gross hyperlipidemia (hyperlipoproteinemia type III). Vertical transmission of dysbetalipoproteinemia represents pseudodominance due to the high frequency of the gene Apo Ed. Dysbetalipoproteinemia is already expressed in childhood. To assess the influence of other genes on the expression of hyperlipidemia in phenotype Apo E-D, comparative studies were carried out in kindreds of hypercholesterolemic (group A) and normo- or hypocholesterolemic probands with dysbetalipoproteinemia (group B). This demonstrated the occurrence of familial (non-type III) forms of hyperlipidemia in group A but not in group B kindreds. Distribution of lipoprotein phenotypes in five of the group A kindreds was consistent with the occurrence of familial combined hyperlipidemia. Apo E phenotypes and hyperlipidemia segregated independently. It is concluded that primary dysbetalipoproteinemia is a frequent monogenic variant of lipoprotein metabolism, but not a disease. Coincidence in one individual of genes for this specific dyslipoproteinemia with any of the genes for monogenic or polygenic forms of familial hyperlipidemia results in hyperlipoproteinemia type III. Hence hyperlipoproteinemia type III is caused by at least two non-allelic genes and is a polygenic disorder.


Long-term administration of vitamin D steroles in incipient and advanced renal failure: effect on bone histology.

In 36 patients with incipient and advanced renal failure (CCr 80--30 ml/min X 1.73 m2), serum chemistry including ionized Ca, serum PTH and fractional intestinal absorption of Ca (whole body counter; two-dose-technique; 47Ca p.o. and i.v. to correct for urinary and endogenous fecal loss were measured. Quantitative bone histology after in vivo tetracycline double labeling was evaluated from undecalcified sections before and 18 months after therapy with vitamin D3 or 5,6-trans-25-OH-CC in a dose sufficient to raise intestinal absorption and/or urinary excretion of Ca. Intestinal absorption of Ca was impaired in some patients at a GFR of 60 ml/min/1.73 m2. After up to 10000 U/d 5,6-trans-25-OH-CC and 8000 IU/d vitamin D3, respectively, fractional intestinal absorption of Ca rose and was normalized in all patients. There was a concomitant rise in urinary Ca. Serum PTH fell, but did not always return into the normal range. Ionized Ca rose in all patients. Bone histology was evaluated in 17 of these 36 patients after informed consent was obtained. The mass of mineralized bone (Vv) rose in 7/17 patients, pointing to a positive calcium balance. Volumetric density of osteoid (Vvos) and surface density of osteoid (Svos) fell in 10/17 patients concomitant with an increase in the fraction of mineralizing seams and a decrease in the number of lamellae in osteoid seams. Osteoclastic resorption (OCl) fell as did the fraction of woven osteoid seams. However, woven osteoid failed to disappear completely and osteoclastic resorption stayed elevated in some patients. 5,6-trans-25-OH-CC and vitamin D3, in doses that normalized intestinal absorption of Ca, failed to restore completely bone histology to normal although mineralization and collagen texture of osteoid were consistently improved. The dose response characteristics to vitamin D of different abnormalities of Ca metabolism appear to be non-uniform.


The effect of prazosin of pre- and post-synaptic alpha-receptors in the pithed rat.

1. Prazosin had a marked inhibitory effect on post-synaptic alpha-receptors in the pithed rat, as measured by the blood pressure response to the cumulative administration of clonidine. 2. Prazosin had no inhibitory effect, however, on the pre-synaptic alpha-receptor, as measured by the clonidine-induced reduction in the tachycardia produced by stimulation of the cardiac nerves. 3. These results suggest that prazosin has a selective antagonistic affinity for post-synaptic alpha-receptors.


The effect of corticotrophin (ACTH) administration on the pressor action of angiotensin II, noradrenaline and tyramine in sheep.

1. Pressor responses to angiotensin II, noradrenaline and tyramine were examined in sheep prior to and during the development of corticotrophin-induced hypertension. 2. Pressor responses to angiotensin II amide did not change with corticotrophin (ACTH) administration. Small significant increases in pressor responses to noradrenaline occurred at low doses only (0.27 and 1.06 mumol/h). Significant increases in response to tyramine occurred after 24h  of ACTH administration, but were not maintained after 6 days of ACTH. These changes are quantitatively small and do not suggest that changes in pressor sensitivity contribute significantly to the rise in blood pressure following ACTH administration. 3. Sodium depletion significantly reduced the pressor responses to angiotensin II amide at all doses and to tyramine in the middle range only, but did not affect the responses to noradrenaline.


Poorly differentiated lymphocytic lymphoma with ectopic parathormone production: visulization of metastatic calcification by bone scan.

Metastatic soft tissue calcification is known to occur in hypercalcemia and is usually present in the kidneys, stomach and lungs. 1--3 This case presents two unusual features: 1) ectopic parathormone production in association with poorly differentiated lymphocytic lymphoma; and and 2) uptake of 99mTc-pyrophosphate in the liver in the absence of demonstrable abnormality at autopsy. The more usual sites of metastatic calcification also showed uptake of the radionuclide. We will discuss metastatic soft tissue calcification, ectopic parathyroid hormone production, hypercalcemia in malignancy and bone scan agent localization in soft tissues.


Paraplegia, syringomyelia tarda and neuropathic arthrosis of the shoulder: a triad.

A casually related triad of syringomyelia tarda, postparaplegia with secondary neuropathic arthrosis of the shoulder has been presented. The development of neuropathic arthrosis of the shoulder has been presented. The development of neuropathic arthrosis of the shoulder in 2 of our paraplegic patients prompted us to look for a correlation and/or a common etiology. Paraplegia secondary to spinal cord injury could be causative or at least an associated factor in a delayed proximal syringomyelia. It is the syringomyelia which can lead to the neuropathic arthrosis in the upper extremity, mainly, in the shoulder. There is an interesting pathological association of paraplegia and syringomyelia with neuropathic arthrosis. Earlier awareness of any neurological changes in the upper extremity of the paraplegic patient could point to recognition of syringomyelic process and portend a tendency toward neuropathic arthrosis of the shoulder. This would require a high index of suspicion, and subsequently a preventative, protective orthopedic approach to minimize the usual functional deformity that occurs when this neuropathy involves the shoulder.


Inhibition of human pulmonary phosphodiesterase activity by therapeutic levels of theophylline.

1. To test the hypothesis that inhibition of cyclic nucleotide phosphodiesterase is the major mechanism of the bronchodilator action of theophylline in reversible airways disease, the effects of therapeutic plasma levels of the drug on human pulmonary phosphodiesterase activity were examined. 2. Therapeutic levels of theophylline inhibited the phosphodiesterase-catalysed hydrolysis of adenosine cyclic 3',5'-monophosphate (cAMP) and guanosine cyclic 3',5'-monophosphate (cGMP), but the percentage inhibition was relatively small. 3. The results, while supporting the assumed contribution of phosphodiesterase inhibition to the overall mechanism of theophylline action, suggest that other presently unknown factors must also be taken into consideration to fully explain the beneficial effects of theophylline in reversible airways disease.


Suprascapular axonotmesis and rheumatoid disease: report of a case treated conservatively.

Axonotmesis of the suprascapular nerve mimics rupture of the musculotendinous cuff of the shoulder. Arthrography of the shoulder joint and electromyographic studies of the shoulder muscles are essential in arriving at the proper diagnosis. Axonotmesis of the suprascapular nerve should be suspected in the patient who presents with signs of ruptured musculotendinous cuff and who has a normal arthrogram.


Decreased cardiac beta-adrenoceptors in hypertensive rats.

1. beta-Adrenoceptors have been investigated in cardiac and renal cell membranes from hypertensive and normal rats. 2. The 125I-labelled beta-adrenoceptor antagonist 1-(4-iodophenoxy)-3-isopropylaminopropan-2-ol was used to measure directly the number and affinity of receptors. 3. Cardiac membranes from hypertensive rats had a lower concentration of beta-adrenoceptors than membranes from control normotensive animals, but the affinities of the receptors remained unchanged. Receptor concentration decreased by half and was similar in each of the three models of experimental hypertension investigated. 4. In contrast, kidney membranes from hypertensive animals had the same beta-adrenoceptor concentration and affinity as membranes from normotensive rats. 5. The decrease in cardiac beta-adrenoceptor concentration may reflect an increase in cardiac sympathetic drive in these experimental hypertensive models.


Poliomyelitis in the fetus and the newborn. A comment on the new understanding of the pathogenesis.

A survey of the literature shows that although poliovirus may be recovered from fetuses whose mothers have paralytic poliomyelitis, there is no evidence that the fetuses themselves are affected. It is suggested that if postnatal poliomyelitis results from an autoallergic response not developed in the fetus, then poliovirus cannot enter the CNS of the fetus. When a mother has paralytic poliomyelitis at delivery the neonate has a 40 per cent chance of poliomyelitis, with a case fatality rate of about 50 per cent. It is suggested that most of these neonates become infected by virus entry into the exposed olfactory and nasal nerve endings after the membranes have burst. This would explain the very short incubation period and the high case and case fatality rates.


Alterations of adrenal and uterine angiotensin II receptors during variation of sodium intake and/or experimental hypertension.

1. Angiotensin II receptors from rat adrenal gland and myometrium were studied during variation of sodium intake. 2. In both target-tissues low Na+ diet increased the number of receptors whereas a high Na+ diet did not modify the adrenocortical receptors but increased the number of uterine receptors. 3. Deoxycorticosterone and one kidney Goldblatt hypertension were associated with a decrease in the number of adrenal receptors. 4. Alterations of angiotensin II receptors alone cannot explain satisfactorily the variations of sensitivity of target-cells to angiotensin II during sodium balance changes.


Tonin--angiotensin II system in hypertension.

1. A single intravenous administration of rabbit tonin antiserum into one-kidney one-clip hypertensive rats restored blood pressure to normal in seven out of ten animals. There was little change in blood pressure in two-kidney one-clip hypertensive, uninephrectomized or sham-operated rats. 2. Infusion of tonin in control rats did not modify arterial blood pressure. However, in indomethacin salt-treated rats a marked increase in arterial blood pressure was observed under tonin infusion. 3. Plasma tonin activity was significantly increased in human essential and renovascular hypertension. 4. These findings strongly suggest that tonin is important in the maintenance of high blood pressure. However, other factors (possibly prostaglandins and sodium) have to be modified in order to activate the tonin--angiotensin II system.


Long-term treatment of hypertension in man by an orally active angiotensin-converting enzyme inhibitor.

1. Captopril or SQ 14 225, administered orally twice a day, reduced the blood pressure of hypertensive patients whatever their clinical diagnosis and even when their plasma renin activity was 'normal' or low. 2. Long-term administration of captopril, either alone or together with diuretics, provides a powerful new tool with which to treat ambulatory hypertensive patients. 3. The renin system may play an important role in maintaining blood pressure in a majority of hypertensive patients.


Altered activity of the sodium-potassium pump in arteries of rats with steroid hypertension.

1. Ouabain-sensitive uptake of 86Rb, a measure of the Na+-K+ pump activity, was studied in tail arteries of rats made hypertensive with deoxycorticosterone and saline. 2. Decreased activity of the ouabain-sensitive Na+-K+ pump supports the hypothesis that the activity of Na+-K+ pump is suppressed in volume expanded hypertension.


Computed tomography of the paranasal sinuses, nasopharynx, and soft tissues of the neck.

A number of cases are presented illustrating the value of computed tomography in the investigation of mass lesions involving the paranasal sinuses, nasopharynx, and soft tissues of the neck. Rapid advances in CT technology now underway will alter our approach to the investigation of certain diseases in these locations.