Changes in muscle blood flow and development of the arm following the Blalock-Taussig anastomosis.

Muscle blood flow was studied in 28 patients after Blalock-Taussig anastomosis using the radioactive xenon clearance method. Insignificant differences in blood flow between the arms were found at rest, but there was a highly significant lowering of the peak blood flow on the operated side. Peak blood flow values in the first years after the anastomosis were close to the values in obliterative artery disease. The difference in maximum blood flow became less apparent beyond the first 5 years after the operation. A prolonged time of onset and duration of hyperemia after ischemic exercise were observed on the side of the operation. Statistically significant differences in circumference and length between the arms were measured and a lower strength of contraction of the affected hand was demonstrated. Abnormally low blood supply to muscles is thought to be responsible for the anatomical changes in the operated extremity.


Operant conditioning of heart rate in free-moving dogs.

The magnitude of changes in heart rate that could be induced by the operant conditioning of beat-to-beat intervals was determined. Shock avoidance methods were employed during 71 sessions conducted over 17 weeks in 3 free-moving mongrel dogs. Experimental periods during which conditioning was done alternated with baseline periods. Heart rate differences between baseline and experimental periods were used as indices of conditioning. Alterations in  heart rate were compared among four types of sessions: control, random shock, decrement conditioning and increment conditioning. Conditioned heart rate decrements averaged 3% while heart rate increments averaged 10%. Thus, operant conditioning carried out over prolonged periods of time resulted in relatively minor changes in heart rate.


Manifestation of the Wolff-Parkinson-White syndrome during isoprenaline infusion and carotid sinus massage.

In a patient with transient WPW syndrome the electrocardiographic pattern of preexcitation could repeatedly be induced by carotid sinus massage during isoprenaline infusion. Adrenergic and cholinergic discharge may favor the conduction of the impulse over an accessory nonfunctional A-V bundle.


Multifascicular block in cardiomyopathy. Incidence, localization by His bundle study and prognosis.

Of 70 patients with cardiomyopathy, nine (13%) had bifascicular block: right bundle branch block with left anterior hemiblock in four, right bundle branch block with left posterior hemiblock in two, and left bundle branch block with left anterior hemiblock in three. Isolated left bundle branch block was present in four, isolated left anterior hemiblock in 13, and isolated left posterior hemiblock in one. No isolated right bundle branch block was found. Five of the patients with bifascicular block were evaluated by His bundle electrocardiography and atrial pacing. Block in the His-Purkinje system (prolonged HV interval) was present in four and atrioventricular conduction (AH interval) was normal in all five. Despite a common prolongation in intraventricular conduction, short term prognosis seems to be favorable since: (1) none had spontaneous progression to more advanced block or experienced Stokes-Adams attacks for an average follow-up period of at least 42 months, and (2) with atrial pacing, one-to-one atrioventricular conduction persisted to rates up to 150/min in all patients with no further prolongation of HV interval.


Clinical utility of the simultaneous recording of heart and Korotkoff sounds.

Heart and arterial sounds and murmurs for a sequence of as many as 100 successive beats are recorded on a single print. The apparatus consists of a cathode ray oscilloscope, a Q-wave trigger to release the beam, a vertical axis which is connected to the blood pressure cuff, and an intensity modulation circuit which determines the intensity of the beam at each instant. This arrangement eliminates the baseline and thereby permits the display of low intensity acoustic signals. Data on normal subjects, and patients with various heart diseases are illustrated. Advantages and limitations of the method are discussed.


[Relation between training status and orthostatic tolerance].

Investigations on orthostatic tolerance and physical fitness prior to and after 6-8 h immersion experiments surprisingly revealed that all untrained subjects endured pre- and postimmersion tilt table tests without subjective complaints, whereas after immersion all trained participants collapsed within the first minutes of erect position displaying symptoms of a vasovagal syncope. Assuming that this impaired orthostatic tolerance of trained subjects can partially be referred to influences of an altered blood pressure control system we decided to record the blood pressure characteristics of both groups. Thereby, the transmural pressure of the carotid artery was changed by applying from outside either reduced or exceeding pressures. The controller sensitivity in the untrained group was almost twice as high as in the trained one. This implies a better regulatory response to disturbing interference in the untrained. Under normal conditions the controlling capacity of both groups is sufficient to cope with body position changes. However, during stress situations--in this case, reduction of aldosterone concentration as well as losses of plasma volume due to the 'Gauer-Henry-Reflex'--blood pressure regulation first fails in the trained subjects.


Use of an elastic garment in the treatment of orthostatic hypotension.

The most common cause of orthostatic hypotension is poor postural adjustment. The most severe form of orthostatic hypotension is of unknown etiology; the primary disorder is a degeneration of the peripheral autonomic nervous system, resulting in a decrease in production of catecholamine. Physiologically, there is a failure of arteriolar and venous vasoconstriction and a lack of cardiac response, both in rate and in contractility to sympathetic stimulation. Total and central cardiopulmonary blood volumes are contracted. The application of hydrostatic counterpressure by the use of an elasticized garment is helpful in the management of orthostatic hypotension, especially when used with sodium-retaining steroids to increase intravascular volume.


Pharmacological basis of the treatment of orthostatic disorders with ergot alkaloids.

Ergot alkaloids increase the tone of isolated canine vein strips dose-dependently in concentrations considerably lower than noradrenaline, but the maximal responses are only about one third of those to noradrenaline. This action can be blocked by prior administration of phentolamine. It is therefore concluded that the long-lasting stimulant action of ergot alkaloids on vascular smooth muscle is mediated mainly by alpha-adrenoceptors. Using the autoperfused hind limb of the cat, it has been shown that dihydroergotamine increases dose-dependently the tone of the capacitance vessels in a manner very similar to electrical stimulation of the sympathetic nerve with increasing frequencies. However, in contrast to sympathetic nerve stimulation, dihydroergotamine elicits only a very weak increase in arteriolar resistance.


[Effect of ergot alkaloids on venous tonus determined in superficial hand veins in situ].

The venoconstrictor effect of dihydroergotamine in man was demonstrated by Rieckert and Pauschinger in 1967. In a study on the influence of local infusions of dihydroergotamine into superficial hand veins in man we found a direct venoconstrictor effect of the drug. In further studies the effects of dihydroergotamine were compared with those of other dihydrogenated and non-dihydrogenated ergot alkaloids and a dose-dependent venoconstrictor effect was observed after local infusion of dihydroergotamine, dihydroergostine, dihydroergovaline, ergotamine and methysergide into superficial hand veins in man. Dihydroergotamine was weight for weight somewhat less active than ergotamine. But as Mellander and Nordenfelt have shown that dihydroergotamine elicits only a weak effect on resistance vessels, this drug seems for the treatment of orthostatic hypotension to be preferable to ergotamine for which Owen and Stürmer have demonstrated a marked constriction of resistance vessels in the skin. Experiment with dihydroergotamine showed that the venoconstriction in these human veins is mainly due to alpha-adrenoceptor stimulation. As in the treatment of orthostatic hypotension drugs are usually given in oral form, the activity of dihydroergotamine was also investigated after oral administration and the venoconstrictor activity confirmed.


Circulatory effects of dihydroergotamine in patients with disturbed sympathetic vasomotor control with special reference to postural hypotension.

In five patients with postural hypotension (disturbed sympathetic vasomotor control) the effect of intravenous and oral administration of dihydroergotamin (DHE) has been studied. The therapeutic effect of oral administration of DHE has also been compared to the effect of treatment with an antigravity suit. Following intravenous administration of DHE, systemic and central venous pressure increased in all cases. The effect on cardiac output was negligible and consequently the calculated total peripheral vascular resistance increased. Orthostatic tolerance increased in all cases. During long-term oral administration the effect was comparable to that obtained with an antigravity suit. The increased orthostatic tolerance is explained by a constrictive effect on both capacitance and resistance vessels. In order to analyze the influence of a functional denervation on the effect of DHE on peripheral circulation, a study has also been performed during epidural anaesthesia of the lower body in 11 patients. In the legs which were deprived of sympathetic tone intravenous administration of DHE caused constriction of resistance vessels. In the intact forearm there was a decrease of tone in the resistance vessels. The results indicate that DHE - apart from its well-known venoconstrictive effect - also may constrict resistance vessels when they are subjected to a low sympathetic nervous outflow.


Neurogenic influences on blood pressure and vascular tone from peripheral receptors during muscular contraction.

In anesthetized dogs, contraction of the muscles of the thigh was elicited either by applying electrodes in the muscles or by stimulation of the spinal ventral roots. The carotid sinuses were isolated; the vagi and the depressor nerves cut. Rhythmic muscular contraction caused hypotension and vasodilatation; tetanic contraction caused hypertension and vasoconstriction. These responses are of reflexogenic origin: the afferent pathway is in the somatic nerves, the efferent pathway in the sympathetic nerves.


[Physiology of orthostatic regulation].

The arterial blood pressure is integrated into a feedback control system and practically depends on three parameters, i. e. blood volume, heart dynamics, and peripheral resistance. When circulation is disturbed in the orthostatic, the correcting elements heart and vessels have to adapt themselves according to this demand. By standing up, the hydrostatic pressure in the arterial vessels increases rapidly in the lower body regions. The initial increase of the hydrostatic pressure will be stopped by the valves in the venous system. By means of a higher arteriovenous pressure gradient the blood flow into the lower body regions increases more than 100%. The blood flow fills the venous system within 1-2 min successively from downward to upward and opens the valves. The venous pressure in the feet can rise to the full hydrostatic value of 100 mm Hg. This change of pressure causes a volume increase of approximately 200 cm3 regarding both legs of healthy persons. In this way the circulatory blood quantity is reduced. The heart is able to put out shortly an increased cardiac output by loading off the central blood volume (lung and end-systolic volume), in order to compensate orthostatic demand. After a long upright position the cardiac output is falling under the starting value and heart frequency increases. By sympathetic stimulation the peripheral resistance increases as well.


[Orthostatic immediate regulation].

A series of well-known orthostatic tests were performed in order to substantiate complaints of patients in respect to blood volume drawn into both legs as an early orthostatic regulation. It could be shown that the blood volume pooled in the legs changed highly from test to test up to 250%. There was evidence that with increasing stagnating blood volume, there was not only an increase of arterial blood influx to the lower extremities, but a maximum of heart rate as well as reaction to the orthostatic stress. Under standardized conditions, 14 orthostatically unstable subjects have been tested. 57% of them collapsed in the course of the tests. Following a 14-day administration of dihydroergotamine, the percentage of collapsing subjects was reduced to 7%. At the same time there was a decrease in the following simultaneously measured circulatory parameters as heart rate, mean arterial pressure and heart volume, especially in cases where these changes of parameters were significantly high at the beginning of the experiment. Suggestions are given when it is necessary to detect disturbance of early orthostatic regulation in patients subjected to orthostatic tests. The importance of the dicroty quotient of the arterial pulse as a diagnostic criterion is emphasized.


Autonomic influences and cardiac conduction in patients with sinus node disease.

18 patients with sinus bradycardia (8 SB) and tachycardia-bradycardia syndrome (10 TBS) were investigated using various vagan reflex manoeuvres, atrial pacing (AP), pharmacological tests and His bundle (HB) recordings, to assess autonomic influences, sinus node function and atrioventricular (AV) conduction. Three types of responses (R) were defined as normal (N), supernormal (SN), poor autonomic (PA). Poor reflex responses to vagal manoeuvres, performed in 12 patients, have no statistical significance. MxCSRT at AP suppression was measured in 13 patients: it was normal (less than or equal to 500 msec) in 8 and increased (from 680 to 4,200 msec) in 5 patients with appearance of junctional escapes. Moreover, AP suppression revealed a sinoatrial block (SAB) in 1 patient with normal MxCSRT. Isoprenaline, administered intravenously (3-6 mug) in 9 patients, showed 4 NR, 3 PAR und 2 SNR (unusual sinus tachycardia, 1; atrial fibrillation, 1). Atropine (0.5-1 mg), used in 15 patients, revealed 5 PAR and 1 SNR. Prostigmine (0.5 mg), used in 11 patients, induced 6 NR and 5 PAR. Lanatosid C (0.08 mg), administered in 16 patients, showed 10 NR, 5 PAR and 1 SNR. Finally, ajmaline (100 mg), given in 13 patients, revealed 5 junctional rhythms and one 2:1 SAB. HB recorded in 17 patients showed an increase of AH interval (greater than 140 msec) in 5 patients and a slight increase (60 msec) of HV interval in 2 patients. Three patients developed 2nd-degree AV block at paced rates greater than or equal to 110/min. Abnormal responses at AP suppression and reduced drug responses were noted in 5 patients suffering from Adam-Stokes attacks; a permanent pacemaker was inserted in 4 of these 5 patients. In conclusion, provocative tests, in 18 patients with SB or TBS, suggest a diffuse process disease involving the autonomic function of both sinus node and AV junction (11 patients) and the AV system (6 patients). These immediate results must be correlated with histological findings in the conduction system, as shown in one of our cases previously reported.


Cell cycle kinetics in an in vitro tumor model.

Cell cycle kinetic parameters of multicell spheroids in vitro have been estimated using thymidine labeling techniques and autoradiography. Both the mitotic index and thymidine labeling index decreased in larger spheroids, whereas the duration of the cell cycle, as determined by two independent methods utilizing labeled mitoses or labeled cells, was essentially independent of spheroid size or age. These results suggest that the tumor-like growth exhibited by spheroids is the result of a decreasing growth fraction and a large apparent cell loss, rather than a general elongation of the cell cycle.


Inner enamel epithelium cell production rates.

Crowning of all four rat incisors slows down their eruption rate, leading to a diminution of cell production by the inner enamel epithelium (IEE). The production rate by 100 progenitor cells is dominated as production rate %. The normal IEE in rats weighing 200 g produces daily 67 cells %. In the crowned incisor this rate drops to 15 cells % while during accelerated eruption rate and IEE cell production rate remains unchanged and an eruption of 6-4 mum reflects a production of 1 cell % by the IEE. A method which relates labelled cell displacement along the IEE with its cell production rate is described and its applicability to other cell renewal systems discussed.


Kinetics of phytohemagglutinin stimulated lymphocytes in chronic lymphocytic leukemia.

The DNA synthesis pattern and several kinetic parameters of in vitro PHA stimulated normal and CLL lymphocytes were determined. The DNA synthesis peak of CLL lymphocytes occurred 2-3 days later than that of normal lymphocytes. The generation time, estimated by the labeled mitoses method, was found to be 28 hr and 20 hr for CLL and normal lymphocytes respectively. This difference was mainly due to longer S and G1 periods. It was also shown that both CLL and normal lymphocytes divide several times. These data were confirmed by the chromatid labeling pattern and by the halving of the grains and the double labeling techniques. By combining continuous and pulse labeling the growth fraction of CLL lymphocytes was found to be progressively increasing, because of the recruitment of new cells in cycle, from the third day of culture. Therefore the delayed peak of DNA synthesis of CLL lymphocytes was caused by a longer cell cycle and by a longer pre-replicative phase.


Effect of various doses of estrogen to BALB/cCrgl neonatal female mice on mammary growth and branching at 5 weeks of age.

High doses of 17beta-estradiol given newborn mice the first 5 days of life had no effect on mammary growth at 3 weeks of age whereas glands measured at 5 weeks of age displayed marked dose-dependent differences in area and branching. The number of duct branches increased significantly as the estrogen dose was raised from the 25 mug threshold to 35 or to 70 mug. Ratios between area and branching remained constant under all experimental conditions. Regional, dose-dependent, differences in gland-pair response were also observed at 5 weeks. Mammary gland pairs differed significantly in several growth parameters. The third gland pair was smaller in area and had fewer branches, but the opposite was true of the second and fourth gland pairs. Systemic injection of high doses of 17beta-estradiol into neonates reduced differences in growth parameters between gland pairs.


Extracellular stimulation of epidermal DNA synthesis.

The removal of the topmost cell layers of the epidermal stratum corneum by stripping initiates a series of biochemical events which alters the normal homeostatic control of and results in the acceleration of the cell cycle in basal cells which are ten to twenty cell layers removed from the site of stripping. One measure of accelerated events is a stimulation of thymidine incorporation into epidermal DNA at time intervals following stripping. Two peaks of maximal stimulation occur between 12 and 24 hr and 48 and 54 hr after stripping. Stimulation of thymidine incorporation into epidermal DNA by limited stripping is a useful technique for studying the stripping-initiated signal at the stratum corneum and its subsequent translation at the proliferative cell receptor site.


Proliferation rate of haemopoietic stem cells after damage by several cytostatic agents.

The haemopoietic tissue of mice was damaged by different cell-cycle-stage specific and cell-cycle-stage non-specific cytostatic agents. The proliferation rate among the surviving pluripotential stem cells, i.e. those cells forming colonies in spleens of lethally irradiated mice (CFUs), was then investigated. The results suggest that, at least in the CFUs population, the cells which synthesize DNA in the S phase of the cell cycle inhibit the entry of the non-proliferating GO cells into cell cycle. This evidence was based on the ability of three cytostatic agents, hydroxyurea, cytosine arabinoside and methotrexate, which are toxic specifically to the S phase cells to increase the proliferation in the CFUs population. This increase was quite out of proportion to the small amount of damage they caused to the population. Colchicine, which kills cells in mitosis, and ionizing irradiation, damaging cells in all stages, proved to be much weaker stimulators of proliferation. It has been suggested that a mechanism for the control of cellular proliferation might be based on the negative feedback in the cell cycle. In this feedback control loop the cells which are preparing for cell division in the S phase of the cell cycle inhibit the entry of the non-proliferating GO cells into cell cycle.


Autoradiographic study of type II-cell hyperplasia in lungs of mice chronicall exposed to urethane.

Hyperplasia of pulmonary alveolar epithelium was induced by exposure of adult mice to 0-1% urethane in drinking water, and their lungs were analysed using combined autoradiographic and morphometric methods. The response of pulmonary epithelium showed three phases: an initial one of 3 weeks in which the number of type II cells was consistently low and, after a transient decrease in 3H-thymidine labeling index, the latter steadily increased. Degenerating or dead type II cells were found, consequently this period was considered one in which cell death was disproportionately increased over cell production. Between the 3rd and 6th weeks the number of type II cells doubled, and thereafter increased at a slower rate. The 3H-thymidine labeling index reached its peak at 6 weeks. The third phase was marked by a decline in labeling index which returned to near-normal levels by the 16th week. The number of type II cells declined slowly after the 10th week and was still elevated at the end of the observation period. Tumors consisting of type II cells continued to arise even during the period of declining labeling index. The dissociation between the proliferative response which was reversible, and the neoplastic response which was not, supports the assumption that the major part of the observed population growth or hyperplasia induced by urethane was a form of regeneration repair secondary to cell death and may be unrelated to tumor growth.


On the intrinsic innervation of normal rat liver. Histochemical and scanning electron microscopical studies.

With the Bodian method stained fibers were observed in the lobules of the rat liver and with the modified Karnovsky and Roots thiocholine method cholinesterase (presumably acetylcholinesterase (AChE))-positive nerve fibers were found in a pattern similar to that of the Bodian-stained fibers. The AChE-positive nerve fibers form a network in the liver lobules in close relation to hepatocytes and sinusoids. Fluorescent varicose nerve fibers demonstrated by the glyoxylic acid and Falck-Hillarp fluorescence methods were found only in the interlobular spaces associated with vessels. As no overlapping of distribution patterns of AChE-positive nerve fibers and fluorescent nerve fibers occurs, the AChE activity of the nerves of the liver lobules probably reflects the associated presence of acetylcholine in the nerve fibers. In consequence we suggest that nerves of the liver lobules belong to the autonomic parasympathetic nervous system. SEM of liver tissue revealed light cords apparently situated in smooth-surfaced channels between adjacent hepatocytes and in the space of Disse, where fibers also cross sinusoids. We tentatively suggest that the cords of the SEM represent the AChE-positive nerve fibers of our LM observations.


Localisation of proteins in coated micropinocytotic vesicles during transport across rabbit yolk sac endoderm.

Rabbit yolk sac splanchnopleur exposed in utero to IgG-HRP and IgG-ferritin conjugates, rabbit and bovine anti-HRP antibodies, free HRP, ferritin and human IgG, was examined ultrastructurally in an attempt to determine whether or not coated micropinocytotic vesicles are involved in selectively transporting immunoglobulins across yolk sac endodermal cells. Human, rabbit and bovine IgG-HRP conjugates, rabbit anti-HRP antibodies, free HRP and human IgG, all become localised in coated micropinocytotic vesicles. Differences were observed in that only human IgG and rabbit anti-HRP antibodies could be located in the intercellular space and bovine IgG-HRP conjugate could not be detected in coated micropinocytotic vesicles in confluence with the lateral and basal plasmalemma. Bovine anti-HRP anti-bodies, IgG-ferritin conjugates, and free ferritin, could not be observed in coated micropinocytotic vesicles. All proteins were detected in macropinocytotic vesicles, and dense bodies resembling phagolysosomes. Results are discussed in the light of a proposal that selection occurs at the cell surface during formation of coated micropinocytotic vesicles and is not linked to intracellular proteolysis.


Ultrastructure of the spermatozoa and spermiogenesis in New Zealand freshwater eels (Anguillidae).

The spermiogenesis and sperm ultrastructure of Anguilla australis schmidtii and A. dieffenbachii is described. During spermiogenesis the chromatin material condenses into the crescent-shaped head of the mature sperm. By elongation of the sperm head the mitochondrion is displaced to the anterior of the head remote from the posterior centriolar apparatus. A ciliary rootlet becomes segregated during spermiogenesis to form a membrane covered, striated, rod-like appendage. From the proximal centriole arise two sets of filaments, each containing three subfibrils. One group of five filaments extends anteriorly along the inner concave side of the head. A group of four modified filaments pass along the outer, convex, side. The middle subfibril B of each triplet in the group of four filaments is flattened and joins the two subfibrils A and B, forming a binocular shaped structure. The motile flagellum has a 9+0 axonemal pattern.


Structure and regeneration of the eyes of strombid gastropods.

The tips of the eyestalks of three species of strombid gastropods were amputated and the structure of the fully developed eye in investigated. The retina contains at least two types of cell: sensory cells bearing long tufts of microvilli with a central cytoplasmic core, and pigment cells with short microvilli. New eyes became visible at the tips of the eyestalk stump 5-16 days after amputation. When the regenerated eyes first appear, they consist of hollow balls of cells with a pigment lined cavity; two types of retinal cells are already distinguishable but their microvilli and cilia are small and sparse. The microvillous tufts and sensory cell contents develop quickly and about 14 days after their first appearance, the eye is a fully formed but miniature organ.


Freeze-etching observations on the characteristic arrangement of intramembranous particles in the apical plasma membrane of the thyroid follicular cell in TSH-treated mice.

Thyroid glands of normal, TSH-treated and Thyradin (powdered thyroid)-treated mice were examined by means of the freeze-etching method. Intramembranous particles on the PF (= A face) face of the apical plasma membrane often form aggregates especially in TSH-treated mice. Each aggregate, about 200 nm in diameter, and consisting of 15-25 large particles, corresponds to a depression of the apical cytoplasm, and the particles sometimes form rosettes. Particle-aggregates are very rare in the apical plasma membrane of the thyroid follicular cell of the Thyradin-treated animal. In the cytoplasm just beneath the particle-aggregate no secretory granules, reabsorbed colloid droplets or other special structures are found. From these facts, the aggregate is considered closely related to an initial site for the micropinocytosis of the luminal colloid.


Gap junctions in hemodichorial and hemotrichorial placentae.

Gap junctions were found to be a constant feature of chorioallantoic placentae with two or three trophoblastic layers. The gap junctions connect layers I and II in hemodichorial and layers II and III in hemotrichorial placentae. Although the gap junctions vary in form and in the packing density of membrane-associated particles, they cover an extensive surface area in all species examined. The gap junctions always connect adjacent membranes of two trophoblastic layers, which show no evidence of micropinocytotic activity; at least one of these trophoblastic layers is syncytial. It is therefore concluded that the gap junctions play an important role in diaplacental transport. We consider that gap junctions act as molecular sieves, resulting in limitations in the transport of large molecules. The passage of small molecules, on the contrary, would be facilitated by the gap junctions.


Structure and function of prothoracic glands and oenocytes in embryos and last larval instars of Oncopeltus fasciatus Dallas (Insecta, Heteroptera).

1. Active prothoracic glands and oenocytes of last larval stage are both characteristized by well-developed smooth and rough endoplasmic reticulum (ER). Prothoracic glands also show plasma membrane infoldings, but not oenocytes which contain a large number of pleomorphic vesicles. 2. The fine structure of embryonic oenocytes corresponds after blastokinesis with that of active larval and adult cells. Thus, an activity in the late embryo can be assumed. Embryonic prothoracic glands reveal no signs of activity: smooth and rough ER are absent. The subcellular structure resembles that of organ anlagen, i.e. not yet fully differentiated tissue. Hormone synthesis is not likely. 3. Ecdysone titer was determined throughout embryonic development and in mature adults. Although prothoracic glands break down during adult ecdysis, imagines contain in the Calliphora-bioassay active factors: females 0.9 CU/g and males 0.5 CU/g. As sites of synthesis the oenocytes are suggested. 4. A relatively high ecdysone titer of 7 CU/g is measured in newly deposited eggs. The hormone is presumably of maternal origin. Subsequent to blastokinesis the hormone content increases dramatically up to about 180 CU/g, apparently due to endocrine function of the embryo. Oenocytes are proposed as the source of ecdysone during late embryonic development. 5. The function of ecdysone during early and advanced embryogenesis, especially in view of "embryonic molts", is discussed.


The first optic ganglion of the bee. II. Topographical relationships of the monopolar cells within and between cartridges.

The arrangement of first and second order neurons in an optic cartridge and the topographical relationships of the second order neurons within a cartridge and to groups of surrounding cartridges have been analyzed in the visual system of the bee, Apis mellifera, from light and electron microscope studies on Golgi preparations. At the level of the monopolar cell body layer, the nine retinula cell fibres of each ommatidium, the six short visual fibres arranged in a circle surrounding the three long visual fibres, become cartridges as a consequence of the appearance of the second order neurons (L-fibres) which join the R-fibre bundles. Two of the four different L-fibre types, L-1 and L-2, remain together in the centre of the cartridge throughout the lamina. The axons of the L-3 and L-4 fibres, however, have their position integrated into the circle formed by the endings of the short visual fibres. On the basis of further examination of light and especially electron microscopical Golgi material, the different L-fibres can be classified into four types which appear in each cartridge. The clear stratification in the first synaptic region (A, B and C) seems to be the best criterion for a morphological classification since such a classification necessarily also includes a functional basis. According to a naming system based on the position of the lateral processes, L-fibres with side branches in strata A, B and C are called L-1 fibres. Fibres with lateral processes in strata A and B are L-2 fibres; monopolar cell fibres with branches only in the second stratum B are L-fibres of type 3; and all monopolar cells with branches only in stratum C are called L-4 fibres. In addition to the branching pattern covering only the parent cartridge, two of the four fibre types (L-2 and L-4) have long collaterals reaching neighbouring cartridges: L-2 in stratum A and L-4 in straum C. These collaterals presumably form a substrate for lateral interactions.


Fine structure of the corpus luteum of the autografted ovary in the rat.

The rat ovary has been transplanted successfully to subcutaneous tissue areas by several investigators. Light microscopy has revealed that corpora lutea in ovarian autografts are formed by luteinization of intact follicles and contain entrapped ova. In the present study, corpora lutea from autografted ovaries in castrate rats were obtained at metestrus and examined electron microscopically to determine whether their cellular morphology correlated with the normal progesterone levels in these animals. Cellular features usually accepted as regressive were apparent. The findings suggest either structural luteolysis is occurring before functional luteolysis or that the adrenal has increased steroidogenic activity in the castrate with ovarian autografts to account for the normal progesterone levels.


Fine structure of nerve-melanophore contacts in the angelfish Pterophyllum scalare.

Contacts between small unmyelinated nerve fibres and dermal melanophores of the angelfish, Pterophyllum scalare, exhibit several features characteristic of synapses, including small synpatic vesicles and dense core vesicles, a narrow synaptic cleft, electron-dense material at the postsynaptic membrane (cell membrane of the melanophore) and, occasionally, presynaptic densities. An analysis of serial thin sections shows that the synapses described here represent varicosities of an otherwise more or less straight nerve fibre. A single axon thereby may form several en passant synapses with a single melanophore. It is suggested that the synaptic contacts described here not only represent sites of transmitter release but also play a role as sites of firm attachment between nerves and melanophores which guarantee a stable arrangement of nerve fibres and melanophores.


Shell attachment in the pond snail Lymnaea stagnalis (L.)

The attachment of the body of the snail Lymnaea stagnalis to the shell was studied by histochemistry and light and electron microscopy. Muscles of the body wall insert into the connective tissue by way of long thin projections of sarcolemma. The muscle cells end under the basement membrane of a specialised area of the epidermis, the adhesive epithelium. The cells of this epithelium are filled with microfilaments and possess characteristic knob-like microvilli. The epithelium is attached to the sheel by way of an adhesive substance containing proteins and mucopolysaccharides.


Direct innervation of the mantle edge gland by neurosecretory axons in Helisoma duryi (Mollusca: Pulmonata).

The mantle edge gland of Helisoma duryi is innervated by neurosecretory axons from the pallial nerves. Synaptoid contacts occur between axons and gland cells, and there is ultrastructural evidence for the release of neurosecretory material. The mantle edge gland contributes to the deposition of periostracum during shell formation, and direct neurosecretory innervation may control shell growth and regeneration.


Myelinated synapse-bearing cell bodies in the central nervous system of Scyliorhinus canicula (L.)

Some neurons of the anterior lateral-line lobe of dogfish (Scyliorhinus) have synapse-bearing perikarya enclosed by layers of compact myelin. The identity of these cells, which have myelinated axons and unmyelinated, synapse-bearing dendrites, is unknown.


Effect of cholecystokinin-pancreozymin (CCK-PZ) on glycoprotein secretion from mouse gallbladder epithelium: an ultrastructural and cytochemical study.

Structural changes in the gallbladder epithelial cells of the mouse were studied following in vivo and in vitro stimulation of the gallbladder with the gastrointestinal hormone cholecystokinin-pancreozymin (CCK-PZ). Signs of increased secretory activity were observed within the first 2-3 min after hormone administration. At the ultrastructural level, best visualized with the PA-CrA-silver technique, granule discharge was observed, as was an overall increase in size of the granules. After prolonged in vitro incubation or repeated in vivo stimulation, there was an almost total depletion of secretory granules. This phenomenon is accompanied by an enhanced uptake of extracellular thorium dioxide by endocytotic vesicles at the apical cell surface. An exocytosis-endocytosis coupling mechanism may be important for membrane conservation in the gallbladder epithelial cells. The findings establish that the hormone CCK-PZ stimulates the secretion of glycoproteins from the mouse gallbladder epithelium.


Thyroid gland in dwarf mice: a stereological study.

Stereological methods were used to compare thyroids of dwarf mice and of their heterozygote littermates. In the thyroid of dwarf mice unorganized cellular masses, adipous tissue and ultimobranchial cysts are abundant. Follicles are small and their distribution function is unimodal. The number of cells per follicle is considerably lowered if compared with the normal. In control mice the distribution function of thyroid follicles is bimodal. These data show that origin of the thyroid anomaly in dwarf mice is due to a drastic diminution of cell divisions, probably resulting from the lack of growth hormone.


Estrogen dependent ciliogenesis in the chick oviduct.

Both the hormone dependency and the morphological details of estrogen-dependent ciliogenesis in the shell gland of the chick oviduct were investigated. Ciliogenesis was initiated on day 3 of estrogen treatment, and progressively more cells became differentiated until, on day 10, approximately 55% ciliation occurred with 17 beta-estradiol (1 mg/day) and approximately 75% ciliation occurred with diethylstilbestrol (1 mg/day). Simultaneous administration of progesterone with diethylstilbestrol (1 mg each/day for 10 days) caused a 50% depression in the number of ciliated cells on day 10. The rate of ciliogenesis was found to be affected by progesterone and the type of estrogen administered. The minimum stimulatory dose of estradiol was found to be between 0.01 mg/day and 0.05 mg/day. Ciliogenic cells were first recognized by the appearance of pro-basal bodies in the apical portion of the cell. Pro-basal body maturation and cilium formation were the same as those described for the chick trachea. Ciliogenesis in the chick was found to be homologous to estrogen-dependent ciliogenesis in various mammalian oviducts.


The subacrosomal granule and its evolution during spermiogenesis in a lizard. Observations about the acrosomal fringe and the spermatid-sertoli cell relationship.

Some aspects of spermiogenesis have been studied in the testis of the teiid lizard Cnemidophorus lemniscatus lemniscatus by electron microscopy. Shortly after the acrosomal vesicle is lodged in a nuclear concavity of the spermatid, a dense granule differentiates in the center of the subacrosomal space. It is cone-shaped and shows a longitudinal striation. Its base applies to the acrosomal membrane and, through this, to the acrosomal granule. Its rounded vertex causes a depression of the nuclear membranes which, initially juxtaposed, separates at this point to form a vesicle. The granule develops and becomes a rod when spermiogenesis is advanced and the subacrosomal space has taken the form of a secondary cap. The rod is cylindrical, retains its original striation and has a convex acrosomal end. It encloses the vesicle formed by the nuclear envelope in its base and follows the apex of the nucleus. Meanwhile, the acrosomal granule loses its identity and the acrosomal cap is filled with a dense substance, in which a fringe of translucent material differentiates. This fringe lies in the dorsal and apical margins of the acrosome and is incompletely divided by longitudinal crests of the dense acrosomal substance. A projection of the Sertoli cell forms an accessory cap which envelops the acrosome and is in turn covered by the cytoplasm of the spermatid, constituting an intricate association. Two reflex membranes underlie the plasmalemma in the outer surface of the projection of the Sertoli cell. They are continuous with one another at their ends and with the cell membrane in the edge of pores. In the peripheral cytoplasm of the spermatid facing the accessory cap, numerous microtubules run longitudinally. By means of thin membranes some are interconnected or connected with the plasmalemma, from which they seem to originate.


Differences in black pigmentation in lepidopteran cuticles as revealed by light and electron microscopy.

Black cuticles of larvae and pupae from various Lepidoptera were studied by light and electron microscopy. There are striking differences in the representation of black pigmentation, especially at the ultrastructural level. Two types may be described: 1. With the light microscope black melanin-like grana, electron-dense electron microscopically, are found in the distal parts of the exocuticle. This type is demonstrated in larvae of Celerio euphorbiae, Papilio machaon, and Phalera bucephala. 2. With the light microscope, a dark homogeneous layer in the distal exocuticle can be recognized, however, electron microscopically no structures correlated with this dark pigment layer. This type of pigmentation was present in pupae of Pieris brassicae and Aglais urticae; in Pieris larvae the dark pigmented layer appeared to be limited to the epicuticle. In Celerio processes of the epidermal cells are involved in transporting precursors to the exocuticle. The conclusion was reached that black pigmentation in cuticles is based on different mechanisms as proposed by structural features. The two likely mechanisms are melanization and sclerotization.


Fine structure of the ocellus of the cabbage looper moth (Trichoplusia ni).

The dorsal ocelli of adult cabbage looper moths were studied by light microscopy, and scanning and transmission electron microscopy. Each ocellus has a cuticular lens located on the distal end of a cuticular cone which encapsulates the receptor cells. There are two distinct types of receptor cells in the ocellus. Seventy large receptor cells from plate-like rhabdoms with several adjacent cells to produce a rhabdom network in the ocellus. Proximally ninety small receptor cells which have a disorganized microvillar rhabdomere are located at the base of the rhabdoms formed by the large cells. Extensive areas of gap junctions which occur between the rhabdoms and the membranes of large and small cells suggest that the cells may be electrically coupled to one another. Axons from both large and small receptor cells leave the base of the ocellus and extend to the protocerebrum to synapse with second order neurons.


Manganese in the shell of Centropyxis (Rhizopodea: Protozoa).

The proteinaceous shells of Centropyxis hirsuta contain a relatively high concentration of manganese in an amorphous state. The concentrations of manganese fill the alveoli which are characteristic of the shell structure. Observations based on cultured animals and subsequently examined by X-ray diffraction, a solid state energy dispersive X-ray analyser, and an analytical electron microscope, lead to the conclusions that manganese is selectively absorbed by Centropyxis hirsuta and deposited in the shell.


Characterization of the 5' termini of hn RNA in mouse L cells: implications for processing and cap formation.

An analysis of the phosphorylated and capped 5' termini of the heterogenous nuclear RNA of mouse L cells has revealed four types of structure: pppXp..., ppXp..., pXp..., and m7/GpppXmp.... The 5'triphosphate termini consists exclusively of pppGp... and pppAp..., whereas a large proportion of the 5' monophosphate termini are pUp.... The 5'diphosphate termini contain all four species of nucleotide in relative proportions that are roughly similar to those found at the Xm position of cap structures. These results indicate that initiation of hnRNA transcription occurre exclusively with purine nucleotides, and consequently that the hnRNA molecules containing pyrimidines at the 5' termini very probably arise by cleavages at internal sites of larger primary transcripts. Taken together with previous results relating cap structures of hnRNA and mRNA, the data favor a model in which some mRNA sequences are located at transcriptionally initiated proportions and others in internal regions of their precursors. According to this model, both the mRNA segments derived from initial 5' end, and those derived by cleavage at internal sites could be converted to diphosphate-terminated derivatives, which then condense with GTP to form cap structures according to the mechanism previously described for vaccinia and reovirus mRNA.


Comparative organization of active transcription units in Oncopeltus fasciatus.

We have analyzed electron micrographs of chromatin-associated fiber arrays from embryos of the milkweed bug, Oncopeltus fasciatus. The analysis has revealed that the arrays have highly ordered patterns of fiber spacings and lengths. These patterns support the interpretation that the fibers are nascent RNA with associated proteins (RNP fibers) which have resulted from transcription of the DNA in the underlying chromatin segment. In particular, the patterns indicate that the chromatin underlying each array is delimited by specific sites for initiation and termination of transcription. We apply the term transcription unit to a chromatin segment thus bounded. The analysis has further revealed that transcription units can be grouped into two principal classes--ribosomal and nonribosomal. Active transcription units of these two classes differ in DNA content, in their proximity to other active transcription units, and in their chromatin morphology. For certain developmental stages, fiber frequencies (that is, the nubmers of fibers per mum of chromatin) are also useful in distinguishing ribosomal from nonribosomal arrays. The most definitive of the above classification criteria is chromatin morphology as observed under our preparative conditions. We propose that term rho chromatin for the unbeaded or smooth chromatin that underlies nascent ribosomal RNP fibers. DNA in rho chromatin has a calculated packing ratio of approximately 1.2 mum of B structure DNA per mum of chromatin. Nu chromatin is used to designate the beaded chromatin for which we calculate a DNA packing ratio of 1.6-2.3 in our preparations. This calculation for nu chromatin is based on the inference that the beads are nucleosomes (nu bodies, PS particles, unit particles). The beaded morphology is observed between fibers of nonribosomal transcription unit as well as for most fiber-free chromatin. The detection of specific sites of transcriptional initiation and termination and the classification of transcription units can provide a basis for further analysis of transcriptional control.


Induction of microtubule protein synthesis in Chlamydomonas reinhardi during flagellar regeneration.

Flagellar regeneration in gametes of Chlamydomonas reinhardi is initiated within 15-20 min after flagellar amputation and proceeds at a rapid but decelerating rate until by 90 min flagellar outgrowth in 80-85% complete. Sufficient flagellar protein reserves exist in the cytoplasm to allow regeneration of flagella 1/3-1/2 normal length. Nevertheless, in vivo labeling with 14C-amino acids shows that microtubule protein and other flagellar proteins are synthesized de novo during flagellar regeneration. To determine whether tubulin is synthesized continuously by gametic cells or whether its synthesis is induced as a consequence of deflagellation, we have isolated polyribosomes from deflagellated and control cells, and analyzed the proteins produced by these polyribosomes during in vitro translation. Two proteins of 53,000 and 56,000 molecular weight which co-migrate with flagellar and chick brain tubulin on SDS-polyacrylamide gels and which selectively co-assemble with chick brain tubulin during in vitro microtubule assembly are synthesized by polyribosomes (or polyadenylated mRNA) from deflagellated cells. No microtubule proteins can be detected in the translation products synthesized by polyribosomes (or mRNA) from control cells, clearly indicating that deflagellation results in the induction ot tubulin synthesis. Kinetics of tubulin synthesis demonstrate that induction takes place immediately after deflagellation; polyribosomes bearing tubulin mRNA can be detected in the cytoplasm in as little as 15 min after removal of flagella. Maximal rates of tubulin synthesis occur between 45 and 90 min after deflagellation when approximately 14% of the protein being synthesized by the cell is tubulin. This estimate of tubulin synthesis based on in vitro translation data agrees well with in vivo measurements of flagellar tubulin synthesis. While high levels of tubulin production extend well beyond the period of rapid flagellar assembly, synthesis begins to decline after 90 min, and by 180 min after deflagellation only low levels of tubulin mRNA are detectable in polyribosomes.


Cross-linking of a major fibroblast surface-associated glycoprotein (fibronectin) catalyzed by blood coagulation factor XIII.

The surface proteins of cultured human skin fibroblasts were iodinated and then exposed to one or more of the following blood coagulation proteins: thrombin, fibrinogen, and factor XIII (plasma protransglutaminase). Radiolabeled polypeptides were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. After exposure to physiological concentrations of activated factor XIII (XIIIa), the band of radioactivity corresponding to the major labeled surface protein (fibronectin, molecular weight = 2.2 x 10(5) daltons) was cross-linked to a very high molecular weight complex. The cross-linking reaction was inhibited by fibrin (which is known to bind the catalytic subunit of XIIIa). Cross-linking of labeled cell surface fibronectin to fibrin could not be demonstrated. The fibrillar pattern of surface fibronectin appeared unaffected by cross-linking when studied by immunofluorescence. Cross-linking of cell surface fibronectin by XIIIa requires highly specific enzyme-substrate and protein-protein interactions, and may be an important physiological reaction.


Studies on the activity of the X chromosomes in female teratocarcinoma cells in culture.

Embryonal carcinoma cells derived from murine teratocarcinomas are able to differentiate into the same variety of tissue types as early embryonic cells. Because embryonal carcinoma cells resemble those of the embryo at a stage before X chromosome inactivation has occurred in females embryonal carcinoma cells containing two X chromosomes were examined to determine whether both are genetically active. The specific activities of X-linked enzymes were measured in embryonal carcinoma cells containing either one or two X chromosomes. The activities in both cell types were similar, suggesting that only one X chromosome was active in the female cells. Further support for this conclusion came from experiments in which azaguanine-resistant mutants were recovered with similar frequencies from embryonal carcinoma cell lines containing one and two X chromosomes. Late replication of an X chromosome DNA was detected in one embryonal carcinoma cell line with two X chromosomes but not in another. This suggests that cells of these two lines were arrested at different developmental stages, and that late DNA replication may not be a necessary adjunct of X inactivation. Evidence is presented which suggests that X chromosome reactivation does not occur during differentiation of the cells in vitro.


Delta-beta-thalassemia is due to a gene deletion.

DNA has been prepared from peripheral blood or cultured skin fibroblasts obtained from three Sicilian and one Greed deltabeta-thalassemia homozygotes. Globin-gene analysis was carried out using a cDNAbeta probe, and the results indicate that deltabeta-thalassemia has arisen from a deletion of the beta-globin genes. A similar result was obtained using DNA prepared from cultured skin fibroblasts from an individual homozygous for the Negro form of hereditary persistence of fetal hemoglobin (HPFH). In both cases, the deletion has spared the Ggamma and Agamma loci directing the gamma chains of hemoglobin F, but it has not been possible to demonstrate any difference between the size of the deletion involved in the production of delta-beta-thalassemia and that which gave rise to HPFH. These experiments provide further direct evidence that deletions of critical areas of the gamma-delta-beta gene cluster result in persistent gamma chain synthesis in adult life.


An electron microscope study of the relative positions of the 4S and ribosomal RNA genes in HeLa cells mitochondrial DNA.

The 4S RNA genes in HeLa mitochondrial DNA (mtDNA) have been mapped by electron microscopy using the electron-opaque label ferritin. This method is based on the high affinity interaction between the protein, avidin,and biotin. 4S RNA, covalently coupled to biotin, was hybridized to single-stranded mtDNA. The hybrids were then labeled with ferritin-avidin conjugates. The positions of ferritin-labeled 4S RNA genes were determined relative to the rRNA genes on both heavy (H) and light (L) strands of mtDNA. This region was recognized as a duplex segment after hybridization either with rRNA in the case of H strands or with DNA complementary to rRNA in the case of L strands. Our studies suggest that at least nineteen 4S RNA genes are present in the HeLa mitochondrial genome. On the H strand, we have confirmed the nine map positions found in a previous electron microscope mapping study (Wu et al., 1972) and obtained evidence for three additional 4S RNA genes. On the L strand, seven 4S RNA genes have been mapped. The nineteen genes are distributed more or less uniformly around the genome. There is a pair of closely spaced genes, approximately 150 nucleotides apart, on the H strand, and another closely spaced pair on the L strand.


The isolation and preliminary characterization of somatic cell mutants resistant to the protein synthesis inhibitor-emetine.

Emetine reversibly inhibits protein synthesis in Chinese hamster ovary (CHO) cells. Stable mutants which are 20-80 fold more resistant to the cytotoxic action of the drug can be isolated in a  single step at a frequency of about 2-5 X 10(-7). The frequency of such mutants is increased 30-50 fold by ethyl methane sulphonate mutagenesis, and the spontaneous rate of mutation to emetine resistance as measured by Luria-Delbruck fluctuation analyses is 4.9 X 10(-7) mutations per locus per generation. Protein synthesis in extracts of the mutant cells is resistant to the inhibitory action of the emetine, indicating that the molecular lesion in these cells lies in the protein synthesis machinery.


Commitment to erythroid differentiation by friend erythroleukemia cells: a stochastic analysis.

A method for the clonal analysis of murine erythroleukemia cells has been developed which allows the precise characterization of the number of progeny produced by each cell and the degree of differentiation of each progeny cell. The potential of almost every cell in the culture can be monitored because a plating efficiency close to 100% has been achieved. The effects of treatment with an inducer of differentiation (DMSO) on the proliferative capacity of the treated cells have been studied with this technique. Cells from a mass culture treated with inducer give rise to colonies of differentiated progeny when subsequently cloned in the absence of inducer. Colonies exhibiting this phenotype represent the progeny of cells committed to the differentiation pathway by treatment with inducer. We observe that the commitment decision limits the subsequent proliferative capacity of the cell to four additional cell divisions. A quantitative analysis suggests that the commitment decision for each cell is made in a stochastic manner. Irreversible commitment to the expression of differentiated functions occurs with discrete probability per cell generation for many cell generations. The value for this probability is a function of the concentration of inducer (DMSO). A correlative biochemical study suggests that an irreversible commitment decision by a significant proportion of the population precedes or accompanies increases in cytoplasmic globin mRNA levels, one of the earliest detectable biochemical markers for erythroid differentiation in this system. A specific kinetic model based on these considerations has been developed to predict clonal phenotypes as a function of time and probability of commitment. Quantitative predictions based on this model are in excellent agreement with experimental observations. The effectiveness of a stochastic model in predicting the behavior of this system is discussed in relation to the stochastic behavior of normal hematopoiesis and the biochemical mechanisms which control these differentiation programs.


Plasminogen activator in early embryogenesis: enzyme production by trophoblast and parietal endoderm.

We have surveyed the early stages in the development and differentiation of cultured mouse embryos for plasminogen activator production. This enzyme is first detectable by the sixth equivalent gestation day. Thereafter, cultured blastocysts produce plasminogen activator with a biphasic time course: in the first phase, enzyme secretion rises to a maximum at about the eighth day and then decreases; a second phase, during which more enzyme accumulates, begins somewhat later and continues to at least the fifteenth day. By fractionating the blastocyst into its constituent cell types, we have identified the trophoblast as the cells responsible for the first phase of enzyme synthesis. The pattern of enzyme production by the trophoblast is closely correlated with the invasive period of these cells in vivo and implies that plasminogen activator is involved in embryo implantation. The second phase of plasminogen activator production is due to parietal endoderm, which initiates enzyme synthesis upon differentiation from the inner cell mass. The properties of the parietal endoderm suggest that plasminogen activator may participate in the migration of these cells and/or in the metabolism of Reichert's membrane which accompanies embryo growth. These results are consistent with the concept, deveolped from work on other cell types, that plasminogen activator may represent a generalized mechanism for tissue remodeling and cell migration.


Two complementary strand-specific termination sites for adenovirus DNA replication.

Adenovirus type 2 DNA, specifically labeled at the termini for DNA replication, was prepared by isolation of viral DNA molecules which were completed during short pulses with 3H-thymidine. The distribution of radioactivity in the two complementary strands at the termini for DNA replication was determined by liquid phase hybridization and gelelectrophoresis. At the right-hand terminus, nearly all radioactivity was found in the viral h strand, whereas at the left-hand terminus, most radioactivity was confined to the viral l strand. The results suggest that both molecular ends serve as origins and termini for replication of adenovirus type 2 DNA.


Mapping DNAase l-susceptible sites in nucleosomes labeled at the 5' ends.

We have used lambda-32P-ATP and polynucleotide kinase to label stoichiometrically the 5' ends of DNA in intact isolated HeLa cell nucleosomes. DNA is not nicked or degraded during the modification reaction. We have used these modified nucleosomes to study both the distribution and the relative availability of sites within the nucleosome which are susceptible to digestion by DNAase l. The results show that the nucleosome contains a potential cleavage site every 10 nucleotides, with the exception of the site 80 nucleotides from the 5' end. Favored cleavage sites are located 20, 40, 50, 100, 120, and 130 nucleotides from the 5' end; sites 30 and 110 nucleotides from the 5' end are strongly disfavored, while the potential site 80 nucleotides from the 5' end is virtually never cleaved. These findings provide constraints for models of histone-DNA interactions within the chromatin subunit.


Influence of lead nitrate on dimethylnitrosamine intoxication.

The effect of lead nitrate, an inhibitor of the hepatic drug-metabolizing enzyme system upon the acute, hepatotoxicity of dimethylnitrosamine (DMN) was studied. Lead pretreatment significantly prevented polysomal disaggregation induced by the nitrosamine. Cell necrosis, evaluated morphologically and by the release of serum glutamic-pyruvic transaminase (GPT), was also diminished. The metabolism of DMN in rats pretreated with lead nitrate was investigated by following its clearance from blood and by determining, in vitro the demethylation of the nitrosamine. Lead increased, although not significantly, the clearance of DMN from blood, but it lowered the activity of DMN-demethylase 24 h after its administration. Finally, lead lowered the lethal effects of DMN. The mechanism by which lead influenced DMN toxicity is discussed.


Effects of N-methyl-N-nitrosourethane on DNA synthesis in the guinea pig pancreas.

The effects of N-methyl-N-nitrosourethane (NMUT) on pancreatic DNA synthesis were investigated at sequential intervals following gavage of Hartley guinea pigs with a single dose of 30 mg/kg. There was a highly significant stimulation of DNA synthesis, as evidenced by increased incorporation of [3H] methyl-thymidine ([3H]TdR), throughout the whole pancreas and particularly in the duodenal segment, at 4 h following NMUT administration, thereafter, DNA synthesis declined sharply up to 24 h, and then recovered gradually to control levels from 24--96 h. DNA synthesis stimulated by NMUT was suppressed by hydroxyurea (HU), and hence is likely to represent replicative, rather than repair, synthesis.


Identification of the persistently bound form of the carcinogen N-acetyl-2-aminofluorene to rat liver DNA in vivo.

In this article the structural analysis of the persistently bound form of the carcinogen N-acetyl-2-aminofluorene (AAF) to rat liver DNA in vivo is described. This compound appears to result from the formation of a covalent bond between carbon-3 of the aromatic ring and the amino group of guanine. Experimental evidence from three different approaches had led to the identification of the structure of the persistently DNA-bound AAF moiety. First, [3-3H, 9-14C]N-acetoxy-AAF was reacted with DNA in vitro. As reported previously, a minor product was isolated from enzymatic digests of the reacted DNA, which had chemical and chromatographic properties identical to those of the persistent--AAF moiety in DNA in vivo. The ratio 3H/14C of this product had diminished to the same extent as 3-CH3S-AAF resulting from the reaction of methionine with [o-3H, 9-14C]N-acetoxy-AAF. Secondly, reaction of [9-14C]N-acetoxy-AAF with DNA, which was tritiated in the C-8 positions of the purines, did not result in removal of tritium in the persistent fraction obtained after acid hydrolysis, thus excluding substitution at C-8 and N-7 of guanine. Finally , by reacting N-OSO3-K-AAF with deoxyguanosine in dimethylsulfoxide-triethylamine, a compound could be isolated, which was identified as 3-(deoxyguanosin-N2-yl)-AAF based on its NMR spectrum and on the mass spectrum of the corresponding guanine derivative obtained after removing deoxyribose by acid hydrolysis. This compound appeared to be identical with the persistently bound form present in DNA hydrolysates from rat liver after injection of [2'-3H]N-hydroxy-AAF.


Involvement of polyphosphorylserine blocks in the Fe(III) binding by phosvitin.

It has been suggested that the binding of iron(III) by phosvitin involves the phosphoric radicals of phosphorylserine residues, many of which are arranged in rows of several consecutive phosphoamino acids. In this paper we present evident that, unlike free phosphorylserine which does not interact with Fe(III), polyphosphorylserine blocks--(Ser-P)n, with n greater than or equal to 4 -- bind Fe(III) like phosvitin, though less actively, to give complexes stable at very acidic pHs. The binding  of iron does not cause any polymerization of the phosphopeptides, and the (Ser-P)n-Fe(III) complexes display an Fe/P ratio significantly lower than 0.5, found in Fe-saturated phosvitin. These findings indicate that polyphosphorylserine blocks play an important role in the binding of iron by phosvitin, and that in the intact protein their binding capacity is optimized by the conformation of the polypeptide chain.


The distribution of [3H]synthanecine A bis-N-ethylcarbamate and its metabolites in the rat.

An intravenous dose of 40 mg/kg [3H] synthanecine A bis-N-ethylcarbamate, sufficient to cause liver lesions similar to pyrrolizidine alkaloid poisoning, was given to male rats. The distribution of radioactivity in body tissues was measured after various times from 2 h to 340 days. About 69% of the dose was eliminated via urine and faeces on the first day, while less than 0.4% was found in expired air. The highest tissue level of radioactivity was initially in liver, but this fell steadily at a rate comparable with most other tissues. High levels were also found in kidneys, spleen and lungs, and elimination from the latter was exceptionally slow. A high level in the erythrocytes probably accounted for a persistence of radioactivity in the spleen. Ehrlich estimations showed that 68% of the radioactivity in the liver 2--6 h after dosing was present as pyrrolic metabolites. A high proportion of the liver activity was not extractable by alcohol or aqueous trichloroacetic acid. Binding in the microsomal and nuclear fractions of liver was surprisingly low. From the low level of radioactivity in stomach tissue it was concluded that 2,3-bishydroxymethyl-1-methyl pyrrole could not have been a major metabolite in the bloodstream, since the latter, when given intravenously, binds strongly to the glandular stomach.


The distribution of radio-activity in rats given [3H]bishydroxymethyl-1-methylpyrrole and its relationship to that from [3H]synthanecine A bis-N-ethylcarbamate.

The distribution of radioactivity was measured in rats various times after single intravenous doses of tritium-labelled 2,3-bis-hydroxymethyl-1-methylpyrrole (BHMP). Over half the dose was excreted in urine during the first day; less than a seventh this amount was found in the faeces. The level in glandular stomach was much higher than in any other organ and there was evidence that this was related to the acidity of this tissue. With this exception, the radioactivity in other tissues was lower than after an equivalent dose of tritiated synthanecine A bis-N-ethylcarbamate, and that in liver tissue was more easily solubilised than after the latter compound. The results indicate it is unlikely that more than a small amount of free BHMP is released into the bloodstreams of rats given synthanecine A bis-N-ethylcarbamate. When [3H]BHMP is given by stomach tube much radioactivity remains within the gut and there is no exceptional binding to glandular stomach tissue. Whereas the tissue binding as well as chemical and toxicological properties of BHMP are similar to those of dehydroretronecine, the binding properties of synthanecine A bis-N-ethylcarbamate are likely to resemble those of monocrotaline and similar pyrrolizidine alkaloids.


Fatty acids. Part 48. 13C nuclear magnetic resonance studies of acetylenic fatty acids.

The 13C NMR spectra of thirty-seven alkynoic acids (C8-C18) and ten alkadiynoic acids (C18 and C20) are reported and interpreted. The influence of COOH, COOCH3, CH3, and C identical to C groups on the chemical shifts of nearby carbon atoms is assessed. These influences are largely additive so that available spectra are readily interpreted and the spectra of new compounds of this type can be predicted. These preliminary results indicate that 13C NMR spectroscopy should be of considerable value in the structural identification of acetylenic compounds.


Fatty acids and long-chain bases of gangliosides of human gastrointestinal mucosa.

The fatty acid and long-chain base composition of five major gangliosides from human stomach and small and large intestine mucosa were analyzed with gas chromatography. All the gangliosides greatly resembled each other in the fatty acid pattern. The main fatty acids were C16:0, C18:0, C22:0 and C24:1. No hydroxy fatty acids could be detected. In all the gangliosides 4-sphingenine was the predominant long-chain base (70-75%). About 15% of the long-chain bases had 20 carbon atoms in their chain. No trihydroxy long-chain bases could be detected.


Solubility of phosphatidylcholine in chloroform. Formation of hydrogen bonding between phosphatidylcholine and chloroform.

The solubility of phosphatidylcholine (PC) was studied by the spectroscopic analysis and the measurement of the solubility. The qualitative analysis of infrared absorption spectra confirmed the existence of two types of hydrogen bondings between chloroform and PC, one between chloroform and the C=O group of PC and the other between chloroform and the phosphorylcholine group of PC. The quantitative analysis of the C-D stretching vibration bands of the chloroform-d solution of PC showed that the latter hydrogen bonding mainly contributes to the solubility and that PC dissolves in chloroform to form a complex consisting of a few or more molecules of chloroform and one molecule of PC. We discussed in this report about the molecular organization of PC in chloroform solution.


Phase transition in a lipid bilayer. II. Influence of adamantane derivatives.

The influence of thirty-four adamantane, protoadamantane, and homoadamantane derivatives on the phase transition characteristics of the bilayer in dipalmitoyl lecithin liposomes has been determined by differential scanning calorimetry. Each of these compounds induces a broadening of the phase transition profile of the lipid bilayer that is dependent upon the concentration of the solute and its molecular structure. The concentration--response curves obtained for these solutes suggest that the cage compound derivatives modify the phase properties and under some conditions may induce a phase separation in the doped bilayer. The relative activity sequences obtained for the compounds examined cannot be accounted for by simple considerations of lipid/water partition coefficients, substitution constants based on free energy relationships, or the relative polarities or sizes of substituent groups. The observations are consistent with the hypothesis that the position and orientation of a solute within the bilayer are critical factors in determining its relative potency. The position of a solute within the bilayer is significantly controlled by the presence of polar substituents and by the relative geometric relationships of these groups. For a given substituent group, the shape and size of the hydrocarbon cage becomes increasingly important. It is apparent that seemingly minor modifications in the structure of a solute can significantly alter its influence on the phase transition behavior of a bilayer.


Changes in cardiac output and total peripheral resistance during development of renal hypertension in the rabbit: lack of confomity with the autoregulation theory.

Serial measurements of cardiac output (CO), mean arterial pressure (MAP), heart rate, and total peripheral resistance (TPR), were made on unanesthetized rabbits with previously implanted Doppler flowmeters. After 2 days of control measurements the rabbits were subjected alternatively to bilateral renal cellophane wrapping (wrap group) or to sham operation and additional measurements were made 1, 2, 4, 8, 16, 25, and 32 days after operation. During the 1st week after operation changes in CO were identical in the wrap and sham-operated groups, with an overall increase to a value of 110% of control on day 4 (P less than 0.05). Thereafter CO fell gradually, reaching 75% of control by day 32 in the wrap group, but only 95% of control in the sham-operated group. When CO was expressed per unit of body weight the latter differences were somewhat reduced, but still were significant. In wrap animals MAP and TPR rose progressively to 155% and 194% of control by day 32. In the sham-operated group the corresponding increases to 108% and 118% of control were significantly smaller. The MAP and TPR of the renal wrap rabbits exceeded the values in sham-operated rabbits, even over the 1st week after operation, by an average for MAP of 8.6 +/- 1.4% (P less than 0.001), and for TPR of 8.0 +/- 2.5% (P = 0.01). The results suggest that the changes in CO during the 1st week were a nonspecific consequence of the preceding wrap or sham operation. They bore no apparent relationship to the subsequent development of the hypertension which was "resistance-mediated" from the earliest stages. We conclude that the present findings for the rabbit differ from those reported for other species and do not conform to the changes predicted by the autoregulation theory of the pathogenesis of hypertension.


Localization of glucocorticoid uptake in normal and ischemic myocardial tissue of isolated perfused cat hearts.

We studied the uptake of labeled dexamethasone (3H-Dex) or methylprednisolone (3H-MP) in isolated perfused cat hearts during the first hour of acute myocardial ischemia. Considerable amounts of 3H-Dex and 3H-MP were taken up by the plasma membrane (F1) fraction in control, border zone, and ischemic myocardial tissue. Lesser amounts were incorporated into the remaining cell fractions. A gradient of glucocorticoid uptake was observed that decreased from control tissue to ischemic tissue in all subcellular fractions (i.e., F1 to F5). Accordingly, supernatant fraction (S) to particulate (P) ratios of labeled glucocorticoid uptake increased from control to ischemic tissue, indicating that myocardial cell damage resulted in a decrease in glucocorticoid-binding capacity in subcellular fractions obtained from ischemic tissue. The activity of 5'-nucleotidase (5'ND), a plasma membrane marker in myocardial cells, also decreased from normal to ischemic tissue. Furthermore, we found that uptake of 3H-MP and 3H-Dex was associated with the retention of 5'ND activity in F1 fractions of both border zone and ischemic tissue. Similar protection of plasma membrane integritg occurred in the supernatant fraction as determined by changes in S/P ratios of 5'ND activity. These data provide support for the concepts that (1) plasma membrane changes occur soon after acute myocardial ischemia, and (2) the mechanism by which glucocorticoids exert a protective effect in myocardial ischemia may be related to membrane stabilization.


Effects of hyperthermic stress on myocardial function during experimental coronary ischemia.

We evaluated hyperthermic influences on ischemic hearts by comparing two groups of intact working swine hearts (n = 20) made globally ischemic. Heart muscle temperature was selectively increased from 37.5 +/- 0.3 degrees C to 39.7 +/- 0.3 degrees C in one group (n = 11) by warming the coronary perfusate. Ischemia in normothermic hearts significantly (P less than 0.05) decreased mechanical function (as reflected by increases in left ventricular end-diastolic pressure [LVEDP]), myocardial oxygen consumption (MVO2), glucose uptake, glycolytic flux, free fatty acid (FFA) uptake and oxidation, and tissue stores of high energy phosphates. Hearing in ischemic hearts further depressed mechanical function at similar reductions in coronary flow and MVO2. Glucose uptake was terminally increased over normothermic values (329 vs. 221 mumol/hr per g) as was glycolytic metabolism, FFA uptake (26 vs. 17 mumol/hr per g), and FFA oxidation (21 vs. 11 mumol/hr per g). However, these changes were not translated into increased energy stores of tissue creatine phosphate and ATP. Thus, in ischemic hearts, hyperthermia neither prevented the development of mechanical deterioration nor improved oxidative phosphorylation despite increases in metabolic substrate utilization. These data suggest that in experimental global ischemia heat is an added energy drain in already burdened myocardium.


Changes in coronary and collateral flows and adequacy of perfusion in the dog following one and three months of circumflex occlusion.

We investigated changes in circumflex, left anterior descending (LAD), and right coronary artery flows as well as changes in collateral flows to these vessels after long-term circumflex occlusion. Coronary and collateral flows of each vessel were determined simultaneously in an isolated heart preparation in which the vasculature was maximally dilated with dipyridamole. The resistances as related to total heart weight of the circumflex, LAD, and right coronary arteries of 16 control dogs were found to be 0.59 +/- 0.06, 0.93 +/- 0.09, and 2.37 +/- 0.17 (mean +/- SEM) mm Hg/[(ml/min)/100 g], respectively. Total minimal coronary resistance was 0.21 +/- 0.01. In 10 dogs subjected to occlusion for 1 month no significant change in circumflex coronary resistance was observed, but the resistance of the unimpaired vessels decreased significantly. The resistances of the LAD and right coronary arteries were 0.66 +/-0.04 and 1.72 +/- 0.13, respectively. Both values were considerably less (P less than 0.01) than control. In nine dogs subjected to occlusion for 3 months the resistance of the unimpaired LAD and right arteries, as well as the circumflex coronary resitance, were not significantly different from control. We also found that retrograde flows for all vessels increased 7-fold after 1 month and 10.5-fold (relative to control) after 3 months of occlusion. From these data we conclude that vascular adaptations, which occurred in response to an ischemic stimulus, are responsible for the long-term regulation of the metabolic needs of the myocardium.


Angiotensin II and its heptapeptide (2-8), hexapeptide (3-8), and pentapeptide (4-8) metabolites in arterial and venous blood of man.

We made separate measurements of angiotensin II (A II) and of its immunoreactive metabolites (2-8 heptapeptide, 3-8 hexapeptide, and 4-8 pentapeptide) in arterial and venous plasma from subjects with widely different plasma levels of the peptides. A II and its three metabolites were extracted from blood, separated by paper chromatography, and measured by radioimmunoassay using an A II antiserum which had a 100% cross-reaction with each metabolite. In contrast to results of previous studies, A II was found to predominate in both arterial (60-100%) and venous (55-100%) blood. The biologically active 2-8 heptapeptide metabolite accounted for only 10% of the activity in arterial plasma. Radioimmunoassay of venous plasma extracts using an A II antiserum which had a low cross-reaction with the 3-8 hexapeptide and the 4-8 pentapeptide confirmed the results obtained using the antiserum which had a 100% cross-reaction with the metabolites. We conclude that radioimmunoassay methods for measuring A II in venous blood may be more accurate and relevant than has previously been recognized. The small difference between A II concentrations in arterial and venous plasma suggests further that there may be significant conversion of angiotensin I (A I) to A II in the limb vasculature.


The myocardial energetic active state. I. Oxygen consumption during tetanus of cat papillary muscle.

The potential role of active state maintenance as a determinant of myocardial oxygen consumption (MVO2) has not been defined. Right ventricular papillary muscles from 15 cats were studied in a polarographic myograph at 23 degrees C in a Krebs-Ringer solution containing 7.5 mM Ca2+ and 10 mM caffeine. MVO2 was determined for isometric tetani at Lmax of 1-5 seconds' duration. Increases in tetanus duration related linearly to increments in both active tension time (delta active tension) and MVO2. In order to examine the oxygen cost of active state maintenance not attributable to associated tension generation, both the same isometric and 2.5- to 10.0-second lightly preloaded isotonic tetani were produced in nine muscles. For each tetanus duration the contribution throughout the contraction of developed force (preload) to MVO2 could be subtracted from overall isotonic MV02. In the absence of the MVO2 associated with force development, the active state duration was related linearly to MVO2, with a mean active state MVO2 of 2.42 +/- 0.29 nl O2/mg dry muscle/sec of isotonic tetanus; this MVO2 is 68% of the value of 3.58 +/- 0.42 nl O2/mg dry muscle/sec that was obtained for isometric tetanus at Lmax. This study identifies active state maintenance as the major determinant of MVO2 during myocardial tetanus and, furthermore, suggests the possibility that alterations in ative state intensity and duration may be the biochemical mechanism by which other determinants of MVO2 act in a more physiological setting.


The effect of an acute increase in renal perfusion pressure on sodium transport in the rat kidney.

We used micropuncture techniques to examine the intrarenal response to an acute elevation of the renal perfusion pressure. In one series of studies (epinephrine, group I) the renal perfusion pressure was acutely increased by intravenous epinephrine infusion; in another series, by bilateral carotid occlusion and vagotomy. A third series of studies (epinephrine, group II) was performed identically to the epinephrine, group I, studies except that the renal perfusion pressure was held constant during the epinephrine infusion by suprarenal aortic constriction. After epinephrine infusion (group I) and following bilateral carotid occlusion and vagotomy the renal perfusion pressure increased, from 119 +/- 1.0 (SEM) to 166 +/- 1.85 mm Hg and from 122 +/- 5.9 to 168 +/- 3.1 mm Hg, respectively. Fractional sodium excretion rose from 2.31 +/- 0.34% to 5.09 +/- 0.58% (P less than 0.001) after epinephrine and from 1.80 +/- 0.71 to 6.40 +/- 1.0% (P less than 0.01) following carotid occlusion and vagotomy. In neither study, however, did we find that the increase in renal perfusion pressure changed the glomerular filtration rate (GFR) (both kidneys) or fractional sodium delivery from the superficial cortical late distal tubule. Furthermore, we found that epinephrine infusion at a constant renal perfusion pressure (epinephrine, group II) did not affect fractional sodium excretion, although a small, but significant, decrease in the GFR and sodium delivery from the superficial late distal tubule occurred. These data suggest that the natriuresis which follows an acute elevation of the renal perfusion pressure cannot be attributed to enhanced sodium delivery from superficial nephrons but must result from (1) inhibition of sodium reabsorption in inner cortical nephrons or (2) an effect on sodium transport in the collecting system.


Reflex vascular capacity reduction in the dog.

The maximum degree and time course of active reflex venoconstriction in chloralose-anesthetized dogs was studied. The mean circulatory filling pressure (Pmc) was measured by fibrillating the heart and rapidly pumping blood from the aorta to the vena cava until the systemic arterial pressure equaled the central venous pressure. Ventricular fibrillation was continued for 1 minute and was assumed to induce maximal sympathetic discharge to the capacity vessels. Blood was removed to maintain the Pmc constant at the level determined at 8 seconds after the start of fibrillation. It was necessary to remove 13.8 ml/kg by the end of 1 minute. After autonomic nervous system blockade by hexamethonium only 4.8 ml/kg were removed to hold the Pmc constant. Thus, in conclusion, the maximal degree of active venoconstriction induced by circulatory arrest was 9.0 ml/kg during the 1st minute of cardiac fibrillation. A basal capacity vessel tone was present, equivalent to 10 ml/kg under the experimental conditions used.


A delayed suppression of the renin-aldosterone axis following saline infusion in human hypertension.

The purpose of this study was to compare the acute suppressibility of the renin-angiotensin-aldosterone (RAA) axis in normotensive (n = 23) and essential hypertensive (n = 62) subjects. Only those hypertensive subjects with normal plasma renin activity (PRA) levels (sodium restricted, upright) were included in the study. Acute suppression of the RAA axis was determined by measuring PRA, plasma angiotensin II (A II), and plasma aldosterone (PA) at frequent intervals during the infusion of isotonic saline (500 ml/hour for 6 hours). Although all parameters fell significantly from control levels by 20-30 minutes in the normotensive subjects, we found that 60% of the hypertensive subjects showed no significant decline in PRA or PA until 120-240 minutes after beginning the infusion. The other hypertensive subjects showed normal RAA suppression. In addition, while there were no significant differences between the three groups in control PRA or PA levels, we found that the PA levels from 30 to 240 minutes during the saline were significantly higher (P less than 0.01) in the hypertensive subjects with delayed suppression. That there were two distinct populations in the hypertensive group was suggested by the bimodality of the frequency response curve, with peaks occurring at 30 and 240 minutes. These studies indicate an abnormality in the acute suppression of the RAA axis in a substantial proportion of subjects with normal renin essential hypertension. Since previous studies in normal subjects have reported that the early phase of response to saline infusion is related to the sodium ion per se and not to intravascular volume expansion, we have come to the conclusion that the present data are consistent with the hypothesis that the delayed suppression hypertensive group has a diminished ability to respond to the sodium ion.


Depression and enhancement of baroreceptor pressor response in cats after intracerebroventricular injection of noradrenergic blocking agents: dependence on supracollicular areas of the brain.

The alpha-adrenergic blocking drugs, phentolamine and Hydergine, both act centrally at different sites to depress and enhance the pressor and sympathetic nerve response to decreased baroreceptor afferent input in anesthetized cats. Depression of the rise in blood pressure and sympathetic nerve discharge during bilateral carotid occlusion (BCO) followed injection of the agents into the 4th cerebral ventricle when the brain was intact but not when connections were interrupted at the midcollicular level by transection or lesion. Enhancement of responses occurred when drug distribution was confined to the brain rostral to the midcollicular level via injection into the 3rd cerebral ventricle with the cerebral aqueduct cannulated. Both agents decreased resting blood pressure and Hydergine decreased heart rate in intact and decerebrate preparations but not in 3rd ventricle-cerebral aqueduct experiments. We found that pretreatment with the noradrenergic precursor. L-dopa consistently prevented depression by phentolamine but was less effective against Hydergine. The results indicate that mechanisms which enhance and suppress the baroreceptor pressor response are normally operative in anesthetized cats and, furthermore, that neural pathways mediating the effects are ones connecting the caudal brainstem with supracollicular levels of the brain. It is further suggested that the pathways may be noradrenergic.


Effect of quinidine and temperature on sodium uptake and contraction frequency of cultured rat myocardial cells.

The effects of quinidine and temperature on Na influx and contraction frequency of synchronously contracting rat myocardial cells in monolayer cultures were studied. Quinidine (10(-6) M to 10(-1) M) produced a prompt reduction in Na influx, maximum after 30 seconds of exposure, and dose-dependent along a sigmoid log dose-response curve. At 37 degrees C, Na influx (mumol/10(11) cells per sec) decreased from 30.19 to 24.70 (P less than 0.001) and 10.49 (P less than 0.001) on exposure to quinidine, 10(-6) and 10(-2) M, respectively. Simultaneously the contraction frequency decreased from a control of 120/min to 105/min and 48/min with 10(-6) M and 5 X 10(-4) M quinidine. At higher concentrations spontaneous contractions ceased. The effects on Na influx and contraction were reversible by washing the cells free of the drug (30 seconds). A temperature-dependent decrease in the Na influx between 37 degrees C and 22 degrees C also induced a decrease in contraction frequency. Between 25 degrees C and 35 degrees C the Q10 values for Na influx and contraction frequency were 2.41 and 2.44 respectively. Under all conditions tested there was a constant linear relationship (r = 0.98) between Na influx and contraction frequency for all values of Na influx greater than 11.82 mumol/10(11) cells per sec. Na influx and contraction frequency were insensitive to tetrodotoxin (10(-5) g/ml) but very sensitive to verapamil and to changes in extracellular Na. Quinidine affected only the verapamil-sensitive Na influx. The results indicate a close relationship between verapamil-sensitive inward Na movement and automaticity in these cells and demonstrate that the quinidine-induced changes in automaticity are closely linked to the effect on Na influx.


Inhibition of feline collateral vessel development following experimental thrombolic occlusion.

We compared development of feline hindlimb collateral circulation after acute occlusion of the terminal aorta by ligation, thrombus formation, and formation of a "closed" aortic loop containing thromboplastin. Collateral circulation development was assessed by aortograms, scintillation scans, neurological signs following occlusion, measurement of hindlimb muscle blood flow, and forelimb and hindlimb temperature. In cats in which aortic occlusion was the result of ligation or thromboplastin in the aortic loop, paralysis was not evident. Aortograms and scintillation scans indicated hindlimb blood flow. Both muscle temperature and blood flow data indicated that the return of blood flow was rapid. The 5th lumbar artery appears to be the origin of the collateral vessels. The mid-zone component is a dorsal and ventral vertebral route and an epaxial muscle route. The reentry components are the 6th or 7th lumbar arteries. The collateral vessels arise from preexisting collateral vessels. Of those cats in which aortic occlusion was the result of a thrombus, all exhibited paralysis. Aortograms, scintillation scans, muscle temperature, and hindlimb blood flow data indicated reduced hindlimb blood flow. The results suggest that the thrombus has an inhibitory effect on the development of collateral circulation.


Idiopathic prolapsed mitral leaflet syndrome. Angiographic-clinical correlations.

Angiographic clinical correlations were made in 59 patients with prolapsed mitral leaflet syndrome. Eight had nonejection systolic clicks (group I), 20 had early, mid or late systolic murmurs with or without a systolic click (group II), and 31 had pansystolic murmurs (group III). Isolated prolapse of posterior leaflet (PL) scallops occurred in 42 and 17 had combined leaflet prolapse. The study demonstrated the following: (I) Group II patients usually had isolated PL prolapse with a predominant biscallop involvement while a high incidence of triple scallop prolapse and combined mitral leaflet prolapse occurred in group III. (II) Severe mitral regurgitation and a greater incidence of atrial fibrillation were seen in patients with triscallop prolapse and combined mitral leaflet prolapse. Mitral regurgitation was milder in patients with single and biscallop prolapse and, when severe, was associated with ruptured chordae. (III) ST-T wave abnormalities in the  inferior leads were most frequent in patients with isolated PL prolapse. (IV) Systolic and diastolic asynergy occurred in 41 patients, most frequently in group II but also relatively frequently in group III (19 of 31). Segmental anterior dysfunction with normal ejection fraction was found in 18 patients, of whom 13 had early anterior wall relaxation. (V) Patients without asynergy were slightly older than those with it. More in the former group had severe mitral regurgitation and were clinically disabled from it.


Mitral valve prolapse. Two-dimensional echocardiographic and angiographic correlation.

In order to define baseline descriptive criteria for the diagnosis of mitral valve prolapse with cross-sectional echocardiography, 49 patients undergoing catheterization were examined by a real-time, two-dimensional phased array echocardiographic imaging system. Angiography was used to separate patients into two distinct groups: 15 with normal mitral valve function and 34 with definite mitral valve prolapse. Systolic mitral leaflet and annulus motion were then observed in each patient and similarities and differences were noted between the two groups of patients. Correlative M-mode echocardiographic data were available in 37 patients. Certain two-dimensional echocardiographic findings restricted to the angiographically proven mitral valve prolapse group were defined: 1) posteriorly displaced coaptation of the leaflets, 2) systolic superior movement of one or both mitral leaflets above the level of the mitral ring, and 3) a systolic curling motion of the posterior mitral ring on its adjacent myocardium. One or more of these abnormalities were found in all 34 patients with angiographic mitral valve prolapse. When mitral valve prolapse does occur, the results of two-dimensional echocardiography would suggest that both leaflets are usually involved.


Distribution of coronary artery disease. Prediction by echocardiography.

To assess the sensitivity of standard echocardiography in detecting ventricular motion abnormalities in patients with coronary artery disease (CAD) without prior myocardial infarction, 56 consecutive patients with a history of angina pectoris were studied during an angina-free period. In the 48 patients with adequate echocardiograms, the amplitude of septal and posterior wall motion in the high, mid, and low left ventricle was determined and used to predict prospectively in a blinded fashion the sites of angiographically-determined CAD. Twenty-eight of 35 patients (80%) with disease of the left anterior descending artery (LAD) had diminished interventricular septal motion (P less than 0.001) and 14 of 27 patients (52%) with disease of posterior vessels had diminished posterior wall motion on echocardiogram. When abnormalties of echocardiographic wall motion were compared with left ventriculography, the results were similar. Echocardiography may aid in predicting the presence and distribution of CAD, especially LAD disease.


Hemodynamic correlates of the normal aortic valve echogram. A study of sound, flow, and motion.

The aortic valve echogram was recorded in open chest dogs simultaneously with aortic flow, acceleration of flow, aortic and left ventricular pressures, and intracardiac phonocardiograms. Comparison of echographic with hemodynamic data showed the following: The aortic valve started its opening with the onset of flow, at the same point that left ventricular pressure exceeded aortic pressure. Complete valve opening preceded peak aortic flow by an average of 43 msec and the cusps started to move toward closure while flow was still accelerating. Final closure of the valve was achieved at the time of zero flow and preceded the aortic second sound by 4-10 msec. The echographically determined "valve orifice area" correlated well with aortic stroke volume (r = 0.94). The intensity of the aortic first sound was related to peak acceleration of aortic flow. The intensity of the second sound was not related to the amplitude of cusp motion but correlated well with the aortic pressure at the time of closure and with peak flow deceleration.


Echocardiographic measurement of cardiac output using the mitral valve and aortic root echo.

The current echo method for measuring cardiac output (CO) has various technical limitations and is based on the assumptions that the left ventricle is not dilated and that it contracts uniformly. We have taken a different approach to measuring CO by echo: CO = cross-sectional area of aortic root X left ventricular ejection time X mean aortic flow velocity X heart rate. Using the Fick method, 34 measurements of CO were made in patients with normal aortic and mitral valves, with simultaneous echo recordings from the aortic and mitral valves. The systolic closure slope of the anterior mitral valve leaflet was assumed to represent the mean aortic flow velocity. CO by Fick and the proposed echo method correlated well, both in patients with (r = 0.94) and those without (r = 0.87) asynergy and the overall correlation was strong (r = 0.90). Using the conventional echo and Fick methods, a much weaker association was found between the two measurements in those patients with asynergy (r = 0.44) as opposed to those without asynergy (r = 0.77).


The posterior aortic wall echocardiogram. Its relationship to left atrial volume change.

The normal posterior aortic wall echocardiogram shows anterior motion during left ventricular systole and predominantly posterior motion in three phases during left ventricular diastole. In six patients undergoing simultaneous left atrial angiograms and posterior aortic wall echocardiograms, there was excellent correlation between the posterior aortic wall motion and the change in the left atrial angiographic area showing the value of the posterior aortic wall echocardiogram in describing the left atrial volume curve. Left atrial and left ventricular pressures were measured with manometer tip catheters and correlated with simultaneous posterior aortic wall and mitral valve echocardiograms in four patients with atrial septal defects. These echocardiographic, angiographic, and hemodynamic correlations, as well as other evidence reported in this paper suggest that a major portion of posterior aortic wall motion is related to left atrial events and describes the left atrial volume curve.