Multifactorial analysis of chronic hypertension induced by electrolyte-active steroids in trained, unanesthetized dogs. Chronic hypertension has been produced in mongrel dogs by the long-term oral administration of metyrapone (100 mg/kg per day). This model is characterized hemodynamically by wide variations in cardiac output and extracellular fluid volume and metabolically, by hypokalemia, suppressed plasma renin activity, and elevated plasma deoxycorticosterone values. Unlike the deoxycorticosterone acetate (DOCA)-saline hypertensive rat, it is associated with decreases rather than increases in plasma catecholamines. Reasons for the raised arterial pressure induced by metyrapone were sought (1) by exploring the role of cardiac output in the initiation and maintenance of hypertension, (2) by an assessment of neural contribution through the study of circulating catecholamines and use of adrenergic blocking drugs, and (3) by determining its relation to dietary sodium. The rise in pressure was not uniformly associated with an increase in cardiac output nor did prevention of rises in output by beta-blockade prevent the development of hypertension. Similarly, pretreatment with either centrally acting (clonidine) or peripherally acting (guanethidine) adrenergic blocking drugs failed to prevent the hypertension or change its pattern. In contrast, salt deprivation was singularly effective in preventing the rise in arterial pressure while increased salt intake led to parallel elevations in both blood pressure and peripheral resistance. These results point to a primary role of arteriolar resistance in determining the initiation and maintenance of hypertension induced by electrolyte-active steroids. In this regard, they agree with other reports of early changes in both membrane and structural properties of vascular smooth muscle that antedate the rise in pressure induced by DOCA and 1% saline.


Caretaking in the first year of life: the role of fathers, and mothers' social isolation. Data are presented from a follow-up study of some 80 normal first or second born children on the extent of father participation childrearing. Though at both 30 and 60 weeks most fathers played regularly with their children, only a minority regularly undertook care-taking activities. Fathers were more participant at 60 weeks than 30 weeks especially with their daughters. No clear social class differences were found. The number of visitors to the home and excursions by mothers showed that social isolation during the day was quite widespread. Social class I and II families were less likely to have relatives in the neighbourhood and so received fewer visits and went out less often. Mothers going out without children showed less marked social class differences. Mothers of children who woke regularly at night and showed more 'difficult' behaviour went out without their children less often than the mothers of children not classified in this way.


Structural and functional changes in vascular resistance and reactivity in the deoxycorticosterone acetate (DOCA)-hypertensive pig. The development of altered vascular resistance and reactivity was characterized in a new model of hypertension, the deoxycorticosterone acetate (DOCA)-hypertensive pig. Terminal hindlimb perfusion studies were carried out on pigs (young uninephrectomized males) approximately 50 days after implantation of DOCA or control Silastic strips. Data from these studies showed that hypertensive vascular beds (n = 5) had significantly greater (P less than 0.005) resistances than controls (n = 9) both before and after maximla vasodilation with papaverine. In response to intra-arterial injections of norepinephrine, hypertensive pigs (n = 8) demonstrated shifts in the dose-response curves to the left and lower vasoconstrictor thresholds than controls (n = 10). Preliminary observations have been made on the effects of "protecting" the hindlimb vascular bed from the increased arterial pressure by external iliac artery ligation. Although this "protection" was incomplete it prevented the development of structurally based changes in resistance and reactivity, but did not prevent the development of increased vascular smooth muscle sensitivity. It is concluded that resistance vessels in the DOCA-hypertensive pig demonstrate changes in both structure and function; structural changes do not occur when the vessels are partially protected from the increased wall stress but this does not prevent the increase in sensitivity. The structural component of resistance makes a greater contribution to the overall increase in resistance in this stage of hypertension than does the smooth muscle contraction; however, since the structural change is secondary to the increase in arterial pressure, it cannot initiate the hypertension.


Specificity of altered electrogenesis of membrane potential in hypertension. Cardiac and hepatic portal venous muscle were used in an attempt to determine the specificity of the altered membrane potential (Em) electrogenesis found in caudal artery of spontaneously hypertensive rats (SHR). Neither the sensitivity to norepinephrine nor the electrogenesis of Em was found to be different in SHR than in their normotensive controls. Thus, ventricular myocardium and portal venous myovascular cells do not show either the increased sensitivity or the Em factor proposed as a contributor to it. A second determination of specificity was stimulation of the caudal artery by Ba2+. Both depolarization and extent of contraction were no different in SHR than in normotensive controls, suggesting that the increased sensitivity of arterial muscle does not extend to all depolarizing agents. These data define the electrogenesis of Em in cardiac and portal venous muscle of SHR and their controls and provide a test of the hypothesis that altered Em electrogenesis contributes to increased arterial norepinephrine sensitivity seen in hypertension. The data support rather than refute the hypothesis, i.e., the Em alteration was specific for both the muscle showing increased sensitivity and the agent used for depolarization.


Role of the renin-angiotensin system in the regulation of aldosterone biosynthesis and arterial pressure during sodium deficiency. The aldosterone and arterial pressure response to long-term infusion of two angiotensin II inhibitory analogues, [Sar1,Ala8]angiotensin II and [Sar1,Ile8]angiotensin II, and the angiotensin I-converting enzyme inhibitor, SQ 20,881, was studied in conscious dogs during sodium deficiency. Plasma aldosterone concentration (PAC), plasma cortisol concentration (PCC), and plasma renin activity (PRA) were determined by radioimmunoassay. In conscious dogs after dietary sodium restriction (5 mEq of Na+/day) for 21 days, PAC averaged 37.5 +/- 8.9 (mean +/- SE) ng/dl, PCC averaged 1.3 +/- 0.5 microng/dl, PRA averaged 3.23 +/- 0.42 ng/ml per hour, and arterial blood pressure (AP) averaged 103 +/- 5 mm Hg. During long-term infusion of [Sar1,Ala8]angiotensin II (5 microng/kg min-1), PAC averaged 34.7 +/- 8.5 ng/dl, PCC averaged 1.5 +/- 0.5 microng/dl, PRA averaged 16.4 +/- 3.1 ng/ml per hour, and AP averaged 88 +/- 5 mm Hg. During long-term infusion of [Sar1,Ile8]angiotensin II (5 microng/kg min-1), PAC averaged 45.8 +/- 12.6 ng/dl, PCC averaged 1.75 +/- 0.5 microng/dl, PRA averaged 13.6 +/- 4.3 ng/ml per hour, and AP averaged 86 +/- 5 mm Hg. During long-term infusion of angiotensin I-converting enzyme inhibitor (5 microng/kg min-1), PAC averaged 14.9 +/- 4.8 ng/dl, PCC averaged 1.75 +/- 0.5 microng/dl, PRA averaged 20.3 +/- 5.3 ng/ml per hour, and AP averaged 76 +/- 5 mm Hg. The intrinsic agonistic properties of the angiotensin II inhibitory analogues on the adrenal cortex negate their use for defining the role of the renin-angiotensin system in the regulation of aldosterone biosynthesis during sodium deficiency. The precipitous fall in aldosterone secretion and arterial blood pressure during long-term infusion of angiotensin I-converting enzyme inhibitor demonstrates the importance of the renin-angiotensin system in mediating aldosterone biosynthesis during sodium deficiency and the essential role of the renin-angiotensin-aldosterone system in the regulation of arterial pressure during sodium deficiency.


The effect of high sodium intake and angiotensin antagonist in rabbits with severe and moderate hypertension induced by constriction of one renal artery. The role of renin was assessed in moderate and severe one-clip, two-kidney hypertension in rabbits during normal and high Na intake. Severe hypertension occurred with high levels of plasma renin and creatinine and with extracellular volume depletion. In this group, hypertension was significantly reduced by the 1-hour intravenous infusion of 1,000 ng/kg of an angiotensin II antagonist or by correcting the volume depletion with a high Na intake, which also decreased renin activity and creatinine. The infusion of the antagonist after a high salt diet failed to decrease blood pressure further. Moderate hypertension occurred with plasma levels of renin that were slightly below normal and with no significant abnormalities in extracellular volume depletion or creatinine. In this group, the administration of an angiotensin II antagonist or a high salt diet did not affect any of the three parameters. In normotensive controls, the blood pressure level was not affected by either the angiotensin II antagonist or the high salt diet, despite a reduction in plasma renin activity. Thus, high levels of renin are important in maintaining severe hypertension, and these increased levels probably are accompanied by a concomitant depletion of extracellular volume. Correction of the extracellular volume depletion by a high salt diet is followed by a decrease in renin activity and in blood pressure. In contrast, renin activity does not seem to be important in maintaining moderate hypertension, the pathogenetic mechanism of which remains to be elucidated.


Mechanisms of hypertension during the acute and intermediate phases of the one-clip, two-kidney model in the dog. The acute and intermediate onset phases of one-clip, two-kidney hypertension were studied in six conscious dogs. Mean arterial pressure (MAP), cardiac output (CO), total peripheral resistance (TPR), serum renin concentration (SRC), plasma aldosterone (PA) and cumulative sodium and water balance were studied prior to unilateral renal artery constriction, at 2, 10, and 24 hours postconstriction, and daily thereafter for 5 days. At 2 hours postconstriction, MAP, CO, TPR, and SRC were elevated, with unchanged fluid balance. At 10 and 24 hours there was a further rise in CO accompanied by positive fluid-sodium balance, with a slight decrease in MAP, TRP, and SRC. During days 2 and 3, MAP, CO, TPR, and SRC remained elevated and stable in the presence of decreasing fluid-sodium balance to preconstriction levels. During days 4 and 5, MAP, SRC, and fluid-sodium balance remained unchanged, TPR demonstrated a secondary increase, whereas CO decreased to preconstriction values. Sequential changes in PA parallel those of SRC, but were significant only at 2 hours postconstriction. These changes suggest that the increased CO is not totally dependent on fluid-sodium balance, and that CO is not the sole determinant of elevated MAP. The temporal relationship between MAP and SRC throughout the study is consistent with renin-mediated hypertension. The secondary rise of TPR may be due to total body autoregulation and/or increased vascular reactivity to high levels of circulating angiotensin. A unitary renin-angiotensin mechanism, therefore, may be responsible for the induction and maintenance of hypertension in this experimental model.


Mechanisms involved in two-kidney renal hypertension induced by constriction of one renal artery. Changes in plasma renin activity (PRA) and sodium balance were studied in hypertensive rabbits and dogs with one renal artery constricted and the other kidney intact (two-kidney hypertension); aldosterone secretion was measured also in the chronic hypertensive rabbits. Both PRA and aldosterone secretion were normal in some chronic hypertensive rabbits but elevated in others. Sodium balance studies revealed that some severely hypertensive rabbits with elevated PRA were in spontaneous negative sodium balance. Unlike the rabbit, PRA was never increased in the chronic hypertensive dog and sodium balance was normal. Infusion of [Sar1, Ala8]angiotensin II (P-113) decreased arterial pressure and aldosterone secretion in those hypertensive rabbits with elevated PRA but not in those rabbits with normal PRA; P-113 also did not decrease arterial pressure in the chronic hypertensive dog unless sodium depletion was superimposed. In the conscious two-kidney dog, acute renal artery stenosis increased both arterial pressure and PRA within minutes, and P-113 blocked the rise in pressure associated with the increase in PRA. Therefore, although apparent species differences between the rabbit and the dog occur, the present data indicate that neither increased PRA nor excess salt retention is essential to the chronic maintenance of two-kidney hypertension in these two species; however, in the dog a role for angiotensin II in the acute phase is indicated.


Plasma prorenin in normal, hypertensive, and anephric subjects and its effect on renin measurements. We have previously shown that a cryoactivatable inactive form of renin, which we have tentatively termed prorenin, occurs in human plasma. Plasma prorenin is measured by subtracting the endogenous plasma renin activity (PRA) from the total renin activity measured after cryoactivation. Cryoactivation is accomplished by shaking plasma at -5 degrees C for 4 days. Circulating prorenin averaged 5.6 +/- 0.8 (SE) ng/ml per hour in a group of 30 normal subjects and 5.1 +/- 1.0 ng/ml per hour in 25 hypertensive subjects during random sodium intake. In these two groups, prorenin ranged from zero to 36 times the endogenous renin level, averaging 2.5-fold in normal subjects and 3.4-fold in hypertensive subjects. During sodium deprivation prorenin more than doubled in 12 hypertensive subjects from a mean of 2.8 to 6.3 ng/ml per hour. In 25 anephric subjects, circulating prorenin was slightly lower than in normal subjects, averaging 3.4 +/- 0.9 ng/ml per hour (P less than 0.05). In contrast to our findings in nephric subjects, the low level of PRA measured in anephric subjects ((0.26 +/- 0.04 ng/ml per hour) was almost always a constant fraction of the measured prorenin. This led to the demonstration that prorenin can be inadvertently activated by chilling blood during processing for renin measurement and this often accounts for the small amount of renin measured in plasmas from anephric subjects. The error in routine PRA measurements due to inadvertent activation averaged +48% in anephric subjects and +17% in those with kidneys. Evaluation of the effect of processing bloods at room temperature revealed that net angiotensin I accumulation is less than 2% of that generated during incubation and can be ignored. Accordingly, to avoid the inadvertent activation of prorenin which can at times lead to a sizable and variable overestimation of PRA we recommend collecting and processing blood samples at room temperature and then storage of plasma completely frozen until the time for analysis.


Mechanisms of hypertension during the chronic phase of the one-clip, two-kidney model in the dog. The mechanism of the chronic phase of one-clip, two-kidney hypertension was investigated in 23 dogs. Mean arterial pressure (MAP), renal hemodynamics, and arterial and renal venous plasma renin activity (PRA) were studied before and at 1 and 3 months after graded, unilateral renal artery constriction. In the dogs that demonstrated sustained hypertension, the magnitude of changes in MAP, renal hemodynamics, PRA, and renal renin secretion correlated with the severity of renal artery constriction. In dogs with severe constriction (group 3, 78-86% renal blood flow reduction), MAP and arterial PRA were significantly elevated throughout. In groups 1 and 2 (0% and 50-56% renal blood flow reduction, respectively), although MAP remained significantly elevated, arterial PRA decreased to baseline values by 3 months. In group 3, there was an initial, apparent redistribution of ipsilateral renal blood flow, and prolonged reductions in ipsilateral renal plasma flow (RPF) and glomerular filtration rate accompanied by significantly increased creatinine clearance (CCreat) in the untouched kidney. Similar but less marked renal hemodynamic changes occurred in the other hypertensive dogs. All hypertensive dogs, regardless of degree of renal artery constriction, demonstrated significantly increased ipsilateral renal renin secretion and contralateral renin suppression. In three dogs that were studied 6, 12, and 16 months postconstriction, the high rate of ipsilateral renin secretion persisted throughout. Ipsilateral nephrectomy consistently resulted in rapid amelioration of the hypertension. These findings are in accord with a renal-dependent and probably renin-mediated mechanism for the maintenance of high blood pressure in the chronic phase of the one-clip, two-kidney dog model.


Intraventricular infusion of angiotensin II on the hemodynamics and renal function of alpha-chloralose anesthetized cats. Intraventricular infusion of angiotensin II (50, 100, and 200 ng/kg per min) produced significant elevations of arterial blood pressure (20-25%) in alpha-chloralose-anesthetized cats. The pressor responses were not accompanied by significant changes in heart rate,cardiac output, or contractility and were chiefly due to significant increases in total peripheral resistance. In contrast, pressor responses to intravenous infusion of angiotensin II (100 ng/kg per min) were accompanied by reflex decrease in cardiac activity. While intravenous angiotensin II caused increases in the resistance of skeletal, mesenteric, and renal vasculature, the intraventricular administration of angiotensin II increased resistance only in the mesenteric and renal vasculature. Further, centrally administered angiotensin II significantly enhanced urinary output and the rate of Na+ excretion both in the intact as well as in the denervated kidneys. However, the diuretic and natriuretic effects were significantly greater in the intact than in the denervated kidneys, indicating a centrally mediated neurogenic mechanism. The significant increase in the urinary concentration of Na+ (mEq/liter) following intraventricular angiotensin appeared to be secondary to the elevation of arterial blood pressure, since this effect was unaltered by acute renal denervation. The results of this investigation are consistent with the hypothesis that an elevation in the concentration of angiotensin II within the cerebrospinal fluid may trigger neurogenic mechanisms resulting in the constriction of glomerular efferent arterioles. Such an effect would be expected to increase glomerular filtration pressure and filtration fraction, and may play a role in the diuretic and natriuretic effects noted.


Relationship between aldosterone and bradykinin. To assess relationships between plasma bradykinin and the individual elements of the renin-angiotensin-aldosterone system 11 normal subjects and five subjects with adrenal insufficiency were studied on an intake of 200 mEq sodium/100 mEq potassium. In adrenal insufficiency subjects, plasma bradykinin did not change when angiotensin II levels were increased by an infusion rate of 3 ng/kg per min for 30 minutes; plasma aldosterone remained undetectabel. In normal subjects, angiotensin II at 3 ng/kg per min or potassium at 20 mEq/hour infused intravenously for 2 hours both significantly increased plasma aldosterone levels, yet neither stimulus altered plasma bradykinin. Thus, acute elevations in angiotensin II and plasma aldosterone were not associated with changes in the levels of plasma bradykinin. In five additional normal subjects on an intake of 10 mEq sodium/100 mEq potassium, a threshold dose of converting enzyme inhibitor (SQ 20881) at 30 microng/kg was given intravenously. Five minutes after administration of the drug, blood pressure and plasma angiotensin II levels had significantly declined and plasma bradykinin levels had significantly increased (P is less than 0.02). Within 10-20 minutes, however, all had returned to control accompanied by a sustained increase in plasma renin activity, and a decrement in plasma aldosterone. Plasma renin activity, which had increased by more than 2-fold after the administration of the converting enzyme inhibitor, remained elevated until the completion of the study at 320 minutes. Eighty minutes after beginning the infusion, plasma aldosterone levels returned to control. Thus, the results suggest that plasma bradykinin correlates more closely with acute changes in plasma renin activity (and angiotensin I) than plasma aldosterone or angiotensin II, suggesting a role for converting enzyme in the regulation of both plasma renin activity and plasma bradykinin. Furthermore, the declines in blood pressure observed with converting enzyme inhibition may be attributable to both changes in plasma angiotensin II and plasma bradykinin  concentrations. However, it is emphasized that these short-term studies may not reflect the role of bradykinin in the long-term control of blood pressure.


Effect of stellectomy and vagotomy on ventricular refractoriness in dogs. The influence of tonic sympathetic and parasympathetic nervous system activity on ventricular refractory period duration was studied in 13 mongrel dogs. Strength-interval curves were obtained from the apex of the right ventricular endocardium with a transvenous bipolar catheter. The effects of sympathetic activity were assessed by unilateral and bilateral stellectomies and tonic vagal effects were studied by inducing cholinergic blockade with atropine (0.2 mg/kg) and by sectioning the cervical vagi bilaterally. Right stellectomy shifted the strength-interval curve 3-5 msec earlier (P less than 0.005). In contrast, left stellectomy shifted the strength-interval curve 4-7 msec later (P less than 0.001). Thus, right and left unilateral stellectomy produced opposite effects. However, when the contralateral ganglion was removed, right and left stellectomy produced qualitatively similar effects, increasing refractoriness. The net effect of bilateral stellectomy was to shift the strength-interval curve 8-10 msec later (P less than 0.001) and this was primarily dependent on left stellectomy. Blockade of vagal efferent activity with atropine shifted the strength-interval curve 3-6 msec earlier (P = .001). Removal of vagal afferent activity by vagotomy during atropine blockade shifted the strength-interval curve 2-3 msec earlier (P = 0.03). It is concluded that both stellate ganglia exert qualitatively similar effects on cardiac refractoriness; however, the left ganglion dominates and unilateral right stellectomy produces a paradoxical decrease in refractoriness. The sympathetic effects are tonically antagonized by vagal activity.


A method for determining segmental resistances in the microcirculation from pressure-flow measurements. On the basis of an electrical analog, open circuit impedance functions were used to analyze the microcirculation. No specific structure need be assumed except a two-port, two-terminal network in which the major artery and vein supplying the tissue represent the input port and the two ends of the microvessel under study are the output port. The open circuit measurements were made by occluding microvessels in the exteriorized omentum of anesthetized rabbits. The pressure upstream and downstream to the occlusion defines the source pressure of a Thévenin's equivalent circuit. The equivalent resistance value was calculated by plotting the flow through a given microvessel against the pressure developed during a gradual occlusion. The changes in pressure vs. the changes in flow during a progressive occlusion were found to be linearly related. The Thévenin's equivalent resistance was maximum downstream to an occluded artery and upstream to the occluded vein. Within the capillary network, source pressures consistently were within a narrow range. Topically applied norepinephrine resulted in marked changes in source resistance and no changes in source pressures. Threshold doses of norepinephrine given intravenously resulted in changes in source pressures, but minimal changes in source resistance, even though a substantial change in vascular resistance was indicated when calculated on the basis of arterial pressure minus micropressure divided by microvessel flow. The present method defines the functional characteristics of the distributing vessels in terms of two pressures and two equivalent resistances and is relatively easy to perform. The technique can be used to determine the vascular components involved in the response to particular stimuli.


Voltage clamp analysis of the effects of dopamine on the transmembrane ionic currents underlying the action potential of sheep cardiac Purkinje fibers. Dopamine shifted the voltage-dependence of the slow outward current that underlies spontaneous diastolic depolarization to a more positive voltage range and increased the rate constants of inactivation of this current at voltages negative to -80 mV. Dopamine increased the magnitude of the slow inward current and the delayed outward current. Dopamine had an inconsistent effect on the positive dynamic current and the time-independent potassium current. These effects were consistent with effects on the slope of diastolic depolarization, plateau voltage, and action potential duration and are similar to those of epinephrine. The effects of dopamine on the slow inward current and on the delayed outward current were abolished by verapamil, a slow channel blocker. This finding substantiates that outward potassium currents are modulated by intracellular calcium.


The effects of hyaluronidase on coronary blood flow following coronary artery occlusion in the dog. In an attempt to determine the mechanism by which hyaluronidase reduces myocardial injury following coronary artery occlusion, myocardial blood flow was studied in 20 open-chest dogs with occlusion of the left anterior descending coronary artery. Ten dogs served as controls, and 10 received hyaluronidase (500 NF units/kg) intravenously 20 minutes after occlusion. At 15 minutes and at 6 hours after occlusion, regional myocardial blood flow in the epicardial and endocardial halves of both ischemic and nonischemic zones were determined with radiolabeled microspheres. Mean arterial pressure, heart rate, and cardiac output were similar in the untreated and treated dogs through the 6 hours of the experiment. Moreover, regional blood flow to nonischemic myocardium (areas without epicardial S-T segment elevation 15 minutes after occlusion) was similar in the two groups 15 minutes and 6 hours after occlusion. Fifteen minutes after occlusion, the flow to the ischemic myocardium subjacent to sites with S-T segment elevation exceeding 2 mV) in the untreated group was: transmural, 28.1 +/- 2.2 (mean +/- SE) ml/min per 100 g; endocardial, 20.7 +/- 1.8; and epicardial, 38.5 +/- 3.1. The endocardial-epicardial flow ratio was 0.56 +/- 0.04. Six hours after occlusion, the untreated group demonstrated a further decrease in blood flow to the ischemic myocardium: transmural, 15.2 +/- 1.4 ml/min per 100 g; endocardial, 6.8 +/- 1.1; and epicardial, 24.3 +/- 1.9. The endocardial-epicardial flow ratio fell to 0.28 +/- 0.04. In contrast, the hyaluronidase-treated dogs showed no further reduction in blood flow to ischemic myocardium 6 hours after occlusion: transmural, 30.3 +/- 3.1 ml/min per 100 g; endocardial, 21.3 +/- 2.5; and epicardial, 38.8 +/- 3.8. These regional myocardial flows were significantly higher than those of the untreated dogs 6 hours after occlusion. Thus, salvage of damaged myocardium by hyaluronidase might be explained by its beneficial effect on collateral blood flow to the ischemic tissue, though this effect on collateral flow could be the consequence rather than the cause of this salvage.


Myocardial function and coronary blood flow response to acute ischemia in chronic canine diabetes. To examine the influence of preexistent diabetes mellitus on left ventricular performance and coronary blood flow responses to acute ischemia, mild normoglycemic diabetes was induced in nine mongrel dogs after three doses of alloxan, (20 mg/kg, iv), at monthly intervals. Hemodynamic measurements and coronary blood flow (85Kr clearance) were obtained before and after the onset of ischemia. This was produced by occlusion of the proximal left anterior descending coronary artery via a balloon-type catheter in nine intact anesthetized diabetic dogs and 10 nondiabetic dogs. During the 1st hour of ischemia in the diabetic group, the end-diastolic pressure rose from 7 +/- 1.1 (mean +/- SE) mm Hg to 23.8 +/- 2.3 without a significant increase of end-diastolic volume. In controls end-diastolic pressure rose from 8.6 +/- 1.1 mm Hg to 15.3 +/- 1.4, and end-diastolic volume was significantly increased, so that the ratio of end-diastolic pressure and volume was significantly higher in the diabetic group (P less than 0.005). Although indices of contractility did not differ, stroke volume and work reductions were significantly greater in diabetics, despite the fact that coronary blood flow was reduced to a similar extent. Size of the ischemic areas appeared comparable as judged by distribution of dye injected distal to the occlusion. Since potassium loss and sodium gain in the inner and outer layers of ischemic tissue did not differ between the two groups, the intensity of ischemia seemed similar. Glycogenolysis was unimpaired in the diabetic ischemic muscle but triglyceride levels remained elevated. Morphologically the diabetic myocardium was characterized by a diffuse accumulation of periodic acid-Schiff-positive glycoprotein in the interstitium, which was thought to limit diastolic filling of the ischemic ventricle and to contribute to the substantial reduction of ventricular performance.


Maximum rate of tension fall during isometric relaxation at end-systolic fiber length in canine papillary muscle. We measured the characteristics of the decline in tension during isometric relaxation of canine papillary muscle. In the intact heart, relaxation begins with the isovolumic phase, but in experiments on papillary muscle previously reported the isotonic phase preceded the isometric phase during the course of relaxation. In our experiments, however, the isotonic bar was locked at the instant the muscle reached the end-systolic fiber length in order to hold the fiber at the length during the succeeding relaxation process. Therefore, we obtained a relaxation process similar to that occurring in the intact heart. The major results of these experiments are: (1) Maximum rate of the decline in tension (-dT/dtmax) is linearly related to the magnitude of total load. (2) -dT/dtmax is augmented by positive inotropic interventions and diminished by negative inotropic interventions. (3) An increase in preload results in only a slight increase in -dT/dtmax. (4) End-systolic fiber length itself is not a principal determinant of -dT/dtmax. (5) -dT/dtmax divided by total load is independent of the amount of muscle shortening. We, therefore, suggest that -dT/dtmax divided by total load cand be a useful index of the relaxation characteristics of cardiac muscle.


Noninvasive assessment of cardiac function and ventricular dyssynergy by precordial Q wave mapping in anterior myocardial infarction. To determine whether multiple lead precordial electrocardiographic recordings offer an improved index for noninvasive estimation of left ventricular hemodynamic function and segmental dyssynergy, precordial mapping was performed in patients with anterior myocardial infarction, and the number of pathologic Q waves (greater than or equal to 0.04 sec) was counted (Q-Index). Left ventricular function was determined by cardiac catheterization and angiography and correlated with the Q-Index. The Q-Index correlated well with dyssynergy extent (r = 0.84) and inversely with ejection fraction (r= -0.87), stroke work index (r = -0.79) and cardiac index (r = =0.66). Three patient groups were defined by Q-Index; group I, 0.04 sec Q complexes less than 15; group II, 15-25; group III, 26-35. Q-Index related closely to functional classification and survival (mean follow-up 12.2 months): group I, 91%; group II, 81%; group III, 40%. Thus 35-lead precordial Q wave mapping with determination of total number of pathologic Q waves permits practical, atraumatic assessment of hemodynamic and functional status and allows prediction of survival in acute and chronic anterior myocardial infarction.


Left ventricular and coronary angiographic anatomy. Relationship to ventricular irritability in the late hospital phase of acute myocardial infarction. Late hospital phase ventricular arrhythmias in acute myocardial infarction (MI) have been associated with a high incidence of sudden death following hospital discharge. Thirty-eight patients were studied 10-24 days following onset of symptoms of MI. Each patient had a 24-hour ambulatory ECG tape recording and left ventricular and coronary angiography performed. Patients with complicated ventricular arrhythmias (multiform, coupled, R on T VPCs or ventricular tachycardia), when compared to those with uncomplicated ventricular arrhythmias (unifocal or no VPCs), had a greater number of proximally narrowed major coronary arteries (P less than 0.001), a higher coronary "score" (P less than 0.001), a greater incidence of previous myocardial infarction (P less than 0.005), a greater percentage of abnormal left ventricular segments 86% vs 69% (P less than 0.001) and lower ejection fractions. These data suggest that late hospital phase survivors of MI with complicated ventricular arrhythmias have more extensive coronary artery disease with greater left ventricular dysfunction than survivors with uncomplicated ventricular arrhythmias. This more extensive disease may result in increased areas of ischemic myocardium and may help explain the refractoriness of these arrhythmias to pharmacologic therapy.


Detection of remote myocardial infarction in patients with positron emission transaxial tomography and intravenous 11C-palmitate. Ischemic myocardial injury has been detected recently in isolated perfused hearts and intact experimental animals with positron-emitting 11C-palmitate and reconstructive tomography providing cross-sectional images of the heart free from superimposed activity in overlying structures. To evaluate the applicability of positron emission transaxial tomography in detecting infarction in man, 10 normal human subjects and 12 patients who sustained documented acute myocardial infarction three to 12 months previously were studied. Tomograms were obtained after intravenous injection of 5 to 10 mCi of 11C-labeled palmitate, a physiological substrate of myocardium. Tomograms from all normal subjects exhibited homogeneous distribution of 11C-palmitate throughout each 1.5 cm thick cross section of the ventricle. Tomograms from all patients with remote anterior or inferior and posterior myocardial infarction exhibited diminished accumulation of 11C-palmitate delineating regions corresponding to the electrocardiographic locus of infarction. The distribution of 11C-palmitate detectable by positron emission transaxial tomography in a series of cross sections from apex to base in the same normal subject or patient with remote myocardial infarction was analogous to that observed in normal dogs and animals with experimentally induced myocardial infarction.


Quantitative assessment of ventricular performance in unstable ischemic heart disease by dextran function curves. The ability to quantitate the amount of permanent left ventricular dysfunction in patients with unstable ischemic heart disease would have important clinical value. Left ventricular function curves were constructed in sixteen patients with acute myocardial infarctions and five patients with unstable angina pectoris syndrome at an average of 56 hours (+/- 8) after the onset of symptoms. Fifty ml increments of low molecular weight dextran were rapidly infused into the right antrium during constant monitoring of the pulmonary artery end-diastolic pressure (PAEDP) via a Swan-Ganz thermodilution catheter. An average of 400 ml (range 200-800) was infused to produce a significant change in the PAEDP (range 3-13 mm Hg). The cardiac index was measured before and after the dextran infusion. The slope of the left ventricular function curve was calculated by dividing the change in the cardiac index by the change in the PAEDP. The sixteen patients with acute myocardial infarction underwent left heart catheterization and left ventricular biplane angiography an average of six months later. The five patients with unstable angina pectoris were studied within one month. The slope value of the left ventricular function curve was compared angiographic ejection fraction by linear regression analysis and the correlation coefficient was 0.80. These data demonstrate 1) the slope of the left ventricular function curve in patients with acute myocardial infarction or unstable angina correlates well with the angiographically calculated ejection fraction; 2) as early as two days post myocardial infarction, the residual impairment of left ventricular function can be estimated.


Graded global ischemia and reperfusion. Cardiac function and lactate metabolism. The effect of global ischemia of different degrees of severity and reperfusion was studied in the isolated working rat heart. Four degrees of ischemia were induced by reducing the control total coronary flow of 8 ml/min to 0, 0.04, 0.4, or 0.8 ml/min for 30 minutes, after which the coronary flow was returned to the control level. After severe ischemia (0 and 0.04 ml/min ischemic coronary flow groups), recovery of contractility was to less than 30% of the control, pre-ischemic value of ventricular developed pressure and dP/dt, and irreversible cardiac contracture and an increased pacing threshold occurred. After moderate ischemia (0.4 and 0.8 ml/min ischemic coronary flow groups), contractile function recovered completely, ischemic contracture was rapidly reversible and the pacing threshold did not increase. The moderately ischemic groups were able to function at a stable, low level of contractility for the 30 minute ischemic period, whereas the severely ischemic groups had no contractile activity. The amount of calculated tissue lactate accumulation correlated with the occurrence of irreversible ischemic injury; the severely ischemic groups which failed to recover with reperfusion accumulated 3-5 times as much lactate as the moderately ischemic groups which recovered completely. The results suggest that relatively small differences in the severity of the ischemic condition can markedly affect the degree of tissue injury.


The effect of propranolol on microvascular injury in acute myocardial ischemia. The purpose of this study was to determine whether propranolol, which has been shown to reduce the extent of myocardial infarction, reduces microvascular injury which may play a role in exacerbating ischemia. Saline (10 dogs) or propranolol (2 mg/kg i.v., 7 dogs) was injected prior to a one hour occlusion of the left anterior descending coronary artery. Carbon black (1 ml/kg), which labels damaged and leaky vessels, was injected 5 min after release of the occlusion and allowed to circulate for two hours. By morphometric analysis of 1 micron thick sections, 75 +/- 12% of vessels and 84 +/- 7% of myocardial cells showed damage in untreated dogs; only 2 +/- 1% of vessels and 9 +/- 8% of myocardial cells showed damage in the propranolol-treated dogs (P less than 0.001). The number of carbon black-labeled vessels/10 fields/biopsy from comparable areas of ischemic tissue was 55 +/- 7 in untreated dogs and 27 +/- 3 in propranolol-treated dogs (P less than 0.001). The results suggest that propranolol not only protects the ischemic myocardial cell, but also significantly decreases the ischemic microvascular changes.


The assessment of mitral stenosis and prosthetic mitral valve obstruction, using the posterior aortic wall echocardiogram. The echocardiographic motion of the aortic root reflects, in part, left atrial filling and emptying. Patients with mitral valve obstruction were studied to determine whether clinically important alterations in patterns of left atrial emptying would alter motion of the posterior aortic wall. Patients with mitral stenosis had a characteristic pattern of slowing of left atrial emptying in early diastole, with loss of the conduit phase in mid-diastole. The atrial emptying index, defined as the fraction of passive posterior aortic wall motion occurring in the first third of diastole, was significantly related to the mitral valve area index (r = 0.86), and thus provides a noninvasive quantitation of the degree of mitral stenosis. Determination of the atrial emptying index also proved useful in the evaluation of patients with prosthetic mitral valve obstruction and in documenting improvement in left atrial emptying after mitral valve surgery.


Decreased frequency of exercise-induced ventricular ectopic activity in the second of two consecutive treadmill tests. Two exercise tests were performed with an intervening rest period of 45 minutes in a group of 13 subjects with previously identified exercise-induced ventricular arrhythmias and no resting arrhythmias. Both normal subjects and patients with heart disease were included in the group. The level of stresss was equal in both tests as judged by similar rate-pressure products at peak exercise. There was a significant decrease (P less than 0.05) in the number of VPCs induced by exercise during and after the second test. When the number of VPCs on test I and test II in the same patients were compared, a regression line fitted the data well (r = 0.92). Analysis of the recovery periods revealed significant (P less than 0.01) decreases in systolic blood pressure at one and three minutes post exercise, comparing the second to the first test. The underlying mechanism may be decreased myocardial oxygen demand during the second test as the lowered rate-pressure products during recovery (P less than 0.01) reflect. The results of this study indicate that tests of effectiveness of an antiarrhythmic drug should not be based solely on a decrease in the amount of severity of ventricular irritability between two successive exercise tests, one immediately before and the other following administration of the drug.


Adipose hypocellularity in cyanotic congenital heart disease. Height, weight, total body fat, adipocyte size (cellular lipid content), total adipocyte number, and lean body mass were studied in two groups of similar aged children with congenital heart disease. One group was comprised of 19 children cyanotic from infancy. The second group was made up of 16 asymptomatic, acyanotic patients and served as the basis for age and sex adjusted comparisons. Cyanotic patients were lighter (P less than 0.001) and had less total body fat (P less than 0.001). Although there was no difference in adipocyte size, total adipocyte number was less in the cyanotic children (P less than 0.001), suggesting that the reduced body fat found with cyanosis is due to adipocyte hypocellularity. The additional observation that cyanotic patients were shorter (P less than 0.003) and had less lean body mass (P less than 0.001) than noncyanotic children implies that early hypoxemia produces widespread abnormalities which impair growth.


De subitaneis mortibus. XXIV. Ruptured interventricular septum and heart block. Five patients with acute posterior myocardial infarction developed varying degrees of atrioventricular (A-V) block prior to rupture of their interventricular septums (IVS). In three of these five the times of septal rupture coincided with the resumption of conduced synus rhythm. During the period of A-V block, the only stable escape rhythm appeared to originate in the A-V junctional region, and in two patients early in their course there was evidence of enhanced A-V junctional automaticity. Although the A-V node contained extensive infarction in all five hearts, its distal part, and the entire His bundle and proximal branches appeared to be spared. The anatomical pattern of dissection within the ruptured IVS was similar in three hearts, extending upward to the crest of the IVS and penetrating into the interatrial septum in two of these. Multiple major coronary narrowing were present in all five hearts, especially of a dominating right coronary artery; recent thrombosis was present in the right coronary artery in four of the five cases. The A-V node artery was markedly narrowed by focal fibromuscular dysplasia in three hearts, compounding the ischemia for the A-V node and eliminating one important source of collateral circulation to the interventricular septum.


Proficiency testing: purification of lactate dehydrogenase 1 and results of its use as a reference material in the New York State program. Highly purified human lactate dehydrogenase 1 has been used in an interlaboratory evaluation and improvement progran in clinical chemistry in New York State since 1971. Although there are difficulties in determining and assigning the most nearly accurate values for test samples in the absence of a reference method and a reference material, we have minimized these difficulties by using human lactate dehydrogenase preparations purified as we describe here and suspended in the same matrix, and by utilizing "reference laboratories" that routinely are doing multiple assays to determine the most nearly accurate value. The lactate dehydrogenase used in the program is stable for longer than 1.5 years. Conversion factors were used to convert all results to U/liter at 30 degrees C. Review of the data for 1972-75 shows a marked improvement in the accuracy of virtually all methods used to determine this enzyme.


Nickel content of human palatine tonsils: analysis of small tissue samples by flameless atomic absorption spectrophotometry. We describe a method for determining the nickel content of small tissue samples by flameless atomic absorption spectrophotometry in this case biopsy specimens from human palatine tonsils. Contact between tissue samples and metallic objects was avoided, except for the use of biopsy forceps (Type No. 8150.00 Wolf, stainless steel) for collecting samples, to imitate the actual procedure when small biopsy specimens are removed from the nasal mucosal membranes in nickel workers for histopathological and chemical investigations. Nickel contamination from this instrument was insignificant at the precision of the present procedures. The mean concentration of nickel in 15 tonsils was 13.5 +/- 7.0 (SD) microng/100g (wet wt). The mean nickel concentration in eight different samples of the same tonsil was 5.6 +/- 2.7 (SD microng/100 g.


Relation between concentrations of free glycerol and triglycerides in human sera. The concentrations of total glycerol, unesterified glycerol, and glycerol deriving from triglycerides (triacylglycerols) were measured in 468 fresh human sera from unselected outpatients of a municipal hospital. The distribution mode of free glycerol was log-normal; that of triglycerides was neither normal nor log-normal. A weak but statistically approved correlation between these variables was demonstrated. A comparison of triglyceride concentrations corrected for unesterified glycerol with those based on total glycerol shows that, for the triglycerides determination, analysis for total glycerol and substraction of 0.11 mmol/liter from the resulting value appears to be justified in clinical routine. Subtraction of individual sample-blank values is unnecessary. In a triglyceride concentration range from 1.71 to 2.85 mmol/liter the 95 percentile for the possible error of this procedure ranges from + 0.04 to - 0.08 mmol/liter.


Evaluation of two commercial kits for serum gastrin assay, and comparison with a conventional radioimmunoassay procedure. We compared two gastrin radioimmunoassay kits ("Immutope" kit, Squibb & Co.; "Gastrin R.I.A." kit, Schwarz/Mann) to the conventional gastrin radioimmunoassay of Yalow and Berson [Gastroenterology 58, 1 (1970)] as run by us and by a second reference laboratory. Although both kits were found to effectively discriminate above-normal and normal values for serum gastrin, they significantly underestimated very high values (greater than 1500 ng/liter). The Schwarz/Mann kit clearly had a superior quality label (lower nonspecific binding and higher specific activity) and a shorter incubation time. However, the 90-min incubation period cited for their kit caused overestimation of gastrin values in the lower range (5-300 ng/liter), which could be corrected by prolonging the incubation to 24 h. The Squibb antibody had fairly good cross reactivity to all gastrin species tested; the Schwarz/Mann antibody had poor affinity for natural human gastrin G34-II. Good correspondence was found for sera run by both reference laboratories (y = 0.96x + 10, r = 0.997), and values obtained with the Schwarz/Mann kit correlated best (+ 0.815) with those from the conventional radioimmunoassay procedure.


Determination of plasma glucose with use of a stirrer containing immobilized glucose dehydrogenase. A stirrer containing immobilized glucose dehydrogenase has been successfully used for determining glucose in plasma. The device is usable for at least two months and for about 500 assays. The reaction was measured kinetically and linearity was observed to 4 g of glucose per liter. Tested with aqueous glucose and with deproteinized plasma, within-day and day-to-day precision were good. Interference and method-comparison (hexokinase method) were examined. The performance of this system makes the technique useful and attractive for routine use in small-volume clinical laboratories.


Amylase activity in serum and urine: comparison of results by the amyloclastic, dyed-starch, and nephelometric techniques. We assayed serum and urine specimens for amylase activity by the nephelometric (I),dyed-starch (Amylochrome) (II), and mayloclastic (III) techniques. For serum, the correlation coefficients of the regression lines were: I vs. II, 0.978 (n = 106); I vs. III, 0.736 (n = 110); and II vs. III, 0.739 (n = 108). For urine, they were I vs. II, 0.938 (n = 49); and I vs. III, 0.752 (n = 46). Because calculation of the Kolmogorov-Smirnov statistic showed the distributions to be nongaussian, Spearman rank correlation coefficients were determined and showed that I and II correlated well but neither method correlated with III. The clinical data show that I and II gave above-normal activities in every case of pancreatitis, but III gave normal values in two of eight cases. In all cases, I and II were more sensitive, giving higher amylase activities (as compared with the upper limit of normal) than did III. The nephelometric procedure is most suitable for routine and emergency testing; the dyed-starch assay is equally sensitive and reliable, but less convenient. The amyloclastic procedure appears to be less reliable.


Calorimetry, a time-honored technique with a potential in analytical work and cellular biology. Different types of calorimeters are briefly reviewed, stressing those instruments likely to be of interest in the clinical chemistry laboratory. The unspecific nature of the measuring procedure is emphasized and various pitfalls likely to cause analytical errors are pointed out. Recent work is reviewed where calorimeters have been used as analytical tools for the determination of glucose, protein, enzymes, and other substances. The results generally compared favorably with those obtained by conventional analytical procedures. In recent reports the time per analysis has been brought down to 2 min, with sample volumes in the micro range. Valuable information on the coagulation process has been obtained by use of calorimetry. I also review studies showing that intact cellular elements such as human blood cells, bacteria, and spermatozoa can successfully be investigated with calorimetric techniques. In particular, studies on human blood cells stimulated with various agents appear to be able to give valuable diagnostic information. I believe that new designs of microcalorimeters have placed an easily handled tool at the disposal of the worker in a clinical laboratory and that use of this tool can contribute to the development of our discipline.


Gas-chromatographic determination of dyphylline in serum and saliva. We describe a simple determination of dyphylline in serum and saliva by gas chromatography after solvent extraction. Dyphylline concentrations were found to be about 36% higher in saliva than in the corresponding serum.


Micromethod for measuring hexachlorophene in whole blood by gas-liquid chromatography. We describe a micromethod for measuring hexachlorophene by use of gas-liquid chromatography with a 63Ni electron capture detector. The procedure requires 100 micronl of blood for extractions of hexachlorophene, and dichlorophene is added as an internal standard. CV is 3.4% over the concentration range of 500 to 1300 microng of hexachlorophene per liter of whole blood. This procedure permits repeated measurements of hexachlorophene in newborns who are being washed with soap containing hexachlorophene.


Determination of acetaminophen and phenacetin in plasma by high-pressure liquid chromatography. We describe a sensitive and precise high-pressure liquid chromatographic method in which acetoacetanilide is used as the internal standard to simultaneously determine acetaminophen and phenacetin in plasma. Therapeutic as well as toxic concentrations can be determined on as little as 0.1 ml of plasma. Sample preparation is rapid and chromatography is complete in 5 min. Quantitation is accurate at 0.5 mg/liter concentration for both drugs. Day-to-day precision within 5% is attainable. Of 36 other drugs tested, only theophylline interfered, with the determination of acetaminophen.


Automated fluorometric analysis of galactose in blood. In galactosemia, prevention of mental retardation depends on early recognition of the disorder and institution of dietary restriction of galactose. We describe an automated fluorometric micromethod for galactose in whole blood spotted on filter paper. Galactose is oxidized by galactose oxidase to D-galacto-hexadialdose and H2O2 and measured as the highly fluorescent condensation product of homovanillic acid formed when H2O2 is acted upon by horseradish peroxidase. The procedure is 10-fold more sensitive than colorimetric procedures for galactose and is not hampered by the nonspecific fluorescence from endogenous NADPH that is encountered in methods in which galactose dehydrogenase is used. At a sampling rate of 40/h with a sample-to-wash ratio of 1/2, carryover is negligible, reproducibility is excellent, and 80% of steady state is achieved. Analytical recovery of added galactose was 95%. The method has the requisite sensitivity and accuracy for quantification of galactosemia and galactosuria in milkfed newborn infants and genetic evaluation of families of patients.


Simple procedure for measuring total protein in urine. We describe a procedure for measuring total protein in urine. The method is simple, sensitive, and free of interference from drugs that are known to affect other commonly used methods. The CV for a 1.1 g/liter urine control in daily, routine use is 4.6%. The procedure involves adsorbing the protein onto cellulose powder, binding of Ponceau S dye by the protein, washing away excess dye, and eluting the bound dye into dilute NaOH for colorimetry. Comparison of results by this method with those by the biuret and turbidimetric procedures showed good agreement in those cases where the specimens were suitable for assay by the comparison methods.


Increased concentrations of serum free fatty acids falsely increase serum thyroxine as determined by competitive protein-binding. Markedly increased concentrations of free fatty acids after a fatty meal and heparin injection already have been shown to falsely increase thyroxine values measured by competitive protein-binding techniques, where ethanol extraction in used. We report here the effect of lesser increases. In 10 patients receiving heparin during hemodialysis we found significant increases in serum thyroxine by competitive protein-binding (mean, 20 nmol/l) and in free fatty acid concentrations (164 micronmol/l). Thyroxine measured as iodine did not change significantly. In seven subjects who had fasted for 27 h, we also noted significant increases in thyroxine by competitive protein-binding (mean 63 nmol/l) and in free fatty acid concentrations (624 micronmol/l). In a single subjects, serial measurements showed a close association of the two variables and the overall correlation between the increases was good. This artefact limits the value of many currently used thyroxine methods because it imposes restrictions on transport of samples and selection of patients.


Specificity and binding capacity of human blood serum for tetrahydropterins. Binding behaviour of labelled biopterin, 6,7-dimethylpterin, 2,4-diamino-6,7-dimethylpteridin, and of their 5,6,7,8-tetrahydro derivatives was studied with human serum proteins. Binding capacity of serum proteins is in the range of 5 x 10-10--10-3 M without saturation for all these pteridines. Preincubation or competition with tetrahydrobiopterin does not influence binding of tetrahydro-6,7-dimethylpterin in these concentrations. The bulk of pteridines is bound through unspecific adsorption. Only at concentrations of less than 0.8 x 10-8 M does high affinity binding seem to be possible, corresponding to about 300 pmol/g protein, whereas physiological biopterin concentration is near 2 x 10-8 M. The binding proteins are very sensitive to ageing and lose their capacity during purification, whereas unspecific binding to serum proteins is only weakly influenced by alteration of salt concentration, pH, or temperature. Attempts to partially purify the binding proteins by ion exchange, dextran gel (Sephadex G-200), or affinity chromatography, demonstrate a specificity of tetrahydropterins for the alpha2-macroglobulin fraction. Due to the high lability of this protein fraction and of pteridine binding, purification of a protein which specifically binds tetrahydrobiopterin was not achieved.


Acid glycosaminoglycans (mucopolysaccharides) in the differential diagnosis of pleural effusion. Pleural fluid glycosaminoglycans (GAG) in 64 patients with various diseases were isolated by anion-exchange chromatography after proteolysis, and characterised by spectrophotometric, electrophoretic and enzymatic techniques. GAG concentrations ranged from 7 to 1178 microng hexuronate/ml pleural fluid. The highest values (1178, 161 and 160 micron/ml) were found in patients with diffuse mesothelioma. Over 90% of the pleural fluid GAG consisted of hyaluronic acid (HA) in these patients. In other types of pleural effusion the relative HA content varied from 42 to 70% of the total GAG. Determination of pleural fluid HA consequently appears extremely valuable in the diagnosis of the form of mesothelioma producing HA. The mean GAG concentration of  pleural fluid was significantly higher in tuberculous pleurisy than in hydrothorax (P less than 0.01), secondary malignant pleural effusion (P less than 0.0005) and idiopathic pleurisy (Pless than 0.03). It was impossible to demonstrate definite correlations between GAG and protein, and GAG and glucose concentrations of pleural fluid.


Accuracy of a calculating system in transcribing output from a reaction rate analyser. A system for the mechanized calculation and transcription of output from a reaction rate analyser has been evaluated in the performance of 8 enzyme assays, many of which show anomalous kinetic behaviour with regard to "lag" or "accelerated" phases or "blank" reactions. For all of these enzymes, a cycle of three consecutive 18-second readings per assay proved optimal, the result being based upon the final output value unless this showed marked discrepancy with the second value, in which circumstances it is helpful to refer to a strip chart recording of the progress curve. Excellent agreement was obtained with manually-derived results and the system functioned without downtime for more than one year. Marked descrepancies between manual and mechanised  results were invariably traced to technician error.


Cramps, thirst and hypertension in hemodialysis patients -- the influence of dialyzate sodium concentration. The effect of increasing dialyzate sodium concentration from 130 to 136 mmoles/l on the incidence of muscle cramps in patients on regular dialysis treatment is assessed. This change was found to be effective in markedly reducing the incidence of cramp at the cost of a slight rise in the complaint of thirst. Mean arterial pressure rose slightly but significantly and exchangeable sodium was increased to levels comparable to those measured during 1969, when with twice weekly dialysis hypertension was a problem. It is concluded that dialyzate sodium concentration may with benefit be increased to 136 mmoles/l, provided that blood pressure is carefully monitored.


Symptoms and course of chronic hypokalemic nephropathy in man. The symptoms and clinical course of chronic hypokalemic nephropathy are described in 21 patients with longstanding potassium deficiency. In 14 patients (group A) the potassium depletion was caused by malnutrition and/or abuse of laxatives and/or diuretics. 7 patients (group B) suffered from primary (6 cases) or secondary (1 case) aldosteronism. The average duration of potassium depletion was 8.8 years in group A and 3.4 years in group B. Depending on the duration of potassium depletion, chronic renal disease develops which may end in terminal renal failure. Urinalysis is non-specific or negative. The clearance of creatinine slowly decreases. Metabolic alkalosis is a constant finding and in group A occurs with a tendency to hyponatremia and hypochloremia, with the development of metabolic acidosis only in advanced renal insufficiency. In contrast to patients of group B, patients of group A have normal or low blood pressures converting to hypertension, if at all only in the late phase. The cases of group A had secondary aldosteronism (and, correspondingly, a hyperplastic juxtaglomerular apparatus). Although urinary tract infection is a regular finding in advanced stages, the clinical, radiological and histological evidence suggests that bacterial pyelonephritis, if occurring at all, is rather a complication than the cause of the disease. In 5 patients 7 instances of acute renal failure of unknown origin were observed which was lethal in one case. Another patient died from terminal renal failure, a third from an intercurrent pneumonia. Renal histology obtained from 13 patients showed the picture of diffuse chronic abacterial interstitial nephritis.


Effect of intraperitoneal diuretics on solute transport during hypertonic dialysis. Hypertonic periotoneal dialysis in New Zealand white rabbits results in increased dialyzate volume, but the sodium content of net ultrafiltrate is 109.5 MEq/l, less than extracellular fluid sodium concentration. With intraperitoneal furosemide, mean net ultrafilrate sodium concentration increased significantly to 121.2 mE1/l while ethacrynic acid had no such effect and both drugs affected dialyzate volume very slightly. Hypertonic peritoneal dialysis increased urea clearance significantly above isotonic dialysis and the addition of ethacrynic acid increased clearances further (P LESS THAN.02). Added furosemide decreased urea clearances suggesting that the effect on sodium transport is not an overall permeability change. During isotonic peritoneal dialysis, furosemide increased peritoneal permeability, i.e. urea and creatinine clearances, but a significantly higher urea clearance resulted from intraperitoneal ethacrynic acid. Furosemide influx clearance average 0.31 ml/kg/min, a mean of 27 percent of the urea clearance. The data suggest that furosemide may be useful to prevent the hypernatremia that may complicate hypertonic peritoneal dialysis, but is not as efficacious as other vasoactive drugs in enhancing peritoneal permeability.


Hyperphosphatemia.

Hyperphosphatemia. Serum phosphorus concentrations are maintained within narrow limits in humans. In the extracellular fluid most of the phosphorus is present in the inorganic form and at the level of the glomerulus greater than 90% of PO4 is ultrafilterable. The kidney plays a key role in PO4 homeostasis. Micropuncture experiments have demonstrated that 60 to 70% of the filtered PO4 is reabsorbed in the proximal tubule; however, there is evidence that a significant amount of PO4 is reabsorbed in the distal tubule. Phosphate secretion probably plays a minor role in the overall renal regulation of phosphate. In normal individuals the amount of PO4 ingested plays a key role in the amount that ultimately will be excreted in the urine. The reabsorption of PO4 along the nephron is regulated by a series of factors of which parathyroid hormone is the most important one. Hyperphosphatemia is seen frequently in clinical medicine and by far, the most common cause is a decrease in urinary PO4 excretion secondary to renal failure. From the practical point of view, the most effective way to treat hyperphosphatemia is to decrease PO4 absorption in the GI tract by the use of PO4 binders.


Hyponatremia and hypernatremia.

Hyponatremia and hypernatremia. The concentration of serum sodium is determined by the external balance of water. Hyponatremia occurs when total body water is in excess of sodium, and hypernatremia develops when body water is relatively decreased in relation to sodium. Both disorders may be present in patients with various disease states in which total body sodium is either decreased, normal or increased. The symptomatology in both disorders is related to the disturbance in central nervous system due to brain edema in patients with hyponatremia and brain dehydration, and cerebrovascular hemorrhages in patients with hypernatremia. The treatment of hypo and hypernatremia is achieved by correcting the abnormalities in body water content.


Digoxin kinetics during furosemide administration. The urinary excretion of a 0.75-mg oral dose of digoxin was assessed in 8 healthy men during a control 5-day period and then, after a washout period of at least 2 wk, during a 5-day period when the subjects also took oral furosemide, 40 mg daily. Analysis of the raw data by a paired Student's test and analysis of variance of log-transformed urinary digoxin excretion data revealed no significant difference attributable to the addition of furosemide (F = 1.16; df = 1,21). The treatment by day interaction showed the slopes to be consistent between the two treatments. It seems that in the presence of normal renal function there is no need to alter loading or maintenance doses of digoxin when furosemide is also given. Analysis of variance with log transformation of data is suggested as a practical approach to handling digoxin urinary excretion results when the transformation linearizes the data through examination by the method of orthogonal polynomials.


Pharmacokinetics and concentration-effect relationships of intervenous and oral clonidine. The kinetics of the disposition of intravenous and oral clonidine in five normotensive subjects have been determined. It is proposed that a two-compartment model adequately describes the disposition of the drug. The drug is rapidly distributed (t1/2alpha = 10.8 +/- 4.7 min) but slowly elimainated (t1/2beta = 8.5 +/- 0.9 hr). The bioavailability of oral clonidine in the tablets tested averaged 75.2% and 40 to 50% of the bioavailable dose is excreted unchanged in urine. Renal clearance of the drug showed considerable intersubject variation (1.82 +/- 0.34 ml/min/kg) and exceed the calculated glomerular filtration rate in some subjects. Oral and intravenous clonidine induced significant falls in blood pressure (greater than 20/15 mm Hg) in our normotensive subjects and consistently caused marked sedation and dryness of the mouth. Sedation and salivary flow correlated with plasma clonidine concentration over the range 0 to 4 ng/ml. Falls in blood pressure were related to plasma concentration to 1.5 to 2 ng/ml but at higher concentrations the hypotensive effect was attenuated.


Variations in the fate of triameterene.

Variations in the fate of triameterene. Triamterene is a pteridine used therapeutically as a diuretic. In order to better understand variations in effect and toxicity of triamterence in individuals, the fate of the drug in man was investigated. Both nonradioactive and 14C-labeled forms of the drug were administered, and specific methods of analysis were used to separate the parent compound from its metabolite. Individual variation in absorption, binding, and elimination was noted. The drug was excreted in bile as well as urine. Rapid and extensive metabolism of the agent occurred after oral and intravenous doses in healthy adult men. The peak plasma levels of the drug after an oral dose (200 mg) were under 0.3 microng/ml, but the concentration of the primary metabolite. (2,4,7-triamino-6-p-hydroxyphenylpteridine) was higher. The urinary excretion of the metabolite was at least three times that of the parent drug.


Aminopyrine metabolism in the presence of hyperbilirubinemia due to cholestasis or hepatocellular disease. Combined use of laboratory tests to study disease-induced alterations in drug disposition. Hepatocellular diseases, such as hepatitis, cirrhosis, or hepatic neoplasm, are associated with impaired metabolism of certain drugs, including aminopyrine, whereas cholestasis produced variable effects on aminopyrine metabolism. Reasons for the variable effects of cholestasis on hepatic aminopyrine metabolism were sought by performing in patients with hyperbilirubinemia the aminopyrine breath test (ABT), which consists of measurements of 14CO2 in breath 2 hr after oral administration of [14C]aminopyrine. Hyperbilirubinemia (total serum bilirubin less than 1.2 mg/100 ml) in these patients was due to hepatocellular disease or cholestasis. The ABT, defined as the percentage of the  administered dose of 14C excreted in breath for 2 hr after [14C]aminopyrine administration, was 6.2 +/- 0.8% (mean +/- SD) in 107 control patients with normal total serum bilirubin. The ABT was severely abnormal (less than 3.1%) in 54 of 77 hyperbilirubinemic patients (70%) with hepatocellular disease and normal (greater than 4.5%) in only 5 of these patients (6%). In contrast, the ABT was severely abnormal in only 1 of 40 cases of  cholestasis with hyperbilirubinemia and normal in 70% of these patients. Thus, aminopyrine metabolism is normal in most cases of hyperbilirubinemia due to cholestasis and is only rarely severely abnormal in these patients. On the other hand, severe abnormality in aminopyrine metabolism occurs in the majority of patients with hyperbilirubinemia due to hepatocellular disease. It therefore appears that the ABT may be useful in hyperbilirubinemia to distinguish patients with hyperbilirubinemia due to cholestasis form most patients with hyperbilirubinemia due to hepatocellular disease.


Practolol peritonitis.

Practolol peritonitis. Barium examination of the small bowel revealed fixation, dilation, separation of bowel loops and delayed small intestinal transit in nine patients with practolol induced peritonitis. The diagnosis may be first suggested by this examination, and a history of prolonged therapy with beta-blocking agents should be sought in patients showing these changes where no other cause for them can be found. Symptoms may develop up to a year following cessation of therapy so that further cases may still come to light despite the withdrawal of practolol for long-term use. Small bowel barium examination is indicated in patients who have received practolol therapy and who develop suspicious alimentary symptoms.


Detection and grading of oesophageal varices by fibre-optic endoscopy and barium swallow with and without Buscopan. Detection of oesophageal varices is important in the diagnosis of portal hypertension. We have, therefore, compared the results of fibre-optic endoscopy and barium swallow in 56 patients with chronic liver disease. Oesophageal varices were graded as small, moderate or gross by independent observers for each technique. In 12 patients varices were not detected by either method, and in six cases varices were detected by endoscopy when the barium swallow was negative. In 15 of the remaining 38 patients varices were found to be one grade larger at endoscopy than on barium swallow. Since it is possible that varices appear larger at endoscopy because of the Buscopan (hyoscine N-butyl bromide) used as a relaxant, we carried out barium examinations after Buscopan 20 mg i.v. in 23 of the patients. In eight cases varices were one grade larger after Buscopan than on the standard barium swallow, and in two were only detected after Buscopan had been given. Eight cases negative after Buscopan were also negative at endoscopy. Although emergency endoscopy has particular value in locating the site of bleeding in patients with portal hypertension and acute gastrointestinal haemorrhage, the results of the present study suggest that a barium meal with Buscopan is as accurate as endoscopy in the detection of oesophageal varices.


The evaluation of dumping and diarrhoea after gastric surgery using a physiological test meal. A test meal consisting of food (Galactomin 18), and a barium preparation (Raybar) was given to 47 patients who had undergone truncal vagotomy combined with either antrectomy or gastric drainage, and to five normal subjects. The radiological findings were correlated with the symptomatology. Total gastric emptying was not significantly different between normal controls, post-operative but asymptomatic cases, or those in whom dumping of diarrhoea was provoked. Small intestinal transit was significantly faster in cases of post-cibal diarrhoea than in other groups, and was also more rapid than normal when dumping was provoked. Colonic entry time was also faster than normal in these two groups. Marked dilution of the meal in the small bowel only occurred in the presence of the dumping syndrome. Jejunal interposition prevented the occurrence of dumping and restored the radiological appearances in the intestine to normal. The test meal gave excellent correlation with the patients' symptomatology. The relationship of the findings to the mechanisms of post-cibal diarrhoea and the dumping syndrome is discussed.


The radiographic patterns of acute mycoplasma pneumonitis. Twenty-two cases of serologically proven pulmonary mycoplasma infection have been reviewed.  All showed abnormality on plain chest radiographs. A definitive diagnosis could not be made on initial or individual films. However, studies of sequential films enabled diagnostic patterns to be distinguished.


Anatomical variations in the articular masses of the seventh cervical vertebra simulating fracture. The articular masses of the seventh cervical vertebra are variable in that about 87% of patients the superior and inferior facets are not parallel. IN ADDITION, THE INFERIOR FACETS ARE OFTEN POSTERIORLY PLACED IN RELATION TO THE SUPERIOR FACETS. These factors contribute to a notched undulating articular mass seen bilaterally on the lateral and oblique projections simulating fracture. The difference in inclination of the superior and inferior facets may cause either the superior or inferior facets to be obscured on the AP VIEW ANGLED CAUDAD--THE POSTERIOR ARCH VIEW. Since these anomalies are bilateral and fractures are almost always unilateral, the differentiation is most easily made on this posterior arch view.


The radiology of the hip joints and pelvis in cerebral palsy. Thw radiological appearances found in the pelvis and hip joints in 79 patients with cerebral palsy, aged 5--16 years, have been studied. These findings have been recorded and correlated with the clinical severity and distribution of the disease. Subluxation of the hip joints was found in 14% and dislocation in a further 6%. Spasticity was the predominant neuromuscular handicap in 69 out of 79 cases (88%). The acetabular angle and femoral neck shaft angle were related to the degree of migration of the femoral head. The acetabular angle, iliac angle, iliac index and femoral neck shaft angle were all significantly increased in the deranged group. In the dislocated group, the mean neck-shaft angle was 154 degrees. When the degree of spasticity was asymmetrical, there was a striking relationship between the laterality of the spasticity and the laterality of the deranged hip, pelvic obliquity and increased acetabular angle. A typical teardrop-shaped femoral head was seen in 48% spina bifida occulta of the lower lumbar and sacral spine in 32% and pseudoarthrosis in 3.8%. No ectopic calcification or ossification of soft tissue was noted. An awareness that these children are at risk from subluxation and dislocation of the hip and the importance of early diagnosis and treatment are emphasised.


The radiology of urinary tract abnormalities associated with hysterectomy. A review of urinary tract complications following hysterectomy shows that these are more likely to occur after radical hysterectomy than after hysterectomy for benign conditions. The role of associated radiotherapy has also been considered and the use of preoperative and postoperative radiological investigations of the urinary tract discussed. Ureteric complications include transient hydroureter and hydronephrosis, ureteric stricture, ureteric fistulae and ureteric severance. The bladder complications which may occur are vesicovaginal fistula, impingement from postoperative haematoma and abscess, the development of bladder calculi, urinary retention and prolaspe of the bladder base. Ureteric complications are often serious especially when there has been fistula or stricture formation, but other abnormalities may be transient. Serious bladder complications such as fistula formation are rare, but urinary retention and prolapse of the bladder base are not uncommon. The purpose and means of radiological investigation have been discussed, as well as some diagnostic pitfalls.


The radiology of the neurological complications of the reticuloses. The application of radiology to the diagnosis of reticulosis involving the nervous system is briefly reviewed, and the pathogenesis of spinal extradural block is discussed. Six cases are added to the existing literature.


Accuracy of grey-scale ultrasonography of liver and spleen in Hodgkin's disease and the other lymphomas compared with isotope scans. Complementary techniques to laparotomy are required to monitor patients with lymphomas both before and after treatment. Our preliminary experience with grey-scale ultrasonography is presented. Fifty-two patients, themajority with Hodgkin's disease or other lymphomas, were examined with ultrasound equipment which was custom built. The essential difference between grey-scale equipment and conventional machines is the ability to display the internal consistency of soft organs. Of 20 spleens examined prior to splenectomy, the ultrasonic scan was considered suggestive of involvement in seven of nine that showed histological evidence of Hodgkin's disease. Threeof 11 histologically negative spleens were considered to be positive preoperatively and two of nine read as negative on the scan contained histological disease. Fifty livers were examined with ultrasound. Of seven patients with histological involvement four were read as positive and three as negative and six of 43 patients with no macroscopic or microscopic evidence of liver infiltration were also considered to have a positive ultrasonic scan. Ultrasonic scanning has proved useful for demonstrating enlarged lymph nodes in the porta hepatis, upper para-aortic lymph nodes and bulky mesenteric nodes. On the basis of these preliminary observations the potential value and application of the technique is discussed.


Cestrum diurnum intoxication in normal and hyperparathyroid pigs. The effect of ingestion of dried leaves of Cestrum diurnum, a plant shown to contain a 1,25-dihydroxycholecalciferol-like principle, was tested in normal pigs fed 1.2% calcium and 1.0% phosphorus for 10 weeks from weaning and in hyperparathyroid pigs fed 0.8% calcium and 1.6% phosphorus for the same periods of time. Addition of 3% Cestrum diurnum leaf meal rapidly resulted in decreased feed consumption and weight gain, hypercalcemia and hypophosphatasemia. In normal pigs, plasma calcium rose to 16 mg/100 ml within one week and remained high for the 4 week experimental period. In hyperparathyroid pigs with hypocalcemia, plasma calcium rose to 12.75 mg/100 ml within one week and later approached 15 mg/100 ml. Ingestion of Cestrum diurnum retarded cell differentiation of growth cartilages. Arrested osteocytic osteolysis was observed within one week with osteopetrosis of epiphyses and metaphyses. The negative effect on the resorbing osteocytes then caused osteonecrosis which, in combination with lack of bone formation because of atrophy of osteoblasts, resulted in osteopenia within 4 weeks. Dystrophic calcinosis occurred within 2 weeks and was widespread after 4 weeks in lungs, kidneys, heart and vessels. Atrophy of parathyroid cells was severe after one week. Hyperparathyroid pigs responded with skeletal lesions, dystrophic calcinosis and parathyroid atrophy more rapidly and severely than normal pigs. The biochemical and anatomical changes in Cestrum diurnum ingestion are closely similar to those in vitamin D3 intoxication in pigs. Whereas pigs can tolerate large amounts of vitamin D3 because of feed-back control of 1 alpha-hydroxylation in the kidney, this control point is by-passed in Cestrum diurnum ingestion and intoxication occurs promptly.


Pine needle toxicity in pregnant mice.

Pine needle toxicity in pregnant mice. Eight groups of pregnant mice were fed various preparations of ground pine needles or aqueous extracts thereof. An additional group was fed only Purina mouse cubes. Ground pine needles were more toxic to both adult mice and embryos than were the various water extracts. Reproductive failure occurred early in the gestational period. A water soluble and heat labile toxin suggestive of a mycotoxin was demonstrated in water extracts. Pine needle fiber contained a heat stable toxin which was insoluble in water. Evidence suggested the possible presence of a third toxin lethal for adult mice. Results suggested that the heat stable toxin is the one of greatest importance as a source of reproductive failure in mice. Toxins produced by fungi were considered to be of secondary importance.


Helminthosporium speciferum as the cause of dermal and nasal maduromycosis in a cow. A 4-year-old Santa Gertrudis cow had epistaxis and loud nasal sounds associated with nasal granulomas. Multiple cutaneous granulomatous nodules were on the ears and single ones on the tail, vulva and thigh. Helminthosporium sp. was isolated from cultured lesions; two isolates from the ear and nasal mucosa were identified as H. speciferum. Tissue sections stained with hematoxylin and eosin contained rounded forms ranging from 5X5 micronM to 12X19 micronM; an indistinct hyaline wall surrounded granules and vacuoles of varying size. Occasional transverse septa were present. Periodic acid-Schiff, Gomori's Methenamine Silver and Gridley's Fungus stain treated sections contained numerous smaller pleomorphic buds and a few branching septate hyphae attached to the rounded forms. Brown pigmentation of the wall was not a consistent finding.


Calcitonin and hibernation bone loss in the bat (Myotis lucifugus). Inhibition of osteocytic osteolysis in bats  given calcitonin injections during late hibernation supported previous morphologic evidence that the bone loss during hibernation is associated with decreased calcitonin and increased parathormone activity.


Intracranial myiasis by Hypoderma bovis (Linnaeus) in a horse. Acute neurologic disease associated with intracranial migration of a first instar larva of a warble fly, Hypoderma bovis (Linnaeus), was observed in a 14-year-old Quarter Horse gelding in western Montana. The disease was characterized by incoordination of gait, circling to the left, head tilt to the right, partial paralysis of the right side of the face, and impaired vision in the right eye. Two and one-half hours after it was first noticed sick, the horse collapsed and was euthanized. Massive hemorrhage unaccompanied by necrosis or significant cellular response was present in the right side of the midbrain and pons.


Naturally occurring respiratory disease in a kennel caused by Bordetella bronchiseptica. The role of Bordetella bronchiseptica as a primary pathogen in nautrally occurring respiratory disease of dogs has been in question since its original isolation in 1911. A study to determine the incidence of B. bronchiseptica in a closed breeding kennel has revealed that the frequency of such isolations is closely associated with mild respiratory disease characterized by a moist, sometimes productive, cough. Infection with B. bronchiseptica usually occurred shortly after weaning and produced illness which lasted for one to two weeks. The organisms, however, continued to be shed for two to three months, and important factor in maintaining the infection in this kennel. Adult dogs sampled at frequent intervals did not harbor B. bronchiseptica in spite of their almost constant exposure to heavily infected pups; immunity to reinfection, therefore, appeared to develop. The involvement of several known canine respiratory viral agents was excluded by virus isolation and serological techniques. It therefore was concluded that B. bronchiseptica was the primary cause of respiratory disease in this large breeding kennel.


Cytogenetics of triplet conjugation in Tetrahymena: origin of haploid and triploid clones. Triplet conjugation occurs frequently when three different mating types of T. thermophila are mixed under appropriate conditions. Genetic studies show that fertilization is usually tripolar, with each conjugant contributing a pronucleus to one mate and receiving a pronucleus from the other. Triplets are less common and less stable when only two mating types are mixed, and nuclear exchange is not always reciprocal. Some conjugants contribute a pronucleus but do not receive one in return. Others receive two pronuclei instead of one. Such triplets characteristically yield one haploid, one diploid and one triploid exconjugant, all of which are able to establish vigorous clones.


Synaptonemal complex karyotyping in spermatocytes of the Chinese hamster (Cricetulus griseus). II. Morphology of the XY pair in spread preparations. The X and Y chromosome axes have a distinctive morphology at pachytene and are clearly distinguishable from autosomal SCs. The X and Y are totally unpaired at late zygotene when most of the autosomes are synapsed; however, their attachment points at the presumptive SC end are closely apposed. The X and Y axes pair to form a length of SC that is somewhat shorter than the unpaired portion of the Y. Unpaired axes may appear thin and sometimes double, or may thicken to form fusiform bulges that are sometimes hollow: two on the X and one on the Y. Discrete differentiations, tentatively identified as kinetochores, are often visible at the proximal end of the SC on the Y axis, and also between the fusiform differentiations on the X axis. Additional differentiations, in the form of loops and densely staining granular excrescences form on the X axis and sometimes on the Y. A further differentiation appears as a cloud-like sheath around the distal end of the X axis, often in association with a bi-lobed dense body. At late pachytene, the XY-SC remains intact, but the unpaired X and Y axes develop side branches. Just before diplotene, while the autosomal SCs are still intact, the XY-SC is lost, although the axes persist and remain together at the attachment point of the SC. The X and Y differentiations form a logical temporal sequence when grouped according to progressive structural complexity. The morphological types may serve as markers for the sub-stages of pachytene.


Lengths and patterns of transcriptional units in the amplified nucleoli of oocytes of Xenopus laevis. Transcriptionally active chromatin from peripheral amplified nucleoli of lampbrush-chromosome stage oocytes of Xenopus laevis was dispersed and spread in various solutions of low salt concentrations (including some with additions of detergents) and examined by electron microscopy. Nucleolar material from oocytes of animals with normal (2-nu) and mutant (1-nu) genetical constitution of nucleolus organizers was compared. Histograms showing the distributions of the length of matrix units, apparent spacer intercepts, and the total repeating units of the rDNA containing chromatin axes revealed a significant degree of heterogeity, with indications of subclasses and predominant repeat unit size classes of 3.3 amd 3.8 micron length. The correspondence of matrix unit length to the molecular weight of the first stable product of rDNA transcription was studied using gel electrophoresis of labelled pre-rRNA under non-denaturing and denaturing conditions. Evaluations of individual strands of nucleolar chromatin further demonstrated the existence of both (i) strands with obviously homogeneous repeating units of and (ii) strands with intra-axial heterogeneity of rDNA subunits. "Prelude complexes", i.e. groups of transcriptional complexes in apparent spacer intercepts, were not infrequently noted. The data are compared with the measurements of lengths of repeating units in fragments of rDNA obtained by digestion with EcoRI endonuclease as described by Morrow et al.(1974) and Wellauer et al. (1974,1976a, b). The results are discussed in relation to problems of variations in the modes of arrangement of the pre-rRNA genes, the state of packing of rDNA during transcription, and possible mechanisms of the amplification process.


Cytogenetic components of reproductive isolation in Trimerotropis thalassica and T. occidentalis. The grasshopper Trimerotropis thalassica (Bruner) has a diploid count of 2n=23 male (XO), 24 female (XX). The two largest autosomes pairs are regularly metacentric, a consequence of fixed pericentric inversions. The X-chromosome is also a fixed metacentric. The remaining nine pairs of autosmes are polymorphic for floating percentric inversions so that the complement consists of a mixture of telocentric and metacentric members. Trimerotropis occidentalis (Bruner) is polymorphic for comparable inversions in only two of its autosome pairs and has a telocentric X. It is however, unique among the species of the genus Trimerotropis in having only 21 chromosomes in its male diploid set in all the populations so far studied. A single male found in a mixed population of these two species at Jasper Ridge, Stanford University, was characterized by the count 2n=22 male. In both this respect of and in its phenotype it was intermediate in character, representing a natural F1 hybrid between the two species. Cytogenetic analysis of this hybrid male indicated that occidentalis is differentiated from thalassica only is respect of a single tandem translocation. This has involved two of the telocentric elements of thalassica which have fused into a single composite telocentric partly homologous with each of the smaller progenitors. Although potentially capable of forming a multiple of three, one or other of the progenitor chromosomes regularly fails to pair with the tandem product in the hybrid so that one or more univalents invariably occur. These, by lagging, prevent cytokinesis and subsequently lead to the formation of macrospermatids which inevitably produce a measure of sterility. It is argued that this sterility provides a basis of reproductive isolation.


The transformation of the Synaptonemal Complex into the 'elimination chromatin' in Bombyx mori oocytes. In Bombyx mori oocytes the synaptonemal complexes are retained in modified from pachytene to metaphase I. At the end of pachytene the length and width of the lateral components of the complex increase, whereafter the complexes become compacted during later stages of the meiotic prophase. Ultimately, at metaphase I the modified synaptonemal complexes of individual bivalents fuse to form a more or less continuous sheet between the homologus chromosomes. This sheet corresponds to the structure historically known as the 'elimination chromatin'. It is concluded that in the absence of crossing over and chiasma formation in Bombyx mori females the retainment and subsequent modification of the synaptonemal complex has evolved as a substitute mechanism to ensure regular disjunction of the bivalents.


The units of DNA replication in the mammalian chromosomes: evidence for a large size of replication units. The replication of chromosomal DNA in human and Chinese hamster cell populations has been studied by means of the DNA fiber autoradiography. It was found that the rate of DNA replication for one fork in human cells varies from 0.2 to 0.9 micron/min, the average being 0.6 micron/min. In the Chinese hamster cells the rate DNA replication is greater, varying from 0.3 to 1.2 micron/min, the average being 0.8 micron/min. There are no clusters containing a great number of replication units in human and Chinese hamster cells. Sequences consisting of two or three replicons which belong to single DNA molecule have been observed, but their frequency was relatively low. The distances between the initiation points in such sequences of replicons vary from 40 to 280 micron, the average value being 130 micron. This value represents the minimum size of the replication units which have completed the DNA synthesis within 3 h of the S-period. The DNA synthesis in most replication units fails to be accomplished within the three hours of labelling. The process can be completed only in the fragments of DNA molecules of 40 to 200 micron (the average value being 100 micron) in human cells, whereas in the Chinese hamster cells the fragments of 40 to 250 micron (the average being about 140 micron) are completely replicated. Provided that the replicaton is bidirectional the complete replicons are supposed to contain two such fragments. Consequently, the greater part of replication units in mammalian cells covers the pieces of a few hundred microns in DNA molecules. The relation between replication process at the DNA molecules level and that at the metaphase chromosome level is discussed.


The diminution of Heterochromatic chromosomal segments in Cyclops (Crustacea, Copepoda). The chromosomes of Cyclops divulsus, C. furcifer, and C. strenuus, like those of several other Copepods, undergo a striking diminution of chromatin early in embryogenesis. The process is restricted to the presumptive soma cells and occurs at the 5th cleavage in C. divulsus, at the 6th and 7th in C. furcifer, and at the 4th in C. strenus. The eliminated chromatin derives from the excision of heterochromatic chromosome segments (H-segments). Their chromosomal location is different in the three investigated species: Whereas in C. divulsus and C. furcifer the H-segments form large blocks-exclusively terminal in the former and terminal as well as kinetochoric in the latter-the germ line heterochromatin in C. strenuus is scattered all along the chromosomes. Extensive polymorphism exists with respect to the length of the terminal H-segments in C. furcifer, and with respect to the overall content of heterochromatin in the chromosomes of C. strenuus. In a local race of C. strenuus an extreme form of dimorphism has been found which is sex limited: females as a fule are heterozygous for an entire set of large (heterochromatin-rich), and a second set of small chromosomes in their germ line. Males are homozygous for the large set. In the first three cleavage divisions the H-polymorphism is solely expressed through differences of chromosome length. Following diminution the differences between homologous have disappeared. Feulgen cytophotometry demonstrates that in the three species the 1C DNA value for the germ line, as measured in sperm, is about twice that measured in somatic mitoses (germ line/soma C-values in picograms of DNA: C. strenuus 2.2/0.9, C. furcifer 2.9/1.44, C. divulsus 3.1/1.8). - The data imply that chromatin diminution is based on a mechanism which allows specific DNA segments, regardless of their location and size, to be cut out from the chromosomes without affecting the structural continuity of the remaining DNA. The mechanism may be analogous to that of prokaryotic DNA excision.