Further identification of a D/E translocation.

An infant previously reported to have psychomotor retardation and multiple congenital anomalies including hypotonia, failure to thrive, peculiar facies, low-set ears, micrognathia, and cryptorchidism in association with monosomy due to a D/E (13-15/17-18) translocation has been reexamined. Further cytogenetic studies, using fluorescent techniques, indicate the translocation involves chromosomes 15 and 17. Only two other patients with partial monosomy due to grossly similar D/E translocations have been reported. On reexamination, the translocation in one has recently been found to involve chromosomes 15 and 18 and that in the other has not been further defined.


Characterization of Hex S, the major residual beta hexosaminidase activity in type O Gm2 gangliosidosis (Sandhoff-Jatzkewitz disease).

Hex S, the major residual beta hexosaminidase activity present in tissues, fluids, and cultured skin fibroblasts of patients with type 0 GM2 gangliosidosis, was isolated and characterized biochemically and immunologically. when appropriate tissue homogenates were tested by electrophoresis on cellulose acetate gels, hex S as well as hex C, the corresponding minor beta hexosaminidase component found in normal visceral tissues, migrated with greater anodic mobilities than hex A. However, a small but reproducible electrophoretic difference was observed between partially purified hex S and hex C components. Hex S and hex C had slightly higher apparent molecular weights than those of hex A or hex G; no major differences were found between hex S and hex A in thermostability, pH optimum, or kinetic properties. Hex S, like hex C from placenta, reacted with an antiserum directed towards the unique antigenic determinants alpha of hex A, indicating that hex S, hex C, and hex A share a common antigenic determinant. No reactivity of hex S was detected with an antiserum directed toward the common antigenic determinant beta of hex A and hex B. These results suggest that further biochemical and immunologic characterization of hex S and elucidation of its relationships with hex A, hex B, and hex C may significantly contribute to the understanding of the molecular defects in the GM2 gangliosidoses.


The role of cytolysin in pneumococcal ocular infection.

A highly purified preparation of pneumococcal cytolysin was responsible for the corneal damage that ensued upon intra-corneal injection of crude cell extract into the rabbit eye. The lysin may produce this pathologic finding by effecting activation or release of corneal degradative enzymes.


Cardiac and respiratory effects of digitalis during chronic hypoxia in intact conscious dogs.

The arrhythmogenic and respiratory effects of ouabain during chronic hypoxia were studied in 10 unanesthetized dogs in a hypobaric chamber (446 mmHg) following 7-19 (mean 14.7) days of continuous exposure at this altitude. Another 15 dogs studied at sea level comprised the normoxic control group. In both groups, a 7.5-mug/kg loading dose of ouabain was followed by infusion of ouabain at 3.0 mug/kg per min to ECG evidence of toxicity. Mean arterial Po2 was 46 +/- 5 mmHg in chronically hypoxic dogs as compared to 86 +/- 7 mmHg in normoxic animals (P less than 0.001). Mean hematocrit was 54 +/- 1% in hypoxic and 43 +/- 2% in normoxic groups (P less than 0.001). In five dogs studied first at sea level and subsequently under conditions of chronic hypoxia, mean maximum left ventricular dP/dt and peak (dP/dt)P-1 were unchanged. Marked hyperventilation during ouabain infusion was observed. In normoxic dogs mean arterial pH rose from 7.43 +/- 0.05 to 7.70 +/- 0.02 U, and Pco2 fell from 41 +/- 4 to 15 +/- 1 mmHg during ouabain administration (P less than 0.001). Similar changes were observed in hypoxic dogs. There was no significant difference in the mean toxic dose of ouabain in chronically hypoxic (71 +/- 11 mug/kg) versus normoxic (78 +/- 12 mug/kg) animals. Thus, in contrast to acute hypoxia, chronic hypoxia in unanesthetized dogs was not associated with a significant reduction in the dose of ouabain required to produce toxic arrhythmias. Chronic hypoxia was also not associated with alterations in left ventricular performance.


Hemodynamics and blood flow distribution following prolonged circulation at 5 degrees C.

Circulation was maintained in profoundly hypothermic dogs for 8 h at temperatures below 10 degrees C. During cooling to 5 degrees C cardiac output and blood pressure decreased and peripheral resistance rose. Thereafter, circulatory dynamics remained relatively stable over the next 6 h with a gradual decline in blood pressure. The proportion of blood flow to the heart and brain increased with cooling and remained elevated throughout the hypothermia period. Despite continued circulation, pulmonary edema developed after 5-7 h and the dogs were nonviable when rewarmed.


Spinal autonomic afferents in elicitation of tachycardia in volume infusion in the dog.

Intravenous infusion of blood (36 ml/kg body wt) elicited tachycardia in artificially ventilated anesthetized dogs with intact autonomic innervation and in dogs with cardiac beta-receptor blockade. In contrast, infusion elicited bradycardia in dogs with section of the spinal cord at C6-C7, and in dogs with combined spinal section and cardiac beta-receptor blockade. The control heart rate was less than or equal to 110 beats/min in all the animals. The presence of infusion-induced tachycardia in dogs with beta-receptor blockade, i.e., dogs in which cardiac sympathetic efferents were blocked, and its absence in dogs with combined spinal section and beta-receptor blockade, i.e., dogs in which spinal autonomic afferents plus cardiac sympathetic efferents were blocked, may be the result of an additional interruption of spinal autonomic afferents by spinal section. It is concluded that tachycardia elicited by infusion may be partly due to a reflex with its afferent pathway in the spinal cord and its efferent pathway in the vagus nerves.


Inotropic responses to digoxin during hypoxia and autonomic blockade.

Inotropic responses to digoxin (0.08 mg/kg) were studied in dogs and compared with responses during hypoxemia and autonomic blockade. Changes in left ventricular contractility (VC) were assessed by constructing function curves relating left ventricular (dP/dt)max and stroke volume to end-diastolic pressure. Augmentation of VC was observed 20 min after digoxin infusion and continued to increase until termination of the experiment after 60 min. In animals subjected to autonomic blockade with practolol, TEAC, and atropine, the increases in VC after digoxin were substantially greater. Equally large increases occurred in blocked dogs during sustained hypoxia (Pao2 = 28 mmHg). However, in animals without blockade there was a progressive fall in VC during hypoxia despite digoxin infusion, although less than in those not given digoxin. Serum digoxin levels were measured by radioimmunoassay and did not differ significantly in blocked compared to unblocked dogs or in hypoxic compared to nonhypoxic animals. These findings indicate that digoxin protects the heart from the decrease in myocardial contractility which occurs during extended hypoxia. This protective effect is more pronounced in animals deprived of autonomic function, possibly reflecting the elimination of reflex sympathetic withdrawal ordinarily induced by digitalis.


Oxygen-linked CO2 transport in sheep blood.

We have analyzed oxygen-linked carbamate formation in sheep hemoglobin B by measuring a) the effect of CO2 on oxygen affinity and Bohr effect in red cell suspensions and dilute (1.3 mM Hb4) and concentrated (5 mM Hb4) hemoglobin solutions at 37 degrees C and b) CO2 binding curves of deoxygenated and oxygenated whole blood and hemoglobin solutions, respectively, at the same temperature. In the presence of CO2 both the Bohr effect and oxygen affinity were significantly lower in 1.3-mM Hb4 solutions than in either red cell suspensions or 5-mM Hb4 solutions, while in the absence of  CO2 Bohr effect and oxygen affinity did not differ significantly in those preparations. Likewise, the fraction of oxygen-linked carbamate obtained from CO2 binding curves was found to be higher in 1.3-mM Hb4 (0.156 M HbCO2/M HbO2) solutions than in 5-mM Hb4 solutions (0.12 M HbCO2/M HbO2) at pH 7.2. We conclude that hemoglobin concentration affects formation of oxygen-linked carbamate. Total oxygen-linked CO2 in sheep whole blood amounted to 0.18 M CO2/M O2 of which 70% is oxygen-linked carbamate. Assuming a respiratory quotient of 0.85, the contribution of oxygen-linked CO2 to carbon dioxide exchange in sheep blood was computed to be 21%.


Effect of norepinephrine on myocardial intracellular hydrogen ion concentration.

The effect of norepinephrine (NE) on the intracellular hydrogen ion concentration [H+]i of isolated rat hearts perfused with a modified Krebs-Henseleit solution (SHS) was determined. The [H+]i was calculated with the [14C]-dimethyloxazolidinedione method. Respiratory or metabolic acidosis was produced by equilibrating the KHS with 20% C02 or decreasing the [HC03-] of the KHS, respectively. Three types of experiments were carried out: 1) beta blockade--MJ 1999 (Sotalol) was added to the KHS; 2) control--no pharmacological treatment; and 3) NE-norepinephrine was added to the KHS. The effective CO2 buffer values (delta[HC03-]i/deltapHi) during respiratory acidosis were: beta blockade, 11; control, 35; and NE, 84. The production of metabolic acidosis resulted in the following [H+]i changes: beta blockade, 52 mM; control, 60 nM; and NE 7 nM. These results suggest that NE markedly attenuates the changes in [H+]i accompanying respiratory and metabolic acidosis and may account in part for previous observations that the effective C02 buffer value of cardiac muscle in vivo is greater than that in vitro.


Increased cardiac contractility in acute uremia: interrelationships with hypertension.

To study the effects of acute uremia on the inotropic state of the rat heart, we subjected rats to bilateral nephrectomy and studied their hearts in the open chest 24 h later. Uremic rats had significantly higher systolic blood pressure than sham-operated animals. Left ventricular systolic pressure and maximum dP/dt, both during ejection and isovolumic contrations, were higher for any given end-diastolic pressure in hearts of uremic rats than in sham-operated animals. This difference in performance charcteristics was not abolished by doses of propranolol that blocked the heart rate response to isoproterenol. The administration of phenoxybenzamine during the 24 h of uremia abolished the blood pressure rise in uremic rats, but the increased contractile state persisted. Treatment of sham-operated animals with methoxamine to produce the same course of blood pressure as observed in uremic rats was also associated with an increased inotropic state. These results indicate that in the rat, acute uremia is associated with an increased inotropic state that is not mediated by beta-adrenergic mechanisms. The systolic hypertension of acute uremia is not the major cause of the increased contractility, although systolic hypertension without uremia can mimic the performance characteristics found in hearts of uremic rats.


Blood oxygen affinity and alveolar ventilation in relation in body weight in mammals.

The validity of the concept relating blood oxygen affinity and alveolar ventilation to body weight in homeothermic mammals was reexamined with blood pH used as the fixed variable. Blood Po2 at 50% saturation at pH 7.4 (P50(7.4)) and PCO2 of oxygenated blood at pH 7.4 (PCO2(7.4)) from a variety of homeothermic mammals were determined at 37 degrees C by in vitro equilibration techniques. In some species, arterial PCO2, PO2, and pH were also measured. PCO2(7.4), which was similar to arterial PCO2, showed a correlation coefficient of +0.33 with body weight over the range of 28-100,000 g and +0.90 among the species under 200 g. P50(7.4) values, which ranged from 26.0 to 38.7 mmHg for all the species, were not well correlated (r = -0.43) with body weight. For the small mammals (less than 200 g) the correlation coefficient was -0.91. In vivo P50 at the body temperature and arterial pH, although different from P50(7.4), showed a similar correlation with body weight. The lack of valid generalization suggests that body weight is only one of the composite factors that influence O2 transport systems.


[Quantitative determination of aqueous humor proteins by electroimmodiffusion in an antiserum containing agarosegel (author's transl)].

An exact quantitation of aqueous humor proteins within a few hours can be attained by an modification of the quantitative estimation of the proteins by electrophoresis in agarose gel established by Laurell. The aqueous humor proteins form long immunoprecipitations in agargel. The length of the precipitats is proportional to the concentration. By determination of albumin, coeruloplasmin and transferrin the application can be pointed out.


Low plasma ionized calcium and response to calcium therapy in critically ill man.

Marked lowering of plasma ionized calcium concentrations [Ca++] occurred in eight patients (2 days to 54 years old) who required extensive pharmacologic support of the circulation. [Ca++]'s ranged from 0.21 to 0.53 mM. Only one patient survived. The hypocalcemia occurred in the absence of massive transfusion of citrated whole blood or well after such transfusions had been discontinued. These abnormally low concentrations of ionized calcium were not readily corrected by intravenous administration of calcium salts in doses generally recommended. The process responsible for inadequate hemodynamic function appeared to be associated with a severe disturbance in calcium metabolism. Contribution of the latter to the severity of hemodynamic deterioration is unclear, and little benefit from intravenous calcium therapy was found. In two patients, normal [Ca++] could not be restored by administration of CaCl2 alone, but [Ca++] rose to normal following continued calcium replacement therapy in conjunction with increased isoproterenol infusion. There was no predictable relationship between total and ionized plasma calcium concentrations. Thus, measurement of total calcium provided no indication of the level of the biologically active moiety. [Ca++] was low with both normal and low plasma pH values. The data suggest that a very high infusion rate of CaCl2 may required to restore [Ca++] to normal and that hypocalcemia occurring during low-flow states often cannot be corrected by calcium therapy alone. It is recommended that calcium replacement therapy be undertaken only with close monitoring of [Ca++].


The effect of local anesthetic, lidocaine, on guinea pig trachealis muscle in vitro.

The effect of lidocaine was studied in guinea pig trachealis muscle by dose-response reversal and protection of agonist-induced contractures in a superfusion system. Lidocaine reversed histamine, acetylcholine, and depolarizing hypertonic potassium contractures with the median effective doses of 2.8, 6.0, and 3.2 mg. Lidocaine presuperfusion shifted in a nonparallel fashion (P less than 0.05) the dose-response of histamine, acetylcholine, depolarizing potassium, and supramaximal electrical stimulation by contact electrodes. Pretreatment with 7 x 10(-6) M atropine did not modify lidocaine's inhibition of the hypertonic potassium contractions. These findings and the decrease in maximal response indicated noncompetitive antagonism. In contrast to isoproterenol, the action of lidocaine was not influenced by beta-blockade induced by superfusate propranolol, 1.0 mug per ml (P = 0.2). Lidocaine's effect on trachealis smooth muscle was facilitated by a decrease in hydrogen ion activity from pH 6.71 to 7.90, consistent with enhanced penetration of the free base. Low bolus dose lidocaine-induced contractures were noted in many studies. The potency of isoproterenol in comparison to lidocaine, as indexed by median effective doses, was 10(5) greater for reversal of histamine contractures and 10(4) greater for acetylcholine. The data were consistent with a nonspecific, reversible antagonism on the smooth muscle cell and may involve an effect on calcium activity.


Association of enteroviruses with natural and artificially introduced colloidal solids in water and infectivity of solids-associated virions.

Encephalomyocarditis viruses adsorb to introducted organic and inorganic solids in water over a wide range of pH and with various concentrations and species of metal cations. Visible flocculation of solids was not a prerequisite for significant virus association. Virus adsorption to natural solids in various types of natural waters was significant but variable. Clay-adsorbed virus retained its infectivity in tissue culture monolayers. These solids-associated viruses also retained infectivity in mice.


Chemostat culture of Escherichia coli K-12 limited by the activity of alkaline phosphatase.

The growth-limiting reaction of a chemostat culture of Escherichia coli K-12 was the hydrolysis of beta-glycerophosphate by alkaline phosphatase. The culture was buffered at pH 5.2 where alkaline phosphatase was unable to supply phosphate to the cell at a rate sufficient to sustain the maximum rate of growth. Alkaline phosphatase activity in this system is discussed in terms of the so-called Flip-Flop mechanism.


Transformation of morphine by resting cells and cell-free systems of Arthrobacter sp.

Morphine can be transformed into 14-hydroxymorphine and a related unidentified material by resting cells of an Arthrobacter species. Cell-free extracts containing the transforming enzyme(s) have been obtained. O2, Fe2+, and reduced nicotinamide adenine dinucleotide stimulate the transformation.


Derivation of high enterotoxin B-producing mutants of Staphylococcus aureus from the parent strains.

Certain pH-sensitive (membrane) mutants of Staphylococcus aureus, strains 14458 and 778, produce significantly more type-B enterotoxin (SEB) than the parent type. Some carbohydrate mutants (car) from these parent strains also are superior to the parent in SEB formation. By isolating car mutants from high-SEB-producing membrane mutants, it is possible to derive a double mutant producing from six to 50 times as much SEB as the parent type. Inversion of the sequence by isolating pH-sensitive mutants from car mutants does not yield clones with strikingly higher SEB production than the parent strain. The successful isolation sequence (pH-sensitive mutant first and car mutants derived from it) is relatively simple and virtually assures detection of a truly high-SEB-producing clone. The total number of clones whose direct assay for SEB formation is necessary for detection of a high-producing mutant is on the order of 50 to 60.


[Development in mosquitoes of 3 filaria of the South American lizards of the genus Oswaldofilaria].

The development of O. petersi, O. belemensis and O. spinosa is similar to that of O. bacillaris: the larvae are in the adipose tissue of various mosquitoes; the infective stages are characterized by the longitudinal salient ridges of the cuticule, the long tail ended by two lappets, the well-developed glandular oesophagus; the female genital anlage lies in the anterior half part of the body, but is not very far from the median line. This character opposes these species to the other viviparous Filariae and stresses the originality of the Oswaldofilariinar.


Extraction of lead from printed matter at physiological values of pH.

In an in vitro laboratory study of the extractability of lead in printed matter it was found that dangerous quantities of lead, up to 200 mug, could be extracted from "small" pieces of printed paper at pH values in the range of human gastric fluid. Lead was not extracted at pH values in the range of human saliva. Children who chew printed matter may not be in danger of absorbing lead, but the pica-prone child who swallows printed material may be at risk of absorbing excessive amounts of this toxic metal. The use of printing inks containing high-lead levels should be discouraged.


Sepsis, resuscitated hemorrhagic shock and "shock lung:" An experimental correlation.

Dogs were submitted to hermorrhagic shock, resuscitated shock and resuscitated shock plus a pulmonary bacterial insult. Pulmonary familure was absent in dogs submitted only to shock or to shock and its resuscitation. The addition of usually sub-lethal amounts of micro-organisms to the shock-resuscitated lung caused rapid death from pulmonary failure. Pulmonary failure was demonstrated by increased lung weight, hypoxemia, decreased compliance and a hemorrhagic destruction of lung tissue. These findings strongly support recent concepts of an infective genesis of "shock lung" in man.


The use of Forane anesthesia for surface-induced deep hypothermia.

The effects of Forane anesthesia for deep surface hypothermia with 30 minutes of total circulatory occlusion were evaluated. With 100% O2 6 of 7 dogs developed motor disorders postoperatively, while 3 of 5 with 98% O2/2% CO2 and none with 95% O2/5% CO2 developed motor disorders. Cooling was uneventful except for 1 episode of ventricular fibrillation in the 5% CO2 group at 23 degrees C. Resuscitation was easy, but the early rewarming period was characterized by repeated episodes of ventricular fibrillation and delayed recovery of cardiac function, especially in the 100% O2 group. Blood lactate levels remained low during cooling and gradually increased during rewarming in all groups, with the highest levels in the 100% O2 group and the lowest in the 5% CO2 group. It is concluded that Forane can be used for surface hypothermia with 30 minutes' circulatory occlusion when administered in 95% O2/5% CO2. A Comparison of these results with previously reported series indicates that Forane is inferior to ether but may be superior to halothane for surface hypothermia.


A critical study on RO-4-1284 antagonism in mice.

Ro-4-1284 is found to produce ptosis, hypothermia, sedation and miosis in mice, and the antagonsim to these symptoms is investigated. The study reports the differential effects of various pharmacologically distinct classes of compounds. The relevance of this type of experiments to possible antidepressant drug activity is discussed.


Effects of chlordiazepoxide, CNS stimulants and their combinations on avoidance behaviour in mice.

Various central stimulant drugs were tested in mice subjected to five daily 100-trial avoidance sessions in the shuttle-box. Facilitation of avoidance responding was observed following i.p. administration of metamphetamine and cocaine, but not following methlyphenidate, caffeine, pentetrazole and strychnine. Some favourable effects were obtained by combining the above stimulant drugs with cholrdiazepoxide. Advantages in the combination with chlordiazepoxide were particularly evident in the case of methamphetamine.


[Age dependence and stimulation of renal excretion of trishydroxymethylaminomethane (Tham)].

In rats of different age (5 to 240 days old) the renal excretion of Trishydroxymethylaminomethane (THAM) was studied. In 5 and in 240 days old rats the renal excretion of THAM was slower than in rats of other age groups. Stimulation of diuresis by i.p. injection of mannitol, thiazide or by oral water load resulted in an increase in THAM excretion in 5 and in 240 days old rats. The renal excretion of THAM was also increased by repeated administration of THAM in all age groups, except in new born rats. Possible mechanisms of action are discussed.


Should tonometry screening be done by technicians instead of physicians?

A random sample of 768 clinic patients 40 years or older were screened by a technician utilizing Schlotz tonometry. All patients with an intraocular pressure of 20 mm Hg or more were referred for ophthalmological evaluation. The prevalence of frank glaucoma and suspected glaucoma in this infirm population was 1.8% and 2.5%, respectively. The cost of detecting frank or suspected glaucoma decreased in the second half of the study due to a decrease in the number of false-positive tests. These findings suggest that all clinic patients 40 years or older should be screened for glaucoma. Because many internists are reluctant to screen for glaucoma and because there are large number of false-positive tests by those who do tonometry episodically, we suggest that technicians do glaucoma screening.


[Kinetics of the proteolytic activity of intact human spermatozoa].

Seminal plasma free intact human washed spermatozoa have hydrolytic activity on N-benzoil-D-L-arginine-naftilamide (BANA). Kinetic activities of this enzymatic activity are similar to those described for acrosine. Therefore, it is likely that it would be the same enzyme. BANA- hydrolytic activity might be liberated to the medium by incubation of pH lower than 4.0 and reincorporates to the membrane after adjustment of the medium to alkaline pH. pH- dependant enzyme liberation curve is similar to titration curve of sialic acid attached to the membrane. Attachment of the enzyme to the spermatic membrane diminishes its specific activity. However, if spermatozoa are previously treated with neuraminidase, its membrane afinity is inhibited. It is thought that this enzyme in the spermatozoon is present as protein ionically bound to the membrane and that sialic acid participates in this union. It is postulated that drastic treatments such as the use of ultrasound or detergents produce redistribution of specific proteins.


Kinetics and properties of beta-ketothiolase from Clostridium pasteurianum.

1. Beta-Ketothiolase of Clostridium pasteurianum was purified 130-fold by ammonium sulphate fractionation and by column chromatography using DEAE-Sephadex A-50 and hydroxylapatite. Subjected to gel electrophoresis beta-ketothiolase revealed two distinct bands; by isoelectric focusing two enzymes with isoelectric points at pH 4.5 and 7.6 were separated. As established by sucrose density gradient centrifugation the molecular weight of both enzymes was found to be 158000. 2. The condensation reaction was measured by a coupled optical test using beta-hydroxybutyryl-CoA dehydrogenase as auxiliary enzyme and either acetyl-CoA or free coenzyme A plus acetyl-phosphate and phosphotransacetylase (regenerating system) or acetyl-CoA plus regenerating system as substrates. Beta-Ketothiolase from C. pasteurianum used only 20% of the chemically synthesized acetyl-CoA; the enzyme from Alcaligenes eutrophus H 16 used 25%. When the regenerating system was added the condensation reaction continued. The enzyme from C. pasteurianum was inactivated by free coenzyme A, while the enzyme from A. eutrophus was inhibited. When acetyl-CoA was added as the substrate the initial velocity determination was impeded by the lack of linearity. With acetyl-CoA as the substrate the Km-value was found to be 2.5 mM acetyl-CoA. If free CoASH (or acetyl-CoA) plus regenerating system was added the Km was 0.44 mM (0.42 mM) acetyl-CoA. 3. The beta-ketothiolase activity was measured in the direction of acetoacetyl-CoA cleavage by an optical assay following the decrease of the enol and chelate form of acetoacetyl-CoA by absorption measurement at 305 nm. The activity was maximal at 24 nM MgCl2. The apparent Km values for acetoacetyl-CoA were 0.133 mM and 0.105 mM with 0.065 and 0.016 mM CoASH, respectively. The Km-values as calculated for only the keto form of acetoacetyl-CoA were 0.0471 and 0.0372 mM, respectively. The cleavage reaction was inhibited by high acetoacetyl-CoA concentrations; the inihibition was partially relieved by CoASH. In the range of low concentrations of acetoacetyl-CoA only a slight inhibition by CoASH was observed. The Km for CoASH was found to be 0.0288 and 0.0189 mM with 0.09 and 0.045 mM acetoacetyl-CoA, respectively. High concentrations of CoASH exerted an inhibitory effect on the cleavage reaction. With respect to enzyme kinetics and sensitivity to inhibitors and metabolites the beta-ketothiolases of C. pasteurianum and A. eutrophus were rather similar.


[Neurologic manifestations following pertussis vaccination].

Twenty cases of acute neurological complications occuring within 7 days of pertussis immunization are reported. Convulsions were present in every case and status epilepticus was observed in five infants. In only 4 cases were neurological or epileptic sequelae lacking. The clustering of neurological complications in the 24 hours following immunization is not consistent with the hypothesis of a mere temporal coincidence. However, the mechanism and incidence of post-immunization encephalopathies remains obscure and epidemiological studies are in order.


Malakoplakia. Defect in digestion of phagocytized material due to impaired vacuolar acidification?

On the basis of light and electron microscopical findings in a case of malakoplakia of urinary bladder, (1) we concur with others that malakoplakia is a hyperplastic accumulation of histiocytes, and that the formation of Michaelis-Gutmann bodies is associated with incomplete digestion of phagocytized cell debris; (2) we hypothesize that digestion within phagolysosomes is impaired because of deficient acidification; and (3) we suggest that the defect in acidification in some cases may be related to treatment with drugs that affect the mechanism of phagocytic vacuolar acidification.


Replantation of avulsed teeth. A review.

Time is a critical factor in successful replantation. The avulsed tooth is simply washed and replaced immediately. Where contamination with soil occurs anti-tetanus prophylaxis is given and some form of splinting is necessary for 3-4 weeks. Endodontic therapy is performed prior to replacement if there is a delay of more than two hours and where apex formation is complete it should be commenced within 10 days. Calcium hydroxide is useful as a root canal dressing and in controlling resorption. The value of immediate replacement and treatment should be publicised.


Resolution of proteases in the keratinolytic larvae of the webbing clothes moth.

The proteases of the larvae of the webbing clothes moth, Tineola bisselliella, were investigated because of this organism's phylogenetic rank as a member of the lower invertebrates, its unique position as one of the relatively few organisms that can digest keratin and its importance as a serious fabric pest. Both the number and nature of different proteolytic enzymes present were investigated and the various activities partially fractionated by ammonium sulphate precipitation and chromatography on DEAE-cellulose and Sephadex G200 columns. A complex mixture of peptidases and proteinases has been found in extracts of whole larvae and has been shown to be associated with the larval digestive tract. The proteinases include metal-chelator-sensitive proteinases (metalloproteinases) and serine proteinases but no SH-proteinases or acid proteinases. The serine proteinases include both trypsin-like and chymotrypsin-like activities. Four major and three minor anionic trypsin-like enzymes and a single major cationic trypsin-like enzyme have been detected. Only a single anionic chymotrypsin-like enzyme appears to be present. The trypsin-like enzymes are unaffected by the naturally occurring proteinase inhibitors, chicken ovomucoid, soybean trypsin inhibitor and lima bean trypsin inhibitor, while the chymotrypsin-like enzyme is inhibited by soybean trypsin inhibitor only. The enzymes resemble the serine proteinases from microorganisms in their pH stability. The peptidases include both aminopeptidase and carboxypeptidase activities and both are present in multiple forms. Sixteen aminopeptidase bands have been detected and all are present in individual larvae. They are not inhibited completely by reagents specific for any of the common active sites, and have different specificity requirements. Two carboxypeptidases have been detected on acrylamide gels and have been completely separated on DEAE-cellulose. No evidence could be found for the existence of any of these proteases as inactive precursors.


The rectal administration of lactulose.

A lactulose colonic washout (pH 4.5) was administered to six male patients with portal systemic encephalopathy.  Estimation of blood ammonia levels and electroencephalography were performed before and after each treatment.  Five patients recorded a significant fall in blood ammonia level as a result of lactulose therapy (P less than .025).  In the sixth subject, an enema with buffered physiological saline, pH 4.5, induced a fall in ammonia which was not increased by subsequent lactulose solution. Improvement in the electroencephalogram was recorded in all periods where a lactulose colonic washout produced a  fall in blood ammonia level.


Volatile organic components in the Skylab 4 spacecraft atmosphere.

The volatile organic components in the spacecraft cabin atmosphere of Skylab 4 were trapped on a solid adsorbent at various times during the mission. In post-flight analyses, more than 300 compounds in concentrations from less than 1 ppb up to 8000 ppb could be detected by high-resolution gas chromatography. In the samples of the 11th, 47th, and 77th day of the mission, approximately 100 components in the molecular weight range of 58 to 592 were identified by mass spectrometry. Besides components known from other environments, such as alkanes, alkenes, and alkylated aromatic hydrocarbons, components typical for the human metabolism such as ketones and alcohols were found. Other typical components in the spacecraft atmosphere are fluorocarbons (freons) and various silicone compounds, mostly normal and cyclic methylsiloxanes.


Autoaggressive reactions and leukemia virus infections: interrelated events in their pathogenesis.

Leukemia viruses can be activated from within murine hosts by immunological reactions against foreign histocompatibility antigens both during graft versus host reactions (GVHR) and host versus graft reactions (HVGR). Presence of both lymphocyte-mediated graft rejection reactions and immuno suppression appears necessary for maximal virus activation and replication in vitro, although viruses can be activated by mixed lymphocyte reactions (MLR) alone in vitro. During HVGR, viruses are first detectable in lymph nodes draining the graft site, and later reach maximal titers in the spleen. Mice infected with murine leukemia virus (MuLV) as neonates or carrying MuLV secondary to milk transmission (carriers) also develop complex autoaggressive responses. By a sensitive in vitro microcytotoxicity assay, neoplastic or pre-neoplastic thymocytes from carrier mice were found to react vigorously against normal uninfected syngeneic embryonic fibroblasts, whereas they reacted much less vigorously with similarly prepared but MuLV-infected fibroblasts. Thymocytes from uninfected animals did not react with infected or uninfected fibroblasts. Peripheral lymphocytes from carriers reacted against MuLV infected fibroblasts, but not against uninfected fibroblasts, a pattern indistinguishable from deliberately-immunized mice. It is proposed that responses analogous to GVHR are also important in the pathogenesis of leukemia following exogenous leukemia virus infection.


[Interactions of some parasympatholytics and structurally similar drugs with bovine serum albumin].

The interactions of the parasympatholytic drugs adiphenin, adiphenin H and propanthelin and the structurally related compounds, diphenhydramine, D-propoxyphene and methadone with bovine serum albumin (BSA) are studied by equilibrium dialysis and ultracentrifugation. The results show that BSA has a few binding sites (n less than 10) for the mentioned drugs. The association between the compounds under study and BSA are relatively weak. The binding constants are in the range of 10(3) l/Mol.


[Pharmacokinetic aspects of tumor tissue impairment by cancerostatics. Ways for the intensification of the induced attack in the multiple step cancer therapy. CMT-Selectine].

After discussion of the causes of natural selectivity of cancerostatics it is pointed out that, thanks to recent advances in cell kinetics (cycle-adequate selection of cancerostatics) combined with microtopography of substrate supply in the intercapillary region of cancer tissue, chemotherapeutic treatment of cancer is developing into a theoretically founded and modifiable therapy. Within the nexus of closely related problems it is especially the studies on pharmacokinetics of cancerostatics that still are in arreals. The discussed course of such an investigation basis oe the carticularly of cancer tissue, i.e., the often inadequate vascularization parameters that deteriorate even further with growing tumor size. -- The given valance equation for a change in the concentration of a cancerostatic in tumor tissue has been evaluated using, for an example, a substance from the alkylating group whose mass number approximately coindices with that of cyclophosphamide in its active form. As far as the better vascularized cancer tissue farther from the capillaries are concerned (e.g. early stages of tumors and metastases) the effective dose only drops from 1 to about 0.7. In poorly vacularized cancer tissue, on the other hand, the respective effective dose is reduced from 1 to approx. 0.25. Unfortunately, however, this drop in effective dose takes place exactly in a location where the most resistant fractions of tumor cells are to be found. A feasible way out this crucial dilemma inherent in the chemotherapy of cancer is to combine a substance of low mass numer that causes a pronounced long-term stimulation of body own defence (e.g. our BA 1, and N-(2-cyanoethylene)-urea) with a cancerostatic like CMT-selectine that -- in optimally hyperacidified cancer tissues -- reaches its active form only at a reduced pH so that (contrary to normal cancerostatics) stimulation of the body-own defence caused by the first mentioned substance is not impaired.


[Blood circulation, oxygen pressure and pH of the cerebral cortex under the influence of bencyclane].

The effect of N-[3-(1-benzyl-cycloheptyl-oxy)-propyl]-N,N-dimethyl-amine (bencyclan-hydrogenfumarate, Fludilat¿ on the cerebral vascular system was studied by means of the following methods: 1. In pigs regional cerebral cortical blood flow was continuously recorded with a heat conduction device, cortical pH with a glass electode in which the flat measuring surface and the reference were close together. Cortical pO2 was recorded with a multiwire platinum electrode. Systemic arterial blood pressure was monitored by means of a Statham transducer. 2. In a second series in cats the perivascular space of small pial arteries or arterioles was perfused with cerebro-spinal fluid (CSF) to which bencyclan had been added. The perfusion was performed by means of micropipettes. Local cortical blood flow increased slightly for some minutes after slow infusion of 1--3 mg/kg bencyclan. Rapid injection caused initially a significant decrease in arterial blood pressure, which was accompanied by a transient decrease in CBF and in cortical pH. After the return of the arterial blood pressure to its initial value, CBF increased. Cortical pO2 and cortical pH returned to initial values. After slow infusion of the substance the pH variation was usually very slight or it was missed completely. Perivascular microperfusion wtih CSF containing bencyclan caused dilatation of those pial sections which were in contact with the drug. High concentration of the drug caused stronger dilatations than did low concentrations. It is concluded that the substance causes dilatations of the cerebral vessels and that this dilatation occurs also during constant perivascular pH.


Toxicological and pharmacological investigations of pinazepam (7-chloro-1-propargyl-5-phenyl-3H-1,4-benzodiazepin-2-one): a new psychotherapeutic agent.

The pharmacological and toxicological properties of 7-chloro-1-propargyl-5-phenyl-3H-1,4-benzodiazepin-2-one (pinazepam) were investigated and compared with those of diazepam. In mouse and rat acute toxicity, in rat motor coordination and in rat metrazol convulsion tests pinazepam was compared with oxazepam too. Pinazepam, which is characterized by the presence of a propargylic side chain, showed a lower toxicity, hypnotic activity and muscular-relaxant activity than diazepam. Pinazepam and diazepam showed, however, similar activity in reducing aggressive behaviour in mice, stimulating the exploratory behaviour in rats and in potentiating hexobarbital narcosis. No clear-cut differences were observed in the anticonvulsant properties of the two drugs when tested against metrazol, strychnine and electroshock induced seizures. Pinazepam differs from diazepam for its longer duration of action. The main metabolic product found in the urine of rats and dogs treated with pinazepam was oxazepam.


The history of controlled hypotension.

As the physiology of deliberate hypotension has been unravelled, and as each new pharmacological agent has become available which either depresses or blocks peripheral vascular tone, depresses myocardial performance, or interferes with the conducting tissues within the myocardium, its possible incorporation into the armamentarium of the anaesthetist who offers induced hypotension has been considered. The result has been a sequence of variations in technique of characteristically recognizable vintage. No matter how the condition of induced hypotension is produced, there is usually vasodilatation, and the characteristic disturbance in physiology is of a loss of postural reactivity in the cardiovascular system. Important lessons have been learned concerning the management of shock states. Though it is difficult to evaluate the morbidity of deliberate induced hypotension, and terms such as "physiological trespass" have been used by its antagonists, it would appear that a well-considered and skilfully managed controlled hypotension is no more of a physiological trespass than anaesthesia, nor indeed than the trespass of the surgeon's knife itself.


An evaluation of Na+, C1- and pH ion-specific electrodes in the study of the electrolyte contents of epidermal transudate and sweat.

Ion-specific electrode detectors have been used to measure electrolytes in various biological fluids, including sweat. As part of a study of skin barrier function, we have used such electrodes to detect the presence of sodium, chloride and hydrogen (pH) in the transepidermal water loss. This paper describes the effects of cellophane tape stripping (of human forearm epidermis in vivo) on surface electrolyte concentrations, in parallel with observations on transepidermal water loss and galvanic skin resistance.


Ocular hypotensive effect of atenolol (Tenormin, I.C.I.). A new beta-adrenergic blocker.

Atenolol (Tenormin or I.C.I. 66082) is a new beta-adrenergic blocking drug, unique in being cardio-selective and in having no intrinsic sympathomimetic or membrane activity. In a controlled double-blind study, a single 50 mg oral dose produced a significant fall in ocular tension for about 7 hours in five patients with definite or suspected glaucoma. The average maximum fall was 35 per cent of the initial pressure; it occurred at 5 hours after oral ingestion. Accordingly neither intrinsic sympathomimetic nor membrane activity can account for all the ocular hypotensive effect of beta blockers in humans. The practical implications for treatment of glaucoma require longer-term investigations some of which are in progress.


Effects of dietary hydrochloric acid on voluntary food intake and metabolism of sheep in relation to the use of mineral acids as silage additives.

1. In Expt I, a pelleted grass-meal diet was supplemented with hydrochloric acid, added to the grass pellets before feeding, at five levels from 0 to 628 mmol/kg dry matter (DM). Each diet was offered ad lib. for 21 d to five sheep in a 5 x 5 Latin-square design. 2. Voluntary food intake decreased rectilinearly with increasing HCl supplementation (P less than 0.001), to 42% of the control value for sheep on the high-HCl diet. The decrease in food intake was related both to dietary pH and to the extent of metabolic acidosis induced by the HCl treatment. Although the pH of rumen fluid decreased slightly with increasing HCl supplementation, effects of the HCl treatment on volatile fatty acid concentrations in rumen fluid were not significant (P greater than 0.05). 3. In Expt 2, palatability and metabolic effects of dietary HCl were studied by comparing its effect when mixed into the pelleted grass meal before feeding, with and without a supplement of an equivalent amount of sodium bicarbonate given intraruminally, or when HCl was given intraruminally while the sheep consumed pelleted grass meal alone. Each of the three treatments was given at two levels of HCl, 280 and 560 mmol/kg DM. At each level of dietary HCl supplementation, the three treatments and the control diet (pelleted grass meal alone) were each given to four sheep, in a Latin-square design, for 11 d. 4. At the low level of supplementation, HCl, when mixed into the pelleted grass meal, reduced food intake by 17%, this effect was not altered by NaHCO3 supplementation, but when HCl was given intraruminally food intake was not reduced. At the high level of HCl supplementation, food intake was reduced by about 40% by each method of HCl supplementation; and NaHCO3 supplementation did not appreciably alter the effect of HCl on food intake, but prevented metabolic acidosis associated with the HCl treatments. Food intakes for the low-HCl treatments were significantly higher than those for the high-HCl treatments (P less than 0.01) and the level of dietary HCl x treatment interaction was also significant (P less than 0.01). DM digestibility, and the pH and volatile fatty acid concentrations of rumen fluid were not significantly affected by the different treatments. 5. It is concluded that at a low level of HCl supplementation the adverse effects of dietary HCl on voluntary food intake of sheep is determined by palatability associated with low dietary pH, whereas at a high level of HCl supplementation the effect is determined by palatability and by a metabolic response.


A study of the effectiveness of calcined magnesite as a neutralizing agent for hydrochloric acid in the diet of sheep.

1. Five experimental diets were offered ad lib. for 21 d to five sheep in a 5 x 5 Latin-square design experiment as follows: pelleted grass meal alone (control); pelleted grass meal plus hydrochloric acid (470 mmol/kg dry matter (OM); pelleted grass meal plus 470 mmol HCl/kg DM and either an equivalent amount of calcined magnesite (MgO) (235 mmol/kg DM) or twice the amount (470 mmol MgO/kg DM); pelleted grass meal plus 470 mmol MgO/kg DM. 2. MgO supplementation partly prevented the reduction in food intake caused by HCl, being more effective at the low than at the high level. When fed alone, the high level of MgO had a slight adverse effect on food intake. 3. There was no significant treatment effect on either the pH or volatile fatty acid concentrations of rumen fluid. MgO supplementation was only slightly effective in preventing the metabolic acidosis caused by HCl supplementation, as indicated by blood and urine acid-base measurements. 4. With the MgO-supplemented diets, values for the faecal and urinary excretion of magnesium were approximately 70 and 10% respectively of Mg intake and were not significantly affected by HCl supplementation. For rumen fluid, the water-soluble Mg concentration as a percentage of the total Mg concentration was similar for each treatment, approximately 90%. For faeces, the corresponding value was also similar for each treatment, approximately 30%. 5. MgO supplementation altered the effect of dietary HCl on faecal calcium excretion and on the balance of Ca, but did not alter its effect on urinary Ca excretion. 6. It is concluded that the beneficial effect of MgO supplementation on the intake of the HCl-treated diet was related more to its influence on dietary pH than on conditions in the rumen or the acid-base balance of the sheep.


Cobalt-cytochrome c. II. Magnetic resonance spectra and conformational transitions.

Between pH approximately 4 and 10 cobaltocytochrome c (Cocyt-c) gives an electron paramagnetic resonance (EPR) spectrum with g parallel = 2.035, g the perpendicular = 2.223, CoA PARALLEL = 61.4 G, CoA the perpendicular = 49.8 G, NA parallel = 15.3 G, and NA THE PERPENDICULAR = 12.5 G. Comparisons with the EPR spectra of deoxycobaltomyoglobin, deoxycobaltohemoglobin, and model compounds and together with other evidence showed cobaltocytochrome c to have Met-80 and His-18 as its axial ligands. The protons of these ligands are seen as resonances shifted by the ring-current field of the porphyrin in the 300-MHZ 1H nuclear magnetic resonance (NMR) spectra of cobalticytochrome c (Cocyt-c+). The methyl and gamma-methylene protons of Met-80 in this molecule occupy positions with respect to heme c which are somewhat different from those in ferrocytochrome c. The 1H NMR spectra also showed that the methyl groups of Leu-32, Ile-75, Thr-63, thioether bridges, and the porphyrin ring in the cobalt protein are in the same state as in native enzyme; the same is also true for Tyr-59, His-26, and His-33 and also possibly Tyr-67, Tyr-74, and Phe-82. Above pH 11, Cocyt-c is converted to a five-coordinated form having g parallel = 2.026, g the perpendicular = 2.325, CoA parallel = 80 G, CoA the perpendicular approximately 10 G, NA parallel = 17.5 G, and NA the perpendicular not resolved. Below pH 1.0 the EPR spectrum of Cocyt-c is also five-coordinated with g parallel = 2.014, g the perpendicular = 2.359, CoA parallel = 93.8 G, and CoA the perpendicular = 38.8 G. The axial ligands in the alkaline and the acidic forms of Cocyt-c are His-18 and Met-80, respectively. New prominent proton resonance peaks are observed in cobalt-cytochrome c which are either absent or weak in native cytochrome c. These are situated at 3.0, 1.7, and 1.44 ppm, attributable, respectively, to the epsilon-CH2, DELTA-CH2 + beta-CH2, and gamma-CH2 of lysyl residues in random-coil-peptides. From the areas of these peaks, it is estimated that one-two lysyl residues in Cocyt-c have been modified; four-five lysyl residues in Cocyt-c+ have been modified. These alterations of surface charged groups are probably responsible for the lowered reactivity of Cocyt-c with cytochrome oxidase and the lack of reactivity of Cocyt-c+ with several cytochrome reductase systems.


Correlation proton magnetic resonance studies at 250 MHz of bovine pancreatic ribonuclease. I. Reinvestigation of the histidine peak assignments.

The deuterium exchange kinetics of the C(2) protons of the four histidine residues of native bovine pancreatic ribonuclease A have been followed at pH 6.5 and 8.0 by proton magnetic resonance spectroscopy (1H NMR). Comparison of the order of exchange of the histidine peaks with tritium exchange rates into individual histidine residues [Ohe, M., Matsuo, H., Sakiyama, F., and Narita, K. (1974), J. Biochem. (Tokyo) 75, 1197] supports the previous assignment of histidine NMR peaks H(1) and H(4) to histidine-105 and histidine-48 but requires reassignment of peaks H(2) and H(3) to histidine-119 and histidine-12, respectively. Ribonuclease A samples having differentially deuterated histidines have been used to verify the existence of crossover points in the histidine proton magnetic resonance titration curves and to observe the discontinuous titration curve of histidine-48. Proton magnetic resonance peaks have been assigned to the C(4) protons of the four histidine residues of ribonuclease A on the basis of their unit proton areas and by matching their titration shifts with the more readily visible C(2)-H peaks of the histidines. The pK' values derived from the C(4)-H data agree, within experimental limits, with those derived from C(2)-H data. The C(4)-H peaks were assigned to histidine-12, -48, -105, and -119 of ribonuclease A on the basis of their pH dependence, pK' values, shifts of their pK' values in the presence of inhibitor cytidine 3'-phosphate, and by comparison with the assignments of the histidine C(2)-H peaks above.


Correlation proton magnetic resonance studies at 250 MHz of bovine pancreatic ribonuclease. III. Mutual electrostatic interaction between histidine residues 12 and 119.

The two adjacent active site histidine residues of bovine pancreatic ribonuclease A (histidine-12 and -119) yield proton magnetic resonance titration curves having Hill coefficients significantly less than unity (0.7 and 0.8, respectively). Three models postulating interactions with other titrating groups in the molecule have been used to approximate these anomalous experimental titration curves. Very good agreement with the data was obtained with models postulating mutual electrostatic interaction between histidine-12 and -119. The additional low pH perturbation of the chemical shift of the C(2)-H peak (but not the C(4)-H peak) of histidine-12 is attributed to a local conformational change with a pHmid of about 3.5.


Limited hydrolysis of bovine plasma albumin at neutral and alkaline pH catalyzed by associated proteinases.

Proteinase contaminants in some plasma albumin samples have previously been shown to produce cleavage of the albumin molecule at acid pH. The F conformer, existing at pH 3.8, is cleaved near erisidue number 400 to yield a large N-terminal fragment of approximately 46,000 daltons. No cleavage was found at pH above approximately 4.4. It is shown in this paper that the proteinase contaminants are active over a broad pH range from 2.5 to 11.4 provided conditions are such as to induce some breakdown of the native conformation of the albumin molecule. Addition of Tris-borate buffer (0.1 M) at pH 7.5-9 is sufficient to permit cleavage. At pH near 9 this occurs predominantly 42,000 and 27,000 daltons. Near neutral pH substantial cleavage occurs in 4-8 M urea solution or in the presence of sodium dodecyl sulfate (AD110 complex). Under these conditions there are two large fragments (42,000 and 47,000 daltons) and essentially two small ones (20,000-27,000 daltons). Under conditions where there is no cleavage at 38-40 degrees, substantial cleavage results at 50-65 degrees but enzyme inactivation also occurs toward the top of this range. The alkaline activity is inhibited by soybean trypsin inhibitor but not by pepstatin; the reverse is true of the low pH activity. Cleavage at neutral or alkaline pH under the various conditions occurs primarily at X-Leu bonds while the low pH activity was already shown to occur at X-Phe. These facts suggest the presence of at least two enzymes. There is surprisingly little pH dependence over the range 7.5-9 in any of the media examined, even though albumin is known to undergo a significant conformational change in this range, the N leads to B transition. This transition is thought to be essentially a tertiary change with little loss of helix content. It is suggested that loss of native secondary structure, especially uncoiling of helical regions, is crucial to permit attack by these enzymes.


Kinetic investigation of unfolding and partial refolding of a crab satellite (dA-dT)n.

Crab (dA-dT)n was isolated from the testes of Cancer borealis by a procedure involving separation of DNA and segregation of the satellite fraction by Hg2+ binding/Cs2SO4 density gradient ultracentrifugation. The titration of crab (dA-dT)n samples at 10 degrees indicated a sharp absorbance change at pH 11.98 in agreement with the pHm value observed for synthetic poly(dA-dT) under identical conditions. The reversal of the titration, however, resulted only in about 50% recovery of the original absorbance (at 260 nm) in marked contrast to the complete reversibility of the synthetic material. pH-jump experiments were carried out for the purpose of characterizing the rates and mechanisms of conformational transitions brought about by changes in the solution environment. It was found that the disintegration of the putative native structure of crab (dA-dT)n starts with a very fast reaction (occurring within the 6-msec deadtime of the instrument and comprising 65% of the total absorbance change) and it is completed via a slower first-order reaction (k = 66 sec minus 1). It is postulated that the first process is due to the rapid untwisting of end regions and, perhaps, some short hairpin-like helical branches present on the macromolecules. The second reaction is believed to be the end-to-end type unwinding of the double-helical backbone of crab (dA-dT)n. In the presence of low concentration (3 mug/ml) of Hg2+ ions the overall rate of disintegration process decreased drastically. pH jumps from pH values above pHm to values below were used to study the rates of absorbance changes corresponding to the refolding of the strands of denatured crab (DA-dT)n. A concentration independent process consisting of two phases was observed. The first phase was a gradual nonexponential process spanning the first second of the reaction, and the other, a very slow first-order process characterized by the rate constant value of 0.053 sec minus 1. It is proposed that the first part of the process (involving about 24% of nucleotide residues) is an intramolecular formation of helical hairpins (frequently interrupted by mismatching bases) and the second part is a manifestation of some association of the extant unpaired bases during the folding of the branched structure. Refolded crab (dA-dT)n samples when subjected again to pH greater than pHm in the stopped-flow apparatus displayed not the disintegration pattern of the native crab (dA-dT)n but rather that of synthetic poly(dA-dT. The marked facility of crab (dA-dT)n macromolecules for rapid conformational transitions induced by slight changes in the solution environment might be relevant to the biological function of this DNA.


Model studies for molybdenum enzymes. The reduction of cytochrome c by molybdenum(V)-cysteine complexes.

The reduction of ferricytochrome c by two molybdenum(V)-cysteine complexes has been investigated as a model for electron transfer in the molybdenum enzymes sulfite oxidase and nitrate reductase. The reduction by the dioxo-bridged Mo(V)-cysteine complex, di-mu-oxo-bis-[oxo(L-cysteinato)molybdate(V)] (I), is relatively slow and its rate is first order in cyt cIII and zero order in I (k = (1.09 +/- 0.10) times 10(-3) sec minus 1, pH 7.5, 20 degrees). The reduction by the monoxo-bridged complex, mu-oxo-bis[oxodihydroxo(L-cysteinato)molybdate(V)] (II), is extremely rapid and its rate is first order in both reactants (k = (2.6 +/- 0.7) times 10(7) M minus 1 sec minus 1, pH 7.0, 25 degrees). Above pH 7.5, the reduction by II follows biphasic kinetics due to the fast reduction of a low pH form of cyt cIII and a slower reduction of a high pH form (at pH 10.0, 25 degrees, k = 2.9 times 10(6) M minus 1 sec minus 1 for the low pH form and k = 7.2 times 10(4) M minus 1 sec minus 1 for the high pH form). Reaction mechanisms for reductions by both I and II are proposed and the biological implications of the results, both for sulfite oxidase and mechanisms of electron transfer to cytochrome c, are discussed.


Effects of pH and thiols on the kinetics of yeast glyoxalase I. An evaluation of the random pathway mechanism.

The disproportionation of alpha-ketoaldehydes, catalyzed by yeast glyoxalase I, has been reported to involve a random pathway mechanism where one branch utilizes the hemimercaptal of glutathione and the alpha-ketoaldehyde in a one-substrate pathway, and the other branch utilizes first glutathione and then the alpha-ketoaldehyde in an ordered two-substrate pathway. The relative importance of the two pathways has been evaluated at 5 degrees in the pH range 3-7, using methylglyoxal and phenylglyoxal as representative aliphatic and aromatic alpha-ketoaldehydes, by comparing initial rates of hemimercaptal formation in the absence of enzyme with initial rates of product formation in the presence of high enzyme concentrations. If the enzyme is not added last, the initial rates of product formation are the same as the initial rates of adduct formation even under conditions where it could be shown that dehydration of the hydrated alpha-ketoaldehyde is not entirely rate determining. If the enzyme is added after hemimercaptal formation, there is a "burst" of product formation equivalent to the amount of hemimercaptal, followed by a slower reaction, consistent with the one-substrate pathway. Additional support for this pathway was obtained from a study of the effects of added thiol reagents on the "burst" kinetics. The broad specificity of yeast glyoxalase I for both aliphatic and aromatic alpha-ketoaldehydes, reflected in Vmax values which are insensitive to the nature of the alpha-ketoaldehyde drops abruptly if the side chain of the alpha-ketoaldehyde is sterically crowded. The hemimercaptal of tert-butylglyoxal has a Vmax 300-fold smaller than Vmax for methylglyoxal; 2,4,6-trimethylphenylglyoxal is essentially inactive as a substrate even though the closely related compound 2,4-dimethylphenylglyoxal is a normal substrate. Analysis of the Vmax and Km (or Ki) values of these alpha-ketoaldehydes suggests that sterically crowded side chains affect both enzyme-substrate formation and the catalytic reaction.


Biodegradative ornithine decarboxylase of Escherichia coli. Purification, properties, and pyridoxal 5'-phosphate binding site.

The biodegradative ornithine decarboxylase of Escherichia coli has been purified to apparent homogeneity. At its pH optimum (pH 7.0), the enzyme exists as a dimer of 160,000 molecular weight. Aggregation of the dimer was promoted by lower pH values. The enzyme requires pyridoxal 5'-phosphate for activity. The coenzyme appears to be bound in Schiff base linkage as suggested by spectral studies and inhibition by NaBH4. The following sequence was determined for the coenzyme binding site: Val-His-(epsilon-Pxy)Lys-Gln-Gln-Ala-Gly-Gln. The properties of this enzyme are compared with the other biodegradative amino acid decarboxylases that have been isolated from E. coli.


Reversible reaction of cyanate with a reactive sulfhydryl group at the glutamine binding site of carbamyl phosphate synthetase.

Carbamyl phosphate synthetase from Escherichia coli reacts stoichiometrically (one to one) with [14C]cyanate to give a 14C-labeled complex which can be isolated by gel filtration. The formation of the complex is prevented if L-glutamine is present or if the enzyme is first reacted with 2-amino-4-oxo-5-chloropentanoic acid, a chloro ketone analog of glutamine which has been shown to react with a specific SH group in the glutamine binding site. The rate of complex formation is increased by ADP and decreased by ATP and HCO3-. The isolated complex is inactive with respect to glutamine-dependent synthetase activity. However, the reaction of cyanate with the enzyme is reversible. The rate of dissociation of the isolated complex is not affected by pH (over the pH range 6-10), is greatly increased by ATP and HCO3-, and is decreased by ADP. The allosteric effectors ornithine and UMP have no effect on either the rate of formation or the rate of dissociation of the complex; however, the apparent affinity of the enzyme for ATP is decreased by UMP and increased by ornithine. The site of reaction of cyanate with carbamyl phosphate synthetase, which is composed of a light and a heavy subunit, is with an SH group in the light subunit to give an S-carbamylcysteine residue. The binding of L-[14C]glutamine to the enzyme and the inhibition of glutamine-dependent synthetase activity by the chloroketone analog are both prevented by the presence of cyanate. The reaction with cyanate is considered to be with the same essential SH group which is located in the glutamine binding site and is alkylated by 2-amino-4-oxo-5-chloropentanoic acid. The bicarbonate-dependent effects of ATP suggest that formation of the activated carbon dioxide intermediate is accompanied by changes in the heavy subunit which functionally alter the properties of the glutamine binding site on the light subunit. The allosteric effects of ornithine and UMP are probably not related to this intersubunit interaction.


Isolation and characterization of papaya peptidase A from commercial chymopapain.

Chromatography on a column of SP-Sephadex shows that commercial chymopapain contains three components with proteolytic activity. Each behaves as a single protein upon rechromatography and electrophoresis on polyacrylamide gels. The major component, which represents 31% of the activity applied to the column and is the most basic protein, was identified as papaya peptidase A. This enzyme has no methionine and isoleucine on its N-terminus. Its molecular weight is about 24,000 as determined by sodium dodecyl sulfate polyacrylamide electrophoresis and sedimentation equilibrium centrifugation. Its fluorescence emission as a function of pH resembles that for unactivated papain. Reduction is required for full activity, and in general it is less active than papain against substrates such as casein, N-benzoyl-L-arginine ethyl ester, N-tosyl-L-arginine methyl ester, N-benzoyl-L-arginineamide, and N-benzoyl-DL-arginine p-nitroanilide. Of the other components isolated from crude chymopapain, the more acidic enzyme contains 20% of the activity applied to the column, has a molecular weight of about 25,000, and N-terminal residues of tyrosine and glutamic acid. The other enzyme represents 26% of the initial activity, has a molecular weight of about 28,000 and tyrosine on its N-terminus. Both proteins have a single residue of methionine per molecule. The more acidic component resembles chymopapain A, and the other enzyme is similar to chymopapain B.


Correlation proton magnetic resonance studies at 250 MHz of bovine pancreatic ribonuclease. II. pH and inhibitor-induced conformational transitions affecting histidine-48 and one tyrosine residue of ribonuclease A.

The microenvironment of histidine-48 of bovine pancreatic ribonuclease A was investigated by proton magnetic resonance spectroscopy (1H NMR) using partially deuterated enzyme in which resolution of the C(2)-H resonance of histidine-48 was simplified. The NMR titration curves at 100 and 250 MHz of histidine-48 of ribonuclease A are discontinuous both for the enzyme alone in 0.3 M chloride and for its complex with cytidine 3'-phosphate. This suggests that titration of histidine-48 occurs only as the result of a slow conformational transition. The sum of the peaks corresponding to histidine-48 in the acid-stable and base-stable forms of the enzyme is less than one proton in the transition region, which indicates that there exists at least one intermediate conformational form of the enzyme. The transition from the acid-stable form to an intermediate form has a pHmid of 5.6, and the transition from an intermediate form to the base-stable form has a pHmid of 6.9. In ribonuclease S and in ribonuclease A in the presence of 0.3 M acetate, the titration curve of histidine-48 is continuous, and the area of the peak is uniform throughout the titration. Proton NMR difference spectra at 100 and 250 MHz reveal a pH-induced conformational change with a pHmid of 5.7 that affects the chemical shift of a single tyrosine residue. This conformational transition is absent in ribonuclease S and is altered in ribonuclease A by the presence of either acetate or cytidine 3'-monophosphate. It is postulated that the same conformational transition is responsible for both the tyrosine perturbation and the disappearance of the histidine-48 peak observed in the acid-stable form of the enzyme. It is proposed that the perturbed tyrosine is tyrosine-25. The transition with pHmid 5.6 is attributed to dissociation of aspartic acid-14, and the transition with pHmid 6.9 is assigned to dissociation of histidine-48. A peak in the aromatic region that moves upfield on addition of the competitive inhibitor cytidine 3'-monophosphate is assigned to a tyrosine, and evidence is presented that this tyrosine is tyrosine-25. Inhibitor binding appears to induce a conformational change in the histidine-48/tyrosine-25 region which is remote from the active site.


The self-association of the reduced ApoA-II apoprotein from the human high density lipoprotein complex.

The molecular properties of the single linear chain form of human apoA-II, i.e., Cm apoA-II, have been evaluated by circular dichroism, polarization of fluorescence, difference absorption, and sedimentation equilibrium. The self-association of Cm apoA-II to a dimer resembles closely that of apoA-II though the free energy change is somewhat smaller. The dimerization of Cm apoA-II is accompanied by major changes in secondary and tertiary structure. The apoA-II molecule, therefore, represents a molecular association where the intramolecular structure is strongly dependent on the quaternary structure.


Relation between redox potentials and rate constants in reactions coupled with the system oxygen-superoxide.

Univalent oxidation-reduction reactions coupled with the oxygen-superoxide system were investigated in the reactions shown in eq 3 and 8, where Q and Q.- stand for p-benzoquinone and p-benzosemiquinone, respectively. From kinetic experiments the following rate constants were obtained at pH 7.0:k3 = 4.5 x 10(4) M-1 sec-1 and k8 = 3 x 10(-2) M-1 sec-1. With known values of k-3 and k-8, and of E0' for the systems Q-Q.- (0.10 V) and Cyt c3+ - Cyt c2+ (0.255 V), the calculated values of E0(O2-O2.-) were found to lie in the range between -0.27 and -0.33 V.


Complete tyrosine assignments in the high field 1H nuclear magnetic resonance spectrum of the bovine pancreatic trypsin inhibitor.

The low-field portions of the 250-MHz 1H nuclear magnetic resonance (NMR) specra of native and chemically modified bovine basic pancreatic trypsin inhibitor (BPTI) have been studied as a function of pH over the range pH 5-13. Resonances associated with the 16 protons of the aromatic rings of the four BPTI tyrosines have been located and assigned to specific tyrosyl residues. Titrations of pH yielded pK's for tyrosines-10, -21, -23, and -35 of 10.4, 11.0, 11.7, and 11.1, respectively. The resonances associated with the nitrotyrosine-10 protons of mononitrated BPTI and the nitrotyrosine-10 and -21 protons of dinitrated BPTI have been similarly located, assigned and titrated yielding pK's for nitrotyrosine-10 and -21 of 6.6 and 6.4, respectively. The high-field NMR spectrum indicates that the aromatic ring of tyrosine-35 rotates less than 160 times per second at 25 degrees for pH's in the range 5-9.


Competitive inhibitors of renin. Inhibitors effective at physiological pH.

Previously we reported the development of competitive inhibitors of renin effective at pH 5.5 (Poulsen, K., Burton, J., and Haber, E. (1973), Biochemistry 12, 3877). At physiologic pH (7.5), the inhibitory constants (Ki) increased and solubility decreased to the point that inhibition could not be demonstrated with these peptides. Modification of the octapeptide sequence, His-Pro-Phe-His-Leu-Leu-Val-Tyr, either by addition of serinol to the carboxyl terminus or by replacement of valine-7 with an isosteric threonyl residue failed to yield peptides active at pH 7.5. Attachment of polyproline sequences to the amino terminus increased solubility from threefold to tenfold and decreased Ki so that competitive inhibition was demonstrable at physiologic pH. In addition, if leucine-6 was replaced in these peptides with a phenylalanyl or tyrosyl residue, Ki decreased (3-12 muM) to give effective competitive inhibitors at physiologic pH in both buffer and in plasma.


Circular dichroism of holo- and apoprotocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa.

Circular dichroism studies have been carried out on both apo- and holoprotocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa, in the absence and presence of competitive inhibitors, protocatechualdehyde and 4-nitrocatechol. The apo- and holoenzyme showed identical spectra in the ultraviolet region between 200 and 250 nm (peptide back bone region), but the low intensity negative bands at 330 and 480 nm of the holoenzyme were completely absent in the apoenzyme. On the side chain region, the positive ellipticity peaks of the holoenzyme change into a lower intensity and broader band indicating the participation of aromatic amino acid residues in the primary binding of iron ion. Under anaerobic conditions, spectral changes were evident in the side chain region for the binary complexes of both the holo- and the apoenzyme with protocatechuate. The presence of iron in the holoenzyme results in an increase in positive ellipticity between 290 and 320 nm. Either with or without the iron, the enzyme protein binds protocatechuate and has a greater positive circular dichroism increase at 240-260 nm. CD difference spectra indicate that the modes of binding to form the binary complexes of holo- or apoenzyme with either substrates or competitive inhibitors are different. The bound iron ion stimulates binding. Spectral changes of the holoenzyme in the aromatic region were also observed in different pH environments of lower enzymatic activity. It is still not established whether these aromatic residues play an active or passive role in the binding of iron and/or substrates and inhibitors.


Enolase catalyzed beta,gamma-alpha,beta isomerization of 2-phospho-3-butenoic acid to (Z)-phosphoenol-alpha-ketobutyrate.

2-Phospho-3-butenoic acid was synthesized and found to be a substrate for both yeast and rabbit muscle enolase (EC 4.2.1.11). Enolase catalyzes the isomerization of 2-phospho-3-butenoic acid to (Z)-phosphoenol-alpha-ketobutyrate, a beta,gamma-alpha,beta isomerization. Nuclear magnetic resonance studies on the product indicate only one isomer is formed. This reaction provides indirect evidence in further support of a carbanion intermediate for the enolase reaction. 2-Phospho-3-butenoic acid is also a good competitive inhibitor of both yeast and rabbit muscle pyruvate kinase (EC 2.7.1.40).


Methylation and ethylation of uridylic acid and thymidylic acid. Reactivity of the ring and phosphate as a function of pH and alkyl group.

At pH 6.8 in aqueous solution (4 hr, 22 degrees), all methylating agents tested, i.e., dimethyl sulfate, methyl methanesulfonate, and methylnitrosourea, react with both the N-3 of the ring and the phosphate of UMP and dTMP. Although the extent of reaction varies from 17 to 76%, the ratio of phosphate/ring methylation is approximately 4. Both the 3-methyl nucleotides and methyl ester of 3-methyl nucleotides are identified, as well as the methyl esters of unmodified UMP and dTMP. At pH 8.2 the extent of total methylation is similar but reactivity of the N-3 is increased and that of the phosphate decreased so that the phosphate/ring ratio is approximately 1. At pH 6 almost all reaction is with the phosphate group. Uridine, under the same conditions, is methylated at pH 6.8 to form 15% 3-methyluridine and, at pH 8.2, the N-3 of uridine and thymidine is methylated to about 50%. Neither uridine nor UMP forms detectable ribose methyl products at any of these pH's. The comparable ethylating agents (diethyl sulfate, ethyl methanesulfonate, and ethylnitrosourea) are less reactive and the total ethylation of UMP or dTMP is about 1/5 that of methylation. There is little ethylation of the N-3 but the phosphate is alkylated to a relatively high extent so that the phosphate/base ratio at pH 6.8 is 10-23, and at pH 8.2 the ratio is 5-8. The fact that ethylating agents have a greater affinity than methylating agents for alkylating phosphates is proposed as the basis for the previously reported analytical data in which ethylating agents, acting on DNA or RNA at neutrality, form more phosphotriesters than the analogous methylating agents.


DNA-dependent protein methylase activity in bull seminal plasma.

The existence of a DNA-dependent protein methylase activity without any concomitant DNA methylase activity was demonstrated in bull seminal plasma. The enzyme utilized S-adenosyl-L-methionine as a methyl donor, and endogenous seminal plasma protein as the substrate. There was no demonstrable enzyme activity when the seminal plasma was preheated at 100 degrees for 10 min, or when the enzyme reaction mixture was incubated at 4 degrees. The protein methylase required a heterologous DNA source, had optimal activity at pH 8.1, and was enhanced in the presence of Mg2+, NH4+, and reduced glutathione. After the methylated protein product was separated from DNA by extraction with 0.2 M HCl, the incorporated radioactivity was shown to be totally solubilized by incubating the protein either with Pronase or 1 M NaOH, while RNase and DNase had no effect. Approximately 70% of the enzymatically synthesized amino acids in the protein product were tentatively identified as O-methylated amino acid ethers by virtue of their elution from a Dowex 50 H+ column with 0.2 M pyridine, and their stability to acid and base hydrolysis. The partially purified methylated product was shown by Sephadex G-50 chromatography to consist of three distinct radioactive proteins with molecular weights of approximately 21,000, 15,000, and 10,000.


The possible relationship between a membrane conformational change and photosystem II dependent hydrogen ion accumulation and adenosine 5'-triphosphate synthesis.

Data are presented which suggest that photosystem II dependent hydrogen ion accumulation and ATP synthesis can occur only after the lamellar membranes have undergone a conformational change. This membrane conformational change is detected by the electron transport dependent incorporation of diazonium benzene[35S]sulfonate into membrane components. Previously it was established that electron flux from the photosystem II primary acceptor to plastoquinone is a necessary event for the occurrence of the diazonium-detected conformational change. These studies indicate that the release of hydrogen ions during photosystem II oxidation of the primary reductant is also a necessary event for the diazonium-detected conformational change. When iodide were substituted for water (or other proton-releasing donors) as the primary reductant of system II the conformational change did not occur even though a substantial rate of electron flow from the primary acceptor to plastoquinone occurred.


Conformational studies on the beta subunits of human hemoglobin and their arginyl-COOH peptides.

The beta subunits of hemoglobin upon alkylation of the cysteinyl residues with iodoacetamide showed a sedimentation velocity with an S20w, near 1.8 as for monomeric subunits. They reacted with alpha chains to give a tetrameric hemoglobin with a sedimentation constant near 4.4. Their CD spectrum was indistinguishable from that of untreated beta chains below 270 nm, otherwise they showed some deviation that became pronounced in the Soret region, where the optical activity of the alkylated subunits was definitely lower than that of the native subunits. Upon removal of the heme the apo-beta subunits showed a decreased optical activity in the far-uv region of the spectrum indicating a substantial loss of helical content. Their sedimentation behavior was consistent with the presence of large aggregates, which dissociates into monomers upon reconstitution with cyanoheme. The apo-beta subunits could be renatured from 6 M guanidine hydrochloride. They showed a stoichiometric reaction with heme in the molar ratio 1:1. Upon reconstitution with the heme their optical activity became similar to that of the native beta chains in the far-uv region of the spectrum, but remained lower in the near-uv and Soret regions. After acylation of the lysyl residues with citraconic anhydride the apo-beta subunits were digested with trypsin and the arginyl-COOH peptides beta(1-30), beta(31-40), beta(41-104), and beta(105-146) were separated by gel chromatography. With the exception of the peptide beta/105-146), which was insoluble at neutral pH, the sedimentation behavior of the other peptides showed the presence of small polymers. The sedimentation behavior of the peptide beta(31-40) was not tested. The percentage of alpha helix, beta conformation, and of random coil (or unordered structure) of the various proteins and peptides was measured fitting their CD spectra in the far-uv region with the parameter published by Y.H. Chen et al. ((1974), Biochemistry 13, 3350) and by N. Greenfield and G.D. Fasman ((1969), Biochemistry 8, 4108). In this way the helical content of the native and reconstituted alkylated beta subunits appeared to be near 76%, a value very near to that present in the same subunits in the hemoglobin crystal. The helical content of the apo-beta subunits in 0.04 M borate buffer at pH 9.6 decreased to a value near 45%. The helical content of the isolated peptides in electrolyte solutions was in any case near 10% indicating an almost complete loss of the structure that they have in the hemoglobin crystal. Cyanoheme reacted with the peptide beta(41-104), however, the reaction was not stoichiometric indicating a low affinity of the heme for the peptide. With the exception of the peptide beta(31-104), all of the other peptides recovered some of their helical structure when dissolved in 50% methanol. Notably also the apo-beta subunits did so suggesting that the loss of structure upon the removal of the heme could be in part due to the exposure of the heme pocket to water.


Purification and properties of an anti-B hemagglutinin produced by Streptomyces sp.

An anti-B hemagglutinin was purified to homogeneity from the culture filtrate of a strain of Streptomyces sp. by affinity chromatography. The Streptomyces hemagglutinin was adsorbed to insolubilized gum arabic and eluted with 1 M NaCl containing 1 M D-galactose. The purified hemagglutinin is thought to be homogeneous judging from sodium dodecyl sulfate-polyacrylamide gel electrophoresis at pH 7.2, disc gel electrophoresis at pH 4.3, isoelectric focusing, and ultracentrifugation. The molecular weight was estimated to be 11,000 from results of gel filtration in 6 M guanidine hydrochloride (Gdn-HCl), sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and sedimentation equilibrium analysis. The amino acid analyses revealed that the hemagglutinin contained large amounts of alanine, glycine, and valine, 47% of the total amino acid residues, and no phenylalanine. Carbohydrate analysis demonstrated that the hemagglutinin might not be a glycoprotein. The circular dichroic (CD) spectrum of the protein is quite different from those of usual proteins in having a large positive peak at 226 nm (theta = 10,000) and a negative band at 212 nm (theta =-2600). The hemagglutinin showed a typical precipitation curve with gum arabic, and agglutinated human blood group B erythrocytes 256 times as strongly as A or O erythrocytes. These activities were not affected by pH (from 4 to 12). The anti-B activity was further confirmed by serological tests. The hemagglutination-inhibition studies indicated that D-galactose was inhibitory, but alpha-D-galactosides were not necessarily better inhibitors than beta-D-galactosides. L-Rhamnose was the best inhibitor among the monosaccharides tested, and L-arabinose and D-fucose were also inhibitory.


Chemical accessibility of tyrosyl and lysyl residues in turnip yellow mosaic virus capsids.

The chemical accessibility of tyrosyl residues in TYMV capsids was studied by spectrophotometric titration and with the nitrating agent tetranitromethane. That of the lysyl residues was probed with trinitrobenzenesulfonate. Attempts to test their accessibility in virions were also made. Since some of these reactions were accompanied by structural changes, degradation of the particles were monitored with ultracentrifugation and light-scattering measurements. Alkaline titration of TYMV capsids induced ionization of two of the three tyrosyl residues per subunit at pH 11.3, but the third tyrosyl ionized with an apparent pK of 12.65, concomitantly with the degradation of the capsids. Reaction with tetranitromethane suggested that one tyrosyl residue per subunit can easily be nitrated and initiates degradation, after which the remaining residues also react. In intact capsids, five out of seven lysyl residues per subunit reacted readily with trinitrobenzenesulfonate. The other two lysyl residues were trinitrophenylated only after degradation of the capsids. On the other hand, all seven lysyl residues per subunit were easily trinitrophenylated in virions, during which reaction the virions disintegrated. The demonstrated  chemical inaccessibility of specific numbers of tyrosyl and lysyl residues in TYMV capsids and the observed structural consequences to the capsids when the residues were made to react are consistent with previously published properties of the cysteinyl and tryptophanyl residues. The findings suggest that in the capsid the central region of the TYMV polypeptide chain is buried and might represent a site of contact between neighboring subunits.


Bovine brain adenosine 3',5'-monophosphate dependent protein kinase. Mechanism of regulatory subunit inhibition of the catalytic subunit.

Adenosine 3',5'-monophosphate (cAMP) dependent protein kinase (EC 2.7.1.37) catalyzes the phosphorylation of serine and threonine residues of a number of proteins according to the following chemical equation: ATP + protein leads to phosphoprotein + ADP. The DEAE-cellulose peak II holoenzyme from bovine brain, which is composed of regulatory and catalytic subunits, is resistant to ethoxyformic anhydride inactivation. After adding cAMP, the protein kinase becomes susceptible to ethoxyformic anhydride inhibition. Ethoxyformic anhydride (2mM) inhibits the enzyme 50% (5 min, pH 6.5, 30 degrees) in the presence of 10 muM cAMP, but less than 5% in its absence. The substrate, Mg2+-ATP, protects against inactivation suggesting that inhibition is associated with modification of the active site. Addition of regulatory subunit or Mg2+-ATP to the isolated catalytic subunit also prevents ethoxyformic anhydride inactivation. These results suggest that the regulatory subunit shields the active site of the catalytic subunit thereby inhibiting it. In contrast to the bovine brain or muscle DEAE-cellulose peak II holoenzyme, the bovine muscle peak I holoenzyme is susceptible to ethoxyformic anhydride inactivation in the absence of cAMP.


On the role of sulfhydryl groups in the structure and function of the Azotobacter vinelandii RNA polymerase.

Exposure of sulfhydryl groups as indicated by titration kinetics is decreased under conditions where RNA polymerase exists as a dimer or higher aggregate (low salt), in the presence of Mn2+, or when bound to d(A-T). Incubation of phenylmercurisulfonate with RNA polymerase above pH 9.0 results in loss of d(A-T) binding ability. Poly(U) binding is more sensitive to sulfhydryl modification and is lost as pH's above 8.0. The presence of 4 mM Mn2+ has an obvious effect in stabilizing the polymerase-poly(U) complex when incubated with 10 muM phenylmercurisulfonate + 1 M urea. Incubation of the enzyme with the mercurial and urea results in disaggregation to subprotomeric forms and release of the alpha subunit. Similar treatment in the presence of 4 mM MnSO4 stabilizes the protomeric structure of the enzyme. During chain elongation the enzyme exists as a ternary d(A-T)n-enzyme-r(U-A)n complex in which the bound d(A-T)n is refractory to the destabilizing effect of the mercurial; however, further phosphodiester bond formation is inhibited. The results are defined in terms of a role which reflects the involvement of polymerase sulfhydryl groups in the various conformations necessary for subunit-subunit interaction, tight template binding and catalytic activity.


Nuclear magnetic resonance studies of residual structure in thermally unfolded ribonuclease A.

A proton nuclear magnetic resonance study of the four histidine residues of thermally unfolded ribonuclease A has provided evidence that two of the residues are in regions of residual structure, whereas the other two are freely exposed to solvent. Histidine-48 and, tentatively, histidine-105 occupy an environment at 69 degrees characterized by residual structure and display a pK value of 5.75 and a spin-lattice relaxation time of about 0.8 sec at pH 5.5. Histidine-12 and, tentatively, histidine-119 are in an environment at 69 degrees which is freely accessible to solvent and show a pK value of 5.96 and a spin-lattice relaxation time of about 1.1 sec at pH 5.5.


Affinity labeling of catechol O-methyltransferase by N-haloacetyl derivatives of 3,5-dimethoxy-4-hydroxyphenylethylamine and 3,4-dimethoxy-5-hydroxyphenylethylamine. Kinetics of inactivation.

In an attempt to elucidate the relationship between the chemical structure and the catalytic function of catechol O-methyltransferase (COMT), several classes of affinity labeling reagents have been synthesized and their interaction with COMT has been studied. Earlier studies have shown that various N-haloacetyl derivatives of 3,5-dimethoxy-4-hydroxyphenylethylamine were effective affinity labeling reagents for this enzyme. In this report we have shown that N-haloacetyl derivatives of the isomeric 3,4-dimethoxy-5-hydroxyphenylethylamine also rapidly and irreversibly inactivate COMT ant they satisfy many of the criteria established for affinity labeling reagents. This latter group of agents appear to modify a nucleophilic residue at the active site of COMT different from that modified by the 3,5-dimethoxy-4-hydroxyphenylethylamine series. Evidence to support this conclusion has been obtained by comparing the kinetics of COMT inactivation and the substrate protection profiles for these two classes of affinity labeling reagents.


Some properties of mitochondrial glutathione.

1. The presence of GSH in rat liver mitochondria is confirmed. GSH diffuses from the suspended particles in the presence of phosphate but respiratory inhibitors inhibit the diffusion. 2. GSH is oxidised in situ by oxidants including t-butyl hydroperoxide. The products formed include GSSG and GSS-protein mixed disulphides. The oxidation occurs at lower oxidant concentrations if phosphate or oxaloacetate are also present. Respiratory inhibitors abolish their effect. 3. With phosphate, the GSSG produced is found chiefly outside the mitochondria whereas with oxaloacetate, it is found chiefly inside. 4. The GSSG formed by the oxidation is reduced by Krebs-cycle acids with the exception of the ketoacids. Exogenous GSSG is reduced by these substrates only after lysis. Intact particles, however, catalyse the reduction of GSSG by either NADH2 or NADPH2.


Studies on the binding of 1-anilino-8-naphthalene sulfonate to very low density and high density himan serum lipoproteins.

Very low density and high density lipoproteins have been isolated from human plasma and their interaction with 1-anilin0-8-naphthalene sulfonate has been studied under different conditions of pH and added salt. Intrinsic fluorescence of bound 1-anilino-8-naphthalene sulfonate was higher for high density lipoproteins then for very low density lipoproteins, but was unaffected by salt in both systems. Binding of 1-anilino-8-naphthalene sulfonate by both these lipoproteins was saturable and was higher in the presence of added NaCl or CaCl2, Ca2+ having a greater effect than Na+ in enhancing fluorescence. The binding data were analyzed by Scatchard plots; the number of binding sites and the affinity of 1-anilino-8-naphthalene sulfonate for the site increased with increasing salt concentration. Fluorescence pH curves were similar to those published for phospholipids. From these and previous observations it is suggested that the phospholipids probably represent the major binding sites for 1-anilino-8-naphthalene sulfonate.


Stimulation and inhibition of pancreatic phospholipase A2 by local anesthetics as a result of their interaction with the substrate.

1. At low concentrations the local anesthetic dibucaine stimulates hydrolysis by pancreatic phospholipase A2 of phospholipids extracted from rat liver mitochondria or microsomes, whereas at higher concentrations it inhibits. The action of this enzyme towards membrane-bound substrates is barely influenced by low, but inhibited by high concentrations of dibucaine. 2. Butacaine, which is a weaker anesthetic, stimulates hydrolysis of extracted phospholipids and inhibits that of membrane-bound substrates, both actions being concentration dependent. 3. The inhibitory potency of dibucaine is several times higher in NaCl than in sucrose solutions and strongly increases with decreasing pH. Neither one of these two effects is the result of a change in binding efficiency of the anesthetic to the substrates. 4. Extracted total membrane lipids bind considerably less anesthetic than an equivalent amount of native membrane. Liver phosphatidylethanolamine is more effective in binding of dibucaine than liver phosphatidylcholine. 5. Binding of dibucaine to the phospholipase, as studied by equilibrium dialysis is at the lower level of detectability. According to the same method dibucaine is unable to displace 45Ca2+ bound to the enzyme. 6. These results are interpreted as to support the view that local anesthetics interfere with pancreatic phospholipase activity by means of interaction with the substrate rather than with the enzyme.


Control of ketogenesis from amino acids. III. In vitro and in vivo studies on ketone body formation lipogenesis and oxidation of tyrosine by rats.

Ketone body formation from tyrosine was studied in rat liver in vitro with special references to the activities of tyrosine aminotransferse (EC 2.6.1.5)  and p-hydroxyphenylpyruvate hydroxylase (EC 1.14.2.2). Liver was obtained from rats which had been given a high protein diet or cortisol to induce various levels of tyrosine aminotransferase. The enzyme activities of the preparations were plotted against the amounts of ketone body formed from tyrosine. It was found that over a low range of tyrosine aminotransferase activities, activity was proportional to the amount of ketone body formed. However, above this range, ketone body formation ceased to increase and p-hydroxyphenylpyruvate started to accumulate. This inhibition of ketone body formation and accumulation of the p-hydroxyphenylpyruvate could be prevented by addition of ascorbate. These results suggest that the primary factor regulating metabolism of tyrosine in vitro is tyrosine aminotransferase and when the activity of this is high so that it is no longer rate limiting, p-hydroxyphenylpyruvate hydroxylase becomes the rat limiting step because its activity is inhibited by the accumulation of p-hydroxyphenylpyruvate. For in vivo studies rats were given a high protein diet or cortisol to induce various levels of tyrosine aminotransferase and then injected with a tracer dose of [U- or 1- 14C]tyrosine. Then their respiratory 14CO2 and the incorporation of 14C into total lipids of liver were measured. The amounts of radioactivity in CO2 and lipids were found to be proportional to the tyrosine aminotransferase activity and were not affected by the free tyrosine concentration in the liver. After injection of [U- 14C]acetate the radioactivities in CO2 and lipids were not proportional to the tyrosine aminotransferase activity. These results indicate that the enzyme activity also regulates tyrosine metabolism in vivo. In vivo studied gave no evidence of the participation of p-hydroxyphenylpyruvate hydroxylase in regulation of tyrosine metabolism.


13C-nuclear magnetic resonance studies of 85% 13C-enriched amino acids and small peptides. pH effects on the chemical shifts, coupling constants, kinetics of cis-trans isomerisation and conformation aspects.

The 13C chemical shifts of several 85% 13C-enriched amino acids and small peptides were studied as a function of pH. The results show that the chemical shifts of carbon atoms of ionizable groups vary significantly within the zone of their pK. Generally with the pH GOING FROM 7 to 1 all the deltaC are shifted more or less upfield with the exception of the carbonyl group carbon of the second last residue which is shifted slightly downfield. This suggests the formation of an hydrogen bond at acid pH involving in a seven-membered ring the C=O in question and the COOH terminal. The percentage of cis and trans conformers of glycyl-L-proline and glycyl-L-prolylglycine were studied as a function of pH. The trans form is always preponderant whatever the pH. The accessibility of the carbonyl group to protonation of the proline residue strongly influences the cis-trans equilibrium. Thus, with the pH varying from 7 to 1, the trans isomer changes from 61 to 85% for glycyl-L-proline and only from 77 to 80% for glycyl-L-prolylglycine. The proton NMR studies underline the important differences existing between the two molecular forms of glycyl-L-proline. The cis conformation is characterized with regard to the trans form by the non-equivalence of the alpha-protons of the glycine residue, by a lower pK(1) and by a larger deltadeltaHalpha of the proline residue as a function of pH. These results could suggest an end-to-end interaction in the cis form of the glycyl-L-proline molecule. The 13C-13C coupling constants were also studied as a function of pH. The results show that J(Co-Calpha) of a C-terminal residue, varying from 5 to 6 Hz and reflecting thhe pK of the carboxylate group, is a linear function of delta(Co) and delta(Calpha) as in the case of the amino acids. The total variation of the electron density of those two carbons in an amino acid is approximately 40% weaker than in a C-terminal residue. The charge distribution along the Calpha-C(o) bond, however, is practically the same in both cases. Finally the ratios of the conversion rate constants of the two isomers cis-trans of glycyl-proline were calculated at different pH values; the relations between the isomer percentages and delta(Co), delta(Calpha) on the one hand and the J(Co-Calpha) on the other were established.


Induction of multiple forms of tyrosine aminotransferase by amino acid mixtures of different compositions.

Hepatic tyrosine aminotransferase (EC 2.6.1.5) was induced in rats by intubation of amino acid mixtures (complete or tryptophan-free). Enzyme activity was increased 4-fold by the complete mixture and 8-fold by the tryptophan-free mixture. The enzyme was analyzed by chromatography on CM-Sephadex. Chromatographic patterns were characteristic of the type of inducer rather than of the chronology of the induction cycle: after induction by the complete amino acid mixture the three forms of the enzyme were equally increased whereas after induction by the tryptophan-free mixture Form I was preferentially increased.