[The role of laparoscopy in pulmonary disease (author's transl)].

Laparoscopy demonstrated sarcoidosis in 79 of 94 proven cases. In addition, it made it possible to assess the involvement of intraabdominal organs which cannot be judged clinically or biochemically and may not be parallel to changes in the thoracic X-ray. Extensive liver sarcoidosis was revealed in 12 and required treatment independent of other organ manifestations. Miliary exudative or cavernous pulmonary tuberculosis in 15 cases revealed gross non-specific changes in one, miliary foci of the liver surface in 13 and coarse nodular infiltration in one. In each case the visual findings were confirmed histologically in the liver biopsy, in 11 before the bacteriological results were known, which retrospectively were positive in only 9 instances. Laparoscopy is, therefore, an important and relatively sparing test, also in confirmation of pulmonary disease (sarcoidosis, certain forms of tuberculosis).


Transfer characteristics of triamterene and its analogs. Central nervous system, placenta, and kidney.

Earlier in vivo studies revealed a low concentration of triamterene in the brain of guinea pigs and baboons, and a transfer of the drug from the fetus to the mother. Additional investigations have been performed to characterize further the transport system(s) for triamterene in the central nervous system (CNS), placenta, and kidney. In guinea pigs a very low brain to free plasma concentration ratio (0.1) was achieved 3.5 min after drug administration and was maintained during 180 min of drug infusion. The cerebrospinal fluid (CSF) concentration was similar to the concentration of the drug in the brain. A higher brain to free plasma concentration ratio was gradually reached in dogs studied with nanogram per ml and microgram per ml concentrations of triamterene in CSF. Administration of triamterene to fetal and maternal sheep revealed a placental extraction (E) from fetal plasma to placenta 20 times greater than that from maternal plasma to placenta. The E from fetal plasma to placenta was unaffected by a triamterene concentration in the maternal circulation 10 times that in the fetus. These findings and studies of renal clearance support an active transfer of triamterene by the CNS, placenta, and kidney; the physiologic substrate for these systems is unknown.


[Rapidly debilitating disease with generalized lymphadenopathy, skin involvement and interstitial pulmonary infiltration (report of three cases)].

Generalized but well-circumscribed lymphadenopathy and rash-like skin changes were observed in three men, aged 58 to 75 years. There was a reticular appearance in the chest X-ray. Dyspnoea, weakness, marked weight loss, changing but marked lymphopenia, markedly increased blood-sedimentation rate, and an always negative Tine test were present in all three. Despite antibiotics, cytostatic drugs and prednisolone the disease quickly ended fatally with high fever, general debilitation and pneumonia. Post-mortem examination revealed diffuse lymphatic hyperplasia with plasma-cell infiltration in the lymph nodes, tonsils and lymphatic tissue of the intestines, and diffuse hyperplasia of the endothelial venules, together with basophilic blast cells, eosimophilic granulocytes and reticulum cells with broad nucleoli. The spleen was normal or enlarged. Spleen, lung tissue and lymphatics, the skin in the area of the small vessels, hair follicles and sweat glands contained lymphocytes, plasma cells and eosinophilic leucocytes. The spleen, if enlarged, also had focal necroses in its periarteriolar septa. In two cases electrophoresis revealed beta-globulin poorly demarkated from gamma-globulin, doubling and increase. In the third case, IgA was markedly increased with one each monoclonal IgG1 und IgA, and corresponding shift in the chi/lambda relationship in serum. Immunohistologically, lymphocytes were made up of about 20 percent IgG-containing and about 25 percent IgA-containing cells. Lymphopenia, if present, was associated with markedly reduced blast transformation of peripheral lymphocytes to phytohaemagglutinin. The granulocytes were defective (intracellular killing of Candida albicans and Staphylococcus aureus), while the nitrobluetetrazolium test was normal. There was RBC phagocytosis in the macrophages of bone marrow and in Kupffer cells of all three cases.


Enterohepatic circulation of bis(p-chlorophenyl)acetic acid in the rat.

After intravenous administration of 14C-bis(p-chlorophenyl)acetic acid (DDA) to biliary cannulated rats, 97% of the dose of 14C was recovered in the bile in 24 hr. When 14C-1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT) was administered orally, approximately 8% of the dose was recovered in the bile in 3 days. The metabolite in bile in each case was a conjugate of DDA, probably with glucuronic acid. Thus less than 10% of a single dose of DDT was metabolized to DDA. The biliary metabolite was shown by two methods to undergo extensive enterohepatic circulation. The role of biliary excretion and enterohepatic circulation in the elimination of DDT metabolites is discussed.


[Consumption coagulopathy and isolated platelet deficiency in childhood septicaemia].

In a retrospective study 40 children were selected out of 53 cases of septicaemia with thrombocytopenia. They were divided into two coincidentally equally large groups of patients with consumption coagulopathy on the one side and patients with isolated thrombocytopenia without consumption coagulopathy on the other side. Both groups were of comparable age and sex distribution. Two-thirds of the children were under three months. For the differential diagnosis of both groups the activated partial thromboplastin time, the thrombotest, the factor V plasma concentration, the serum concentration of fibrin (fibrinogen) degradation products as well as control coagulation studies can be considered to have the greatest diagnostic value. The results of the study permit the following conclusions: 1. Platelet deficiency in sepsis does not prove the presence of consumption coagulopathy. 2. Consumption coagulopathy and isolated thrombocytopenia differ statistically significantly according to the bacteria cultured from the blood, the circulatory state and the pH of the blood. 3. The finding of thrombocytopenia in a patient with shock, acidosis and gramnegative septicaemia justify the suspicion of consumption coagulopathy.


Correlation of aryl hydrocarbon hydroxylase activity of human lymphocyte cultures and plasma elimination rates for antipyrine and phenylbutazone.

A high correlation was observed between the aryl hydrocarbon hydroxylase activities in short-term lymphocyte cultures of 23 individuals and their plasma half-lives of antipyrine and phenylbutazone. Individuals with low inducibility of aryl hydrocarbon hydroxylase activities had very long plasma half-lives of antipyrine and phenylbutazone, whereas subjects with high inducibility of aryl hydrocarbon hydroxylase activites had relatively short plasma half-lives. Individuals with intermediate aryl hydrocarbon hydroxylase activities displayed intermediate half-lives for both drugs. The observed correlation indicates determinants which are common to the elimination of antipyrine or phenylbutazone, and aryl hydrocarbon hydroxylase metabolism of hydrocarbons. The differences in rates of drug elimination are probably due to genetic differences and may have pharmacological and therapeutic  significance.


In vitro studies on irreversible binding of halothane metabolite to microsomes.

Incubation of rat liver microsomal suspensions with [14C] halothane resulted in the binding of a metabolite of halothane to microsomal phospholipids and, to a lesser extent, to microsomal proteins. The binding was inhibited by carbon monoxide, required NADPH, and was greatly enhanced under anaerobic conditions. The binding to lipids occurred only to phospholipids, particularly those with characteristics of phosphatidylcholine and phosphatidylethanolamine. The binding was dependent on the time of incubation as well as on the amount of microsomes present.


[Cellular immunity in actively conditioned recipients of renal allotransplants (author's transl)].

The presence of circulating "killer" cells was demonstrated in actively "conditioned" rats with permanently accepted renal allotransplants. Immunocompetant cells of these animals were able to develop a normal graft-versus-host reaction 100 and 200 days after the transplantation. The ability of these recipients simultaneously to accept secondary donor-specific skin transplants without demonstrable immunological rejection is compatible with the concept that the cytotoxic potential of these cells in vivo is inhibited by enhancing antibodies.


[Cardiac side-effects of bencyclan].

Bencyclan (Fludilat), used therapeutically as a vasodilator drug, exerts a distinct negative inotropic and chronotropic action on myocardium, in contrast to papaverine.  It prolongs the functional refractory period in the isolated heart preparation.  In isolated myocardial mitochondria it decreases the velocity of oxidative phosphorylation and the rate of calcium uptake.  These results indicate that heart function should be checked if bencyclan is applied at high dosage, especially if other cardio-depressive substances such as narcotics, antidepressive and antiarrhythmic agents as well as beta-adrenergic blockers are used at the same time.  On the other hand, the results suggest that bencyclan should be tested for possible use in the treatment of ischaemic heart disease as well as ventricular and supraventricular tachycardia.


Cardiac output in the conscious and anaesthetised horse.

Cardiac output in the horse was measured before and at predetermined times during 2-hour periods of thiopentone-halothane and thiopentone-diethyl ether anaesthesia. Left ventricular stroke volume was decreased to a similar extent during anaesthesia with each volatile agent, but a greater reduction in cardiac output occurred during halothane anaesthesia. This finding reflected the differing effects of halothane and ether on heart rate, a slight bradycardia occurring with the former agent while ether produced a small degree of tachycardia. The latter effect was attributed to enhanced sympathoadrenal activity. Changes in cardiac output and stroke volume were considered in relation to other factors, including arterial blood pH and tensions of oxygen and carbon dioxide. Positive correlations between some of these variables and cardiac function were established. With both volatile agents the reductions in stroke volume and cardiac output were related to the duration of anaesthesia, being greatest during the early stages. Possible reasons for the tendency of stroke volume and cardiac output to return towards control levels are discussed.


The effect of exercise on blood metabolite levels in the horse.

The effects of exercise of different intensities on blood concentrations of glucose, lactate, pyruvate, free fatty acids and glycerol were studied in a group of clinically normal horses. Blood lactate, pyruvate and lactate/pyruvate ratio increased during exercise, particularly during galloping. These changes occurred within the first 12-15 seconds of exercise indicating that anaerobic metabolic pathways are brought into use very quickly in the strenuously exercising horse. Since blood glycerol levels were significantly increased during exercise body lipids were also mobilised. At the same time, free fatty acid levels increased during cantering but decreased during galloping indicating increased fat oxidation during strenuous exercise. It was concluded that both lipids and carbohydrates are as important energy sources in the exercising horse as in other species.


Ventilation and cardiovascular studies during mechanical control of ventilation in horses.

Eleven out of 12 horses were underventilating while breathing spontaneously during halothane anaesthesia with high arterial carbon dioxide tensions. In addition, large alveolar to arterial oxygen tension gradients were found to be present. Mechanically, controlled ventilation with an intermittent positive pressure of 20-30 cm H2O reduced arterial carbon dioxide levels to normal. The alveolar to arterial oxygen gradients did not increase and in some cases decreased. These (A - a) Po2 gradients were due mainly to true shunt of the order of 30 per cent and not to ventilation perfusion inequality.


The effects of thioketo substitution upon uracil-adenine interactions in polyribonucleotides. Synthesis and properties of the alternating polynucleotides poly(r(A-s2U)) and poly(r(A-s2s4U)).

The polynucleotides poly[r(A-s-2U)] and poly]r(A-s2s4U)] have been synthesized and characterized by nearest-neighbour analysis, sedimentation analysis as well as spectroscopic techniques. Absorption-temperature profile and absorption-pH profile of poly[r(A-s-2U)] did not reveal a structural transition between 10 and 95 degrees C even at low ionic strength, although a variety of properties indicated a helical structure of poly[r(A-s-2U)]: remarkable hyperchromicity of the absorption spectrum, circular dichroic spectrum displaying extrema of large amplitudes, resistance against hydrolysis by ribonuclease and interaction with ethidium bromide in a manner which is characteristic of helical polynucleotides. Our results show that interactions of the type A-s-2U and A-s-2s-4U do in fact exist in helical polynucleotides. The properties of poly]r(As-2U)] furthermore demonstrate the general stabilizing effect of 2-thioketopyrimidine bases in helical polynucleotides by virtue of vertical stacking interactions with neighbouring pyrimiding and purine bases.


3Alpha-hydroxysteroid dehydrogenase from Pseudomonas testosteroni: kinetic properties with NAD and its thionicotinamide analogue.

The kinetics of 3alpha-hydroxysteroid : NAD oxidoreductase (EC 1.1.1.50) from Pseudomonas testosteroni (ATCC 11996) have been investigated. The kinetic analysis based on initial activity measurements and product inhibition studies, indicates that the addition of substrate to the enzyme and the release of products from it, follows an obligatory order (ordered bi bi mechanism). The ability of the enzyme to utilize the thionicotinamide analogue of NAD (sNAD) as cofactor has been investigated using various 3alpha-hydroxysteroids from both the C19, C21, and C24 series. The results show that the reaction velocity with sNAD as the cofactor is generally lower than with NAD. The decrease, however, varies considerably, being negligible with some steroids such as litocholic acid and deoxycholic acid and very pronounced with other such as tetrahydrocortisol and tetrahydrocortisone. The introduction of an 11beta-hydroxy or an 11-oxo group into the steroid molecule significantly reduces the ability of the enzyme to attack the 3alpha-hydroxy group. No such effect could be seen when the 11-hydroxy group was in the alpha-position. The results also indicate that, whereas NAD can serve as cofactor for both the monomeric and the dimeric forms of the enzyme, sNAD only acts as cofactor for the monomeric form. Thus sNAD is a valuable tool for the study of the reversible, concentration-depenedent monomeric-dimeric transition of the 3alpha-hydroxysteroid dehydrogenase.


[Synthesis of 2,3-diazaphenothiazine. IX. Derivatives of 1- and 4-alkylamine, triazole and N-10-dialkylaminoalky 2,3-diazaphenothiazine].

Methods of synthesis are described for 10H-pyridazino[4,5-b] [1,4]benzothiazine with alkylamine groups at the 1 and 4 position, triazole derivatives of the same heterocycle containing a pentadecanoic chain and derivatives with dialkylaminoalkyl groups at N-10. The results of pharmacological and microbiological examination are reported.


Effect of rising intragastric pH induced by several antacids on serum gastrin concentrations in duodenal ulcer patients and in a control group.

The interrelationship of the effects of antacids and of rising intragastric pH on serum gastrin levels was examined by comparing the effect of three antacids (Mg(OH)2Al(OH)3, and CaCO3), and their nonbuffering chloride compounds (MgCl2, AlCl3, and CaCl2), on serum gastrin and intragastric pH in duodenal ulcer patients. In the case of calcium, the effect of CaCl2 with a pH of 10, and in another group, CaCl2 with a pH of 2, was studied. In the case of magnesium, a control group was also investigated. In another group, the effect of a nonionized suspension (BaSO4) was studied in order to assess the contribution of intragastric volume. Serum gastrin was determined radioimmunologically, and pH measurements were performed in vitro using a glass electrode. Serum gastrin concentrations rose significantly whenever intragastric pH was raised. Serum gastrin also rose after MgCl2, AlCl3, and CaCl2 with a pH of 2, when intragastric pH was not significantly altered. This rise, however, was significantly smaller than after administration of the antacids, except in the MgCl2-Mg(OH)2 and in the CaCl2 pH 10-CaCO3 groups. No significant rise of serum gastrin levels was observed after BaSO4. In nonulcer subjects, gastrin response was smaller than in the duodenal ulcer patients. The results suggest that administration of nonbuffering Mg-, Al-, and Ca-chlorides leads to elevated serum gastrin levels in duodenal ulcer patients; rising intragastric pH, however, exerts an additional serum gastrin response.


Effect of optimum therapeutic dose of poldine on acid secretion, gastric acidity, gastric emptying, and serum gastrin concentration after a protein meal.

An oral optimum therapeutic dose of poldine was established in 5 normal subjects. Acid secretion in response to a protein meal was measured for 3 hr by continuous intragastric titration with sodium bicarbonate. Poldine 30 min before the meal reduced food-stimulated acid secretion from zero to 60% in the 5 subjects (average inhibition 32%). Poldine inhibited histamine-stimulated acid secretion to approximately the same extent. In separate experiments, gastric acidity after the meal was allowed to seek its natural level (i.e., there was notitration with bicarbonate). Poldine reduced average hydrogen concentration of the gastric contents by 85 to 50% from 1.5 to 3 hr after the meal. Since poldine did not alter the volume or the buffer content of the stomach, poldine inhibition of gastric acidity is due entirely to reduction of acid secretion and not to delayed emptying of food buffer. Poldine had no consistent effect on serum gastrin concentration after the meal when pH was maintained at a constant level by titration with bicarbonate; therefore, poldine inhibition of acid secretion is not mediated by a reduction of serum gastrin concentration.


Effect of p-chloromercuribenzene sulfonate on gastric parietal and surface cell function in the dog.

p-Chloromercuribenzene sulfonate, a polar organic mercurial which presumably penetrates cell membranes with great difficulty, led to a profound decrease in acid recovered from exteriorized segments of dog gastric fundus stimulated by exogenous histamine. Trapping of secreted hydrogen ions by Tris buffer did not significantly reduce the back-diffusion component of apparent inhibition. Sodium-for-hydrogen exchange was close to unity before and after inhibition.


Electron-microscopic study of a Mycoplasmatales virus, strain MV-Lg-pS2-L 172.

Morphology and adsorption of a globular virus, lysing Acholeplasma laidlawii were studied in ultrathin sections of plaques in a lawn of the host strain. The virus was globular, about 50 to 90 nm in diameter, with a clearly defined membrane, 6.5 to 8 nm thick. A protuberance about 25 to 35 nm long and 12 to 20 nm thick was observed on numerous virus particles. The evenly granulated, electron-optically dense content of the cells became clearer in cells affected by the viruses. Fibrillar structures of different thickness and small dense areas appeared in cells assumed to be in the preliminary stages of lysis. The interactions in the virus-host system and possible development stages of the virsu are discussed.


Studies on xylanase from Basidiomycetes. Selection of strains for the production of xylanase.

Formation of extracellular xylanase was studied in 10 strains of wood-destroying fungi belonging to Basidiomycetes during their submerged cultivation with willow sawdust. The highest enzyme activity was found in the fungus Trametes hirsuta (Wulf.) Pilát. The effect of sources of carbon and nitrogen, cultivation time and initial pH of the cultivation solution on the formation of xylanase by the fungus Trametes hirsuta was investigated. The highest production of the enzyme was reached during cultivation  in the presence of willow sawdust, asparagine and at the initial pH of 5.0. The presence of xylanase, cellulase, mannanase and amylase as well as of beta-xylosidase, beta-glucosidase, beta-mannosidase and beta-galactosidase was demonstrated in the enzyme preparation obtained after a 10-day submerged cultivation of Trametes hirsuta under optimal conditions.


Volume, adherence properties, membrane antigens and mitogen responsiveness of rabbit lymphoid cell subpopulations.

Subpopulations of rabbit spleen cells which respond to T and B mitogens, respectively, can be distinguished by sedimentation velocity in the earth's gravitational field. T cell subpopulations which differed in their responsiveness to Con A and to PHA could be identified by differences in adherence properties and by their sensitivity to complement mediated cell kill with RTLA-antiserum.


Assay, characterization, and localization of an enterotoxin produced by Salmonella.

An enterotoxic factor isolated from cultures of Salmonella yielded reproducible results in the suckling mouse model in contrast to other animal models. The enterotoxin appears to possess properties similar to both the heat-stable and heat-labile enterotoxins of Escherichia coli. Preliminary results indicate that the toxin is a protein, is located in the cell wall or outer-membrane fraction, and is difficult to separate from other cell wall constituents.


Phagocytosis of Francisella tularensis by Rhesus monkey peripheral leukocytes.

Phagocytosis of Francisella tularensis by rhesus monkey peripheral neutrophils (PMN, was assessed by autoradiography, electron microscopy, and biochemical techniques. PMN, in contrast to mononuclear phagocytes, were unable to phagocytize F. tularensis in vitro in the absence of immune serum. It is postulated that in the nonimmune host the innate inability of PMN to phagocytize F. tularensis deletes one of the possible mechanisms of host defense to bacterial infection and thus permits the early dissemination of F. tularensis to intracellular sites of specific target tissues.


Pathophysiology of infectious hematopoietic necrosis virus disease in rainbow trout: hematological and blood chemical changes in moribund fish.

Infectious hematopoietic necrosis (IHN) is a rhabdoviral disease of rainbow trout (Salmo gairdneri). Trout were injected with IHNV, and various hematological and biochemical measurements of clinically ill fish were compared to uninfected controls. Infected fish had reduced corpuscular counts, hemoglobin, and packed cell volume, but normal mean corpuscular volume, mean corpuscular hemoglobulin, and mean corpuscular hemoglobin concentration. The percentage of immature erythrocytes was increased, but the percentage of leukocytes was unchanged. Differential leukocyte counts showed a significant decrease in neutrophils, increase in lymphocytes, but no change in monocytes. Unidentifiable necrobiotic cells were prevelant in blood smears and hematopoietic tissue imprints. Plasma bicarbonate, chloride, calcium, phosphorus, bilirubin, and osmolality were signigicantly reduced, but plasma glucose and anterior kidney ascorbate were unchanged. Plasma pH increased and the alpha fractions of the serum proteins were altered. No change was found in plasma enzymes, except that a LDH isozyme was significantly increased. The alkali reserve was diminished and alterations in acid-base and fluid balance occurred. Death probably resulted from a severe electrolyte and fluid imbalance caused by renal failure.


Liver glutathione and glutathione reductase response of endotoxin-treated mice.

The decrease in the level of liver glutathione (GSH) in endotoxin-treated mice was in part due to formation of glutathione disulfide (GSSG). An electron-generating system (EGS) had no effect when incubated with soluble liver extracts from normal controls but resulted in recovery of GSH amounting to 25% in endotoxin-treated animals. Incubation in the absence of the EGS caused a decline of 16% in the GSH in extracts from normal animals compared with a 50% decrease in endotoxin-treated animals. Exclusion of nicotinamide adenine dinucleotide phosphate (NADP) from the EGS resulted in a slight decline in the GSH of the extract from the normal controls but 25% for the endotoxin-treated animals. Reduction of exogenous GSSG by the liver extracts required that exogenous NADP be added to ghe incubation mixtures.


Effect of Corynebacterium acnes on interferon production in mouse peritoneal exudate cells.

Corynebacterium acnes, an organism closely related to C. parvum, has been recognized to have a striking effect on the reticuloendothelial system, as well as on both humoral and cellular immunity. In mice previously exposed to C. acnes, serum interferon levels induced by injection of Newcastle disease virus (NDV), Chikungunya virus (CV), and polyinosinic-polycytidylic acid are suppressed. When peritoneal macrophages and lymphocytes from animals exposed to C. acnes were cultivated in vitro, their capacity to produce interferon in response to NDV and CV was reduced. Furthermore, the interferon-producing capacity of these cells in tissue culture was inhibited after exposure to C. acnes to vitro. Exposure of separated populations of peritoneal macrophages and lymphocytes to C. acnes in vitro demonstrated that the interferon response to NDV by both cell types is inhibited. Peritoneal macrophages appear to be the major contributor to the interferon response in this system. Finally, this inhibitory effect was shown to occur after exposure to a purified cell wall preparation of C. acnes organisms, as well as a lipid extract of this preparation.


Temporal appearance of opsonizing antibody to Francisella tularensis: detection by a radiometabolic assay.

The burst in oxidative metabolism that is mediated through activation of the hexose monophosphate shunt and accompanies particle ingestion by polymorphonuclear leukocytes was used as the indicator in an in vitro radiometabolic assay for detection of specific opsonizing antibody to Francisella tularensis. Release of 14CO2 from radiolabeled glucose was increased significantly when specific immune serum added to suspensions of monkey polymorphonuclear leukocytes and F. tularensis. With this method, opsonizing antibodies to F. tularensis were detected in monkey serum 3 days after vaccination. Significantly increased opsonic activity in these monkeys preceded the appearance of, and persisted longer than, antibody activity as determined by conventional serological techniques. In addition, sera from 11 of 12 humans that were immunized 1 month to 13 years previously and had nondiagnostic agglutinating antibody titers demonstrated significant opsonizing activity.


Dental treatment for handicapped adults.

Handicapped adults can be grouped into those who are mentally subnormal, those with major organic disease, those who are emotionally disturbed and those for whom local anaesthesia is unsuccessful. At the first visit the degree of handicap and the state of the mouth are assessed and a plan of treatment is formulated. In most of the patients, satisfactory conditions for operating can be achieved by the use of intravenous diazepam and local anaesthesia, though some may require a full general anaesthetic. The results of treatment of a group of 139 patients are analysed and it is shown that the average DMF score in handicapped adults is appreciably higher than in normal adults of the same age. The reason for the inability of some of these patients to seek and accept dental treatment under normal surgery conditions is discussed and it is shown that with suitable initial treatment many of them become able to accept treatment in the ordinary circumstances of a dental surgery.


The success of restorative dentistry?

Direct statistical evidence of the value of routine, periodic dental examination and treatment is lacking and inferences can only be drawn from surveys of population samples and groups of people. Except perhaps in areas of exceptionally favourable dentist: population ratios the restorative philosophy makes too high a demand on scarce professional resources for it to answer the treatment needs of populations. However, it was originally developed to benefit individual patients who sought dental care and the evidence at present suggests that it may postpone the loss of teeth. It is hypothesized that the restorative philosophy is palliative rather than remedial.


Cell-mediated immunity in the uremic rat.

The popliteal node assay of the graft-versus-host reaction in the inbred rat provides a sensitive measure of cell-mediated immunity. Spleen cells from uremic Lewis rats as well as normal cells cultured in uremic serum demonstrated significant immunosuppression which was proportional to the severity of the uremia.


Assay of chemotaxis by a reversible Boyden chamber eliminating cell detachment.

A modified in vitro assay for measuring chemotaxis of leucocytes is presented. A closed chamber is used in preference to an open one thus allowing reversal of the chamber during the incubation. The responding cells which at the time the chamber is turned over are inside the filter, then migrate upwards and are consequently retained on the filter by gravity. This chamber is compared with the conventional type and various parameters are examined using guinea pig eosinophils and other cell types. Loss of cells in the conventional Boyden technique is negligible for rabbit peritoneal neutrophils but is considerable for human and guinea pig neutrophils and eosinophils. This is circumvented by the proposed method; accordingly it allows more accurate quantitation in clinical and experimental work.


Histamine in the tissues of the marsupial Setonix brachyurus (the quokka).

Passive cutaneous anaphylactic (PCA) reactions in the marsupial Setonix brachyurus (the quokka) were completely inhibited by the histamine antagonist mepyramine maleate, but were unaffected by disodium cromoglycate or the serotonin antagonist, methysergide. Histological examination of quokka skin indicated that mast cell degranulation occurred during the PCA reaction in this marsupial and animals whose skin was relatively deficient in mast cells were poor PCA recipients. In contrast to many eutherian (placental) species, this marsupial was found to lack histamine in blood leukocytes and platelets. Also, while the peritoneal mast cells of rats and mice contain large quantities of histamine, this amine was not detected to quokka peritoneal washings, even after the induction of a peritoneal exudate or the regular intraperitoneal injection of antigen. Immunologic challenge of quokka blood or peritoneal cells did not induce the synthesis of histamine, but histamine release was elicited from sensitized quokka lung by antigenic challenge.


Incidence and degree of hypoxemia after gynecological surgery.

Arterial blood gases and pH values were determined before and after performing abdominal hysterectomies in 32 women. Four to five liters of oxygen were given by nasal cannula to 15 patients, while 17 patients breathed only ambient air. Falls of arterial oxygen tension averaging 10 torrs were seen up to 30 minutes after surgery in the latter group, but no apparent hypoxemia occurred in the patients who received nasal oxygen. Postoperative mild hypoxemia present in patients undergoing pelvic surgery can be obviated by prophylactic administration of oxygen at low flows.


Prostaglandin-like activity in the aqueous humor following alkali burns.

Following application of 20 mul of 2 N sodium hydroxide to the rabbit cornea, the prostaglandin-like activity in the aqueous humor increased from undetectable levels to around 30 ng, per milliliter, a level which was sustained for up to 24 hours. This increase correlated with the previously reported sustained elevation of intraocular pressure following a 20 mul alkali burn. Pretreatment with aspirin or indomethacin virtually abolished the increase in intraocular pressure and elevation of prostaglandin-like activity in the aqueous humor following a 20 mul burn. Marked ocular hypotension following 50 mul and 100 mul sodium hydroxide burns correlated with very low prostaglandin-like activity in the aqueous humor. The low prostaglandin-like activity probably resulted from alkaline hydrolysis of aqueous prostaglandins and massive cellular death in iris and ciliary body tissues.


Drugs affecting the cholinergic system in the intact mammalian eye. I. Evaluation of the miotic activity of acetylcholine-like drugs in the mouse eye.

The effect of concentration and pH on the miotic activity of five acetylcholine-like drugs were studied in the intact mouse eye. The data suggests that it is mainly the nonionized form of the drug which contributes to its miotic activity. Acetylcholine-like drugs, degradable by cholinesterases, exert their miotic activity at higher concentrations than do those which are resistant to enzymatic hydrolysis. More active acetylcholine-like drugs appear to be less sensitive to concentration changes.


Evaluation of clinical disorders of acid-base balance.

An  understanding of acid-base disturbances depends on the interpretation of changes in blood pH, P-co-2, and bicarbonate concentration and a comprehension of the physiologic mechanisms that control them. The physiologic relationships between Pco-2 and bicarbonate can be visualized by means of the balance nomogram and this visualization facilitates determination of any excess or deficit for both respiratory and metabolic factors.


The relationship between angiography, intraerythrocytic pH and hemoglobin oxygen equilibrium.

The acute effects of an injection of contrast material (Renovist) on intraerythrocytic and extracellular pH was studied at cardiac catheterization in 72 patients with congenital heart disease. A decrease in mean extracellular (plasma) pH (7.444 plus or minus .006 to 7.419 plus or minus 0.009) and an increase in mean intraerythrocytic pH (7.204 plus or minus .005 to 7.232 plus or minus .006) were observed within minutes after injection (p smaller than .01). In 17/72 patients, simultaneous measurements of oxygen affinity for hemoglobin as characterized by P50 (oxygen tension at 50% O-2 saturation) corrected to in vivo arterial pH decreased from a mean of 26.4 to 25.2 mm Hg (p smaller than .01). It is postulated that the acute increase in intraerythrocyte pH and increased affinity of hemoglobin for oxygen are due to a decrease in intracellular hydrogen ion concentration induced by the increase in plasma osmolality with subsequent shift of hemoglobin oxygen equilibrium via the Bohr effect.


Solute excretion during intravenous urography: a comparison of sodium and methylglucamine iothalamates.

The sodium and methylglucamine salts of iothalamic acid were compared as urographic agents. The two drugs were administered intravenously to dogs at a dose of 600 mgI/kg. The outputs and concentrations of the major urinary solutes and the injected solutes were measured to define the differences in renal excretion of sodium and methylglucamine iothalamates. The action of methylglucamine as a relatively inert osmotic diuretic was reconfirmed. When sodium iothalamte was adminstered the tubular reabsorption of sodium and chloride was greater than when methylglucamine iothalamate was used. It is suggested that this reabsorption explains the advantage of sodium-linked agents over methylglucamine-linked agents in urography.


Factor 420-dependent pyridine nucleotide-linked hydrogenase system of Methanobacterium ruminantium.

Methanobacterium ruminantium was shown to possess a nicotinamide adenine dinucleotide phosphate (NADP)-linked factor 420 (F420)-dependent hydrogenase system. This system was also shown to be present in Methanobacterium strain MOH. The hydrogenase system of M. ruminantium also links directly to F420, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), methyl viologen, and Fe-3 plus. It has a pH optimum of about 8 and an apparent Km for F420 of about 5 x 10-6 M at pH 8 when NADP is the electron acceptor. The F420-NADP oxidoreductase activity is inactive toward nicotinamide adenine dinucleotide (nad) and no NADPH:NAD or FADH2(FMNH2):NAD transhydrogenase system was detected. Neither crude ferredoxin nor boiled crude extract of Clostridium pasteuranum could replace F420 in the NADP-linked hydrogenase reaction of M. ruminantium. Also, neitther F420 nor a curde "ferredoxin" fraction from M. ruminantium extracts could substitute for ferredoxin in the pyruvate-ferredoxin oxidoreductase reaction of C. pasteurianum.


Factor 420-dependent pyridine nucleotide-linked formate metabolism of Methanobacterium ruminantium.

Methanobacterium ruminantium was shown to possess a formate dehydrogenase which is linked to factor 420 (F420) as the first low-molecular-weight or anionic electron transfer coenzyme. Reduced F420 obtained from the formate dehydrogenase can be further linked to the formation of hydrogen via the previously described F420-dependent hydrogenase reaction, thus constituting an apparently simple formate hydrogenlyase system, or to the reduction of nicotinamide adenine dinucleotide phosphate via F420:nicotinamide adenine dinucleotide phosphate oxidoreductase. The results indicate that hydrogen and formate, the only known energy sources for M. ruminantium and many other methanogenic bacteria, should be essentially equivalent as sources of electrons in the metabolism of this organism.


Control of lysine biosynthesis in Bacillus subtilis: inhibition of diaminopimelate decarboxylase by lysine.

Diaminopimelate decarboxylase has been characterized in extracts of Bacillus subtilis and resolved from aspartokinases I and II. Under certain conditions, the enzyme is specifically inhibited by physiological concentrations of L-lysine, but less specificity and altered kinetics of inhibition are observed if lower ionic strengths are employed in the assay procedure. Diaminopimelate decarboxylase can be desensitized to lysine inhibition by either lowering the pH or diluting the enzyme in Tris buffer in the absence of pyridoxal phosphate. Evidence is presented to incidate that, under proper conditions, lysine inhibition involves an interaction of the amino acid with the enzyme rather than competition for available pyridoxal phosphate in the assay. Lysine, by affecting the level of meso-diaminopimelate, may thus regulate its biosynthesis through  sequential feedback inhibition. Analysis of the diaminopimelate decarboxylase of 15 revertants of mutants that had originally lacked diaminopimelate decarboxylase activity indicates that as little as 5% of the specific activity of enzyme observed in the wild-type strain is sufficient to permit normal growth rates. In the growing cell, diaminopimelate decarboxylase may therefore exist largely in an inhibited state.


Metabolic function and properties of 4-hydroxyphenylacetic acid 1-hydroxylase from Pseudomonas acidovorans.

The enzyme 4-hydroxyphenylacetate, NAD(P)H:oxygen oxidoreductase (1-hydroxylating) (EC 1.14.13 ...; 4-hydroxyphenylacetate 1-monooxygenase; referred to here as 4-HPA 1-hydroxylase) was induced in Pseudomonas acidovorans when 4-hydroxyphenylacetate (4-PHA) was utilized as carbon source for growth; homogentisate and maleylacetoacetate were intermediates in the degradation of 4-HPA. A preparation of the hydroxylase that was free from homogentisate dioxygenase and could be stored at 4 C in the presence of dithioerythritol with little loss of activity was obtained by ultracentrifuging cell extracts; but when purified 18-fold by affinity chromatography the enzyme became unstable. Flavin adenine dinucleotide and Mg2+ ions were required for full activity. 4-HPA 1-hydrocylase was inhibited by KCl, which was uncompetitive with 4-HPA. Values of Ki determined for inhibitors competitive with 4-HPA were 17 muM dl-4-hydroxymandelic acid, 43 muM 3,4-dihydroxyphenylacetic acid, 87 muM 4-hydroxy-3-methylphenylacetic acid, and 440 muM 4-hydroxyphenylpropionic acid. Apparent Km values for substrates of 4-HPA 1-hydroxylase were 31 muM 4-HPA, 67 muM oxygen, 95 muM reduced nicotinamide adenine dinucleotide (NADH); AND 250 muM reduced nicotinamide adenine dinucleotide phosphate (NADPH). The same maximum velocity was given by NADH and NADPH. A chemical synthesis is described for 2-deutero-4-hydroxyphenylacetic acid. This compound was enzymatically hydroxylated with retention of half the deuterium in the homogentisic acid formed. Activity as substrate or inhibitor of 4-HPA 1-hydroxylase was shown only by those analogues of 4-HPA that possessed a hydroxyl group substituent at C-4 of the benze nucleus. A mechanism is suggested that accounts for this structural requirement and also for the observation that when 4-hydroxyphenoxyacetic acid was attacked by the enzyme, hydroquinone was formed by release of the side chain, probably as glycolic acid. Only one enantiometer of racemic 4-hydroxyhydratropic acid was attacked by 4-HPA 1-hydroxylase; the product, alpha-methylhomogentisic acid (2-(2,5-dihydroxyphenyl)-propionic acid), exhibited optical activity. This observation suggests that, during its shift from C-1 to C-2 of the nucleus, the side chain of the substrate remains bound to a site on the enzyme while a conformational change of the protein permits the necessary movement of the benzene ring.


Identification of the structural gene for glutamine synthetase in Klebsiella aerogenes.

Mutations at two sites of the Klebsiella aerogenes chromosome, unlinked by transduction with phages PW52 and P1, result in the lack of enzymatically active glutamine synthetase. A mutation in the glnB site leads to a marked decrease in the formation of an apparently normal enzyme. Some of the mutations in the glnA site lead to the production of enzymatically inactive material capable of reacting with anti-glutamine synthetase serum. The revertant of a glnA mutant was found to produce a glutamine synthetase with less activity and less stability to heat than the enzyme of the wild type. These results locate the structural gene to the production of enzymatically inactive glutamine synthetase antigen, not subject to repression by exogenously added ammonia. This observation suggests that glutamine synthetase is itself involved in the regulation of the synthesis of glutamine synthetase.


Genetic control of glutamine synthetase in Klebiella aerogenes.

Mutations at two sites, glnA and glnB, of the Klebsiella aerogenes chromosome result in the loss of glutamine synthetase. The locations of these sites on the chromosome were established by complementation by episomes of Escherichia coli and by determination of their linkage to other genetic sites by transduction with phage P1. The glnB gene is located at a position corresponding to 48 min on the Taylor map of the E. coli chromosome; it is linked to tryA, nadB, and GUA. The glnA gene is at a position corresponding to 77 min on the Taylor map and is linked to rha and metB; it is also closely linked to rbs, located in E. coli at 74 min, indicating a difference in this chromosomal region between E. coli and K. aerogenes. Mutations in the glnA site can also lead to nonrepressible synthesis of active glutamine synthetase. The examination of the fine genetic structure of glnA revealed that one such mutation is located between two mutations leading to the loss of enzymatic activity. This result, together with evidence that the structural gene for glutamine synthetase is at glnA, suggests that glutamine synthetase controls expression of its own structural gene by repression.


Protoplast formation and leakage of intramembrane cell components: induction by the competence activator substance of pneumococci.

Treatment of pneumococci with activator (a protein that induces bacterial "competence" to absorb deoxyribonucleic acid molecules and undergo genetic transformation) can cause either protoplast formation or leakage of intracellular components to the medium depending on postincubation conditions. The leaked intracellular components include nucleoside phosphates, beta-galactosidase, deoxyribonuclease, autolysin, and hemolysin. Leakage and protoplast formation are induced by the electrophoretically pure activator, and these phenomena require the same conditions as induction of competence for genetic transformation, namely, genetic capacity for competence, protein synthesis, incorporation of choline, and the optimal pH for activation. It is suggested that the activator protein accelerates a normal process of transport (leakage) of autolysin molecules into the periplasmic space. The activity of these autolysin molecules from within would then unmask deoxyribonucleic acid binding sites located on the plasma membrane.


Fine structure of mesosomal involvement during Bacillus macerans sporulation.

The ultrastructure of endospore formation in Bacillus macerans ATCC 8244 is characterized by the examination of thin sections of cells grown synchronously in a defined medium. For the most part, sporulation in this organism proceeds as described in other Bacillus species. However, unusually extensive mesosomal involvement occurs during the early stages of sporulation, through the completion of engulfment. A large mesosome is associated with spore septum formation and a portion of this mesosome is included in the developing forespore. As engulfment continues, the forespore mesosome moves to the apex of the cell and participates in the completion of the double forespore membrane. This participation is morphologically similar to mesosome involvement in division and spore septation and seems to comprise a second sporal septation process. Based on this study, it is suggested that the mesosome functions to facilitate the "fusion" of membranes thought to occur during cell division and sporulation.


Further evidence for a partially folded intermediate in penicillinase secretion by Bacillus licheniformis.

Protoplasis of Bacillus licheniformis 749/C (a mutant constitutive for penicillinase production) continued to synthesize and release penicillinase in hypertonic growth medium in the presence of trypsin and chymotrypsin at 25 mug each per ml. When the protoplasts were stripped of about half of their membrane-bound penicillinase by pretreatment at pH 9.5 or with a higher level of trypsin, penicillinase activity no longer increased in the presence of the proteases. This effect was immediately eliminated after addition of soybean trypsin inhibitor. These proteases do not significantly inhibit general protein synthesis. Stripped protoplasts of strain 749/C and of uninduced strain 749 (unable to synthesize penicillinase) were incubated with 50 mug of chymotrypsin per ml, and the supernatent fluids were examined immunochemically for peptides derived from the penicillinase chain. Such fargments were found only with the protoplasts capable of synthesizing penicillinase (strain 749/C). The direct detection of the products of protease degradation of a susceptible form of penicillinase provides strong evidence that, in stripped protoplasts of B. licheniformis 749/C, penicillinase synthesis continues in the presence of trypsin or chymotrypsin and that, in these modified membranes, the protease is able to act on an early form of the enzyme that has not yet attained the protease-resistant conformation characteristic of the membrane-bound and exopenicillinases. This finding is discussed in terms of the current models of penicillinase secretion.


Halide uptake by the filamentous ascomycete Neocosmospora vasinfecta.

The uptake of Cl minus by the ascomycetes Neocosmospora vasinfecta was investigated. Intramycelial concentrations of more than 55 mM (890-fold the external concentration) were reached. Accumulation was as inorganic Cl minus and nystatin induced total loss of mycelial Cl minus without extensive protein loss, implying that Cl minus retention was not due to binding to macromolecules. Cl minus transport was largely unidirectional with efflux being low under all conditions. Uptake was temperature dependent (maximal Arrhenius activation energy of 18.0 kcal/mol) and was severely reduced by KCN, dicyclohexylcarbodiimide, and anaerobiosis. A comparison with the inhibition of oxygen uptake under the same conditions implied that Cl minus transport was not directly coupled to aerobic electron transport. Cl minus uptake was a saturatable function of the external Cl minus concentration, and apparent Km values of 6.4 times 10-6 M and 10-4 M were calculated. Of the anions tested, only Br minus effectively inhibited Cl minus uptake and I minus, NO3 minus, SO4 minus 2, HCO3 minus, and H2PO4 minus were without effect. Cl minus uptake did not require concomitant cation uptake.


Polyphosphate levels in nongrowing cells of Saccharomyces mellis as determined by magnesium ion and the phenomenon of "Uberkompensation".

Magnesium ion enhances the maximum amount of polyphosphate that resting phosphate-starved cells of Saccharomyces mellis can store by increasing the length of time the cells will continue assimilating phosphate. The divalent cation has no effect on the rate of formation of polymer. As much as 12 times more polyphosphate is formed in cells incubated in reaction mixtures containing 0.3 M MgCl2 than in the absence of Mg2+. Potassium ion also has an influence on the amount of polyphosphate that phosphate-starved cells can accumulate but the degree of stimulation is not very large. Mg2+ and K+ have no effect on polyphosphate formation or storage in phosphate-satiated cells. Apparently, then, there are two systems for polyphosphate accumulation in S. mellis. Each system is stable in nondividing cells. The one present in phosphate-starved cells seems to be repressible by growth of the organism in media containing orthophosphate. The shift from the derepressed state to the repressed state, or vice versa, occurs only in exponentially dividing cells in appropriate media with 100% of the cells in the new physiological state by the time the cell mass has doubled. It is suggested that the word to describe the phenomenon of the accumulation of higher amounts of polyphosphate in phosphate-starved cells than the steady-state level of phosphate-satiated cells be changed from "uberkompensation" to "magnesium ubertriebung," or "magnesium enhancement."


Dihydrofolate reductases from the wild type and aminopterin-resistant mutants of Diplococcus pneumoniae.

Dihydrofolate reductase from the wild type and aminopterin-resistant mutants of Diplococcus pneumoniae has been compared. Specific activity, optimum pH, Km, thermal stability, and inhibition by aminopterin are identical for both strains. Aminopterin resistance for such mutants is, therefore, not due to an alteration of the dihydrofolate reductase.


Regulation of the L-lactase dehydrogenase from Lactobacillus casei by fructose-1,6-diphosphate and metal ions.

The lactate dehydrogenase of Lactobacillus casei, like that of streptococci, requires fructose-1,6-diphosphate (FDP) for activity. The L. casei enzyme has a much more acidic pH optimum (pH 5.5) than the streptococcal lactate dehydrogenases. This is apparently due to a marked decrease in the affinity of the enzyme for the activator with increasing pH above 5.5; the concentration of FDP required for half-maximal velocity increase nearly 1,000-fold from 0.002 mM at pH 5.5 to 1.65 mM at 6.6. Manganous ions increase the pH range of activity particularly on the alkaline side of the optimum by increasing the affinity for FDP. This pH dependent metal ion activation is not specific for Mn2+. Other divalent metals, Co2+, Cu2+, Cd2+, Ni2+, Fe2+, Fe2+, and Zn2+ but not Mg2+, will effectively substitute for Mn2+, but the pH dependence of the activation differs with the metal ion used. The enzyme is inhibited by a number of commonly used buffering ions, particularly phosphate, citrate, and tris (hydroxymethyl) aminomethane-maleate buffers, even at low buffer concentrations (0.02 M). These buffers inhibit by affecting the binding of FDP.


Purification and properties of gentisate 1,2-dioxygenase from Moraxella osloensis.

Gentisate:oxygen 1,2-oxidoreductase (decyclizing) (EC 1.13.11.4; gentisate 1,2-dioxygenase) from Moraxella osloensis was purified to homogeneity as shown by polyacrylamide gel electrophoresis. The enzyme has a molecular weight of about 154,000 and gives rise to subunits of molecular weight 40,000 in the presence of sodium dodecyl sulfate. Gentisate 1,2-dioxygenase showed broad substrate specificity and attacked a range of halogen- and alkyl-substituted gentisic acids. Maleylpyruvate, the product formed from gentisate, was degraded by cell extracts supplemented with reduced glutathione, but substituted maleylpyruvates were not attacked under these conditions.


Relation between reduced nicotinamide adenine dinucleotide oxidation and amino acid transport in membrane vesicles from Bacillus subtilis.

The rate of reduced nicotinamide adenine dinucleotide (NADH) oxidation by membrane vesicles from Bacillus subtilis W23 increases three- to fourfold during logarithmic growth, reaching maximal levels in early stationary phase. Initial rates of L-proline, L-alanine, and L-glutamate transport energized by NADH closely parallel the increase in NADH oxidation. In vesicles prepared at different stages of growth, a constant number of NADH molecules varying from 150 to 260 have to be oxidized to transport one molecule of amino acid. Membrane vesicles from B. subtilis aroD (strain RB163), a mutant defective in menaquinone synthesis, do not transport amino acids in the presence of NADH. Ascorbate plus phenazine methosulfate, however, energizes amino acid transport equally well as in vesicles of B. subtilis W23. NADH oxidation and NADH-driven amino acid transport can be restored instantaneously by the addition of menadione (vitamin K3).


Degradative acetolactate synthase of Bacillus subtilis: purification and properties.

A degradative acetolactate synthase (acetolactate pyruvate-lyase [carboxylating], EC 4.1.3.18) from Bacillus subtilis has been partially purified and characterized. The synthesis of the enzyme was induced by growth of cells in minimal medium plus isobutyrate or acetate. The enzyme was partially purified by ammonium sulfate fractionation, gel filtration, and hydroxyapatite chromatography. The pH optimum of the purified enzyme was 7.0 in phosphate buffer. When assayed in phosphate buffer (pH 7.0), activity was stimulated by acetate and inhibited by sulfate. When assayed in acetate buffer (pH 5.8), activity was inhibited both by sulfate and phosphate. Michaelis-Menten kinetics was observed when the enzyme was assayed in phosphate buffer (pH 6.0 or 7.0), and inhibition by sulfate was competitive and activation by acetate was noncompetitive. When assayed in acetate buffer (pH 5.8), nonlinear Lineweaver-Burk plots were obtained; inhibition by phosphate appeared to be competitive and that by sulfate was of the mixed type. The approximate molecular weight of the purified enzyme was 250,000 as determined by gel filtration.


Purification and properties of a serine protease from Pseudomonas matophilia.

The extracellular protease of Pseudomonas maltophilia was partially purified by ammonium sulfate precipitation and chromatography on Sephadex G-75 and Bio-rex 70. Gel electrophoresis revealed minor impurities. The enzyme exhibited the following properties: (i) molecular weight, 35,000; (ii) A see article; 10.8; (iii) isoelectric point, 9.3; (iv) pH optimum, 10.0; (v)s20, w equal 3.47. The enzyme was rapidly inactivated by ethylenediaminetetracetate, but activity could be partially restored with divalent cations. Of those tested, Ca2+, Sr2+, Ba2+, Co2+, Cu2+, Mg2+, and Zn2+ were all effective. Both phenylmethylsulfonylfluoride and diisopropylfluorophosphate were powerful inhibitors of protease activity, but L-1-tosylamide-2-phenylethylchloromethyl ketone, iodoacetic acid, and iodoacetamide were without effect. The enzyme hydrolyzed the esters N-acetyl-L-tyrosine ethyl ester and alpha-N-benzoyl-L-arginine ethyl ester (BAEE) with Km values of 10.4 and 3.4 mM, respectively. The hydrolysis of BAEE was also inhibited by phenylarsonic acids. The kinetics of inhibition by m-nitrophenylarsonate were of the mixed type, and the K1 was 1.8 mM. The data followed a theoretical curve for a 1:1 enzyme-inhibitor complex with a dissociation constant of 1.8 mM. Inhibition by m-nitrophenylarsonate was pH dependent and followed a theoretical curve for the titration of a protonated group with a pKa of 7.0.


Induction of hepatic tyrosine aminotransferase in vivo by derivatives of cyclic adenosine 3':5'-monophosphate.

A number of 8- and N6-SUBSTITUTED DERIVATIVES OF CYCLIC ADENOSINE 3':5'-MONOPHOSPHATE-DEPENDENT PROTEIN KINASE, AND AS SUBSTRATES FOR RAT LIVER CYCLIC NUCLEOTIDE PHOSPHODIESTERASE. All of the analogs tested were able to induce the transaminase. The induction by the analogs was shown to be the result of an actual increase in the amount of enzyme, and the mechanism of induction was an increase in the rate of synthesis of the transaminase. The induced enzyme appeared to be immunologically similar to the non-induced enzyme. A good correlation was found to exist between the dose that produced 50% of maximal induction and a combination of the activation constant for cyclic adenosine 3':5'-monophosphate-dependent protein kinase by the analog and its susceptibility to hydrolysis by cyclic nucleotide phosphodiesterase. These data suggest that the phosphorylation of some site is involved in the mechanism by which cyclic adenosine 3':5'-monophosphate affects the rate of synthesis of tyrosine aminotransferase.


The essential role of Ca2+ in the activity of bovine pancreatic deoxyribonuclease.

DNase requires Ca2+ for activity against DNA with Mg2+. The Ca2+ selective chelating agent, ethylene glycol bis(beta-aminoethyl ether)-N, N'-tetraacetic acid, (EGTA) inhibits DNase completely at pH 7 or 8, and subsequent addition of excess Ca2+ reverses inhibition in less than one second. DNase action can be stopped at any point by the addition of excess EGTA over Ca2+. Ca2+ is required for DNase to bind substrate. Gel filtration experiments fail to show DNase binding to 0.2 mg per ml of DNA at 5 mm Mg2+ and 10-4 M EGTA. The concentration of Ca2+ needed for half of maximum DNase activity decreases with increases DNA concentration, from 1.2 times 10-5 M Ca2+ at 2.3 times 10-5 M DNA-P to about 4 times 10-7 M Ca2+ at 2.3 DNA-P. Kinetic analysis by the titrametic assay of protons releases shows that V max is independent of Ca2+ concentration while Km increases from 7.7 times 10-5 M DNA-P at 5 times 10-4 M Ca2+ to 3.4 times 10-4 M DNA-P at 5 times 10-6 M Ca2+. Both of these results are predicted by a rate equation which is derived from the assumption that DNase must bind Ca2+ before it can bind DNA. The essential Ca2+ atom probably binds to the one of two high affinity Ca2+ binding sites on DNase which cannont bind Mg2+ or Mn2+. The only other divalent metal ions which can bind to this site, Sr2+ and Ba2+, are also the only metal ions which can substitute for Ca2+ in DNase action against DNA with Mg2+. Some DNase activity is obtained in the absence of added Ca2+ with Mg2+ at pH 6 or below and with Mn2+ or Co2+ at pH 8. These assay solutions are contaminated by 1 to 3 muM Ca2+, which may be sufficient to account for the observed activity.


The role of metal in liver alcohol dehydrogenase catalysis. Spectral and kinetic studies with cobalt-substituted enzyme.

The kinetic and spectral properties of native and totally cobalt-substituted liver alcohol dehydrogenase have been compared. Based on titrimetric determinations of enzyme active site concentration, the turnover number at pH 7.0 for cobalt enzyme was the same as for native enzyme. At pH 10, however, the turnover number was slower for cobalt-substituted enzyme, 3.14 s-1 as compared with 4.05 s-1 for native enzyme. A comparison between native and totally cobalt-substituted enzyme showed a blue-shifted enzyme-NADH double difference spectrum and a splitting and red-shifted enzyme-NAD+-pyrazole double difference spectrum in the near-ultraviolet. The 655-nm peak of the cobalt-substituted enzyme was perturbed by the formation of enzyme-NADH binary complex, enzyme-NAD+-trifloroethanol ternary complex, and enzyme-NAD+ binary complex formation. At pH 7.0, the only observable step in the reaction sequence with a significantly different rate constant for cobalt enzyme was the catalytic hydrogen-transferring step. The rate constant for this step is 92 s-1 for totally cobalt-substituted enzyme as compared with 138 s-1 for native liver alcohol dehydrogenase. The results of this study indicate that zinc is involved in catalysis alcohol and NADH.


Effects of temperature on diphosphopyridine nucleotide-linked isocitrate dehydrogenase from bovine heart. Aspects of the kinetics, stability, and quarternary structure of the enzyme.

A temperature-dependent conformational change of the active DPN-linked isocitrate dehydrogenase was observed. When initial reaction kinetic data were examined between 35 and 5 degrees, the Hill number (n) varied from 2 at higher to n approaching unity at lower temperatures, with an inflection point at 17 degrees. The presence of manganous isocitrate in the incubation media shifted the transition temperature for enzyme inactivation by 5,5'-dithiobis(2-nitrobenzoate) from 8-16 degrees. These temperature-dependent transitions were paralleled by progressive changes in sedimentation velocities from s20, w of 10.4 at 25 degrees to 7.3 at 10 degrees as measured by active band centrifugation. The linear Arrhenius plot for apparent V max and the constancy of S0.5 for the substrate manganous isocitrate between 35 and 5 degrees suggest that this temperature-dependent conformational change may not be solely related to manganous isocitrate. Further indications of equilibria between different species of enzyme in solution and effects of substrates and cofactors on conformation came from studies of specific activity of enzyme diluted into buffers at 3 and 25 degrees. Dilution to concentrations between 10 and 25 mum enzyme resulted in relatively rapid protein concentration-dependent inactivation which could be prevented and fully reversed by manganous isocitrate. No further substantial inactivation was found subsequent to this phase at 25 degrees. Lowering the temperature of the dilution buffer to 3 degrees favored formation of enzyme species exhibiting a further time and pH-dependent loss of activity which became independent of protein concentration below 7 mum enzyme. The rate of cold inactivation was reduced by raising the ionic strength of the buffer and its progress could be arrested by manganous isocitrate; however, the substrate did not restore the original activity.


Complexation of iron hexacyanides by cytochrome c. Evidence for electron exchange at the exposed heme edge.

Electrostatic binding of at least two anionic iron hexacyanides to cationic horse heart cytochrome c was demonstrated by equilibrium dialysis measurements. No binding was detected following trifluoroacetylation of all of the 19 lysine residues. Replacement of the natural heme iron ligand methionine 80 by the alternative intrinsic ligand lysine 79 but not the extrinsic ligand imidazole resulted in the loss of one hexacyanide binding site. It is proposed that this site is located at the exposed heme edge and is functional in electron exchange.


Phosphate-independent glutaminase from rat kidney. Partial purification and identity with gamma-glutamyltranspeptidase.

Phosphate-independent glutaminase can be quantitatively solubilized from a microsomal preparation of rat kidney by treatment with papain. Subsequent gel filtration and chromatography on quaternary aminoethyl (QAE)-Sephadex and hydroxylapatite yield a 200-fold purified preparation of this glutaminase. The purified enzyme also hydrolyzes gamma-glutamylhydroxamate and exhibits substrate inhibition at high concentrations of either glutamine or gamma-glutamyhydroxamate, which is partially relieved by increasing concentrations of maleate. Rat kidney phosphate-independent glutaminase reaction is catalyzed by the same enzyme which catalyzes the gamma-glutamyltranspeptidase reaction. The ratio of glutaminase to transpeptidase activities remained constant throughout a 200-fold purification of this enzyme. The observation that the phosphate0independent glutaminase and gamma-glutamyltranspeptidase activities exhibit coincident mobilities during electrophoresis, both before and after extensive treatment with neuraminidase, strongly suggests that both reactions are catalyzed by the same enzyme. This conclusion is strengthened by the observation that maleate and various amino acids have reciprocal effects on the two activities. Maleate increases glutaminase activity and blocks transpeptidation, whereas amino acids activate the transpeptidase but inhibit glutaminase activity. In contrast, the addition of both maleate and alanine resulted in a strong inhibition of both activities. Both activities exhibit a similar distribution in the various regions of the kidney. Recovery of maximal activities in the outer stripe region of the medulla is consistent with previous quantitative microanalysis which indicated that this glutaminase activity is localized primarily in the proximal straight tubule cells. The glutaminase and transpeptidase activities have different pH optima. Examination of the product specificity suggests that decreasing pH also promotes glutaminase activity and that below pH 6.0, this enzyme functions strictly as a glutaminase. Because of the localization of this activity on the brush border membrane, these resuts are consistent with the possibility that the physiological conditions induced by metabolic acidosis could convert this enzyme from a broad specificity transpeptidase to a glutaminase. Therefore, this enzyme could contribute to the increased renal synthesis of ammonia from glutamine which is observed during metabolic acidosis.


Kinetic studies on pig heart cytoplasmic malate dehydrogenase.

Kinetic studies on the pig heart cytoplasmic malate dehydrogenase have been performed over a wide range of conditions using the full time course of the reaction and computer simulation to obtain the kinetic parameters. The maximum velocity and Michaelis constants for the oxidation of reduced coenzyme have been determined as a fundtion of pH in 0.05 M phosphate buffer at 15 degrees. At pH 7.5 and at low substrate concentrations, the kinetic data are consistent with a sequential addition of substrates, coenzyme binding first, and involving the formation of at least one ternary complex. No oxalacetate binding to the enzyme was observed. The rate constants for the dissociation of coenzyme from the enzyme-coenzyme complex are small enough to define the maximum velocity in either direction of the reaction. These data, plus data using deuterated reduced coenzyme, indicate that the chemical transformation step is not rate determining. It is also shown that DPNH binding can be tight enough to practically exclude the possibility of obtaining initial velocities when measuring the reduction of DPN. Kinetic abnormalities do appear at higher substrate or product concentrations, but these do not appear to be related to the formation of inactive abortice, complexes.


The nonspecific binding of Fe3+ to transferrin in the absence of synergistic anions.

An obligatory role for barbonate (or other synergistic anions) in the specific binding of Fe3+ by transferrin has been a point of controversy for two decades. There are an equal number of confirmatory and negative reports of specific Fe3+-transferrin binary complexes. A criticism of previous studies is the use of only one synthetic route, and limited product testing. This study reports the development of several preparative routes aimed at the formation of a specific Fe3+-transferrin complex, and the characterization of the products by spectrophotometry and chemical reactivity. The preparative routes described include: (a) displacement of carbonate from Fe3+-transferrin-CO32- at low pH followed by removal of CO2 by several techniques; (b) addition of FeCl3 to apotransferrin under CO2-free conditions; (c) oxidation of Fe2+ in the presence of apotransferrin under CO2-free conditions; (d) reaction of apotransferrin with nonsubstituting Fe3+ complexes in the absence of CO2; and (e) attempts to displace anions from weak Fe3+-transferrin-anion complexes. The product were examined with regard to their visible spectra, and their examined with regard to their visible spectra, and their reactivity with: (a) NaHCO3, (b) Fe3+-nitrilotriacetic acid in NaHCO3, and (c) citrate. The results are compared with the characteristics of Fe3+-transferrin-anion complexes and nonspecific Fe3+, transferrin mixtures. The data indicate that in the absence of synergistic anions the affinity of the specific metal binding sites of transfe-rin for Fe3+ is so low as to not compete favorably with hydrolytic polymerization and nonspecific binding effects.


Identification of a folate binder in hog kidney.

A macromolecular binder of folic acid (pteroylglutamic acid) and folic acid derivatives has been identified in extracts of hog kidney. With partially purified preparations, binding of [3H]pteroylglutamate was competed for by unlabeled pteroylglutamate, 5-methyltetrahydrofolic acid and its triglutamate derivative, by tetra- and dihydrofolic acid, and by N-10-formyltetrahydrofolic acid. The partially purified extract did not bine [3H]methotrexate nor could methotrexate or 5-formyltetrahydrofolic acid compete for [3H]folic acid-binding sites. The rate of binding of pterolyglutamate at 37 degrees was approximately 3%/s, was independent of pteroylglutamate concentration, and was essentially irreversible between pH 6.0 and 9.0. Below pH 6.0 binding was reversible, and at pH 3.5 the folic acid-binder complex completely disassociated. Based upon Sephadex gel filtration, the molecular weight of the folate-binder complex is 35,000 to 40,000. Binding activity was unaffected by pretreatment with ribonuclease or deoxyribonuclease but was completely destroyed by trypsin. The initial, unfractionated extract showed gamma-glutamyl carboxypeptidase (conjugase) activity which was lost in subsequent steps of purification of the folate binder.


The effects of polyamines on a residue-specific human plasma ribonuclease.

A ribonuclease, purified some 2700-fold from human plasma, exhibited a strong predilection for the hydrolysis of internucleotide bonds containing cytidylic acid. Analysis of [3'-32P]- and [5'-32P]phosphoryl-terminal fragments obtained after enzymic digestion of rabbit liver and yeast RNA indicated that the nucleotide found at the 3' terminus of the fragments was invariably cytidylic acid. The nucleotide at the 5' terminus varied between cytidylic and uridylic acids in a ratio of 9:1. When characterized by DEAE-cellulose chromatography, approximately 70 per cent of the digest consisted of oligonucleotides from 4 to 8 nucleotides in length. Enzyme activity, when measured in low ionic strength buffer, could be increased severalfold above control levels by the addition of either of the polyamines, spermidine or spermine. These substances also restored nucleolytic activity to preparations inhibited by such ordered synthetic polyribonucleotides as polyguanylic acid. Estimations of the molecular weight of the enzyme, both by Sephadex gel filtration and sucrose density centrifugation, indicate that the weight may vary, depending on the presence or absence of certain cations. Of the cations examined, spermidine and spermine appear to have the greatest effect, causing an alteration in molecular weight from greater than 150,000 to approximately 32,000.


PH dependence of the Adair constants of human hemoglobin. Nonuniform contribution of successive oxygen bindings to the alkaline Bohr effect.

In order to solve the problem of an apparent discrepancy between the pH variance of oxygen equilibrium curve and the linear relation between the number of released Bohr protons and the degree of ligation, precise oxygen equilibrium curves of human hemoglobin were determined at a number of pH values from 6.5 to 8.8. From the equilibrium data individual steps (Adair constants), ki (i equals 1, 2, 3, 4), were obtained and the number of Bohr protons (deltaHi+) released on the ith stage of oxygenation was estimated. The pH dependence of k4 was very small, while the other ks strongly depended on pH over the pH range examined. As a consequence, the contribution of each step of oxygen binding to the alkaline Bohr effect nonuniform: deltaH4 was very small compared with deltaH1+, deltaH2+, and deltaH3+. In spite of this, calcuation has shown that the fractional number of released protons is essentially proportional to fractional oxygen saturation because of cooperative effects in hemoglobin. Thus, the present study indicates that the linear relationship between the fractional number of released protons and the degree of ligation, as obtained from titration experiments, is not necessarily incompatible with the pH variance of the shape of the oxygen equilibrium curve. The nonuniform pH depencence of the Adair constants implies that the two-state allosteric model of Monod, J., Wyman, J., and Changeus, J.P. (1965) J. Mol. Biol. 12, 88-118 is not adequate to describe the heterotropic effect caused by protons.


Transport and metabolism of folates by bacteria.

Transport of labeled folic acid (PteGlu), pteroylpolyglutamates (PteGlu3-5), 5-methyl-tetrahydrofolate (5-methyl-H4PteGlu), and methotrexate in late-log phase cells of Lactobacillus casei was active, and subject to inhibition by unlabeled pteroylmonoglutamates, pteroylpolyglutamates, and iodoacetate, but not glutamate or glutamate dipeptides. Pteroylpolyglutamates were transported without prior hydrolysis and shared a common uptake system with pteroylmonoglutamates. The affinity and maximum velocity of PteGlun uptake decreased with increasing glutamate chin length (Km:PteGlu1, 0.03 mum; PteGlu3, 0.32 mum; PteGlu4, 1.9 mum; PteGlu5, 3.7 mum) and comparisons with growth response curves suggested that polyglutamates were more effectively utilized by L. casei, once transported, than monoglutamate. No concentration of 5-methyl-H4PteGlu3-8 inside the cells was observed. The major folate metabolites found in L. casei preloaded with high levels of [3H]PteGlu (0.5 mum) were 10-formyl-H4PteGlu2 and 10-formyl-PteGlu. Both compounds were released, the monoglutamate more rapidly. Pteroyltriglutamate formation appeared to be a rate-limiting step in intracellular metabolism. No 10-formyl-Pte-Glu was found in iodoacetate-treated cells and efflux was inhibited. Cells preloaded with low levels of [3H]PteGlu (7 nm) metabolized the vitamin to polyglutamate forms, the major derivatives being H4PteGlun. First order exit rates of labeled folate from preloaded L. casei indicated an inhibition of PteGlu uptake with time. Exit rates dropped from 0.05 min-1 to greater than 0.002 min-1 as intracellular folate was metabolized from monoglutamate to polyglutamate derivatives (n larger than or equal to 3). In the latter case, materials lost by efflux were breakdown products and no folate of glutamate chain length greater than two was released. Pediococcus cerevisiae actively transported 5-methyl-H4PteGlu but did not take up to 5-methyl-H4PTeGlu3-8. No active accumulation of 5-methyl-H4PteGlu was observed in Streptococcus faecalis.


The molecular behavior of apoA-I in human high density lipoproteins.

The properties of apoA-I in human high density lipoprotein (1.063 smaller than d smaller than 1.210 gm per ml) have been examined by fluorescence and difference absorption spectroscopy, while the behavior of the total complement of apoproteins has been evaluated by far ultraviolet circular dichroism. Marked increases in stability to temperature, pH, and guanidine hydrochloride were observed for apoA-I in the native particle as compared to the isolated state.


On the structure-function relationship of acyl carrier protein of Escherichia coli.

The conformations of Escherichia coli acyl carrier protein (ACP) and acetylated ACP have been studied as a function of pH and salt concentration by circular dichroism measurements. The results show that the amino groups of ACP in their protonated form  are important for maintaining the native conformation of the protein at physiological pH. However, externally added cations (divalent more effectively than monovalent ones) can substitute for the ammonium groups in maintaining the ordered structure pf ACP. It is suggested that both the ammonium groups of ACP and externally added cations reduce the repulsion between carboxylate groups of ACP and thereby prevent the unfolding of the protein. A reduction of the number of negatively charged carboxylate groups by either protonation or chemical modification abolished the requirement for either ammonium groups or other cations. A qualitative agreement between the effect of salt on the conformation and on the biological activity of acetylated ACP has been observed. The single arginine residue of acetylated ACP has been modified by treatment with a trimer of 2,3-butanedione with the resulting derivative of ACP retaining most of its biological activity.


Ketopantoic acid and ketopantoyl lactone reductases. Stereospecificity of transfer of hydrogen from reduced nicotinamide adenine dinucleotide phosphate.

The stereospecificity of hydrogen transfer from NADPH to the appropriate carbonyl substrate catalyzed by ketopantoic acid and ketopantoyl acid and ketopantoyl lactone reductases of yeast (Saccharomyces cerevisiae) and Escherichia coli has been determined. Yeast and E. coli ketopantoic acid reductases are B-specific enzymes which transfer hydrogen from [4B-3H]-NADPH to ketopantoic acid to form [3H]pantoic acid. In contrast to the usual observations on the stereospecificity of functionally similar dehydrogenases from different species, yeast and E. coli ketopantoyl lactone reductases exhibit opposite stereospecificities. Both of two forms of yeast ketopantoyl lactone reductases are A-specific enzymes which form [3H]pantoyl lactone from ketopantoyl lactone and [4A-3H]NADPH, whereas, two forms of E. coli ketopantoyl lactone reductases are B-specific enzymes.


Catalytic acitivity of Ntau-carboxymethylhistidine-12 ribonuclease.

Ntau-Carboxymethylhistidine-12 RNase is active with both RNA and uridine cyclic 2':3'-monophosphate as substrates. Experimental evidence is presented to show that the activity cannot be due to contaminating RNase A, or other RNase-type protein, to the presence of a mixed dimer between 12- and 119-substituted RNases, or to the presence of trace amounts of Ntau-carboxymethylhistidine-12 RNase. The carboxymethyl derivative has approximately 1 and 13 per cent the specific activity of native enzyme against cyclic 2',3'-UMP at pH 5.0 and 8.5, respectively.


Long chain fatty acid binding to human plasma albumin.

The binding of six physiologically important long chain fatty acids to defatted human plasma albumin was measured at 37 degrees in a calcium-free Krebs-Ringer phosphate buffer, pH 7.4. The data were analyzed in terms of multiple stepwise equilibria. With the saturated acids, the magnitude of the equilibrium (association) constants, Ki, increased as the chain length increased: laurate smaller than myristate smaller than palmitate smaller than stearate. Oleate was bound more tightly than stearate; by contrast, linoleate was bound less tightly than stearate. The equilibrium constants, K1 through K12, ranged from 2.4 times 10-6 - 3.5 times 10-3 m-1 for laurate to 2.6 times 10-8 - 3.5 times 10-5 m-1 for oleate. Successive values of Ki decrease for each of the acids, indicating that major cooperative binding effects do not occur over the physiological range of fatty acid concentrations. In no case could the Ki be segregated into distinct classes, suggesting that any grouping of albumin binding sites is somewhat arbitrary. The results were inconclusive concerning whether premicellar association of unbound fatty acid occurs. Although corrections for premicellar association produced very little change in the Ki values for myristate, they raised the Ki for palmitate and stearate by 300 to 700 per cent. A sigmoidal relationship was obtained when the logarithm of Ki was plotted against chain length for the saturated fatty acids containing 6 to 18 carbon atoms, indicating that the binding energy is not simply a statistical process dependent only on the fatty acid chain length. This selectivity that albumin contributes to the binding process may be due to varying degrees of configurational adaptability of its binding sites as the fatty acid increases in length.


Chromatin-associated protein phosphokinases of rat ventral prostate. Characteristics and effects of androgenic status.

Protein phosphokinase activity endogenous to rat ventral prostate chromatin was assayed by using edphosphophosvitin as an exogenous substrate. For maximal activity of the kinase reaction, the presence of 200 mM NaCl, 5 mM MgCl2, and 1 mM dithiothreitol was essential. Two apparent pH optima were observed, a broad one between pH 7 and 7.4, and one at pH 7.89. At pH 7.4 the apparent Km for 31% dephosphophosvitin was 0.3 mg per ml. With respect to ATP, two apparent Km values (0.04 and 0.41 mM) were found. The kinase activity was minimal toward exogenous histones when used as substrates (3% for lysine-rich and 0.3% for arginine-rich (f3) histones, compared with dephosphophosvitin controls). The protein phosphokinases were not significantly stimulated by cyclic adenosine 3':5'-monophosphate (cyclic AMP) when histones used as substrate. With dephosphophosvitin as substrate, cyclic AMP produced a small inhibition (5 to 15%). Orchiectomy of adult rats resulted in a rapid decline in the chromatin-associated protein phosphokinase activity assayed using optimal experimental condition described above. At 9 hours postorchiectomy, a 30% decline in the activity was observed; this was further reduced to about 50% of the control by 18 hours. This decrease in the kinase activity (e.g. at 9 hours postorchiectomy) appears to precede measurable changes in the protein and RNA complements of chromatin. Testosterone replacement following orchiectomy abolished this decline in the chromatin-associated activity. The chromatin-associated protein phosphokinase activity toward lysine-rich and arginine-rich histones was also sensitive to androgenic status of the animals and declined rapidly postorchiectomy. The results suggest the presence of multiple and androgen-sensitive protien phosphokinases associated with rat ventral prostate chromatin, which may modulate the phosphorylation of nuclear nonhistone phosphoproteins with changing gene action mediated by testosterone in this target tissue.


Spectroscopic evidence for the generation of singlet oxygen in the reduced nicotinamide adenine dinucleotide phosphate-dependent microsomal lipid peroxidation system.

In the presence of Fe3+-ADP and NADPH, washed rat liver microsomes at 37 degrees and at pH 7.5 emitted ultraweak light with the peroxidative cleavage of endogenous lipid. The main light-emitting species of this system was analyzed spectroscopically in the visible region and estimated to be singlet oxygen. The addition of superoxide dismutase or catalase did not inhibit the light emission significantly. It is unlikely therefore that superoxide anion and OH are involved in the generation of singlet oxygen in the present system.


Injuries of the midtarsal joint.

Injuries involving the midtarsal joint, which are frequently misdiagnosed, have been studied to clarify the mechanism, classification and treatment. The necessity for routine antero-posterior, lateral and oblique radiographs is emphasised. Seventy-one injuries have been classified according to the direction of the deforming force:medial, longitudinal compression, lateral, plantar and crush types are described. Included in the medial and lateral types is a hitherto undescribed tarsal rotation or "swivel" injury. The mechanism whereby longitudinal compression causes fractures of the body of the navicular is described, and two varieties having different prognoses are defined: one due to purely longitudinal compression and the other due to longitudinal compression with a medial component. The results of treatment have been assessed clinically and radiologically. Reduction, open if necessary, with internal fixation, is recommended for displace fractures: primary arthrodesis is not indicated. For severe persistent symptoms from medial and longitudinal force injuries triple arthrodesis is recommended, and from lateral force injuries, calcaneo-cuboid arthrodesis.


Computer analysis of amino acid chromatograms.

A procedure is described for the automatic off-line analysis of amino acid chromatograms of protein hydrolysates, using a small computer. The data requirements are basic, and, unlike previous programs, the present system allows the separation and identification of bands, as well as the quantitative determination of composition. With minor modification, the program could be extended for use with most types of chromatographic data. The validity of the application of the program to experimental data is discussed.


Separation of tricyclic psychosedative drugs by high-speed ion-pair partition and liquid-solid adsorption chromatography.

The application of high-speed ion-pair partition and liquid-solid adsorption chromatography to the separation of twenty common tricyclic tranquilizers and antidepressant drugs is described. In the ion-pair system, amine-perchlorate ion-pairs were extracted from an aqueous stationary phase supported on 10-mum silica gel by organic eluents containing a chloromethane and a higher aliphatic alcohol, and chromatographic parameters for elution by eight eluent mixtures are presented. Using 5 mm times 120 mm columns good separations, according to chemical class, were achieved. For adsorption chromatography, the components were eluted from 20-mum spherical alumina using eluents containing methylene chloride, n-hexane or n-pentane, and acetic acid. Chromatographic parameters are given for eight eluent compositions. Components differing little in structure are well separated by liquid-solid adsorption chromatography. Compared with ion-pair partition chromatography, adsorption chromatography is much more selective for compounds of the same chemical type. The two methods are therefore complementary. Both methods gave plate heights in the range of 0.1 to 0.3 mm.


Chromatographic separation of conformers of substituted asymmetric nitrosamines.

The syn and anti conformers of N-nitrosoproline, N-nitrososarcosine and N-nitroso-2-(ethylamino)-ethanol, have been separated by liquid chromatography. These conformers result from hindered rotation about the N-N bond. Separation was achieved using adsorption, reversed-phase, and ion-exchange modes. For the nitroso-amino acids, a shift in the equilibrium conformer concentration was observed with changes in pH.


Aromatization of androgens by human abdominal and breast fat tissue.

The ability of human abdominal, breast and axillary fat to convert androgens into estrogens was investigated by incubating labeled substrates in the presence of NADPH with a variety of cell preparations. The incubation products were subjected to phenolic partition, paper chromatography, methyl-ether formation, repeat chromatography and crystallization with cold carrier reference standards to constant specific activity. Androstenedione was converted to estrone and, to a lesser extent, to 17beta-estradiol by crude homogenates, minces, fat-free particulate fractions (1,000-100,000 time g) and isolated fat cells obtained from abdominal, breast or axillary fat. Testosterone was found to be aromatized as actively as androstenedione, but inthis case more 17 beta-estrodiol was formed than estrone. 19-Hydroxyandrostenedione-2 also served as substrate, givingresults similar to those obtained with androstenedione. Fat tissue obtained from cancerous breasts was found to be as active as normal breast fat (1-4 pg/g fat/90 min) and within the range found for abdominal fat (1-27 pg/g fat/90 min). In each case in which axillary fat was compared to breast fat from the same subject, the activity of the axillary fat was 5 to 10 times higher. The results indicate a possible role of adipose tissue as a significant extra-gonadal source of estrogens.


Chromatographic heterogeneity of insulin extracted from insulomas.

On gel filtration of acid-ethanol extracts from three pancreatic beta-cell adenomas 1.4% to 1.8% of total immunomeasurable insulin (IMI) eluted ahead of proinsulin. This high molecular IMI was resolved into three components. The presence of urea in the dilute acetic acid solutions of extracted tumor tissue did not influence the pattern of gel filtration. High molecular IMI dissolved in dilute acetic acid showed to be stable if immediately rechromatographed, but a partial dissociation to insulinlike and proinsulinlike components (ILC and PLC) was found if rechromatography was performed after 48 h of incubation. Mainly ILC and PLC were found on rechromatography provided high molecular IMI was dissolved and incubated briefly in 0.04M phosphate buffer, pH 7.4. It proved improbable that the proteolytic action of some protein being extracted with the hormones caused a splitting of high molecular IMI at pH 7.4. We conclude from our findings that the components of high molecular IMI are not precursors of proinsulin and insulin but are either self-associated products of the hormones or associations of insulin and proinsulin to other proteins extracted from insuloma tissue.


Studies on the human testis. VI. NADH-linked reactions of microsomal steroid 20alpha-and 20beta-hydroxysteroid dehydrogenase and 17alpha-hydroxylase.

NADH-linked 20alpha- and 20beta-hydroxysteroid dehydrogenase and 17alpha-hydroxylase activities were demonstrated in the microsomal fraction of the human testis. The microsomal 20alpha-hydroxysteroid dehydrogenase showed substrate affinity to pregnenolone and progesterone and not to 17alpha-hydroxyprogesterone and preferred NADH to NADPH as a hydrogen donor. In the presence of NADH, the optimal pH for the enzyme was 7.7 and the apparent Michaelis constants of the enzyme for progesterone and pregnenolone at 37 C and pH 7.4 were 6.9-7.1 X 10-6M and in the order of 10-5M, respectively, 17alpha, 20beta-Dihydroxypregn-4-en-3-one was the only significant metabolite produced from 17alpha-hydroxyprogesterone by microsomal fraction of the human testis in the presence of NADH. The apparent Michaelis constant of microsomal 20beta-hydroxysteroid dehydrogenase for 17alpha-hydroxyprogesterone in the presence of NADH was in the order of 10-5M at 37 C and pH 7.4. The microsomal 17alpha-hydroxylase catalyzed the metabolism of pregnenolone and progesterone at a similar rate in the presence of NADH. The optimal pH and the apparent Michaelis constant at 37 C and pH 7.4 of the NADH-linked reaction of 17alpha-hydroxylase for progesterone were 7.7 and 5.3-5.4 X 10-7M, resepctively. The NADH-linked enzyme activity for progesterone was competitively inhibited by both pregn-5-ene-3beta, 20alpha-diol (inhibition constant: 1.7 X 10-7M) and 20alpha-hydroxypregn-4-en-3 one (inhibition constant: 6.6 X 10-7M), and was resistant to poor oxygen supply during incubation. The results indicate that the microsomal 20alpha-hydroxysteroid dehydrogenase is a different enzyme from the one in the soluble fraction of the human testis and that microsomal 17alpha-hydroxylase in the human testis is activated by NADH as well as NADPH.


Role of adrenergic stimuli in parathyroid hormone secretion in man.

The role of adrenergic stimuli in the secretion of parathyroid hormone (PTH) in man was evaluated. Intradermal injections of isoproterenol, 0.15 mg, or epinephrine, 0.3 mg, caused significant prompt increases in serum PTH levels. These increases were not accompanied by any changes in serum calcium (Ca) during the period of observation. Phenylephrine, 1.5 mg, intradermally, did not cause any significant changes in serum PTH or serum Ca. Propranolol infusion alone significantly inhibited the basal secretion of PTH. This inhibition by propranolol was overcome by isoproterenol administration. The results indicate that 1) beta adrenergic agents increase PTH secretion whereas alpha adrenergic agents have no effect, 2) beta adrenergic stimuli probably play an important physiological role in basal PTH secretion in man.


Action of (1-des(aspartic acid), 8-isoleucine) angiotensin II upon the pressor and steroidogenic activity of angiotensin II.

The vascular and steroidogenic responses to (1-Sarcosine, 8-Isoleucine)-, and to (1-Des (Aspartic acid), 8-Isoleucine) angiotensin II were compared in bilaterally nephrectomized, ACTH-suppressed dogs receiving constant infusions of angiotensin II. Aldosterone secretion rate was significantly inhibited by pretreatment with 200 ng/Kg/min of the heptapeptide, (1-Des(Aspartic acid), 8-Isoleucine) ang II, but not by similar doses of the octapeptide, (1-Sarcosine, 8-Isoleucine) ang II. In contrast, the pressor action of ang II was unaffected by (1-Des (Aspartic acid), 8-Isoleucine) ang II though significant inhibition occurred with relatively small doses of (1-Sarcosine, 8-Isoleucine) ang II. This study suggests that: (a) angiotensin receptors in adrenal cortex and vascular smooth muscle are functionally different, and (b) (1-Des(Aspartic acid),8-Isoleucine) angiotensin II is a specific antagonist of steroidogenic effect of ang II.


Hemoglobin Syracuse (alpha2beta2-143(H21)His leads to Pro), a new high-affinity variant detected by special electrophoretic methods. Observations on the auto-oxidation of normal and variant hemoglobins.

Family members from four generations were found to have polycythemia and increased whole blood O2 affinity (P50; 11 mm Hg; normal, 27 mm Hg). No abnormal hemoglobin bands were seen after electrophoresis on starch gel at pH 8.6 or agar gel at pH 6.0. Analysis of the oxygenated hemolysate by isoelectric focusing on polyacrylamide gel revealed two closely spaced bands. When deoxygenated hemolysate was analyzed in oxygen-free gels, the two components were more widely separated. About 40% of the patient's hemoglobin focused at a more acid pH than hemoglobin A, indicating a hemoglobin variant with impaired Bohr effect. Chromatography of globin in 8 M urea revealed two beta-chain peaks, the first of which was eluted at a lower buffer molarity than normal beta chain. Fingerprints of tryptic digests of the aminoethylated chains were done on silica gel thin-layer plates. Tp 14 from the abnormal beta chain had slower electrophoretic mobility and a greater Rf value. Amino acid analyses of this peptide gave values identical with those of betaTp 14, except that it contained one proline residue and no histidine. Since the one His in betaTp 14 is in position 143, hemoglobin Syracuse in alpha2beta2-143 His leads to Pro. Native Hb Syracuse could be separated from hemoglobin A on a carboxymethylcellulose column. The inclusion of 0.1 mM EDTA in the preparative buffers proved very useful in reducing the formation of methemoglobin. Oxygen equilibria of purified hemoglobin Syracuse showed high oxygen affinity (P50 value 12% that of hemoglobin A) and lack of cooperativity between subunits (Hill's n equals 1.1). The alkaline Bohr effect was about half that of hemoglobin A. The proline substitution at betaH21 disrupts the helical configuration and probably prevents the formation of salt bonds that are important in stabilizing the deoxy structure and contribute to the alkaline Bohr effect. Since beta143 His is a binding site for 2,3-diphosphoglycerate (2,3-DPG), it is not suprising that hemoglobin Syracuse had markedly impaired reactivity with 2,3-DPG. Hemoglobin Syracuse auto-oxidized more slowly than hemoglobin A, probably reflecting a slower rate of dissociation of oxygen from fully liganded hemoglobin.


The effect of an H2-receptor antagonist on food-stimulated acid secretion, serum gastrin, and gastric emptying in patients with duodenal ulcers. Comparison with an anticholinergic drug.

The purpose of the present series of experiments was to measure and compare the effects of an anticholinergic drug (isopropamide) and an antagonist of the histamine H2 receptor (metiamide) on food-stimulated acid secretion. Patients with duodenal ulcers were stimulated by a steak meal, and acid secretion was measured by in vivo intragastric titration. The largest dose of isopropamide that can be taken clinically without producing intolerable side effects (maximum tolerated dose) suppressed food-stimulated acid secretion by 35%. By contrast, metiamide in a 400-mg dose produced no side effects and almost completely abolished food-stimulated acid secretion. A dose-response curve revealed that a 50-mg dose of metiamide was required to suppress food-stimulated acid secretion by 50%. Further studies showed that metiamide and isopropamide are additive in suppressing food-stimulated acid secretion, and that metiamide has no effect on serum gastrin concentration or on gastric emptying.


Laser fusion of selected dental casting alloys.

Laser-fused precious, nonprecious, and mixed metal welds were investigated for optimum weld energy requirement as determined by tensile strength tests. These strengths were compared to soldered unions. Laser fusion is as good as, or better than, unions created by conventional dental joining methods.


Effects of phosphates in acid-containing beverages on tooth erosion.

Rat studies demonstrated that low-pH powdered beverage mixes containing monocalcium phosphate caused less molar erosion than reconstituted frozen orange juice and grape juice when these beverages were consumed at excessive levels. A number of sodium phosphate supplements in low-pH beverages were not as effective as monocalcium phosphate in preventing molar erosion. Total acidity or pH values of the beverages could not be used with reliability to predict their potential to cause molar erosion.


Studies of fluorapatite: II. The solubility behavior.

The solubility product of fluorapatite has been determined by equilibrating a very pure mineral specimen in KCl solutions at ionic strengths up to 0.1. At pH's less than 5.0 to 6.0 the solubility product of fluorite, and not that of fluorapatite, is the governing principle under the experimental conditions used.