Collagen cross-linking. Purification and substrate specificity of lysyl oxidase. Lysyl oxidase is a specific amine oxidase that catalyzes the formation of aldehyde cross-link intermediates in collagen and elastin. In this study, lysyl oxidase from embryonic chick cartilage was purified to constant specific activity and a single protein band on sodium dodecyl sulfate acrylamide gel electrophoresis. This band had an apparent molecular weight of 62,000. The eluted protein cross-reacted with inhibiting antisera developed against highly purified lysyl oxidase. The highly purified enzyme was active with both insoluble elastin and embryonic chick skin or bone collagen precipitated as reconstituted, native fibrils. There was low activity with nonhydroxylated collagen, collagen monomers, or native fibrils isolated from lathyritic calvaria. The maximum number of aldehyde intermediates formed per molecule of collagen that became insoluble was two. These results indicate that lysyl oxidase has maximum activity on ordered aggregates of collagen molecules that may be overlapping associations of only a few collagen molecules across. Formation of aldehyde intermediates and cross-links during fibril formation may facilitate the biosynthesis of stable collagen fibrils and contribute to increased fibril tensile strength in vivo.


Collagen cross-linking. Synthesis of collagen cross-links in vitro with highly purified lysyl oxidase. In this paper, the synthesis of collagen cross-links in vitro was investigated in a defined system consisting of highly purified chick cartilage lysyl oxidase and chick bone collagen fibrils. Cross-link synthesis in vitro was quite similar to the biosynthesis of collagen cross-links in vivo. Enzyme-dependent synthesis of cross-link intermediates and cross-linked collagen derived from lathyritic collagen occurred. The concentration of the two principal reducible cross-links, N6:6'-dehydro-5,5'-dihydroxylysinonorleucine and N6:6'-dehydro-5-hydroxylysinonorleucine, increased to a peak value of approximately two cross-links per molecule and then decreased. Synthesis of histidinohydroxymerodesmosine and a second polyfunctional cross-link of unknown structure began after synthesis of bifunctional cross-links was largely completed and proceeded linearly afterwards. Inhibition of lysyl oxidase after the bulk of bifunctional cross-link synthesis had occurred did not alter the rate of decrease in reducible cross-link concentration but did inhibit further histidinohydroxymerodesmosine synthesis. These results indicate that lysyl oxidase and collagen fibrils are the only macromolecules required for cross-link biosynthesis in vivo. It is likely that the decrease in reducible cross-links observed during fibril maturation results from spontaneous reactions within the collagen fibril rather than additional enzymatic reactions.


Stimulation of guanylate cyclase of fibroblasts by free fatty acids. The membranous guanylate cyclase of Balb 3T3 fibroblasts was stimulated by a fraction of calf serum extracted by ether. Stimulation was observed with Mg2+ as the only bivalent cation in the presence of Lubrol PX. The activator co-chromatographed with free fatty acids, and several of these were found to stimulate guanylate cyclase. Among the saturated fatty acids, myristic acid had the highest activity. Stimulating activity diminished as the hydrocarbon chain of the fatty acid was lengthened or shortened. Introduction of an unsaturated bond enhanced the activation by the longer fatty acids. This pattern of specificity is similar to that observed for the effect of fatty acids on many other membranous functions. Under appropriate conditions fatty acids were found to stimulate guanylate cyclase activity in the absence of Lubrol PX. The relationship among the effects of Mg2+, Mn2+, Lubrol PX, and fatty acids on enzyme activity was examined. On the basis of these studies, it appears that fatty acids stimulate the enzyme by a mechanism different from nonionic detergents or Mn2+.


Identification of a functional arginine residue involved in coenzyme binding by the NADP-specific glutamate dehydrogenase of Neurospora. The NADP-specific glutamate dehydrogenase (EC 1.4.1.4) of Neurospora crassa is inhibited by reaction with 1,2-cyclohexanedione which binds to arginine residues. With the 14C-labeled reagent, a peptide was isolated with the sequence: Gly-Gly-Leu-Arg-Leu-His-Pro-Ser-Val-Asn-Leu, corresponding to residues 78 through 88 in the protein. The arginine, residue 81, was present as N7,N8-(1,2-dihydroxycyclohex-1,2-ylene)-arginyl (or DHCH-arginine). Present evidence indicates that this arginine residue resides at or near the nicotinamide binding domain of the enzyme. Similar sequences are present in the bovine liver enzyme (EC 1.4.1.3) and the NAD-specific glutamate dehydrogenase of Neurospora (EC 1.4.1.2).


X-ray and functional studies of hemoglobins Nancy and Cochin-Port-Royal. The mutations in hemoglobin Nancy beta145(HC2) Tyr leads to Asp and hemoglobin Cochin-Portal-Royal beta146(HC3) His leads to Arg involve residues which are thought to be essential for the full expression of allosteric action in hemoglobin. Relative to the structure of deoxyhemoglobin A, our x-ray study of deoxyhemoglobin Nancy shows severe disordering of the beta chain COOH-terminal tetrapeptide and a possible movement of the beta heme iron atom toward the plane of the porphyrin ring. These structural perturbations result in a high oxygen affinity, reduced Bohr effect, and lack of cooperatively in hemoglobin Nancy. In the presence of inositol hexaphosphate (IHP), the Hill constant for hemoglobin Nancy increases from 1.1 to 2.0. But relative to its action on hemoglobin A, IHP is much less effective in reducing the oxygen affinity and in increasing the Bohr effect of hemoglobin Nancy. This indicates that IHP does not influence the R in equilibrium T equilibrium as much in hemoglobin Nancy as in hemoglobin A, and this probably is due to the disordering of His 143beta which is known to be part of the IHP binding site. IHP is also known to produce large changes in the absorption spectrum of methemoglobin A, but we find that it has no effect on the spectrum of methemoglobin Nancy. In contrast to the large structural changes in deoxyhemoglobin Nancy, the structure of deoxyhemoglobin Cochin-Port-Royal differs from deoxyhemoglobin A only in the position of the side chain of residue 146beta. The intrasubunit salt bridge between His 146beta and Asp 94beta in deoxyhemoglobin A is lost in deoxyhemoglobin Cochin-Portal-Royal with the guanidinium ion of Arg 146beta floating freely in solution. This small difference in structure results in a reduced Bohr effect, but does not cause a change in the Hill coefficient, the response to 2,3-diphosphoglycerate, or the oxygen affinity at physiological pH.


A stereochemical method for detection of ATP terminal phosphate transfer in enzymatic reactions. Glutamine synthetase. An isotope scrambling method is described for the detection of transient [Enz:ADP:P-X] formation from [18O]ATP in ATP-coupled enzyme reactions. The method makes use of torsional symmetry of the newly formed (see article) group in ADP. [18 O]ATP labeled in the betagama bridge oxygen was incubated with enzyme and reversible cleavage of the PbetaO -- Pgamma bond was detected by the appearance of 18O in the beta nonbridge oxygens of the ATP pool. Experiments with sheep brain and Escherichia coli glutamine synthetases show that cleavage of ATP of enzyme-bound ADP and P-X requires glutamate. The exchange catalyzed by the E. coli enzyme with glutamate occurs in the absence of ammonia and is partially inhibited by added NH4Cl, as expected if the exchange is in the mechanistic pathway for glutamine synthesis. The results provide kinetic support for a two-step mechanism where phosphoryl transfer from ATP to glutamate precedes reaction with ammonia.


Steroid 5alpha-reductase in cultured human fibroblasts. Biochemical and genetic evidence for two distinct enzyme activities. Various properties of the steroid 5alpha-reductase have been examined in cell-free extracts of skin and of fibroblasts cultured from genital and nongenital skin from control subjects and from patients with several forms of male pseudohermaphroditism. When 20alpha-hydroxy-4-[1,2-3H] pregnen-3-one was used as substrate, two 5alpha-reductase activities could be demonstrated in intact skin and cultured fibroblasts. The major activity, previously described for microsomes from human prepuce and extracts of cultured foreskin fibroblasts, is characterized by a narrow pH optimum near 5.5 and is limited to fibroblasts derived from genital skin. A second activity, not limited by the site of biopsy, has been demonstrated over a higher and broader range of pH (from 7 to 9); this enzyme activity is found in both genital and nongenital skin and in fibroblasts cultured from all skin regions. Whereas there is wide variability in the activity at pH 5.5 in genital skin fibroblasts, the activity at pH 7 to 9 is similar in fibroblasts derived from all anatomical sites. The two activities exhibit different kinetics with respect to steroid substrate and are also dissimilar in their subcellular distributions. Other properties, such as coenzyme requirement, steroid substrate specificity, and instability with increasing temperature, appear to be similar.


The NADH dehydrogenase of the respiratory chain of Escherichia coli. II. Kinetics of the purified enzyme and the effects of antibodies elicited against it on membrane-bound and free enzyme. The purified respiratory chain NADH dehydrogenase of Escherichia coli oxidizes NADH with either dichlorophenolindophenol (DCIP). ferricyanide, or menadione as electron acceptors, with values for NADH are similar with the three electron acceptors (approximately 50 muM). The purified enzyme contains no flavin and has an absolute requirement for FAD, with Km values around 4 muM. The pH optimum of the enzyme appears to be between 6.5 and 7; the optimum is difficult to establish because of nonenzymatic reduction of DCIP at the lower pH values. Potassium cyanide stimulates the DCIP reductase activity about 2-fold, but has no effect on ferricyanide reductase. The enzyme exhibits hyperbolic kinetics with respect to NADH concentration in both the ferricyanide and DCIP reductase assays, but cooperatively is seen in the menadione reductase reaction. NAD+ is an effective competitive inhibitor of the reaction (Ki congruent to 20 muM); in the presence of NAD+, the NADH saturation curve becomes cooperative, even in the DCIP reductase assay. Many adenine containing nucleotides are competitive inhibitors of the enzyme. The apparent Ki values for these nucleotides as inhibitors of the purified enzyme, the membrane-bound NADH dehydrogenase, and the NADH oxidase are equivalent. An examination of inhibitory effects of a series of adenine nucleotides suggests that the inhibitors act as analogues of NAD+, which is the true physiological inhibitor. The results suggest that the enzyme in situ is always partially inhibited by the levels of NAD- in the E coli cell, and thus behaves in a cooperative fashion to changes in the NAD+/NADH ratio. An antibody has been elicited against the purified NADH dehydrogenase. Immunodiffusion and crossed immunoelectrophoresis show that the antibody is directed principally against the NADH dehydrogenase, with some activity against minor contaminants in the purified preparation. The antibody inhibits NADH dehydrogenase activity 50% at saturating levels. When this antibody preparation is used to examine solubilized membrane preparations, two major immunoprecipitates are found. A parallel inhibition of the membrane-bound NADH dehydrogenase and NADH oxidase activities is seen, supporting the hypothesis that the purified enzyme is indeed a component of the respiratory chain-dependent NADH oxidase pathway.


Aspartate transcarbamylase of Escherichia coli. Heterogeneity of binding sites for carbamyl phosphate and fluorinated analogs of carbamyl phosphate. Some preparations of both native aspartate transcarbamylase from Escherichia coli and catalytic subunit have fewer tight binding sites per oligomer for carbamyl-P than the number of catalytic peptide chains. In contrast, the number of sites for the tight-binding inhibitor N-(phosphonacetyl)-L-aspartate does equal the number of catalytic chains in each case. Binding of the labile carbamyl-P was determined using rapid gel filtration, with conversion to stable carbamyl-L-aspartate during collection. Native enzyme (six catalytic chains) obtained from cells grown under the conditions of J.C. Gerhart and H. Holoubek (J. Biol. Chem. (1967) 242, 2886-2892) has 5.4 tight sites for carbamyl-P at pH 8.0 (KD = 9.9 muM), whereas native enzyme from cells grown with higher concentrations of glucose, uracil, and histidine (to yield more enzyme per unit volume of culture) has only 1.9 tight sites at pH 8.0 (KD = 4.6 muM) and only 2.3 tight sites at pH 7.0 (KD = 2.6 muM). At pH 8.0, catalytic subunit (three catalytic chains) obtained from the former native enzyme has 2.2 tight sites for carbamyl-P (KD = 2.4 muM) and the number of sites is 2.3 in the presence of 35 mM succinate, whereas catalytic subunit obtained from the latter native enzyme has 1.8 tight sites (KD = 3.6 muM) in the absence of succinate and 2.3 tight sites in its presence. The number of tight binding sites is also less than the number of subunit peptide chains in 19F nuclear magnetic resonance experiments performed with catalytic subunit and two fluorinated analogs of carbamyl-P at comparable concentrations of analogs and active sites. A model is proposed in which incomplete removal of formylmethionine from the NH2 termini of the enzyme under conditions of extreme depression affects affinity for ligands.


Evidence from 13C NMR for protonation of carbamyl-P and N-(phosphonacetyl)-L-aspartate in the active site of aspartate transcarbamylase. Nuclear magnetic resonance has been used to study the binding of [13C]carbamyl-P (90% enriched) to the catalytic subunit of Escherichia coli aspartate transcarbamylase. Upon forming a binary complex, there is a small change in the chemical shift of the carbonyl carbon resonance, 2 Hz upfield at pH 7.0, indicating that the environments of the carbonyl group in the active site and in water are similar. When succinate, an analog of L-aspartate, is added to form a ternary complex, there is a large downfield change in the chemical shift for carbamyl-P, consistent with interaction between the carbonyl group and a proton donor of the enzyme. The change might also be caused by a ring current froma nearby aromatic amino acid residue. From the pH dependence of this downfield change and from the effects of L-aspartate analogs other than succinate, the form of the enzyme involved is proposed to be an isomerized ternary complex, previously observed in temperature jump and proton NMR studies. The downfield change to chemical shift for carbamyl-P bound to the isomerized complex is 17.7 +/- 1.0 Hz. Using this value, the relative ability of other four-carbon dicarboxylic acids to form isomerized ternary complexes with the enzyme and carbamyl-P has been evaluated quantitatively. The 13C peak for the transition state analog N-(phosphonacetyl)-L-aspartate (PALA), 90% enriched specifically at the amide carbonyl group, is shifted 20 Hz downfield of the peak for free PALA upon binding to the catalytic subunit at pH 7.0. In contrast, the peak for [1-13C] phosphonaceatmide shifts upfield by about 6 Hz upon binding. Since PALA induces isomerization of the enzyme and phosphonacetamide does not, these data provide further evidence consistent with protonation of the carbonyl group only upon isomerization. The degrees of protonation is strong acids of the carbonyl groups of PALA, phosphonacetamide and urethan (a model for the labile carbamyl-P) have been determined, as have the chemical shifts for these compounds upon full protonation. From these data it is calculated that the amide carbonyl groups of carbamyl-P and PALA might be protonated to a maximum of about 20% in the isomerized complexes at pH 7.0. The change in conformation of the enzyme-carbamyl-P complex upon binding L-aspartate, previously proposed to aid catalysis by compressing the two substrates together in the active site, may be accompanied by polarization of the C=O bond, making this ordinarily unreactive group a much better electrophile. A keto analog of PALA, 4,5-dicarboxy-2-ketopentyl phosphonate, also binds tightly to the catalytic subunit and induces a very similar conformational change, whereas an alcohol analog, 4,5-dicarboxy-2-hydroxypentyl phosphonate, does not bind tightly, indicating the critical importance of an unhindered carbonyl group with trigonal geometry.


Oxidation of p-cresol by horseradish peroxidase compound I. Rate constants for the reaction between horseradish peroxidase compound I and p-cresol have been determined at several values of pH between 2.98 and 10.81. These rate constants were used to construct a log (rate) versus pH profile from which it is readily seen that the most reactive form of the enzyme is its most basic form within this pH range so that base catalysis is occurring. At the maximum rate a second order rate constant of (5.1 +/- 0.3) x 10(-7) M-1 s-1 at 25 degrees is obtained. The activation energy of the reaction at the maximum rate was determined from an Arrhenius plot to be 5.0 +/- 0.5 kcal/mol. Evidence for an exception to the generally accepted enzymatic cycle of horseradish peroxidase is presented. One-half molar equivalent of p-cresol can convert compound I quantitatively to compound II at high pH, whereas usually this step requires 1 molar equivalent of reductant. The stoichiometry of this reaction is pH-dependent.


Stoichiometry of the reaction between horseradish peroxidase and p-cresol. Over a wide range of pH horseradish peroxidase compound I can be reduced quantitatively via compound II to the native enzyme by only 1 molar equivalent of p-cresol. Since 2 molar equivalents of electrons are required for the single turnover of the enzymatic cycle, p-cresol behaves as a 2-electron reductant. With p-cresol and compound I in a 1:1 ratio compound II and p-methylphenoxy radicals are obtained in the transient state. Compound II is then reduced to the native enzyme. A possible explanation for the facile reduction of compound II involves reaction with the dimerization product of these radicals, 1/2 molar equivalent of 2,2'-dihydroxy-5,5'-dimethylbiphenyl. If only 1/2 molar equivalent of p-cresol is present, than at high pH the reduction stops at compound II. The major steady state peroxidase oxidation product of p-cresol (with p-cresol in large excess compared to the enzyme concentration) is Pummerer's ketone. Pummerer's ketone is only reactive at pH values greater than about 9 where significant amounts of the enol can be formed via the enolate anion. Therefore, in alkaline solution it is reactive with compound I, but not with compound II, which is converted into an unreactive basic form. These results indicate that Pummerer's ketone cannot be the intermediate free radical product responsible for reducing compound II in the single turnover experiments. It is postulated that Pummerer's ketone is formed only in the steady state by the reaction of the p-methylphenoxy radical with excess p-cresol.


Active site chlorination of D-amino acid oxidase by N-chloro-D-leucine. N-Chloro-D-leucine is an irreversible inhibitor or D-amino acid oxidase on a time scale of seconds. Studies with N-[36C]chloro-D-leucine, N-chloro-D-[1-14C]leucine and N-chloro-D-[4,5-3H]leucine show that the modified enzyme has been chlorinated at a site, or sites, on the apoenzyme. The 36Cl measurements agree with titrations of catalytic activity in showing that two chlorine equivalents are incorporated per active site flavin. Kinetically, the interaction with N-chloro-D-leucine behaves in a manner which is consistent with consecutive chlorinations of an amino acid residue, or residues, in the active site region by the first 2 molecules of N-chloro-D-leucine to be processed by the enzyme. The effect of chlorination of the enzyme on the steady state parameters for oxidation of D-alanine is entirely explained by a single perturbation, namely, a 1000-fold reduction in the specific rate of flavin reduction as measured directly by rapid reaction techniques.


Spore germination promoter of Dictyostelium discoideum excreted by Aerobacter aerogenes. The nutrient medium in which Aerobacter aerogenes was grown, contains a spore germination promoter (SGP) for the cellular slime mould Dictyostelium discoideum. SGP can cuase synchronous spore germination in a short time, and triggers the germination process in just a few minutes. Germination-promoting capacity of SGP decreases as it comes in contact with increasing number of spores. When spores activated by SGP are stored at 4 degrees C, they gradually return to the dormant state. SGP is comparatively heat-stable, but is unstable at pH above 10 or under 3.


Surgical treatment of dissecting aortic aneurysm. Twenty years' experience. Early and late results of 40 cases of dissecting aortic aneurysm were reported. 1. Result of 6 cases with acute dissection was as follows: two of three surgical patients survived, whereas all three with drug therapy died. Surgical treatment is preferred for the dissection of the ascending aorta or the arch. Some problems of the postoperative morbidity were discussed: 2. Whether medically treated or surgically, chronic dissection of the descending aorta or the abdominal aorta had better prognosis than that of the other site. Chronic dissection with no saccular enlargement had good prognosis. More than 10 years survival is to be expected. The operative result for the chronic aortic dissection was poor, especially in case of the involved ascending aorta and arch. For the saccular enlargement of the pseudolumen wrapping method with or without the resection might be the preferable procedure to avoid early rupture.


Aortitis syndrome due to Takayasu's disease. A guideline for the surgical indication. Seven cases of aortitis syndrome were surgically treated with good results: Abdominal aortic aneurysm 1, atypical coarctation of aorta 2, aortic valve insufficiency 2, renovascular hypertension 2. Several attentions were paid as following: 1. Operation should be avoided during acute phase of aortitis. 2. Synthetic graft material should be avoided if possible. Autogenous vein is advisable for reconstruction of small-sized artery. 3. Surgical intervention should be performed before the loss of organ function. 4. Hematological consideration is worthwhile to prevent hypercoagulopathy due to aortitis.


Effect of glass on pH-dependent stability of typhoid vaccine. Vaccines made from the Ty-2 strain of Salmonella typhosa were tested periodically for stability of pH and of potency. The acetone-treated cultures prepared in buffered saline solutions retained potency beyond 30 months of storage at 0 to 5 C. Similar vaccines in unbuffered saline solutions lost potency coincident with increase of alkalinity. Vaccines packaged in United States Pharmacopeia borosilicate glass vials retained potency and pH stability, whereas those in Type III United States Pharmacopeia soda-lime glass vials were less stable.


Latex agglutination in the diagnosis of pneumococcal infection. A latex agglutination (LA) method for detection of pneumococcal antigens was evaluated and compared with counterimmunoelectrophoresis (CIE). LA was 2 to 10 times more sensitive than CIE for the detection of purified capsular polysaccharides in defined media, but only when a 1+ or 2+ agglutination reaction was interpreted as positive. LA was much less sensitive than CIE with clinical samples. In 50 cases of pneumococcal pneumonia, antigen was detected in the serum almost twice as often with CIE (40%) as with LA (22%). LA was positive in six cases of pneumonia where CIE was negative; however, in three of these cases, antigen was detected only in undiluted sera, which raised some question about the specificity of the result. With 18 samples of cerebrospinal fluid (CSF) from 11 patients with pneumococcal meningitis, the CIE test was positive more frequenlty (14 samples) than was LA (11 samples). Moreover, antigen was detected in CSF by LA in only one additional patient than was positive by CIE alone. There was one false-positive LA reaction among 45 samples of CSF from patients without pneumococcal infection. Although LA is a less complicated method than CIE, it is not a sensitive test for pneumococcal antigens and would be of little value as a routine diagnostic method.


Optimal conditions for elution of hepatitis B antigen after absorption onto colloidal silica. Hepatitis B surface antigen (HBSAg) adsorbed from sera onto colloidal silica could be completely eluted through the use of 0.25% sodium deoxycholate in 0.01 M borax, pH 9.3, at 56 degrees C. The HBSAg recovered in the eluate represented 100% of that present in the original serum, and it was contaminated by only trace amounts of serum proteins (in decreasing amounts: beta-lipoprotein, immunoglobulin G, albumin). This preliminary step greatly facilitates purification of large amounts of HBSAg and provides small volumes of highly concentrated material for subsequent purification by density gradient centrifugation.


Immune response to acute otitis media: association between middle ear fluid antibody and the clearing of clinical infection. Clearing of the middle ear fluid in patients with acute otitis media due to Streptococcus pneumoniae or Haemophilus influenzae was significantly associated with the presence and concentration of specific antibody in the middle ear fluid at the time of diagnosis.


Metalloproteases of human articular cartilage that digest cartilage proteoglycan at neutral and acid pH. Extracts of human articular cartilage contain proteases capable of degrading the proteoglycan component of cartilage matrix at neutral and acid pH. These enzymes have been partially purified by ion exchange chromotography and characterized by disc electrophoresis, inhibition patterns, and action of proteoglycan. Three distinct metalloproteases are described. A neutral protease that digests proteoglycan subunit optimally at pH 7.25 has been purified up to 900-fold. It is strongly inhibited by o-phenanthroline, alpha-2-macroglobulin, and egg white, and to a lesser extent by D-penicillamine and EDTA. Inhibition by chelating agents is reversed by cobalt, zinc, and ferrous ions. Two acid metalloproteases, distinct from cathespins B1, D, and F, digest proteoglycan subunit at pH 4.5 and 5.5. Both are inhibited by o-phenanthroline and activity is restored by cobalt, zinc, or ferrous ions. With electron microscopy, it was found that cartilage slices were depleted of ruthenium red-staining matrix proteoglycan after incubation in vitro with a partially purified cartilage extract at neutral pH. Sedimentation, gel chromatography, sodium dodecyl sulfate-gel electrophoresis, and immuno-diffusion studies of digests of isolated proteoglycan fraction produced by the partially purified cartilage extract at neutral and acid pH confirmed that the cartilage enzymes act only on the protein component of proteoglycan subunit, producing fragments with 5 to 12 chondroitin sulfate chains. The link proteins were not digested.


The particulate superoxide-forming system from human neutrophils. Properties of the system and further evidence supporting its participation in the respiratory burst. Studies were performed to characterize the previously reported particulate O2--forming system from human neutrophils. Of eight reducing agents examined, including glutathione, ascorbic acid, and intermediates of the glycolytic and hexose monophosphate shunt pathways, only the pyridine nucleotides could serve as electron donors. At 0.1 mM pyridine nucleotide, O2- production was relatively independent of pH. The Km for NADH was approximately 0.7 mM regardless of pH, while with NADPH the Km varied from 0.02 mM at pH 6.0 to 0.3 mM at pH 7.5. The molar ratio of NADPH oxidized to O2- produced was consistent with the reaction: NADPH + 2 O2- leads to NADP+ H+; the product nucleotide was shown enzymatically to be NADP. O2- production was not inhibited by CN-, Na-, EDTA, or 1,10-phenanthroline. Particulate O2- production accounted for 35% of the oxygen taken up during the respiratory burst by an equivalent number of intact neutrophils. Greatly diminished O2- production was seen with particles prepared from cells obtained from three patients with chronic granulomatous disease, with 2.5 mM NADPH as electron donor. With 5.0 mM NADH similar observations were made with particles from two of the patients, but with this nucelotide, O2- production was only slightly reduced in the third case. The evidence available suggests that this particulate O2- -forming system is the one responsible for the respiratory burst in activated neutrophils. The relationship between this system and other O2- -forming system found in human neutrophils is discussed.


Characterization and modification of permselective properties of apatite membranes. Compressed apatite disks were used as models for tooth enamel. Their permselective properties were studied as functions of the pH of the gradient solutions, the composition of the apatites, and the pretreatments with anionic phosphate compounds and cationic proteins. The results are discussed in relation to the caries mechanisms.


Capacity of buffers to inhibit acid production within dental plaque. An oral radiotelemetric technique was used to determine whether a range of efficient buffers had the capacity to control the levels of hydrogen ions within dental plaque in the presence of sucrose. It was found that the buffers failed to control both the intensity and duration of the acid produced.


Comparison of dextranases for their possible use in eliminating dental plaque. Dextranases produced by P lilacinum NRRL 896 and NRRL 895 and by P funiculosum NRRL 1768 were studied for their possible incorporation into a dental plaque elimination system. The following properties of the enzymes were compared: effect of the pH level on the activity and the stability of the enzymes on the acid side of the pH range; molecular weight; affinity to Sephadex G-25 which served as a model for insoluble dextran in plaques; and the extent of hydrolytic action on dextrans containing alpha-1,3, alpha-1,4 and alpha-1,6 bonds in various proportions. The enzyme of P funiculosum NRRL 1768 certainly has its limitations as a plaque-degrading enzyme, for example, diminished activity at a high pH level and lack of activity on alpha-1,3 bonds. However, from our studies, and from a survey of the relevant literature with respect to the aforementioned properties in other dextranases, the enzyme of P funiculosum NRRL 1768 emerges as a suitable choice for incorporation as dextranase, possibly together with other enzymes, into an enzymatic dental plaque elimination system.


Histamine hypersensitivity in mice induced by Bordetella pertussis or pharmacologic beta adrenergic blockade. Effects of adrenergic, cholinergic, and other drugs. The effects of prostaglandin E1, E2, F2alpha (PGE2 PGF2alpha), isoproterenol, epinephrine, norepinephrine, salbutamol, practolol, atropine, aminophylline, and corticosterone on the hypersensitivity to anaphylaxis, histamine, and serotonin in Bordetella pertussis-treated mice and propranolol-treated mice were investigated. Female HLA-SW (ICR) mice, 27-29 gm, were injected with pertussis vaccine intravenously 4 days before challenge with antigen, histamine, or serotonin. Alternatively, instead of pertussis vaccine, propranolol was injected intraperitoneally 45 min before histamine challenge. Test drugs were administered intraperitoneally 15 min before challenge. PGE1 and PGE2 at a narrow range of between 10 and 100 mug and epinephrine at 100 mug protected both pertussis- and propranolol-treated mice. Isoproterenol (25 mug) and aminophilline (800 mug) protected beta-blocked mice, but did not protect pertussis-treated mice even with very high doses (1,000 and 3,2000 mug, respectively), although salbutamol (500 mug) did. PGF2alpha, norepinephrine, and atropine were not protective at all. Practolol, a beta 1-blocker, given intraperitoneally 30 min before histamine neither sensitized normal mice nor changed the effect of isoproterenol or salbutamol in pertussis-treated mice. Corticosterone 10 mg/kg reduced the number of deaths from histamine in beta-blocked mice, but not in pertussis-treated mice. The protective effect is discussed in connection with probable effects of the drugs on intracellular cyclic adenosine monophosphate (cAMP) levels.


A comparison of ATP-degrading enzyme activities in rat incisor odontoblasts. In active odontoblasts from the rat incisor, used as a model system for biologic calcification, two distinguishable enzyme activities capable of degrading adenosine monophosphate (ATP) exist. Once can be inhibited ny 1-tetramisole, (+/-)-2,3,5,6,-tetrahydro-6-phenylimidazo (2.1B) THIAZOLE HYDROCHLORIDE (Levamisol) and (+/-)-6(m-bromophenyl)-5.6-dehydroimidazo (2.1-b) thiazole oxalate (R823) and is probably identical with nonspecific alkaline phosphatase (EC 3.1.3.1). The activity of the other enzyme, named Ca2+-ATPase, is dependent on the presence of Ca2+ or Mg2+ and is activated by these ions. The pH optimum of Ca2+-ATPase is 9.8. The Ca2+-ATPase is unaffected by Levamisole, R 8231, ouabain, ruthenium red, Na+ and K+ ions. Maximal activity was found against ATP, whereas adenosine diphosphate, guanosine triphosphate, inosine triphosphate and adensoine monophosphate were hydrolysed at lower rate. It may be speculated that the Ca2+-ATPase is concerned with the transmembranous transport of Ca2+ ions to the mineralization front.


Detection of A1A2 and A2AAm1 heterozygotes among human A blood group phenotypes. From the variations of alpha-N-acetylgalactosaminyltransferases activities with the pH, evidence was obtained for the recognition of A1A2 heterozygotes in normal A blood group sera. Besides, unusual transferase properties associated with two A2 sera from individuals out of AAm1 siblings, lead to the identification of the very infrequent A2AAm1 genotypes. These results strongly support the simultaneous coexistence of both A1 and A2 transferases in heterozygotes' sera, and bring some new information on the genetical background of the Am phenotype. The meaning of transferase properties directly determined on whole sera is briefly discussed.


Adrenergic mechanisms and the adenyl cyclase system in atopic dermatitis. Patients with atopic dermatitis have abnormal autonomic responses of the arterioles, pilomotor smooth muscle, and sweat glands. Their lesions have been reported to contain increased amounts of the neurohumors, acetylcholine and norepinephrine, as well as increased activity of acetylcholinesterase and catechol-O-methyltransferase. In vitro studies of epidermis show that beta adrenergic agonists fail to evoke the normal inhibition of mitosis of basal cells of patients with atopic dermatitis. Epidermis removed not only from the lesions, but also from normal-appearing skin, responded abnormally. The increase in intracellular levels of cAMP after exposure to catecholamines was similar in normal and atopic epidermis. Lymphocytes and PMN leukocytes isolated from patients with atopic dermatitis show both a decreased physiologic response (glycogenolysis and inhibition of lysosome enzyme release) and a decreased rise in intracellular levels of cAMP upon incubation with beta agonists, but a normal response to PGE1. Cortisol increases the response of lymphocyte adenyl cyclase to both agonists and, in the case of the patients with atopic disease, more than overcomes the depressed response to beta agonists. Because the  leukocytes respond normally to PGE1 and because others have reported normal activities of skin and adenyl cyclase, phosphodiesterase, and protein kinases, we conclude that the step responsible for the diminished beta adrenergic response lies antecedent to the catalytic site of adenyl cyclase.


Coenzyme A derivatives of bile acids-chemical synthesis, purification, and utilization in enzymic preparation of taurine conjugates. Synthesis of the coenzyme A derivatives of bile acids by the mixed anyhydride method results in a product that is contaminated by unreacted CoASH and bile acid. These compounds can be purified by Sephadex LH-20 chromatography. In each case, the purified product is free of starting materials and exhibits an equimolar ratio of bile acid, coenzyme A, and thioester bond. Millimolar extinction coefficients were calculated for these compounds as A259 nm, 15.03 +/- 0.58; A232 nm, 7.60 +/- 0.17; and A232 nm for the thioester bond, 4.12 +/- 0.17. These CoA derivatives were hydrolyzed in strongly alkaline solution, but were stable at physiologic temperature and pH. Utilization of these compounds in the enzymic preparation of taurine conjugates of bile acids indicated 94% activity. These purified CoA derivatives may be useful in studying the enzymic conjugation of bile acids.


Sphingomyelinase activity at pH 7.4 in human brain and a comparison to activity at pH 5.0. A hitherto undescribed sphingomyelinase (sph'ase 7.4) of human brain has been studied in crude and partially purified (3- to 4- fold) extracts of grey matter, and compared to the known sphingomyelinase with an acid pH optimum (sph'ase 5.0). Its specificity for sphingomyelin as substrate is similar to that of sph'ase 5.0, but it differs from sph'ase 5.0 in its pH optimum (7.4 vs 5.0) and in a requirement for Mg2+ for optimal activity. Other properties of sph'ase 7.4 that distinguish it from sph'ase 5.0 include (a) its lack of appreciable solubilization during dialysis of crude homogenates (b) a more marked concentrations in grey matter than in white matter (9- to 13- fold vs 1.5- to 2-fold for sph'ase 5.0); (c) inhibition by Ca2+ and Cd2+ ions, and by EDTA; (D) stimulation by dithiothreitol, and inhibition by cysteine, N-ethylmaleimide, and p-hydroxymercuribenzoate; (e) lack of inhibition by nucleotides (AMP.ADP, and ATP) and by NAD plus NADH; and (f) relative instability to storage or manipulation between -20degrees C and 40degrees C. These differences indicate the SPH'ASE 7.4 is a different enzyme protein from sph'ase 5.0. Unlike sph'ase 5.0, which is widely distributed in mammalian tissues, sph'ase 7.4 occurs predominantly in grey matter and little activity was observed is spleen, liver, or leukocytes. The high levels of this enzyme in brain suggest a role related to the specific functions of this organ or to the need for a more stringent control of sphingomyelin catabolism in brain as compared to other organs.


A stable, radioactive substrate emulsion for assay of lipoprotein lipase. A method is described for the assay of lipoprotein lipase, using a stable, radioactive substrate emulsion. Fatty acid-labeled trioleoylglycerol was emulsified by homogenization in glycerol with lecithin as detergent. This anhydrous emulsion was stable for at least six weeks. Substrate solutions for enzyme assay were prepared by diluting the emulsion with buffer containing serum and albumin. The fatty acid produced on hydrolysis was isolated in a one-step liquid-liquid partition system. Incubations with extracts of acetone powders from adipose tissue displayed characteristics of lipoprotein lipase activity, i.e., serum dependence and inhibition by NaCl and protamine. The method is rapid (less than 1 hour), sensitive and reproducible, and suitable for routine use.


A new method for the determination of gamma-glutamyltransferase in serum. A new, simple and sensitive method is described for assay of serum gamma-glutamyltransferase activity based on the hydrolysis of the substrate L-gamma-glutamyl-3-carboxy-4-nitranilide which offers the advantage of producing directly its own chromogen. The substrate is highly soluble and the method can therefore easily be adapted to any equipment for the automated assay of gamma-glutamyltransferase. The activities at measured optimum substrate concentration are slightly higher than with the Szasz ((1969), Clin. Chem. 15, 124-136) method in which L-glutamyl-p-nitroanilide is used as substrate.


A reversible, hydrogen ion blockade of spontaneous oocyte maturation in the starfish: locus of action. Starfish (Asterias forbesi) oocytes encased within their follicle cells mature spontaneously during a portion of the normal reproductive period when released from the ovary into seawater. A previous report has shown that oocytes isolated in acidic seawater do not mature spontaneously but retain the capacity to do so when returned to normal seawater. The object of this study was to determine the mechanism by which acidic pH reversibly blocks spontaneous oocyte maturation in isolated follicles. Incidence of spontaneous oocyte maturation in follicles isolated in acidic seawater decreased as pH decreased from 7 to 4. Oocytes in which spontaneous maturation was inhibited (ASW at pH 4.7 TO 5.4) underwent germinal vesicle breakdown with the addition of 1-methyladenine. Oocytes isolated in acidic seawater (pH 4 or 5) with intact follicle cells matured spontaneously when transferred immediately to normal seawater pH 8); after four hours, 60-65% of the follicles incubated in seawater at pH 5 matured spontaneously when returned to normal seawater as compared to less than 10% of the follicles maintained at pH 4. Inhibition of spontaneous maturation was not reversible in the absence of the follicle cells. Oocytes isolated in acidic seawater with their follicle cells did not spontaneously mature when transferred to calcium-free seawater at pH 8. The results obtained support the hypothesis that acidic seawater reversibly inhibits spontaneous oocyte maturation by interfering with the release of meiosis-inducing substance from the follicle cells.


Production of retarded, albino, regenerates in newts by alpha-methyl-p-tyrosine. Inhibition of tyrosine hydroxylase with alpha-methyl-p-tyrosine retarded growth and prevented melanization of limb regenerate in adult newts (Triturus cristatus).


On Ho's "modern logic and schizophrenic thinking". The purpose of this note is to point out that conclusions drawn in the title paper about the role of logic in the schizophrenia thought process are not reliable, since they are based on patients medicated with antipsychotic drugs. The substance of this note is drawn from the title paper and related literature. The principal conclusion is that Dr. Ho's deductions about the role of logic in schizophrenia follow only if one confuses psychiatric diagnosis with psychotic behavior.


Medication, logic, and schizophernic thinking: a reply to Marini. This article attempts to answer Marini's critique of my paper and to clarify the issue of medication in the study of the role of logic in the production of schizophrenic thought. It is argued that clinical observations do not lead one to believe that either the form or the content of schizophrenic thought disorder changes in any fundamental way as a result of medication. Consequently, the fact that most of the schizophrenic Ss were medicated at the time of the original research does not negate the conclusion that the thinking pathology found in schizophrenics cannot be accounted for by positing a basic defect in their deductive reasoning process.


Analysis of passive and active electrophysiologic properties of neurons in mammalian nodose ganglia maintained in vitro. 1. We studied the passive and active electrical properties of the soma membrane of neurons in nodose ganglia removed from cats and rabbits and maintained in vitro. The ganglia were superfused at 37 degrees C with a solution formulated to approximate the extracellular fluid of each species. The solution was buffered to pH 7.34, continuously equilibrated with 95% O2 and 5% CO2, and contained dialyzed calf serum and glucose. We also examined these properties in nodose ganglion neurons in vivo. Intracellular recordings were obtained with glass micropipettes filled with either 3 M KCl or 5 M K acetate. 2. We determined mean values for a variety of passive and active electrophysiologic properties. Values obtained in vitro did not differ significantly from those obtained in vivo. Based on the passive electrical properties of the soma membrane, neurons in the nodose ganglion appear to be a uniform population, despite the different sensory modalities conveyed by the afferent fibers. 3. Cell bodies of neurons generated action potentials in response to impulses in their afferent fibers. Somatic spikes could be evoked by stimulation of either the supranodose or infranodose vagus nerve, and an inflection point could be seen on their rising phase. When the vagus nerve was stimulated at frequencies greater than 10-20 Hz, the generation of somatic spikes often became progressively delayed and then failed completely, leaving a smaller potential (IS spike) which was apparently generated in the initial complex. The afterhyperpolarization was associated only with the somatic spike. 4. Many neurons, both in vitro and in vivo, developed a persistent hyperpolarization when repetitive action potentials occurred in the soma. This hyperpolarization was apparent at frequencies as low as 1-2 Hz, persisted for up to 5 s after the occurrence of the last somatic spike, and sometimes caused failure of somatic spikes to be generated. 5. Neurons in both species differed in their responses to suprathreshold depolarization applied through the recording electrode. Some neurons produced a train of action potentials which lasted for the duration of the depolarizing pulse, the frequency of the train being related to the magnitude of depolarization. The trains were characterized by gradually decreasing spike amplitudes and increasing interspike intervals. Other neurons responded with only a single spike or brief burst of action potentials at the beginning of depolarization to threshold. 6. It is suggested that the adaptive properties of the soma membrane of a peripheral sensory neuron are similar to those of its sensory ending, and that electrophysiological studies of the soma membrane may provide an opportunity to examine mechanisms of receptor adaptation.


Influence of molasses lignin-hemicellulose fractions in rat nutrition. Previous reports have shown that fractionation of the non-sugar, non-dialyzable components of cane molasses yielded a fraction designated as a black phenolic-carbohydrate complex. Incorporation of 0.03% of this complex into diets fed weanling male rats significantly increased the growth rate above that of rats fed the basal diet alone. This study was conducted to determine the chemical nature and growth stimulating action of the black phenol-carbohydrate complex. Alkaline cleavage under nitrogen yielded a mixture of phenols and a carbohydrate fraction which was recovered by precipitation at pH 6 in four volumes of ethanol. The alkaline cleaved, free hemicellulose was non-diayzable and stimulated growth when incorporated into rat diets at the 0.03% levels. Acid hydrolysis of the complex yielded an insoluble product identified as lignin and found to represent 18% to 20% of the entire complex. The chemical nature of this ligninhemicellulose fraction and the previously reported growth-enhancing acid resistant hemicellulose fraction isolated from various plant sources were found to be similar.


Effects of alkali-treated proteins: feeding studies with free and protein-bound lysinoalanine in rats and other animals. To find out whether alkali-treated proteins posses nephrotoxic properties, feeding studies were conducted with drastically treated soybean protein and casein, and also with lysinoalanine (LAL), the amino acid known to be formed in protein subjected to high pH at elevated temperature. The feeding of synthesized LAL to rats at dietary levels of 100 ppm and above induced typical renal changes, called nephrocytomegalia. No such changes or any other indications of toxicity were observed, however, upon feeding much higher levels of LAL (up to 6,000 ppm) when provided as the protein-bound compound in alkali-treated casein or soybean protein. When set free by complete acid hydrolysis, LAL induced considerable renal activity, comparable to that of the synthetic compound. These results indicate that alkali treatment of proteins does not induce nephrotoxic properties provided that the compound remains protein-bound. Some nephrotoxic activity was observed, however, with peptide-boound LAL in break-down products (molecular weight less than 5,000) of alkali-treated casein, but considerably less than that of the free compound. LAL-analyses in blood, urine, and feces of rats fed free or protein-bound LAL indicated a positive correlation between intestinal absorption and nephrotoxic potential. No renal changes were encountered upon feeding diets with 1,000 ppm synthetic LAL to mice, hamsters, rabbits, quail, dogs or monkeys, which suggest a species specificity of LAL-induced renal changes in rats.


Effects of divalent cations on vitamin B12 adsorption to brush borders of rat intestine. A brush border preparation from rat intestine was incubated with rat intrinsic factor-vitamin B12 complex in 0.01 M Tris-HCl buffer, pH 7.4. The 57Co-B12 uptake to brush borders was proportional to the amount of protein or to alkaline phosphatase activity in the preparations. The uptake increased with time of incubation. At 37degreesC, the uptake after incubation for 15 min was 80-85% of that for one hr. The uptake at 4degreesC was approximately 70% of that at 37degreesC. Ther was no difference as a result of adding glucose to the incubation medium. The uptake was observed in the alkaline environment above pH 6.3. Maximum uptake occurred at pH 8.0. Brush borders washed with Krebs-Ringer bicarbonate buffer (pH 7.4) exhibited no difference in B12 uptake, whether in the presence or absence of calcium ion. But brush borders washed with ethylenediaminetetraacetate exhibited no uptake when incubated in calcium-free medium. The uptake reached a maximum by addition of calcium ion at a concentration of 0.3 mM, and was not alter up to 10 mM. Addition of magnesium ion exhibited no uptake. Calcium-dependent B12 uptake was markedly inhibited by manganese ion. Magnesium ion seemed to slightly inhibit the calcium-dependent uptake.


Purification and properties of the dihydrofolate synthetase from Serratia indica. The dihydrofolate synthetase (EC 6.3.2.12) responsible for catalyzing the synthesis of dihydrofolic acid from dihydropteroic acid and L-glutamic acid was purified about 130-fold from extracts of Serratia indica IFO 3759 by ammonium sulfate fractionation, DEAE-Sephadex column chromatography, Sephadex G-200 gel filtration, and DEAE-cellulose column chromatography. The enzyme preparation obtained was shown to be homogeneous by DEAE-cellulose column chromatography and ultracentrifugal analysis. The sedimentation coefficient of this enzyme was 3.9 S, and the molecular weight was determined to be about 47,000 by Sephadex G-100. The optimum pH for the reaction was 9.0. The enzymatic reaction required dihydropteroate, L-glutamate and ATP as substrates, and Mg2+ and K+ as cofactors. gamma-L-Glutamyl-L-glutamic acid cannot replace L-glutamic acid as the substrate. Neither pteroic acid nor tetrahydropteroic acid can be used as the substrate. ATP was partially replaced by ITP or GTP. The enzyme reaction was inhibited by the addition of AD, but not by AMP. One mole of dihydrofolate, 1 mole of ADP and 1 mole of orthophosphate were produced from each 1 mole of dihydropteroic acid, L-glutamic acid, and ATP by the following equation: 7,8-Dihydropteroic acid ml-Glutamic acid matp Mg2+, K+ leads to Dihydrofolic acid + ADP + Pi. These results suggest that the systematic name for the dihydrofolate synthetase is 7,8-dihydropteroate: L-glutamate ligase (ADP).


The use of microsurgery in the treatment of the undescended testis. An experimental method of orchidopexy for high inguinal or abdominal testes, in the dog, using microsurgical anastomosis of the testicular vessels to vessels of the thigh or inferior epigastric vessels, is described. Experimental results suggest that this is a feasible method of treatment of such testes. A clinical trial of the method has commenced.


The significance of the time interval in twin delivery. In order to evaluate the influence of the time interval on the second twin in twin deliveries, we have used more precise criteria than have been used in the literature to date. In the period from July 1, 1970 to December 31, 1974, 35 twin deliveries in our hospital were analyzed, in which both twins fulfilled the following criteria: 1. vertex presentation 2. vaginal deliveries 3. birth weight above 2000 g. We analyzed various parameters in the clinical and acidity score of the second twin alone as well as in comparison to the first twin. The following points of view were considered: 1. The more sensitive aspect of morbidity was utilized by including the assessment of the state of the newborn rather than just mortality. 2. Since Caesarean sections in twins are often performed for a fetal indication, short time intervals between the birth of both infants correlate intrinsically with a higher risk for the second twin. Therefore we considered only vaginal deliveries in order to exclude this intrinsic correlation. 3. By excluding infants below a birth weight of 2000 g, the influence of the low birth weight of the second twin was eliminated. 4. The frequently occurring positional anomalies of the second twin will deteriorate the conditions independent of the time interval. therefore we have considered only births from vertex presentations. A comparison of the acidity state demonstrated that the actual pH values in the umbilical artery blood of these second twins (Fig. 1) decrease statistically significantly with increasing time intervals. this is also true for metabolic acidity (pHqu40). The differences of the actual pH values (Fig. 2) and the pHqu40 values between the first and second twin decreases significantly also with an increasing time interval. Our results indicate that obstetricians, according to the clinical conditions, should accomplish the birth of the second twin as soon as possible after the birth of the first twin.


Evaluation of the fetal state by automatic analysis of the heart rate. 2. Deceleration areas and umbilical artery blood pH. Fetal heart rate (FHR) deceleration areas were studied to obtain by objective measurement of the FHR, their prognostic value of the new-born state. 1. There is a reasonably good correlation between FHR deceleration areas and UApH (Tab. II). Such a correlation was found by SHELLEY and TIPTON [6] for the whole deceleration area, and by TOURNAIRE et al. [10] for areas divided in a slightly different way. The correlation coefficients between FHR deceleration areas and Apgar score at 1 minute are within a close range of those of the FHR deceleration area and UApH (Tab. I and II). 2. According to the time relationship between deceleration areas and uterine contractions the best correlation coefficient was obtained surprisingly for total, followed by residual and then simultaneous areas. These results agree with those of SHELLEY and TIPTON [6] suggesting that in practice a simple measurement of the whole deceleration area, regardless of the uterine contractions is a sufficient method in evaluating FHR patterns. 3. The special purpose computer built by the BAUDELOCQUE research group can be used on-line, thus in clinical practice. It was not the case for the manual method [4] or the method using a large programmed computer [10]. 4 The evaluation of deceleration areas appears to have several advantages: 1. It provides objective measurements. 2. The unit used is independant of factors such as display speed or scale of the strip-chart. 3. The data is reduced: A few numbers replace the long descriptions of the usual clinical classifications.


Dc Polarographic assay of tetracyclines. The inhibitory effect of dioctyl sodium sulfosuccinate on hog pepsin activity was investigated over the pH 1.5-3.0 range. The inhibitory effect was studied using a natural substrate, hemoglobin, and a synthetic substrate, N-acetyl-L-diiodotyrosine. The mechanistic studies revealed that a substrate-inhibitor interaction was the major mechanism of inhibition with hemoglobin. However, some direct enzyme inhibition also was involved. With the synthetic substrate, the inhibition las due to a competition between the substrate and the inhibitor molecules for the enzyme. The possible therapeutic significance of the inhibitory effect of the medicinal surfactant is discussed.


Isolation and characterization of the cardiotonic polypeptide anthopleurin-A from the sea anemone Anthopleura xanthogrammica. A highly potent cardiotonic polypeptide, anthopleurin-A, was isolated from the sea anemone, Anthopleura xanthogrammica (Brandt), using solvent partition, gel permeation chromatography, and cation-exchange chromatography. It is a pure basic polypeptide wtih a molecular weight of about 5500.


Solid-state anomalies in IR spectra of compounds of pharmaceutical interest. Solid-state anomalies in the IR spectra of lysine monohydrochloride, etoxadrol hydrochloride, thiamine hydrochloride, and L-histidine in a potassium bromide matrix were noted. With the first three compounds, the anomalies were due to metathetical exchange of the halide anion between the compound and the matrix. The anomaly seen with L-histidine was related to the crystal structure.


Calcium-binding protein in bull seminal vesicle secretion and seminal plasma. A protein which showed high affinity for calcium ions was isolated from bull seminal vesicle secretion and seminal plasma. Its calcium-binding activity depended on the ionic strength and pH of the medium. The dissociation constant was 7-7 X 10(-7) M and there were 14 binding sites per protein molecule. The molecular weight of calcium-binding protein from bull seminal vesicle secretion, estimated by the gel filtration method, was 110,000. The protein may be involved in the regulation of the calcium ion level in seminal plasma.


Clonidine and related analogues. Quantitative correlations. Twenty-two structural derivatives of clonidine [2-(2,6-dichlorophenylimino)imidazolidine] have been synthesized and their main physicochemical parameters (log P, deltaRM, pKa) determined. Quantitative correlations between the peripheral alpha-mimetic action (pithed rats) and physicochemical parameters pointed out the critical role of the steric effect in the ortho positions. On the other hand, attempted quantitative correlations between physicochemical parameters and central hypotensive activity were unsuccessful. These results are discussed in the light of the postulated mechanism of action of clonidine.


Synthesis and pharmacology of novel anxiolytic agents derived from 2-[(dialkylamino)methyl-4H-triazol-4-yl] benzophenones and related heterocyclic benzophenones. A series of novel [(dialkylamino)methyl-4H-1,2,4-triazol-4-yl]benzophenones and related compounds has been prepared via total synthesis from substituted aminodiphenylmethanes or by hydrolysis and subsequent methylation of triazolobenzodiazepines. These new triazole compounds were found to have potent sedative and muscle relaxing activity in mice (i.e., these compounds depressed the traction and dish reflexes). In addition, the title compounds antagonized the clonic convulsions induced in mice by the administration of pentylenetratrazole (Metrazol, 85 mg/kg), with ED50's varying from 2.0 to 23.0 mg/kg, and the lethality induced by thiosemicarbazide, with ED50's varying from 0.02 to 9.0 mg/kg. In several biological tests, the potency of seven new benzophenone derivatives approached or exceed that of diazepam (35a) or its glycylaminobenzophenone analogue 36.


Na+ transport by rabbit urinary bladder, a tight epithelium. By in vitro experiments on rabbit bladder, we reassessed the traditional view that mammalian urinary bladder lacks ion transport mechanisms. Since the ratio of actual-to-nominal membrane area in folded epithelia is variable and hard to estimate, we normalized membrane properties to apical membrane capacitance rather than to nominal area (probably 1 muF approximately 1 cm2 actual area). A new mounting technique that virtually eliminates edge damage yielded resistances up to 78,000 omega muF for rabbit bladder, and resistances for amphibian skin and bladder much higher than those usually reported. This technique made it possible to observe a transport-related conductance pathway, and a close correlation between transepithelial conductance (G) and short-circuit current (Isc) in these tight epithelia. G and Isc were increased by mucosal (Na+) [Isc approximately 0 when (Na+) approximately 0], aldosterone, serosal (HCO-3) and high mucosal (H+); were decreased by amiloride, mucosal (Ca++), ouabain, metabolic inhibitors and serosal (H+); and were unaffected by (Cl-) and little affected by antidiuretic hormone (ADH). Physiological variation in the rabbits' dietary Na+ intake caused variations in bladder G and Isc similar to those caused by the expected in vivo changes in aldosterone levels. The relation between G and Isc was the same whether defined by diet changes, natural variation among individual rabbits, or most of the above agents. A method was developed for separately resolving conductances of junctions, basolateral cell membrane, and apical cell membrane from this G--Isc relation. Net Na+ flux equalled Isc. Net Cl- flux was zero on short circuit and equalled only 25% of net Na+ flux in open circuit. Bladder membrane fragments contained a Na+-K+-activated, ouabain-inhibited ATPase. The physiological significance of Na+ absorption against steep gradients in rabbit bladder may be to maintain kidney-generated ion gradients during bladder storage of urine, especially when the animal is Na+-depleted.


Perturbational effects of inorganic cations on human erythrocyte membranes. The perturbational effects of monovalent and divalent cations on human erythrocyte membranes were analyzed by examining their influence on kinetic and structural characteristics of trinitrobenzenesulfonic acid (TNBS) incorporation into the amino groups of protein and phospholipid structural components. The stimulatory effects of monovalent cations on TNBS incorporation, which were size-independent and attributed to nonspecific membrane alterations resulting from ionic strength factors, contrasted with the more pronounced stimulatory properties of divalent cations which were markedly size-dependent. These stimulatory effects of cations on TNBS incorporation were associated with alterations not only in rate but also in activation energy in incorporation. Changes in activation energy produced by divalent cations paralleled their ability to perturb membrane protein components and probably reflected changes in probe permeation. The rate of TNBS incorporation exhibited a dependence on divalent cation ionic radius which paralleled ion-induced perturbations in the labelling of the membrane amino phospholipid phosphatidylethanolamine. Divalent cations differed both in the relative extent and in the characteristics of protein and phospholipid perturbation. Alkaline earth cations behaved as a rather homogeneous group while Ni++, Co++ and Mn++ constituted a second heterogeneous group. The influence of monovalent and divalent cations on the hemolytic behavior of intact erythrocytes paralleled their effects on TNBS incorporation into isolated membranes rather closely. It is suggested that TNBS incorporation may provide a valuable means of analyzing functionally relevant cation-induced alterations in biological membranes in general.


Proton fluxes associated with erythrocyte membrane anion exchange. Transient extracellular pH changes accompany the exchange of chloride for sulfate across the erythrocyte membrane. The direction of the extracellular pH change during chloride efflux and sulfate influx depends on experimental conditions. When bicarbonate is present, the extracellular pH drops sharply at the outset of the anion exchange and tends to follow the partial ionic equilibrium described by Wilbrandt (W. Wilbrandt, 1942. Pfluegers Arch. 246:291). When bicarbonate is absent, however, the anion exchange causes the pH to rise, indicating that protons are cotransported with sulfate during chloride-sulfate exchange. The pH rise can be reversed by the addition of HCO(-3) (4 muM) or 2,4-dinitrophenol (90 muM). This demonstrates that the proton-sulfate cotransport can drive proton transport uphill. The stoichiometry of the transport is that one chloride exchanges for one sulfate plus one proton. These results support the titratable carrier model proposed by Gunn (Gunn, R.B. 1972, In: Oxygen Affinity of Hemoglobin and Red Cell Acid-Base Status. M. Rorth and P. Astrup, editors. p. 823. Munksgaard, Copenhagen) for erythrocyte membrane anion exchange.


Bacteriology and antibiotics in acute suppurative otitis media. One hundred and forty-seven patients with acute suppurative otitis media, were divided into three groups and treated with antibiotics (azidocillin, ampicillin, and cephalexin). The therapeutic effect was assessed bacterioloically by swabbing from the aural discharge and from the nasopharynx on the first, second, third, and seventh day after initiation of treatment. In addition, the concentration of antibiotic in the aural discharge and in the nasopharynx was determined. As compared with other published materials, there was a common occurrence of Haemophilus influenzae and S. Aureus. Hemolytic streptococci are less common than prior to the advent of antibiotics. Pneumococci disappeared in all cases from the aural discharge in the course of the first three days. The effect upon Haemophilus was slower. In the nasopharynx the effect was questionable, and no effect was obtained upon other bacteria. The clinical course could not be correlated to the bacterial findings except that resistant bacteria were found in all cases with persisting discharge.


Enzymatic action of coliphage omega8 and its possible role in infection. The receptor of coliphage omega8 is the O-specific mannan of Escherichia coli O8 in which the trisaccharide alpha-mannosyl-1,2-alpha-mannosyl-1,2-mannose is joined through alpha-mannosyl-1,3-linkages. Coliphage omega8 produces an endo-alpha-1,3-mannosidase which destroys the receptor, liberating a series of oligosaccharides (repeating trisaccharide and multiples). The enzyme is an integral part of the phage particles and also occurs in a free form in the lysates. Phage particles hydrolyze alpha-1,3-mannosyl linkages in the lipopolysaccharide, the polysaccharide (mannan) moiety, and higher oligosaccharides with an efficiency decreasing in this order. No transmannosylation could be detected. Phage particles also degrade the receptor mannan on whole bacteria, as determined with 14C-labeled E. coli O8. The values of Km and Vmax were determined with omega8 particles and free enzymes using native lipopolysaccharide and its triethylammonium salt. The latter, which was obtained after electrodialysis, has a micellar weight of 2.5 X 10(5), whereas the native lipopolysaccharide forms supermicelles with micellar weights of several millions. With coliphage omega8 as enzyme and supermicellar lipopolysaccharide as substrate Km=5 X 10(-8) M was obtained. This, together with the fact that omega8 attaches irreversibly to E. coli O8, was used in proposing a hypothesis for the possible role of the enzyme in the first steps of infection with coliphage omega8.


Alpha-adrenoceptor-mediated coronary artery spasm. Selective coronary arteriography performed on a 41-year-old woman with angina pectoris demonstrated proximal stenosis of the right and left main coronary arteries that was unaffected by nitrate therapy. To exclude coronary artery spasm, the study was repeated, and a striking increase in the narrowing of the right coronary artery was observed. This 90% stenosis was virtually abolished by pretreatment with intravenously given phentolamine hydrochloride. Prolonged alpha-adrenoceptor blockade with phenoxybenzamine hydrochloride improved the patient's exercise tolerance and postexercise electrocardiographic abnormalities when compared to therapy in matched controls given placebo. These observations suggest that alpha-adrenoceptor-mediated coronary artery spasm may mimic organic lesions at coronary arteriography and may be a factor in the pathogenesis of angina pectoris in some patients.


[In vitro antibacterial effects of spectinomycin and penicillin G on Neisseria gonorrhoeae singly and in combination (author's transl)]. The antibacterial effects of spectinomycin and penicillin G on clinical isolates of Neisseria gonorrhoeae were studied. The concentrations of penicillin G at which the isolates showed drug sensitivity ranged widely from 0.011 to 6.25 mug/ml. Some of the isolates were resistant to penicillin G. Sensitivity to spectinomycin was observed at the drug concentrations ranging from 3.13 to 12.5 mug/ml. About 60% of the isolates were sensitive to 6.25 mug/ml of spectinomycin, and those isolates which were resistant to penicillin G showed good sensitivity to spectinomycin. No correlation in sensitivity was noted between the two drugs. Tests for their bactericidal activities on bouillon media revealed that the addition of spectinomycin at the concentration of 12.5 mug/ml or over produced a marked bactericidal effect in a short time while penicillin G exhibited a bactericidal or bacteriostatic effect depending upon the concentration used. A synergistic effect of a penicillin and an aminoglycoside antibiotic was observed in these isolates of N. gonorrhoea as was in the isolates of Pseudomonas aeruginosa. Where penicillin G and spectinomycin were used in combination, a simultaneous addition of both the drugs produced the most marked synergistic effect. Morphology of N. gonorrhoeae cells exposed to either of these drugs was examined under a scanning electron microscope. Exposure to spectinomycin at the level of 6.25 mug/ml resulted in almost no morphological change. At 6.25 mug/ml of the drug, however, a roughened cell surface, a bleb-like structure or a state suggesting the loosening of such a bleb-like structure was noted. The addition of penicillin G at 0.19 mug/ml led to an impairment of cell division at one hour of exposure and to cell swelling and lysis with further exposure. At 1.19 mug/ml of the drug, these processes of cell swelling and lysis took place early.


Absence of blocking effect of quinidine on responses to norepinephrine in the isolated dog atrium. The isolated right dog atrium was perfused through the sinus node artery with blood led from a heparinized support dog under constant perfusion pressure of 100 mmHg. The positive chronotropic and inotropic responses to norepinephrine were not significantly suppressed by treatment with large amounts of quinidine or procainamide, but completely blocked by propranolol. From these results, it was demonstrated that quinidine and procainamide have no adrenergic beta-blocking activity on chronotropism and on inotropism in the heart.


[Intra-renal arterio-venous aneurysm after renal puncture biopsy. Supra-selective embolization. Cure (author's transl)]. This case emphasizes the value of: 1. Treatment of malignant hypertension with the combination of hemodialysis and beta-blockers. 2. Supra-selective arterial embolization in inaccessible intra-renal aneurysms or those difficulty accessible to surgery.


Effect of capsular polysaccharide of Klebsiella pneumoniae on host resistance to bacterial infections. I. Induction of increased susceptibility to infections in mice. When Klebsiella pneumoniae capsular polysaccharide (CPS-K) from type 1, Kasuya strain, was injected intraperitoneally (i.p.) immediately before i.p. bacterial challenge, the survival time of mice infected with Salmonella enteritidis NUB 1 (virulent strain) was shortened and the mortality rate for mice infected with S. enteritidis NUB 31 (avirulent strain) was enhanced. The promotion of infection with S. enteritidis NUB 1 by CPS-K depended upon its dose, the effect of CPS-K being demonstrable up to as little as 0.2 mug per mouse. In the case of S. enteritidis NUB 31, the effect of CPS-K was detectable only when more than 20 mug per mouse was injected. As a result of enumeration of bacterial populations in the peritoneal washing, blood, liver and spleen, it was revealed that CPS-K promoted in vivo growth of S. enteritidis NUB 1 and NUB 31. In addition, CPS-K enhanced the mortality rate in mice infected with Streptococcus pyogenes or Streptococcus pneumoniae. The peak CPS-K effect on infection with S. enteritidis NUB 1 was seen when given immediately before bacterial challenge. The active substance responsible for the infection-promoting effect of CPS-K was neutral CPS-K, which is distinct from the O antigen and from acidic CPS-K (the type-specific capsular antigen). Preparations of neutral CPS-K isolated from the other three strains of K. pneumoniae exhibited a marked infection-promoting effect comparable with that of preparations from the Kasuya strain. Neutral CPS-K, with identical antigenicity to that from the Kasuya strain, has already been found to exert a strong adjuvant effect on antibody responses to various antigens in mice. No parallelism exists between infection-promoting activity and adjuvant activity of neutral CPS-K.


A critical examination of some current assumptions in the treatment of alcoholism. Recent scientific research challenges a number of traditional assumptions in the treatment of alcoholism. Two major tenets, the disease conception of alcoholism and mandatory abstinence as a goal of treatment are reviewed, and insufficient evidence is found to support a dogmatic position on either.


[Biological properties of Cl. perfringens type A, isolated from human subjects inhabiting a hermetic chamber]. This paper gives the results of quantifying and studying the biological properties of Cl. perfringens strains isolated from feces of test subjects kept in an enclosure for 34 days. During the experiment the total number of Cl. perfringens increased. It was especially true of the strains with an elevated toxigenic activity. This activity correlated with an increase of the titer of alpha-hemolysines and proteolytic enzymes.


Interactions of phospholipase D with 1,2 diacyl-sn-glycerol-3-phosphorylcholine, dodecylsulfate, and Ca2+. Some properties of the pure, soluble phospholipase D (phosphatidycholine phosphatido hydrolase, EC 3.1.4.4) interactions with phosphatidyl choline (1,2 diacyl-sn-glycerol-3-phosphoryl choline) in a system also containing dodecylsulfate and Ca2+ ions were studied. Concentrations of Ca2+ greater than 50 mM were necessary both for activity and adsorption of the enzyme to the "supersubstrate." Ethylenediamine tetraacetic acid caused inhibition of activity, greater than one would expect from its chelating capacity. A nonlinear increase in activity with the increase of enzyme protein was observed, suggesting a subunit aggregation into a higher mol wt protein, catalytically more active. Upon centrifugation of the supersubstrate-enzyme complex at 4.5 X 10(5) g-min at 30 C, most of the substrate molecules sedimented regardless of the pH. The reverse was true when centrifugation was done at 1 C. Phospholipase D hydrolyzed phosphatidylcholine molecules present in the supersubstrate at temperatures around 0 C at a rate 1/5 that of a maximal value measured at 30 C. The Arrhenius plot was linear in the range from 0 to 30 C, and at that temperature the curve broke with a smaller slope. Activation energy of 9.1 Kcal/mol, below 30 C, was calculated. Adsorption of the enzyme to the sedimentable supersubstrate occurred at pH 8.0, regardless of temperature. At pH 5.6, a considerable portion of phosphatidylcholine was degraded at 30 C, thus minimizing the capacity of the supersubstrate to adsorb the enzyme. Although Mg2+ could replace Ca2+ in the formation of sedimentable supersubstrate, it neither assists in adsorption of the enzyme nor in activation of the phosphatidylcholine hydrolysis.


Studies on drug-induced lipidosis: VII. Effects of bis-beta-diethyl-aminoethylether of hexestrol, chloroquine, homochlorocyclizine, prenylamine, and diazacholesterol on the lipid composition of rat liver and kidney. 4,4'-Bis (beta-diethylaminoethoxy)-alpha, beta-diethyldiphenylethane (DH), which had been shown to induce a type of lipidosis resembling Niemann-Pick disease, was given to rats at a dose of 20, 50, 100, and 150 mg/kg body weight per day for 1 or 2 weeks. An enlargement of the liver with marked increases in free cholesterol, total phospholipids, and phosphatidylinositol took place by administration of a larger dose. The increase in bis (monoacylglyceryl) phosphate (BMGP), which is peculiar to this kind of drug-induced lipidosis, was dependent upon the dose of the drug as well as the length of time. Similar changes were also observed in kidney. Among several other drugs tested, chloroquine and diazacholesterol brought on as much increase in BMGP as treatment with DH.


Role of the parietal cell in gastric damage induced by aspirin and related drugs: implications for safer therapy. The proton-secreting parietal cell is shown, from recent evidence, to be the principal focus for the actions of aspirin (and related drugs) in damaging the gastric mucosa. It is suggested that the selective damage to the parietal cells is due to the ph gradient favouring a high rate of aspirin uptake and subsequent entrapment of drug anions inside these cells. This concept may serve as a useful basis for developing procedures to minimize the gastric damage by aspirin and related acidic drugs.


Schizophrenic psychology, associative learning and the role of forebrain dopamine. It is suggested that schizophrenic thinking can be explained as a lowering of levels of significance for acceptance of conclusions based on inductive logic. The formal similarity between inductive logic and operant, or classical conditioning is pointed out. It is thus possible to explain the therapeutic effects of neuroleptic drugs by referring to the effect of these, and related drugs, and of lesions of ascending dopamine pathways, on acquisition of conditioned responses. It is tentatively suggested that recognition of association of related features of the environment, whether in humans or animals occurs in the basal ganglia by a dopamine dependent process. A role is suggested for neocortical noradrenaline in consolidaton of newly acquired associations. Implications and tests of this hypothesis are discussed.


Two weeks' treatment with chlorpromazine, thioridazine, sulpiride, or bromazepam: actions and interactions with alcohol on psychomotor skills related to driving. Subacute effects of C, T, S, or B, alone or in combination with A, were tested against P in two double-blind crossover trials with 37 healthy students. The drugs were given in capsules t.i.d. for 2 weeks each and the psychomotor performance (choice reaction, coordination, attention) was measured on the 7th and 14th days of treatment. At each session the subjects swallowed a capsule together with 0.5 g/kg of A or P drink, and the measurements were done, 30, 90, and 150 min thereafter. T alone did not differ from placebo in the doses used (10 mg t.i.d. for 7 days and 20 mg t.i.d. for the next 7 days). After C (dosing as above) and S (50 mg t.i.d.) both reactive and coordinative skills were slightly impaired. B (6 mg t.i.d.) clearly impaired both reactive skills and attention. T + A had no major combined effect on skills while C interacted with A resulting in impaired reactive and coordinative skills. After C + A the subjects were unable of compensating their coordination mistakes by slow driving. The interaction of S with A was mild, whereas B + A strongly impaired coordination and divided attention. No alterations were recorded in flicker fusion after any treatment.


[Longterm therapy of angina pectoris patients with a new beta-receptor blocker (author's transl)]. The beta-receptor blocker Timolol was investigated for 28 weeks in a double blind trial and then for up to 100 weeks in an open trial in patients with angina pectoris. In both phases of the investigation. Timolol caused a quite marked regression in the frequency of attacks and in the pulse rate at rest. The evaluation of therapeutic success by doctor and patient was positive for Timolol.


[Angina pectoris. Results of investigations to date (author's transl)]. Among the most important coronary risk factors are a diet rich in saturated fatty acids, cholesterol and calories, hypercholesterolemia, high blood pressure and excessive smoking. The typical pain can be elicited by putting a strain on the patient, e.g. climbing stairs. The disappearance of the pain after treatment with nitroglycerine is one of the most important pointers. Treatment is with nitroglycerine which, moreover, remains the drug of choice, also with nitrates such as pentaerythritol tetranitrate and with betablockers like propranolol. Favorable results have also been reported with a combination of isosorbide dinitrate and propranolol.


[Self cleaning of natural waters].

[Self cleaning of natural waters]. The planting of the waters left the question open as to whether the necessary expenditure of work is really worthwhile and should be encouraged. The investigations reported here with higher and particularly emersed plants, show what an astonishing influence the higher plants exert on organic and inorganic matter, on pH regulation, destruction of pathogenic bacteria, worms' eggs, and viruses, and also how they influence the waters and waste waters.


Lichens as indicators of air pollution.

Lichens as indicators of air pollution. Field and laboratory studies have demonstrated that lichens are extremely sensitive to sulfur dioxide and that lichens are fairly sensitive to hydrogen fluoride and heavy metals. Further studies are necessary to assess lichens' relative sensitivity to oxidant air pollutants.


Molecular sensory physiology of Euglena. After a short description of the motile physiology of Euglena gracilis the responses to light stimulation are analyzed and a new nomenclature is proposed concerning flagellar motility variations. A discussion follows about the characteristics of the photoreceptive system of Euglena and about the flavinic nature of its phototactic pigments.


[Apudomas].

[Apudomas]. Apudomas, i.e. tumours deriving from apud system cells, comprise already known forms located in widely different organs at considerable distances one from the other. They present great cytochemical, biochemical and ultrastructural similarities and possess a common derivation in the neuroectodermal cells of the neural crest. They thus form part of that tumour group known as neurolophomas. They are also responsible for typical clinical pictures connected with the production of hormone-type substances, and are classified among multiple endocrine adenomatoses and in the ectopic hormone syndromes.


[Critical observations on changes in gamma-glutamyl-transpepdidase in hepatopathies]. The behaviour of gamma-glutamyl transpeptidase was compared with other serum enzyme activities and functional parameters in a carefully selected and relatively extensive series of patients with liver disease, including alcoholics, in an investigation of the underlying pathogenesis and its clinical expression. Reference. to the literature and to personal data showed that increased gamma-glutamyl transpeptidase levels could be attributed to enzyme induction (caused by drugs or alcohol), liver damage in the broad sense, and intra- or extrahepatic cholestasis. These causes were individually predominant, or nearly so, on occasions, though their concomitance was more common. High levels, however, were not pathognomonic for a given disease. In alcoholism, they were highly indicative, especially if accompanied by GLD changes. They were a virtually constant, early, and typical finding in intra- and extra-hepatic cholestasis, and tended to persist for a time after the resolution of icterus. Lastly, they were an aid in the early diagnosis of aggressive hepatitis and liver cancer.


[Changes in partial pressures of gases and of pH in the arterial blood of hypocapnic and hypercapnic subjects subjected to oxygen therapy]. The behaviour of partial tensions of respiratory gases and pH in the arterial blood was evaluated in anoxiaemic and hypercapnic (1st group) and hypo-eucapnic (IInd group) subjects undergoing oxygen therapy. The following phenomena were observed: 1) normalization in paO2 values in both groups; 2) statistically significant diminution in paCO2, especially as regards hypercapnic subjects; 3) statistically significant diminution in blood pH; 4) statistically significant diminution in bicarbonates. In the light of these findings, it is considered that other variables, such as the electrolytes, might play an important role in pH diminution during oxygen therapy and that further research should be carried out to look into the possibility.


[Changes in acid-base equilibrium of the arterial blood in hypoxemic-eucapnic subjects subjected to oxygen therapy with and without an antiphosphodiesterase drug]. The behaviour of the partial tensions of respiratory gases and the main parameters of acid base balance in hypoxaemic and eucapnic chronic bronchopneumopathics undergoing oxygen therapy has been assessed. The patiests were subdivided into two groups, the first consisting of subjects undergoing oxygen therapy alone, the second of subjects having oxygen and i.v. theophylline at the same time. Two subgroups were also distinguished. These consisted respectively of normochloraemic and hypochloraemic patients, the purpose being to reveal whether electrolytic changes could be attributed to the  concentration of chlorine evaluated in basal conditions. The results obtained showed a disappearance of the hypoxaemic state without appreciable changes in paCO2 in all patients; in those undergoing oxygen therapy only, also observed were: 1) increase in plasmatic H+ and reduction in pH; 2) diminution in HCO3- and plasma Na+; 3) increase in plasma concentration of Cl-; 4)  statistical significance of these data; 5) tendency of all values in the parameters considered to return to basal values upon suspension of oxygenation.


[Gasometric changes and acid-base equilibrium of venous blood oxygenated in vitro and incubated with or without a phosphodiesterase inhibitor]. Changes in partial tension of the respiratory gases and in acid-base balance were studied in venous blood subjected to oxygenation, with or without incubation with theophylline. The following modifications were noted: 1) increased pO2; 2) decreased CO2 due to true wash-out caused by tonometry in oxygen; 3)decreased hydrogen ion concentration and bicarbonates following CO2 washing in both experimental models. A smaller reduction in pH and HCO3 was statistically significant in specimens treated with theophylline. This is seen as proof of the view that theophylline inhibits red cell membrane ion exchanges, such as to oppose or at any rate limit metabolic changes in acid-base balance induced by oxygenation.


[Acid-base equilibrium of the arterial blood of subjects with grave hypercapnia subjected to mechanical ventilation with the iron lung (concerning 2 cases of post-hypercapnic alkalosis)]. Two cases of severe chronic respiratory insufficiency in severely acute phase with disturbances in acid base balance characterized by serious gaseous acidosis are reported. Therapy was based essentially on controlled ventilotherapy using an iron lung and considerable improvements were achieved clinically with practically total normalization of the acid base imbalance after only a few hours of treatment. In the following days, however, a picture of metabolic alkalosis established itself and this is discussed and interpreted as an expression of post-hypercapnic hypochloraemia.


Measurement of pituitary hormones: clinical applications. 5. Prolactin. A sensitive and specific assay for human prolactin has been developed using human prolactin and antiserum distributed by the United States National Pituitary Agency. Plasma prolactin concentrations in control subjects ranged from 0-20 ng/ml. No sex difference in prolactin concentration was observed. A brisk increase in plasma prolactin levels occurred in normal subjects during the administration of chlorpromazine and thyroid stimulating hormone releasing factor (TRH). These stimulatory tests of prolactin release should therefore be useful in the assessment of hypothalamic pituitary function. Basal plasma prolactin values were raised in most patients who were being treated with phenothiazines and were helpful diagnostically in patients with amenorrhoea, galactorrhoea, hypogonadism, cranio-pharyngioma, "non-functioning" pituitary tumours and acromegaly. In many of these disorders a significant reduction in the plasma prolactin concentration was observed following oral administration of bromocriptine. Plasma prolactin determinations should be useful in evaluating the response to medical treatment or pituitary ablation.


Correlation of fetal heart rate patterns and fetal pH with neonatal outcome. Fetal monitoring data in 200 consecutive cases was evaluated in the second hour prior to delivery to determine how neonatal outcome could be most accurately predicted. It was found that the correlation between ominous fetal heart rate-uterine contraction (FHR-UC) patterns and a depressed Apgar score at 1 minute was, at best, 37%. However, where the last fetal scalp blood pH (FSB-pH) was less than or equal to 7.2, neonatal depression occurred in 88% of cases. FSB-pH, therefore, provided a more reliable means of diagnosing fetal distress.


The detection of toxin production by Corynebacterium diphtheriae in diagnostic laboratories. In vitro tests for assessing the toxigenicity of strains of C. diphtheriae using the original agar plate precipitin method of Elek are unreliable. At high concentrations of antitoxin (500-1000 U/ml) multiple non-specific precipitin lines occur making interpretation difficult. Increased specificity was obtained by diluting the antitoxin but this in turn caused a delay in the appearance of toxin-antitoxin lines. Also, the failure of lines to appear on media enriched with unsuitable serum may lead to false-negative results. Final assessment of the toxigenicity of strains of C. diphtheriae can probably only be made reliably by guinea-pig tests.


Defective polymorphonuclear leukocyte chemotaxis and bactericidal capacity in a boy with recurrent pyogenic infections. A 13-year-old boy with a history of recurrent pyogenic infections had abnormalities of polymorphonuclear leukocyte (PMN) function which probably accounted for his susceptibility to infection. PMN phagocytosis and nitroblue tetrazolium dye reduction were normal but glucose 14C oxidation was abnormally increased in resting cells. The patient's PMNs possessed decreased bactericidal activity against Staphylococcus aureus and Escherichia coli. Also documented were decreased PMN chemotactic activity and leukocyte accumulation in Rebuck skin windows. PMN random motility, PMN bone marrow reserve, PMN kinetics, lymphocyte blastogenesis, and delayed cutaneous hypersensitivity were normal. This patients represents another of the growing number of distinct granulocytopathies currently being recognized which may result in increased susceptibility to infection.


Polynucleotides. XL. Synthesis and properties of poly 2'-azido-2'-deoxyadenylic acid. Poly 2'-azido-2'-deoxyadenylic acid (Poly Az) was synthesized from 2'-azido-2'-deoxyadenosine diphosphate by polynucleotide phosphorylase. Poly (Az) has U.V. absorption properties similar to poly (A) and hypochromicity of 40% at 0.1 M Na+ and neutrality. CD curve also resembled to that of poly (A), but has smaller ellipticity. Titration of poly (Az) with HCl gave a transition at pH 5.5, but exact structure of the acid-form complex was not elucidated. Upon mixing with poly (U), poly (Az) forms a 1:1 and 1:2 complexes having Tm's somewhat higher than that of poly (A)- poly (U) complex in the same condition.


Properties of DNA ligase from uninfected and virus-infected HeLa cells. HeLa cells contain a high M.W. form of DNA ligase which can be completely converted to a low M.W. form. Stokes radius, frictional ratio, sedimentation coefficient, molecular weight, pH dependence, and heat inactivation rate of the two forms have been studied. The major properties of the two forms of DNA ligase in HeLa cells (in particular molecular weights and pH dependence) resemble those of the "dimer" and "monomer" structures described in cultured human cells (Pedrali, G., Spadari, S., Ciarrocchi, G., Pedrini, M., Falaschi, A. (1973) Eur. J. Biochem., 39 343) . In synchronized HeLa cells, the DNA ligase shows a two fold increase during S phase and parallels the increase in the DNA synthesis rate. DNA ligase increases in parallel with viral DNA synthesis after infection of HeLa cells with vaccinia and Herpes virus but its cofactor requirements and physical properties (including the dimer leads to monomer conversion) are unchanged, suggesting that the newly formed ligase is not virus-coded.


Histones bind more tightly to bromodeoxyuridine-substituted DNA than to normal DNA. Using a membrane filter assay, we have obtained results from both kinetic and competition experiments indicating that histones bind more strongly to bromodeoxyuridine-substituted DNA than to normal DNA. At 37 degrees C in our standard buffer of 0.2 M ionic strength, the rate of dissociation of histones H1, H2, and h4 from BrdU-substituted DNA is respectively 7, 4, and 2 times slower than it is from normal DNA. Competition experiments show an even greater difference between BrdU-substituted and normal DNA with respect to histone binding. The tighter binding of histones to BrdU-substituted DNA is of interest because of the known effects of BrdU on eukaryotic chromosome condensation and staining, virus induction, and the inhibition of differentiation.


The reaction between thiols and 8-azidoadenosine derivatives. Thiols react at room temperature in dilute solution with 8-azidoadenosine and its nucleotides to give the corresponding 8-aminoadenosine derivatives. The reaction which takes place in the dark is base-catalysed and is particularly rapid when dithiols, e.g. dithiothreitol are used.


Novikoff hepatoma deoxyribonucleic acid polymerase. Sensitivity of the beta-polymerase to sulfhydryl blocking agents. Unlike other beta-class eukaryotic DNA polymerases, the enzyme purified from the Novikoff hepatoma is inhibited by both sulfhydryl blocking agents N-ethylmaleimide (NEM) and p-hydroxymercuribenzoate (pHMB). The degree of sensitivity varies depending on the enzyme purity, pH of the reaction, and the presence of sulfhydryl reducing agents. Novikoff beta-polymerase activity is unaffected by the presence of 2-mercaptoethanol (2-Me) or dithiothreitol (DTT); however, the combination of 2-mercaptoethanol and NEM or pHMB acts to reverse the inhibition of the sulfhydryl blocking agent. The reversal of inhibition involves more than just a titration of NEM with 2-mercaptoethanol since a) the combination of these two reagents actually stimulates the DNA polymerase, and b) dithiothreitol did not reverse the inhibition. Binding of the polymerase to DNA did not affect the enzyme sensitivity to NEM.


The relationship between beta-blockade, hyporeninaemic and hypotensive effect of two beta-blocking agents. In two consecutive series of hypertensive patients the hypotensive effect, the hyporeninaemic effect and the blockade of cardiac beta-receptors was studied using weekly increasing doses of propranolol or atenolol. With both beta-blockers, cardiac blockade and hypotensive effect increased in a parallel fashion when the dosage was increased suggesting that the hypotensive effect is related to cardiac beta-blockade. On the other hand lack of parallelism between the hypotensive effect and the hyporeninaemic effect suggests that the hypotensive effect was not related to a major extent to the hyporeninaemic effect of the drugs in the dosage range studied here.


Ahlquist and the development of beta-adrenoceptor antagonists. The power and fecundity of Ahlquist's original concept of alpha- and beta-receptors can now be seen in dramatic contrast to the obfuscation and sterility of the then contemporary theory of two "sympathins". But when his famous paper appeared in 1948, pharmacologists apparently were not ready for the new idea. Receptor theory was perhaps too esoteric (Dale, one of the great pharmacologists of his time, studiously ignored it) and pharamacological taxonomy was virtually non-existent. The relationship of the new idea to Ehrlich's older ones, with consequent utilitarian implications, was overlooked.


The properties of beta-adrenoceptor antagonists. After the clinical studies with pronethalol and propranolol confirmed Black's hypothesis that drugs which block beta-adrenoceptors were of value in the treatment of patients with angina pectoris and cardiac arrhythmias, other compounds have been described which block beta-adrenoceptors. Detailed pharmacological studies with propranolol and comparison of its properties with those of other beta-adrenoceptor blocking drugs have indicated that these drugs may possess properties in addition to their effect in blocking beta-adrenoceptors. These properties have been termed intrinsic sympathomimetic activity, membrane stabilizing activity and cardioselectivity. These properties have all been described in detailed observations in laboratory animals but not to the same extent in normal man or in patients. It appears that the therapeutic effectiveness of these drugs results from their beta-adrenoceptor blocking activity and that intrinsic sympathomimetic activity and membrane stabilizing activity do not contribute to the effects of these drugs in patients, although cardioselectivity may be of value in some patients.


Beta-blockade and mechanisms of disease. In this review an attempt has been made to define the contribution of beta-antagonists to out understanding of the mechanisms of disease. It is concluded that beta-antagonists, and propranolol in particular, have clarified mechanisms in angina pectoris, cardiac arrhythmias and some aspects of essential hypertension as well as hypertrophic obstructive cardiomyopathy. The study of beta-antagonists in anxiety states shows that they can help to define the somatic component but give no clue as to central mechanisms involved. There is no substantial evidence that beta-antogonists are of value in the psychoses and these negative findings suggest that disturbances of brain catecholamines contribute little to the pathogenesis of these conditions.