Molecular analysis of the histone gene cluster of Psammechinus miliaris: I. Fractionation and identification of five individual histone mRNAs. The electrophoretic separation of labeled "9S" histone mRNAs obtained from cleaving sea urchin polysomes was found at first to be highly unreproducible. It became evident that the secondary structure of the individual mRNAs had a greater effect on their relative electrophoretic mobilities than did their molecular weight differentials. We determined the parameters affecting electrophoretic mobility by the novel method of running the labeled polysomal RNA in slab gels across polyacrylamide and urea gradients. The initially complex and species-specific electrophoretic pattern could then, by a judicious choice of denaturing conditions, be simplified to yield five well defined classes of labeled mRNAs. Using optimal conditions for the separation of the RNA components, five messengers were isolated from Psammechinus embryos by preparative disc electrophoresis, four of which, after two electrophoretic separations, exhibited a unimodal distribution. Each of the mRNAs was translated in vitro, four of the five fractions promoting the synthesis of one major protein. The in vitro products were characterized by comparison of their electrophoretic mobilities with those of known sea urchin histones. It was thus possible to correlate individual mRNAs with specific histones. We propose that the five mRNAs designated a-e in order of decreasing electrophoretic mobility code for the histones H4, H2A, H2B, H3, and H1.


Molecular analysis of the histone gene cluster of psammechinus miliaris: II. The arrangement of the five histone-coding and spacer sequences. Histone DNA of Psammechinus miliaris was obtained in an enriched form by buoyant density gradient centrifugation and was cleaved into 6 kb repeat units (Birnstiel et al., 1975a) by the action of the specific endonucleases EcoRI and HindIII. Since it was suspected that the 6 kb unit harbored all five histone-coding sequences, the histone DNA unit was subdivided into five segments with the aim of providing five fragments carrying just one coding sequence each. This was achieved by the combined use of EcoRI Hindll, Hindlll, and Hpa I. A physical map was constructed from the overlaps arising in these restriction experiments. Each of the five segments was shown to hybridize uniquely with just one of the five highly purified histone mRNAs (Gross et al., 1976a). By this procedure, the order of the mRNA sequences on the histone DNA was found to be a, c, d, b, e (Gross et al., 1976a), and hence of the protein coding sequences H4, H2B, H3, H2A, and H1. Further evidence is presented that the 6 kb repeat unit, amplified by means of a Murray lambda vector phage, contains AT-rich DNA sequences which would be expected not to code for histone proteins.


Molecular analysis of the histone gene cluster of Psammechinus miliaris: III. Polarity and asymmetry of the histone-coding sequences. Fragments of histone DNA produced by restriction endonucleases contain 5' and 3' termini with defined topologies relative to the histone-coding sequences. After limited resection with lambda-exonuclease, the 6 kb Hindlll histone DNA fragment (see Schaffner et al., 1976) hybridizes to H4 histone mRNA whether or not the DNA has been denatured. This shown that the coding sequence for the histone H4 is proximal to the 3' terminus of the Hindlll restriction fragment. By contrast, lambda-exonuclease digestion of the 6 kb EcoRI histone DNA fragment drastically reduces hybridization of the H4 mRNA. Hence in this molecule, the H4 DNA sequence is near a 5' terminus of the EcoRI restriction fragment. From these results and those described in the preceding paper (Schaffner et al., 1976), the polarity of the H4 gene is therefore 5' H2B leads to H4 leads to H1 3'. Cloned Psammechinus histone DNA (S. G. Clarkson, H. Smith, W. Schaffner, K. Gross, and M. Birnstiel, manuscript submitted for publication) may be strand-separated by electrophoresis. Highly purified histone mRNAs (Gross et al., 1976) all hybridize almost exclusively to the strand of lesser electrophoretic mobility. It follows that all coding sequences are arranged in tandem within the same DNA strand, and that hence they share the same polarity as the H4 DNA sequences. Transcription therefore proceeds in the gene cluster in the direction H4 leads to H2B leads to H3 leads to H2A leads to H1.


Biosynthesis and stability of globin mRNA in cultured erythroleukemic Friend cells. Biosynthesis and stability of the mRNA population in DMSO-induced Friend erythroleukemic cells were studied after labeling the RNA with 3H-uridine and then chasing it with nonlabeled uridine. Globin RNA metabolism was studied by hybridization to excess complementary DNA convalently coupled to oligo(dT)-cellulose. After a labeling period of 120 min, 2-4% of the poly(A)-containing labeled RNA was in globin RNA; it decayed with a half-life of 16-17 hr. The rest of the poly(A)-containing RNA was composed to two kinetic populations: 85-90% decayed with a half-life of about 3 hr, while 10% decayed with a half-life of about 37 hr. The portion of globin RNA in labeled poly(A)-containing RNA behaved in an unexpected fashion during the chase period. During the initial chase period, the percentage of globin RNA increased rapidly, reaching a maximum of about 15% at 20 hr, but it subsequently declined gradually. Based on these findings, a model was built that describes the changes in the proportion of globin mRNA in poly(A)-containing RNA during continuous synthesis and after chase of the labeled RNA. It appears that if the parameters described remain constant during the maturation of erythroblasts, then this model would not account for the almost exclusive presence of globin RNA in the reticulocyte. By far the most effective way to achieve this high level of globin RNA is the destabilization of the mRNA population which is more stable than globin RNA, and not the stabilization of globin RNA itself.


Globin mRNA sequences in polyadenylated and nonpolyadenylated nuclear precursor-messenger RNA from avian erythroblasts. Nuclear RNA from immature duck erythrocytes was fractionated into polyadenylated and nonpolyadenylated fractions, and globin mRNA sequences were determined by hybridization to DNA complementary to globin mRNA. 80--90% of labeled nuclear RNA is found to be nonpolyadenylated, and 70--80% of the globin mRNA sequences present in the nucleus are found in nonpolyadenylated molecules. These data suggest that polyadenylation does not specifically select for globin mRNA sequences. The nonpolyadenylated globin mRNA sequences present in the nucleus are found mostly in molecules of small size, close to the size of polyribosomal globin mRNA, suggesting that polyadenylation is a later event in globin mRNA formation.


Induction of globin mRNA accumulation by hemin in cultured erythroleukemic cells. The role of heme in erythroid development is investigated in erythroleukemic (Friend) cells. Exogenous hemin induces the accumulation of globin mRNA and globin protein in T3-C12 erythroleukemia cells to levels comparable to those induced by polar solvents, such as dimethylsulfoxide (DMSO). The hemin concentration required for maximal induction (10(-4) M) is the same as that which stimulates globin message translation in reticulocytes or cell-free reticulocyte lysates. Hemin and DMSO together cause T3-C12 cells to accumulate 8-9 fold more globin mRNA than either inducer individually. The kinetics of globin mRNA induction in hemin as compared to DMSO are very different: globin message accumulation begins 4 hr after hemin addition, but not until 30--40 hr after DMSO addition. Biliverdin induces 20--40 fold less hemoglobin than hemin; delta-aminolevulinic acid and porphobilinogen do not induce.


Tissue-specific forms of actin in the developing chick. Actin has been identified in nonmuscle and muscle tissues as a highly conserved homogeneous protein. We have identified and characterized actin from embryonic and adult chick brain and muscle, and have compared these actins by SDS and urea/SDS gradient polyacrylamide gel electrophoresis. In the presence of SDS alone, embryonic or adult brain and muscle actin co-migrate as homogeneous polypeptides. Electrophoresis of both actins in the presence of urea and SDS, however, reveals that brain and muscle actins migrate with distinctly different mobilities. Actin from embryonic thigh muscle at different stages of development migrates as two separate components. In early muscle development, only the "brain" type actin is present. As muscle development progresses the "muscle" type actin becomes relatively more abundant, so that by day 20 of embryonic development, "muscle" actin becomes predominant. These results may be interpreted as due to differences in the primary structure of actin.


Evidence for the formation of hybrid DNA during mitotic recombination in Chinese hamster cells. Direct evidence is provided for the formation of hybrid DNA during mitotic recombination in CHO cells. The cells were labeled for one round of replication in medium containing BUdR, so that the density of the DNA was heavy light (HL) and then returned to light medium. Further DNA synthesis, during either repair or chromosome replication, can only result in HL or fully light (LL) DNA; however, the formation of hybrid DNA as part of the process of recombinational repair will produce some fully heavy (HH) DNA. A small fraction of DNA containing regions of HH DNA has been detected on neutral CsC1 gradients, and the amount of this DNA is increased by treatment of the cells with mitomycin C. Increasing doses of mitomycin C produce smimlar increases in both the amount of HH DNA and the frequency of sister chromatid exchanges measured cytologically. This correlation provides evidence that the HH DNA is hybrid DNA, formed as an intermediate in recombinational repair.


[Electron microscopy of hepatocytes in voluntary blood donors with positive finding of Australia antigen]. In twenty blood-donors without clinical symptoms of any hepatic lesion whatever there were ascertained positive serum findings of Australian antigen. In three samples of hepatobiopsy worked up in these carriers of HB Ag we found specific alterations; once it was the case of virus-like dense particles found only in the liver, and twice there were similar dense spherical corpuscles in the cisterns of smooth endoplasmatic reticule. Once we found a relatively great number of filamentous adjustments in a distended endoplasmatic reticule, simultaneously with less numerous spherical dense corpuscles which may be considered, however, as most likely the modified covering material of the virus.


[Clinically unrecognized valvular defects in high age groups]. The study has been based on an autopsy series of 474 unrecognized valvular defects of the left heart in high age groups beyond 65. The series comprised 186 males and 288 females with an average age of 77. The incidence of valvular defects was 4.02%. There was a significantly higher incidence of mitral valve insufficiency than in lower age groups of clinically recognized valvular defects (10.75%). The ratio of mitral insufficiency to the stenosis of the left venous ostium and combined mitral defect with prevalence of stenosis was 1:4. The prevalence of males in the series of isolated stenosis of the left arterial ostium attained only 1.35:1.


[Cytocarcinogenesis of the rat hepatoma induced by N-nitrosomorpholine]. In experiments carried out on Wistar rats ultrastructrual charges of hepatocytes were studied during long-term carcinogenesis due to N-Nitrosomorpholine administered as a 20 mg% solution in drinking water. Carcinoma usually developed after the 20th week of the experiment. The development of the tumour was preceded by early dystrophic changes and, later, by a precancerous posttoxic "glycogenosis". The structural alterations as observed during the precancerous stage were rather noncharacteristic so that they did not permit to disclose the exact point of neoplastic change of the hepatocytes. Nevertheless, the model of carcinogenesis as demonstrated here appears to be a rather standard one and seems to be suitable for studying oncological problems of general character.


The interaction of acetoxy-dimethylnitrosamine, a proximate metabolite of the carcinogenic amine, and bacteriophages R17 and T7. The biological and physicochemical effects of reacting bacteriophages R17 and T7 with acetoxy-dimethylnitrosamine (ADMN) have been studied. The rate-determining step in the reactions appeared to be the loss of the acetoxy group by hydrolysis, the hydroxymethyl-methylnitrosamine generated decomposing rapidly to give a methyldiazonium ion and formaldehyde. In experiments with bacteriophage suspended in phosphate buffer the biological inactivation observed was the sum of the effects of the formaldehyde and of alkylation by the methylcarbonium ion produced from the diazonium ion. In experiments with bacteriophage suspended in Tris--HCl buffer the effects of formaldehyde were eliminated by its reaction with the buffer component. Alkylation by the carbonium ion produced unstable phosphotriesters in the bacteriophage RNA which on hydrolysis led to degradation of the molecule. In phosphate buffer the formaldehyde cross-linked the protein coat of the bacteriophage blocking the extraction of the RNA. Estimates of the mean lethal dose and of the extent of degradation of the RNA following reaction in Tris--HCl buffer were fairly close to those observed in experiments with N-methyl-N-nitrosourea (MNUA).


Effects of methylation  of the beta-galactosidase genome upon in vitro synthesis of beta-galactosidase. A template DNA from phage lambdah80dlacp5 coding for the in vitro synthesis of beta-galactosidase was used to study the effect of DNA methylation by the alkylating agent, dimethyl sulfate (DMS). Increasing the levels of DMS up to 50 mM concentration in the incubation medium led to an increase of DNA methylation. When incubated for 10 min at 37 degrees C, 3-4% Of nucleotides were methylated. The increase was linear to about 0.6% nucleotide methylation level. A higher yield was obtained at 37 degrees C incubation temperature than at 20 degrees C. Methylation of lambdah80dlacp5 DNA alone without methylation of other factors in the incubation mixture caused inhibition of the synthesis of beta-galactosidase in vitro. Increasing levels of DNA methylation caused greater inhibition of the newly synthesized enzyme activity. Total protein and RNA synthesis was inhibited by the methylated DNA to a much lesser extent than the inhibition of enzyme activity. When the level of nucleotide methylation was 0.74%, only 2% of enzyme activity remained, but total protein and RNA synthetic activities were found to be 72% and 44%, respectively.


The 7-methylbenz(a)anthracene deoxyribonucleoside products isolated from DNA after metabolism of the carcinogen by rat liver microsomes in the presence of DNA. Metabolism of 7-methylbenz[a]anthracene (7MeBA) by 3-methylcholanthrene-induced rat liver microsomes in the presence of added native or denatured DNA resulted in covalent binding of the hydrocarbon to the nucleic acid. Enzymatic degradation and column chromatographic fractionation showed that the hydrocarbon-deoxyribonucleoside products were separable from the products similarly obtained from DNA having 7MeBA bound following treatment of mouse embryo cells in culture with this hydrocarbon. Comparison of the microsome catalysed hydrocarbon-deoxyribonucleoside products with those obtained by reaction with DNA of 7MeBA-5,6-oxide suggested that this K-region epoxide made a significant contribution to the liver microsome-induced DNA binding.


PHA response and methylazoxymethanol acetate. Several reports have indicated the mutagenicity of the cycasin aglycone, methyl azoxymethanol (MAM). Van Den Berg and Ball (1972) have demonstrated the inhibitory effects of MAM acetate (MAMac), a more stable form of the aglycone, on DNA synthesis and cell proliferation in HeLa cells. The purpose of this study was to observe the effects of MAMac on blastogenesis in the human short-term leucocyte culture system. A depression in blastogenesis by MAMac was observed cytologically derived from two individuals. The same effect was observed utilizing [3H] thymidine as an indicator of blastogenesis in a series of cultures from 11 male individuals exposed to varying doses of MAMac, ranging from 5 through 800 mug/ml.


Metabolic interaction of secondary amines and tertiary amino propiophenones with monoamine oxidase systems. The metabolic interaction of three secondary amines and three nitrogen-containing metabolites of safrole (tertiary amino propiophenones) with rat liver mitochondrial monoamine oxidase systems was studied in vitro employing [7-14C] benzylamine--HCl as a substrate. The two cyclic secondary amines, piperidine and pyrrolidine, showed hyperbolic competitive inhibition while pure competitive inhibition was observed in case of dimethylamine--HCl and three safrole metabolites. Inhibition characteristics for rat liver, kidney and brain mitochondrial monoamine oxidase with two cyclic secondary amines and tertiary amino with two cyclic secondary amines and tertiary amino propiophenones of safrole and elemicin were investigated manometrically using tyramine and serotonin as the substrates.


The binding of ethyl carbamate to DNA of mouse liver in vivo: the nature of the bound molecule and the site of binding. Ethyl carbamate, labelled at C1 with 14C, bound in vivo to liver DNA of intact and partially hepatectomised mice. Isotope (18O) enrichment was not detected in the oxygen of liver DNA of mice injected with [18O] ethyl carbamate, C2H5--18O--CO--NH2. This suggests that it was the ethyl group and not the ethoxy group which bound to DNA. Chromatographic analysis of acid hydrolysates of liver DNA from mice treated with [1-14C] ethyl carbamate provided no evidence of alkylation or other form of binding to purine or pyrimidine bases. On relatively mild acid hydrolysis the alkyl group remained bound to the "apurinic acid" fraction, while more vigorous hydrolysis lead progressively first to its separation as highly ionisable hydrophilic non-volatile compounds and then to its loss as a volatile compound. DNAase I followed by phosphodiesterase hydrolysis also split off the 14C-containing group as a volatile compound. The volatile compound was identified as ethanol. These results suggest that the alkyl group was bound as an ester to a phosphate group in the DNA chain. Results with DNA from partially hepatectomised mice did not differ from those with DNA from intact mice.


Biotransformation of 4-dimethylaminophenol: reaction with glutathione, and some properties of the reaction products. 4-Dimethylaminophenol (DMAP) forms ferrihemoglobin by catalytic transfer of electrons from ferrohemoglobin to oxygen. In solutions of purified human hemoglobin, quick binding of oxidized DMAP to the globin moiety of hemoglobin terminates this reaction. Reduced glutathione in high concentrations, as in the red cell, substantially diminished binding of oxidized DMAP to hemoglobin by formation of S,S,S-(2-dimethylamino-5-hydroxy-1,3,4-phenylene)-tris-glutathione (tris-(GS)-DMAP), which does not form ferrihemoglobin. In the presence of reduced glutathione, DMAP disappeared more rapidly from hemoglobin solutions than in its absence. The formation of tris(GS)-DMAP in red  cells was found to be of importance for the termination of catalytic ferrihemoglobin formation by DMAP in vivo. With low concentrations of GSH, DMAP in hemoglobin solutions formed another conjugate, (GS)-DMAP, S,S(2-dimethylamino-5-hydroxy-1,3-phenylene)-bis-glutathione. Similar to DMAP, bis(GS)-DMAP catalyzed the formation of ferrihemoglobin. As the oxidized bis(GS)-DMAP was bound to hemoglobin more slowly and to a lesser extent, it produced more ferrihemoglobin than DMAP. In contrast to the reactions of DMAP with hemoglobin, hydrogen peroxide and superoxide radicals are involved in the ferrihemoglobin formation by bis(GS)-DMAP. The radicals accelerate the oxidation of bis(GS)-DMAP and thereby the ferrihemoglobin formation.


Platinum complexes of substituted ethylenediamines and their anti-tumour activity. A large number of substituted ethylenediamine complexes of platinum (II) and platinum(IV) have been prepared and characterised, and their anti-tumour activity tested against sarcoma 180, leukemia L1210, and ADJ/PC6A plasma cell tumours. In general these complexes are less active than simpler amine analogues, though several complexes show activity against all of the screens. Difference in activity of platinum(II) and platinum(IV) analogues leads us to cast doubt on the "in vivo reduction" theory hitherto used to describe the action  of platinum(IV) complexes.


The time course of DNA repair following methyl nitro-nitrosoguanidine (MNNG) treatment of P388F lymphoma cells in culture. I. Alkali dissociable DNA lesions and their restitution. The pattern of formation and rejoining of P388F cell DNA single strand breaks revealed by alkaline sedimentation centrifugation studies following treatment with methyl nitro-nitrosoguanidine (MNNG) has been studied. Three periods of single strand break formation have been recognised, an initial period during the first 4 h where breaks are rapidly formed and rejoined, a second (medial) time range of 10-15 h following treatment and a final stage where the formation of breaks appears to be related to cell killing processes. The initial phase is considered to be due to the production and repair of X-ray like DNA lesions whereas those of the medial time range are more comparable to the enzymatic processes associated with the repair of ultraviolet light-induced damage.


The time course of DNA repair following methyl nitro-nitrosoguanidine (MNNG) treatment of p388f lymphoma cells in culture. II. DNA repair synthesis and its correlation with strand breakage. Repair synthesis has been followed in P388F cell DNA following treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) using 5-iodo-deoxyuridine (IUdR) and [3H] thymidine combination. A pattern of repair synthesis was obtained, similar in its timing to the initial and medial stages of single-strand break formation and rejoining within the first 24 h after treatment. A higher base insertion frequency in relation to single-strand breaks occurs during the medial stage of repair. However, a fundamental problem with the use of iododeoxyuridine and [3H] thymidine ([3H] TdR) as a suitable measure of repair synthesis was encountered and a potential source of error in such experiments was observed.


Effects of actinomycin D and puromycin on colchicine-induced endoreduplication in Chinese hamster cells in vitro. Experiments were carried out to study the events through which the process of endoreduplication occurs after treatment with colchicine in Chinese hamster cells cultivated in vitro, and to analyse the differences between the processes leading to normal mitosis and those leading to endoreduplication. Chinese hamster cells (line C-125) were treated with colchicine (10(-4) M) for 1 h, in order to induce endoreduplication. Before and after colchicine treatment the cultures were treated at various intervals with actinomycin D and puromycin. For preparation of mitosis specific RNA synthesis is required, and also synthesis of a specific protein, which is not required for endoreduplication. Moreover this suggests not only that the cells that endoreduplicate do not need new RNA and protein synthesis, but that inhibition of their synthesis apparently favours the processes of endoreduplication.


Actinomycin D-induced enhancement of ubiquinone biosynthesis in rat. Administration of actinomycin D to fasted rats induces an enhancement of the labeling of hepatic ubiquinone by [2-14C] acetate both in vivo and in vitro. The incorporation of [2-14C] mevalonate into ubiquinone is also increased, although to a significantly lesser extent; this, however, presumably results from greater uptake of the labeled precursor by liver of drug-treated rats. The drug-administered animals show increased activity of liver microsomal mevalonate: NADP oxidoreductase, the rate-limiting enzyme in isoprenoid biogenesis. The incorporation of [u-14C] benzoic acid and CH3-[14C] methionine into ubiquinone in liver slices, however, reveals a decrease in actinomycin D administered rats. This appears to be due to a specific inhibition of the pathway leading to the benzoquinone moiety of ubiquinone and not to an increase in the pool-size of the precursors. The stimulatory effect of the drug on ubiquinone biosynthesis is also observable in cholesterol-fed rats. The actinomycin D-induced increase in ubiquinone biosynthesis is dependent on new protein synthesis since the effect is abolished by treating the animals with either cycloheximide or puromycin.


Action of a platinum complex [cis-dichlorobis (cyclopentylamine)-platinum (II)] on Chinese hamster ovary cells in vitro. cis-Dichlorobis(cyclopentylamine)platinum (II) (DBCP) treatment of Chinese hamster ovary (CHO) monolayer cell cultures was found to cause: (1) growth inhibition due to an impaired G1 leads to S transition and formation of a non-cycling compartment; (2) delayed cell death (with a maximum at 72 h after treatment); (3) decrease of [14C] TdR incorporation into DNA. Part of the surviving cell population was subject to non-lethal damage. The Do values of the dose--survival curves were 9.2 and 14 mug/ml in two kinds of media differing in Na+ and Cl- concentrations. There was no sparing effect of dose fractionation and no cell cycle phase specificity. In the dose range corresponding to the shoulder region of the dose-survival curve,the lethal effect of DBCP treatment was directly related to the decreased rate of DNA synthesis in DBCP-treated cells.


Inhibition of viral multiplication by homologous methylated ribonucleic acids. V. Inhibition of myxoviruses in mouse. Mice respond to intravenous injection of homologous methylated RNA by the production of a circulating interferon-like substance. The treatment with modified RNA induces a significant protection against viral infection. This effect is optimum when the treatment occurs a few hours before viral infection. The protective effect is a peculiar property of homologous methylated RNA as heterologous RNAs do not induce any resistance to the infection with influenza virus.


Distribution and cytogenetic test of 2,4-D and 2,4,5-T phenoxyacetic acids in mouse blood tissues. The phenoxyacetic acids 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), extensively used as herbicides, were tested for cytogenetic effects by means of induced micronuclei in erythrocytes of mouse bone marrow.  Because of the hig experimental resolution power this is a particularly suitable test system for the detection of weak chromosome breaking activity in mammals.  The cytogenetic tests were supplemented with chemical analyses of the concentration the the test substances reaching the target cells...


The effect of pre-treatment with phenobarbitone on the activation of aflatoxin B1 by rat liver. Microsomes prepared from rats pre-treated with phenobarbitone are more effective in activating aflatoxin B1 in vitro to a metabolite which inhibits RNA polymerase than are microsomes obtained from control animals. This result is in contrast with the situation in vivo where pre-treatment with phenobarbitone protects against inhibition of RNA synthesis by aflatoxin B1. The hypothesis is advanced that, in vivo, the activation of aflatoxin B1 which is significant in terms of inhibition of nucleic acid synthesis largely occurs on the outer nuclear membrane, and that by increasing activation by the microsomes, phenobarbitone pre-treatment reduces the amount of aflatoxin B1 available for the nuclear activation.


Urinary metabolites of 3,3-dimethyl-1-phenyltriazene. Urinary metabolites excreted after a subcutaneous injection of 3,3-dimethyl-1[14C] phenyltriazene (DM[1-14C]PT) to rats accounted for 82% of the applied radioactivity. We have isolated aniline (1-2%), 2-hydroxyaniline (5-7%), 3-hydroxyaniline (about 1%) and 4-hydroxyaniline (31-37%) from ethyl acetate extracts of acid-hydrolysed urine, UV spectrometric determination of 4-hydroxyaniline, using the indophenol reaction, showed that the most abundant metabolite accounted for 56 to 61% of the applied dose. We have also demonstrated the excretion of metabolites containing the intact triazene structure (0.9-1.1%) by cold acid cleavage of these compounds, followed by coupling of the released arenediazonium cations with N-ethyl-1-naphthylamine (EN). The coloured derivatives of these metabolites, 4-benzeneazo-N-ethyl-1-naphthylamine (BAEN) (0.6-0.7%), 4-(2-hydroxybenzeneazo)-N-ethyl-1-napthylamine (2-HO-BAEN) (0.02%) and 4-(4-hydroxybenzeneazo)-N-ethyl-1-naphthylamine (4-HO-BAEN) (0.3-0.4%) were isolated. The identification of BAEN as the principal azo derivative of the excreted triazene metabolites is in full agreement with the proposed in vivo activation of 3,3-dimethyl-1-phenyltriazene (DMPT) to a carcinogenic methylating agent. The hydroxylation of the methyl group at N-3 yields the corresponding aminol, some of which is covalently bonded to a water-soluble compound.


The effect of oral doses of a polythiol resin on the excretion of methylmercury in mice treated with cysteine, D-penicillamine or phenobarbitone. Mice dosed with methylmercury chloride (0.5 to 2.0 mg Hg/kg i.p.) labeled with the 203Hg isotope were given polythiol resin in their food (2% w/w). The effect of pretreatment with phenobarbitone, or co-administration of D-penicillamine or L-cysteine, was tested on the efficacy of the resin to accelerate the excretion of methylmercury. The effect of the polythiol resin in enhancing fecal excretion was dependent on the dose of methylmercury, it being significantly greater at doses of 1.0 and 2.0 than at 0.5 mg Hg/kg. In animals given the resin and phenobarbitone (0.1% w/w drinking water), fecal excretion was approx. 36% greater than in animals given resin alone. Penicillamine (2.5 mg/ml drinking water) actually retarted elimination of methylmercury in animals given the resin. Despite the enhanced efficacy of the resin plus phenobarbitone, the brain levels of methylmercury were not reduced beyond those seen in animals given resin alone. The mechanisms and implications of these effects are discussed.


A new class of inhibitory cytochrome P-450 complexes formed during metabolism: a comparison with amphetamine and SKF 525-A type complexes. During the metabolism of p-chloroaniline, sulfanilamide, 2-methylindoline, and dapsone, complexes with cytochrome P-450 are formed which absorb maximally at 448, 450, 451, and 452 nm, respectively. These complexes are similar to the complexes from amphetamines (456 nm) and SKF 525-A (452 nm) in the conditions for, and rate of their formation, and their inhibition of ethylmorphine N-demethylase activity. Similarly, the formation is mainly a property of phenobarbital-induced cytochrome P-450 and is inhibited by comparable concentrations of metyrapone and 2-O-iodophenoxymethylimidazole. Unlike the complexes from amphetamine and SKF 525-A, those formed from p-chloroaniline, sulfanilamide, 2-methylindoline, and dapsone are unstable in the presence of sodium dithionite.


Cadmium-thionein and the protection by cadmium against the nephrotoxicity of mercury. Uptake of Hg2+ into the renal and hepatic metallothioneins of rats is increased by pretreatment with Cd2+. This increased uptake occurs both by displacement of Cd2+ (and of Zn2+) from the presynthesized cadmium-thionein, and by further synthesis of thionein. The former mechanism predominates in the kidney of the male rat, which is more sensitive than the female to Hg2+. The latter mechanism, which occurs particularly in the kidney of the female, also is considered to involve an initial displacement of Cd2+ from cadmium-thionein, but is followed by further synthesis of the metalloprotein, which is induced by the liberated cation. Pretreatment with Cd2+ increases not only the incorporation of Hg2+ into the renal metallothionein, but also the uptake of Hg2+ into other components of the kidney. At dose levels of Hg2+ at which Cd2+-pretreatment gives complete protection against the nephrotoxicity in male and female rats, the increase in Hg2+-uptake into both the particulate components and into the soluble fraction of the kidney is greater than into metallothionein. It is concluded, therefore, that binding of Hg2+ by pre-induced cadmium-thionein alone cannot explain the protection by Cd2+ against the nephrotoxicity of Hg2+.


"Paired reading" tuition: a preliminary report on a technique for cases of reading deficit. A simple and flexible reading tuition procedure is described, incorporating simultaneous reading and verbally reinforced individual reading, utilizing any reading material of the child's choice, and maximizing continuous adaptation to individual reading behaviour. Three reading-deficient children tutored by "paired reading" are presented, indicating a clear increase in the percentage of words correctly read and reaching statistical significance over twelve hours of tuition.


The effect of hypoxia on the regional distribution of cardiac output in the dog. Twenty-one dogs were studied under conditions of normal oxygenation and hypoxia with the microsphere distribution method to determine the effect of arterial oxygen saturation on the regional distribution of cardiac output. The dogs were anesthetized and artifically ventilated. Cannulas were placed in the left ventricle to administer microspheres and in a peripheral artery to determine cardiac output. Each dog received two microsphere injections: (1) while normally oxygenated (room air), and (2) under hypoxia (10% oxygen-90% nitrogen in 10 dogs and 5% oxygen-95% nitrogen in 11 dogs). Absolute cardiac output increased from 87 +/- 15 ml/min per kg to 101 +/- 14 ml/min per kg during mild hypoxia (10% oxygen) (P less than 0.05), and from 73 +/- 17 ml/min per kg to 120 +/- 24 ml/min per kg during severe hypoxia (5% oxygen) (P less than 0.01). Absolute blood flows increased to all organs except skin and muscle during hypoxia, although there were decreases in the fractional distribution of cardiac output to the splanchnic bed and kidney. Striking changes were found in coronary, hepatic, and cerebral circulation, and the organ with, greatest response to hypoxia was the heart, with increased coronary flow of 37% and 285% during exposure to 10% and 5% oxygen, respectively. Hence, low oxygen levels in blood cause redistribution of cardiac output and arterial content plays an important role in blood flow regulation.


Action potential changes under varied [Na+]0 and [Ca2+]0 indicating the existence of two inward currents in cells of the rabbit atrioventricular node. In the perfused rabbit heart,the upstroke of the transmembrane action potential of fibers of the atrioventricular (AV) node presents two distinct components. The first depends strongly on extracellular sdoium concentration, but the degree to which it is activated is influenced by extracellular calcium, as indicated by the correlation between its Vmax and [Ca2+]0. The second component depends on calcium and sodium concentrations and is blocked by Mn ions. An analysis comparing action potentials from atrial (A), atrionodal (AN), and nodal (N) fibers shows that the second component of the upstroke of the action potential contributes 12%, 27%, and 34% to the total depolarization. The results suggest that the upstroke of the nodal action potential results from the activation of two inward currents, as in ordinary cardiac fibers. We postulate that (1) the degree of steady state inactivation of gNa is larger in N than in A fibers because of the low resting potential of the former, and (2) the contribution of the second channel to the upstroke depends on the time course of the previous depolarization and the potential level at which this component is activated.


Low renal papillary plasma flow in both Dahl and Kyoto rats with spontaneous hypertension. Abnormally low plasma flow to renal papilla characterizes Dahl hypertension. When eating a normal Na diet (0.3% NaCl) both hypertension-sensitive (S) rats and hypertension-resistant (R) rats, 16 weeks old, have fairly normal blood pressure (BP), averaging 144 and 129 mm Hg, respectively. However, even in this barely hypertensive state, 18 S rats had a 31% lower papillary plasma flow (Lilienfield method) than 22 R rats, 19.2 ml/100 g of papilla per min compared to 25.6 (P less than 0.001). When a high (8%) NaCl diet was fed for 7 days, R rats increased papillary plasma flow from 25.6 on 0.3% NaCl to 33.8 on 8% NaCl, a 32% rise (P less than 0.001). S rats increased papillary flow from 20.4 to 24.8, a 22% rise (P less than 0.05). When a high (8%) NaCl diet was fed for 4 weeks, R rats increased papillary plasma flow from 25.7 ml/100 g per min on 0.3% NaCl to 29.5 ml/100 g per min on 8% NaCl, a 15% rise (P less than 0.025). S rats increased papillary flow from 17.7 to 20.0 ml/100 g per min (not significant). S rats on 8% NaCl had a papillary flow 32% lower than R rats on 8% NaCl (P less than 0.001). BP of S rats rose to 162 mm Hg after 4 weeks on 8% NaCl; in R rats, BP did not rise at all. S rats on 0.3% NaCl have a low papillary flow even in a borderline hypertensive state. When challenged with 8% NaCl, R rats increased papillary flow, an adaptation possibly important for the natriuresis. S rats failed to achieve this same high papillary flow. Lacking this adaptation, hypertension may then conceivably occur in S rats to accomplish natriuresis through a "pressure natriuresis" mechanism. Papillary flow also decreased by 11% in 26 Kyoto 17-week-old spontaneously hypertensives (BP, 182 mm Hg) compared to 24 Kyoto normotensives (BP, 118 mm Hg), 29.5 vs 33.2 ml/100 g per min (P less than 0.001). Thus, low papillary flow exists in both hypertensions.


Follow-up ?at II years of 46 children with severe unilateral hearing loss at 7 years. In the ongoing NCDS, 44 children of the 46 identified at 7 years as having a serious unilateral hearing loss were followed up at II years. Half of them had recovered normal bilateral hearing, the remainder still had serious deafness in one ear. Although at 7 years the 46 children as a group had shown backwardness in oral ability, speech and reading, at II years both the "recovered" and the "persistent" were similar to their age peers in scholastic attainment. Despite their original apparent difficulty it was encouraging to find that several children in both subgroups were noted as possessing outstanding academic ability. It is concluded that with prompt follow-up by an alerted school doctor, children with unilateral deafness at age 7 years are likely to progress satisfactorily in later childhood.


The effect of the pattern of cardiac sympathetic activity on myocardial contractile force and norepinephrine overflow in the dog heart. The left of right cardiac sympathetic nerves in open-chest, anesthetized dogs were stimulated at mean frequencies of 2 or 4 Hz. The stimuli were applied intermittently, in patterns with repetition rates or either 60/min or 15/min, to simulate the spontaneous patterns of sympathetic neural activity that occur synchronously with the cardiac or respiratory cycles, respectively. With either repetition rate, intermittent stimulation of the left sympathetic nerves was about 10-20% less effective in enhancing myocardial contractile force (CF) and about 10% less effective in increasing coronary sinus blood flow than was steady stimulation at the same mean frequency. With right-sided stimulation, there was no appreciable difference between steady and intermettent stimulation patterns with respect to the effect on heart rate. With either left-or right-sided stimulation, the rate of norepinephrine (NE) overflow into the coronary sinus blood was 20-40% less with intermittent than with steady stimulation. Cocaine administration did not materially affect this difference in NE overflow. It was concluded that the higher instantaneous frequencies that prevail during intermittent stimulation result in a reduction in the rate of NE release at the sympathetic postganglionic nerve endings in the heart.


Differential methods of enhancing cognitive growth in urban preschool children. The investigation was carried out to design and test more purposeful and structured methods of promoting intellectual growth in culturally deprived preschool children. The relative merits of providing specific cognitive training both in a nursery setting and in the home through the influence of the mother were assessed. Four groups each of 15 children were involved in the experiment, which extended over a period of four months. Gains were confirmed for both general intelligence and linguistic competence. Both the two nursery groups and the experimental "home" group all showed considerable improvement, the latter being comparable to the nursery control group.


Interaction of capillary and tissue forces in the cat small intestine. We measured steady state capillary hydrostatic pressure (P c,i), plasma and lymph protein concentrations, lymph and blood flow, and capillary filtration coefficients in an in situ loop of cat small intestine at venous outflow pressures (PV) of 0, 5, 10, 15, 20, 25, and 30 mm Hg. The data were used to calculate colloid osmotic pressure of lymph and plasma, interstitial fluid pressure (Pt), pre- and postcapillary resistances, and a tissue pressure-volume curve of the intestinal interstitium. When PV was elevated from 0 to 30 mm Hg, lymph protein concentration decreased from 3.8 to 1.9 g/100 ml (representing a change in colloid osmotic pressure of 6.2 mm Hg), lymph flow increased 7-fold (or an equivalent imbalance in Starling forces of 4.3 mm Hg), and the calculated PT increased from 1.8 to +5.3. Because lymph flow draining the loop decreased during the determination of Pc, i at venous pressures between 15 and 30 mm Hg, the corresponding calculated PT may be in error by 1-2 mm Hg. The tissue pressure-volume relationship calculated from the data indicates that the intestinal interstitial volume expands nonlinearly and this expansion is characterized by two distinctly different compliant components: (1) tissue compliance is low at PV between 0 and 15 mm Hg (0.4 ml/mm Hg), and (2) at PV greater than 15 mm Hg the tissue compliance is relatively high (4 ml/mm Hg). We found that when PV was elevated from 0 to 15 mm Hg, increases in PT are the major tissue adjustments that oppose the increased filtration pressures. Furthermore, at Pv of 20-30 mm Hg, tissue protein concentration decreases, lymph flow relative to the filtration coefficient (deltaP DROP) increases and, to a much lesser extent, PT increases. Finally, the combination of these changes in tissue force at high filtration pressures represent a maximum tissue edema "safety factor" of 10 mm Hg; further increases in filtration pressures result in large volume movements into the intestinal lumen.


Sound reward toys.

Sound reward toys. The multiplicity of disciplines involved in a comprehensive treatment and assessment centre provides fertile ground for the design of new apparatus of practical use in the field of therapy. Such projects encourage team work, are of considerable benefit to the patients and can lead to new approaches to problems. The construction and use of three sound reward toys for the diagnosis and treatment of speech disorders is described.


Bed wetting and emotional disorder: an "interactionist" model. Twenty-two of 94 4-year-olds in a nursery schools sample in a working class area wet their beds at least once a week. The enuresis was significantly associated with other behavioural items which could be interpreted as representing general emotional immaturity. A malaise score of the enuretics' mothers was higher than that of the remaining mothers. It is suggested that from these results can be seen the beginning of a damaging interaction which could lead to emotional disorder in the child.


Transcapillary escape rate of albumin and right atrial pressure in chronic congestive heart failure before and after treatment. The transcapillary escape rate of albumin (TERalb), i.e., the fraction of intravascular mass of albumin that passes to the extravascular space per unit of time, was determined from the disappearance of intravenously injected 125I-labeled human serum albumin during the first 60 minutes after injection in 10 subjects with chronic right heart failure. The investigation was repeated after sodium and water depletion. Before treatment TERalb was significantly elevated (mean 8.3 +/- 1.6% (SD)/hour, in comparison to values for normal subjects (mean 5.4 +/- 1.1%/hour, P less than 0.001). With treatment TERalb decreased significantly (mean 5.9 +/- 1.2%/hour, P less than 0.01). Right atrial pressure decreased from an average of 10 mm Hg to 6 mm Hg during treatment. A statistically significant, positive correlation was found between TERalb and right atrial pressure (r = 0.77, P less than 0.001). Our results best can be explained by increased filtration, mainly through the venous end of the microvasculature, due to the increased venous pressure in heart failure.


Quantification of collateral resistance in acute and chronic experimental coronary occlusion in the dog. The resistance to coronary blood flow in various parts of the myocardium was studied with the tracer microspheres technique before and immediately after an acute coronary occlusion and several weeks after a more slowly occurring coronary occlusion by Ameroid constrictor. All experiments were carried out in the isolated, metabolically supported, empty, beating dog heart at maximal coronary vasodilation induced with adenosine. Coronary resistance of the normal empty beating heart at maximal coronary vasodilation was 0.20 mm mm Hg/(ml/min) per 100 g of tissue (subepicardium) and 0.16 mm Hg/(ml/min) per 100 g of tissue (subendocardium). After acute coronary occlusion the perfusion of the subtended myocardium was maintained at a much lower level by way of collateral vessels, which showed a resistance to flow of 3.52 mm Hg/(ml/min) per 100 g. If coronary artery occlusion proceeded more slowly the collateral vessels became more functional and myocardial infarction was avoided. During collateral enlargement collateral resistance fell from 3.52 to 0.22 mm Hg/(ml/min) per 100 g within a period of 8 weeks after implantation of the constricting device. The degree of compensation by collaterals for the loss of the occluded native coronary artery was 33% of its former conductance.


Effects of treatment with pyruvate and tromethamine in experimental myocardial ischemia. Failure of glycolysis to increase sufficiently to supply optimal levels of energy production in ischemic heart muscle is due in part to the cummulative restrainst of acidosis on rate-limiting enzymes, particularly glyceraldehyde-3-phosphate dehydrogenase. In an effort to modify this inhibition and salvage jeopardized myocardium, treatment with excess levels of pyruvate and tromethamine (Tris), designed to buffer intracellular hydrogen ion accumulations and improve the oxidation-reduction ratio, NAD+/NADH, was tested in 59 swine hearts in two separate preparations of global and regional ischemia. Global ischemia, per se, caused hemodynamic deterioration and shortened survival time (44.3 +/- 3.1 minutes). Myocardial oxygen consumption, fatty acid oxidation, and glucose uptake were all significantly (P less than 0.001) reduced as were estimates of glycolysis and tissue stores of creatine phosphate and ATP (P less than 0.01). Although treatment with Tris alone was inconclusive, administrations of pyruvate (40-50 mM) buffered with Tris (added directly into the coronary perfusate) effected an improvement in mechanical function and a significant prolongation in survival time (56.9 +/- 2.6 minutes. P less than 0.01). Glycogenolysis was enhanced and levels of key glycolytic intermediates were reduced, suggesting an acceleration of glycolytic flux. Excess levels of pyruvate (1.52 +/- 0.48 mumol/ml of coronary perfusate) provided added substrate for oxidation and led to a greater than 5-fold incrase in rates of pyruvate decarboxylation as compared to untreated ischemic hearts...


The decay of the potentiated state in sheep and calf ventricular myocardial fibers. Influence of agents acting on transmembrane Ca2+ flux. Paired pulse stimulation increases the contractile strength of mammalian myocardium. If stimulation is discontinued the "potentiated state" takes several minutes to decay, as shown by the first contraction following resumption of stimulation. From earlier experiments it has been inferred that contractile force depends on Ca2+ released from intracellular stores rather than on an influx of Ca2+ associated with a given action potential. This view now receives support from the following findings: (1) Ni2+ and Co2+, known to inhibit Ca2+ influx during the action potential, when applied during a 2-minute rest period, are practically without effect on the strength of the first beat but strongly depress steady state contractions. (2) Caffeine, known to enhance Ca2+ efflux from and inhibit Ca2+ uptake into intracellular stores, greatly accelerates the decay of the potentiated state during a rest period. (3) Na+ -poor solution, known to inhibit Ca2+ efflux, has a strong positive inotropic effect. Paired pulse stimulation fails to increase contractile strength in Na+ -poor solution, and a rest period of many minutes is practically without effect on the amplitude of the first beat after resumption of stimulation. The results indicate that contraction is due to Ca2+ released from internal stores whose degree of filling can be altered.


Chronic one-kidney hypertension in rabbits. II. Evidence for a new factor. The arterial pressure of rabbits with chronic one-kidney hypertension can be lowered to normotensive levels by direct immunization with preparations made from the cortex of hog kidneys. Hypertensive rabbits that are immunized with large amounts of renin may develop high plasma antirenin titers without affecting their blood pressure. Removal of renin by chromatography on columns of immobilized antirenin yields preparations with little or no renin. Such preparations may be effective in lowering blood pressure although they do not elicit the formation of plasma antirenin and the rabbits remain sensitive to challenging doses of rabbit renin. The evidence suggests that an unknown substance is responsible for the development of a cross-reacting antibody that neutralizes an unknown factor that is essential for the maintence of an elevated blood pressure level in rabbits with chronic one-kidney hypertension.


Age-related changes in ouabain pharmacology. Ouabain exhibits a different volume of distribution in adult and young dogs. To better understand why one must administer much higher doeses of digitalis glycosides to immature than to mature humans and animals, we studied ouabain pharmacokinetics in adult and young dogs. Consistent with reported observations that ouabain-binding, metabolism, and excretion do not change with age, we found no significant differences in the transfer coefficients in a linear two-compartment open model for ouabain pharmacokinetics following a bolus of 0.05 mg/kg. We did find, however, that young dogs had nearly twice the ouabain volumes of distribution per kilogram of body weight as adults (155.4 +/- 1.2 (SE) ml/kg vs. 80 +/- 0.6, P less than 0.0005) and that one could account for this difference with the fact that young dogs had nearly twice the plasma volume 108 +/- 9.8, vs. 68 +/- 7.3, P = 0.001) and interstitial fluid space (318 +/- 35 vs. 190 +/- 6.5, P = 0.006) as the adults. For the same dose per kilogram, left and right ventricular ouabain concentrations were inversely related to the volume of distribution, with the adults having significantly higher tissue levels and incidence of arrhythmias. One must give more ouabain to a young dog to get the same plasma concentration as in an adult because the mass of ouabain in rapid equilibrium with the plasma is diluted in a larger volume of distribution.


Effect of exercise conditioning on the intrinsic contractile state of cat myocardium. Fourteen adult cats were exercised on a motor-driven treadmill 5 days each week for 6 weeks to determine the effect of exercise conditioning on the intrinsic contractile state of the myocardium. The exercise program was sufficient to produce a cardiovascular training effect manifested by slower exercising heart rates and resting heart rates after atropine by the end of the 6th week. The mechanical function of the isolated right ventricular papillary muscle from exercised cats was compared with that of 17 sedentary adult cats. There were no significant differences between exercised and control cats in in heart weight-body weight ratios, resting and active lenght - tension relations, maximal rate of isometric force development at the peak of the length-tension curve (Lmax), time to peak force at Lmax, maximal force development with paired stimulation and norepinephrine, or force-velocity relations. These results indicate that the intrinsic state of feline myocardium is unaffected by exercise conditioning.


Effects of cardiac sympathetic stimulation and ablation of canine ventricular anodal strength-interval curves. We studied the effects of cardiac sympathetic stimulation and ablation on characteristics of the anodal-strength interval curve in 10 pentobarbital-anesthetized open-chest dogs. We gave special attention to the form and particularly the depth of early dips of the anodal strength-interval curve. When the central connections of the cardiac sympathetic nerves were intact, left stellate stimulation decreased threshold for excitation during the dip only slightly (from 883 +/- 442 muA to 774 +/- 326 muA P less than 0.1). However, after left stellectomy and upper thoracic ganglionectomy, stimulation of the cut ends the ansa subclavia decreased the dip threshold from an average of 952 +/- 321 muA to 707 +/- 210 muA, A less than 0.01. Dip thresholds in strength-interval curves determined 1-1.5 hours after ganglionectomy average 1164 +/- 296 muA compared to an average dip threshold of 791 +/- 269 muA in strength-interval curves measured immediately prior to stellectomy. This increase is statistically significant (P less than 0.005). In addition to the effects of sympathetic tone on disparity of refractory period and ventricular fibrillation threshold, the effects of sympathetic tone on the form of the strength-interval curve may be another factor influencing vulnerability to arrhythmias and particularly in patients with abnormal autonomic tone.


Cell permeability, sodium transport, and the hypertensive process in the rat. The tail artery of the spontaneously hypertensive rat (SHR) (Carworth Farms), excised rapidly and immersed immediately in cold (2 degrees C) Li-substituted physiologic salt solution (LiPSS), continues to exchange cell Na+ and K+ for Li+, this exchange is negligible to the control (Carworth Farms normotensive) CFN). In the incubated artery at 37 degrees C, when the vascular smooth muscle cell is slack, the leakiness of the cell membrane in the SHR is more than offset by increased Na+ pumping activity, so that cell Na+ is subnormal. A high precision technique with ion-specific electrodes was developed to follow the passive downhill and active uphill phases of Na+-K+ exchange in the perfused artery exposed to K+-free physiologic salt solution (K+-free PSS) followed by physiological salt solution (PSS). The exchange was found to be fully reversible and sufficiently equimolar to be definable in terms of movements of K+ alone. The rates of ionic movement across the vascular smooth muscle cell were found to be about 6 times faster for the vessel perfused at low pressure (less than 3 mm Hg) than for the slack incubated artery. The rate of passive downhill movement was significantly accelerated in the mature SHR compared with CFN, and the net active transport activity much enhanced. Similar changes were seen as early as 3 weeks after treatment with DOCA and were pronounced at 8 weeks. It is proposed that conditions favoring a sustained accumulation of Na+ in the vascular smooth muscle cell are countered by an enhanced synthesis of transport protein, of contractile protein, and of paracellular matrix protein which progressively restructure the wall.


Hormonal and nutritional substrate control of cardiac lysosomal enzyme activities. Prolonged starvation is known to induce significant alterations in several cardiac lysosomal enzymes, particularly the acid proteinase cathepsin D. To determine what specific factors might mediate these changes, fetal mouse hearts in organ culture were maintained in media designed to simulate selected hormonal or nutritional substrate changes that accompany starvation. Reduced concentrations of glucose caused an increase in the activity of beta-acetylglucosaminidase but had no effect on cathepsin D or acid phosphatase activites (i.e., effects opposite from those of starvation). Also, high concentrations of free fatty acid, acetoacetate, and beta-OH-butyrate induced an increase in cathepsin D (+18%) and a simultaneous decrease in glucosaminidase (-19%), with little change in acid phosphatase. Furthermore, glucagon had no effect on any of the enzymes, whereas growth hormone caused a small (6%) increase in cathepsin D activity. In addition, insulin deprivation caused significant increases (7-25%) in the activities of all three enzymes. Insulin deprivation and excess ketones, but not the other interventions, increased the proportion of enzyme activity which was nonsedimentable. These results suggest the possibility that lysosomal alterations during starvation may be related in part to prolonged insulin deficiency and exposure to high concentrations of ketones and free fatty acids.


Red cell velocity and plasma transit time in the cerebral microcirculation of spherocytic deer mice. Spherocytic deer mice provide a model of human spherocytosis. Their erythrocytes are abnormal in shape and are more rigid than normal red blood cells (RBC). Like their human counterparts, spherocytic mice are anemic. Measurements of RBC velocity in microvessels on the cerebral surface failed to reveal a difference between the velocity of cells in spherocytic as compared to normal deer mice. However, plasma transit, as measured by fluorescein microangiography, was faster than normal. Both decreased plasma transit time and increased RBC velocity are expected in nonspherocytic, anemic mice. Since the former, but not the latter, was found in the spherocytic, anemic mice, it appears that increased RBC rigidity has a greater effect on RBC movement than on plasma movement within the cerebral microcirculation. Thus iit would seem that this increased RBC rigidity prevents the increased RBC velocity that otherwise would be observed in anemia but does not prevent the decrease in plasma transit time.


Effect of different lysing and washing methods on free amino acid concentrations of sheep whole blood and erythrocytes. 1. A study was conducted to determine the effect of different washing and lysing methods on the free amino acid concentrations of sheep erythryocytes (RBC) and whole blood. Methods of lysis included laking, freeze-thawing or sonication. RBC were washed with isotonic solutions of either saline or sucrose. 2. Washing the RBC with isotonic sucrose resulted in significantly greater concentrations for ten of the amino acids. Greater amino acid concentrations were realized when washed RBC were sonicated and when the RBC of whole blood were lysed by laking. 3. Suggestions are made for future application to whole blood and RBC amino acid determinations.


The effects of anti-rheumatoid drugs on the in vitro activity of human serum hyaluronidase. Fourteen antirheumatoid drugs were tested for their effects on the in vitro hyaluronidase activity of normal human serum. Four drugs produced significant changes in enzyme activity. Different results were obtained with ovine testicular hyaluronidase when diluted with either saline or inactivated human serum. No increase in serum hyaluronidase activity was found in patients with rheumatoid arthritis. There was no evidence for the existence of tissue specific isoenzymes of hyaluronidase in the serum of either normal subjects or patients with rheumatoid arthritis.


The quantitation of urinary and spinal fluid proteins by a modified biuret method. A method is presented for the quantitation of urinary and CSF protein. In the presence of 0.10 N NaOH the peptide bonds of the protein remove and bind copper from an ion exchange resin. The resulting copper-protein complex is separated from low molecular weight substances by gel filtration and the copper in the eluted complex is determined colorimetrically with diethyldithiocarbamic acid. The method requires only 100mul of sample, has biuret specificity and uses a single prepacked column. The limit of sensitivity is 2 mg of protein per deciliter.


A comparative study of micro-ammonia determinations in plasma using two different methods. Two commercially available kits for determining blood ammonia were compared, an enzymatic method requiring a sample volume of 100mul and a nonenzymatic method requiring 1 ml of sample. Calibration curves for both kits were linear to 270 mumol/l, however correlation studies on plasma samples revealed a lack of agreement between methods (r equal to 0.825, Y equal to 0.76X +51). Improved correlation occurs when protein interference is eliminated from the enzymatic procedure. Finally, the nonenzymatic method is modified to accommodate 100 mul of sample and compared to the original method.


A simple method for "reference range" estimation from routine laboratory data. A simplified method is presented to estimate reference ranges from hospital laboratory data. It is based on a combination of graphical estimation of relative sizes of normal and abnormal populations and the "mode-center" concept in which the mode of the total population centers on the 50% cumulative frequency of the normal population. This method can be applied to determine reference ranges even though the data source contains abnormally high and/or low values. The reference ranges obtained for BUN and calcium from in-patient and out-patient sources by this method were found to be similar to those reported for "healthy" subjects.


Critical evaluation of the logit-log plot for the radio assay of vitamin B12. The dose-response calculation for radio assay of vitamin B12 is carried out using a straight line and a third degree curve to fit the experimentally obtained calibration points. It is shown that high dispersion of the calibration points with respect to the regression line can lead to errors in the calculation of the response. The calculation of the mean dispersion sx with respect to the regression line turns out to be a good criterion for checking the accuracy of the results.


On the interpretation and potential diagnostic value of the measurements related to lecithin:cholesterol acyltransferase activity. At least one reliable method is available for measuring LCAT activity in human subjects(25), and this assay may help to probe even subtle changes in plasma lipids and lipoproteins. Since plasma lipids and lipoproteins are known to be present in abnormal amounts in a large number of metabolic diseases, the method of measuring the rate of cholesterol esterification in plasma could assume a substantial role in the diagnosis and (or) monitoring of treatment of pathologic states.


Comparison of two radioimmunoassay kits for aldosterone determination. Two commercially available radioimmunoassay kits for aldosterone determination not requiring preliminary chromatographic purification were examined. Aldosterone standard curve solutions prepared from the CIS kit, Nuclear International Corp., (x-axis) and Diagnostic Products kit (y-axis) were assayed with both kits and the results yielded a regression equation of y = 0.834x + 0.08 nmol/liter. Analytical recovery experiments with aldosterone added to urine and serum showed 120% and 101% recovery with the CIS kit, respectively, and 92% and 92% recovery with the Diagnostic Products kit, respectively. Both kits demonstrated good parallelism with urine and serum. Antibody specificity was tested with six structurally related steroids and each kit showed virtually no cross-reactivity at clinically normal serum concentrations. Serum aldosterone values were compared before (y-axis) and after (x-axis) column chromatography on Sephadex LH-20, with the following regression equations: y = 1.070x + 0.092 nmol/liter for the CIS kit and y = 1.023x + 0.093 nmol/liter for the Diagnostic Products kit. Regression equations comparing patient samples between kits, CIS (x-axis) and Diagnostic Products (y-axis), were: y = 0.832x + 0.007 mumol/24 h with urine and y = 0.850x + 0.097 nmol/liter with serum.


Kinetic urine creatinine determination with the Gemsaec analyzer. A rapid automated method for urinary creatinine with the Jaffé reaction is described. Interferences by protein, glucose and ketones are circumvented by the reaction rate kinetics. The precision and accuracy of this centrifugal method on the ENI-Gemsaec analyzer are comparable to the manual end-point Jaffé reaction with deproteinization and to the common Auto-Analyzer technique.


A minicomputer-automated array spectrometer for liquid-chromatographic detection of metabolites. A third-generation multiwavelength array spectrometer was developed as a detector for the high-resolution liquid-chromatographic characterization of metabolites. Components include a PDP-8/e minicomputer, matched pair of linear photodiode arrays, holographically-ruled gratings, fiber optics, flow cells, and high intensity xenon light source. The wavelength range is 256 nm differential with 1-nm resolution and can be adjusted from 200 to 800 nm. The system is capable of storing 20 spectra per second (200-456 nm) in a dual-beam mode. Special features include minicomputer-driven signal enhancement via integration as a function of signal strength. The display output includes presentation of the total absorption chromatogram vs. elution time in both real and post-run time as well as selectable single absorption band vs. elution time (post-run time). Application of this dedicated system is illustrated by the separation and charcterization of the metabolites of a carcinogen, 4-ethylsulfonyl-1 napthalenesulfonamide.


Ion chromatography: a new technique for clinical chemistry. Ion chromatography is a new analytical technique in which the ions to be analyzed are separated on a special pellicular ion-exchange column. Once separated, the effluent from the separating column passes into a second ion-exchange column, which removes the eluent or buffer ions from the moving phase without removing the sample ions, which are detected and measured by electrical conductivity on a background of low conductance. Trace quantities can be so determined. This technique has been applied to four biological fluids: blood serum, tissue extract, urine, and cerebrospinal fluid. It is shown to be a practical analytical tool, particularly for ions having no chromophores, such as sodium, ammonium, potassium, magensium, calcium, chloride, nitrite, phosphate, and sulfate.


An immobilized-enzyme flow-enthalpimetric analyzer: application to glucose determination by direct phosphorylation catalyzed by hexokinase. A novel flow-enthalpimetric analyzer is described and its use demonstrated by an analysis in which glucose is determined by its hexokinase-catalyzed phosphorylation reaction. The method depends on measurement of the temperature differential across a column packed with glass-supported immoblized enzyme. Sample volumes of 120 mul can be used to obtain a calibration curve that is linear up to 25 mmol of glucose per liter. A precision (within-day) of 5% is generally observed in the optimum concentration range where glucose is quantitatively phosphorylated. Results by the technique correlate reasonably with those by the o-toluidine and the hexokinase/glucose-6-phosphate dehydrogenase methods: Other sugars--including fructose, glucosamine, and mannose--will interfere; galactose does not. The technique is amenable to both routine and emergency analyses.


The free-falling drop detector--a nover fluorescence detector for high-performance liquid chromatography. A novel fluorescence detector for high-performance liquid chromatography has been designed to minimize the instrumental limitations of conventional flow-through, fluorometers. This unit contributes less than 5 mul to the "dead volume" of the chromatographic system. Sensitivity comparable to that of existing flow-through fluorometers has been attained without optimizing instrumental parameters. Performance of the unit has been evaluated with reference samples containing saccharides typical of glycosphingolipids. Future improvements are discussed.


A propagation of error analysis of the enzyme activity expression. A model for determining the total system random error of a kinetic enzyme analyzer. We present a total system error evaluation of random error, based on a propagation of error analysis of the expression for the calculation of enzyme activity. A simple expression is derived that contains terms for photometric error, timing uncertainty, temperature-control error, sample and reagent volume errors, and pathlength error. This error expression was developed in general to provide a simple means of evaluating the magnitude of random error in an analytical system and in particular to provide an error evaluation protocol for the assessment of the error components in a prototype Miniature Centrifugal Analyzer system. Individual system components of error are measured. These measured error components are combined in the error expressiion to predict performance. Enzyme activity measurements are made to correlate with the projected error data. In conclusion, it is demonstrated that this is one method for permitting the clinical chemist and the instrument manufacturer to establish reasonable error limits.


Modified nucleosides in urine: selective removal and analysis. Because excretion of certain nucleosides increases in all types of cancer, detection and treatment of the disease would be enhanced by rapid, simple quantitation of such nucleotides. To facilitate these analyses in urine, we have prepared a specific adsorbent for nucleosides. The affinity adsorbent gel contains an immobilized phenylboronic acid group that specifically binds cis-diols, as in ribonucleosides, which can then be quantititatively recovered by elution with acetic acid. The advantage of this boronate gel over other preparations lies in its high capacity in the swollen state (0.26 mmol/ml), so that only small volumes (less than 1 ml) of gel are required for assays. Nucelosides containing a free diol group on the ribosyl group are retained at pH 8.8 in 0.25 mol/liter salt solution with a retention volume of 15 or larger, as compared with values of 1 to 2 for the free bases. Negatively charged species (not including the boronate complex) are eluted earlier than neutral or cationic species. Added nucleosides (neutral, cationic, or anionic) are quantitatively recovered. Because pseudouridine is eluted in a unique position, this property has been used to measure it in urine. The procedures used are chemically mild. For example, we have confirmed the observation of Fink and Adams [Arch. Biochem. Biophys. 126, 27 (1968)] that urine contains N1-methyladenosine and not N6-methyladenosine, the alkaline rearranged product.


Instrumental sample-preparation technique for an automated immunohematology assay system. In the course of the development of an automated immunohematology assay system, we have produced, in prototype form, an instrumental device capable of accepting an unmeasured quantity of whole, anticoagulated blood and deriving from it multiple fixed-volume aliquots of plasma and cells.


Multicomponent analysis in clinical chemistry by use of rapid scanning fluorescence spectroscopy. To be useful in the clinical laboratory, multicomponent fluorescence analysis requires both the rapid measurement of the fluorescence intensity at a variety of excitation and emission wavelengths and the unambiguous reduction of the data by efficient algorithms. The Video Fluorometer, which exploits the multi-channel capability of a low-light-level television sensor to simultaneously acquire excitation and emission spectra, can meet the first requirement. For example, a complete set of emission and excitation spectra for perylene can be obtained in less than 2 s at concentrations of 10(-10) mol/liter. To meet the second need, we present two types of data-reduction strategies: (a)a least-squares fit to the data, with use of the spectra of previously determined compounds likely to be present; and (b)a determination of the eigenvalues and eigenvectors fo the fluorescence matrix, from which the number of components and the possible spectra of each can be estimated. Computer simulations of the least-squares fitting algorithms show that five strongly overlapping components can be determined in the presence of noise with an accuracy of better than 5%. Also, a fluorescent sample containing two species with very similar but unknown spectral properties can be resolved to obtain the spectrum of each.


Centrifugal system for affinity chromatography with eluate monitoring. A prototype centrifugal system has been developed that permits parallel photometric monitoring of eluate streams from a radial array of chromatographic columns. The modular rotor design consists of a discoidal center insert for eluent and sample apportionment, the chromatographic columns, and flow-through cuvetts-all mounted on an aluminum base plate. A common sample is introduced simultaneously to each column; a single eluent stream is used for all columns. The goal is to assay simultaneously for eight to 16 serum proteins separated by affinity chromatography from a single sample. The system is here exemplified by the use of immunosorbents (consisting of antibodies to human immunoglobulins covalently bound to alumina particles) to allow simultaneous determinations of IgG AND IgM from a single human serum sample in less than 16 min.


Automated approach to radioimmunoassays of somatotropin (human growth hormone) and insulin. The adaptation of human somatotropin and insulin assays to the automated Centria radioimmunoassay system [Clin. Chem. 21, 1305 (1975)] is reported. For the somatotropin assay, reaction conditions include a borate/bovine serum albumin buffer (pH 8.4) AND 20-H Incubation at 4 degrees C. Assay results for clinical samples compared favorably (correlation coefficient = 0.930) with values obtained from a reference laboratory. The means determined for 92 patients' samples were 4.3 mug/liter (reference laboratory) and 5.1 mug/liter (Centria). Intra- and inter-run precision ranged from 3.2 to 15.9%. For the insulin assay, a phosphate/bovine serum albumin buffer (pH 7.4) is used, with a 20-h incubation at 4 degrees C. Previously analyzed insulin samples from a reference laboratory were determined by the Centria analyzer with excellent correlation (r = 0.965). Means for patients' samples were 43.0 milli (USP) units of insulin per liter (reference laboratory) and 47.5 milliunits of insulin per liter (Centria). In both assays an anionicexchange gel is used in the separation step. The criterion of parallelism, an indication of the validity of a radioimmunoassay, was satisfied in both assays. The Centria radioimmunoassay system offers the advantage of automating all the critical steps of these radioimmunoassays.


Profiles of ultraviolet-absorbing components of urine from autistic children, as obtained by high-resolution ion-exchange chromatography. Urines from 19 autistic children (3 female, 16 male) were analyzed by high-resolution, high-pressure anion-exchange chromatography. The results showed abnormalities in the excretion of hippuric acid, 4-hydroxyhippuric acid, and N1-methyl-2-pyridone-5-carboxamide, the end product of the nicotinic acid pathway. Considerations as the metabolic origin on the 4-hydroxyhippuric acid are discussed and related to the possibility of bacterial action and malabsorption.


A simple and rapid determination of the lecithin/sphingomyelin (LS) ratio in amniotic fluids and in pharyngeal aspirates of newborn infants. A simple, rapid method for the routine determination of the lecithin/sphingomyelin (L/S) ratio in amniotic fluid and/or in pharyngeal aspirate of the newborn has been developed. In 33 samples of amniotic fluid at various gestational ages, an L/S ratio corresponding to the fetal age was found: in 16 samples of biochemically immature amniotic fluid (gestation age less than 34 weeks) the mean of the ratio was 1.26 and in 17 samples of mature amniotic fluid (gestation age greater than 35 weeks) the mean value was 3.02. Pharyngeal aspirates of 55 non-selected newborn were examined by the same method. The L/S ratio of 39 infants (gestation age greater than 38 weeks) gave a mean value of 7.3 and that of 16 premature infants (gestation age less than 37 weeks) a mean value of 5.5. In none of these cases did RDS develop after birth. The results suggest that the method is useful for the determination in both amniotic fluid and pharyngeal aspirate.


A study of the Bratton and Marshall hydrolysis procedure utilizing high performance liquid chromatography. Most analytical methods for conjugated aromatic amines require hydrolysis to free the amine group. The rate of conversion of N-acetyl-p-aminohippuric acid to its deacetylated and deglycinated derivatives is directly related to the hydrolysis reaction time and the concentration of hydrochloric acid used in the reaction. Variations in methodology involving either of these hydrolysis parameters produced artifacts which were not detectable by the standard colorimetric procedures. High performance liquid chromatography offers an improved tool for detecting and quantitating the changes produced during hydrolysis.


Creatine kinase activity in serum and uric acid solution. Skeletal muscle creatine kinases from man and rabbit, activated to measured specific activities by thiol agents, were inactivated in human serum and in uric acid solution, and the extent of their subsequent reactivation by dithiothreitol was determined. Both enzymes were completely reactivated from uric acid solution but were only 60-70% reactivated from serum. No inactivation occurred within 14 days if serum samples contained dithiothreitol (0.01 M) and were stored at -17 degrees C.


Studies of intracellular distribution of acid phosphatase 5 in the spleen cells of leukemic reticuloendotheliosis by isopycnic gradient centrifugation. A fresh spleen sample obtained from a patient with leukemic reticuloendotheliosis was homogenized and subjected to centrifugation on a sucrose density gradient. A major portion of acid phosphatase band 5 was observed in the lysosome, confirming that the elevated phosphatase activity in the neoplastic spleen is a lysosomal enzyme. However, a significant amount of brand 5 was also observed in the microsome. The microsomal and lysosomal enzymes have different affinity to CM-cellulose. The relationship between lysosomal and microsomal enzymes has not been established.


A method for performing a calcium balance study in man and the interpretation of results. Methods for the measurement of calcium in the diet, urine and faeces for the performance of a calcium balance study was described, along with experiments on analytical procedures including recovery values. A means of calculating the inherent "technical error" in such a balance is given, and a method for determining the significance of any change in a patient's balance is described. These are illustrated by worked examples of data from a patient suffering from Paget's disease and one with osteoporosis before and after treatment with calcitonin.


The mass spectrometric identification of dipeptides in the urine of a patient suffering from chronic skin ulceration and oedema. The identification of a complex ninhydrin positive mixture present in the urine of a child suffering from chronic skin ulceration and oedema by direct chemical ionisation mass spectrometric analysis is described. The compounds were shown to be dipeptides, of which glycylproline was the major constituent. At least 15 dipeptides were identified in the urine, most of which contained proline or hydroxyproline in the carboxy terminal position. The results suggested that the patient suffered from a defect in collagen metabolism. This hypothesis was subsequently confirmed by a grossly diminished level of prolidase in cultured fibroblasts and erythrocytes.


Relationship of plasma uric acid to plasma lipids and lipoproteins in subjects with peripheral vascular disease. Hyperuricaemia and hyperlipidaemia (elevated fasting plasma cholesterol or triglycerides) were frequently found in 219 males and 63 females with peripheral vascular disease (PVD). The subjects were divided into sexes and the uric acid, cholesterol and triglyceride concentration adjusted for the effects of age and obesity by multiple regression analysis. Followig this no significant relationship was found between uric acid and cholesterol or triglyceride. When the males with PVD were divided into lipoprotein types it was found that those who were normo-lipoproteinaemics or who had type IV hyperlipoproteinaemia (HLP) had a significantly higher mean uric acid level. The other types had a mean uric acid concentration similar to that found in 25 healthy normolipoproteinaemic males. The discrepancy between this result and the lack of correlation between uric acid and triglyceride noted above is presumably due to the complex effects of age and obesity. In the females no significant increase in the mean uric acid concentration was found in any of the lipoprotein groups.


The assessment of 125I treatment of thyrotoxicosis. 257 patients have been reviewed 1-5 years (mean 3 years 2 months) after receiving one of five dose regimes of 125I for thyrotoxicosis. The cumulative incidence of hypothyroidism was 34% and of persistent thyrotoxicosis 17%. The group receiving doses between 351 and 500 muCi/g had the highest proportion of euthyroid patients (65%) with the lowest requirement for repeat therapy (46%). In the euthyroid patients, increasing dose of 125I was associated with progressive decline in mean thyroxine (T4) level and free thyroxine index (FTI) within the respective normal ranges, and increase in mean thyroid stimulating hormone (TSH) level to above the normal range. Euthyroid patients with elevated TSH levels had significantly lower T4 and FTI values compared with those with normal TSH, and showed a 3-4-fold increased rate of development of hypothyroidism over 1 year. Euthyroid patients with elevated T3 levels remained euthyroid during the subsequent year and mean T3 levels declined significantly, suggesting that abnormally elevated T3 levels after 125I do not generally indicate impending relapse of thyrotoxicosis. It is concluded that the potential admantages of 125I therapy for thyrotoxicosis in reducing the incidence of hypothyroidism have not been realized in practice.


Response of plasma testosterone, urinary 17-oxosteroids, oestrogens, and androsterone plus aetiocholanolone to human chorionic gonadotrophin in dexamethasone-suppressed men. The administration of human chorionic gonadotrophin (HCG) to dexamethasone-suppressed men caused parallel changes in the concentration of plasma testosterone and in the urinary output of androsterone+aetiocholanolone, total 17-oxosteroids and oestrogens. Discrepant results occurred in only four of the thirty-seven men tested. With these exceptions, the response to HCG could by followed as well by measuring androsterone+aetiocholanolone, 17-oxosteroid or oestrogen excretion rates as by following plasma testosterone levels. The most sensitive index of response was the rate of appearance of oestrogens in urine, and the next that of androsterone+aetiocholanolone.


Galactrorrhoea, hyperprolactinaemia and pituitary tumours in the female. A series of sixty-two women with pituitary tumours, but having neither acromegaly nor Cushing's disease, has been analysed with respect to mode of presentation. Thirty-six (58%) presented with amenorrhoea and galactorrhoea and a further fifteen (24%) presented with amenorrhoea only. Pretreatment prolactin levels were available in thirty-four of these patients and in twenty-seven (79%) hyperprolactinaemia was found, this occurring in the absence of a history of galactorrhoea in four out of ten cases. In addition, among a series of twenty-five unselected women with unexplained galactorrhoea, we have found twelve pituitary tumours, amenorrhoea being a feature of all twelve cases. We conclude that the incidence of hyperprolactinaemia in women with apparently functionless pituitary tumours is much higher than previously estimated and suggest that serum prolactin should be assayed in all patients with such tumours even in the absence of a history of galactorrhoea. All patients with unexplained galactorrhoea should have careful radiological investigation for a possible pituitary tumour.


Effect of acute and chronic neuroleptic therapy on serum prolactin levels in men and women of different age groups. A single fasting level of serum prolactin was measured in each of sixty control subjects and eighty-three psychiatric patients of both sexes who had been on neuroleptic therapy for 2-4 weeks (acute treatment) or at least 5 years (chronic treatment) and who were aged either 17-45 or 48-85 years. All groups of patients had significantly higher mean prolactin levels than controls. Gender, age group of women, and exposure to acute or chronic treatment were significant variables determining the magnitude of neuroleptic-induced elevation of prolactin. In some of the groups, dose, duration of chronic therapy, and concomitant administration of anticholinergic drugs also influenced prolactin levels. Whereas all acutely treated women had prolactin values above the control range, one out of twelve (8-3%) of the women aged 17-45 years and six out of fourteen (42-9%) of the women aged 48-85 years who were under chronic treatment had normal values. Normal prolactin levels were also found in five out of sixteen (31-2%) of the acutely treated and nine out of twenty-four (37-5%) of the chronically treated men aged 17-85 years.


Clinical use of glycine intravenous load for diagnosis of growth hormone deficiency. Intravenous glycine injection (250 mg/kg of body weight) resulted in growth hormone release in normal children but not in those with growth hormone deficiency diagnosed by insulin-induced hypoglycaemia. In the latter significantly higher peak concentrations of serum alpha-amino nitrogen were also found. False negative responses to glycine (no GH release) were observed in two patients of short stature but normal pituitary function. In them the peak levels of serum alpha-amino nitrogen were lower than in those with hypopituitarism. We propose the clinical use of glycine as an inexpensive and innocuous procedure for the detection of GH deficiency in children. A post-glycine GH peak greater than 10-0 mu/l seems to be a good index of an intact GH reserve.


The effect of varying doses of cerebral irradiation on growth hormone production in childhood. The radiation dose, delivered to the hypothalamic-pituitary region, has been calculated in thirty-nine children irradiated for brain tumours and in seventeen children who had received prophylactic cranial irradiation for acute leukaemia. All subjects had an insulin tolerance test at least 2 years after their radiotherapy. There is a significant inverse correlation between radiation dose and peak GH response. Thirty-seven of the fifty-six patients showed an impaired GH response and thirty-six of these received more than 2900 rads. Only five patients who received such a dose showed normal GH responses and four of these were aged over 13 years when treated. In conclusion, cerebral irradiation is a potent cause of GH deficiency in children.


Recurrent hormone dependent chorea: effects of oestrogens and progestogens. A case of hemi-chorea is described in a young woman, initially following administration of an oral contraceptive pill and recurring in a subsequent early pregnancy. The chorea ceased with the removal of the aetiological factor on each occasion. Subsequent challenge with a combined oestrogen/progestogen pill resulted in recurrence of the chorea, but the patient has been successfully maintained on a progestogen without any further symptoms. Thus it would appear that oestrogen component was the precipitating factor for the chorea.


Quantitation of the accelerating effect of metyrapone on cortisol metabolism. Quantitation of the accelerating effect of metyrapone on cortisol metabolism has been made by determination of the metabolic clearance rate (MCR) of exogenous cortisol during a metyrapone load. Six adrenalectomized patients were studied. The slope of cortisol concentrations in plasma was determined after intravenous administration of 0-3 mg cortisol/kg b.w. with or without metyrapone 17-5 mg/kg b.w./h. In all six patients studied, the MCR of cortisol increased during metyrapone load from an average of 12-3 +/- 5-0 (SD) l/h to 29-6 +/- 15-7 (SD) l/h or corrected for body weight 3-0 +/- 1-1 (SD) ml/kg b.w./min to 7-1 +/- 2-5 (SD) ml/kg b.w./min. The discrepancy discovered in clinical practice between the relatively small rise in plasma total corticosteroid concentration and the greater increase in urinary excretion of corticosteroid metabolites during the metyrapone test may be explained by the findings in this study.


The Wolf-Hirschhorn (4p-) syndrome.

The Wolf-Hirschhorn (4p-) syndrome. In a review of 43 cases, the phenotypic spectrum of the Wolf-Hirschhorn syndrome is analyzed and the frequency of clinical anomalies is tabulated. The characteristic features are intrauterine growth retardation, severe psychomotor retardation, typical facies, and various major and minor congenital anomalies suggestive of a midline fusion defect. Diagnosis is established by karyotyping- deletion of the short arm of chromosome No. 4. All cases so far reported are de novo occurrences with no sibling involvement and normal parents. Prognosis is poor, with death in the first 2 years of life in 34% of cases, usually due to cardiac decompensation or infection. Psyhomotor retardation is profound, so that heroic medical efforts probably need to be reconsidered.


Human chromosome polymorphism and congenital malformations. Several authors have suggested that heterochromatin polymorphism influences the origin and/or development of different malformations. In this investigation special consideration was given to the A1qh + variant. Several families with this variant are reported in which the incidence of otherwise rare malformations is surprisingly high. The possibility that the A1qh+ varient or, more probably, interactions between all types of heterochromatin polymorphism are of pathogenetic significance is considered.


Altered isoenzyme patterns of liver alpha-L-fucosidase in cystic fibrosis. The isoenzyme pattern of alpha-L-fucosidase was studied by isoelectric focusing in livers from seven patients with cystic fibrosis and in normal and pathological (GM1-gangliosidosis, Type II and Sanfilippo disease) controls. The controls had very reproducible patterns consisting of seven isoenzymes of alpha-L-fucosidase with the most neutral from (I) representing a small proportion of the total activity. All seven of the cystic fibrosis livers had altered alpha-L-fucosidase isoenzyme patterns. The chemical relationship of the seven isoenzymes of normal liver alpha-L-fucosidase was investigated using neuramindase. The five most acidic forms of alpha-L-fucosidase appear to be related to the most neutral form by sialic acid residues. Since the isoenzymes of liver alpha-L-fucosidase appear to be related by sialic acid residues, it is possible that the altered alpha-L-fucosidase isoenzyme patterns found in cystic fibrosis livers may result from aberrant sialylation.


A man with presumptive Y/Y translocation, observed in a forensic psychiatric department. A man charged with a sexual offence was found to have complex karyotype anomaly, including a Y-chromosome of aberrant structure. Other features were: very small stature, skeletal deformities and obscure neurological defects. There were no gross psychiatric symptoms, nor was there mental retardation. Q- and G-banding and photometric scanning of the chromosomes of the propositus and his three healthy brothers indicated that the aberrant Y-chromosome probably arose from a reciprocal translocation, which may be written: t(Y: Y) (qter leads to p11: : q11 leads to qter). Fibroblast cultures differed from the lymphocytes: in the former the majority of the cells lacked the abnormal Y-chromosome, their karyotype being 45,X0.


Familial short atrioventricular conduction time (P-R interval). Five sibs with related parents are presented. All the sibs have short atrioventricular conduction time (P-R interval). The short P-R interval is probably an inherited feature in the family.


Genetic lipoprotein variation and lipid levels in man. Analyses of 12 population samples have revealed that the total serum cholesterol level is significantly higher in Lp(a +) than in Lp(a -) individuals. The difference is more pronounced in middle-aged and other people than in young persons, and the difference between Lp(a +) and Lp(a -) individuals is larger when LDL cholesterol rather than total serum cholesterol is considered. Lp(a -) individuals have a significantly higher triglyceride level than Lp(a +) individuals. The Lp(a) variation is genetically controlled. Thus, the present study has revealed a small but significant effect of a single, autosomal locus on lipids implicated in atherosclerosis. An association between the phenotype Lp(a +) and coronary heart disease has been demonstrated previously.


Detection of circulating antigen-antibody complexes by their inhibitory effect on the agglutination of IgG-coated particles by rheumatioid factor of Clq. The agglutination of Ig-coated particles by human RF or Clq can be inhibited by Ig aggregates or AgAb complexes. The effect of Ig class was studied by means of agarose-linked human monoclonal Igs. RF was inhibited by all subclasses of IgG and IgA but not by IgM, whereas Clq reacted with IgM, IgG3 and IgG1. Heat-aggregated IgG3 was fractionated by gel-filtration on Ultrogel. Inhibition was restricted to certain fractions of aggregates, viz (IgG3) approximately 7 and (IgG3) approximately 21 for RF, and (IgG3) approximately 10, (IgG3) approximately 14 and (IgG3) approximately 27 for Clq. In a precipitin curve experiment, it was found that RF was inhibited by soluble complexes over an extended range of AgAb ratios, the inactivation of Clq being limited to complexes with 2-5 times antigen excess. Inhibiting factors were found in patients with various diseases and, at low titres, in 22% of healthy people. In 27% of patients' sera, the inhibitors were demonstrable by Clq only after removal of endogenous RF by adsorption on insolubilized IgG. In several patients endogenous agglutinating activity and direct inhibitory activity tended to alternate during the course of the disease. Sera from various patients were also filtrated on Ultrogel and the elution was monitored by immunoassay of IgA, IgM and IgG, as well as by the two inhibition tests. The inhibiting factors were distributed over several peaks which only partially coincided with the elution profiles of IgG and IgM.


Immunoglobulins and transient paraproteins in sera of patients with the Wiskott-Aldrich syndrome: a follow-up study. Immunoglobulin levels of individual classes and IgG subclasses and the occurrence of homogeneous immunoglobulins--paraproteins--were studied longitudinally in the sera of three patients with the Wiskott-Aldrich syndrome; Common findings in all three patients were great variations in the immunoglobulin levels, restricted heterogeneity of the immunoglobulins, the frequent appearance of transient homogeneous immunoglobulins and the presence of serum antibodies against bovine milk proteins. A partial and selective deficiency involving mainly the T immune system is postulated as an explanation for these findings.


Chemotaxis of mitogen-activated human lymphocytes and the effects of membrane-active enzymes. Following incubation with polyclonal activators such as PHA or concanavalin A, human peripheral blood lymphocytes show directional migration towards chemotactic factors such as endotoxin-activated plasma, casein or denatured proteins. This migration is inhibited by treatment of the cells with phospholipase C and sphingomyelinase C, but little affected by proteases or glycosidases. In addition, these lymphocytes migrate towards PHA and other lymphocyte activators when these reagents are used as chemoattractants at concentrations well below their mitogenic doses. They also migrate towards staphylococcal protein A. Migration towards PHA and protein A is reduced by pretreating the lymphocytes with proteases but not with phospholipase C. These results suggest two independent membrane interactions which initiate lymphocyte chemotaxis, one acting directly via the lipid bilayer, the other involving the binding of ligands to membrane proteins.


Molecular size of IgG in patients with focal glomerulosclerosis. Nineteen patients with focal glomerulosclerosis and with anomalously high clearance of IgG into the urine have been investigated by G.200 column chromatography. The findings are compared with normal sera, with 14 patients with minimal change disease and with 12 patients with proliferative glomerulonephritis. No significant reduction in the molecular size of IgG was found as compared with normal sera and the other diseases in 18 of the patients. In patients with minimal change disease and focal glomerulosclerosis an increased proportion of the IgG eluted in the void volume