Peptide inhibitors of ADP-ribosylation by pertussis toxin are substrates with affinities comparable to those of the trimeric GTP-binding proteins.

Pertussis toxin (PTX) ADP-ribosylates alpha subunits of GTP-binding proteins (G proteins) when they are in association with beta gamma dimers, and free alpha subunits are thought not to be substrates under standard assay conditions. We now report the rather unexpected discovery that synthetic peptides encompassing the last 10-20 amino acids of alpha subunits of PTX-sensitive G proteins are substrates for PTX by themselves and in the absence of beta gamma dimers. As determined for G13, the Km of PTX for the 20-amino acid carboxyl-terminal peptide is 10-fold higher than that for the trimeric G protein. Interestingly, PTX ADP-ribosylates the free full length alpha 13 subunit with a Km not different from that of the trimer but with a Vmax that is only 1% of that with which it ADP-ribosylates the trimer. It follows that the primary role of beta gamma dimers in ADP-ribosylation of G proteins is one of increasing the Vmax of the reaction without affecting the Km of the substrate for the toxin. Mutant peptides lacking the ADP-ribose acceptor site act as competitive inhibitors.


Indirect effect of guanine nucleotides on antagonist binding to A1 adenosine receptors: occupation of cryptic binding sites by endogenous vesicular adenosine.

Guanine nucleotides such as guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) have been found to increase the binding of antagonists to adenosine A1 receptors. This response can be attributed either to a direct effect of GTP on receptors to increase antagonist affinity or to an indirect effect to decrease the affinity of receptors for a pool of endogenous adenosine that cannot be readily removed from membranes. In this study, adenosine content was measured in preparations of membranes and 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS)-solubilized receptors by a sensitive radioimmunoassay. In both preparations, pools of adenosine (2.5-10 pmol/mg of protein) were detected that were resistant to deamination by added adenosine deaminase (0.5-3 units/ml) unless membrane lipids were first dissolved in acetone. Electron microscopic examination of crude CHAPS-solubilized receptors revealed the existence of small vesicles (< 1 microns in diameter). Furthermore, most "solubilized" receptors were retained by a 0.1-microns filter. The effects of GTP gamma S were evaluated on the binding of an antagonist, 3-(4-amino-3-125I-phenethyl)-1-propyl-8-cyclopentylxanthine (125I-BW-A844U), to A1 receptors of bovine brain membranes, receptors solubilized in CHAPS (crude solubilized), or receptors partially co-purified with G proteins by agonist affinity chromatography (partially purified). GTP gamma S (10 microM) increased antagonist binding to membranes (20-50%) and crude CHAPS-solubilized receptors (> 200%) but increased binding to partially purified receptors by only 10-15%. GTP gamma S decreased agonist (125I-N6-aminobenzyladenosine) binding and increased antagonist Bmax, but did not significantly decrease (5%) the dissociation rate of the antagonist. Omission of Mg2+ mimicked the effects of GTP gamma S on agonist and antagonist binding and increased both the association and dissociation rates of 125I-BW-A844U. These data suggest that a Mg(2+)-dependent GTP gamma S-induced increase in antagonist binding to membranes and solubilized receptors is primarily due to unmasking of cryptic binding sites occupied by contaminating vesicular adenosine. These findings are consistent with the observation that adenosine receptor antagonists have been found to have little or no inverse agonist physiological effects in well oxygenated tissues.


Isolation and characterization of imidazoline receptor protein from bovine adrenal chromaffin cells.

We sought to isolate and partially purify proteins corresponding to the binding element of the imidazoline receptor (IR) from adrenal chromaffin cell membranes. These cells express IRs of the I-2 subclass and not alpha 2-adrenergic receptors. Proteins were solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate-containing buffer and were assayed by binding of [3H]idazoxan, an imidazoline radioligand. Two ligand affinity resins, p-aminoclonidine-Trisacryl GF-2000 (PAC-ReactiGel) and idazoxan-PharmaLink agarose (IDA-agarose), were synthesized. These allowed purification by single-step affinity chromatography of a major receptor binding protein component of 70 kDa, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and [3H]idazoxan binding assay. The purified imidazoline-binding proteins from IDA-agarose and PAC-ReactiGel had similar affinities for the radioligand [3H]idazoxan (Kd = 3.7 and 4.9 nM, respectively) and a displacement profile, showing sensitivity to imidazoline agents (cirazoline > clonidine) and insensitivity to catecholamines and adrenergic agents (epinephrine approximately rauwolscine), that was similar to that of the intact membrane receptor. The imidazoline-binding protein did not bind to concanavalin A, suggesting that it may not be glycosylated or that the sugar moieties present are not recognized by this lectin. The results indicate that IR and alpha 2 receptor proteins may be biochemically distinct and that IDA-agarose and PAC-ReactiGel columns are useful for purification of sufficient quantities of imidazoline-binding proteins to allow for structural and functional studies of the IR.


Prolonged D2 antidopaminergic activity of alkylating and nonalkylating derivatives of spiperone in rat brain.

Alkyl and arylalkyl derivatives of the dopamine (DA) D2 antagonist spiperone were prepared and characterized chemically and pharmacologically. They included the N-methyl, N-phenethyl (NPS), and N-p-aminophenethyl (NAPS) derivatives, as well as the alkylating isothiocyanato (NIPS), bromacetamido, and ethylfumaramido p-substituted N-phenethylspiperones. These compounds showed high lipophilicity (log P up to 6.0 with NIPS), as well as very high in vitro D2 affinity (Ki = 35-280 pM) and D2 versus D1 selectivity (540-9000-fold) in radioreceptor assays with corpus striatum of rat brain. Of the alkylating series, NIPS showed the highest D2 affinity (57 pM) and D2 versus D1 selectivity (2040-fold) and so was selected for further evaluation. NPS, NAPS, and NIPS showed little or no affinity for 34 non-DA binding sites defined by radioligand assays for monoamine, amino acid, and peptide neurotransmitters, ion channels, peptide growth factors, and transmission mediators but did show low alpha 2 and moderate alpha 1 and 5-hydroxytryptamine (5-HT2) affinity with rat forebrain tissue in vitro; NIPS showed a marked gain in D2 versus 5-HT2 selectivity, compared with spiperone (1520- versus 26-fold). Systemic injections of NIPS induced marked decreases in rat striatal D2 binding sites 24 hr later, with little effect on D1, 5-HT2, or alpha 1 sites; NIPS and NAPS lowered apparent Bmax values at D2 receptors with little change in ligand affinity, ex vivo as well as in vitro. NPS, NAPS, and NIPS all induced dose-dependent lowering of D2 binding ex vivo (ID50 = 1-9 mumol/kg, intraperitoneally) and blocked the behavioral effects of the DA agonist apomorphine (0.9 mumol/kg) potently (ID50 = 0.3-0.5 mumol/kg) at 24 hr. Recovery from these anti-DA actions required about 1 week after equimolar (15 mumol/kg) and similarly effective doses of NPS and NAPS, as well as NIPS. Thus, highly selective and avidly bound lipophilic D2 affinity ligands with similarly avid in vitro and prolonged in vivo anti-DA activities can be derived from N-phenethylspiperones with or without an alkylating moiety present. Such affinity ligands may represent useful additions to previously used, generally less selective, D2 affinity ligands.


Depletion of 5,10-methylenetetrahydrofolate and 10-formyltetrahydrofolate by methotrexate in cultured hepatoma cells.

The effect of the inhibition of dihydrofolate reductase by methotrexate on the cellular folates involved in de novo purine and thymidylate biosynthesis has been measured in H35 hepatoma cells grown in 4 microM folic acid or 20 nM folinic acid. The major cellular folate species in cells from medium with folate or folinate is 10-formyltetrahydrofolate (approximately 5 microM), with lesser amounts of 5,10-methylenetetrahydrofolate and tetrahydrofolate. Cultures were exposed to a pulse dose of methotrexate, resulting in the accumulation of nearly exclusively methotrexate polyglutamates (predominantly Glu3, Glu4, and Glu5), or a continuous exposure to the poorly glutamylated analog threo-4-fluoromethotrexate, resulting in 93% intracellular monoglutamate. At 4 hr and 18 hr after exposure to either compound there was extensive depletion of the reduced folate coenzymes, which generally corresponded to the extent of inhibition of glycine and deoxyuridine incorporation. This was accompanied by an increase of the cellular dihydrofolate and 10-formyldihydrofolate. In the H35 cells the effect of methotrexate polyglutamates on the reduced folate coenzyme pools was restricted to dividing cultures, because the reduced folate coenzymes were not depleted in confluent cultures. The results demonstrate that the methotrexate and methotrexate polyglutamates that initially accumulate within dividing H35 cells readily inhibit dihydrofolate reductase but are not adequate to inhibit thymidylate synthase and prevent the depletion of reduced folate coenzymes. Thus, inhibition of de novo glycine and deoxyuridine incorporation into DNA as a result of dihydrofolate reductase inhibitors appears to be closely related to a reduction in the intracellular concentration of 10-formyltetrahydrofolate and 5,10-methylenetetrahydrofolate, the respective folate coenzymes for de novo purine and thymidylate synthesis.


Mechanism of inhibition of aldose reductase by menadione (vitamin K3).

Incubation of human placental aldose reductase (EC 1.1.1.21) with menadione (0.5-3.0 mM) resulted in time-dependent loss of the catalytic activity of the enzyme. Kinetic analysis of the data suggests that the inactivation process follows a single apparent rate constant that displays hyperbolic dependence on menadione concentration, indicating that menadione forms a kinetically significant, dissociable complex with the enzyme before the formation of an inactive enzyme-menadione complex. The inactivation of the enzyme with menadione was reversed upon dialysis of the inactivated enzyme against buffer containing 10 mM dithiothreitol suggesting that menadione reacts with enzyme sulfhydryl residue(s). Inactivation of the enzyme was significantly prevented by dithiothreitol (5 mM), NADPH (0.1 mM), and DL-glyceraldehyde (10 mM). Correlation of the fractional remaining activity with the extent of modification indicates that loss of catalytic activity corresponds to the modification of a single amino acid residue of the enzyme protein. Recombinant human aldose reductase, obtained by overexpression in Escherichia coli, and aldose reductase in which Cys-80 or Cys-303 was replaced by serine were also inactivated by menadione. However, enzyme in which Cys-298 was replaced by serine was insensitive to menadione. On the basis of these observations, it is suggested that menadione forms a thiodione-like adduct with Cys-298, leading to inactivation of the enzyme.


Nuclear magnetic resonance spectroscopy and pattern recognition analysis of the biochemical processes associated with the progression of and recovery from nephrotoxic lesions in the rat induced by mercury(II) chloride and 2-bromoethanamine.

Nephrotoxic lesions were induced in Fischer 344 rats using HgCl2, a proximal tubular toxin, and 2-bromoethanamine (BEA), a medullary toxin. Biochemical effects of these toxins on urinary composition were observed by high resolution 1H NMR spectroscopy over 9 days after dosing. The onset of, progression of, and recovery from the induced toxic lesions were also followed histopathologically and related to the perturbed urinary biochemistry. Urinary concentrations of 20 endogenous substances were measured simultaneously by NMR at eight time points, to provide a time-related 20-dimensional description of the urinary biochemistry for each rat. Principal components analysis and nonlinear mapping were used to reduce the biochemical parameter spaces for each rat to two or three dimensions for display and classification purposes. An investigation of alternative data-presentation methods was made, and taking interanimal means of the map coordinates at each time point yielded a novel type of metabolic trajectory diagram with which the biochemical abnormalities associated with the HgCl2 and BEA lesions could be related to the progression and recovery phases of the toxic lesions. The time-course trajectories showed characteristically different paths for each toxin. These trajectories allowed the time points at which there were maximum metabolic differences to be determined and provided the visualization of net movements of the treatment group populations in time in relation to interanimal variation. Control animal urine samples subjected to this analysis showed simple clustering, with no evidence of metabolic trajectory. The trajectory for BEA showed different routes for onset of and recovery from toxicity, whereas for HgCl2 the outward trajectory (onset) mapped a space similar to the inward trajectory (recovery phase). This suggests that the NMR-detectable biochemical abnormalities after mercury toxicity mainly reflect the proportions of functional cells lining the nephron, whereas the biochemical abnormalities associated with renal medullary insult probably relate to functional integrity. An examination has been made for those metabolites that are most responsible for defining the trajectories, i.e., the discrimination of renal cortical and medullary toxicity from each other and from controls. These discriminatory metabolites (using paired t test, p < 0.001) included valine, taurine, trimethylamine N-oxide, and glucose for HgCl2 and acetate, methylamine, dimethylamine, lactate, and creatine for BEA, whereas citrate, succinate, N-acetyl resonances from as yet unidentified metabolites, hippurate, alanine, and 2-oxoglutarate played an important role in defining the biochemically perturbed trajectory of both toxins.(ABSTRACT TRUNCATED AT 400 WORDS)


Metabolite intermediate complexation of microsomal cytochrome P450 2C11 in male rat liver by nortriptyline.

Antidepressant drugs that contain alkylaminoalkyl substituents have been associated with serious pharmacokinetic interactions in humans that may be related to the inhibition of cytochrome P450 (P450) enzymes. In this study, the propensity of the tricyclic antidepressant nortriptyline (NOR) to inhibit individual microsomal P450 enzymes in rat liver was investigated to provide a mechanistic explanation for these pharmacokinetic interactions. Enzyme kinetic studies revealed that NOR inhibited steroid 2 alpha-, 6 beta, 7 alpha-, and 16 alpha-hydroxylation in untreated rat liver with Km/Ki ratios of 0.53, 0.59, 0.25, and 0.29, respectively. When the drug was preincubated with microsomes and NADPH before testosterone hydroxylation was conducted, marked increases in the Km/Ki ratios were observed (to 8.8, 3.9, 0.62, and 13, respectively). Thus, enzymic oxidation of NOR enhanced its inhibition capacity against P450 activities. Indeed, the altered Km/Ki ratios indicate 17-, 6.6-, 2.5-, and 47-fold increases in inhibition of the four pathways of testosterone hydroxylation after the biotransformation of NOR to its metabolites. From these experiments it was apparent that testosterone 2 alpha- and 16 alpha-hydroxylations, catalyzed predominantly by P450 2C11, were subject to the most pronounced increase in inhibition. Under these conditions, the apparent content of microsomal P450 was decreased, thus suggesting the formation of a NOR metabolite intermediate (MI) complex with the cytochrome. Further, optical difference spectroscopy of NADPH-supported metabolism of NOR in microsomes and in a reconstituted system incorporating purified P450 2C11 indicated the appearance of an absorbance peak near 454 nm, similar to those produced by triacetyloleandomycin, SKF 525-A, and orphenadrine. Formation of this absorbance peak in microsomes was inhibited by an antibody raised against the male-specific P450 2C11. Because oxidative metabolism of NOR to inhibitory products would not necessarily involve MI complexation, additional experiments were undertaken in which NOR-related free metabolites produced in microsomal incubations were removed on Sep-Pak mini-C18 columns before estimation of testosterone hydroxylation. The principal finding from this experiment was that P450 3A2-dependent steroid 6 beta-hydroxylase activity was inhibited to a much lesser extent after removal of unbound NOR metabolites on Sep-Pak columns (25% inhibition after Sep-Pak extraction, compared with 82% inhibition observed when all NOR metabolites were present during subsequent testosterone hydroxylation); inhibition of P450 2C11-mediated 2 alpha- and 16 alpha-hydroxylation was not noticeably different after Sep-Pak treatment.(ABSTRACT TRUNCATED AT 400 WORDS)


Control analysis of rat liver glycolysis under different glucose concentrations. The substrate approach and the role of glucokinase.

Control Analysis has been carried out in the first steps of a rat liver glycolytic system. Attention has been focused on the effect of several glucose concentrations on the control, particularly regarding the role of glucokinase. From kinetic studies of the whole metabolic system we have obtained information on the flux variation under different glucose concentrations. This information together with the kinetics of glucokinase has allowed us to calculate Flux Control and Elasticity Coefficients for glucokinase and the Response Coefficient of the system with respect to glucose. The changes in of the value of Flux Control Coefficients demonstrates that in conditions of low glucose concentration, glucokinase is the main enzyme in controlling the flux through the pathway, but at high glucose concentration the control moves to phosphofructokinase. Next, we have compared our results with those obtained with the shortening and titration method, previously described (Torres, N.V., Mateo, F., Meléndez-Hevia, E. and Kacser, H., (1986) Biochem. J. 234, 169-174; Torres, N.V. and Meléndez-Hevia, E. 1991. Molec. Cell. Biochem. 101, 1-10). Furthermore, from knowledge of the enzyme kinetics of the system we have been able to build a model of the pathway that allows us computer similation of its behavior and calculation of the Flux Control Coefficient profile at different glucose concentrations. By the three methods the results correlate, supporting the use of the pathway substrate as external modulator of the metabolic system as a tool for practical application of Control Analysis.


The association of the human epsilon-globin gene with the nuclear matrix: a reconsideration.

The association of the human epsilon-globin gene with the nuclear matrix was studied in erythroid and non-erythroid cell lines. Using a high salt method to prepare histone depleted nuclei we studied the association of variety of fragments covering a 7.8 kb region which contains the human epsilon-globin gene. We furthermore studied the association of a set of DNA fragments covering the 13 kb human G gamma/A gamma-globin gene domain, the 16 kb psi beta/delta-globin gene domain and the 10 kb beta-globin gene domain with the nuclear matrix of K562 and Raji cells. The results show that all fragments studied are easily released from the nuclear matrix, indicating no specific association. Summarizing our results we could say that a region starting 5.7 kb 5' to the human epsilon-globin gene and ending 4 kb 3' to the human beta-globin gene seems to contain no attachment sites with the nuclear matrix of both erythroid and non-erythroid cells.


Release of anchored membrane enzymes by lipoamidase.

Lipoamidase is found to be able to release various membrane-anchored enzymes from the membrane compartment of pig brain. Released enzymes revealed their intact enzyme activities in the soluble fraction. Lipoamidase could release at least two types of anchored enzymes, i.e. glycosyl-phosphatidylinositol-bonded and myristoylated enzymes, but not integral membrane bound enzymes. The reaction was competitively inhibited by lipoyllysine. This releasing mechanism found in the membranes may play important roles in the secretory mechanism of extracellular enzymes and also in the cellular signal-transduction system through topological changes in cellular enzymes.


Effect of spermine on peptide-bond formation, catalyzed by ribosomal peptidyltransferase.

The effect of spermine on the binding of AcPhe-tRNA to poly(U)-programmed ribosomes (step 1) and on the puromycin reaction (step 2) has been studied in a cell-free system, derived from E. coli. In the absence of ribosomal wash (FWR fraction) and at suboptimal concentration of Mg++ (6 mM), spermine stimulated the binding of AcPhe-tRNA at least five fold, while at 10 mM Mg++ there was a three fold stimulation. The above stimulatory effect was decreased at 6 mM Mg++, or was abolished at 10 mM Mg++ by the presence of FWR during the binding. Beside the stimulatory effect, spermine enhanced the stability of initiation complex AcPhe-tRNA-poly(U)-ribosome. In step 2, spermine affected the final degree of puromycin reaction and the activity status of peptidyltransferase. Both stimulatory and inhibitory effects have been observed, depending on the experimental conditions followed during the binding of the donor and during the peptide bond formation.


Transient elevation of aldose reductase mRNA in lens of rats developing galactose cataracts.

Aldose reductase (AR), a major enzyme in the polyol pathway, is thought to be responsible for accumulation of polyols in lenses exposed to high doses of galactose or glucose, and it may be linked to some of the complications found in diabetes. In this report we examined the level of expression of AR mRNA in lens epithelia undergoing development of galactose cataracts in vivo. The AR mRNA was quantitated by Northern blot hybridization with a [35S]-RNA transcript from a previously described AR cDNA clone. This was done on normal lens epithelia and on epithelia from lens of rats fed a diet of Purina Chow containing 50% galactose for periods of from 6 hr to 20 days. We found AR mRNA to elevate to about 5-fold the control levels by 12-24 hr on galactose, then decrease to the control levels by day 4. The increase in AR mRNA appears to be transitory. The high abundance in AR mRNA by 24 hr on galactose was confirmed by in situ hybridization. At later periods, from 8 to 20 days on galactose, a slow increase in AR mRNA took effect, as we have previously reported. Changes in the levels of galactose and dulcitol between 0 and 96 hr were also quantitated by gas chromatography, showing that there was a significant increase in both galactose and dulcitol occurring throughout the experimental period.(ABSTRACT TRUNCATED AT 250 WORDS)


Arachidonic and palmitic acid utilization in aged rat brain areas.

We have previously demonstrated that the arachidonic acid (20:4) incorporation into brain lipids differs according to the age of the animals used and the experimental conditions adopted. These differences led to a further investigation of arachidonic acid uptake in both aged and adult rat brains, its transformation into CoA derivatives, its incorporation into diacyl-glycerols and polar lipids, and finally its oxidation to CO2. These metabolic parameters were then compared with those obtained after using the saturated fatty acid palmitate (16:0). In both cases slices or mitochondria from different brain areas of 24-month-old and 4-month-old rats were examined. The results obtained indicate that the uptake of the fatty acids into cells is not modified by age. However, the successive metabolic transformations of the acids are altered to a considerable extent. In particular, in 24-month-old animals (compared with 4-month-old rats) there is a significant decrease of 20:4 in its incorporation into lipids as well as its oxidation to CO2, while arachidonoyl-CoA content increases by about 50%. This increased amount of CoA derivative, which has a potent detergent effect, may interfere with membrane structure and affect membrane physiological functions. Furthermore, because the free arachidonate pool is maintained in a dynamic equilibrium with its esterified forms, the final result may be a perturbation of this equilibrium.


A kinetic description of sequential, reversible, Michaelis-Menten reactions: practical application of theory to metabolic pathways.

Equations are presented which describe a linear coupled system of reactions that utilize a single substrate and convert it to product by way of several intermediate enzyme catalysed steps. The present analysis extends previous results by assuming that the enzymes obey reversible Michaelis-Menten kinetics. In order for the system to reach steady state one must assume that the initial substrate concentration and the final product concentration are buffered to a constant value. Using the present analysis it can be shown that the system will not enter a steady state if the maximal velocity of any forward reaction is less than the steady state flux through the system. This condition represents a practical test for determining if a system will enter steady state but is valid only when the rate of the primary enzyme is not affected allosterically be intermediates in the pathway. The equations are used to analyse a portion of the rat liver glycogenic pathway that catalyses the conversion of glucose to fructose 1,6-bisphosphate.


Detection of ischemia-reperfusion cardiac injury by cardiac muscle chemiluminescence.

Various methods have been used in the past to assess the implication of oxygen free radicals (OFR) in ischemia-reperfusion-induced cardiac injury. Luminol-enhanced tert-butyl-initiated chemiluminescence in cardiac tissue reflects oxidative stress and is a very sensitive method. It was used to elucidate the role of OFR in cardiac injury due to ischemia and reperfusion. Studies were conducted on perfused isolated rabbit hearts in three groups (n = 8 in each): I, control; II, submitted to global ischemia for 30 min; III, submitted to ischemia for 30 min followed by reperfusion for 60 min. The heart tissue was then assayed for chemiluminescence (CL); content of malondialdehyde (MDA), an indicator of OFR-induced cardiac injury; and activity of tissue levels of antioxidants [superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px)]. The control values for left and right ventricular CL and malondialdehyde were 81.1 +/- 15.4 (S.E.) and 182.4 +/- 50.3 (S.E.), mv.min.mg protein-1; and 0.024 +/- 0.006 (S.E.) and 0.324 +/- 0.005 (S.E.) nmoles.mg protein-1 respectively. Ischemia produced an increase in the cardiac CL (3.3 to 4.4 fold) and MDA content (2 to 2.6 fold). Reperfusion following ischemia also produced similar changes in CL and MDA content. The control values for activity of left ventricular SOD, catalase, and GSH-Px were 45.77 +/- 1.73 (S.E.) U.mg protein-1, 5.35 +/- 0.51 (S.E.) K.10(-3).sec-1.mg protein-1, and 77.50 +/- 7.70 (S.E.) nmoles NADPH.min-1.mg protein-1 respectively. Activities of SOD and catalase decreased during ischemia but were similar to control values in ischemic-reperfused hearts. The GSH-Px activity of left ventricle was unaffected by ischemia, and ischemia-reperfusion. GSH-Px activity of the right ventricle increased with ischemia, and ischemic-reperfusion. These results indicate that cardiac tissue chemiluminescence would be a useful and sensitive tool for the detection of oxygen free radical-induced cardiac injury.


Retardation of cell aging by lipid peroxidation.

Different concentrations of Fe2+/vitamin C mixtures were used as initiators of lipid peroxidation in diploid fibroblasts from cultured human embryonic lung. Malondialdehyde (MDA) formation in the cell cultures was correlated directly with the concentrations of Fe2+ and vitamin C. Lipid peroxidation was associated with an increase in life-span, decrease in the population doubling time and increase in cellular DNA synthesis. The effects of lipid peroxidation varied inversely with the MDA level. These data showed that low levels of lipid peroxidation retarded several biological properties of cultured cells that are associated with cell aging.


Escherichia coli heat-labile enterotoxin preferentially interacts with blood group A-active glycolipids from pig intestinal mucosa and A- and B-active glycolipids from human red cells compared to H-active glycolipids.

The capacity of cholera toxin (CT) and of the heat-labile enterotoxin produced by Escherichia coli isolated from humans (LTh) to interact with glycolipids bearing ABO(H) blood group determinants isolated from different sources and separated by thin layer chromatography was studied. Toxin binding to the ABO(H)-related glycolipids depends on the glycolipid source, the type of the blood group activity, and the toxin. LTh and CT were capable of interacting with several blood group-active glycolipids from pig intestinal mucosa and both toxins preferentially recognize glycolipids isolated from animals carrying A-blood group antigenic determinants compared to those isolated from animals lacking these antigens. In contrast, LTh but not CT was able to interact with ABO(H)-active glycolipids from human erythrocytes. LTh preferentially binds to glycolipids isolated from A, B, and AB compared to O red cells. Results from competition experiments between CT and LTh for binding to the blood group-active glycolipids suggest that the carbohydrate structure requirements for the interaction of each toxin are different. The present findings may help to understand the results of clinical studies indicating an association between ABO(H) blood groups and the severity of diarrheal diseases produced by some toxigenic enterobacteria.


Identification of calcium-dependent phospholipid-binding proteins (annexins) from guinea pig alveolar type II cells.

A new group of calcium-regulating proteins, called annexins or Ca(++)-dependent phospholipid-binding proteins (PLBP), have been detected in different species, organs and cell types. In the present study, we have identified and quantitated PLBP from guinea pig lung, lavage fluid and alveolar type II cells to elucidate the possible role of PLBP in lung surfactant biogenesis and secretion. Lungs were lavaged and type II cells from lavaged lung were isolated by elastase digestion and purified by centrifugal elutriation. For the quantitative identification of PLBP, we performed ELISA assays and Western blot analysis by using an antiserum raised in guinea pigs against a pure rabbit lung 36 kDa PLBP. The lavage fluid, cytosol from lung and type II cells contained 784, 167 and 435 ng per mg protein, respectively, of PLBP. The SDS-PAGE electrophoretic pattern and Western blot confirmed that all lung samples have band corresponding to a 36 kDa protein. This indicates that both alveolar type II cells and lavage fluid have higher levels of PLBP than whole lung cytosol.


Vanadate-induced gene expression in mouse C127 cells: roles of oxygen derived active species.

An underinvestigated aspect of the mitogenic and cell regulatory actions of vanadium is the regulation of gene expression. Among the fifteen cellular genes studied in cultured mouse C127 cells, vanadium (as 10 microM sodium vanadate) increased levels of mRNA of the actin and c-Ha-ras to four times control values. These increases represented de novo synthesis of mRNA, since they were inhibited by actinomycin D. Vanadate did not increase mRNA corresponding to c-src, c-mos, c-myc, p53, HSP70, pODC or RB genes, and expression of c-erb A, c-erb B, c-sis and c-fes genes was undetectable whether vanadium was present or not. Expression of a third gene affected by vanadium, c-jun, was augmented by addition of a reductant or oxidant together with the vanadate. Addition of NADH (marginally effective on its own) or H2O2 (effective alone) dramatically enhanced the effect of vanadate on c-jun gene expression. Catalase inhibited the effect of NADH partly. The vanadate-stimulated expression of actin and c-Ha-ras mRNA were unaffected by oxidants, reductants, metal chelators, or anti-oxidant enzymes. Evidently vanadate acts by two separate mechanisms on these two categories of genes. The alternate hypothesis that the actions of vanadate on actin and c-Ha-ras were mediated by a protein kinase cascade was inconsistent with the following observations. Neither insulin nor epidermal growth factor increased mRNA levels of c-Ha-ras or actin gene. Neither genistein (a tyrosine kinase inhibitor) nor pretreatment with 12-O-tetradecanoylphorbol-13-acetate blocked the actions of vanadate on these genes. Clearly the biological actions of vanadium depend in part on altered expression of genes. Since two of the genes are proto-oncogenes, this mechanism is potentially relevant to the mitogenic responses of cells to vanadium.


[Modified nucleosides and nucleotides inhibiting HIV replication: analysis of the situation and potential prospects].

The results of an eight year search of blockaders of human immunodeficiency virus (HIV) among groups of modified nucleosides and nucleotides are reviewed. The molecular mechanism of action of these compounds is based on the inhibition of DNA polymerases activity. Attempts of systematic analysis of structure--anti-HIV activity relationship for modified substrates of DNA biosynthesis are made. In this analysis attention is focused on the evaluation of general properties of enzymes of the phosphorylation cascade and DNA polymerases. Such properties are analyzed on the basis of empiric rules which summarize electronic, steric and conformational properties of substrate analogs. The ability of phosphorylating nucleosides to block HIV reproduction are analyzed. The interdependence of structure of several most known inhibitors with their action on nucleic acids components metabolism as well as the structure--therapeutic properties relationship are discussed.


[Spatial organization and conformational mobility of DNA complexed with nucleoproteins].

The importance of spatial organization of DNA for the regulation of genome activity is discussed. The main problem reviewed in this paper includes cooperative interactions of proteins with DNA, formation of DNA loops in the regulatory domains of the genome and conformational mobility of DNA in chromatin.


[Coding part of the son gene small transcript contains four areas of complete tandem repeats].

Overlapping cDNA clones for the smaller son gene transcript were isolated by several steps of human placenta cDNA library walking. Here we report the structure of repeats that exist at the long open reading, present in the structure of this smaller (5.6 kb) transcript of human gene son. We were able to identify in this transcript four areas of complete tandem repeats, formed by several steps of duplication. For three of them the extent of homology between amino acid sequences of repeated elements is higher than between their nucleic acid sequences. It means that amino acid sequences of repeated elements are conserved, that could reflect the structural significance for the function of hypothetical protein product of human gene son. On the basis of the results of analysis we proposed the model for the formation of repeats by processes of duplication, mutation and natural selection pressure at the expressed protein level.


[Expression of orthopoxvirus DNA sequences in Escherichia coli cells].

We observed the expression of recombinant plasmids genes containing ectromelia virus DNA fragments in E. coli minicells. Using plasmids with vaccinia or ectromelia viruses DNA fragments inserted upstream of lacZ gene we showed that certain orthopoxvirus genome fragments carry out a promoter-like function in bacterial cells.


[Affinity modification of 80S ribosomes from human placenta with mRNA analogs--derivatives of oligouridylates with alkylating groups at the 3'-end].

2',3'-O-[4-(N-2-chloroethyl-N-methylamino)]benzylidene derivatives of oligoribonucleotides (Up)5U[32P]pC and (Up)11U[32P]pC were used for affinity labeling of 80S ribosomes from human placenta in the presence of cognate Phe-tRNA(Phe). The derivatives retained the ability of tRNA(Phe)-dependent binding with 80S ribosomes and within the corresponding complexes were cross-linked to 40S subunits (preferentially to 18S rRNA). Sites of the mRNA analogs attachment were identified by blot-hybridization of the modified rRNA with restriction fragments of corresponding rDNA. They are located within positions 1610-1747 and 1748-1865 for both analogs. These fragments are situated at the 3'-end region of 18S rRNA in a highly conserved part of eukaryotic small subunit rRNA secondary structure.


[Crystals of free aspartate aminotransferase].

The crystals of free cytosolic chicken aspartate aminotransferase were subjected to X-ray investigation at 2.7 A. One subunit of the dimeric molecule crystalline enzyme is in the open conformation and the other is in the closed conformation.


[Conformational stimulation in DNA type macromolecules].

A concept for the description of structural elements mobility of a DNA-type macromolecule is suggested. The possible existence of conformational excitations weakly sensitive to a macromolecule heterogeneity in DNA is shown. The dependence of the macromolecule low-energy excitation spectrum on the nucleotide content is found and its sensitivity to thermal action is predicted.


[The human son gene: the large and small transcripts contains various 5'-terminal sequences].

Human son gene was previously identified by cloning and hybridization to GC-rich rat genomic fragment. After several cycles of human placenta library walking we were able to identify and clone two alternative transcripts of this gene. Recently we have determined nucleotide sequence of small son gene transcript, coding region of which contains four areas of complete tandem repeats. Here we report the structure of the part (2.9 kb) of large transcript of son gene from close to its 5'-end to the end of sequence, unique for this transcript. Comparison of nucleotide sequences of two transcripts indicates that they have common 3'-ends and, probably, arise from alternative splicing. 5' proximal part of large transcript contains two areas of complete tandem repeats not present in small transcript. Results of analysis of this area support our model for forming son gene tandem repeats structure by duplication, mutation and natural selection at the protein level.


[Design of hybrid metalloproteinases from Bacillus].

Possibility of using recombination mechanisms for the construction of hybrids between relative proteins containing highly homologous regions has been demonstrated. In order to design hybrid neutral proteases (NPr) NPr B. amyloliquefaciens--NPr B. brevis genes encoding these enzymes have been cloned into the same plasmid in tandem orientation with subsequent recombination between them. With the help of sequential inactivation we have managed to ensure efficient selection at intermediate stages as well as at the completion stage of construction when the desirable hybrid forms exhibited proteolytic activity. Constructions containing expressed genes of the hybrid neutral proteases NPr B. amyloliquefaciens--NPr B. brevis were obtained. The presence of several regions of high homology between the genes of the two Bacilli neutral proteinases determines the possibility of obtaining of various variants of hybrid proteins with different properties.


Alanine-scanning mutagenesis of human prolactin: importance of the 58-74 region for bioactivity.

We have generated 10 alanine mutants of human PRL (hPRL), a member of the PRL/GH family, to investigate the involvement of the highly conserved 58-74 region in the biological behavior of the protein. When treated with polyclonal anti-hPRL antibodies, all mutants were immunologically indistinguishable from the unmodified hPRL, and circular dichroism analyses indicated that their alpha-helix content was similar to that of the unmodified hormone. Mutations C58A, K69A, and, to a lesser extent, P66A affected drastically the ability of hPRL first to bind to the lactogenic receptor and second to stimulate the proliferation of Nb2 lymphoma cells, proving the importance of the 58-74 peptide segment for hPRL bioactivity. Binding affinities of these mutants to the Nb2 lactogenic receptor have been compared to lactogenic binding data previously obtained for several mutants of hGH. The comparison reveals that the residues involved in the biological properties of the two proteins are not at topologically equivalent positions. Hence, we suggest that the binding of these hormones to the lactogenic receptors occurs through a different molecular mechanism having distinct requirements at the residue level.


Prohormone convertase-1 will process prorelaxin, a member of the insulin family of hormones.

Relaxin is a polypeptide hormone involved in remodeling of the birth canal during parturition. It is synthesized as a preprohormone precursor, which undergoes specific processing to form the mature two-chain disulfide-linked active species that is secreted by the cell. A major part of this processing requires endoproteolytic cleavage at specific pairs of basic amino acid residues, an event necessary for the maturation of a variety of important biologically active proteins, such as insulin and nerve growth factor. Human type 2 preprorelaxin was coexpressed in human kidney 293 cells with the candidate prohormone convertase-processing enzymes mPC1 or mPC2, both cloned from the mouse pituitary tumor AtT-20 cell line, or with the yeast kex2 alpha-mating factor-converting enzyme from Saccharomyces cerevisiae. Prorelaxin expressed alone in 293 cells was secreted into the culture medium unprocessed. Transient coexpression with mPC1 or kex2, but not with mPC2, resulted in the secretion of a low mol wt species with an electrophoretic mobility very similar, if not identical, to that of authentic mature relaxin purified from human placenta. This species was precipitable by monoclonal antibodies specific for relaxin and had a retention time on reverse phase HPLC comparable to that of relaxin. Its analysis by both electrospray and fast atom bombardment mass spectrometry generated mass data that were consistent only with mature relaxin. The basic residues required for mPC1-dependent cleavage of prorelaxin are defined by site-directed mutagenesis.


[Molecular cloning of the gene for secreted beta-galactosidase of the filamentous fungus Penicillium canescens].

A secreted beta-galactosidase gene from P. canescens was cloned using synthetic oligonucleotide probes and its nucleotide sequence was partially determined. The gene was shown to be present in the genome as a single copy. The deduced primary structure of the signal peptide and the first 25 amino acid residues of the mature protein was established. The structure analysis of the beta-galactosidase gene revealed at least one short intron 64 bp long. The transcription start point was found to be located 89 bp upstream the initiation codon. Some peculiarities of the signal peptide cleavage mechanism are discussed.


[Localization of conserved and variable segments in amino acid sequences of homologous proteins using a personal computer].

A set of aligned homologous protein sequences is divided into two groups consisting of the most related sequences m and k. The value of the position variability of homologous protein sequences is defined as a number of failures to coincide in the intergroup comparison of all possible k x m pairs of amino acid residues in that position divided by k x m. The position variability value plotted vs the sequence position number with a window of 10 positions gives the intergroup local variability profile. The area S of the figure included between the local variability profile and the straight line corresponding to the mean local variability value is compared with the average area S(r) for 1000 random homologous protein families. If S is greater than S(r) by more than 2 standard deviation units sigma r the local variability profile is assumed to contain peaks and hollows corresponding to significant variable and conservative regions of the sequences. The profile extrema containing the area surplus delta S = S-(S(r) + 2 sigma r) are cut off by two straight lines to locate significant regions. The numerical experiment on the family of homologous phospholipases A2 revealed the linear dependence of the values S(r) and sigma r upon the position variability standard deviation sigma v of the homologous sequences. Furthermore, it was shown for protein families of various length (rhodopsins, aspartate aminotransferases, cytochromes b, L- and M-subunits of photosynthetic bacteria photoreaction centre and alpha-subunits of Na, K-ATPase), that delta S = S - n(S'r + 2 sigma r), where S - the area of the local variability profile, n = L/l (L - the length of the given protein family and l - the length of the hypothetical protein domain). If l = 250 then S'r = -1.42 + 62.56 sigma v and sigma'r = -0.14 + 7.46 sigma v.


[Regulation of the expression of the c-fos gene in cells, reverting from being transformed to the pseudonormal phenotype].

Regulation of c-fos expression in mice sarcoma cell lines CBA and C3H was investigated. Each of the cell lines was represented by a pair of clones: the tumorigenic and the one, which was produced from it by cloning. It was found, that c-fos expression in cells of the pseudonormal phenotype was similar to that in the normal fibroblasts. Experiments with cells reverted to pseudonormal phenotype transfected transiently or permanently with an indicator plasmid fos-cat have shown, that a 600 bp sequence of the c-fos promotor including the TATA site, provides the expression level of the chloramphenicol acetyltransferase, correlating with the level of the c-fos mRNA expression. In the tumorigenic cells, permanent high activity of the cat gene expression was observed which was comparable to that in the normal fibroblasts stimulated by the embrionic serum or TPA. Activity of the transcription factors interacting with regulatory elements SRE, DSE, TRE did not correlate with the c-fos expression level in all the cells.


Modulation of glucocorticoid receptor function by protein kinase A.

Protein kinase A (PKA) has been shown to modulate the pattern of gene expression via transcription factors such as cAMP response element binding protein. However, in F9 embryonal carcinoma cells which lack endogenous functional cAMP response element binding protein, we have found that PKA is still able to control gene transcription through the glucocorticoid receptor (GR) by up-regulating its hormone-dependent trans-activation. Dose-response analysis indicates that PKA does not alter the ligand binding affinity of GR. PKA seems to act through the DNA binding domain of GR, since GR mutants which lack either the amino-terminal or the ligand binding domain are still able to be up-regulated by PKA. In support of this proposal, we demonstrate that PKA can enhance the DNA binding activity of GR. Our results suggest a novel mechanism by which PKA modulates the steroid sensitivity of a target cell by enhancing the DNA binding activity of GR for its cognate hormone response elements.


[Study of transcription of the human cytochrome P450IID6 gene by polymerase chain reaction].

Polymerase chain reaction was used to study the expression of the drug metabolism gene. Primers complementary to the 2070-2090 and 2912-2930 sites within exons 4 and 6 of the gene CYP2D6 were synthesized. The amplification of the cDNA from total human liver mRNA was achieved. The length of the fragment obtained (238 bp) was in accordance with the distance between the primers binding sites in cDNA. The amplification of the DNA from the same source led to the longer fragment due to the presence of introns. The total RNA from the blood cells of the extensive metabolizers was shown to contain the mRNA transcribed from the CYP2D6 gene. The Taq polymerase reaction in the presence of cDNA derived from a poor metabolizer did not lead to the synthesis of the 238 bp fragment.


[Comparison of the state of the area between B- and Z-segments of superhelical plasmids in vitro and in situ].

The structure of B-Z junction in a cloned plasmid pGC20 containing a (dG-dC)10 insert at the SmaI site has been studied in vitro and in situ by modifying the DNA with O-beta-diethylaminoethylhydroxylamine (OHA). The latter is an analog of hydroxylamine possessing specificity with respect to unpaired cytidine. Experiments in vitro showed a complicated pattern of inhibiting the restriction hydrolysis of the OHA-modified DNA within the polylinker region of the plasmid. As the duration of the DNA reaction with OHA grows, a gradual increase in the inhibition of restriction is observed at the BamHI site neighboring the Z-insert and at the HindIII site at a distance of about 30 bp from the insert, while an intact segment (containing the SalGI site) is retained in the intermediate region. On passing to the cell level, only the region immediately adjacent to the Z-insert appears to be modified. According to estimates, about 30 to 40% of pGC20 molecules have the (dG-dC)10 insert in the Z-form when modified in situ in 1M OHA, pH 5.0.


[Interaction of mRNA of the p53 protein gene with ribosomal RNA].

By blot hybridization we found that DNA fragments of eukaryotic 18 and 28S rRNAs bind specifically with mRNA. In these experiments the in vitro transcribed mRNA of mouse gene p53 was used. In addition we found that both 18 and 28S rRNAs were able to form intermolecular complexes with mRNAs of several genes 18S rRNA-mRNA; 28S rRNA-mRNA, but fail to bind the antisense RNAs of the same genes. The experimental data allow to suppose that 18S and 28S rRNAs carry several fragments that are complementary to some fragments in the mRNA sequences.


Positive and negative thyroid hormone response elements are composed of strong and weak half-sites 10 nucleotides in length.

The steroid-thyroid hormone receptors bind to imperfect repeats of two or more half-sites. It is generally accepted that a T3 response element (TRE) half-site consists of a six-nucleotide core motif (5'-AGGT(C/A)A-3'). It is less widely appreciated that the nucleotides flanking this core motif also have a major influence on the affinity of T3 receptor (TR) for its response element. We analyzed TR-DNA interactions under conditions in which the affinity of receptor monomers for individual TRE half-sites of the rat GH (rGH) gene was measured. These studies avoided the effects of half-site spacing and orientation on receptor binding. Variations in the nucleotides flanking the core sequence can modulate receptor binding by more than 15-fold. Systematic mutational analysis of TRE half-site structure demonstrated that at least two nucleotides flanking either side of the half-site core motif strongly influence TR binding affinity and activity, indicating that half-sites are approximately 10 nucleotides long. Thus, the half-sites of most TREs overlap, and mutations in one half-site may affect the activity of its partner. The TRE half-site sequence 5'-CTGAGGTAACG-3' was bound with highest affinity by TRs. The negatively T3-responsive promoter of the rGH gene was used to investigate the functional significance of the nucleotides flanking the core motif in vivo. A promoter consisting of only 22 rGH nucleotides, containing two functional TRE half-sites which overlap the rGH TATA box, directed T3-inhibited transcription. Mutation of nucleotides flanking the core sequence of the weaker half-site dramatically reduced the activity of the element, demonstrating that the flanking sequences of the half-sites can profoundly affect TRE activity.


[Theoretical analysis of the amino acid sequence of receptors for growth hormones and prolactins. Prediction of ligand-binding segments].

At present the growth hormone and prolactin receptors were cloned along with their variant forms from human, rat, mouse, rabbit, bovine and sheep tissues. The functional topography of receptors is practically unknown. Because of the high price and difficulty of protein's total mutagenesis, it is reasonable to carry on a theoretical analysis of structures of receptors to predict the most probable ligand-binding sites. We studied the primary structures of known prolactin and growth hormone receptors using theoretical methods proved to be powerful in earlier structure--activity relationship investigations. We analyzed the secondary structure, conservative positions, hydrophilicity profiles of the growth hormone and prolactin receptors, and used the original method based on information theory to predict the sites which are promising for mutagenesis or peptide synthesis as probable ligand-binding sites. Three segments corresponding to the main conservative, hydrophilic and rare sites were predicted to form the ligand-binding determinant.


[Affinity modification of the 40S subparticle from human placenta with derivatives of pAUG and pAUGU3].

Affinity labeling of 40S subunits from human placenta with 4-(N-2-chloroethyl-N-methylamino)benzylmethyl-[32P]phosphoamide s of oligoribonucleotides pAUG and pAUGU3 was studied. Covalent attachment of these derivatives to 40S subunits within the complexes with 40S subunits, formed in the presence of Met-tRNAf.eIF-2.GTP, was detected. Both rRNA and ribosomal proteins were modified. Fragments of 18S rRNA, containing sites of the reagent attachment were identified: 1058-1164 for pAUG derivative and 976-1057--for pAUG and pAUGU3 ones. The data obtained allowed to conclude that the presence of the neighbouring codon at the A-site, regardless of the presence of the tRNA in it, affects significantly the arrangement of the trinucleotide template in the codon-anticodon interaction region. The large subunit does not cause significant alterations in the structural organization of the codon-anticodon interaction region.


Expression and regulation of a proenkephalin beta-galactosidase fusion gene in the reproductive system of transgenic mice.

A fusion gene containing 3 kilobases of human proenkephalin 5'-flanking sequences and 1 kilobase of human proenkephalin 3'-flanking sequence and the easily visualized histochemical marker, Escherichia coli beta-galactosidase, was used to study the function of cis-regulatory elements within the human proenkephalin gene in transgenic mice. Here data are presented on expression and regulation of this fusion gene in the reproductive system of three independent lines of transgenic mice. Within the male reproductive system, the fusion gene is expressed in the proximal epididymis and in developing germinal cells but not in mature or elongating spermatids. In the female reproductive system, the transgene was expressed at low basal levels, but expression was dramatically stimulated in the ovary and oviduct by hormonal stimulation and pregnancy; additionally, expression was induced at the uteroplacental junction in pregnant mice. Taken together these observations suggest that critical sequences for expression and regulation of the proenkephalin gene within the reproductive system are contained within sequences of the construct.


[Human genetics and transplantation].

Transplantations represent an important element of treatment in end-stage chronic disease both inborn (mostly genetic) and acquired (degenerative, neoplastic). They are supposed to establish durable coexistence of cells with different genetic origin (in most cases). This union is contradictory to immunological properties of the tissues involved and can only succeed in case of sufficient histocompatibility to be identified by the diagnostic tests of immunogenetics. This review discusses the current approach used in choosing the most appropriate donor for an individual patient, and in monitoring the maintenance of "chimerism" established by the transplantation focussing on bone marrow transplantation. Initially a general outline of indications for organ transplantation is given with emphasis laid on genetic disorders as the outlook of conservative treatments in inborn diseases is generally very poor.


[Monoclonal hybrid antibodies. New perspectives for basic research and tumor therapy].

Monoclonal hybrid antibodies are bi-specific constructs of two monoclonal antibodies with defined specificity. Hybrid antibodies can be used to force interactions between cell populations that under normal circumstances would not react with one another. Applications include the field of basic research as well as employment in therapy of malignancies and infectious diseases. The first part of this review describes the characteristic properties of hybrid antibodies and gives a short introduction into basic techniques for the production of these molecules. In the second part we present our observations on T-lymphocyte development studied with hybrid antibodies in fetal thymic organ cultures. Finally, we show that hybrid antibodies encompassing a binding site to the T-cell receptor and a binding site to a surface marker on tumor cells can be used to recruit cytotoxic T-cells to eliminate efficiently the malignant cells. Our data demonstrate that monoclonal hybrid antibodies are useful in the future development of new therapeutical principles, through facilitation of the immune response.


[Pertussis--an illness with typical clinical symptoms?].

There has been a noticeable increase in the incidence of pertussis in West Germany over the last decade. Since the availability of adequate bacteriological diagnosis a much broader clinical spectrum can be attributed to infections with B. pertussis. Three patients with an unusual clinical presentation of pertussis are presented. A three month old infant presented with severe apneic spells without cough as the sole clinical symptoms of the infection. B. pertussis was isolated in the nasopharyngeal swab. A nine month old premature infant with bronchopulmonary dysplasia after long time intubation and artificial ventilation presented with apneic spells, pulmonary and cardiac decompensation and required ventilatory support. The diagnosis was suggested by a massive leucocytosis with lymphocytosis. The diagnosis on the patient was established by serologic methods. Adult contacts of this patient developed longstanding cough and clinical signs of pertussis. The diagnosis of pertussis in these persons was established by nasopharyngeal culture. The third patient with trisomy 21 and a corrected AV canal suffered from nonspecific cough and gradually developed signs of congestive heart failure with pneumonia. B. pertussis was isolated from the nasopharynx. This patient showed neither the typical paroxysmal coughing spells nor disclosed the typical lymphocytosis in his white blood count. Microbiological investigations of patients with symptoms of respiratory tract infections should include the isolation of B. pertussis. Thus, additional cases of pertussis not suspected on the basis of their initial clinical presentation will be detected.


[Embolism of the aortic bifurcation. Complication of bacterial endocarditis in childhood].

Acute occlusion of the aortic bifurcation by a sattle embolus, as a complication of infective Endocarditis, is a very rare, extremely serious event. The treatment of choice consist in an immediate simultaneous bilateral femoral embolectomy. The indication for operation must be realised without delay based on anamnesis and physical examination.


[Effects of passive smoking on the pediatric respiratory tract].

To explore the risk of parental smoking to the respiratory health of their children data of a cross sectional study on fourth-grade schoolchildren in Munich and Southern Bavaria were analysed. Allergic and asthmatic diseases and symptoms, risk factors like family history, indoor pollution and parental smoking were evaluated by a questionnaire. Pulmonary function tests were performed in 7284 school children aged (9-11 years). Lung function values were adjusted for height, weight, sex and other confounders. The children, whose parents smoke at home, had significantly lower levels of peak flow, MEF75, MEF50 and MEF25 compared to children from non-smoking families, with a dose-response relationship. Smoking of more than 20 cigarettes at home is associated with a mean decrease in MEF75 of 5.7%, in MEF50 of 4.9% and in Peak Flow of 4.9% (p < 0.001). The prevalence of cough and wheezing increased with increasing smoking rates of the parents. Passive exposure to smoke has direct measurable dose-dependent effects on the respiratory system of children.


[Hepatitis B virus DNA. Detection with polymerase chain reaction in liver tissue of children with chronic hepatitis B].

Detection of hepatitis B virus DNA is a reliable evidence of the presence of the viral agent and its replication. Conventional hybridization techniques are limited to detect about 30,000 virions. With the polymerase chain reaction it became possible to extend the sensitivity by amplification of viral sequences. In our study we intended to test whether viral sequences could be found in liver tissue specimens negative for hepatitis B virus DNA by conventional hybridization techniques. Hepatitis B virus DNA was detected by PCR in liver tissue of 37 children with chronic hepatitis B, negative for hepatitis B virus DNA by Southern blot hybridization. PCR was performed in a thermal cycler using Taq-polymerase and oligonucleotide primers within the hepatitis B core region. Hepatitis B virus DNA was visualized by ethidium bromide staining and subsequent Southern blot hybridization. 20 patients were HBeAg- and 17 anti-HBe-seropositive. Viral sequences were present in each of the 20 HBeAg positive HBsAg carriers and in 10 patients with anti-HBe. No hepatitis B virus DNA could be found in 7 children, all of them positive for anti-HBe. Our results confirm polymerase chain reaction to be a more sensitive method to detect hepatitis B virus DNA in the liver compared with conventional hybridization techniques. Every HBeAg positive carrier as well as the majority of anti-HBe positive patients present viral DNA in their liver. Polymerase chain reaction will be suitable to monitor viral replication in spontaneous course and treated patients.


[Does breast feeding prevent asthma and allergies? Results of the Munich asthma and allergy study].

The relationship of breast feeding to atopic diseases is studied in a population-based cross sectional study in Munich and Bavaria 1989/1990 in 6,535 german ten year old children. According to the questionnaire answers 1914 (29.3%) children were not breast fed, 2,368 (36.2%) shorter than 2 months, 1,744 (26.7%) 2 to 6 months and 509 (7.8%) more than 6 months. Compared to controls without any allergic disease the relative risk of later asthma, hayfever, atopic dermatitis and of allergic skin sensitization was not diminished by breast feeding. The same result pertained to high risk subgroups defined by positive family history of asthma or hayfever. It is concluded that breast feeding alone has no long term protective effect against atopic diseases.


[Chronic hepatitis B. Treatment in childhood with alpha-interferon].

In adults several trials of successful therapy for chronic hepatitis B using alpha-interferon with rates of seroconversion from HBeAg to anti-HBe of 30-40% have been reported. Despite the experiences in children are limited, alpha-interferon seems to be a promising drug in this age group as well. We report on our results in the treatment of chronic hepatitis B virus carrier using the recombination interferon alpha-2b. 24 children aged 0.6-16 years with chronic active or chronic persistent hepatitis B were included in the study. 12 children received 9 million units of alpha-interferon/m2 body surface area three times a week during four months. 12 control patients were not treated. The follow-up period was 9-12 months after the beginning of therapy. HBsAg, anti-HBs, anti-HBe and Hepatitis-B-Virus-DNA were assessed during this time on a regular basis. Only seroconversion of HBe-Ag to anti-HBe was considered as response to interferon treatment. During the follow-up period anti-HBe could be detected in 5 (41.6%) of the treated and in one (8.3%) of the untreated children. In one case additional seroconversion of HBsAg to anti-HBs due to virus elimination was observed. In all children a marked reduction of viral replication could be shown. 9 patients cleared Hepatitis-B-Virus-DNA at least for one time during therapy. Alpha-interferon was well tolerated and no severe side effects were observed. Our results demonstrate that alpha-interferon can be successfully applied to a considerable number of children with chronic hepatitis B. In patients responding to alpha-interferon usually serum transaminases become normal and infectivity of the disease is markedly reduced. alpha-Interferon treatment should be primarily recommended for children with chronic active inflammation.


[Idiopathic epilepsy with generalized seizures in early childhood].

Idiopathic epilepsies with generalized seizures of early childhood are based on a genetic predisposition. The onset takes place between the first and fifth years of age, boys are affected more often than girls. Dependent on the clinical symptomatology you have to distinguish: myoclonic seizures; atonic-astatic seizures; myoclonic-astatic seizures; absences; tonic-clonic seizures. In more than half of the cases a combination of these seizures can be observed. The differentiation of epilepsies with generalized seizures of multifocal origin (infantile spasms, Lennox-Gastaut syndrome and Pseudo-Lennox syndrome [atypical benign epilepsy]) may be difficult but is essential. Therapy of choice is valproate, often in combination with ethosuximide (in children with minor seizures) or with kaliumbromide or phenobarbital (in children with tonic-clonic seizures). Generally the prognosis is more unfavourable if epilepsy starts in the first year of life with afebrile and febrile generalized tonic-clonic or clonic seizures, if children are suffering from longlasting states of seizures and if development is disturbed before beginning of epilepsy.


[Transient, prolonged, combined sicca syndrome of the eyes and mouth. Occurrence along with a common cold in an 8-year-old girl].

A girl of eight years fell ill with signs of common cold and bronchitis. Already on the second day of illness, before any treatment was started, she showed symptoms of a sicca-syndrome, beginning in the eyes, later the mouth. Ophthalmologic examination showed keratitis filiformis, histology of the salivary glands in the mucosa of the mouth was normal. Local substitution therapy led to subjective improvement. The disorder of secretion persisted for four months only, then rapid restitutio ad integrum of tears and saliva production occurred. Pathogenetically a parainfectious immunologic mechanism seems probable. During the observed period there were significant changes of antibody titers against RS-virus and streptococci, but it is not possible to identify the cause of the illness really. We conclude that a sicca-syndrome with normal histology of mouth-mucosa-glands is not necessarily prognostically infavorable.


[Percutaneous Silastic catheters in newborn and premature infants. A report of experiences with 497 catheters in 5 years].

Central catheters are an important prerequisite for adequate parenteral nutrition in preterm infants. However, a variety of complications have been shown to be associated with central lines: septicemica, thrombotic complications, mechanical complications. In this retrospective analysis we summarize our recent experience with central silastic catheters. Within a five-year-period (1986-1990). 497 silastic-catheters were inserted in 366 high risk neonates (mean birthweight 1360 g; 1060-1740 g, 25.-75. percentile) treated at the NICU, Department of Pediatrics, University of Göttingen. 451 catheters which were placed in a central position, were removed after an average duration of 11 days (mean; 8-18 days, 25.-75. percentile). During the observation period, 62.8 percent of the catheters were purposely removed. Making use of the Kaplan-Meier-curve, we calculated how long the catheter could stay without complications; 50% of all catheters could be expected to be in place for 25 days. The incidence of septicemia was 1.9%, bacterial contamination of the catheters was evident in 22% of all central lines. The most predominant microorganisms responsible for catheter-contamination were coagulase-negative staphylococci. In addition, catheters were removed because of signs of phlebitis or suspected thrombotic complications (11.1%), and mechanical complications (dislocation, occlusion; 11.7%). Due to malposition of the central catheter two preterm infants developed pericardial effusions. There was no correlation between the site where the catheter was inserted and these complications. Central silastic catheters wherever clinically indicated are a valuable adjunct in the parenteral nutrition on high risk neonates.


[In vivo studies of residual allergen activity of hydrolysate feeding].

The capacity of food proteins to induce IgE-mediated reactions can be reduced by hydrolysis, heat treatment or ultrafiltration. It was the aim of our study to investigate the capacity of hydrolysates used for dietary purposes in cow's milk allergy to induce allergic symptoms in cow's milk sensitive children. Six different hydrolysates were tested by skin prick test and oral provocation test in 17 cow's milk sensitive children. Our data indicate that certain hydrolysates induce positive skin reactions as well as allergic symptoms after oral challenge. Casein hydrolysates were found to have the least residual allergenic activity. From our data we conclude that hydrolysates should be tested by titrated oral challenges before used in the diet of cow's milk sensitive children.


[Intrapulmonary pressure measurements in high frequency ventilation of extremely small premature infants. The triggered airway occlusion method].

The interrupter technique is an established method for measuring the intrapulmonary pressure in mechanically ventilated patients. We have developed a computer controlled method enabling us to interrupt the respiratory flow at precisely reproducible occlusion times (triggered by the respirator). It is therefore possible to determine the intrapulmonary pressure course in inspiration and expiration and the time constant for the upper respiratory tract (including the intratracheal tube) even in high-frequency ventilation. In 16 premature infants (mean birth weight 741 +/- 138 g, gestational age 26.1 +/- 1.9 weeks) we measured time constants between 58 and 190 msec for the intrapulmonary pressure decrease in expiration. An inadvertent PEEP between 1 and 4.5 cmH2O was found in 6 out of 23 examinations (ventilation frequency between 67 and 150/min, expiration times between 250 and 500 msec). The relation of expiration time to time constant was less than 4 in these cases only. The triggered interrupter technique is a useful, noninvasive method for monitoring the intrapulmonary pressure in neonates without disturbing the patient. Particularly in critically ill infants the measurements are the basis for optimal respirator settings.


[Children with psychiatric disorders examined by established pediatricians].

Studies concerning children with psychological disorders presented in the paediatric practice are rare. We therefore investigated rates of attendance and rates of referral to other specialists from theses practices. The data were collected by the paediatricians and recorded according to a list of psychosocial symptoms. Frequencies were compared with results of a similar study concerning general practitioners. The chi-square seemed to be appropriate as test of significance. 18 paediatricians were included in the study. 8.3% of children seen by these professionals were thought to show signs of psychological deviance. The accompanying physical disorders hereby were the more prominent presenting symptoms. Only in 1.3% of the cases were psychological disorders the sole reason for the visit. In 30% of the cases were psychological problems a follow up referral to another institution was recommended. Rates of attendance and of follow-up referral were more frequent in rural practices than in practices of a university town. In general practices, 20.3% of children were noted as psychologically disturbed. The study reveals the important role played by paediatric practices in the mental health care, especially in rural regions. The low rate of psychological disorders as the only reason for attending a paediatric practice indicates that there may be an attendance pattern in which physical symptoms are initially used as a vehicle for subsequently presenting psychological disturbance.


[Epigastric colic after ceftriaxone therapy].

The following article describes a girl with right upper quadrant abdominal colic following Ceftriaxon therapy for purulent meningitis. Ultrasound made it possible to demonstrate sludge-balls, floating in the gallbladder, a follow up examination was normal. Moreover the features of gallbladder precipitations following Ceftriaxon therapy will be described, and the clinical consequences will be discussed.


[Has post-streptococcal glomerulonephritis disappeared?].

We report on 21 children with poststreptococcal glomerulonephritis (PSGN). The diagnosis was based on clinical and laboratory criteria. 19/21 had a clinically apparent throat infection initially, but only 6 received antibiotic treatment. Initial symptoms were: macrohematuria (20/21), edema (10/21), arterial hypertension (6/21), reduced creatinine-clearance (11/21), and proteinuria (18/21). No Patient was dialyzed. After an interval of 0.3 to 25 months 19 children are asymptomatic. All patients have normal creatinine-clearances; none is hypertensive. The present data show that PSGN has not disappeared in Mid-Europe. Remarkable acute disturbances occurred, which healed in almost all cases. The importance of accurate bacteriologic diagnosis and adequate therapy is emphasized.


[Grand mal epilepsy in childhood].

Generalized tonic-clonic seizures are the most common type of convulsive disorders in children. They are always a sign of an epileptogenic cerebral dysfunction and present either acutely, predominantly as a single event with detectable origin, or chronically, recurring as an epileptic syndrome. In view of the etiology and classification of convulsions it is important to differentiate between primarily and secondarily generalized seizures. This distinction is first of all based on an exact description of the very beginning and of the course of the seizures, on the EEG findings and on any connection between the seizures and a particular time of day. Primarily generalized tonic-clonic seizures with and without associated petit mal seizures are manifestations of an idiopathic epilepsy and are most probably genetically determined, secondarily generalized seizures on the other hand are often signs of a central nervous lesion or of another symptomatic form of epilepsy. Benign idiopathic partial seizures, however, take the from of secondarily generalized convulsions during the morning sleep. Prolonged tonic-clonic seizures of any origin require vigorous treatment with anticonvulsants, if necessary in an intensive care unit. Recurrent seizures are treated with long-term anticonvulsant medication. The first-line treatment is valproic acid or phenobarbitone (or if necessary, a bromide) in primarily generalized seizures and carbamazepine or phenytoin in secondarily generalized convulsions. The recommended duration of this therapy and the risk of recurrence of seizures vary widely with the underlying etiology and the type of epilepsy.


[Post-traumatic epilepsy].

There might be 3 different forms of epilepsy following head injury: earliest seizures with occasional characteristics, early seizures prompting considerations of differential diagnosis, and, late seizures which might have a chronic course = posttraumatic epilepsy proper. The risks to have posttraumatic epilepsy for a child suffering from head injury are: penetrating cranial trauma, early seizures, intracranial hemorrhage, unconsciousness > 24 hours, depressed fracture with dural laceration and unconsciousness > 24 hours, fractures on the base of the skull, focal synchronous activity in the EEG which is related to the site of brain injury. If a or > or = 2 of b-g are given we recommend prophylactic antiepileptic therapy: in the younger (< 5 years) with a low dose of phenobarbital and in the elder (> 5 years) carbamazepine for at least 2 years of treatment.


[Rupture of a renal artery aneurysm. Fulminant course, fatal complication of type IV Ehlers-Danlos syndrome in childhood].

A 12 year old girl with joint laxity, hyperelastic and vulnerable skin and tall stature was labeled as "Marfan syndrome". She died unexpectedly from a ruptured aneurysm of the right renal artery. All these features are typical for Ehlers-Danlos syndrome type IV. The diagnostic difficulties and the classification of connective tissue disorders are discussed and further possible complications are reviewed from the literature. The importance of an exact diagnosis is stressed in order to assess the prognosis and start possible therapeutic measures as early as possible.


[Experiences with surfactant therapy of adult respiratory distress syndrome].

We report on 2 patients with ARDS, who underwent a therapy with surfactant. In both cases the underlying reason for the lung disease probably was a viral pneumonia. In both patients the gas exchange improved after tracheal instillation of surfactant. This improvement however was much less than we know it from therapy of the respiratory distress syndrome of the premature babies. Reasons for these differences in response to surfactant are discussed.


[Candida infections in premature infants weighing less than 1,500 g. Mucocutaneous colonization and incidence of systemic infections].

An increasing incidence of systemic candidiasis has been reported in low birth weight infants requiring intensive care. We have retrospectively analyzed mucocutaneous Candida-colonization and infection rate in 422 preterm infants with a birthweight < 1,500 g. All infants were treated at the NICU, University of Göttingen, from 1/1985-5/1991. 359 neonates (85%) were on mechanical ventilation, no prophylactic antimycotic regimen was applied. Mucocutaneous swabs and cultures from various anatomic sites were regularly obtained from all infants. 37/422 preterm infants (8.8%) had mucocutaneous colonization with candida, none of our patients developed systemic candidiasis. In 7 mechanically ventilated patients (1.9%) Candida albicans or Candida tropicalis was repeatedly detected in the bronchial secretions; 1 patient who had invasive Candida-pneumonia was effectively treated with 5-Fluocytosin and Fluconazol. 4/352 (1.1%) central silastic catheters were colonized with Candida albicans; none of these patients required specific treatment. The low rate of mucocutaneous Candida-colonization and invasive infection found in our patients may be explained--at least in part--by epidemiological and obstetrical factor as well as by the procedures of the neonatal management.


[IgA endomysium antibodies. Detection in children with celiac disease].

Epidemiology of coeliac disease gives rise to the search for a non-invasive, reliable screening test. We looked for IgA anti-endomysial antibodies (IgA-EmA) in 103 sera of 90 children (age: 2 months-13.9 years) using an indirect immunofluorescence method on monkey oesophagus sections. In 44 patients, the diagnosis of coeliac disease was confirmed fulfilling new criteria of the European Society of Pediatric Gastroenterology and Nutrition. All 24 coeliac disease patients with an initial flat mucosa had IgA-EmA (sensitivity 100%). In 36% of coeliac disease patients adhering to a gluten-free diet we found IgA-EmA. None of 46 patients in whom coeliac disease had been excluded by jejunal biopsy had IgA-EmA (specificity 100%). The sera of 102 blood-donors were used as controls and showed a test specificity of 99%. In coeliac disease patients, the titer of IgA-EmA declined during gluten-free diet by 0.66 steps/month on average, and rose 1.76 steps/month during gluten challenge. These results confirm the diagnostic significance of IgA-EmA for patients with suspected coeliac disease and their value for monitoring treatment even in young children (below 2 years).


[Experiences with triple antibiotic combination in neonatal infection].

In-vitro and clinical efficacy of a combination therapy consisting of 3 antibiotic agents was to be assessed in neonatal septicemia. From 1980 to 1987, 152 newborns with septicemia as proven by blood culture were treated with an initial antibiotic regimen consisting of azlocillin (150 mg/kg bw), cefotaxime (100 mg/kg bw), and tobramycin (5 mg/kg bw). According to the microbiologic testing, antimicrobic therapy was effective in each of the 152 organisms: 101/152 bacteria were susceptible to all 3 agents; resistance to 1 or 2 of the antibiotics was evident in 33/152 and in 18/152 organisms, respectively. Mortality due to septicemia was 7.2%. As no difference was observed in the frequency in which one of the three antibiotic substances was the only effective drug, each of the 3 agents seemed to be necessary for clinical effectiveness of this antibiotic combination.


[Perinatal calcium metabolism. Physiology and pathophysiology].

During pregnancy, calcium is continuously transferred directly from the maternal intestine to the fetal bone, a transfer that is mainly induced by the interrelated actions of the calcium-regulating hormones parathyroid hormone (PTH), 1,25-dihydroxyvitamin D (1,25(OH)2D) and calcitonin. It has recently been demonstrated in animals that PTH-related protein (PTHrP) is the fetal equivalent of PTH. Human PTHrP, originally described as a product of a human lung cancer cell line and implicated in the pathogenesis of humoral hypercalcemia of malignancy, is a protein with 141 amino acids, and it has biochemical actions similar to PTH. It is believed that fetal PTHrP is mainly derived from the placenta during early gestation and from the fetal parathyroid glands during further development and that this protein has the role of maintaining the maternal-fetal calcium gradient either alone or in concert with 1,25(OH)2D. With birth, the placental supply of calcium ceases abruptly, stimulating the increase of PTH and 1,25(OH)2D, which are the main regulators of postnatal calcium metabolism. Alterations in the placental calcium (and phosphate) gradient may be caused by maternal hypo- or hypercalcemia and placental insufficiency and may be followed by transient disorders of calcium metabolism in the newborn. Due to abrupt cessation of the calcium and phosphate supply after delivery at a time when mineral demands are the highest, preterm infants are especially prone to hypocalcemia and osteopathy. If bone disease of prematurity is to be prevented, the amounts of calcium and phosphate must be adequate, as demonstrated by laboratory tests, the most important being calcium and phosphate in urine and alkaline phosphatase activity in serum.


[Calcium, phosphorus and vitamin D administration in infancy. Unsolved questions].

In premature infants calcium and phosphate supplementation should be continued until the infants weigh more than 2000 g, because osteopenia of prematurity has been described after discontinuation of the supplements at body weights of < 1800 g. Premature infants generally receive substantially more vitamin D than their daily requirements (> 400 IU), but then are no studies on the side effects of high vitamin D intake (hypercalciuria?). The extremely high calzitriol concentrations in plasma of premature infants who receive Ca/P supplements indicate that maximal stimulation of Ca/P absorption is necessary and present supplementation could still be inadequate. The daily vitamin D requirements of term infants are between 100 and 400 IU and no supplements are necessary if the infants are fed vitamin-D-fortified formulas. The high prevalence of rickets in infants on macrobiotic or vegetarian diets indicates that those infants should receive calcium and vitamin D supplements.


Mineral nutrition and bone mineralization in full-term infants.

Bone mineralization is an intricate and tightly regulated process. Calcium, magnesium and phosphorus are the main minerals and play a principal role in skeletal mineralization. The following conclusions can be derived from different clinical studies. The large differences in Ca/P ratio between different formulas and between formulas and human milk suggest that most healthy full-term infants can adjust to a wide range of Ca/P ratio in their diet. The differences in serum levels of mineral and of mineral-regulating hormones are rarely clinically significant and most probably reflect continued compensatory mechanisms activated in response to dietary differences to maintain these levels within clinically normal ranges. Thus in most cases, these compensatory mechanisms are sufficient to reverse both short-term and long-term consequences and to prevent clinical disease. In the case of neonatal tetany, the compensatory mechanisms are overwhelmed, resulting in clinical signs and disease. Vitamin D is known to play an essential role in bone mineralization. Our studies have shown significant differences in vitamin D status in breast-fed infants with and without vitamin D supplementation and in infants fed various "humanized" formulas, whether cow milk-based or soy protein-based. The major variables affecting bone mineralization are Ca/P ratio and mineral-regulating hormones. However, factors such as season, geography (i.e. sun exposure), race and sex may have a significant long-term influence on bone mineralization and mineral metabolism. Some biological differences such as differences in serum vitamin D metabolite level may directly effect Ca/P absorption and retention and thus bone mineralization and growth.(ABSTRACT TRUNCATED AT 250 WORDS)


[A new method for determining mineral content of bones using ultrasound].

With conventional methods radiation exposure is relatively high, due to frequent examinations. For this reason an alternative method for measuring bone mineral content was sought. Like X-rays, sound waves are subject to an exponential law of attenuation. For this reason the attenuation coefficient of the sound waves was used to describe the sound-conducting medium. With the help of this transmission sonographic method thickness dependence and increase in attenuation were shown as a function of increasing calcium apatite concentration using a reference system and also femoral sections of varying thickness. Examination of the reflected portion indicated that for this type of measurement the reflection at the bone is negligible at least in the first approximation. In the case of bone specimens a linear dependence was demonstrated between half-value thickness and mineral content in the area of bone calcium concentrations of 50-300 mg/cm. For practical employment of the method, a number of patients were examined in whom bone-mineral-content fluctuations were to be expected on the basis of their disease and therapy. As the examinations indicate, fluctuations in vivo can be detected with the help of this method.


[The clinical relevance of phosphorus and calcium in infant nutrition].

This paper is an introduction to the clinical part of the symposium and deals with the question of whether and under which circumstances the calcium and phosphorus content in baby formula can provoke pathological conditions. In a healthy baby, high or low mineral intake is efficiently compensated for by Ca-P homeostasis. Both nutritional calcium deficiency and calcium excess are the exception with modern baby feeding practices. However, P-deficiency states resulting in phosphopenic rickets might occur in premature babies and in children with familial hypophosphatemic rickets. These two conditions should be treated and prevented by an alimentary P-supplement. On the other hand, formula with a rich P-content might be a cause of the late form of neonatal hypocalcemia. Therefore, a relatively low-phosphate formula preparation, similar to human milk, is recommended for the first 2 weeks of life of full-term newborns, as well for infants with hyperphosphatemic renal failure.


Why is the phosphorus content of human milk exceptionally low?

The phosphorus supply of children and adults is adequate, and usually in excess. Therefore, it is surprising that in breast-fed infants phosphorus intake is very low. This is very rare among mammals. In infants three pathophysiological mechanisms argue in favour of a low, but adequate phosphorus intake in the presence of a balanced ratio of calcium to phosphorus. A low intestinal phosphorus concentration is an essential condition of an acid pH of the faeces, inhibiting the growth of potentially pathogenic germs. Owing to the characteristic renal physiology of the newborn, a small metabolic phosphorus surplus results in a high serum phosphorus level, a well-known risk factor for several disorders, e.g. hypocalcaemic tetany. During infections, impairment of intestinal calcium but not of phosphorus absorption results in an increased phosphorus and renal net acid excretion. Considering the low renal capacity for acid excretion in newborns, a high intake of calcium and phosphorus is a risk factor for the development of metabolic acidosis. It is thought that all three pathophysiologic mechanisms were effective in the biochemical evolution of humans, selecting women with a low phosphorus milk and infants with a constant high intestinal absorption rate of phosphorus.


[Phosphate concentration. Does reduction in infant formula feeding modify the micro-ecology of the intestine?].

The potential influence of phosphates in formulas on intestinal microflora was studied in 25 infants, aged 8 days to 12 weeks. The babies were either fed an infant formula with the usual phosphate concentration (n = 10) or an infant formula with reduced phosphate and protein concentrations (n = 7). The microbiological findings were compared with those obtained from breastfed infants (n = 8). Low-phosphate concentrations did not correlate with a predominance of bifidobacteria or suppression of putrefactive bacteria in the feces. The fecal excretion of phosphates and fat was found to be significantly lower with mother's milk compared to formulas both rich and poor in phosphate. Protein synthesis and breakdown rates, as well as the net protein gain, did not have a significant correlation with protein intake.


Evaluation of a low-phosphate cow's milk diet on growth and bone mineralization of full-term infants.

Growth, bone mineralization and intestinal absorption were studied in 16 infants fed low-phosphate cow's milk and compared with a group of 15 infants fed conventional cow's milk. In the low-phosphate-fed infants the intestinal calcium absorption was as high as in infants fed human milk, the main characteristics of growth and bone mineralization being similar to those of infants fed conventional cow's milk. Urinary calcium was elevated with a low cAMP excretion. The relative hypercalciuria might be the result of the amount absorbed in excess of that used for the bone accretion.


Technical aspects of phosphorus reduction and mineral adaptation in infant formulae.

Adapted milk-based formulae are complex products which simulate the protein and mineral composition of breast milk. The desired casein-to-whey protein ratio (40:60) in these formulae could be obtained by mixing cow's milk and whey. Unfortunately, this would yield formulae with too high a mineral content and a very unfavourable Ca:P ratio. The adjustment of the mineral content and the Ca:P ratio is the most sophisticated step in manufacture of such formulae. For this purpose, whey is demineralized with ion-exchange resins, electrodialysis or a combination of both processes. The demineralized whey (DW) obtained must be standardized in minerals by the addition of well-selected salts. After the standardization, whey is pasteurized, concentrated and dried, to give finally, the demineralized and standardized whey powder (DWPS). Addition of this ingredient to whole milk or skim milk leads to the standard adapted milk-based formula. Partial replacement of milk in the standard formula by caseinate leads to a low-phosphate formula with a Ca/P ratio equivalent to that found in breast milk. Because DWPS is quantitatively the most important ingredient of adapted milk-based formulae, the second most important being cow's milk, the nutritional and processing quality of DWPS strongly affects the heat stability and the nutritional and final quality of such formulae.


Calcium and phosphorus metabolism in full-term infants.

Appropriate intake of dietary calcium and phosphorus is essential for the maintenance of mineral homeostasis and support of adequate bone mineralization in growing infants. Disturbances of calcium homeostasis such as newborn hypocalcemia, have been shown to be secondary to high oral phosphate intake. The incidence of hypocalcemic tetany has changed from epidemic proportions in newborns, who were formerly fed evaporated cow's milk formula, to the present sporadic occurrence in newborns who are now fed humanized cow's milk formula, with a calcium concentration of about 50 mg/dl and a phosphorus concentration of about 35 mg/dl. Human milk, provided vitamin D intake is adequate, has been shown to maintain calcium-phosphorus homeostasis and support adequate bone mineralization at least in the first 6 months of life. After this, the phosphorus content of breast milk could limit mineral accretion in exclusively breast fed infants. The present concentration of calcium and phosphorus in the humanized cow's milk formulas support adequate mineral homeostasis and bone mineralization throughout the first year of life. As bone calcium content is proportional to calcium intake, feeding whole cow's milk from early infancy onward is associated with abnormal bone mineralization. The adequacy of bone mineralization in infants fed soy formula is still controversial.


Enzymes of the thymidylate cycle as targets for chemotherapeutic agents: mechanisms of resistance.

This brief review should serve to indicate that it is possible to assess tumor sensitivity to antifolates using fresh human tissue in short-term culture. The assays described differ from general assays of tumor sensitivity, such as the clonogenic assay or assays that measure 3H-thymidine incorporation as an indicator of cell viability (32-34), in that the effect of a drug on a human specific target (dihydrofolate reductase) is measured. These specific assays also may prove to be extremely useful in the detection of acquired drug resistance and for new analog drug development. In a general sense, this type of assay may eventually also be useful for guiding and selecting treatment for individual patients with other drugs with known mechanisms of action. Knowledge of the basis of tumor resistance is essential to develop new approaches to treatment, such as the use of other folate analogs that may still be effective, and to devise ways in which to selectively inhibit tumor cell growth using new analogs (trimetrexate) and leucovorin.


Tamoxifen and endometrial cancer.

Tamoxifen is a nonsteroidal antiestrogen employed frequently in the treatment of breast cancer. An association between this drug and endometrial neoplasia has been reported. We report on 11 postmenopausal women with breast cancer who developed endometrial cancer while undergoing tamoxifen therapy and recommend aggressive investigation of vaginal bleeding in all women being treated with this agent.


N10-propargyl-5,8-dideazafolic acid (CB3717): inhibitory effects on human leukemia cell lines resistant to methotrexate or trimetrexate.

The inhibitory effects of N10-propargyl-5,8-dideazafolic acid (CB3717), a quinazoline antifolate and a potent thymidylate synthase inhibitor, were evaluated in human leukemia cell lines resistant to methotrexate (MTX) and trimetrexate (TMQ). MTX-resistant MOLT-3 cell lines, MOLT-3/MTX200 and MOLT-3/MTX10,000, were cross-resistant to CB3717; however, the degree of resistance was only tenfold for both cell lines, and increased dihydrofolate reductase activity in MOLT-3/MTX10,000 had little influence on the degree of CB3717 resistance. The MOLT-3 cell line made resistant to TMQ, MOLT-3/TMQ200, was as sensitive to CB3717 as the parent line. The cell growth inhibitory effect of CB3717 on MOLT-3 was reversed by the addition of thymidine. Leucovorin also partially reversed CB3717-induced growth inhibition. Cellular uptake of MTX and 5-methyl-tetrahydrofolate was hindered by the presence of a high concentration of CB3717, whereas TMQ uptake was not influenced by CB3717. CB3717 appears to enter the cells not only through reduced folate transport system, but by other route(s). CB3717 does not share the transport pathway with TMQ. Our observations that MTX-resistant cells with increased dihydrofolate reductase are not more resistant than cells without increased enzyme activity, and that TMQ-resistant cells are not cross-resistant to CB3717, may have clinical relevance.


Peripancreatic lymph node gastrinoma in a patient with Zollinger-Ellison syndrome.

The case of a patient who had Zollinger-Ellison syndrome caused by a primary peripancreatic lymph node gastrinoma is presented. Accompanying diffuse pancreatic islet cell hyperplasia, a well-documented occurrence in hypergastrinemia, was present. A perforated esophageal ulcer in a Barrett's esophagus led to right-sided necrotizing pleuritis, septicemia, and death. The diffuse parathyroid hyperplasia also noted in the patient is thought to be secondary to chronic hypertensive renal failure rather than MEN-I syndrome.


Treatment of a large high-grade neurofibrosarcoma with concomitant vinblastine, doxorubicin, and radiotherapy.

A patient with neurofibromatosis developed a large inoperable malignant schwannoma on the posterior neck. The tumor underwent complete local regression following combined-modality treatment with radiotherapy, vinblastine, and doxorubicin. Vinblastine may be effective in combined-modality therapy.