Conformational adaptability of the terminal regions of flagellin. Secondary structure formation in the disordered terminal regions of flagellin were studied by circular dichroic (CD) spectroscopy, Fourier transform infrared spectroscopy, and x-ray diffraction. The terminal regions of flagellin are known to form alpha-helical bundles upon polymerization into flagellar filaments. We found from comparative CD studies of flagellin and its F40 tryptic fragment that a highly alpha-helical conformation can be induced and stabilized in the terminal regions in 2,2,2-trifluoroethanol (TFE) containing solutions, which is known to promote intra-molecular hydrogen bonding. Two oligopeptides, N(37-61) and C(470-494), each corresponding to a portion of terminal regions and predicted to have a high alpha-helix forming potential, were synthesized and studied. Both peptides were disordered in an aqueous environment, but they showed a strong tendency to assume alpha-helical structure in solutions containing TFE. On the other hand, peptides were found to form transparent gels at high concentrations (> 15 mg/ml) and all three methods confirmed that the peptides become ordered into a predominantly beta structure upon gel formation. Our results show that large segments of the disordered terminal regions of flagellin can adopt alpha-helical as well as beta structure depending on the environmental conditions. This high degree of conformational adaptability may be reflecting some unique characteristics of the flagellin termini, which are involved in self-assembly and polymorphism of flagellar filament.


Teaching macromolecular modeling.

Teaching macromolecular modeling. Training newcomers to the field of macromolecular modeling is as difficult as is training beginners in x-ray crystallography, nuclear magnetic resonance, or other methods in structural biology. In one or two lectures, the most that can be conveyed is a general sense of the relationship between modeling and other structural methods. If a full semester is available, then students can be taught how molecular structures are built, manipulated, refined, and analyzed on a computer. Here we describe a one-semester modeling course that combines lectures, discussions, and a laboratory using a commercial modeling package. In the laboratory, students carry out prescribed exercises that are coordinated to the lectures, and they complete a term project on a modeling problem of their choice. The goal is to give students an understanding of what kinds of problems can be attacked by molecular modeling methods and which problems are beyond the current capabilities of those methods.


Distinct conformers of alkylchrysene diol epoxide-deoxyguanosine adducts detected by proton NMR. Proton NMR spectra, obtained in MeOH-d4, of the major DNA adduct of 5,7-dimethylchrysene-1,2-diol 3,4-epoxide, identified as 1(R),2(S),3(S)-trihydroxy-4(S)-(N2-deoxyguanosyl)-1, 2,3,4-tetrahydro-5,7-dimethylchrysene, showed the presence of two distinct conformers. One conformer, similar to those observed previously in spectra of peracetates of related DNA adducts of anti-diol epoxides of polynuclear aromatic hydrocarbons, had a chair-like conformation of the tetrahydrobenzo ring. The other conformer, which has not been previously observed, had a boat-like conformation of the tetrahydrobenzo ring. This conformer was converted to the chair-like conformer upon addition of D2O or trifluoroacetic acid to the MeOH-d4 solutions of the adduct. The new conformers were also observed in proton NMR spectra of major DNA adducts of 5-methylchrysene- and 5,6-dimethylchrysene-1,2-diol 3,4-epoxides.


Oxidation of cyclopenta[cd]pyrene by human and mouse liver microsomes and selected cytochrome P450 enzymes. The metabolism of the environmental pollutant and suspected human carcinogen, cyclopenta[cd]pyrene (CPP), was investigated. Human liver microsomes from three individuals were examined, as well as CD-1 mouse liver microsomes. Five new metabolites recently identified in our lab, 4-hydroxy-3,4-dihydroCPP, 3,4-dihydroCPP-cis-3,4-diol, 4-oxo-3,4-dihydroCPP, 3,4,9,10-tetrahydroCPP-trans-3,4-trans-9,10-tetrol, and trans-3,4-dihydroCPP-3, 4,x-triols, as well as the known major metabolite, 3,4-dihydroCPP-trans-3,4-diol, were all observed from the incubations of human liver microsomes and CPP. Even though all three human samples were capable of producing all the metabolites identified from the mouse liver microsomal incubations of CPP, the quantity of each metabolite varied among the microsomal samples. In an attempt to explain the variation among human liver samples, the microsomes derived from genetically engineered cells containing specific cytochrome P450 isozyme cDNAs were employed. It was found that the 3,4-cyclopenta double bond can be oxidized by the cytochrome P450 enzymes 1A1, 1A2, and 3A4. The 9,10 K-region double bond was not efficiently oxidized by cytochrome P450 1A1, but by P450 1A2 either from CPP or from the t-3,4-dihydrodiol. The lack of catalytic activity of 3A4 toward the t-3,4-dihydrodiol, despite its high activity toward CPP oxidation to tetrahydrotetrols, suggests the possibility of two dihydrodiol epoxides, 3,4-dihydrodiol 9,10-epoxide and 9,10-dihydrodiol 3,4-epoxide, of CPP.


Evidence of involvement of multiple sites of metabolism in the in vivo covalent binding of dibenzo[a,h]pyrene to DNA. The in vivo formation of dibenzo[a,h]pyrene-DNA adducts in mouse skin was assessed by laser-excited fluorescence spectroscopy at 77 and 4.2 K. Two adducts were identified with fluorescence origin bands at approximately 383.5 and 407.2 nm, and these were shown to possess pyrene and benzo[a]pyrene (B[a]P) chromophores, respectively. Both DNA-bound chromophores displayed considerable electron-phonon coupling and likely assume a highly base-stacked or quasi-intercalated configuration within DNA duplexes. The presence of B[a]P and pyrene aromatic systems indicates that two-electron or monooxygenation metabolism occurred on either the a or h benzo moieties (which are equivalent) in the former case, and on both these rings in the latter case. The presence of two adduct species agrees with 32P-postlabeling analysis of the DNA, which showed the presence of two major adducts in both thin-layer and high-performance liquid chromatographic separations.


Direct synthesis and identification of benzo[a]pyrene diol epoxide-deoxyguanosine binding sites in modified oligodeoxynucleotides. Adducts derived from the reaction of the benzo[a]pyrene metabolite model compound (+)-anti-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene [(+)-BPDE] with the single-stranded oligodeoxynucleotide 5'-d(TATGCGTAT) were obtained according to direct synthesis techniques described earlier [Cosman, M., Ibanez, V., Geacintov, N. E., and Harvey, R. G. (1990) Carcinogenesis 11, 1667-1672]. Four major adducts, involving trans and cis addition (trans/cis adduct ratio approximately 4.5) of (+)-BPDE to the exocyclic amino groups of guanines G4 and G6 (the numbers denote the positions of the guanines counted from the 5'-side) were obtained. These adducts can be separated from one another by reverse-phase high-performance liquid chromatography methods. The site of BPDE binding on either G4 or G6 can be determined from the electrophoresis band patterns on 20% polyacrylamide gels of the BPDE-modified oligonucleotides subjected to the G+A and G Maxam-Gilbert strand cleavage reactions [Maxam, A. M., and Gilbert, W. (1980) Methods. Enzymol. 65, 499-560]. The electrophoresis gel band patterns are different for unmodified DNA and the two different BPDE-modified oligonucleotides because (1) the strand cleavage fragments bearing BPDE residues migrate slower than the corresponding fragments derived from the unmodified oligonucleotide and (2) strand cleavage tends to be inhibited on the 5'-sides of BPDE-modified guanines in the G+A, but not the G reaction.


S-(2-hydroxy-3-buten-1-yl)glutathione and S-(1-hydroxy-3-buten-2-yl)glutathione are in vivo metabolites of butadiene monoxide: detection and quantitation in bile. Administration (ip) of butadiene monoxide, a toxic metabolite of 1,3-butadiene, to rats caused the appearance of two new biliary peaks when analyzed by HPLC chromatography. These peaks were isolated and identified as the regioisomeric glutathione conjugates, S-(2-hydroxy-3-buten-1-yl)glutathione (I) and S-(1-hydroxy-3-buten-2-yl)glutathione (II), by comparison of their HPLC retention times and fast atom bombardment mass spectra to those of synthetic standards. S-(4-Hydroxy-2-buten-1-yl)glutathione, a rearrangement product formed during chemical synthesis or storage of I, was not detected. Whether butadiene monoxide was given at a dose of 14.3 or 143 mumol/kg, the amount of conjugates excreted in 30 min was at least 85% of that excreted in 120 min. Conjugate excretion in 60 min did not exhibit saturation when the butadiene monoxide dose was varied between 14.3 and 286 mumol/kg; the total amount of the butadiene monoxide dose excreted as combined I and II averaged only 7.6 +/- 4.2% (mean +/- SD, n = 12), with approximately a 3:1 ratio of isomers I:II being excreted at all butadiene monoxide doses. Whereas these results indicate a role for glutathione S-transferase-catalyzed reactions in butadiene monoxide metabolism in vivo, biliary excretion of I and II can only account for a small fraction of the butadiene monoxide dose given.


Oxidative conversion by rat liver microsomes of 2-naphthyl isothiocyanate to 2-naphthyl isocyanate, a genotoxicant. The present study investigated the oxidative metabolism of 2-naphthyl isothiocyanate catalyzed by rat liver microsomes. Incubation of 2-naphthyl isothiocyanate, microsomes, and NADPH yielded either N,N'-di-2-naphthylurea or, on inclusion of 2-aminofluorene in the incubations, N-2-naphthyl-N'-2-fluorenylurea. These ureas were formed by the production of 2-naphthyl isocyanate, which reacted with its hydrolysis product, 2-aminonaphthalene, to yield the symmetrical urea or, with 2-aminofluorene, to form the mixed urea. Formation of N,N'-di-2-naphthylthiourea was also observed, since 2-aminonaphthalene reacted with the substrate. Urea formation was dependent on microsomes, NADPH, and O2. Use of microsomes from rats previously treated with Aroclor increased urea formation > or = 10-fold. The enzyme activity was inhibited by alpha-naphthoflavone, flavone, or CO and slightly inhibited by metyrapone, 7-ethoxycoumarin, or SKF-525A. It was not inhibited by methimazole or paraoxon. These data are consistent with a cytochrome P-450-dependent, oxidative desulfuration of the isothiocyanate to yield an isocyanate.


Fluoranthene metabolism: human and rat liver microsomes display different stereoselective formation of the trans-2,3-dihydrodiol. The metabolism of the environmental carcinogen fluoroanthene by human liver microsomes was compared to that by liver microsomes from rats treated with Aroclor 1254. Although the human-derived system gave primarily one product, similar metabolites were noted from each system. Enantiomers of the major metabolic product, in both cases the trans-2,3-dihydrodiol, were separated by chiral stationary-phase chromatography. Absolute configurations were assigned by application of the benzoate exciton chirality rules to the CD spectra of the 4-(dimethylamino)benzoyl esters. Liver microsomes from Aroclor 1254-treated rats produced the R,R enantiomer of the diol in 75-78% enantiomeric excess, while human liver microsomes produced this enantiomer in only 6-12% excess. The activities of these enantiomers were compared in Salmonella typhimurium strain TM677 mutagenicity assays employing the 9000g supernatant of Aroclor 1254-induced rat liver homogenates. Both the syn- and anti-2,3-dihydrodiol 1,10b-epoxides, which had only been inferred to be metabolites in previous studies, were isolated from the microsomal incubations by preparative reverse-phase HPLC. The evident exceptional aqueous stabilities of these diol epoxides were further examined by half-life determination experiments. Their tetrahydrotetrol hydrolysis products were also noted in the metabolite HPLC profiles. The structures of the tetrahydrotetrols were confirmed by total synthesis.


Identification and characterization of deoxyguanosine-crotonaldehyde adducts. Formation of 7,8 cyclic adducts and 1,N2,7,8 bis-cyclic adducts. Crotonaldehyde, a chemically reactive alpha,beta-unsaturated carbonyl compound, is an important industrial chemical and a ubiquitous environmental pollutant. It has been shown to be carcinogenic and mutagenic. We have studied the reaction of crotonaldehyde with nucleosides and 5'-mononucleotides and found three different types of adducts with deoxyguanosine and 2'-deoxyguanosine 5'-monophosphate. No adducts could be isolated either with nucleosides other than deoxyguanosine or with nucleotides other than 2'-deoxyguanosine 5'-monophosphate. With crotonaldehyde, deoxyguanosine produced 1,N2 and 7,8 adducts as well as 1,N2/7,8 bis-adducts. The 1,N2 adducts were mixtures of diastereomers: one pair in which the substituents in the newly formed ring were trans [adduct Ia (6S,8S) and (6R,8R)], about 94%, and another pair Ib in which they were cis. In the case of the 7,8-adducts IIa,b, the ribose was cleaved and a mixture of isomers in which the substituents were cis-IIa and trans-IIb (2:1) in the newly formed tetrahydropyrrole ring was observed. A 3:2 cis-IIIa and trans-IIIb mixture of 1,N2,7,8 bis-adducts was found with the isomerism in the newly formed tetrahydropyrrole ring in analogy to the 7,8 adducts IIa,b. The corresponding bis-adduct with the cis form in the newly formed tetrahydropyrimidine ring was not observed.


Simultaneous modelling of mexiletine and hydroxy-methyl-mexiletine data after single- and multiple-dose administration of a sustained-release mexiletine formulation. The pharmacokinetics of mexiletine and its metabolite hydroxy-methyl-mexiletine have been investigated following single-dose and during multiple-dose administration of a sustained-release form of mexiletine to six post-myocardial infarct patients. Comparison of single-dose and washout pharmacokinetics, after short-term multiple-dose administration, showed significant (p < 0.005), but not systematic, modifications in mexiletine apparent clearance for three patients. Furthermore, for these patients, simulation with both sets of parameters indicated that the steady-state was achieved before washout experiment in two cases. The fraction of mexiletine metabolized to hydroxy-methyl-mexiletine was lower for multiple-dose administration (about 18 per cent) than for the single dose (about 42 per cent). The hydroxy-methyl-mexiletine elimination rate constant was about four times that of mexiletine. Mexiletine clearance could be accounted for by other metabolic pathways. In one patient, hydroxy-methyl-mexiletine was undetectable even during multiple-dose administration, despite a significant increase in mexiletine clearance. However, the observed changes in mexiletine disposition had no therapeutic implications and active plasma levels were achieved by the third day of administration and maintained in the therapeutic range (0.75 to 2 micrograms ml-1) in all patients after a twice daily dosage regimen.


The enzymatic formation and chemical reactivity of quinone methides correlate with alkylphenol-induced toxicity in rat hepatocytes. The effects of o-alkyl substituents on both the cytochrome P450-catalyzed oxidation of phenols to p-quinone methides (QM's; 4-methylene-2,5-cyclohexadien-1-ones), and on the rates of nucleophilic additions to the 4-methylene carbon of QM's were investigated. The derivatives of 4-methylphenol studied were BHT (2,6-di-tert-butyl), BHTOH [6-tert-butyl-2-(hydroxy-tert-butyl)], BDMP (2-tert-butyl-6-methyl), BMP (2-tert-butyl), TMP (2,6-dimethyl), and DMP (2-methyl). QM formation was estimated to be in the range 0.17-0.70 nmol/(nmol of P450.min) in rat liver microsomes and 16-62 pmol/(10(6) cells.min) in isolated rat hepatocytes. QM's derived from BHT (BHT-QM), BHTOH (BHTOH-QM), BDMP (BDMP-QM), and TMP (TMP-QM) were synthesized and their rates of reaction with water and reduced glutathione (GSH) determined. BDMP-QM and TMP-QM were the most reactive, BHT-QM was consumed relatively slowly, and BHTOH-QM displayed intermediate reactivity. These variations in rate were rationalized by differences in hydrogen bonding with the carbonyl oxygen, which affects positive charge density at the site of nucleophilic attack. The loss of hepatocyte viability during incubations with BMP, BDMP, and BHTOH was preceded by GSH depletion. Pretreatment of hepatocytes with diethyl maleate exacerbated alkylphenol toxicity, and metyrapone protected the cells. These data, together with information on the formation and reactivity of QM's, strongly support the proposal that QM's mediate the toxicity of alkylated 4-methylphenols in rat hepatocytes.


Characterization of (+/-)-7,8,10-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-9-sulfonate. The genotoxicity of certain benzo[a]pyrene (BP) derivatives is significantly enhanced in strains of Salmonella typhimurium following addition of sulfite to the incubations. The interaction between sulfite and those BP derivatives also results in the formation of isomeric BP sulfonates. As these trihydroxy sulfonates are formed in incubations of BP derivatives and sulfite in which a marked potentiation of bacterial mutagenicity occurs, we have investigated the properties of these novel intermediates. The compound (+/-)-7,8,10-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-9-sulfonate (BPT-9-sulfonate) was isolated and characterized in terms of its chemical and biological activity. This BPT sulfonate isomer is formed by the addition of the sulfite anion radical to the 9,10-double bond of the known promutagen, (+/-)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol). Evidence for the free radical character of this addition includes the initiation of the reaction by either peroxidase-catalyzed or chemical one-electron oxidation of sulfite, the inhibition of the reaction by phenolic antioxidants, and the isolation and characterization of the chain termination product, 7,8-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-9,10-disulfonate (BPD-disulfonate). Analysis of incubations of S. typhimurium strain TA98 with BP-7,8-diol and sulfite, which resulted in a 10-fold increase in revertant bacterial colonies above control levels, showed that BPT-9-sulfonate and BPD-disulfonate were the only isolable products derived from BP-7,8-diol. This prompted a further investigation of the chemistry of these products. BPT-9-sulfonate was found to be quite stable in aqueous media, being refractory to acid- or base-catalyzed hydrolysis over a pH range of 3-11.(ABSTRACT TRUNCATED AT 250 WORDS)


Bioavailability of lithium from lithium citrate syrup versus conventional lithium carbonate tablets. The bioavailability of lithium citrate syrup was compared with that of regular lithium carbonate tablets in 18 healthy male human volunteers. Blood samples were collected up to 48 h after dosing. Lithium serum concentrations were determined by means of AAS. The absorption rate following oral administration of the syrup was greater (tmax 0.8 h) than following administration of regular tablets (tmax 1.4 h). Maximum lithium serum concentrations, however, were only about 10 per cent higher after syrup dosing and serum concentrations resulting from syrup and tablets were almost superimposable from 2 h after dosing. The terminal half-life of lithium was found to be 22 h after syrup as well as after tablet dosing. No side-effects were observed during the study. The bioavailability of lithium from syrup relative to tablets was found to be bioequivalent with respect to the maximum lithium serum concentration and the extent of drug absorption (AUC).


Mutagenic decomposition products of nitrosated 4-chloroindoles. 4-Chloro-6-methoxyindole, a constituent of fava beans, forms a potent direct-acting mutagen, 4-chloro-6-methoxy-2-hydroxy-1-nitrosoindolin-3-one oxime, when nitrosated. In order to better understand the properties of this mutagen, we have studied a readily-available analog, 4-chloro-2-hydroxy-1-nitrosoindolin-3-one oxime, prepared by nitrosation of 4-chloroindole. This analog is also mutagenic, and both mutagens decompose rapidly at neutral or higher pH to yield in each case a new, less potent mutagen which then reacts further to form a nonmutagenic final product. The two products arising from 4-chloro-2-hydroxy-1-nitrosoindolin-3-one oxime, on the basis of comparison of spectroscopic and chromatographic evidence with that from authentic standards, are 4-chloro-N-nitrosodioxindole and 4-chloroisatin; those arising from 4-chloro-6-methoxy-2-hydroxy-1-nitrosoindolin-3-one oxime appear to be the corresponding 6-methoxy analogs. The interplay of these pathways with respect to net biological activity, especially under gastric conditions, remains to be described.


Effect of congestive heart failure on clentiazem pharmacokinetics in a dog model. Clentiazem, 8-chloro diltiazem, is a calcium channel blocker currently undergoing evaluation for the treatment of stable angina and hypertension. As patients with ischaemic disorders often present some degree of heart failure, the aim of this study was to investigate the effect of congestive heart failure on clentiazem (200 micrograms kg-1, i.v. bolus) pharmacokinetics in a canine model. Congestive heart failure was induced in six dogs by rapid ventricular pacing (240 beats min-1) for 3-5 weeks. Clentiazem pharmacokinetics was studied in each dog under the control condition and after the development of clinical signs of heart failure (ascites, dyspnea, fatigue). Blood samples were collected up to 480 min post-dose. Clentiazem plasma concentrations were determined by high performance liquid chromatography. The area under the plasma concentration versus time curves (AUC0-infinity) was significantly increased in congestive heart failure dogs (8.8 +/- 1.6 vs 21.8 +/- 1.4 micrograms min ml-1) (mean +/- SEM). These changes were related to a reduction of the volume of distribution of the central compartment (0.9 +/- 0.1 vs 0.2 +/- 0.11 kg-1) and total body clearance (1.9 +/- 0.4 vs 0.7 +/- 0.21 h-1 kg-1). It is concluded that, in our model, congestive heart failure significantly modifies clentiazem disposition. These results suggest that caution should be exercised when clentiazem is given to patients with a low ejection fraction and a compromised cardiac function. Reduced loading and maintenance doses might be recommended in patients with severe congestive heart failure.


Formation of 3,N4-ethenocytidine, 1,N6-ethenoadenosine, and 1,N2-ethenoguanosine in reactions of mucochloric acid with nucleosides. Mucochloric acid, a genotoxic hydroxyfuranone found in chlorinated drinking water, was reacted with cytidine, adenosine, guanosine, and uridine. HPLC analyses with UV detection at 290 nm showed that one major product peak was formed in the reactions of cytidine, adenosine, and guanosine. The products formed in reactions carried out at 90 degrees C for 24 or 45 h were isolated by HPLC and characterized by 1H and 13C NMR spectroscopy, direct inlet chemical ionization mass spectrometry, and UV absorbance spectroscopy. The structures of the products were assigned to 3,N4-ethenocytidine, 1,N6-ethenoadenosine, and 1,N2-ethenoguanosine. In reactions performed at 37 degrees C and pH 7.0 the same compounds were detected after a reaction time of 7 days. No observable reaction took place between mucochloric acid and uridine.


Nickel(II)-mediated oxidative DNA base damage in renal and hepatic chromatin of pregnant rats and their fetuses. Possible relevance to carcinogenesis. DNA base damage was studied in renal and hepatic chromatin of nickel(II)-injected pregnant female F344/NCr rats and their fetuses under conditions leading to initiation of sodium barbital-promotable renal tumors, but not liver tumors, in the male offspring. Pregnant rats were given a total of 90 or 180 mumol of nickel(II) acetate/kg body wt in a single ip dose on day 17 or in 2 or 4 ip doses between days 12 and 18 of gestation. Control rats received 180 mumol of sodium acetate/kg body wt. The animals were killed 24 or 48 h after the last injection. Chromatin was isolated from livers and kidneys from both adults and fetuses and analyzed by gas chromatography/mass spectrometry with selected ion monitoring. Eleven products derived from the purine and pyrimidine bases in DNA bases were identified and quantified. These were the following: 5-hydroxy-5-methylhydantoin, 5-hydroxyhydantoin, 5-(hydroxymethyl)uracil, cytosine glycol, thymine glycol, 5,6-dihydroxycystosine, 4,6-diamino-5-formamidopyrimidine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 8-hydroxyadenine, 2-hydroxyadenine, and 8-hydroxyguanine (8-OH-Gua). Nickel(II) exposure increased the content of these products, especially those derived from purines, in both renal and hepatic chromatin of pregnant rats. The major difference between these two organs was the content of 8-OH-Gua, which increased greatly in the kidney but remained unchanged in the liver.(ABSTRACT TRUNCATED AT 250 WORDS)


In vitro metabolism and DNA adduct formation from the mutagenic environmental contaminant 2-nitrofluoranthene. The metabolism and DNA adduct formation by the mutagenic environmental contaminant 2-nitrofluoranthene (2-NFA) were studied. Incubation under aerobic conditions with liver microsomes of rats pretreated with 3-methylcholanthrene yielded trans-7,8-dihydroxy-7,8-dihydro-2-nitrofluoranthene, trans-9,10-dihydroxy-9,10-dihydro-2-nitrofluoranthene, and 7-, 8-, and 9-phenolic metabolites. When the epoxide hydrolase inhibitor 3,3,3-trichloropropylene was present in the incubation, only phenolic metabolites were detected. Under hypoxic conditions, 2-aminofluoranthene was obtained, together with a trace of the ring-oxidized metabolites. The activated metabolite, N-hydroxy-2-aminofluoranthene, was prepared in situ and reacted with calf thymus DNA. Upon enzymatic hydrolysis of the DNA and purification by HPLC, a C8-substituted deoxyguanosine adduct, N-(deoxyguanosin-8-yl)-2-aminofluoranthene, was identified by mass and proton NMR spectral analysis. This adduct was also formed at a level of 10 pmol/mg of DNA when 2-NFA was metabolized by xanthine oxidase, 6 pmol/mg of DNA from incubation with liver microsomes of rats pretreated with 3-methylcholanthrene, and 3-pmol/mg of DNA from metabolism by liver microsomes of rats pretreated with phenobarbital.


Metabolism of the food mutagen 2-amino-3-methylimidazo[4,5-f]quinoline in nonhuman primates undergoing carcinogen bioassay. 2-Amino-3-methylimidazo[4.5-f]quinoline (IQ) is a potent bacterial mutagen and rodent carcinogen which also produces hepatocellular carcinoma in monkeys. The metabolism and disposition of this procarcinogen were investigated in monkeys undergoing carcinogen bioassay and in monkeys given an acute dose of IQ. Analysis of urine, feces, and bile revealed that IQ was extensively metabolized. A number of metabolites in urine were purified by high-performance liquid chromatography and characterized by 1H NMR and mass spectroscopy. Metabolites resulted from cytochrome P450-mediated ring oxidation at the C-5 position or N-demethylation. These metabolites could be further transformed by conjugation to sulfate or beta-glucuronic acid. Glucuronidation and sulfamate formation at the exocyclic amine group were other major routes of metabolism. Enteric bacteria also contributed to IQ biotransformation by forming the 7-oxo derivatives of IQ and N-demethyl-IQ. The metastable N2-glucuronide conjugate of the carcinogenic metabolite, 2-(hydroxyamino)-3-methylimidazo[4,5-f]quinoline, was found in urine. This indicates that metabolic activation through cytochrome P450-mediated N-oxidation occurs in vivo and that glucuronidation is a means of transport of the carcinogenic metabolite to extrahepatic tissues.


The effects of chronic oral diltiazem and cimetidine dosing on the pharmacokinetics and negative dromotropic action of intravenous and oral diltiazem in the dog. The kinetics and negative dromotropic action of intravenous (1 mg kg-1) and oral (5 mg kg-1) diltiazem were studied in dogs after acute doses, after treatment for 3 days with oral diltiazem (5 mg kg-1, t.i.d.), and after 3 days' treatment with oral diltiazem (5 mg kg-1 t.i.d.) and cimetidine (200 mg t.i.d.). Plasma concentrations of diltiazem and two of its metabolites, desacetyldiltiazem and desmethyldiltiazem were measured by HPLC. Chronic oral dosing significantly lowered both the systemic and oral clearance of diltiazem, with no changes in either the volume of distribution or blood binding of diltiazem. Cimetidine treatment resulted in a significant reduction in diltiazem oral clearance from chronic control with no effect on its systemic clearance. The AUCs of both metabolites increased by greater than threefold from acute to chronic oral dosing; however, the ratio of each metabolite's AUC to that of diltiazem AUC was not significantly altered. Cimetidine treatment significantly lowered these ratios. The negative dromotropic potency of diltiazem after the acute oral dose was three times greater than that after intravenous or chronic control dosing. Cimetidine treatment resulted in further lowering chronic oral diltiazem potency. These data indicate that the disposition and negative dromotropic action of diltiazem is dependent both on the route of administration and the duration of treatment, and can be altered by co-administration with cimetidine.


Simultaneous monitoring of bemoradan pharmacokinetics and hemodynamics in mongrel dogs. This study was designed to determine the relationship between the hemodynamic responses to bemoradan, a novel cardiotonic agent, and its plasma levels after single administration of an oral dose or a 15 minute i.v. infusion to mongrel dogs, and to demonstrate that the clinical capsule formulation of bemoradan elicits a pharmacological response in the dog. Four conscious, instrumented mongrel dogs received each of four bemoradan treatments (30 micrograms kg-1 i.v., 100 micrograms kg-1 i.v., 100 micrograms kg-1 suspension p.o., or 1 mg capsule p.o.) in a Latin square cross-over design. Plasma levels of bemoradan up to 24 h post-dosing were determined by HPLC. Cardiac contractility (dP/dt), heart rate, and arterial blood pressure were continuously monitored for 8 h and again at 24 h. Results of the study indicate that there was a significant correlation between bemoradan plasma levels and dP/dt. The 1 mg clinical capsule formulation was well absorbed when compared to an oral suspension and an i.v. dose. Peak increases in dP/dt of 64 per cent at 15 min for the suspension and 53 per cent at 1 h for the capsule were observed.


In vivo evaluation of the effect of the size and opsonization on the hepatic extraction of liposomes in rats: an application of Oldendorf method. In the hepatic uptake of large particles such as liposomes, a serum component called opsonin plays an important role. In this study, the 'Oldendorf method' is introduced to evaluate the hepatic extraction under the condition of single passage, which enabled examination of the effect of opsonization on liposome uptake by the intact liver. 14C-labelled liposomes and, an internal reference, 3H-H2O were injected as a bolus into portal vein. Liver uptake index (LUI) was calculated from the ratio of the extraction of 14C to that of 3H. The effect of liposome size (mean diameter of 0.8, 0.4, 0.2, and 0.05 micron) and opsonization (preincubation with fresh blood for 5 min) on liposomal hepatic uptake were investigated using this method. LUI increased with size significantly (p < 0.001), and opsonization enhanced LUI only for the large liposomes (0.8 micron). This result suggests that the critical diameter of opsonization for these liposomes lies between 0.4 and 0.8 micron.


A thermospray liquid chromatography/mass spectrometry method for analysis of human urine for the major malondialdehyde-guanine adduct. A method is described for detection and quantitation of the major malondialdehyde-guanine adduct (M1G) based on thermospray liquid chromatography/mass spectrometry. A stable isotope analog of M1G ([2H2]M1G) was used as an internal standard. Thermospray mass spectra of M1G and [2H2]M1G showed intense protonated molecular (MH+) ions that were suitable for use in quantitation of M1G. M1G was purified from human urine and reduced with NaBH4 to a dihydro derivative that was cleanly separated from the contaminants in the urine. The detection limit of reduced M1G by thermospray liquid chromatography/mass spectrometry in the selected ion monitoring mode was 250 fmol on column. Six human urine samples were analyzed, and the concentrations of M1G were below the limit of detection of the assay (500 fmol/mL).


Extrahepatic production of the lignocaine metabolite monoethylglycinexylidide (MEGX). The extrahepatic production of monoethylglycinexylidide (MEGX) from lignocaine was measured in a 20-year-old female rendered anhepatic while awaiting liver transplantation 4 days after a paracetamol overdose. Following hepatectomy and during continuous arteriovenous dialysis, cardiac stability and control over rising intracranial pressure was restored. Lignocaine (1 mg kg body weight-1 intravenously over 2 min) reached a subtherapeutic peak serum concentration of 0.89 mg l-1 and was rapidly and exponentially cleared, reaching the lower limit of detection after 5 h (cf. around 2 h in normal subjects). There was significant production of MEGX at extrahepatic sites with serum concentrations rising from undetectable levels to 15 micrograms l-1 at 15 min and to a peak of 30 micrograms l-1 at 2 h and falling thereafter. MEGX concentrations were similar in arterial, venous, and pulmonary arterial blood, suggesting minimal MEGX production in the heart, lungs or skeletal muscle. Extrahepatic production of MEGX may contribute to total MEGX formation and should be considered when interpreting test results.


Genetic disease: effects on human health. Genetic variation (chromosomal, single gene, multifactorial) is a significant cause of morbidity and mortality in human populations. It increases in relative importance along with improvement in socioeconomic and environmental conditions and in association with prudent lifestyle. It affects longevity, development, reproductive capability, schooling, work and appearance of individuals. Its evidence is ubiquitous in the health care disciplines. Health care systems in general do not provide resources to meet the demand for genetics. Prevention of mutations, avoidance of their effects by reproductive counseling and treatment to offset mutant phenotypes are increasingly relevant scientific initiatives with broad social implications.


Delivery of genetic screening services.

Delivery of genetic screening services. This paper describes the genetic services in the United Kingdom and how the evolution of genetic screening services is taking place. Since these Community Genetic Services depend on the offer of a screening test that affects the whole population, it is essential that the community is given genetic education and an opportunity to discuss the issues before services are initiated. In this way, the differing beliefs and needs of individual communities are appropriately taken into account. The development of screening services for cystic fibrosis will show whether this community-orientated model can be successful.


Absenteeism as a symptom of occupational ill-health in hospitals and its repercussion on quality assurance. The purpose of the present study was to evaluate the importance of absenteeism due to illness in the hospital organization. A measurement of absenteeism due to illness among hospital professionals of the National Health System in the Alicante province (Spain) during the years 1988, 1989 and 1990 was carried out, as well as its repercussion on quality assurance in health care. The absenteeism index (frequency) was 5.39, 6.38 and 6.79 in 1988, 1989 and 1990, respectively. The extent of absenteeism (duration) in 1988 was 19.7 days per worker, and was 24.23 in 1989 and 26.45 in 1990. Hospitals with more than 400 beds presented during these periods poor absenteeism self-control. Maintenance personnel, nurses, administrative personnel and graduates had, in this order, the highest scores of days lost. We show that the dimension of Intrinsic Job Satisfaction, Extrinsic Job Satisfaction and Job Monotony are interrelated with absenteeism.


Outcomes of care for anesthesia services: a pilot study. This paper describes a pilot study involving 4347 patient abstracts collected by anesthesia providers during the first quarter of 1991 under the auspices of the American Association of Nurse Anesthetists. Descriptive statistics are presented on surgical site, type of provider and utilization of anesthetic agents as well as the prevalence of 87 preexisting conditions and the incidence of 103 adverse events.


Why do cardiology out-patient appointments fail in Saudi Arabia? In a retrospective study, 534 appointments for a cardiology clinic were analysed to determine the frequency of no-show and to identify contributory factors. The overall rate of no-show was 30.1%, which is higher than the 18% and 20% reported from other teaching hospitals. Variables with the strongest univariate association with no-show were nationality (Saudi 35%, non-Saudi 22%; p = 0.0015), gender (males 34%, females 25%; p = 0.03), heart failure (present 44%, absent 27.9%; p = 0.005) and valvular heart disease (present 23.4%, absent 32.6%; p = 0.04). Turning to a stepwise logistic regression to predict no-show behaviour, we found that nationality, gender and heart failure were significant, while valvular heart disease was not. We conclude by recommending that physicians and mass media should share in the responsibility of stressing the importance of keeping out-patient department (OPD) appointments to all patients especially those who are more prone to no-show behaviour, namely male nationals. Telephone and mailed reminders have been used successfully to improve attendance at the OPD, and can be evaluated in our community.


An evaluation of the mathematical skills of beginner Swedish nursing students. During the autumn of 1989 a mathematical test was given to 997 students attending a 2-year program of nursing education and 188 students on a 3-year program of nursing education. The test consisted of 65 items. Apart from the four fundamental rules of arithmetics, the test included calculations concerning fractions, scales, transformation of units and percentages. The level of the test was comparable to the 9-year compulsory school curriculum. The main purpose of the present study was to investigate whether nursing students had adequate knowledge and skills in mathematics when they started their studies. The result showed that approximately 50% of the items were solved correctly. The nursing students attending a 3-year nursing education program had higher average scores than the group attending a 2-year education program. The result is discussed in connection with medical errors. The Swedish Medical Responsibility Board supervises nurses' professional work and penalizes those who give wrong doses of drugs to patients.


"Avoidable" causes of death in Sweden 1974-85. Mortality from potentially avoidable causes of death in Sweden 1974-85 for ages 0-64 years was analysed, based on a list published by Rutstein et al., [N Engl J Med 294: 582, 1976] of conditions that were suggested to serve as negative indicators of the quality of health care. In females 22% of deaths and in males 18% of deaths had underlying causes that were included in the list. Deaths from avoidable causes in the Rutstein list were concentrated to a limited number of causes of death and cause of death groups, such as neoplasms and diseases of the respiratory system. Both preventable and treatable conditions were found among the most common avoidable causes of death. There were differences in the causes of death that were most common between different age groups. A large number of the avoidable causes of death were relatively rare in Sweden. Areas for continued methodological development are suggested concerning the choice of avoidable causes of death and the definition of standards.


Comparison of information: a way to improve the quality of health care. Quality of care is a growing concern of health care providers, financers and consumers, as regards both the cost and the outcome. Uncertainty about what is appropriate health care has triggered interest in comparing the outcomes of different health care practices. This paper addresses the necessity to compare information on variations in practice, utilization and cost of health care delivery. However, for general improvements in the outcome of health care, a variety of partners, such as ministries of health, non-governmental organizations, professional societies, third party financers and consumer groups, must be actively involved. The Regional Office for Europe of the World Health Organization (WHO/EURO) regularly collects data from member states. Such data collection is part of the monitoring of the European regional strategy for Health for All by the year 2000. These data can and actually are being used for the purpose of comparison of achievements in accordance with the European Health for All policy in member states. In addition, other indicators, information systems and longitudinal databases are being developed in collaboration with different professions, and provider and community groups to serve as instruments for quality assurance in the European region primarily at the level of health care services. However, this approach, which has been promoted over the past decade by the Quality of Care and Technologies programme of the WHO/EURO, is gradually gaining momentum at all levels of health care as a useful tool for quality assessment and development.


Mouse embryo stem cells: their identification, propagation and manipulation. The early mouse embryo contains a transient population of pluripotential stem cells which are responsible for generating both the foetal primordia and extraembryonic membranes. The characterisation of murine embryo stem cells and their isolation and propagation in culture provides the first instance in which pure populations of normal stem cells are directly accessible to the researcher. This marks a considerable advance in stem cell biology which may pave the way to the dissection of general stem cell control mechanisms and the identification of key regulatory factors. In addition, the genetic manipulation of embryo stem cells affords a unique avenue for experimental intervention in mammalian development and for controlled modification of the mouse germ line.


Multipotential stem cells in the vertebrate CNS. The functional differences between the many mature neuronal types make the origins of cellular diversity in the nervous system one of the most intriguing problems in biology. This paper discusses recent experiments which explore the mechanisms generating cell diversity in the vertebrate central nervous system, focusing particularly on the immediate precursor to neurons and on neuronal differentiation. Transplantation experiments have been used to explore regional and temporal differences in the cells of the neuroepithelium. The effects of specific genes on neuronal development have been examined by mutational analysis in transgenic mice and in established neuronal stem cell lines. The combination of methods now available allow increasingly powerful dissection of the molecular and cellular mechanisms that generate the large number of different neurons in the vertebrate brain.


The O-2A(adult) progenitor cell: a glial stem cell of the adult central nervous system. Systematic comparison of the properties of oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells derived from optic nerves of perinatal and adult rats has revealed that these two populations differ in many fundamental properties. In particular, O-2A(perinatal) progenitor cells are rapidly dividing cells capable of generating large numbers of oligodendrocytes over a relatively short time span. Oligodendrocyte differentiation generally occurs synchronously in all members of a clone, thus leading to elimination of that clone from the pool of dividing cells. However, some O-2A(perinatal) progenitors are also capable of giving rise to O-2A(adult) progenitors. These latter cells express many of the characteristics of stem cells of adult animals, including the capacity to undergo asymmetric division and differentiation. We suggest that precursors which function during early development give rise to terminally differentiated end-stage cells and to a second generation of precursors with properties more appropriate for later developmental stages. It is this second generation of precursors which express the properties of stem cells in adult animals, and we therefore further suggest that our work offers novel insights into the possible developmental origin of stem cells.


The haemopoietic stem cell: properties and control mechanisms. All the cells of the immuno-haemopoietic system derive ultimately from a single pluripotent stem cell through processes of commitment and differentiation. The stem cell is also likely to be capable of extensive self renewal. Many factors which potentially control these processes have been identified and characterised both in vitro and in vivo. We discuss the nature of the haemopoietic stem cell and also the factors which have been identified as potential stem cell regulators. We also draw parallels from embryonal stem cell differentiation to derive a model of intrinsic determination of the haemopoietic stem cell. The possible role of developmental regulators, such as homeobox containing genes, are discussed in the context of differentiation commitment of the haemopoietic stem cell.


Epidermal keratinocyte stem cells: their maintenance and regulation. The epidermis is a stratified epithelium consisting of interfollicular regions and appendages (hair follicles, sweat glands, sebaceous glands). The dominant cell type (the keratinocyte) is arranged in groups of cells termed epidermal proliferative units (EPUs), and one centrally-located clonogenic stem cell is ultimately responsible for replacing the remainder of the cells in the unit. Evidence is reviewed which indicates that the epidermal Langerhans' cell (ELC), and the cells comprising the dermis, may modify the keratinocyte microenvironment to create stem cell 'niches' and cellular diversity within the basal layer.


Epithelial stem cells in gastrointestinal morphogenesis, adaptation and carcinogenesis. Epithelial stem cells have a central role in the physiological maintenance of epithelial tissues in a steady state. In addition it is becoming clear that these cells may also be implicated in the generation of metaplastic epithelia and ultimately the generation of neoplastic epithelia. This review aims to compare and contrast stem cells and their functions in different areas of the gastrointestinal tract in an attempt to emphasise unifying concepts. New methods of 'organotypic' cell culture can potentially be applied to the study of gastrointestinal stem cells and are, therefore, also discussed.


What do patients want to know about their inflammatory bowel disease? Being informed is not only a patient's right, it is also important in order to improve compliance and management. Apart from verbal consultations, informative material is often produced without prior analysis of what patients consider important to know. Patient information needs were defined in order to plan future educational programmes. A 44-item questionnaire was given to 100 Inflammatory Bowel Disease (IBD) out-patients (50 Crohn's Disease (CD) and 50 Ulcerative Colitis (UC). Sixty-two per cent of those with UC and 78% of those with CD consider themselves insufficiently informed about their disease. CU and CD patients indicated different priorities concerning areas where further information is requested: CD patient needs were aetiology, diet, symptoms, history, new treatments, risks deriving from treatment, cancer risk and consequences on work. UC patients indicated a different priority order: risk of cancer, new treatments, symptoms, psychological and diet factors and aetiology. The media preferred by patients were: specifically prepared books (73%), video-cassettes (20%) and leaflets (25%). Ninety per cent think that educational material prepared according to their needs could be very useful; however 35% think that knowledge of the possible severity of their disease might increase their anxiety.


Animal models for heat stroke studies.

Animal models for heat stroke studies. Heat stroke is a medical emergency where quick diagnosis and management of victims are essential for positive prognosis. Several biochemical, physiological and hematological changes were observed in heat stroke. It seems that all of these changes are a consequence of induced tissue damage, or may have been a compensatory action by the body. Induction of hyperthermia and temperature measurement are important components in heat stroke studies to determine the stage of progression or regression of heat stroke. Several animal models have been established by investigators in heat related studies. Rats, dogs, monkeys, baboons, cows, rabbits, sheep and chicks have all been used in such studies that allow manipulation of exposure conditions and various designs of experiments. Amongst these species, rats, rabbits and sheep are the most suitable models because of their similarity to man in response to high temperature and in relation to their availability, cost and simplicity of handling. Such models may be used to study various pharmacological and biochemical parameters and functions concurrently. Further informations could also be obtained from isolated organ studies. The present review is to analyse and compare the available methodology for heat stroke studies.


Primary sclerosing cholangitis: an analysis of 37 retrospective cases. The clinical and laboratory findings of 37 patients with primary sclerosing cholangitis (PSC) were reviewed. Mean age was 43.8 years, sex ratio between males and females was 3:1; IBD was present in 91% of patients with 51% having ulcerative colitis, 23% unclassified colitis and 17% Crohn's disease. Twenty-seven patients (73%) were symptomatic presenting most commonly with fatigue, pruritus and hepato-splenomegaly. Cholangiography revealed abnormalities affecting both extrahepatic and intrahepatic biliary ductal systems in 51.8% of cases, and only the intrahepatic or extrahepatic biliary tree, respectively in 11.1% and in 37% of cases. The last prevalence was very high compared with that previously known. Clinical and biochemical data, when compared between asymptomatics and symptomatics, demonstrated a significant difference only for alkaline phosphatase which increased in the symptomatic group and for prothrombin activity which decreased among symptomatic patients. Nevertheless, predictive value of sALP for the presence of PSC was high when pts were pooled together with a randomly selected group of 36 non-affected persons that underwent ERCP for suspected primary sclerosing cholangitis: sensitivity was 94% and specificity 78%.


4-Trimethylammonium antipyrine: a quaternary ammonium nonradionuclide marker for blood-brain barrier integrity during in vivo microdialysis. The well-controlled microdialysis (MD) study of substance permeation into brain extracellular fluid (ECF) and cerebrospinal fluid requires consideration of blood-brain barrier (BBB) integrity, which might be compromised by microdialysis probe implantation. Others have assessed BBB integrity with radionuclide markers. A nonradionuclide marker may be desirable in many studies. A charged antipyrine analogue may be useful to determine BBB integrity with concomitant antipyrine characterization of probe efficiency (Yokel et al., 1992, J Pharmacol Toxicol Methods 27:135-142), and may not require another analytical technique. We synthesized, validated, and evaluated 4-trimethylammonium antipyrine (4TMA-AP) as a BBB integrity marker. BBB permeation was determined by calculation of a BBB integrity percentage (Pi) from brain/blood concentrations. The PiS of Evan's blue, which does not permeate the intact BBB, and 4TMA-AP were not significantly different in rats without known BBB disruption, suggesting a lack of 4TMA-AP permeation through the intact BBB. When MD probes were slowly implanted into the frontal cortex, 4TMA-AP PiS were usually zero. Intracarotid oleic acid injection to open the BBB significantly increased 4TMA-AP PiS, suggesting that 4TMA-AP entered brain ECF when the BBB was compromised. Rapid probe implantation produced increased 4TMA-AP PiS, suggesting BBB disruption. The predicted appearance of 4TMA-AP in brain ECF suggests that it is a BBB integrity marker.


In vitro measurement of endogenous norepinephrine release from small blood vessels with short stimulation trains. We have previously developed a low-volume perfusion-superfusion system for studying neurotransmitter release in small blood vessels. We now extend this technique to allow for the measurement of neurotransmitter release with short stimulation trains in order to examine modulating factors such as activation of prejunctional receptors or the effects of altered external calcium. Segments of rat tail artery were perfused in the presence of deoxycorticosterone and cocaine (10(-5) M). For short trains, nerves were activated with five sequential trains, each 4 sec in length (total of 160 pulses) at 8 Hz. For long trains, nerves were activated continuously for 3 min for a total of 1440 pulses. Norepinephrine in the perfusate and tissue norepinephrine were quantitated with high-performance liquid chromatography (HPLC) and electrochemical detection. Fractional norepinephrine release was significantly greater with short as compared to long trains when extracellular calcium was maintained at 1.6 mM. When nerves were stimulated with short trains, fractional norepinephrine release was very sensitive to altered extracellular calcium, with a significant decline when extracellular calcium was reduced to 1 mM and a significant increase when extracellular calcium was increased to 5 mM. In contrast, with long-stimulation trains increases or decreases in calcium had no effect on fractional norepinephrine release. This unique method facilitates the study of norepinephrine release with short trains or low-frequency activation and will also make it easier to study presynaptic receptor function as well as interactions between extracellular calcium and presynaptic effectiveness.


Assessment of changes in vasomotor tone in vivo using intravascular ultrasound. This study tested the capability of high-frequency, two-dimensional real-time intravascular ultrasound (IVUS) in the detection of dynamic changes of large vessel diameter in vivo. An IVUS-catheter (4.8 French, 20-MHz mechanical transducer) was inserted via the femoral vein, and advanced to the inferior vena cava of anesthetized rabbits (n = 7). The depth of field of the transducer allowed for visualization of the entire cross-sections of both the inferior vena cava (IVC) and the adjacent aorta. Changes in vessel diameter were induced pharmacologically using norepinephrine (NE) and glyceryltrinitrate (GTN), which were injected intravenously before and after the administration of L-NG-nitro-arginine methyl ester) L-NAME, a specific inhibitor of endothelium-derived relaxing factor (EDRF)-biosynthesis. Vasoconstriction and -dilation could be observed continuously from the two-dimensional real-time recordings of vessel cross-sections. Vessel diameters and cross-sectional areas (CA) were measured from still frames at given time intervals of drug infusion, and blood pressure and heart rate were recorded continuously. Following NE, an increase of aortic and a simultaneous decrease of venous CA were observed, while GTN elicited the opposite responses. Inhibition of EDRF was followed by an augmentation of the vascular responses. It is concluded that IVUS is capable of detecting changes in vascular dimensions in vivo. Thus in large vessels, IVUS may become a method for the direct assessment of vasomotion in vivo.


Computed tomography detection of gastrointestinal neoplasms. Gastrointestinal (GI) tract neoplasms can be detected, evaluated and staged by computed tomography (CT), especially from high-resolution examinations, but routine CT scanning may occasionally discover GI neoplasms in patients with non-specific symptoms. We therefore evaluated the sensitivity and main signs of CT examinations resulting from routine sessions. In 46 out of 1560 patients who underwent abdominal CT, it was possible to obtain a diagnosis of primitive gastric or colo-rectal tumour by means of barium X-ray and/or endoscopy. The sensitivity rate of CT obtained from the present study was 86% in gastric tumours and 87% in colonic tumours. In gastric lymphoma, infiltrating carcinoma of stomach and rectal carcinoma, the sensitivity rate was 100%. The CT diagnosis of GI neoplasms was based on diffuse or local thickening of the gut on a solid dishomogenous mass showing contrast enhancement. The prevalence of various patterns depends on site of origin and on macroscopic features of the neoplasia. In conclusion the results suggest that abdominal CT, even if performed with a method not specifically devoted to GI tract, has high sensitivity rates and then a high probability of detecting GI neoplasms when unsuspected at time of the examination. In our series 11% of GI neoplasms were initially diagnosed by routine CT examination.


Early events in human colorectal carcinogenesis. Aberrant crypts and microadenoma. Experimental studies have allowed the identification of foci of aberrant crypts on the fixed methylene-blue-stained mucosal surface of rodent colons after colon carcinogens administration. Similar lesions can also be observed and quantified on the mucosal surface of the human colon, using the same technique. Several foci showed dysplasia at histological examination, thus allowing the identification of microadenomas. Microadenoma may thus be a precursor lesion of adenomas and colorectal cancer. The identification of aberrant crypt foci and microadenomas in vivo could provide a new end-point for studies on the effect of environmental and genetic factors in the early stages of cancer of the large intestine. Further studies are needed to define the natural history of these lesions. Moreover, a critical evaluation of current colon cancer prevention strategies should be considered.


Removal of venous endothelium with air.

Removal of venous endothelium with air. Much research on the activity and half-life of endothelium-derived substances has entailed the removal of endothelium from arteries by mechanical or enzymatic processes. It has been observed that the technique used for the removal of arterial endothelium may profoundly affect smooth muscle function and release of prostanoids by the vessel wall. The function and patterns of regeneration of arterial endothelium have been extensively described, but there is a relative paucity of information about the venous endothelium, due in part to the difficulty of its removal. We developed a technique for removal of the endothelium of rabbit femoral veins by passing a stream of air through the lumen of the vessel to dry and remove the endothelium. The effectiveness of endothelium removal was verified by the lack of in vitro reactivity to endothelium-dependent relaxing substances, examination of frozen sections of vessels, labeled with fluorescent-tagged acetylated low-density lipoprotein, with fluorescent light microscopy and scanning electron microscopy of vessel segments. Air drying effectively removed the endothelium and abolished mechanical responses to endothelium-dependent vasodilators but did not affect the function of the smooth muscle. We propose the use of air to remove endothelium from veins to be used to study endothelium-derived factors since this method achieves complete removal of endothelium without causing detectable damage (morphological or functional) to the remainder of the vessel wall.


Determination of acetazolamide in human serum by enzymatic assay. Carbonic anhydrase (CA) inhibitors, such as acetazolamide (AZ), formerly used as diuretics, still play a role in the treatment of glaucoma, epilepsy, and altitude sickness. There is now hard evidence from both in vitro and in vivo studies in animals that carbonic anhydrase plays a vital function in bone loss. Acetazolamide blocks bone resorption in these experimental models. We have postulated that acetazolamide has potential for the treatment of human conditions associated with bone loss. In preparation for a clinical trial of acetazolamide's effectiveness in this regard, we developed an enzymatic method for determining the total concentration of acetazolamide in human serum. Acetazolamide is stripped from binding to serum proteins by adding 10(-6) M salicylic acid and adjusting the pH to 2.5, followed by ultrafiltration through a membrane (10 kD cutoff). The latter permits the free acetazolamide to enter the filtrate but retains any carbonic anhydrase (31 kD) which may contaminate the serum from hemolysis. The carbonic anhydrase inhibitory activity in the filtrate, representing the acetazolamide, is determined in a carbonic anhydrase assay using acetazolamide as the standard. Recoveries of acetazolamide added to human serum ranged from 83% to 94% depending on the concentration. Precision, as judged by the coefficient of variation, was 10.5%.


Assay for expression of methotrexate-resistant dihydrofolate reductase activity in the presence of drug-sensitive enzyme. A simple, continuous spectrophotometric assay for dihydrofolate reductase (DHFR) activity was adapted for determination of drug-resistant enzyme activity expressed from transfected genes in cells containing drug-sensitive enzyme. Methotrexate inhibition characteristics in this assay system were assessed for the murine wild-type (WT) enzyme as well as variant genes encoding amino acid substitutions at codon positions 22 (arg22) or 31 (trp31) expressed in DHFR-deficient Chinese hamster ovary (CHO) cells and in mouse 3T3 cells. Methotrexate concentrations were thus identified which maximized inhibition of the wild-type enzyme while maintaining substantial arg22 or trp31 activity. Mixing experiments were conducted to determine the minimum amount of drug-resistant enzyme distinguishable from a constant amount of wild-type enzyme in the presence of methotrexate. Mixtures of enzymes from a variety of sources (WT, arg22, or trp31 expressed in CHO or 3T3 cells) demonstrated a detection limit of 0.03 to 0.06 nmol/min. Assay of methotrexate-resistant arg22 DHFR appeared to be limited by the low level of activity associated with this enzyme variant, whereas assay of the trp31 variant was limited by enzyme inhibition at lower concentrations of methotrexate. The assay was thus applicable to two quite diverse DHFR variants and may be useful for assaying the expression of other drug-resistant DHFR genes as well after introduction into cells containing drug-sensitive enzyme.


Real-time measurement of microcirculation of skin by reflectance spectrophotometry. A reflectance spectrophotometric measurement was developed to analyze the microcirculation of the skin in real time. The relative absorption (RA) spectra were obtained from skin tissue 10 times per second using a spectro-multi-channel-photodetector system. In this system, white light was projected onto the skin of the back of the anesthetized rat and the spectrum of the reflected light between 450 and 643 nm was analyzed. Two absorption peaks at wavelengths of about 540 and 577 nm were observed, corresponding to the absorption peaks in the oxyhemoglobin (HbO2) spectrum. Either phlebotomy or the i.v. injection of norepinephrine (NE) reduced the RA spectrum. Peaks of the reduction in spectrum [(RA spectrum before the treatment) - (RA spectrum after the treatment)] were observed at about 540 and 577 nm. Injection of NE (0.75-48 ng/100 g B.W., i.v.) reduced the RA value at 577 nm dose-dependently, suggesting a decrease in the skin blood flow due to vasoconstriction. In addition, the content of HbO2 and capillary permeation were measured at the same time after the i.v. injection of Evans blue (EB) dye. As indices of HbO2 content and capillary permeation, RA changes at 540 and 610 nm (the latter is an absorption peak in the EB spectrum) were measured in real time, respectively. Both RA values increased dose-dependently after the intradermal injection of histamine (0.3-100 micrograms/site), suggesting the presence of vasodilation and an increase in permeability. The time at which the RA value for HbO2 reached a maximum was shorter than that for EB. These observations suggest that the method described here can detect changes in the HbO2 content and permeation of skin microcirculation at the same time, and in real time.


Laparoscopic versus open cholecystectomy and the need for a randomized trial: a survey of surgeons and ethical committees in the British Isles. A questionnaire on the necessity and ethics of a randomized trial to compare laparoscopic cholecystectomy and open cholecystectomy was sent to 200 randomly selected surgeons in the British Isles, of whom 117 replied. Of these, 58% thought that a trial was needed to compare laparoscopic cholecystectomy with open cholecystectomy by either minilaparotomy or the conventional approach. Nearly half (45%) expressed interest in participating in such a trial. A hypothetical protocol for a trial was sent to 40 ethical committees; 25 gave approval and only 3 considered that a trial was unethical (12 refused to comment). This survey shows wide support for a trial comparing laparoscopic cholecystectomy with open cholecystectomy.


Extraperitoneal endoscopic inguinal hernia repair. The endoscopic extraperitoneal hernioplasty as reported in this study is a similar repair to that achieved by the conventional preperitoneal repair as described by Stoffa, Nyhus, and Rignault. However, this new repair is completed via a totally extraperitoneal approach. Thus, it eliminates all early and late complications related to the violation of the peritoneal cavity as proposed by other intraperitoneal laparoscopic approaches to hernia repair. This report demonstrates the safety and feasibility of this procedure while offering the patient the advantages of a minimally invasive surgical procedure which can be performed under regional anesthesia.


A comparative analysis of adhesion reduction, tissue effects, and incising characteristics of electrosurgery, CO2 laser, and Nd:YAG laser at operative laparoscopy: an animal study. In this study, the relative efficacy and tissue effects of lasers and electrosurgery at operative laparoscopy were evaluated. Thirty rabbits underwent surgical procedures to create extensive intraperitoneal adhesions. The animals were then randomly assigned to laparoscopic adhesiolysis utilizing either electrosurgery, CO2 laser, or Nd:YAG laser exclusively for the assigned group. Each surgical tool was utilized at its optimal power density to achieve the best results. The depth of thermal injury on ovarian and uterine tissues, and the speed at which various segments of the uterine horn were transected were also compared. All three modalities significantly reduced (p < 0.01) intraperitoneal adhesions by approximately 50%. The depth of thermal injury was threefold greater with the Nd:YAG laser than either electrosurgery or the CO2 at both ovarian and uterine tissues (p < 0.001). The speed of transection across the uterine horn was significantly slower (p < 0.001) with the Nd:YAG (2.6 +/- 0.3 sec) than either the CO2 laser (1.4 +/- 0.2 sec) or electrosurgery (1.5 +/- 0.2 sec). From this study, the authors conclude that the Nd:YAG laser causes more tissue damage and is less efficient at incising tissue than either CO2 or electrosurgery, but that all three modalities are equally effective for laparoscopic adhesiolysis.


Laparoscopic cholecystectomy: an initial community experience. The initial 950 consecutive laparoscopic cholecystectomies performed in one city at four hospitals by 30 general surgeons are reported, covering a period from April 4, 1990 to April 3, 1991. There were two operative deaths (0.2%), three common bile duct lacerations (0.3%), two subhepatic abscesses, two bowel perforations, and three bile leaks, two requiring laparotomy. Seven episodes of bleeding occurred, of which five required laparotomy, but none involved a major vessel. Sixty-five procedures were converted to open (6.7%). The mean operative time was 85.4 min. Intraoperative cholangiography was adequately completed in 49.8% and not attempted in 30.3%. Thirteen patients (2.7%) were found to have common duct stones. The pathologic diagnoses were chronic cholecystitis in 784 patients (82.5%), acute cholecystitis in 145 (15.3%), and cancer of the gallbladder in one (0.1%). Hospital stays ranged from 4 h to 31 days (mean 49.5 h). This procedure can be learned and performed safely in a community setting.


Preliminary experience with thoracoscopic surgery. Thoracoscopic surgery was accomplished in 12 patients utilizing thoracoscopic instruments and a stapler. Five patients were treated for recurrent, spontaneous pneumothoraces, for which blebectomies were done; three patients for pulmonary nodules, for which wedge resections were done; one patient for cryptogenic pleural effusion; one patient for debridement of an empyema cavity; one patient for traumatic bronchopleural fistula; and one patient with AIDS for interstitial lung disease. All patients were done under general anesthesia in the lateral decubitus position and were prepped and draped for a standard thoracotomy. They underwent endobronchial double-lumen ventilation so that the ipsilateral lung could be deflated to create a working space. In addition, insufflation of 4 to 5 mmHg was also used. Trocars were placed using a blunt technique. The mid-axillary trocar was at about the eight intercostal space and was used for the endoscope, and then additional trocars were placed, usually on the anterior axillary line and posterior axillary line at about the fifth intercostal space. If adhesions were encountered, the lung was grasped atraumatically with a clamp or retractor used to give counteraction, and the adhesions were lysed with shears or electrocautery. After a thorough exploration of the hemithorax involved, the area of pathology was grasped with the clamp, which was used for countertraction. Through a 12 mm trocar, a stapler was introduced and fired. This staples and also transects on a 3 cm length. Several firings were usually necessary to remove the pathology, which, if malignant, was placed in a retrieval bag.(ABSTRACT TRUNCATED AT 250 WORDS)


Comparison of laparoscopic cholecystectomy versus elective open cholecystectomy. Laparoscopic cholecystectomy has essentially replaced open cholecystectomy as the procedure of choice for gallbladder disease. This rapid shift to laparoscopic cholecystectomy, however, has resulted more from marketing forces than from prospective clinical trials. To evaluate the safety and efficacy of laparoscopic cholecystectomy, the first 486 laparoscopic cholecystectomies at two institutions were studied. These results were then compared to the results of the last 6 months of elective open cholecystectomy cases prior to the introduction of laparoscopic surgery. The age, sex, height, and weight were similar in both groups. The mean operative time was 78.8 +/- 1.8 min for laparoscopic cholecystectomy and 62.7 +/- 2.6 min for open cholecystectomy (p < 0.01). The mean time for tolerating a regular diet was 1.23 +/- 0.04 days in the laparoscopic group versus 2.44 +/- 0.07 days in the open group (p < 0.01). Laparoscopic cholecystectomy patients required only oral pain medications by a mean of 1.22 +/- 0.03 days postoperatively compared to 2.55 +/- 0.07 days postoperatively for those undergoing open cholecystectomy (p < 0.01). The mean length of hospitalization was 1.58 +/- 0.07 days for laparoscopic patients and 3.55 +/- 0.11 days for open patients (p < 0.01). Thirty-one patients undergoing laparoscopic cholecystectomy were converted to open cholecystectomy (6.4%). The most common reasons for conversion to open cholecystectomy were acute inflammation, adhesions, and bleeding. For the laparoscopic patients, the morbidity rate was 8.4% and the mortality rate 0.2% (1 death). In the open cholecystectomy group the morbidity rate was 8.0% and there were no deaths. The most troublesome complication in laparoscopic cholecystectomies continues to be bile leaks and bile duct injuries.(ABSTRACT TRUNCATED AT 250 WORDS)


Operative laparoscopy in the management of tubal ectopic pregnancy. One hundred and seventeen consecutive patients with diagnosis of ectopic pregnancy admitted to Temple University Hospital between October 1989 and March 1992 were divided into two groups. Group 1 consisted of 56 patients with operative laparoscopy and Group 2 consisted of 61 patients treated by laparotomy. The two groups were similar for age, race, parity, gestation, presentation, and location of the ectopic gestations. Fifty seven percent of patients in the laparoscopy group were treated by salpingectomy and 43% by salpingostomy, compared to 84% and 16% respectively in the laparotomy group. Mean operative time for laparoscopy was 58 min and 42 min for laparotomy. Complication rates were similar in the two sub-groups. Only two patients in the laparoscopy group required subsequent laparotomy, one to assure hemostasis and one, 5 weeks following surgery, for persistent trophoblastic disease. Operative laparoscopy was associated with a significantly shorter length of hospital stay (1.25 v. 4.39 days). This reflected in a lower cost of hospital stay ($10,105 vs. $13,608). The present data demonstrates that operative laparoscopy is not only safe and effective, but also more economical than open laparotomy in the treatment of ectopic pregnancies. This procedure is expected to replace laparotomy for the treatment of most cases of tubal ectopic pregnancy.


Laparoscopic closure of perforated duodenal ulcer. Simple patch closure is a treatment option for perforated duodenal ulcer. A 46-year-old male was admitted with evidence of perforation of a duodenal ulcer. The decision was made to carry out laparoscopy to confirm the diagnosis and to close the perforation. This was accomplished without difficulty by suture closure of an anterior perforation and application of an omental patch. The patient had an uneventful recovery. Laparoscopy can be of value in the diagnosis and treatment of perforated duodenal ulcer. The principles of management should be essentially similar to those adhered to during open surgery.


Laparoscopic resection of benign stromal tumor of the stomach. Laparoscopic surgery has recently become commonplace in the general surgeon's armamentarium of surgical approaches to many problems. This has been seen most notably with laparoscopic cholecystectomies. Herein is reported a case of an incidental benign stromal tumor of the stomach being resected laparoscopically.


Alternative management of choledocholithiasis. This paper is presented to clarify the role of ERCP in conjunction with laparoscopic cholecystectomy. As the field of surgery moves more toward less invasive procedures, our management schemata may also need revision. With the popularity of laparoscopic cholecystectomy came a large increase in the number of patients treated with cholelithiasis, and therefore an increase in patients with choledocholithiasis. The authors believe that treatment options exist to avoid open common bile duct procedure in most cases.


Laparoscopy through an inguinal hernia for diagnosis of intraperitoneal pathology. During the repair of an incarcerated inguinal hernia, a 68-year-old gentleman was found to have unsuspected ascites. Upon palpation of the peritoneal cavity through the hernial sac, superficial nodules could be appreciated, suggesting abnormal pathology. A laparoscope was placed through the hernia sac after establishing a pneumoperitoneum through the same approach. This allowed visualization of the entire peritoneal cavity and appropriate exploration of the abdominal viscera. Biopsies confirmed unsuspected metastatic renal cell carcinoma. The authors describe this technique and suggest possible indications for its use.


Laparoscopic appendectomy: a simplified technique. Laparoscopic appendectomy can easily be performed by a surgeon familiar with laparoscopic cholecystectomy. The technique described employs the same spatial relationships which are used in laparoscopic cholecystectomy. By maximizing the similarities between the two operations, surgeon and assistants can rapidly become comfortable with laparoscopic appendectomy. Of the laparoscopic appendectomies attempted 88% were successfully completed. The technique and results are presented and discussed.


A simplified technique for open laparoscopy using disposable trocars. An easily accomplished method for performing open laparoscopy with disposable trocars is presented. This technique provides for safe entry into the peritoneum and subsequently facilitates removal of the gallbladder and any larger stones at the completion of the procedure.


A comparative ultrastructural study of the cardiac and skeletal striated muscles of the skink. Ultrastructural differences between cardiac and striated muscle fibres observed in the ocellated skink (Chalcides ocellatus ocellatus) indicate that functional differences have a structural correlate in the muscle cells. Although the myofibrils of both the cardiac and skeletal muscles displayed the same typical band pattern, differences in mean nuclear diameter and sarcomere length and in the A, I, Z, H, and M bands (discs) were observed. In addition, differences were found in the number, size and distribution of the mitochondria in the two types of muscle fibres. The Golgi complex was indistinguishable in skeletal muscles, but small Golgi vesicles could (with difficulty) be identified in cardiac muscle fibres. Skeletal striated muscle fibres were also found to contain significantly more glycogen granules, smooth sarcoplasm vesicles and heterochromatin than cardiac muscles. Three types of junctional complexes were identified in the cardiac muscles.


Effect of adriamycin on the morphology of the erythrocytes and blood parameters of the toad Bufo regularis. The effect of the anticancer drug adriamycin on the morphology of the erythrocytes as well as some blood parameters were studied in the Egyptian toad Bufo regularis. Injecting the toads subcutaneously in the dorsal lymph sac with adriamycin at a dose level of 2 mg/kg body weight once every three weeks for 12 weeks induced the formation of abnormalities in the cytoplasm and nuclei of the erythrocytes. Its administration also caused severe anaemia, since the number of red blood corpuscles, haemoglobin content and haematocrit values were obviously decreased throughout the whole experimental period. The total leucocytic count was also diminished. On the other hand, the erythrocyte sedimentation rate was increased all over the experiment.


Abnormal delayed-type hypersensitivity in mice born to females treated with vitamin A during pregnancy. The effects of vitamin A (retinolum palmiticum) administered to pregnant mice on the cell-mediated immunity of the offspring were studied in random-bred ICR mice. Delayed-type hypersensitivity (DTH) to sheep red blood cells was assayed after immunization of the control and experimental young in the 4th postnatal week. The administration of vitamin A, in doses of 5,000 IU, on days 15-17 of pregnancy depressed DTH of the female offspring. On the other hand, the administration of single doses of 5,000 and 10,000 IU on the 16th day of pregnancy increased DTH in the offspring. The effects were correlated to the sex and body weight of the offspring, the highest DTH being observed in the female offspring with a low body weight.


Biochemical properties of cartilage and multinucleate chondroclast formation. This study demonstrates a relationship between the biochemical properties of the extracellular matrix of a cartilaginous model of long bone and the fusion of mononuclear clastic elements into multinucleate chondroclasts. The results suggest that the chondroitin sulphate level in the extracellular matrix has an inhibitory effect on this process.


Modulation by vitamin C of tumour incidence and inhibition in oral carcinogenesis. This study reports the mechanism of involvement of vitamin C in the pathogenesis of induced oral carcinogenesis in the hamster cheek pouch epithelium. This site was exposed to either DMBA singly or in combination with vitamin C or DMBA for 7 weeks followed by only vitamin C till tumour induction. Macroscopically, vitamin C reduces the epithelial tumour incidence in the hamster cheek pouch. Microscopically tumours induced by DMBA alone were well differentiated squamous carcinomas in all the animals, whereas those induced by DMBA plus vitamin C were papillary epidermoid carcinomas with minimal invasion. These observations suggest that vitamin C is capable of restricting the growth of the initiated cells and does not allow the invasion of the subepithelium. The mechanism of action of vitamin C in the process of restricting growth and invagination was clearly shown in ultrastructural pathology too. In vitamin C exposed tumours the dermal epidermal junction of the cheek pouch epithelium maintains basement membrane integrity, with the presence of cellular organelles and cytoskeletal structure of the cells.


Developmental, histological and ultrastructural studies on the harderian gland of the Egyptian toad, Bufo regularis Reuss. The present study shows that the Harderian gland of the toad Bufo regularis starts to appear during development at stage 57 concomitant with the development of the nictitating membrane. In the adult toad, the gland was found to be formed of one lobe and occupying the antero-ventral aspect of the orbital cavity. No anatomical differences were noted between the male and female glands. The histological and ultrastructural studies showed that the Harderian gland is a compound tubular gland composed of rounded tubules lined by a single layer of epithelial cells and having a meshwork of myoepithelial cells surrounding the gland tubules. Differences were observed in the epithelial cells of female and male toads Bufo regularis. While females have a dilated rough endoplasmic reticulum and several secretory granules throughout the cytoplasm, males have a compact rough endoplasmic reticulum and numerous secretory granules and vacuoles.


Malformations of the eye in the exencephalic tail-short mice during its development. Exencephalic tail-short (Ts/+) embryos were obtained from mating normal mice of Patch strain with Ts/+ mice from Babl/c strain. The most common eye malformations in the exencephalic embryos were exophthalmy, microphthalmy, eye ball irregularity and corneal opacity. Squint eye and unilateral anophthalmy occurred at a lower frequency. The eye abnormalities were recognized histologically by 11-days of gestation. The retina was retarded in development. Lens fibres were swollen and some of them appeared degenerated. Lens epithelium was protruded anteriorly, detached from the underlying lens and joined the corneal endothelium. The lens remained in close contact to the ectodermal layer, forming a thick lens stalk. At the 16-day stage, the corneal epithelium became irregular in thickness and the corneal stroma was discontinuous, having disarranged collagenous fibrils. In a few embryos the corneal endothelium was absent. In the squint-eyed embryo, the cornea bulged through the opened eyelids, while in an anophthalmic embryo it was thick and underdeveloped. Generally, the upper and lower eyelids of the exencephalic embryos were asymmetrical, and the lower lid appeared covered by a thick irregular peridermal layer.


The importance of prenatal period to the new-borns population quality. Approximately 60% of all human pregnancies terminate spontaneously before birth, mostly before being recognized. Exceptionally high prevalence of abnormalities has been found in this reproduction output. Original frequency of chromosomal (ChA) and developmental anomalies (DA) is not known, but may be very high. From 100 spontaneously aborted fetuses about 50 are ChA, approximately 70 are DA and only about 20 are without any pathological finding. Using data on ChA and DA distribution in newborns and those in abortuses--together with the knowledge on abortion rates in various gestation phases--the prenatal population structure may be reconstructed: in the pregnancy recognition phase about 7% ChA and 10-15% DA conceptuses remain in the population of surviving fetuses. Prenatal selection decreases gradually the frequency of anomalies to the values that are usually found in new-borns. The disappearance of particular anomalies is nonrandom, changing the malformation spectrum continually. The risk of prenatal elimination of the anomaly is not invariably proportional to the degree of phenotype damage of the fetus. Study of the hidden part of prenatal population offers the possibility to cover a considerably larger fraction of the whole reproduction output and supplies new valuable knowledge.


The rearrangement of the cranial part of embryonal body in chick studied by linear marking. II. Relations of the neck, thorax and shoulder. The linear marking method was employed for studying morphogenetic movements of tissues involved in formation of the cranial part of the chick embryonic body. A total of 120 chick embryos marked on days 3-5 were used in this study with the following results: The cranial shift of axial structures and somites with respect to the other components of the cranial part of the embryonic body, described in our earlier papers, has been proven. This shift is considered crucial from the point of understanding and proper interpretation of the following formative processes: The prospective mesenchyme of the wall of the thorax shifts continuously in the cranial direction. The rate of the shift of the mesenchyme in the dorsal part of the developing body wall appears considerably lower than that of the somites. The cranial shift of the mesenchyme of the ventral part of the body wall is directed ventromedially and proceeds more rapidly than in the dorsal part of the body wall. The ventromedial deviation of the cranial shift is gradually increased in the craniocaudal sequence. In this way, the mesenchyme of the lateral body wall replaces step by step the membrana reunions to cover the heart, rapidly growing lungs and the liver. The presumptive shoulder shifts cranially at the rate similar to that of the somites. In this way, the shoulder slides over the body wall mesenchyme formerly located in the more cranial position. The shoulder undergoes rotation by the mechanism which is discussed.


Structure of the coronary arteries during the prenatal period in man. The coronary arteries of human foetuses from the 4th to 10th month of gestation were examined in the light and the electron microscope. Marked changes were demonstrated in the course of prenatal development, particularly in the media and adventitia (thickening, an increase in the number of collagen fibrils). In regions of coronary artery branching, here and there we found intimal pads or diffuse thickening of the intima, which were separated from the media by a mostly thin or discontinuous inner elastic membrane with an accumulation of smooth muscle cells and a smaller quantity of elastic fibrils. The intimal thickenings, which occurred mostly in the region of bifurcation of the left coronary artery and at sites where branches sprang from the r. ventricularis anterior of the left coronary artery, were already discernible at the beginning of the period in question (i.e. in the 4th month). In some cases, oedematous changes were found in the pads, where they were characterized by dilatation of the elastic membranes and smooth muscle cells by proliferating structureless matter.


Effects of growth hormone on thymocyte development from progenitor cells in the bone marrow. The effect of growth hormone (GH) on T cell differentiation was studied in young and old mice, employing in vivo and in vitro experimental approaches. Injections of GH during a period of 3 months to young and old mice resulted in a significant increase in the cell number and the percentage of CD3+ cells in the thymus of the old, but not in the young mice. Treatment of intact fetal thymus (FT) lobes with recombinant human GH (hGH) had no significant effect on cell numbers or on the values of CD4/CD8 thymocyte subsets. When partially depleted FT (10 Gy) were colonized with bone marrow (BM) cells and subsequently cultivated on monolayers of GH3, a GH-secreting cell line, the values of T cells deriving from the donor BM cells were elevated. Treatment with hGH to cocultures of lymphoid-depleted FT (dGUA) with BM lent further support to the idea that GH affects the newly emigrating BM cells, rather than the resident thymocytes. The results suggest that GH affects the thymocyte progenitors in the BM at the early stage of their development in the thymus.


In vivo effects of growth hormone on thymus function in aging mice. It is well demonstrated that the normal functioning of the thymus gland is under neuroendocrine control. Thus, steroid, thyroid, and pituitary hormones can affect distinct structural and/or functional thymic parameters. Particularly growth hormone (GH) was shown to be capable of restoring some thymus functions in old individuals. This prompted us to carry out a multiparametric analysis of the thymus in young, middle-aged, and old mice, subjected to GH treatment lasting 3 or 6 weeks. For that, we treated animals with daily injections of ovine GH (2 micrograms/g BW). Although the general microarchitecture of the thymus remained unchanged following in vivo GH treatment, there was a clearcut increase in thymulin production, independent of the age group analyzed. Regarding the lymphoid compartment, we could not find evidence of changes in total thymocyte numbers nor in the subsets phenotypically defined by the expression of CD3, CD4, and CD8 antigens. Nonetheless, in GH-treated middle-aged and old mice, the concanavalin A-dependent proliferative response of thymocytes, as well as IL-6 production were enhanced compared to age-matched controls. These findings support the notion that GH has a pleiotropic effect upon the thymus, functionally affecting both microenvironmental and lymphoid compartments of the organ.


The production of growth hormone and insulin-like growth factor-I by the same subpopulation of rat mononuclear leukocytes. In the present study, we evaluated the subpopulation of lymphoid cells from normal and hypophysectomized rats producing GH and IGF-I in vitro. The data show that removal of the pituitary results in depression of GH production in spleen, thymus, and bone marrow and an increase in the peripheral blood leukocytes. The changes in the percentage of cells producing GH in hypophysectomized animals are not due to a single cell type but appears to influence the T-helper, T-cytotoxic, and B-cell subsets. Interestingly, no significant changes in the levels of GH RNA were detected between control and hypophysectomized animals after the in vitro culture. We also found that the increase in GH production in spleen cell cultures after mitogen stimulation could be accounted for by an increase in the percentage of T cells producing GH. Lastly, we demonstrated that the cells positive for GH production were also positive for IGF-I production. This later finding coupled with our previous results suggest that an autocrine regulatory circuit may be important for the production of leukocyte-derived irGH and irIGF-I within the immune system.


Insulin and IGF-I stimulate normal and virally transformed T-lymphocyte cell growth in vitro. We used normal and HTLV-II-transformed T-lymphocytes as target cells to study clonal proliferative responses to physiologic and supraphysiologic concentrations of insulin and IGF-I. Responses of both growth factors were measured in the presence and absence of alpha IR-3, and IGF-I receptor-blocking antibody. A biphasic response to insulin was noted in all cell lines with the first peak [78 +/- 6.6% (mean +/- SE) above control] occurring at 1.4 or 1.6 nmol/liter and a second peak (84 +/- 4.9% above control) occurring at 18.0 nmol/liter. Following preincubation with alpha IR-3, the overall clonal profile in response to insulin was significantly reduced [F(7,56) = (10.4, p < .0001] as a result of blunting at high physiologic and supraphysiologic insulin concentrations, i.e., > or = 1.6 nmol/liter. As expected, the overall clonal profile in response to IGF-I was blocked by alpha IR-3 [F(4,32) = 11.6, p < .0001]. These data show that insulin at both physiologic and supraphysiologic concentrations, as well as IGF-I, stimulate virally transformed T-lymphoblast growth. The significant inhibition of growth responses to high concentrations of insulin and to IGF-I by alpha IR-3 suggests mediation of these effects through the IGF-I receptor. Similar studies were performed using freshly isolated, phytohemagglutinin (PHA)-stimulated T-lymphocytes. The overall response to insulin was significantly reduced compared to the profile of transformed T-lymphoblasts [F(7,70) = 4.9, p = .0002] as a result of blunting at physiologic insulin concentrations < 1.8 nmol/liter. In response to IGF-I, the clonal profile of PHA-stimulated T-lymphocytes was slightly reduced compared to that of virally transformed T-lymphoblasts [F(4,40) = 3.4, p = .0174]. Thus, both insulin and IGF-I receptor-effector mechanisms are involved in the growth of virally transformed T-lymphoblasts, whereas the IGF-I receptor-effector mechanism appears to play a more significant role in the growth of normal, mitogen-activated T-lymphocytes.


In vivo activation of circulating monocytes by exogenous growth hormone in man. Animal studies have shown that administration of growth hormone improves wound healing. Monocyte activation is a prerequisite for optimal repair of damage. In vitro, human recombinant growth hormone was shown to be a potent human monocyte chemoattractant. It induced random migration and chemotaxis at picomolar concentrations of recombinant human growth hormone; combinations of growth hormone with other chemoattractants deactivated the chemotactic response. Other functions of monocytes that are activated by growth hormone include release of superoxide anion and production of cytokines. In order to test activation of human monocytes by growth hormone in vivo, we investigated the effects of recombinant human growth hormone administration on monocyte migration in nine healthy young adults. After a single dose of recombinant human growth hormone (4 IU subcutaneously injected), random migration of circulating monocytes significantly increased, whereas chemotaxis of monocytes that was maximally stimulated with f-Meth-Leu-Phe decreased (p < .05). The alterations paralleled the concomitantly measured plasma levels of growth hormone. After recombinant human growth hormone administration, no changes were seen in plasma levels of proinflammatory cytokines. These in vivo data on monocyte migration are comparable to effects of growth hormone on monocyte migration in vitro and strongly suggest that recombinant human growth hormone can activate circulating monocytes in man.


Modulatory effect of prolactin on the resting and mitogen-induced activity of T, B, and NK lymphocytes. Prolactin (PRL) has been shown to contribute to the development of lymphoid tissues and maintenance of physiological immune function. Here we show that the role of the hormone extends to the control of the effector phase of the immune response. In addition to triggering resting lymphocytes to cell division, the hormone can also control the magnitude of their response to polyclonal stimuli. Concentrations of PRL in the physiological range increased the [3H]thymidine, [3H]uridine, and [3H]leucine incorporation of unstimulated NK cells cultured in serum-free conditions. The same concentrations of the hormone increased the response of NK, T, and B cells to the mitogenic stimuli interleukin 2 (IL2), phytohemagglutinin (PHA), and staphylococcus aureus cowan, respectively, the effect being maximally evident in the presence of suboptimal concentrations of the mitogens. By contrast concentrations of PRL five- to tenfold the physiological levels inhibited the mitogenic response to IL2 and PHA. These data indicate a double-faceted regulatory role of this hormone in vivo.


The stabilizing system of the spine. Part I. Function, dysfunction, adaptation, and enhancement. Presented here is the conceptual basis for the assertion that the spinal stabilizing system consists of three subsystems. The vertebrae, discs, and ligaments constitute the passive subsystem. All muscles and tendons surrounding the spinal column that can apply forces to the spinal column constitute the active subsystem. The nerves and central nervous system comprise the neural subsystem, which determines the requirements for spinal stability by monitoring the various transducer signals, and directs the active subsystem to provide the needed stability. A dysfunction of a component of any one of the subsystems may lead to one or more of the following three possibilities: (a) an immediate response from other subsystems to successfully compensate, (b) a long-term adaptation response of one or more subsystems, and (c) an injury to one or more components of any subsystem. It is conceptualized that the first response results in normal function, the second results in normal function but with an altered spinal stabilizing system, and the third leads to overall system dysfunction, producing, for example, low back pain. In situations where additional loads or complex postures are anticipated, the neural control unit may alter the muscle recruitment strategy, with the temporary goal of enhancing the spine stability beyond the normal requirements.