Cloning, sequencing, and expression in Escherichia coli of the Clostridium tetanomorphum gene encoding beta-methylaspartase and characterization of the recombinant protein. The gene encoding methylaspartase (EC 4.3.1.2) from Clostridium tetranomorphum has been cloned, sequenced, and expressed in Escherichia coli. The open reading frame (ORF) codes for a polypeptide of 413 amino acid residues (M(r) 45,539) of which seven are cysteine residues. The size of the ORF indicates that methylaspartase is a homodimer rather than an (AB)2 tetramer. The deduced primary structure of the protein shows no homology to enzymes that catalyze similar reactions or, indeed, any convincing homology with any other characterized protein. The recombinant protein is identical to the enzyme isolated directly from C. tetanomorphum as determined by several criteria. The enzyme is obtained in a highly active form (approximately 70% of the activity of the natural enzyme) and migrates as a single band (M(r) 49,000) in SDS-polyacrylamide gels. The kinetic parameters for the deamination of (2S,3S)-3-methylaspartic acid by the natural and recombinant proteins are very similar, and the proteins display identical potassium ion-dependent primary deuterium isotope effects for V and V/K when (2S,3S)-3-methylaspartic acid is employed as the substrate. In accord with the activity of the natural enzyme, the recombinant protein is able to catalyze the slow formation of (2S,3R)-3-methylaspartic acid, the L-erythro-epimer of the natural substrate, from mesaconic acid and ammonia. Earlier work in which the cysteine residues in the protein were labeled with N-ethylmaleimide had indicated that there were eight cysteine residues per protein monomer. One cysteine residue was protected by substrate. Here evidence is forwarded to suggest that the residue that was protected by the substrate is not a cysteine residue but the translation product of a serine codon. Kinetic data indicate that this serine residue may be modified in the active enzyme. The implications of these findings on the mechanism of catalysis are discussed within the context of a few emerging mode of action for methylaspartate ammonia-lyase.


Mechanistic studies on the human matrix metalloproteinase stromelysin. To probe the mechanism of stromelysin (SLN)-catalyzed peptide hydrolysis, we determined the pH dependence of kc/Km and solvent deuterium isotope effects on kc and kc/Km. pH dependencies of kc/Km were determined for the SLN-catalyzed hydrolysis of three peptides: Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Nle-NH2,Arg-Pro-Ala-Pro-Gln-Gln- Phe-Phe - Gly-Leu-NleNH2, and N-acetyl-Arg-Pro-Ala-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Nle-NH2 (cleavage at Gln-Phe bond). The pH dependencies are all bell-shaped with shoulders that extend from pH 7.5 to 8.5. The existence of a shoulder indicates that the reaction mechanism involves at least two routes to products. These curves are governed by three proton ionizations with pKa values of 5.4, 6.1, and 9.5. The solvent isotope effect measurements provided the following values: D(kc/Km) = 0.80 +/- 0.05 and D(kc) = 1.58 +/- 0.05. That D(kc/Km) and D(kc) are different suggests that the rate-limiting transition states for the processes governed by kc/Km and kc cannot be the same. We use these results, together with analogy to thermolysin catalysis, to develop a mechanism for SLN catalysis.


Haloperoxidase activity of Phanerochaete chrysosporium lignin peroxidases H2 and H8. Monochlorodimedone (MCD), commonly used as a halogen acceptor for haloperoxidase assays, was oxidized by hydrogen peroxide in the presence of lignin peroxidase isoenzymes H2 and H8. When oxidized, it produced a weak absorption band with an intensity that varied with pH. This absorbance was used as a simple method for the product analysis because it disappeared when MCD was brominated or chlorinated. We assessed the activity of the lignin peroxidases for oxidation of bromide by measuring the bromination of MCD, the formation of tribromide, the bromide-mediated oxidation of glutathione, and the bromide-mediated catalase-like activity. We analyzed the reaction products of MCD and the halide-mediated oxidation of glutathione when bromide was replaced by chloride. These enzymes demonstrated no significant activity for oxidation of chloride. Unlike other peroxidases, the lignin peroxidases exhibited similar pH-activity curves for the iodide and bromide oxidations. The optimum pH for activity was about 2.5. Surprisingly, this pH dependence of lignin peroxidase activity for the halides was nearly the same in the reactions with hydrogen donors, such as hydroquinone and guaiacol. The results suggested that protonation of the enzymes with pKa approximately 3.2 is necessary for the catalytic function of lignin peroxidases, irrespective of whether the substrates are electron or hydrogen donors. These unique reaction profiles of lignin peroxidases are compared to those of other peroxidases, such as lactoperoxidase, bromoperoxidase, chloroperoxidase, and horseradish peroxidase. Isozyme H2 was more active than isozyme H8, but isozyme H8 was more stable at very acidic pH.


Cleavage of the Lys196-Ser197 bond of prolyl oligopeptidase: enhanced catalytic activity for one of the two active enzyme forms. Prolyl oligopeptidase, a representative of a new family of serine proteases, is remarkably sensitive to ionic strength and has two catalytically active forms, which interconvert with changing pH [Polgár, L. (1991) Eur. J. Biochem. 197, 441-447]. To reveal whether conformational changes are associated with these effects, prolyl oligopeptidase was digested with trypsin. SDS gel electrophoresis studies demonstrated that tryptic digestion of the 75-kDa native protein generated two fragments, one having a molecular mass of 51 kDa and the other of 26 kDa. The digestion was markedly dependent on the ionic strength. Specifically, the digestion proceeded more rapidly in 0.05 M Hepes buffer than in 0.05 M Hepes buffer containing 0.5 M NaCl. Moreover, the nicked enzyme formed at low ionic strength was not stable but degraded and inactivated during an extended incubation. The digestion experiments suggested that alteration in the ionic strength elicits conformational changes in native prolyl oligopeptidase, and this may account for the enhanced catalytic activity observed at higher ionic strength. The two fragments of the nicked prolyl oligopeptidase did not separate during size-exclusion chromatography under nondenaturing conditions on a Superose 12 column and eluted in place of the native enzyme, indicating that they were strongly associated. The reactive serine residues of the nicked enzyme was labeled with tritiated diisopropyl phosphofluoridate, and the fragments were separated by size-exclusion chromatography in urea.(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of dipyridophenazine complexes of ruthenium(II): the light switch effect as a function of nucleic acid sequence and conformation. Spectroscopic parameters for two novel ruthenium complexes on binding to nucleic acids of varying sequences and conformations have been determined. These complexes, Ru(bpy)2dppz2+ and Ru(phen)2dppz2+ (bpy = 2,2'-bipyridine; phen = 1,10-phenanthroline; dppz = dipyrido[3,2:a-2',3':c]-phenazine) serve as "molecular light switches" for DNA, displaying no photoluminescence in aqueous solution but luminescing intensely in the presence of DNA. The luminescent enhancement observed upon binding is attributed to the sensitivity of the excited state to quenching by water; in DNA, the metal complex, upon intercalation into the helix, is protected from the aqueous solvent, thereby preserving the luminescence. Correlations between the extent of protection (depending upon the DNA conformation) and the luminescence parameters are observed. Indeed, the strongest luminescent enhancement is observed for intercalation into DNA conformations which afford the greatest amount of overlap with access from the major groove, such as in triple helices. Differences are observed in the luminescent parameters between the two complexes which also correlate with the level of water protection. In the presence of nucleic acids, both complexes exhibit biexponential decays in emission. Quenching studies are consistent with two intercalative binding modes for the dppz ligand from the major groove: one in which the metal-phenazine axis lies along the DNA dyad axis and another where the metal-phenazine axis lies almost perpendicular to the DNA dyad axis. Ru(bpy)2dppz2+ and Ru(phen)2dppz2+ are shown here to be unique reporters of nucleic acid structures and may become valuable in the design of new diagnostics for DNA.


DNA recognition and binding by the Euplotes telomere protein. The 51-kDa telomere protein from Euplotes crassus binds to the extreme terminus of macronuclear telomeres, generating a very salt-stable telomeric DNA-protein complex. The protein recognizes both the sequence and the structure of the telomeric DNA. To explore how the telomere protein recognizes and binds telomeric DNA, we have examined the DNA-binding specificity of the purified protein using oligonucleotides that mimic natural and mutant versions of Euplotes telomeres. The protein binds very specifically to the 3' terminus of single-stranded oligonucleotides with the sequence (T4G4) > or = 3 T4G2; even slight modifications to this sequence reduce binding dramatically. The protein does not bind oligonucleotides corresponding to the complementary C4A4 strand of the telomere or to double-stranded C4A4.T4G4-containing sequences. Digestion of the telomere protein with trypsin generates an N-terminal protease-resistant fragment of approximately 35 kDa. This 35-kDa peptide appears to comprise the DNA-binding domain of the telomere protein as it retains most of the DNA-binding characteristics of the native 51-kDa protein. For example, the 35-kDa peptide remains bound to telomeric DNA in 2 M KCl. Additionally, the peptide binds well to single-stranded oligonucleotides that have the same sequence as the T4G4 strand of native telomeres but binds very poorly to mutant telomeric DNA sequences and double-stranded telomeric DNA. Removal of the C-terminal 15 kDa from the telomere protein does diminish the ability of the protein to bind only to the terminus of a telomeric DNA molecule.


Enhanced stability of histone octamers from plant nucleosomes: role of H2A and H2B histones. Gel filtration and sedimentation studies have previously established that the vertebrate animal core histone octamer is in equilibrium with an (H3-H4)2 tetramer and an H2A-H2B dimer [Eickbush, T. H., & Moudrianakis, E. N. (1978) Biochemistry 17, 4955-4964; Godfrey, J. E., Eickbush, T. H., & Moudrianakis, E. N. (1980) Biochemistry 19, 1339-1346]. We have investigated the core histone octamer of wheat (Triticum aestivum L.) and have found it to be much more stable than its vertebrate animal counterpart. When vertebrate animal histone octamers are subjected to gel filtration in 2 M NaCl, a trailing peak of H2A-H2B dimer can be clearly resolved from the main octamer peak. When the plant octamer is subjected to the identical procedure, there is no trailing peak of H2A-H2B dimer, but rather a single peak containing the octamer. A sampling across the octamer peak from leading to trailing edge shows no change in the ratio of H2A-H2B to (H3-H4)2. Surprisingly, the plant octamer shows the same stability at 0.6 M NaCl, a salt concentration in which the vertebrate animal octamer dissociates into dimers and tetramers. Equilibrium sedimentation data indicate that the assembly potential of the wheat histones in 2 M NaCl is very high at all protein concentrations above 0.1 mg mL-1. In order to disrupt the forces stabilizing the plant histone octamer at high histone concentrations, the concentration of NaCl must be lowered to approximately 0.3 M.(ABSTRACT TRUNCATED AT 250 WORDS)


Probing the primary donor environment in the histidineM200-->leucine and histidineL173-->leucine heterodimer mutants of Rhodobacter capsulatus by light-induced Fourier transform infrared difference spectroscopy. Light-induced P+QB-/PQB FTIR difference spectra of reaction centers (RCs) have been obtained from chromatophores lacking light-harvesting B800-850 antenna for Rhodobacter capsulatus wild type (WT) and for the two mutants HisM200-->Leu and HisL173-->Leu. The primary donor (P) in both mutants consists of a bacteriochlorophyll-bacteriopheophytin heterodimer. The most prominent difference between the WT and the mutant spectra is in the 1600-1200-cm-1 region. The WT spectrum displays large positive bands at approximately 1290, 1500-1430, and 1580-1530 cm-1. These three bands are either small or altogether absent in the heterodimer spectra. In addition, both heterodimer spectra compare well with the electrochemically generated BChla+/BChla spectrum [Mäntele, W.G., Wollenweber, A. M., Nabedryk, E., & Breton, J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8468-8472]. These observations indicate that the positive charge is localized on the monomeric BChl in the heterodimers. The overall shape of the ester and keto C = O signals in the BChla+/BChla spectrum is maintained in the in situ spectra although significant differences are observed in the frequency, width, and splitting of the bands. The shape of the signal at 1757/1744 cm-1 in HisL173-->Leu is comparable to the 1751/1737-cm-1 signal of BChla+/BChla in tetrahydrofuran, indicating a free 10a ester C = O of PM in HisL173-->Leu. The reduced amplitude of the negative 1740-cm-1 feature in both HisM200-->Leu and WT spectra suggests a hydrogen-bonded 10a ester C = O for PL.(ABSTRACT TRUNCATED AT 250 WORDS)


Role of the carboxy terminus of polypeptide D1 in the assembly of a functional water-oxidizing manganese cluster in photosystem II of the cyanobacterium Synechocystis sp. PCC 6803: assembly requires a free carboxyl group at C-terminal position 344. The D1 polypeptide of the photosystem II (PSII) reaction center is synthesized as a precursor polypeptide which is posttranslationally processed at the carboxy terminus. It has been shown in spinach that such processing removes nine amino acids, leaving Ala344 as the C-terminal residue [Takahashi, M., Shiraishi, T., & Asada, K. (1988) FEBS Lett. 240, 6-8; Takahashi, Y., Nakane, H., Kojima, H., & Satoh, K. (1990) Plant Cell Physiol. 31, 273-280]. We show here that processing on the carboxy side of Ala344 also occurs in the cyanobacterium Synechocystis 6803, resulting in the removal of 16 amino acids. By constructing a deletion strain of Synechocystis 6803 that lacks the three copies of the psbA gene encoding D1, we have developed a system for generating psbA mutants. Using this system, we have constructed mutants of Synechocystis 6803 that are modified in the region of the C-terminus of the D1 polypeptide. Characterization of these mutants has revealed that (1) processing of the D1 polypeptide is blocked when the residue after the cleavage site is changed from serine to proline (mutant Ser345Pro) with the result that the manganese cluster is unable to assemble correctly; (2) the C-terminal extension of 16 amino acid residues can be deleted with little consequence either for insertion of D1 into the thylakoid membrane or for assembly of D1 into a fully active PSII complex; (3) removal of only one more residue (mutant Ala344stop) results in a loss of assembly of the manganese cluster; and (4) the ability of detergent-solubilized PSII core complexes (lacking the manganese cluster) to bind and oxidize exogenous Mn2+ by the secondary donor, Z+, is largely unaffected in the processing mutants (the Ser345Pro mutant of Synechocystis 6803 and the LF-1 mutant of Scenedesmus obliquus) and the truncation mutant Ala344stop. Our results are consistent with a role for processing in regulating the assembly of the photosynthetic manganese cluster and a role for the free carboxy terminus of the mature D1 polypeptide in the ligation of one or more manganese ions of the cluster.


Analysis of human red cell spectrin tetramer (head-to-head) assembly using complementary univalent peptides. The mass-driven assembly of spectrin dimers to form tetramers involves two equal head-to-head alpha-beta associations and requires at least 30 degrees C for interconversion to occur readily. In this paper, the properties of tetramer formation were investigated using two complementary univalent peptides (the alpha I domain and beta monomers). Since the alpha I domain lacks an essential nucleation site required for side-to-side (lateral) heterodimer assembly [Speicher et al. (1992) J. Biol. Chem. 267, 14775-14782], these two peptides can only assemble head-to-head at a single site. This head-to-head assembly readily occurs at lower temperatures, indicating the temperature barrier for dimer-tetramer interconversion is caused by a conformational constraint of the dimer. This constraint, a closed hairpin loop, is released when the laterally associated partner is removed. The univalent alpha I-beta binding affinity at 37 degrees C (Ka = 1.4 x 10(5) M-1) is similar to the dimer-tetramer association constant at the same temperature. As the temperature is decreased from 37 to 0 degrees C, the alpha I-beta binding affinity increases about 32-fold. In contrast with head-to-head associations involving dimers, the second-order rate constants of two complementary univalent peptides (i.e., alpha I and beta) are dramatically higher, and the estimated activation energy (about 50 kJ mol-1) is about 5-fold lower. An open dimer conformation is an obligatory high-energy intermediate required for dimer-tetramer interconversion, and opening the dimer hairpin loop contributes about 190 kJ mol-1 to the activation energy for tetramer association.(ABSTRACT TRUNCATED AT 250 WORDS)


Amphiphilic forms of butyrylcholinesterase in mucosal cells of rat intestine. The properties of a cholinesterase from mucosal cells of rat intestine have been characterized. The enzyme was identified as butyrylcholinesterase because it was more sensitive to iso-OMPA (IC50 = 1.0 x 10(-6) M) than to BW284C51 (IC50 = 5.5 x 10(-5) M) and was not inhibited by substrate excess. It displayed a higher affinity for acetylthiocholine than for butyrylthiocholine. A major molecular form was observed sedimenting at 5.9 S. Two other minor molecular forms were identified as a hydrophilic tetramer (G4, sedimenting at 10.5 S) and a monomer (G1, sedimenting at 4.3 S). The 5.9 S component was referred to as "G" form (G for globular) and not "G2" as usual dimers for the following reasons: (i) the G form was unaffected by the reducing agents, beta-mercaptoethanol and dithiothreitol, which converted disulfide-linked dimers of acetylcholinesterase into monomers, (ii) the G form was shifted from 5.9 to 3.4 S when the sucrose gradient contained Triton X-100. This value of 3.4 S (in Triton X-100) appeared too low for a typical G2 form. The shift in the S value was partly reversible: the 3.4 S form resedimented at 5.2 S in the absence of detergent. The behavior of the G form in sucrose gradients indicated that it was amphiphilic. This was confirmed in nondenaturing electrophoreses and also by quantitative binding of the G form to octyl-Sepharose. The hydrophobic domain of the G form was not a glycolipid, as shown by its insensitivity to Bacillus thuringiensis phosphatidylinositol-specific phospholipase C and its nonaggregating properties in the absence of nondenaturing detergent.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of adenyl nucleotides and carbachol on cooperative interactions among G proteins. Muscarinic agonists and adenyl nucleotides are noncompetitive modulators of sites labeled by [35S]GTP gamma S in washed cardiac membranes from Syrian golden hamsters. Specific binding of the radioligand and its inhibition by either GTP gamma S or GDP reveals three states of affinity for guanyl nucleotides. In the absence of adenyl nucleotide, carbachol promotes an apparent interconversion of sites from higher to lower affinity for GDP; the effect recalls that of guanyl nucleotides on the binding of agonists to muscarinic receptors. In the presence of 0.1 mM ATP gamma S, the binding of [35S]GTP gamma S is increased at concentrations up to about 50 nM and decreased at higher concentrations. At a radioligand concentration of 160 pM, binding exhibits a bell-shaped dependence on the concentration of both ATP gamma S and AMP-PNP; with ADP and ATP, there is a second increase in bound [35S]GTP gamma S at the highest concentrations of adenyl nucleotide. ATP gamma S and AMP-PNP also modulate the effect of GDP, which itself emerges as a cooperative process: that is, binding of the radioligand in the presence of AMP-PNP exhibits a bell-shaped dependence on the concentration of GDP; moreover, the GDP-dependent increase in bound [35S]GTP gamma S is enhanced by carbachol. The interactions among GDP, GTP gamma S, and carbachol can be rationalized quantitatively in terms of a cooperative model involving two sites tentatively identified as G proteins. Both GTP gamma S and GDP exhibit negative homotropic cooperativity; carbachol enhances the homotropic cooperativity of GDP and induces or enhances positive heterotropic cooperativity between GDP and [35S]GTP gamma S. An analogous mechanism may underlie the guanyl nucleotide-dependent binding of agonists to muscarinic receptors. The data suggest that the binding properties of G proteins and their associated receptors reflect cooperative effects within heterooligomeric arrays; agonist-induced changes in cooperativity may facilitate the exchange of GTP for bound GDP and thereby constitute the mechanism of G protein activation in vivo.


Molecular cloning and characterization of rKlk10, a cDNA encoding T-kininogenase from rat submandibular gland and kidney. We have cloned and determined the nucleotide sequence of a novel kallikrein-like mRNA, designated rKlk10*, from rat submandibular gland and kidney with the aid of the polymerase chain reaction (PCR). This cDNA contains 737 base pairs comprising the sequence encoding a mature protein of 235 amino acid residues, partial zymogen peptide, and 3' noncoding sequence. Sequence comparisons showed that rKlk10 mRNA shares 87 and 88% sequence identity with rat tissue kallikrein at nucleic acid and amino acid levels, respectively. It encodes a 26,428-Da acidic protein whose derived amino acid sequence matches completely with the partial amino acid sequence of a kallikrein-like enzyme designated as T-kininogenase, K10 protein, or antigen-gamma purified from rat submandibular gland [Xiong et al. (1990) J. Biol. Chem. 265, 2822-2827; Gutman et al. (1991) Eur. J. Biochem. 784, 1-5; Berg et al. (1991) Biochem. J. 280, 19-25]. The protein encoded by rKlk10 retains the key amino acid residues determining kallikrein cleavage specificity. Northern blot analysis with an rKlk10-specific oligonucleotide probe showed that its mRNA level in the submandibular gland is decreased dramatically by administration of the beta agonist isoproterenol. Tissue-specific expression of rKlk10 was analyzed by Northern blotting and Southern blotting of PCR-amplified cDNA, which showed that rKlk10 is expressed at high levels in the submandibular gland and low levels in the kidney but not in seven other tissues including prostate, liver, heart, adrenal gland, testes, pituitary, and pancreas. rKlk10 cDNAs cloned from the kidney and submandibular gland show sequence identity.(ABSTRACT TRUNCATED AT 250 WORDS)


Antibody and peptide probes of interactions between the SH1-SH2 region of myosin subfragment 1 and actin's N-terminus. The negatively charged residues in the N-terminus of actin and the 697-707 region on myosin subfragment 1 (S-1), containing the reactive cysteines SH1 and SH2, are known to be important for actin-activated myosin ATPase activity. The relationship between these two sites was first examined by monitoring the rates of SH1 and SH2 modification with N-ethylmaleimide in the presence of actin and, secondly, by testing for direct binding of SH1 peptides to the N-terminal segment on actin. While actin alone protected SH1 from N-ethylmaleimide modification, this effect was abolished by an antibody against the seven N-terminal amino acids on actin, F(ab)(1-7), and was greatly reduced when the charge of acidic residues at actin's N-terminus was altered by carbodiimide coupling of ethylenediamine. Neither F(ab)(1-7) nor ethylenediamine treatment reversed the effect of F-actin on SH2 reactivity in SH1-modified S-1. These results show a communication between the SH1 region on S-1 and actin's N-terminus in the acto-S-1 complex. To test whether such a communication involves the binding of the SH1 site on S-1 to the N-terminal segment of actin, the SH1 peptide IRICRKG-NH2(4+) was used. Cosedimentation experiments revealed the binding of three to six peptides per actin monomer. Peptide binding to actin was affected slightly, if at all, by F(ab)(1-7). The antibody also did not change the polymerization of G-actin by the peptides. The peptides caused a small reduction in the binding of S-1 to actin and did not change the binding of F(ab)(1-7).(ABSTRACT TRUNCATED AT 250 WORDS)


Alzheimer's disease and control brain contain soluble derivatives of the amyloid protein precursor that end within the beta amyloid protein region. The 39-43 amino acid beta amyloid protein (A beta) that deposits as amyloid in the brains of patients with Alzheimer's disease (AD) is encoded as an internal sequence within a larger membrane-associated protein known as the amyloid protein precursor (APP). In cultured cells, the APP is normally cleaved within the A beta to generate a large secreted derivative and a small membrane-associated fragment. Neither of these derivatives can produce amyloid because neither contains the entire A beta. Our study was designed to determine whether the soluble APP derivatives in human brain end within the A beta as described in cell culture or whether AD brain produces potentially amyloidogenic soluble derivatives that contain the entire A beta. We find that both AD and control brain contain nonamyloidogenic soluble derivatives that end at position 15 of the A beta. We have been unable to detect any soluble derivatives that contain the entire A beta in either the AD or control brain.


Partner and grandmother contact in black and white teen parent families. Contact of teen mothers with grandmothers and partners was studied as a predictor of maternal and middle childhood developmental outcomes. Mothers were interviewed yearly beginning in 1979 until 1986. Each child was given a developmental assessment in 1986. Black teens lived longer with their parents after the child's birth and white teens married earlier and had more disruptions in their relationships. Grandmother assistance with childcare was positively associated with the mother's ability to pursue her education. The findings suggested, however, that child developmental problems may occur when mothers routinely rely upon grandmother assistance into middle childhood, particularly for white families. In general, a male partner who had regular contact with the mother's child, more stimulation and support in the home environment, and higher educational level of the mother were associated with better child developmental outcomes.


Young motherhood and infant hospitalization during the first year of life. We studied the relationship of young maternal age with infant hospitalization using data from the National Longitudinal Survey of Youth for 3,130 infants born between 1979 and 1983 to mothers aged 14-25 years. Data on the mothers were first collected in 1979 and yearly thereafter. Data on their children were collected starting in 1982. Logistic regressions of infant hospitalization rates were estimated for first and second and higher births. The odds of infant hospitalization during the first year of life increased with decreasing maternal age, even with adjustment for sociodemographic characteristics, preventive health-care practices, and newborn health status, factors hypothesized to explain the maternal age effect. The maternal age relationship with hospitalization differed by birth order; among second and higher births, the odds of hospitalization was increased only for infants of mothers aged 20-22 years. Male infants, infants with a first well-baby visit after the first month of life, with birth weights between 1501 and 2500 g, and with nursery stays longer than 1 week also had increased odds of hospitalization. Ethnicity, grandmother's education, poverty status, mother's school enrollment, and family composition were not related to the odds of hospitalization, nor was smoking during pregnancy when adjustment was made for birth weight and length of nursery stay.


Maternal weight status and term birth weight in first and second adolescent pregnancies. The relationship of maternal weight status to birth weight was evaluated retrospectively for the first and second pregnancies of 72 younger (age 12-15 years at first conception) and 80 older (age 16-19 years at first conception) low-income adolescents (76% black, 24% white). Mean birth weight increased during the second pregnancies of both groups (277 g and 132 g, respectively). Multiple regression analysis (controlling for potentially confounding variables) indicated a positive relationship between second and third trimester rate of maternal weight gain (kg/week) and birth weight for younger adolescents during their second pregnancy (p = 0.014), and for older adolescents during their first pregnancy (p = 0.047). Mean birth weight increased with each increase in maternal weight-for-height (W/H) category near term from the lowest (< 100% of standard) to the highest (> or = 140% of standard), for both age groups during both pregnancies. Multiple regression analysis indicated that among older adolescents birth weight increased 4.2 +/- 2.0 g (X +/- SE) for each 1% increase in maternal W/H near term in their first pregnancy (p = 0.038) and 7.1 +/- 1.8 g for each 1% increase in maternal W/H near term in their second pregnancy (p = 0.0003). Among younger adolescents these relationships, while in the same direction, were not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)


Beliefs and behavioral intentions regarding human immunodeficiency virus testing among New York City runaways. From 1988 to 1991, 139 runaways aged 11-19 years in the New York City area (n = 70 males, 69 females) were recruited from four shelters. Each runaway participated in a semistructured interview assessing beliefs and behavioral intentions regarding human immunodeficiency virus (HIV) testing. When asked how they would respond to being seropositive for HIV, 29% of runaways reported that they would engage in self-destructive acts and/or harm others (e.g., suicide, unprotected sex), 80% anticipated extreme distress, 47% expected difficulty securing housing and food, and 61% believed that friends were likely to avoid them. When presented with specific alternatives, fewer runaways anticipated self-destructive acts. Drug use, rather than sexual behaviors, would lead runaways to get tested for HIV. These results suggest that health-care providers must anticipate emotional distress and potential self-destructive behavior following receipt of documentation of HIV positive serostatus among runaways. Furthermore, prior to testing, youths' access to food, shelter, medical care, and social support must be secured.


An evaluation of a school-based AIDS/HIV education program for high school students. The effect of a 1-hr school-based AIDS/HIV education program on the knowledge and attitudes of high school students was evaluated with a modified version of the Centers for Disease Control Health Risk Survey. One urban and one suburban school each were randomly assigned to an educational intervention (n = 535) or a control group (n = 659). All students received a posttest 2 weeks after the intervention. Knowledge was based on responses to 12 true-false questions (pretest alpha = .76, posttest alpha = 0.81). Principal components analysis was used to develop three attitude scales and risk-taking behavior was assessed by self-report. Data were analyzed with Kruskall-Wallis analysis of variance (ANOVA) and multivariate ANOVA. The groups did not differ in knowledge level at pretest. At posttest the education group had significantly (p < or = 0.006) higher knowledge even after controlling for the effects of previous AIDS education (p < or = 0.019), gender (p < or = 0.007), and Hispanic ethnicity (p < or = 0.048). After the education program, students were less worried about exposure to the AIDS virus, but were more worried (p < or = 0.048) about AIDS acquisition during their adult life. Although single school-based AIDS/HIV education programs may increase knowledge, more extensive education may be needed to change the behavior and attitudes of older high school students.


Gender differences in knowledge, intentions, and behaviors concerning pregnancy and sexually transmitted disease prevention among adolescents. Gender differences in knowledge, intentions, and behaviors regarding preventing pregnancies and sexually transmitted diseases were studied. Data for the study were collected from 1,033 students in 13 California high schools. Females in this sample were more likely than males to have discussed sexuality topics with parents, to have engaged in sexual intercourse more frequently, to have experienced a pregnancy scare, to have used oral contraceptives during their last sexual encounter, to perceive that a larger proportion of their peers were engaging in sex and using birth control, to obtain birth control from health facilities, and to report intentions to abstain or use protection in hypothetical situations placing them at risk for unprotected sex. In contrast, males reported that they were more likely to have always used birth control, to have used birth control during their first sexual encounter, and to have used a condom during their last sexual encounter. Furthermore, males were more likely to obtain birth control from a store or a friend. Finally, males knew more about using condoms correctly and their role in preventing sexually transmitted diseases. The efficacy of interventions designed to reduce unintended pregnancy and sexually transmitted diseases among adolescents may be increased by addressing these gender differences. Understanding gender differences may also facilitate an increased role for males in the overall prevention scheme. Further research is clearly needed to increase knowledge about these gender differences.


Prepubertal initiation of sexual experiences and older first partner predict promiscuous sexual behavior of delinquent adolescent males--unrecognized child abuse? Sexual experiences of 1580 delinquent adolescent males ages 9-19 years admitted to a juvenile detention center from January 1985 to July 1990 were determined through a structured health history interview. A total of 89.6% admitted to prior sexual activity; 26.8% claimed sexual experience by age 11 years. Only 3.2% reported sexual abuse or incest. Of 334 sexually active adolescents who revealed both age of their first partner and their age of first intercourse, 42% had their first experience with a female 2 or more years older. Males who had their first sexual experience before age 11 years or with a female 2 or more years older, reported significantly larger numbers of sexual partners in their lifetime and during the year preceding their incarceration. Girls with similar first sexual experiences in terms of age of initiation and age difference from partner are considered to be abused. Practitioners who work with children and adolescents need to recognize the possibility of sexual activity among pre-adolescent males and to be aware of its implications.


Leisure-time physical activity and aerobic fitness in African-American young adults. This cross-sectional study identified the leisure-time physical activity (LTPA) and aerobic fitness levels of 189 African-American young adult college freshmen. LTPA was measured with the Lipid Research Clinics (LRC), Godin Leisure-Time Exercise, and the College Alumnus physical activity questionnaires. The Physical Activity Index (PAI), an index of walking, stair climbing, and recreational sports participation, was obtained from the College Alumnus questionnaire. Aerobic fitness was measured indirectly with the Cooper 12-Minute Walking/Running Test. More women (82%) than men (53%) were classified as inactive (strenuous exercise or labor < 3 days/week and much less active than peers) or low active (strenuous exercise or labor < 3 days/week and as active or more active than peers) on the LRC Questionnaire. The PAI scores were moderately low in men (1,521 +/- 1,634 kcal.week-1) and very low in women (706 +/- 868 kcal.week-1). The majority of men (71%) and women (82%) were classified as 'very poor' in aerobic fitness levels. Body mass index (BMI), a measure of body fatness, was classified as 'overweight' or 'obese' for 39% of men and 37% of women (BMI = 25.9 +/- 5.7 kg/m2), reflecting inactive LTPA habits. These findings are consistent with studies showing low LTPA in middle-age African-American adults. School and community-level interventions are recommended to increase LTPA and aerobic fitness in adolescent and young adult African-Americans.


Use of nonmedical treatment by cystic fibrosis patients. To determine frequency, perceived effectiveness, and cost of religious, physical, and other nonmedical practices, we interviewed all (n = 402; 100% participation) of our cystic fibrosis patients/families [age, 0-45 (median, 18) years]. At least one type of nonmedical therapy was used by/on 264 (66%), two-thirds of which was religious. Most respondents perceived benefit (e.g., group prayer, 95%; religious articles, 81%; chiropractic, 69%). Only 17 (4.2%) invested over $200 lifetime (minimal compared with medical costs). These treatments, apparently used by most patients/families, rarely interfered with medical care. Physician openness and empathy toward patients' beliefs might enhance rapport and facilitate discussion of possible helpful or adverse effects of nonmedical treatment.


Physician attitudes toward confidentiality of treatment for adolescents: findings from the Upper Midwest Regional Physicians Survey. The provision of confidential medical services to adolescents is an enduring health policy issue in the United States, and the focus of policy statements by several professional medical organizations. Physician attitudes toward confidential service provision to teenagers were examined in the Upper Midwest Regional Physician Survey, a representative sample of community-based pediatricians and family physicians. Overall, three-quarters of participants favored confidential service provision for youths. Multivariate analysis revealed that the most salient reasons for favoring confidentiality were perception of unique needs among adolescents, year of licensure, high self-assessed competency in addressing sexual concerns of adolescence, adequacy of training in interpersonal and sexual issues, frequency of addressing interpersonal issues, and lower self-assessed adequacy of training in traditional medical problems of youths. Implications for state and federal legislation are discussed.


Sexual intercourse, contraception, and risk-taking behavior among unselected French adolescents aged 11-20 years. An epidemiological study was carried out among 4,255 adolescents, aged 11-19 years, randomly selected from secondary schools in a northern urban area of France. The questionnaire concerned sexual activity, contraception, health status, licit and illicit drug consumption, and other risk-taking behavior. A total of 31% of the adolescents had had sexual relations (43% of the boys, 20% of the girls). Sexual activity increased with age. A large majority (70%) of the sexually active adolescents used some form of contraception. The study confirms the relationship between smoking, drinking, illicit drug consumption, and sexual activity, but shows that contraceptive behavior is not related to problem behavior.


Oral contraceptives and venous thromboses in adolescents undergoing elective surgery: a case report, and review of the literature. Oral contraceptive (OC) use is a risk factor for the development of deep venous thromboses (DVT) in adolescents, particularly for those patients undergoing surgery. Adolescents have received little attention in the literature on this subject. We describe a 17-year-old female who developed a DVT while receiving OCs at the time of elective surgery. We recommend that adolescent females undergoing elective surgery discontinue OCs at least 1 month before and after the surgery and attendant immobilization.


Fiber transformation and fiber replacement in chronically stimulated muscle. Chronic low-frequency stimulation (CLFS) of fast-twitch muscles induces fast-to-slow fiber-type transitions that differ in their extent in rat and rabbit. Fast-twitch muscle of the normal rat responds to CLFS with sequential transitions in myosin heavy chain (HC) expression in the order HCIIb --> HCIId --> HCIIa. However, in rabbit muscle the changes proceed beyond the state of HCIIa and include, as a final step, the expression of the slow myosin HCI. The time course for the transitions in myosin HC expression at both the messenger ribonucleic acid and protein levels suggests that fiber-type conversions occur asynchronously in a sequential manner. Thus type IIB fibers convert first to type IID fibers; these fibers then transform into type IIA fibers, which in rabbit muscle ultimately change into type I fibers. This sequence and the related changes in myosin isoforms point to different thresholds of the genes encoding the various fast and slow myosin HC isoforms. Although transformation of existing fast fibers into slower fibers is the major process that underlies the stimulation-induced fast-to-slow conversion, fiber replacement may also contribute. In rabbit muscle CLFS may cause metabolic exhaustion and subsequent deterioration in a fraction of the fast-twitch glycolytic fibers. For the most part these are replaced by satellite cell-derived, newly formed slow-twitch fibers.


Significance of the biopsy site of the latissimus dorsi muscle for fiber typing. The use of the latissimus dorsi muscle for cardiomyoplasty requires accurate assessment of the outcome of methods used to convert fast fibers to slow fibers. A knowledge of the normal distribution pattern of slow fibers within the latissimus dorsi is necessary for this endeavor. Fresh latissimus dorsi and teres major muscle tissues from seven pigs, one rabbit, two sheep, one monkey, and four dogs were studied with myosin adenosine triphosphatase staining. Fiber counts were made visually. With the exception of the rabbit, the distribution patterns were similar for all species: (1) intersegmentally--the number of slow fibers decreased steadily from the teres major to the anterior edge of the lateral segment; (2) intrasegmentally--slow fibers were more frequent in the deep layer than the superficial layer; and (3) intrasegmentally--the slow fibers tended to cluster in rosette formations around the neurovascular bundles. These patterns of distribution indicate the need for careful location of biopsies to ensure valid comparison of the amount of slow fibers in tissue before and after treatment.


Evaluation of cardiomyoplasty and skeletal muscle ventricle procedures in a clinically realistic animal model. Cardiomyoplasty and skeletal muscle ventricle procedures have shown increasing promise in the treatment of cardiomyopathy with associated chronic congestive heart failure. More than 100 patients have now received such procedures worldwide. The clinical results to date have been mostly anecdotal with subjective improvement but without firm objective data to collaborate the subjective impression. A clinically realistic animal model would have tremendous advantages to help elucidate the mechanisms by which cardiomyoplasty or skeletal muscle ventricles improve the sense of well-being of patients who have congestive heart failure. Several possible animal models exist, including pharmacologically induced congestive heart failure models and postischemic injury cardiomyopathy models. The most intriguing, however, is a spontaneously occurring cardiomyopathy in large dogs. This idiopathic cardiomyopathy that appears mainly in large-breed dogs (Great Danes, Dobermans, and Saint Bernards) has a rapidly progressive course; it has a 6-month mortality rate of 75% and a 12-month mortality rate of 95% to 100%. The most efficacious use of such an experimental model would include the evaluation of cardiomyoplasty and skeletal muscle ventricle procedures in a multidimensional fashion. Experimental endpoints should include mortality, exercise tolerance, systolic and diastolic ventricular function, and arrhythmia occurrence. Sophisticated techniques now exist for the evaluation of systolic and diastolic ventricular function. Such evaluation may well provide additional insight into how such experimental procedures benefit those with congestive heart failure.


Blood flow to the latissimus dorsi muscle pouch during chronic counterpulsation stimulation. The cyclic contraction of a skeletal muscle ventricle (SMV) stimulated in counterpulsation results in phasic perfusion of the muscle. Perfusion will occur primarily during cardiac systole when the muscle is relaxed. However, the resting preload of the SMV will be systolic arterial pressure, which will impede blood flow to the relaxed muscle. To determine the effect of chronic counterpulsation stimulation on the blood flow to an SMV and identify stimulation regimens that prevent the risk of chronic ischemia, SMVs were created in four mongrel dogs by implementing an implantable mock circulation device. The SMV was stimulated in counterpulsation for 4 weeks after a 2-week vascular delay period and 2 weeks of low-frequency muscle conditioning. During biweekly studies, the muscle was stimulated in four modes against preloads varying from 20 to 120 mm Hg. Resting blood flow decreased significantly at preloads greater than 60 mm Hg. Normalized blood flow increased between 10% and 30% during stimulation; greater increases corresponded to more demanding stimulation modes. The elevated blood flow during stimulation, however, decreased with increasing preload. Stroke work increased with increasing preload until preload exceeded 100 mm Hg. The decreased blood flow and increased stroke work occurring at higher preloads indicate that the supply/demand ratio becomes compromised with increasing preload. A hyperemic response occurred during the resting beats after a stimulated beat, increasing the volume blood flow by as much as 80%. This response occurred regardless of preload or stimulation rate. If the SMV relaxed before the onset of systole, a hyperemic response occurred within the stimulated beat.(ABSTRACT TRUNCATED AT 250 WORDS)


Skeletal muscle ventricles in circulation long-term: one hundred ninety-one to eight hundred thirty-six days. It has been shown previously that skeletal muscle ventricles fashioned from canine latissimus dorsi muscle can function as effective blood pumps. We describe four dogs that have been reported on before but have now had skeletal muscle ventricles functioning as effective aortic diastolic counterpulsators for 191 to 836 days. One dog remains well with a skeletal muscle ventricle that has been pumping in circulation for 194 days; there has been no evidence of thrombus in the skeletal muscle ventricle cavity or thromboembolism. None of the three dogs that died had evidence of peripheral embolization, although two of those dogs had thrombus within the cavity of the skeletal muscle ventricle. This study proves that skeletal muscle ventricles can function long-term in the circulation and maintain diastolic pressure augmentation beyond 2 years. To the best of our knowledge, the dog whose skeletal muscle ventricle pumped in circulation for 836 days represents the longest living laboratory animal or human with a functioning heart assist device.


A new skeletal muscle linear-pull energy convertor as a power source for prosthetic circulatory support devices [corrected]. Previous studies show that stimulated skeletal muscle wrapped around the heart or a blood pumping pouch can provide partial circulatory assistance. However, skeletal muscle is accustomed to pulling in direct tension, and power obtained from using the muscle in wraparound configurations is very inefficient. Therefore we have developed a new skeletal muscle-powered, linear-pull energy convertor for powering a wide range of implanted devices, including circulatory support blood pumps such as counterpulsation devices or complete prosthetic ventricles. This energy convertor system is powered by a skeletal muscle such as the latissimus dorsi, which is stimulated with a pulse generator. With the muscle left intact and in situ as much as possible, its insertion at the humerus is removed and reattached to a small-cylinder hydraulic energy convertor that is firmly attached to the ribs. Mechanical force in direct tension from the muscle is converted to high-pressure, low displacement; hydraulic energy (at approximately 200 psi). The output of this hydraulic energy convertor is connected by a small-diameter tube to a hydraulic actuator to drive the pusher plate of an implantable ventricular assist device located in the thoracic cavity or abdomen or another suitable blood pump. Preliminary in vitro tests from an engineering model of the Thoratec muscle-powered ventricular assist device show flow outputs of 5.2 L/min at a mean arterial pressure of 99 mm Hg. The muscle-powered ventricular assist device is a specific application designed to provide completely implantable circulatory support as an alternative to heart transplantation. It will enable patients to experience a quality of life free from batteries and the electrical power-conditioning hardware required with electromechanical systems.


Voltage-dependent performance of skeletal muscle pouches: implications for cardiomyoplasty. Cardiomyoplasty, a new therapy for heart failure, uses autologous skeletal muscle to mechanically assist the heart. The success of dynamic cardiomyoplasty is critically dependent on the contraction strength of the assisting skeletal muscle. Unlike cardiac muscle, skeletal muscle contracts in a graded response to electrical stimulation. However, in current cardiomyoplasty practice, no systematic technique exists to set the stimulating voltage effecting skeletal muscle contraction. The stimulating voltage is simply set to some multiple of the "threshold" voltage. Furthermore, researchers do not consider the role of stimulating voltage when they determine the amount of assistance afforded during cardiomyoplasty. To more accurately assess the value of this heuristic voltage-setting technique, we investigated the role of stimulating voltage on the strength of contraction of the latissimus dorsi muscle. Six New Zealand white rabbits had isovolumic hydraulic pouches constructed from the latissimus dorsi muscle. The muscles were wrapped around a compliant balloon in which isovolumic pressure development was measured during tetany-inducing burst (pulse-train) stimulation. The tetanic plateau of the pouch pressure record was used to measure the effects of stimulating voltage on skeletal muscle contraction. Results indicated that (1) increasing stimulating voltage from two to four times the "threshold" voltage increased normalized pouch pressure from 0.38 +/- 0.21 to 0.78 +/- 0.12 (mean +/- SD) (p < 0.05); (2) the threshold-normalized voltage necessary to cause maximal muscle contraction varied widely (5.7 +/- 2.0, mean +/- SD; range, 3.1 to 9.3); and (3) the current achieving maximal pressure development varied from 5.6 to 31.4 mA (19.9 +/- 10.4 mA).(ABSTRACT TRUNCATED AT 250 WORDS)


Successful lung transplantation for posttraumatic adult respiratory distress syndrome after extracorporeal membrane oxygenation support. A severe adult respiratory distress syndrome after bilateral lung contusion was successfully treated by extracorporeal membrane oxygenation and subsequent double-lung transplantation in a 19-year-old man. The patient is fully rehabilitated 1 year after transplantation.


Infections in heart transplant recipients: the experience of the Italian heart transplantation program. Italian Study Group on Infections in Heart Transplantation. Infectious complications are an important cause of morbidity and mortality in heart transplantation. In this report we describe their incidence in the Italian Heart Transplantation Program and their relationship to induction immunosuppressive protocols used. A total of 662 heart transplantations in 651 patients had been performed by March 1, 1990, in 10 Italian centers, with a follow-up of 12,022 patient-months. Actuarial survival at 54 months was 80%. There were 115 deaths, 28 as a result of infection (24%); 30 were perioperative. Mortality from infection was 15% in the first 30 postoperative days and 35% thereafter. During follow-up, 256 life-threatening infectious episodes occurred in 180 patients, for a mean of 1.42 infections/patient. Of these episodes, 28 (10.9%) were lethal. The rate at 54 months, excluding perioperative deaths, was 0.021 episodes/patient-month, with a mean of 0.39 infections/patient in the 651-patient series. At 6 months, 72% of patients were free from infection. This value did not change significantly during subsequent follow-up. Bacterial infections were most frequent (49.2%); fungal (10.5%) and protozoan (5.9%) infections had the worst outcome (lethality 26% and 20%, respectively). A primary cytomegalovirus infection was observed in 23 cases (12 mismatches). Lung involvement (99 episodes) and bacterial sepsis (47 episodes) were most common. Eighty episodes of infection (31.2%) were preceded by a supplementary treatment for rejection with steroid pulses (69%), prednisone boost (20%), or cytolytic therapy (11%). Treatment had preceded the infection by less than 15 days in 55% of cases.(ABSTRACT TRUNCATED AT 250 WORDS)


Differences in left ventricular filling patterns in heart and heart-lung transplant recipients as assessed by Doppler echocardiography of transmitral flow. The conventional surgical technique of orthotopic heart transplantation involves reconstruction of atria from donor and recipient components. The mechanical function of the atria is thus likely to be altered either by this anatomic disruption or by the autonomic denervation, which also occurs as a result of transplantation. We investigated 18 subjects with Doppler echocardiography of transmitral flow, to assess the contributions of passive and active filling of the left ventricle in six normal subjects, six heart transplant recipients, and six heart-lung transplant recipients. The ratio of passive transmitral flow to active flow was significantly higher in isolated heart transplant recipients than in normal subjects (p < 0.001), suggesting a reduced active, late diastolic component to left ventricular filling. This was not due to denervation, because the ratio in heart-lung recipients was lower than that in normal subjects (p < 0.01). These data suggest that anatomic rather than neural dysfunction is responsible for a major change in transmitral flow characteristics after isolated heart transplantation; whether the resulting change in pattern of left ventricular filling is clinically important deserves further investigation.


Quantitative analysis of nitroglycerin-induced coronary artery vasodilation in transplanted hearts. Quantitative serial coronary angiograms performed annually over a 3-year period after transplantation in 26 orthotopic heart transplant recipients showed persistently normal nitroglycerin-induced vasodilation. The vasodilator response to nitroglycerin did not predict development of graft arteriopathy; the presence of graft arteriopathy did not prevent a substantial vasodilator response to nitroglycerin.


Impact of proximal or midvessel discrete coronary artery stenoses on survival after heart transplantation. To assess survival after the development of transplant coronary artery disease, annual angiography reports from 353 heart transplant recipients were reviewed. Fifty-four patients who survived beyond 1 year and in whom moderate-to-severe proximal or midvessel coronary artery disease developed were identified. Moderate-to-severe proximal or midvessel coronary disease was defined for this study as a 40% or more stenosis in 1 or more primary or secondary epicardial arteries. Actuarial survival (Kaplan-Meier) from the time of disease detection in those 54 patients was 67% at 1 year, 44% at 2 years, and 17% at 5 years. Actuarial survival was reduced proportionate to disease severity. Survival for single-vessel disease (> or = 40% stenosis) was 64% at 1 year, 36% at 2 years, and 22% at 5 years. Survival was significantly worse with triple-vessel disease (13% at 2 years; p = 0.01) and intermediate for double-vessel disease (41% at 2 years; p = 0.01). Of 19 patients who underwent retransplantation for coronary artery disease, 13 patients (68%) died at a mean of 24 +/- 20 months (range, 1 to 59), and of 15 patients from whom postmortem or angiographic data were available, 11 patients (73%) showed recurrence of significant coronary artery disease in the new graft. Identification of moderate or severe proximal or midvessel coronary disease at angiography predicts an overall mortality rate of more than 50% at 2 years. The poor survival rate in those who underwent retransplantation (around 50% at 4 years) and the high rate of redevelopment of coronary disease suggest that retransplantation should be reserved for selected candidates with angiographically severe disease, if used at all.


Echocardiographic evaluation of left ventricular wall motion before and after heart transplantation. Forty transplanted hearts were retrospectively investigated before, immediately after, and 15 +/- 12 months after heart transplantation by two-dimensional echocardiography for the presence and course of left ventricular myocardial wall motion abnormalities. Fourteen heart donors who were brain dead because of subarachnoid hemorrhage formed group 1 (mean age, 35 years); 21 heart donors who were brain dead because of head injury formed group 2 (mean age, 29 years), and five heart donors who were brain dead because of head injury with an additional chest trauma formed group 3 (mean age, 28 years). Myocardial wall motion was examined in six different myocardial segments (inferior, septal, anterior, posterior, posterolateral, apical) and was quantitatively assessed by a modified score index system (score index 0 = normal wall motion; score index 1 = diffuse hypokinesia). Overall, 27 of the 40 heart donors showed mild to severe (9 of the 40) wall motion abnormalities, which improved shortly after heart transplantation (score index: 0.36 vs 0.18, p < 0.01), and remained improved 15 months after heart transplantation (score index: 0.15). Among the different study groups, a significant improvement occurred in the myocardial wall motion score index on a short-term and long-term basis in all the groups, except for group 2, regarding the long-term follow-up. This study concluded that brain-dead, potential heart donors often reveal mild-to-severe left ventricular wall motion abnormalities, which are readily detected and semiquantitated by two-dimensional echocardiography.(ABSTRACT TRUNCATED AT 250 WORDS)


Global and regional left ventricular function in heart transplant patients: an angiographic follow-up study. We studied the global and regional left ventricular function, its determinants and its modification with time, in orthotopic heart transplant recipients. We reviewed the left ventricular cineangiography performed 1 (50 patients), 2 (33 patients), 3 (18 patients), and 4 (seven patients) years after operation. Regional wall motion was quantitatively evaluated by the area method. All patients had angiographically normal coronary arteries and no evidence of acute rejection at the time of the study. One year after heart transplantation, cardiac index and left ventricular ejection fraction were mildly but significantly lower than normal. Cardiac index was more than 2.5 L/min/m2 in all but one patient, and ejection fraction was more than 50% in all patients. Only previous acute cardiac rejection necessitating therapy and arterial hypertension showed some influence on the left ventricular function. Two years after operation, the left ventricular end-diastolic volume was increased, and left ventricular mass-volume ratio decreased compared with year 1. Three years after operation, an increase of left ventricular end-diastolic pressure and of left ventricular ejection fraction was also evident. Four years after operation, the heart rate was higher, compared with previous years. Even if the changes in the parameters of left ventricular function were significant, they were small in terms of absolute value. Regional hypokinesia was detected 1 year after heart transplantation in eight patients, involving one segment in six patients and two segments in two patients.(ABSTRACT TRUNCATED AT 250 WORDS)


Total lymphoid irradiation in the treatment of early or recurrent heart rejection. Total lymphoid irradiation (TLI) has been shown experimentally to induce a state of partial tolerance when administered before organ transplantation. Anecdotal reports in clinical transplantation have suggested efficacy of TLI in the treatment of recurrent rejection after heart transplantation. To further assess the safety and efficacy of TLI, 19 patients were entered into a protocol of TLI for the treatment of recurrent or early severe rejection despite conventional therapy. Rejection rate decreased from 1.3 episodes/month before TLI to 0.53 during TLI and 0.07 after TLI (p < 0.0001). Infections increased during TLI (possibly related to recent augmented immunosuppression before TLI), but all infections were successfully treated. One death occurred after TLI from acute allograft rejection. White blood cell (WBC) and platelet counts were depressed during and after (3 months) TLI. Frequent adjustments of dosing interval and, occasionally of the dosage were required to control WBC and platelet counts. Five patients experienced transient WBC of less than 1000/ml. More rejection episodes (and thus greater overall immunosuppression) before TLI and a lower tolerated dose of azathioprine before TLI predicted (by multivariate analysis) a lower WBC during TLI. (1) TLI is an effective adjunct for the intermediate control of early or recurring acute allograft rejection. (2) Close surveillance of WBC and platelets with appropriate adjustment of TLI dose and interval is necessary during TLI therapy. (3) The long-term benefits, possible late deleterious effects, and potential role of TLI as induction therapy remain to be elucidated.


Reevaluation of steroid tapering after steroid pulse therapy for heart rejection. A retrospective analysis was conducted to determine the efficacy and complications resulting from steroid pulse therapy, with or without a steroid taper, in 93 episodes of heart transplant rejection that occurred in 72 patients (58 men, 14 women; mean age, 47.6 years). Each rejection episode was classified according to severity (Texas Heart Institute endomyocardial biopsy scale) and the treatment. Group 1 included 25 episodes of grade 7, 8, 9, or 10 rejection (International Society for Heart Transplantation [ISHT] grade IIIB or IV) that were treated with high-dose methylprednisolone (2.5 to 3.0 gm) and a steroid taper of 1.75 gm over 30 days. Group 2 included 16 episodes of rejection, with the severity of rejection and methylprednisolone pulse therapy being similar to that in group 1, but without a steroid taper. The results of treatment in group 1 were compared with those in group 2. Group 3 included 12 episodes of grade 5, 6, or 7 rejection (ISHT grade IIIA or IIIB) that were treated with moderate-dose methylprednisolone (1.0 to 2.0 gm) and a steroid taper, as described. Group 4 included 40 episodes of rejection, with the severity of rejection and methylprednisolone therapy being similar to that of group 3, but without a steroid taper. The results of treatment in group 3 were compared with those in group 4. No statistically significant differences were found among the groups regarding subsequent episodes of rejection or infection within 3 months of treatment. No statistically significant difference was noted among the groups in the number of rejection episodes requiring additional therapy to control the rejection.(ABSTRACT TRUNCATED AT 250 WORDS)


Heart transplantation in patients over 54 years of age with triple-drug therapy immunosuppression. Between April 1, 1986, and December 31, 1989, 206 patients received orthotopic heart transplants with triple-drug therapy immunosuppression (cyclosporine, azathioprine, prednisone). Forty-six patients were aged 55 years or more at the time of transplantation and 160 patients were less than 55 years of age; these two groups were compared. Selection criteria and treatment regimen were the same in both groups. In our experience, although both groups had good postoperative survival, older patients had higher mortality rates early after transplantation, particularly from rejection and infection. Patients over 55 years of age had similar nonfatal rejection rates and were no more likely to have infectious episodes than younger patients. As demand for heart transplantation increases and waiting lists lengthen, the age of potential recipients should be an important factor in deciding how to allocate scarce donated organs, although age should not be exclusive.


Stability of long-term renal function in heart transplant patients treated with induction therapy and low-dose cyclosporine. Long-term renal function was evaluated in heart transplant recipients who were treated with antilymphocyte globulin induction therapy and low-dose cyclosporine therapy. Although an initial 16% drop in the glomerular filtration rate occurred, long-term follow-up revealed stability of renal function. Four-year patient survival was 77.6%. Use of induction therapy with low-dose cyclosporine may preserve renal function without compromising long-term patient survival.


Neurologic complications of heart transplantation in children. Neurologic complications can add significant morbidity to otherwise successful orthotopic heart transplantations in children. Complications have been reported to occur in up to 50% of children undergoing heart transplantation. The purpose of this study was to identify the prevalence and outcome of neurologic complications of heart transplantation in children. We reviewed all children who received orthotopic heart transplantation at Texas Children's Hospital from November 1984 to November 1990. Twenty-two patients (ages, 3 weeks to 17 years; mean, 8.5 years) underwent heart transplantation using cardiopulmonary bypass with moderate hypothermia. For analysis, we compared results during the first 3 years of our experience, 1984 through 1987 (group 1), to 1987 through 1990 (group 2). Survival was 45% (5 of 11 patients) for group 1 and 73% (8 of 11 patients) for group 2. A neurologic complication was defined as a change in the neurologic examination and/or status. Neurologic complications included seizures (6 of 22 patients), strokes (3 of 22 patients), unresponsiveness (3 of 22 patients), and change in mental status (2 of 22 patients). Early (within 2 weeks after operation) neurologic complications occurred in 45% (10 of 22 patients), were persistent (sequelae lasting more than 4 months) in 27% (6 of 22 patients), and resulted in death in 9% (2 of 22 patients). Late (after 2 weeks after operation) neurologic complications occurred in 23% (5 of 22 patients), were persistent in 9% (2 of 22 patients), and have occurred in only two survivors. Neurologic factors were not responsible for the cause of death in group 2. No neurologic complications (early or late) were seen in 1 of 11 patients in group 1 as compared with 7 of 11 patients in group 2 (p < 0.015). Serious neurologic morbidity decreased between the two groups after preoperative cyclosporine was avoided and postoperative hypertension was controlled. All survivors are functioning at age-appropriate levels. Although neurologic complications may be frequent, long-term neurologic disability in survivors is rare.


Long-term results in diabetic patients undergoing heart transplantation. We conducted a retrospective study of 305 nondiabetic patients and 37 diabetic patients who underwent heart transplantation from July 1982 to May 1990. Actuarial survival was similar for both groups of patients at 1 year (76.4% versus 81.3%) and at 2 years (69.6% versus 73.0%). Because we were interested in long-term results, we further analyzed only those patients surviving more than 1 year after transplantation (214 nondiabetic patients and 29 diabetic patients). Mean follow-up for the nondiabetic patients was 31.8 +/- 16.2 months and for the diabetic patients, 32.9 +/- 4.1 months. The respective mean age in each group was 50.4 +/- 10.3 years and 51.6 +/- 9.1 years. No difference was observed between the nondiabetic patients and diabetic patients regarding the rejection rate per patient-month (0.040 +/- 0.041 versus 0.045 +/- 0.051 episodes per patient-month), the infection rate per patient-month (0.056 +/- 0.081 versus 0.081 +/- 0.102 episodes per patient-month), or renal function as evidenced by mean creatinine levels at 1, 2, and 3 years. Twelve patients were insulin-dependent before transplantation; and 1 year after transplantation, they required an insulin dose 2.12 times greater than the dose before operation. Coronary artery disease developed in 32.8% of the nondiabetic patients, compared with 31.0% of the diabetic patients by the fourth year of follow-up. Despite the need for increased insulin doses, the diabetic patients had similar long-term survival to that of the nondiabetic patients, without an increased risk of rejection, infection, renal dysfunction, or coronary artery disease. Our experience supports the feasibility of heart transplantation in selected diabetic recipients.


Different patterns of cell volume regulation in hyposmotic media between attached and suspended HeLa cells. Both attached and suspended HeLa cells swelled in a medium of a hypotonic osmolality of 235 mosmol/kg H2O. When the osmolality was further decreased to 166 mosmol/kg H2O, attached cells instantly swelled and then rapidly lost water and K+, followed by slow gains of them. Suspended cells instantly swelled and then K+ loss and regulatory volume decrease (RVD) occurred. Neither 0.1 mM ouabain nor 10 mM TEA changed the water loss of attached cells, whereas ouabain inhibited RVD of suspended cells. Quinine (1 mM) inhibited water losses from both cells and comparison of the losses implies stronger activation of K+ channel in attached cells than in suspended cells. Omission of medium Ca2+ or addition of 10 mM BaCl2 inhibited RVD in part. These results suggest that hyposmotic stress induces net water loss from attached cells, associated with K+ release through the Ca(2+)-dependent K+ channel. Suspended cells osmotically swell, followed by RVD with K+ and Na+ releases through the K+ channel and Na(+)-pump, respectively. The different patterns of volume changes may relate to the difference of activity or time of activation of the K+ channel between both cells.


The effects of interferon-alpha/beta in a model of rat heart transplantation. Interferons have multiple immunologic effects. One such effect is the activation of expression of cell surface antigens. Interferon alpha/beta enhance expression of class I but not class II histocompatibility antigens. Contradictory information has been published regarding the effect of interferon-alpha/beta administration in patients with kidney transplantation. In a model of rat heart transplantation we demonstrated that administration of interferon-alpha/beta accelerated rejection in a dose-dependent fashion in the absence of maintenance cyclosporine. Animals treated with maintenance cyclosporine had evidence of increased rejection at 20 days that was resolved completely at 45 days with cyclosporine alone.


Cardiac xenografting in the pig-to-rhesus monkey model: manipulation of antiendothelial antibody prolongs survival. Transplantation of immediately vascularized grafts across species barriers in which preformed cytotoxic antibodies exist, otherwise known as discordant combinations, has uniformly resulted in hyperacute rejection. We studied how well plasma exchange and perfusion through organs removes preformed immunoglobulin M cytotoxic antibodies and prolongs survival of a porcine heart heterotopically transplanted into a rhesus monkey. With the use of plasma exchange or absorption of antibodies by porcine kidney perfusion with or without immunosuppression, graft survival was prolonged, although antibody-mediated rejection ultimately occurred. In one case in which plasma exchange, kidney perfusion, and immunosuppression were combined, a functioning pig heart survived in a rhesus monkey for 8 days without evidence of rejection. The animal was killed on day 8 according to protocol because of a wound dehiscence. With this animal we were able to demonstrate that circulating antibodies against graft endothelium had bound to the graft endothelium without inducing rejection, a process referred to as accommodation. In this case, despite the presence of antiendothelial antibodies, complement did not appear to be activated, and fibrin thrombi did not form. Although we have achieved this rejection-free survival only in one animal, this case suggests that it may be possible to maintain xenotransplants in discordant species without rejection if preformed antibodies are appropriately lowered or altered during the initial period of graft implantation.


Cyclosporine A and prednisone-associated osteoporosis in heart transplant recipients. Immunosuppressive therapy with cyclosporine A or prednisone produces bone loss in some animal models. Although we have clinically observed osteoporotic fractures in our heart recipients, the effects of cyclosporine and prednisone on bone density in transplant populations has not been fully elucidated. This study was undertaken to examine indexes of mineral metabolism and bone mineral density (BMD) in heart transplant recipients referred for evaluation of possible bone disease. Twenty of 93 patients who underwent heart transplantation at our institution were evaluated for osteoporosis. Sixteen of these patients (eight men; eight women) were included in this cross-sectional study (two patients were excluded because of hyperparathyroidism, and two patients were excluded because severe fractures prevented BMD from being measured). The mean age of the heart transplant recipients was 52.4 +/- 2.2 years, and the study was conducted a mean of 33.4 +/- 4.6 (men) and 19.0 +/- 7.0 (women) months after heart transplantation. Forty-four percent of these heart transplant recipients were seen clinically with fractures. Biochemical tests of skeletal homeostasis and BMD measurements with dual energy x-ray absorptiometry were performed. In male and female patients, the indexes of mineral metabolism showed (mean +/- sem) osteocalcin levels of 9.60 +/- 2.3 micrograms/L and 9.46 +/- 1.9 micrograms/L (normal: men, 6.39 +/- 0.69 micrograms/L; women, 5.87 +/- 0.71 micrograms/L) and intact parathyroid hormone levels of 48.8 +/- 10.3 ng/L and 63.4 +/- 10.7 ng/L (normal: men, 26.8 +/- 3.3 ng/L; women, 30.7 +/- 2.1 ng/L), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)


TPEN, a transition metal chelator, improves myocardial protection during prolonged ischemia. In view of the hypothesis that free radicals induced damage during ischemia and reperfusion is mediated by transition metals, we investigated the effect of the potent metal chelator TPEN (N,N,N'N'-tetrakis(-)[2-pyridylmethyl]-ethylenediamine) on cardiac function after prolonged myocardial ischemia. Isolated working rat hearts were subjected to 12 hours of cold ischemic arrest followed by reperfusion for 1 hour. The study was carried out on five groups (nine hearts in each): (1) St. Thomas' Hospital cardioplegic solution; (2) St. Thomas' Hospital cardioplegic solution with 7.5 mumol/L TPEN; (3) protection conditions as in group 2, but with TPEN administration during preischemic and reperfusion periods; (4) University of Wisconsin solution; and (5) the same conditions as in group 4 with TPEN administration during the preischemic and reperfusion periods. Significant enhancement of hemodynamic recovery was observed in the presence of TPEN throughout the experiment. The recovery of cardiac output was 24% +/- 4% in group 3, as compared to 12% +/- 4% in group 1 (p < 0.01). The postischemic left ventricular pressure recovery was 57% +/- 4% in group 3, as compared to 18% +/- 7% in group 1 (p < 0.005). The hearts in group 5 recovered, reaching 29% +/- 2% of the preischemic cardiac output and at 65% +/- 2% of the left ventricular pressure recovery (p < 0.05 versus group 3). Lactate dehydrogenase was released throughout the reperfusion. TPEN addition to groups 2 and 3 did not significantly reduce lactate dehydrogenase release; however, TPEN in University of Wisconsin solution and throughout the experiment significantly decreased lactate dehydrogenase release.(ABSTRACT TRUNCATED AT 250 WORDS)


Permeability of lipid bilayers to amino acids and phosphate. Permeability coefficients for amino acid classes, including neutral, polar, hydrophobic, and charged species, were measured and compared with values for other ionic solutes such as phosphate. The rates of efflux of glycine, lysine, phenylalanine, serine and tryptophan were determined after they were passively entrapped in large unilamellar vesicles (LUVs) composed of egg phosphatidylcholine (EPC) or dimyristoylphosphatidylcholine (DMPC). The following permeability coefficients were obtained for: glycine, 5.7 x 10(-12) cm s-1 (EPC), 2.0 x 10(-11) cm s-1 (DMPC); serine, 5.5 x 10(-12) cm s-1 (EPC), 1.6 x 10(-11) cm s-1 (DMPC); lysine, 5.1 x 10(-12) cm s-1 (EPC), 1.9 x 10(-11) cm s-1 (DMPC); tryptophan, 4.1 x 10(-10) cm s-1 (EPC); and phenylalanine, 2.5 x 10(-10) cm s-1 (EPC). Decreasing lipid chain length increased permeability slightly, while variations in pH had only minor effects on the permeability coefficients of the amino acids tested. Phosphate permeability was in the range of 10(-12)-10(-13) cm s-1 depending on the pH of the medium. The values for the polar and charged amino acids were surprisingly similar to those previously measured for monovalent cations such as sodium and potassium, which are in the range of 10(-12)-10(-13) cm s-1, depending on conditions and the lipid species used. This observation suggests that the permeation rates for the neutral, polar and charged amino acids are controlled by bilayer fluctuations and transient defects, rather than partition coefficients and Born energy barriers. The results are relevant to the permeation of certain peptides into lipid bilayers during protein translocation and membrane biogenesis.


Prevention of loss of vertebral bone density in heart transplant patients. Seventy-six patients (63 men, 13 women) have been followed up by vertebral bone density (VBD) studies from 3 to 36 months. VBD was measured by single-energy computerized tomographic scan. Before transplantation, VBD was found to be lower than in age-matched controls (less than 40 years of age [group 1], 96% of controls: 40 through 49 years of age [group 2], 77%; 50 to 60 years of age [group 3], 87%; more than 60 years of age [group 4], 76%). After transplantation, despite oral calcium supplements, VBD fell further in all but two patients (97%), which was almost certainly related to maintenance steroid and cyclosporine therapy, and was most marked in the older groups (group 2, 67% compared with age-matched controls at 6 months; group 3, 60%; group 4, 50%). Intensive therapy with synthetic salmon calcitonin (in 29 of 76 patients [38%]), testosterone (in 33 of 63 men [52%]), or estrogen (in 12 of 13 women [92%]) limited, but did not totally prevent, further loss in VBD; in patients who had shown an approximate 45% loss of VBD from pretransplantation levels, further loss was reduced to between 4% and 10%. Five patients increased bone density after calcitonin therapy. Despite significantly reduced VBD in several older patients, minor vertebral bone compression developed in only one patient. We recommend that all patients undergoing heart transplantation, particularly those over the age of 50 years, should be followed by VBD studies, and therapy should be administered to prevent VBD loss.


Donor heart preservation survey.

Donor heart preservation survey. A questionnaire requesting information on donor heart preservation technique and outcomes during the first 6 months of 1990 was circulated to heart transplantation centers worldwide. Seventy-nine usable replies representing 1371 clinical transplant operations were received. Twenty-seven percent of the respondents reported using some form of donor pretreatment. Most (90%) used single flush cardioplegic induction with the use of eight different types of cardioplegic solutions, only 5% of which were oxygenated. Six different types of storage media were used, and the coolant was melting ice in 66% of the centers. Storage temperatures between 0 degrees C and 7 degrees C were reported, with 78% of the respondents using 4 degrees C storage. Fifty-five percent of the centers used some form of reperfusion modification. No statistically significant associations were noted between outcome and technique, apart from the use of storage medium in which the use of cardioplegic solution conferred a 2.5 times increase in deaths compared with cold saline. The results of this questionnaire provide evidence for the diversity of techniques currently used for donor heart preservation, reflecting the lack of any one optimal method.


Localization of the binding domain of the inhibitory ligand forskolin in the glucose transporter GLUT-4 by photolabeling, proteolytic cleavage and a site-specific antiserum. The binding domain of forskolin in the adipocyte/muscle-type glucose transporter (GLUT-4) was localized with the aid of the photoreactive derivative, [125I]IAPS-forskolin (3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetyl-forskolin). Plasma membranes from insulin-treated rat adipocytes containing predominantly the GLUT-4 isoform were irradiated with UV light in the presence of [125I]IAPS-forskolin. The covalently labeled glucose transporters were isolated by immunoprecipitation with specific antiserum and partially digested with trypsin and elastase. The fragments were separated by gel electrophoresis, transferred on to nitrocellulose membranes, and identified by direct autoradiography and by immunoassay with antiserum against a peptide sequence corresponding to the C-terminus of GLUT-4. Digestion with a high-purity grade trypsin generated two photolabeled fragments with apparent molecular weights of 21 and 16 kDa. Since the antiserum detected two fragments with identical electrophoretic mobility, both labeled fragments appeared to contain the intact C-terminus of GLUT-4. In contrast, digestion with elastase generated only one photolabeled fragment with intact C-terminus at 21 kDa, and a smaller unlabeled fragment with intact C-terminus at 15 kDa. A less pure trypsin preparation generated two labeled (21 and 17 kDa) and one unlabeled (15 kDa) fragment with intact C-terminus. These data suggest that the site of covalent binding of IAPS-forskolin in the GLUT-4 is located within a region of 1-6 kDa defined by the difference between the unlabeled C-terminal fragment (15 kDa) and the labeled fragments (21, 17 and 16 kDa). Based on a tentative allocation of the fragments to the sequence of the GLUT-4, it is suggested that the covalent binding site of IAPS-forskolin is located between the membrane spanning helices 7-9, possibly in the proximity of helix 9.


Histologic changes in donor bronchi may explain the reduced mucosal blood flow seen during acute lung allograft rejection. We assessed the histologic changes in donor and recipient bronchi from 15 dogs that had received a lung transplant and related these changes to the severity of acute rejection seen in the parenchyma of these same lungs. Minimal rejection was associated with no abnormal change in either the donor or the recipient bronchi. In mild lung rejection, mononuclear cell cuffings were seen around bronchial arteries in donor bronchial adventitia, although few mononuclear cell infiltrates were seen in the submucosa. Moderate lung rejection was associated with more prominent mononuclear cell cuffings in the donor bronchial adventitia and mucosal infiltrates of mononuclear cells in the membranous portion. In cases of severe lung rejection, the mononuclear cell infiltrates were also apparent in the cartilaginous portion of the donor bronchial mucosa. Moreover, submucosal edema and detachment of bronchial epithelium were seen. These histologic changes were not observed in the recipient bronchi during acute rejection, nor were they seen in the donor and the recipient bronchi during lung infection without rejection. They might, therefore, reflect acute rejection in the donor bronchus. These results might provide the histologic support for our previous observation of decreased bronchial mucosal blood flow measured by the laser Doppler flowmeter in relation with the extent of acute lung rejection.


Exchange efflux of [3H]palmitate from human red cell ghosts to bovine serum albumin in buffer. Effects of medium volume and concentration of bovine serum albumin. [3H]Palmitate, PA, exchange efflux kinetics is recorded from human erythrocyte ghosts to buffer with bovine serum albumin, BSA, at 0 degrees C. The effects have been investigated of three medium/ghost volume ratios: 36, 80 and 500, of six BSA concentrations, [BSA]: 0.01, 0.02, 0.05, 0.2, 1 and 2% (1.5, 3.0, 7.5, 30, 150 and 300 microM) and of various v, molar ratios of palmitate to BSA, between 0.15 and 0.94. Data are analyzed in terms of a virtually closed three-compartment model. In theory, the tracer efflux is biexponential and the rate coefficients differ at least 20 fold [1]. The efflux rate at 2% BSA is monoexponential beyond our resolution time of about 1 s, but nearly biexponential at or below 0.2% BSA with a well-defined smallest-rate coefficient beta. beta depends strongly on [BSA] but is remarkably v independent. The medium/ghost volume ratio has no effect on beta when [BSA] > or = 0.2%, although beta measured at 2% BSA is almost 2-fold higher than at 0.2%. This suggests the presence of an unstirred layer, USL. According to our model, the observations are understood quantitatively on basis of our previously published dissociation rate constants of the PA-BSA complex, as well as PA equilibrium bindings to ghost membranes (Bojesen, I.N. and Bojesen, E. (1991) Biochim. Biophys. Acta 1069, 297-307). Essentially, beta is theoretically a function of two terms, one comprising the membrane transport parameters and the other the medium-dependent variables. Most important is the clearance with respect to monomer concentration adjacent to the membrane. The clearance is calculated on basis of quasi-stationary diffusion in USL. The data are compatible with a planar USL of 6 microns depth and with the same area as a ghost but not with a spherical USL.


Alteration of the passive electrical properties of lymphocyte membranes induced by GM1 and GM3 glycolipids. The electrical conductivity of normal human lymphocyte suspensions has been measured in the frequency range from 10 kHz to 100 MHz, where a well-pronounced conductivity dispersion occurs, caused by the surface polarization at the interface between the cell membrane and the extracellular solution. We have investigated the alteration of the passive electrical properties of the cytoplasmatic cell membrane induced by two different gangliosides (GM1 and GM3) inserted, at various concentrations, into the outer leaflet of membrane double layer. The alterations observed in the dielectric parameters (the membrane conductivity and the membrane permittivity) derived on the basis of a 'double-shell' model, result in an overall increase of the ion permeation across the membrane and an enhanced polarizability of its hydrophilic region for both gangliosides investigated. The relevance of these alterations is discussed.


A new diffusion chamber system for the determination of drug permeability coefficients across the human intestinal epithelium that are independent of the unstirred water layer. A new method for determining permeability coefficients, that are independent of the unstirred water layer (UWL), has been developed. The method was used to determine the cellular permeability coefficient of the rapidly absorbed drug testosterone in monolayers of the human intestinal epithelial cell line, Caco-2. Using a new diffusion cell with an effective stirring system based on a gas lift, the cellular permeability coefficient for testosterone was (1.98 +/- 0.13).10(-4) cm/s which is 3.5-times higher than the permeability coefficient obtained in the unstirred system. The thickness of the UWL obtained with the well stirred diffusion cell was 52 +/- 4 microns. This value is much lower than those previously reported in various well stirred in vitro models. The calculated cellular permeability of testosterone was 13-23-times lower than that for an UWL of the same thickness as the epithelial cell (17-30 microns). We conclude that the permeability of the epithelial monolayer must be included in calculations of the thickness of the UWL.


Influence of staphylococcal delta-toxin on the phosphatidylcholine headgroup as observed using 2H-NMR. The interaction of the 8-toxin peptide isolated from Staphylococcus aureus with the headgroup region of lipid bilayer membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was investigated using deuterium (2H) and phosphorus (31P) nuclear magnetic resonance (NMR) spectroscopy. At relatively low peptide/lipid ratios (P/L < 0.10), all 2H- and 31P-NMR spectral lineshapes at 25 degrees C were indicative of a single population of liquid-crystalline lipids in a bilayer arrangement. At these P/L ratios, delta-toxin had only marginal effects on the size of the quadrupole splitting measured from POPC labelled at either the alpha-methylene (POPC-alpha-d2) or the beta-methylene segment (POPC-beta-d2) of the choline headgroup and, similarly small effects on the magnitude of the chemical shift anisotropy (CSA) of the 31P-NMR spectrum. With increasing amounts of delta-toxin (0.10 < P/L < 0.15) the size of the 2H quadrupole splitting from POPC-alpha-d2, as well as the magnitude of the 31P-CSA, decreased progressively and rapidly. The quadrupole splitting from POPC-beta-d2, however, remained relatively unaffected. At yet higher levels of delta-toxin (P/L > 0.15), all 2H- and 31P-NMR spectra indicated the presence of multiple lipid populations experiencing varying degrees of increased conformational disordering. The spectral lineshapes of these apparently nonbilayer spectral components reverted to bilayer-type lineshapes upon lowering the measuring temperature to 5 degrees C. At the utmost highest level of delta-toxin measured here (P/L = 0.20), all 2H- and 31P-NMR spectra consisted of a single, broad, apparently nonbilayer-type component, indicative of hindered but virtual isotropic motional averaging of the POPC headgroups. In this case no reversion to bilayer-type spectra could be obtained by decreasing the temperature. We could obtain no evidence that the conformation of the choline headgroup of POPC was responding to any specific influence of delta-toxin on bilayer surface electrostatics.


Expression of GLUT-2 cDNA in human B lymphocytes: analysis of glucose transport using flow cytometry. The molecular characterization of transport proteins is often limited by transient functional expression or the need for a simple method to select functional cDNA clones. We used a mammalian expression system to obtain long-term expression of GLUT-2, an isoform of glucose permease. Rat GLUT-2 cDNA was ligated into an EBV vector (pLPP) and transfected into B lymphocytes which lack GLUT-2. Northern and Western analyses confirmed expression of GLUT-2 protein in membranes of transfected cells. Two functional assays using flow cytometry were developed to distinguish GLUT-2 transfectants from control/pLPP transfectants. Uptake of NBD-glucosamine, a fluorescent analogue of glucose, was increased in GLUT-2 transfectants. In addition, when exposed to hypertonic glucose medium, GLUT-2 transfectants and control/pLPP transfectants exhibited a difference in forward-angle light scatter (FALS), an index of cell volume, indicating a difference in glucose permeability. Independent measurements of glucose uptake (isotopic) and cell volume (video microscopy) confirmed the flow cytometry observations. This expression system used in combination with flow cytometry is useful for studying the functional properties of glucose and other solute transporters.


Effect of cationic cholesterol derivatives on gene transfer and protein kinase C activity. Four different cationic derivatives of cholesterol were synthesized which contain either a tertiary or a quaternary amino head group, with and without a succinyl spacer-arm. Their ability to inhibit protein kinase C (PKC) activity was measured in a detergent mixed micellar solution. Derivatives containing a quaternary amino head group were effective inhibitors (Ki approx. 12 and 59 microM) of PKC and derivatives containing a tertiary amino head group were approx. 4-20-fold less inhibitory. Liposomes containing an equimolar mixture of dioleoylphosphatidylethanolamine (DOPE) and a cationic cholesterol derivative were tested for the DNA-mediated transfection activity in mouse L929 cells. Highest activity was found with the derivative with low PKC inhibitory activity and with a succinyl spacer-arm. The transfection activity of this tertiary amine derivative, N,N-dimethylethylenediaminyl succinyl cholesterol was dependent on DOPE as a helper lipid; liposomes containing dioleoylphosphatidylcholine and this derivative had little activity. The transfection protocol of this new cationic liposome reagent was optimized with respect to the ratio of liposome/DNA, dose of the complex and time of incubation with cells. Several adherent cell lines could be efficiently transfected with this liposome reagent without any apparent cytotoxicity. However, the transfection activity was strongly inhibited by the presence of serum components.


The mechanism of the solute-induced chain interdigitation in phosphatidylcholine vesicles and characterization of the isothermal phase transitions by means of dynamic light scattering. A new method is introduced for the detection of chain interdigitation in phospholipid bilayers. The same method is used to measure the hydrocarbon tilt in the dipalmitoylphosphatidylcholine membranes as a function of the bulk concentration of the interdigitation-inducing solutes, such as ethanol. The hydrocarbon tilt in the phosphatidylcholine bilayers is demonstrated to be limited to angles below approx. 51 degrees. The need for higher tilt values leads to bilayer interdigitation. Solute-induced chain interdigitation is shown to be a cooperative process provoked by the excessively large lateral repulsion in the interfacial region and the concomitant excessive chain tilt. Ethanol-induced phosphatidylcholine interdigitation, for example, proceeds via interdigitated domains formation and finally gives rise to the bilayers with fully intercalated chains tilted by at least 30 degrees (and sometimes as much as 50 degrees) with respect to the membrane normal.


Retinal and retinol promote membrane fusion. Disk membranes from the bovine retinal rod outer segments (ROS) were found to fuse with vesicles made of lipids extracted from unbleached ROS disk membranes, using a lipid mixing assay for membrane fusion (relief of self-quenching of R18, octadecylrhodamine B chloride). If the retinal chromophore of rhodopsin was reductively linked to opsin before lipid extraction, the vesicles made of the extracted lipids were not suitable targets for fusion of the disk membranes. The addition of retinal and retinol to these vesicles restored their ability to fuse. Therefore, the presence of all-trans retinal was implicated in promoting membrane fusion in this system. To test this possibility, the ability of retinal and retinol to influence the phase behavior and the fusion capability of large unilamellar vesicles (LUV) of N-methyl dioleoylphosphatidylethanolamine (N-methyl-DOPE) was examined. Both retinal and retinol stimulated the fusion of vesicles of N-methyl-DOPE (contents mixing with ANTS, 1-aminonaphthalene-3,6,8-trisulfonic acid; DPX, p-xylylene bis(pyridinium bromide)). Both compounds reduced the onset temperature for isotropic resonances in the 31P-NMR spectra of N-methyl-DOPE dispersions and the onset temperature, TH, for formation of hexagonal II phase. These results were consistent with previous studies in which the onset temperature for the 31P-NMR isotropic resonances were correlated with stimulation of membrane fusion. These data suggested that both retinal and retinol may stimulate membrane fusion by destabilizing the bilayers of membranes.


Cocaine induces intracellular free Mg deficits, ischemia and stroke as observed by in-vivo 31P-NMR of the brain. 31P-NMR spectroscopic studies were performed in vivo on brains of rats administered cocaine. Cocaine.HCl (1-5 mg/kg) administered systemically to lightly anesthetized rats resulted in significant and progressive deficits in whole brain intracellular free Mg ([Mg2+]i). Intracellular pH (pHi) also fell in a progressive manner but only after a significant fall in brain [Mg2+]i was noted. Both [Mg2+]i and pHi returned to normal in most rats. Brains of rats that exhibited stroke-like events, however, demonstrated continued intracellular acidosis associated with progressive loss of phosphocreatine and elevation of Pi up until death. These observations are consistent with the tenet that injection of cocaine can result in severe cerebral vasospasm, ischemia and rupture of cerebral blood vessels as a consequence of depletion of brain [Mg2+]i.