Comparative analysis of Pax-2 protein distributions during neurulation in mice and zebrafish. Members of different vertebrate species share a number of developmental mechanisms and control genes, suggesting that they have similar genetic programs of development. We compared the expression patterns of the Pax-2 protein in Mus musculus and Brachydanio rerio to gain a better understanding of the evolution of developmental control genes. We found that the tissue specificity and the time course of Pax-2 expression relative to specific developmental processes are remarkably similar during the early development of the two organisms. The brain, the optic stalk, the auditory vesicle, the pronephros, and single cells in the spinal cord and the hindbrain express Pax-2 in both species. The Pax-2 expression domain in the prospective brain of E8 mouse embryos has not been described previously. Expression appears first during early neurulation at the junction between the midbrain and hindbrain. However, there are some differences in Pax-2 expression between the two species. Most notable, expression at the midbrain/hindbrain boundary is no longer detectable after E11 in the mouse. Using monoclonal antibodies, we could exclude that primary neurons express Pax-2 in the zebrafish spinal cord. Our results confirm that Pax genes are highly conserved both in sequences and in expression patterns, indicating that they may have a function during early development that has been conserved during vertebrate evolution.


An early molecular component of the wound healing response in rat embryos--induction of c-fos protein in cells at the epidermal wound margin. The spreading of epithelial sheets plays a pivotal role in normal embryonic morphogenesis but the cellular and molecular mechanisms underlying these processes are not very well understood. One way of directly examining epithelial spreading in the embryo is to excise a piece of tissue and follow the epithelial wound healing process. In this paper we report our studies of the healing of a simple excisional lesion to the E12.5 rat embryo hindlimb. The wounded, living embryo is cultured in a roller bottle and under such conditions the lesion is completely re-epithelised by 24 h. We find that epidermal cells specifically at the margin of the wound rapidly (within 15 min of wounding) and transiently express the nuclear transcription factor c-fos. This induction occurs whether or not serum is included in the culture medium. We speculate that local induction of c-fos at the wound site may play a role in regulating transcription of genes involved in the healing process.


Computer-aided classification of malignancy in astrocytomas. I. The value of nuclear parameters obtained by automated black and white image analysis. Ninety-three selected cases of astrocytomas including glioblastomas (astrocytomas grades 1-4) were evaluated by means of Feulgen-stained microscopic slides for nuclear parameters obtained by automated black and white image analysis (ABWIA). The goal was to determine to what extent nuclear features evaluated by ABWIA were applicable as classifiers for the computer-aided numerical classification of malignancy in astrocytomas. Before the automated evaluation, all tumours had been subjectively graded according to the Mayo Clinic grading rules as delineated by Ringertz. Twenty-three nuclear parameters were evaluated and tested for their classification impact. With a model of five parameters (number of nuclei per area, mean of the convex form factor, extinction sum, extinction variation, and full-width-half-maximum of the extinction distribution) the highest reclassification rate of 75% correctly reclassified cases was obtained. Although this is a good result for a classification using only nuclear parameters, it is too poor for practical application. Thus, nuclear parameters evaluated by ABWIA alone are insufficient for numerical classification models assessing the malignant expression of astrocytomas.


Computer-aided classification of malignancy in astrocytomas. II. The value of categorically evaluated histologic and non-histologic features for a numerical classifier. The present study was carried out in order to obtain a numerical classifier for the assessment of the malignancy in astrocytomas including glioblastomas ('astrocytomas grade 4'). The attempt resulted in 'TESTAST 268', a classifier based on a reference sample of 268 tumours, 67 in each of four malignancy classes. TESTAST 268 aids the identification of astrocytomas with one of four malignancy classes by means of eight classification variables, five histologic and three non-histologic. Identification is achieved with the aid of linear discriminant functions, both according to Bayes' decision rule (BAYTEST) and by canonical discriminant analysis (CANTEST) using the squared Mahalanobis distance. The discriminant functions with the calibration of the reference sample of the 268 tumours may be implemented on personal and even small pocket computers for practical application.


Can chromatin texture predict structural karyotypic changes in diploid cells from thyroid cold nodules? To assess the effect of a single chromosomal translocation on the nuclear phenotype of human cells, seven diploid adenomas and five diploid carcinomas of the thyroid gland were studied using quantitative nuclear morphometry. Four adenomas and three carcinomas were cytogenetically normal, whereas three adenomas and two carcinomas had a unique chromosomal translocation. A densitometric parameter discriminated adenomas from carcinomas (skewness of the optical density histogram, SODH), and tumours with and without chromosomal translocation (standard deviation of the optical density, SDODH). These results demonstrate that single chromosomal structural rearrangements produce quantifiable alterations of nuclear organisation, but that other nuclear features which do not express an aneuploid DNA content or an abnormal karyotype differentially characterise benign and malignant conditions.


A morphometric filter improves the diagnostic value of morphometric analyses of frozen histopathological sections from mammary tumours. Frozen sections of 202 consecutive breast tumour cases were analyzed by morphometric quantitation of nuclear features. Nuclei were selected at random. Conventional light microscope examination of the paraffin-embedded specimens revealed 144 cases of cancer and 56 benign tumours. Using multivariate discriminant analysis of morphometric features, all but two of the benign cases and 79% of the malignant tumours were correctly classified. When a morphometrically based dynamic filter set to exclude 'non-diagnostic' nuclei was used, the correctly classified malignant cases rose to 86% Morphometry is a fast, reproducible and efficient method that can be used in conjunction with the histomorphological diagnosis of mammary frozen sections. The combination of systematic sampling and an objective dynamic filter may be a powerful approach to quantitative analyses of tumours from other sites. However, it is also likely that efficiency can be improved by combining nuclear morphometric features with structural, histochemical and molecular biological data. The combination of traditional histomorphological examination with quantitative information may well increase the diagnostic accuracy in individual patients.


How often is dysplasia diagnosed by biopsy or smear examination? Application of a maximum likelihood based method to the assessment of detection rates in the nasal mucosa of nickel workers. In view of the known increased risk of nasal carcinoma and the high prevalence of dysplastic lesions of the nasal mucosa among nickel workers, regular screening for the existence of possibly precancerous dysplastic lesions is offered to workers in a Norwegian nickel refinery. Unfortunately, available sampling techniques do not allow the identification of all subjects in whom dysplastic changes are present. Independent histological and cytological (brush cytology) diagnoses, obtained for each of a group of 90 workers, have been used to estimate, by a maximum likelihood method, the probabilities that existing dysplastic lesions will be detected by each of these two screening methods. In the group studied, cytology performed rather less well than histology in unambiguously detecting dysplasia. However, when cytological specimens showing irregular (possibly dysplastic) epithelial cells were grouped with those showing clear dysplastic changes, detection probabilities were estimated at 0.52 by histology and 0.57 by cytology. Detection probabilities were estimated to be higher among subjects with a previously known history of dysplasia, particularly by histology (P < 0.01), probably due to larger dysplastic areas. In view of both its greater facility and speed of sampling, and its greater acceptability, brush cytology may be preferable to biopsy sampling for the screening of large numbers of workers at risk.


The determination of the steady-state pharmacokinetic profile of fluphenazine decanoate by gas chromatography/mass spectrometry detection. This study uses the highly sensitive method of gas chromatography/mass spectrometry to compare the basic steady-state pharmacokinetic parameters of two fluphenazine decanoate formulations. Sixteen stable outpatients participated in a two-way crossover design study of the bioavailability of a new formulation of FPZ Dec, i.e., 10 mg/ml, to the standard 25 mg/ml formulation. When compared to a 1 ml injection of the standard formulation (25 mg/ml) over a two-week, steady-state period, we found bioequivalence as evidenced by similar mean areas under the curve (hrs x ng/ml). We did find that the injection volume of the same dose (2.5 ml of a 10 mg/ml formulation) results in a statistically significantly higher maximum serum level of parent fluphenazine. A tendency toward faster time to peak level was observed with the 10 mg/ml formulation but the difference was not statistically significant. Both of these differences are considered too small to be clinically significant. In a subgroup of 10 patients, pre-injection serum fluphenazine levels correlated significantly (Pearson r = 0.78, p < 0.05) with serum prolactin levels.


Repetitive behaviors in chronically institutionalized schizophrenic patients. Repetitive dysfunctional behaviors (e.g., polydipsia, bulimia, hoarding, mannerisms) are frequently observed in chronically institutionalized schizophrenics, cause significant morbidity and are readily reproduced in animal models. The goal of this study was to assess the frequency and severity of these behaviors. Thirty-two chronic schizophrenics on an extended treatment unit were rated on the Elgin Behavioral Rating Scale, which includes eight repetitive behaviors and eight positive and negative symptoms. Forty-seven percent of the patients exhibited at least one severe, or 2 moderate, repetitive behaviors, while 63% exhibited at least one severe or 2 moderate positive or negative symptoms. The mean total score (+/- SD) on the eight repetitive behaviors (10.3 +/- 6.1) was about 2/3 that for the eight positive and negative symptoms (15.3 +/- 8.9, t = 4.1, p = .0001). Interrater reliability for the repetitive behaviors was similar to that for the positive and negative symptoms. Repetitive behaviors were positively related to male gender, white race and total length of hospitalization. Repetitive dysfunctional behaviors are frequently observed and can be reliably rated in chronically institutionalized schizophrenics.


Natural killer cell activity in schizophrenia and schizoaffective disorder: a pilot study. Natural killer cell activity was prospectively studied in 15 patients with chronic schizophrenia and in seven patients with schizoaffective disorder, depressed type. These patients were compared to individually matched normal controls. No mean differences in natural killer cell activity between the patient groups and their controls were observed.


Deficits in social schemata in schizophrenia. Several recent studies have examined the manner in which social information is processed in an attempt to better understand the interpersonal functioning deficits of schizophrenia. In this study, the manner in which schizophrenic subjects represent social information, and the relationships between these social representations and measures of information processing, are examined. Specifically, 30 DSM-IIIR patients with schizophrenia and 15 normal controls were assessed on measures of social schema processing, information processing, and symptomatology. Results showed that schizophrenic patients earned significantly lower schema processing scores than the normal comparison group. Schema deficits of the schizophrenic group were significantly associated with recall memory and vigilance. These findings suggest that deficits in the representation of social information provides a unique perspective for understanding the interpersonal dysfunctions of schizophrenia.


Selecting controls for schizophrenia research studies: the use of the National Adult Reading Test (NART) is a measure of premorbid ability. The National Adult Reading Test (NART) has achieved popularity as a measure of pre-morbid intellectual ability, based on the premises that pronunciation of irregular words is unaffected in many clinical disorders and that performance is highly correlated with general intellectual ability. Recently, schizophrenia research studies have begun to appear in the literature, where the NART has been used to estimate pre-morbid ability. However, this use has preceded the basic required demonstration that, in fact, NART performance is unaffected by the schizophrenic process. In the present study, NART performance was compared across three groups; 20 acutely ill unmedicated DSM-IIIR schizophrenics, 10 other unmedicated acute psychotics, and 20 control subjects. When demographic variability between the groups was controlled for, there were no group differences in terms of NART performance. NART performance was not correlated with Brief Psychiatric Rating Scale scores, and in all three groups, no significant differences emerged when demographically predicted intelligence quotients were compared with NART estimated intelligence quotients. NART performance (predicted on the basis of demographic variables) was not significantly different from observed NART performance in any of the three experimental groups. However, within the sample with schizophrenia, NART estimated pre-morbid IQ was significantly higher than currently measured intellectual abilities. These results suggest that the National Adult Reading Test provides a reasonable estimate of pre-morbid ability in acutely ill, unmedicated schizophrenic patients.


Positive and negative symptoms in the psychoses: multidimensional scaling of SAPS and SANS items. Recently, the validity of the simple dichotomy between positive and negative symptoms in psychosis has been questioned. A newly admitted group of 114 DSM-III patients with psychotic disorder were assessed using Andreason's positive and negative symptoms scales. Multidimensional scaling, augmented by cluster analysis, was applied to the full item set of these scales and showed clearly that there are three major, independent groups of symptoms: Hallucinations/Delusions, Positive Thought Disorder and Negative Symptoms. Within the Hallucinations/Delusions and Negative Symptoms groups there was some additional structure which does not conform to the SAPS and SANS sub-scales. In particular there was considerable heterogeneity within the Hallucinations/Delusions group, and delusions of persecution may represent a fourth independent dimension of psychopathology which is under-represented in these scales.


Procedural learning of cognitive and motor skills in psychotic patients. Two kinds of procedural learning, viz. learning of a sequence of simple motor responses and learning to solve a rather complex problem (Tower of Hanoi), as well as declarative learning (word list learning) were investigated in a group of psychotic inpatients (n = 67) and a control group of non-psychotic psychiatric inpatients (n = 19). Within the psychotic group, correlations of the task variables with positive and negative symptoms were explored. There was no difference between both groups in motor procedural learning. Psychotic patients were less efficient than controls in solving the Tower problem, but both groups again showed an equal amount of procedural learning. Consistent with the literature, however, a clear difference between both groups was found in declarative learning. The memory tasks did not correlate significantly with psychotic symptoms. These findings are interpreted as another indication that automatic information processing in psychotic patients is intact. The results are discussed with reference to neuropsychological research on procedural learning in neurological patients.


Semantic priming of word pronunciation and lexical decision in schizophrenia. Experimental assessments of semantic memory structure and function in schizophrenic subjects can be a useful approach for delineating some of the information processing deficits in schizophrenia. In this study, a pronunciation and a lexical decision semantic priming experiment were conducted with 19 schizophrenic subjects and 20 normal controls. A short stimulus-onset asynchrony (250 msec) and a relatively low proportion of related prime-target pairs were used in order to examine automatic priming and in order to avoid the contribution of attentional, controlled processes. On the pronunciation task, schizophrenic subjects showed a significant priming effect, equal to the priming shown by normal controls. However, on the lexical decision task, schizophrenics, unlike normal controls, did not show a priming effect which is significantly greater than zero, even though the group difference in priming effect (interaction of priming effect by group) was nonsignificant. The lack of priming on the lexical decision task is consistent with the hypothesis that schizophrenic subjects may show abnormalities in the realm of post-lexical, controlled information processing. The equal-to-normal priming for schizophrenic subjects indicates that the basic structure of the semantic network, including associations among related concepts, is intact in schizophrenia, and that spreading activation also occurs normally.


Carboxy-terminal-extended variant of the human fibrinogen alpha subunit: a novel exon conferring marked homology to beta and gamma subunits. Similarities between the N-terminal regions of the three subunits of the clotting protein fibrinogen--(alpha beta gamma)2--suggest that they evolved from a common progenitor. However, to date no human alpha chain has been found with the strong C-terminal homology shared by the beta and gamma chains. Here we examine the natural product of a novel fibrinogen alpha chain transcript bearing a separate open reading frame that supplies the missing C-terminal homology to the other chains. Additional splicing leads to the use of this extra sequence as a sixth exon elongating the alpha chain by 35%. Since the extended alpha chain (alpha E) is assembled into fibrinogen molecules and its synthesis is enhanced by interleukin-6, it suggests participation in both the acute phase response and normal physiology.


Helical structure and orientation of melittin in dispersed phospholipid membranes from amide exchange analysis in situ. A trapping method combined with high-resolution nuclear magnetic resonance spectroscopy is described for the measurement of hydrogen-deuterium exchange rates for individual amides of polypeptides bound to fully hydrated, dispersed phospholipid bilayers. Exchange rates were measured for 22 of the 24 amide hydrogens of bee venom melittin bound to bilayers composed of egg phosphatidylcholine/phosphatidylserine (88:12, mol/mol) dispersed in 20 mM sodium acetate, pH 4.0. Amides of residues 5-11 and 16-22 had exchange rates suppressed by between 30- and 1000-fold, and the rate suppression exhibited a helical periodicity with amides on the hydrophobic helix face up to 20-fold more stable than those on the hydrophilic face of the helix. These results demonstrate that under the conditions studied melittin adopts a helical conformation with stable helical hydrogen bonds extending to residue 22 and that the helix is oriented with the hydrophobic face directed toward the membrane interior.


Structure and dynamics of transmembrane signaling by the Escherichia coli aspartate receptor. The structure of the cytosolic extension of the first transmembrane region (TM1) of the Escherichia coli aspartate receptor (residues 3, 4, and 5) and conformational changes within that region have been characterized by targeted cross-linking studies and by measurement of the effect of aspartate binding on cross-linking and methylation rates and compared with the periplasmic extension of the same helix. These experiments show that (1) the cytosolic extension of TM1 is helical, with residues 4 and 4' closest together at the dimer interface; (2) the helix is more solvent-exposed at the cytosolic side of the membrane than on the periplasmic side; and (3) aspartate binding enhances the rate of cross-linking at Cys 4, and the resulting cross-linked receptor displays aspartate-induced transmembrane increases in methylation by the cytoplasmic methylase (the CheR protein). We conclude that aspartate induces a conformational change that does not involve large intersubunit movements that lead to an increase in distance between the cytosolic ends of the first membrane-spanning helices; rather, the motion involved is largely contained within individual subunits, possibly resulting in a small movement between positions 4 and 4'.


Structure-function analysis of Escherichia coli translation initiation factor IF3: tyrosine 107 and lysine 110 are required for ribosome binding. Translation initiation factor IF3 is required for peptide chain initiation in Escherichia coli. IF3 binds directly to 30S ribosomal subunits ensuring a constant supply of free 30S subunits for initiation complex formation, participates in the kinetic selection of the correct initiator region of mRNA, and destabilizes initiation complexes containing noninitiator tRNAs. The roles that tyrosine 107 and lysine 110 play in IF3 function were examined by site-directed mutagenesis. Tyrosine 107 was changed to either phenylalanine (Y107F) or leucine (Y107L), and lysine 110 was converted to either arginine (K110R) or leucine (K110L). These single amino acid changes resulted in a reduced affinity of IF3 for 30S subunits. Association equilibrium constants (M-1) for 30S subunit binding were as follows: wild-type, 7.8 x 10(7); Y107F, 4.1 x 10(7); Y107L, 1 x 10(7); K110R, 5.1 x 10(6); K110L, < 1 x 10(2). The mutant IF3s were similarly impaired in their abilities to specifically select initiation complexes containing tRNA(fMet). Toeprint analysis indicated that 5-fold more Y107L or K110R protein was required for proper initiator tRNA selection. K110L protein was unable to mediate this selection even at concentrations up to 10-fold higher than wild type. The results indicate that tyrosine 107 and lysine 110 are critical components of the ribosome binding domain of IF3 and, furthermore, that dissociation of complexes containing noninitiator tRNAs requires prior binding of IF3 to the ribosomes.


Backbone dynamics of the glucocorticoid receptor DNA-binding domain. The extent of rapid (picosecond) backbone motions within the glucocorticoid receptor DNA-binding domain (GR DBD) has been investigated using proton-detected heteronuclear NMR spectroscopy on uniformly 15N-labeled protein fragments containing the GR DBD. Sequence-specific 15N resonance assignments, based on two- and three-dimensional heteronuclear NMR spectra, are reported for 65 of 69 backbone amides within the segment C440-A509 of the rat GR in a protein fragment containing a total of 82 residues (MW = 9200). Individual backbone 15N spin-lattice relaxation times (T1), rotating-frame spin-lattice relaxation times (T1 rho), and steady-state (1H)-15N nuclear Overhauser effects (NOEs) have been measured at 11.74 T for a majority of the backbone amide nitrogens within the segment C440-N506. T1 relaxation times and NOEs are interpreted in terms of a generalized order parameter (S2) and an effective correlation time (tau e) characterizing internal motions in each backbone amide using an optimized value for the correlation time for isotropic rotational motions of the protein (tau R = 6.3 ns). Average S2 order parameters are found to be similar (approximately 0.86 +/- 0.07) for various functional domains of the DBD. Qualitative inspection as well as quantitative analysis of the relaxation and NOE data suggests that the picosecond flexibility of the DBD backbone is limited and uniform over the entire protein, with the possible exception of residues S448-H451 of the first zinc domain and a few residues for which relaxation and NOE parameters were not obtained. in particular, we find no evidence for extensive rapid backbone motions within the second zinc domain. Our results therefore suggest that the second zinc domain is not disordered in the uncomplexed state of DBD, although the possibility of slowly exchanging (ordered) conformational states cannot be excluded in the present analysis.


Cloning, nucleotide sequence, and chromosome localization of the human pleiotrophin gene. Pleiotrophin (PTN), midkine (MK), and retinoic acid-induced heparin-binding (RI-HB) protein are members of a recently discovered family of developmentally regulated cytokines. We report here the cloning, sequencing, chromosomal localization, and structural organization of the genomic version of the human PTN gene and its comparison to the mouse MK gene. The PTN gene was found to be arranged in five exons and four introns, in a fashion similar to that of the mouse MK gene. Exon 1, as for MK, does not appear to encode amino acid sequence. As in the case of the MK gene, exon 2 encodes the hydrophobic leader sequence of PTN, which constitutes the beginning of gene translation. The signal peptide cleavage site of both genes lies toward the 3' end of exon 2. Exons 3 and 4 of PTN were most closely related to exons 3 and 4 of the MK gene; in particular, six of the ten cysteine residues were coded for in exon 3 and the remaining 4 in exon 4. The intron-exon splice junctions of both genes occurred through the same residues. The two genes were found to be less closely related in the fifth exon which encodes the highly basic C-terminal domains, the translation termination codon, and the polyadenylation signal of both cDNAs. We also report approximately 2000 bp of the 5' untranslated sequence of the PTN gene and the site of initiation of transcription in human placenta. PTN was localized to human chromosome 7q33-34 by fluorescence in situ hybridization. These data confirm the existence of a new gene family of developmentally regulated cytokines.


Comparative circular dichroism studies of an anti-fluorescein monoclonal antibody (Mab 4-4-20) and its derivatives. This study presents circular dichroism (CD) spectra of a high-affinity monoclonal anti-fluorescein antibody (Mab 4-4-20), its Fab fragments, and corresponding single-chain antibody (SCA). In the region 200-250 nm, the differences in the CD spectra between these proteins reflect the uneven distribution of chromophores (tryptophan and tyrosine) rather than a major conformational change. On the basis of near-UV CD spectra, binding of the hapten fluorescein to these protein antibodies elicits an increased asymmetry in the microenvironment of the chromophoric residues in contact with the hapten and also perturbs the interface between VL and VH domains. The hapten-binding site provides a chiral microenvironment for fluorescein that elicits a pronounced induced fluorescein CD spectrum in both the visible and UV regions. In contrast to the parent molecules, SCA is thermolabile. Our results demonstrate that (1) UV CD spectra are useful for assessing the chromophoric microenvironment in the binding portion of antibodies and (2) the extrinsic fluorescein hapten CD spectra provide information about the interaction of hapten with the binding pocket.


Characterization of recombinant HIV-1 Tat and its interaction with TAR RNA. Recombinant HIV-1 Tat (Tat 1-86) has been purified from the cytoplasmic fraction of Escherichia coli without the use of protein denaturants or chaotropic agents. Chloroquine-mediated uptake of the purified protein into cells resulted in transactivation of the HIV LTR promoter. Tat retains 1.64 mol of Zn2+/mol of protein by atomic absorption spectroscopy. Circular dichroism measurements indicated that the structure of recombinant Tat contains 15-20% alpha-helix. Filter binding assays showed that Tat binds to a 63-nucleotide target TAR RNA with a dissociation constant (Kd) of 10 nM at 25 degrees C, 0.05 M ionic strength, pH 7.5, in a 1:1 Tat-TAR RNA stoichiometry. Nonelectrostatic interactions provide the principal source of free energy of association. While the pH optimum occurs over a wide H+ concentration, the salt dependence of Kd indicates formation of a single ion pair. UV-induced protein-RNA cross-linking produced a labeled Tat-TAR RNA adduct, indicating that direct contact occurred between the Tat protein and TAR RNA.


DNA sequence selectivities in the covalent bonding of antibiotic saframycins Mx1, Mx3, A, and S deduced from MPE.Fe(II) footprinting and exonuclease III stop assays. DNA sequence selectivities in the covalent binding of the antitumor antibiotic saframycins Mx1, Mx3, A, and S have been determined by complementary strand MPE.Fe(II) footprinting and exonuclease III stop assays on two different 545 and 135 base pair long HindIII/RsaI restriction fragments of pBR322 DNA. Saframycins Mx1, Mx3, A, and S recognize primarily 5'-GGG sequences. All four antibiotics also recognize 5'-GGPy sequences, however a cytosine is preferred over a thymine at the 3'-end of this recognition site in all cases. Saframycins Mx1, Mx3 and S, which possess the OH leaving group, also recognize the 5'-CCG sequence, in contrast to saframycin A, which contains the CN leaving group. In contrast, the OH-containing saframycins also recognize the 5'-CTA sequence. Saframycins Mx2, B and C, which lack the critical CN or OH leaving group, do not show any footprints on the restriction fragments examined in this study. The measured binding site size for all four antibiotics is three base pairs. The exonuclease III stop assay independently confirmed the formation of a covalent bond and the strong preference of the antibiotics for 5'-GGG and 5'-GCC sequences. The latter enzyme assay also suggests that the 5'-terminal or central G of the triad binding site is the base to which reversible covalent attachment of the antibiotic takes place.


Very stable mismatch duplexes: structural and thermodynamic studies on tandem G.A mismatches in DNA. We have used ultraviolet melting techniques to compare the stability of several DNA duplexes containing tandem G.A mismatches to similar duplexes containing tandem A.G, I.A, and T.A base pairs. We have found that tandem G.A mismatches in 5'-Y-G-A-R-3' duplexes are more stable than their I.A counterparts and that they are sometimes more stable than tandem 5'-Y-T-A-R-3' sequences. This is not the case for tandem G.A mismatches in other base stacking environments, and it suggests that tandem G.A mismatches in 5'-Y-G-A-R-3' sequences have a unique configuration. In contrast to tandem 5'-G-A-3' mismatches, tandem 5'-A-G-3' mismatches were found to be unstable in all cases examined.


Influence of an exocyclic guanine adduct on the thermal stability, conformation, and melting thermodynamics of a DNA duplex. As part of an overall program to characterize the impact of mutagenic lesions on the physiochemical properties of DNA, we report here the results of a comparative spectroscopic study on pairs of DNA duplexes both with and without an exocyclic guanine lesion. Specifically, we have studied a family of four 13-mer duplexes of the form d(CGCATGYGTACGC).d(GCGTACZCATGCG) in which Y is either the normal deoxyguanosine residue (G) or the exocyclic guanine adduct 1,N2-propanodeoxyguanosine (X), while Z is either deoxycytosine (C) or deoxyadenosine (A). Thus, the four duplexes studied, which can be designated by the identity of their central Y.Z base pair, are a Watson-Crick duplex (GC), a duplex with a central mismatch (GA), and two duplexes with exocyclic guanine lesions (X), that differ only by the base opposite the lesion (XC and XA). The data derived from our spectroscopic measurements on these four duplexes have allowed us to evaluate the influence of the exocyclic guanine lesion, as well as the base opposite the lesion, on the conformation, thermal stability, and melting energetics of the host DNA duplex. To be specific, our circular dichroism (CD) spectra show that the exocyclic guanine lesion induces alterations in the duplex structure, while our temperature-dependent optical measurements reveal that these lesion-induced structural alterations reduce the thermal stability, the transition enthalpy, and the transition free energy of the duplex.(ABSTRACT TRUNCATED AT 250 WORDS)


Solution structure of the TnAn DNA duplex GCCGTTAACGCG containing the HpaI restriction site. The solution structure of the self-complementary DNA duplex [d(GCCGTTAACGGC)]2, which contains the HpaI restriction site GTTAAC, has been elucidated by two-dimensional NMR, distance geometry (DG), and NOE back-calculation methods. Initial distance constraints were determined by polynomial fitting the two-spin initial NOE rates; backbone constraints from NOE and J-coupling observations (Kim et al., 1992) were included. RMSDs between initial-distance-refined structures derived from random-embedded DG, A-DNA, and B-DNA starting structures were all in the range 0.5-1.0 A, indicating good convergence properties of the algorithm, regardless of the starting structure. A semiautomatic back-calculation refinement procedure was developed and used to generate more refined structures for which the BKCALC-simulated NOE volumes matched the experimental data. The six final structures refined from various starting structures exhibit very good agreement with the experimental data (R values = 0.18) and converge well to within 0.8-A RMSD differences for the central 8 base pairs. The torsion and pseudorotation phase angles were found to be well determined by the data, and the local helical parameters for each base step converged quite well. The final structures show that the central T6-A7 step is somewhat underwound (twist angle ca. 29 degrees), with a large negative cup and a normal (wide) minor groove width, while the T5-T6 and A7-A8 steps have a partially narrowed minor groove.


Site-directed analysis of the functional domains in the factor Xa inhibitor tick anticoagulant peptide: identification of two distinct regions that constitute the enzyme recognition sites. Recombinant tick anticoagulant peptide (rTAP) is a highly selective inhibitor of blood coagulation factor Xa. rTAP has been characterized kinetically as a slow, tight-binding, competitive inhibitor of the enzyme. We used an approach consisting of both recombinant, site-directed mutagenesis and solid-phase chemical synthesis to generate 31 independent mutations in rTAP to identify those regions of the molecule which contribute to the specific, high-affinity binding interaction with factor Xa. Our results demonstrate that the four amino-terminal residues of rTAP constitute the primary recognition determinant necessary for the formation of the high-affinity enzyme-inhibitor complex. The Arg residue in position three is probably not interacting with the S1-specificity pocket of factor Xa in a substrate-like manner since substitution at this position with a D-Arg amino acid produced only a modest decrease in affinity (5-fold). An additional domain in the rTAP molecule located between residues 40 and 54 was identified as a probable secondary binding determinant. Interestingly, this region in rTAP shares significant amino acid sequence homology with a sequence in prothrombin immediately amino-terminal to the factor Xa cleavage site that generates meizothrombin. These observations indicate that specific segments within two different regions of the rTAP molecule contribute to the potent binding interaction between rTAP and factor Xa.


Expression of platelet-derived endothelial cell growth factor in Escherichia coli and confirmation of its thymidine phosphorylase activity. Platelet-derived endothelial cell growth factor (PD-ECGF) has been expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The fusion protein was purified by one-step affinity chromatography on glutathione-agarose beads, and recombinant PD-ECGF was proteolytically cleaved with thrombin from its GST leader peptide to yield pure protein. Recombinant PD-ECGF stimulated [3H]methylthymidine uptake by endothelial cells in vitro; however, we were unable to detect stimulation of cell proliferation under a wide variety of conditions. We confirm that in accord with the recent report that PD-ECGF and human thymidine phosphorylase are products of the same gene [Furukawa, T., Yoshimura, A., Sumizawa, T., Haraguchi, M., & Akiyama, S. I. (1992) Nature 356, 668] recombinant PD-ECGF has thymidine phosphorylase activity comparable to that of E. coli thymidine phosphorylase. Further, E. coli thymidine phosphorylase was able to mimic the activity of recombinant PD-ECGF in the [3H]methylthymidine uptake assay, and it appears that recombinant PD-ECGF's effect on the uptake of thymidine by endothelial cells may be due to modulation of cellular thymidine pools. The mechanism by which PD-ECGF stimulates angiogenesis remains to be elucidated.


Identification of topaquinone and its consensus sequence in copper amine oxidases. The nature of the active site cofactor and the amino acid sequence flanking this structure have been determined in a range of copper amine oxidases. For enzymes from porcine plasma, porcine kidney, and pea seedlings, proteolytic digestion was performed on phenylhydrazone or p-nitrophenylhydrazone derivatives. Thermolysin treatment leads to relatively small active site peptides, which have been characterized by Edman degradation and by resonance Raman spectroscopy. Resonance Raman spectra of peptides show identical peak positions and intensities relative to each other and to a model p-nitrophenylhydrazone derivative of topaquinone hydantoin, establishing topaquinone as the cofactor in each instance. Edman degradation of peptides provides active site sequences for comparison to previous determinations with bovine serum and yeast amine oxidases. The available data establish a consensus sequence of Asn, Topa, Asp/Glu. Trypsin leads to significantly longer peptides, which reveal a high degree of sequence identity between plasma proteins from bovine and porcine sources (89%), with significantly decreased identity between the porcine serum and intracellular amine oxidases (56%). A lower degree of identity (45%) is observed between the pea seedling and mammalian enzymes. As an alternative to the isolation of active site peptides for topaquinone identification, visible spectra of intact proteins have been investigated. It is shown that p-nitrophenylhydrazone derivatives of native enzymes, active site-derived peptides, and a topaquinone model exhibit identical behavior, absorbing at 457-463 nm at neutral pH (pH 7.2) and at 575-587 nm in basic solution (1-2 M KOH). These spectral properties, which appear unique to topaquinone, provide a rapid and simple test for the presence of this cofactor in intact enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)


Orotidylate decarboxylase: insights into the catalytic mechanism from substrate specificity studies. Pyrimidine nucleotides were tested as substrates for pure yeast orotidylate decarboxylase in an attempt to gain insight into the nature of the catalytic mechanism of the enzyme. Substitutions of the 5-position in the pyrimidine ring of the orotidylate substrate resulted in compounds that are either excellent inhibitors or substrates of the enzyme. The 5-bromo- and 5-chloroorotidylates are potent inhibitors while the 5-fluoro derivative is a good substrate with a turnover number 30 times that observed with orotidylate. When carbon 5 of the pyrimidine ring is replaced by nitrogen in 5-azaorotidylate, the resulting compound is unstable in solution with a half-life of 25 min at pH 6. However, studies with freshly generated 5-azaorotidylate show that an enzyme-dependent reaction occurs, presumably decarboxylation. This enzyme reaction follows simple Michaelis-Menten kinetics. Because the 5-aza group is not electrophilic, an enzyme mechanism utilizing a nucleophilic addition of the enzyme at the 5-position is ruled out. We also present studies that are not compatible with a mechanism requiring the formation of a Schiff's base prior to decarboxylation. The enzyme is tolerant of modest substitution at the 4-position, for the 4-keto group can be replaced with a thioketone. However, no catalysis is observed when the same substitution is made at the 2-position. Similarities in the substrate specificity of orotate phosphoribosyltransferase and orotidylate decarboxylase led us to compare the amino acid sequences of the two enzymes; significant (20%) sequence homology was observed.(ABSTRACT TRUNCATED AT 250 WORDS)


A unique catalytic and inhibitor-binding role for Lys93 of yeast orotidylate decarboxylase. The presence of a proton-donating catalytic amino acid side chain in orotidylate decarboxylase (ODCase) was sought by site-directed mutagenesis. Replacement of yeast ODCase Lys93 with a cysteine resulted in a mutant protein (K93C) with no measurable activity, representing a decrease in activity by a factor of, at most, 2 x 10(-8) times the activity of the wild-type enzyme. Treatment of this mutant protein with 2-bromoethylamine, designed to append Cys93 to yield S-(2-aminoethyl)cysteine, restored activity by a factor of at least 5 x 10(5) over the untreated mutant protein. Activity could not be restored by treatment with other brominated reagents designed to replace the epsilon-amino of S-(2-aminoethyl)Cys93 with a different functional group. The overall architecture of the K93C protein was not significantly changed, as judged by the similar dimerization properties (in the absence of ligands) of the mutant enzyme compared to the wild-type enzyme. The binding affinity of the substrate orotidylate was not measurably changed by the mutation, indicating that Lys93 has an essential role in catalysis which is mechanistically distinguishable from substrate binding. Apparently the mutation removes an integral portion of the active site and does not drastically affect the structural or substrate binding properties. However, the affinities of the mutant protein for the competitive inhibitors 6-azauridylate (6-azaUMP) and UMP are significantly altered from the pattern seen with the wild-type enzyme. The K93C protein has an affinity for the neutral ligand UMP which is greater than that for the anionic 6-azaUMP, in clear contrast to the preference for 6-azaUMP displayed by the wild-type enzyme. Lys93 is apparently critical for catalysis of the substrate to product and for the binding of anionic inhibitors; the data are discussed in terms of previously existing models for transition-state analogue inhibitor binding and catalysis.


Characterization by infrared spectroscopy of the interaction of a cardiotoxin with phosphatidic acid and with binary mixtures of phosphatidic acid and phosphatidylcholine. The effect of cardiotoxin IIa from Naja mossambica mossambica, a small basic protein extracted from snake venom, on dimyristoylphosphatidic acid (DMPA) and on equimolar mixtures of DMPA and dimyristoylphosphatidylcholine (DMPC) has been studied by Fourier transform infrared spectroscopy. The interaction of cardiotoxin with DMPA dispersions decreases both the cooperativity of the phase transition of the lipid and the molecular order of the lipid acyl chains in the gel phase. This effect increases with the proportion of the toxin in the complexes and leads to the total abolition of the phase transition of DMPA at a lipid-to-protein molar ratio of 5. Small-angle X-ray results demonstrate that the structure of the lipid-protein complexes is poorly ordered and gives rise to broad diffusion peaks rather than to well-resolved diffraction patterns. Infrared spectra of oriented cardiotoxin-DMPA films show that the protein is not homogeneously oriented with respect to the bilayer surface. The destabilization of the gel-phase structure of DMPA by cardiotoxin also results in a deeper water penetration in the interfacial region of the lipid since more carbonyl ester groups appear to be hydrogen bonded in the presence of the toxin. The infrared results on the phosphate group vibrations also indicate clearly that the basic residues of cardiotoxin interact strongly with the phosphate group of DMPA that becomes partly ionized at a pH as low as 6.5. The results obtained on the interaction of cardiotoxin with an equimolar mixture of DMPA and DMPC clearly demonstrate the ability of this toxin to induce lateral phase separation in this mixture with one phase containing DMPA-rich domains perturbed by cardiotoxin while the second phase is composed of regions enriched in DMPC. Comparison of the results of the current study with those obtained on other basic proteins and polypeptides suggests that charge-induced phase separation occurs only when the charge density on certain regions of the protein structure is high enough to lead to efficient electrostatic interactions with anionic phospholipids. This condition occurs only when the conformation of the protein or polypeptide is well-ordered at the lipid interface.


The major glycolipid recognized by SP-D in surfactant is phosphatidylinositol. Surfactant protein D (SP-D), a multimeric calcium-dependent lectin isolated from pulmonary alveolar lavage, has been previously shown to interact reversibly with crude surfactant [Persson et al. (1990) J. Biol. Chem. 265, 5755-5760]. In this study, SP-D is shown to interact reversibly with a preparation of organelles enriched in lamellar bodies, in a manner inhibited by calcium-chelating agents and by competing saccharides. An interaction with an endogenous glycoprotein could not be identified by electrophoresis of surfactant or lamellar body-associated proteins followed by electrotransfer of the separated proteins to nitrocellulose and then probing with radioiodinated SP-D via lectin overlay. Separation of the surfactant or lamellar body lipids on two-dimensional thin-layer chromatography (2D-TLC) followed by probing with radioiodinated SP-D via lectin overlay demonstrated binding to a single lipid. This interaction was dependent on the presence of calcium and was inhibited by competing saccharides. By assaying column fractions for the ability to bind radioiodinated SP-D after TLC, the glycolipid was purified to homogeneity and identified as phosphatidylinositol (PI). Identification was confirmed by mass spectrometry. We further demonstrate the ability of radiolabeled SP-D to bind to PI presented in a lipid bilayer through separation of free SP-D from liposome-bound SP-D on density gradients of Percoll. The interaction of SP-D with PI is dependent on calcium and inhibited by competing saccharides. SP-D binds with similar efficiency to liposomes with mole fractions of PI ranging from 2.5% to 30%, thereby demonstrating the lectin's ability to recognize mole fractions of PI available in surfactant.(ABSTRACT TRUNCATED AT 250 WORDS)


Gangliosides: differentiation markers for murine T helper lymphocyte subpopulations TH1 and TH2. On the basis of the pattern of lymphokines they secrete, murine T helper clones can be divided into two subsets, TH1 and TH2. This concept of two different T helper effector cells helps to explain the diversity of immune reactions occurring in different parts of the body. The in vivo localization of T helper subtypes is of great interest, but up to now no biochemical or surface markers were available to distinguish between them. We analyzed the glycolipids from altogether 12 murine TH1 and TH2 cell lines or clones. A comparison of the gangliosides by thin-layer chromatography showed differences between the TH1 and TH2 cells. Binding studies with specific antibodies to asialo backbone structures after degradation by neuraminidases showed that the main gangliosides from these lymphocytes shared a common GgOse4 backbone and thus differed only in their degree or position of sialylation. Two disialogangliosides appeared to be characteristic. They were isolated from the D10.G4.1 TH2 cell clone and identified by fast atom bombardment mass spectrometry as IVNeuAc,IINeuAc-GgOse4Cer (GD1a) and IVNeuAc,IIINeuAc-GgOse4Cer (GD1 alpha), respectively. GD1a was characteristically only detected in TH2 cells, whereas GD1 alpha was preferably, but not exclusively, expressed by TH1 lymphocytes. Although GD1a was also found in the lung, heart, kidney, and spleen, its expression within the murine immune cells under investigation was unique to TH2 lymphocytes. Scarcely any GD1a was found in thymocytes, B cells, or CD8 positive (cytolytic) T cells, but significant expression was seen in CD4 positive (helper) T cells which include the TH2 subpopulation.(ABSTRACT TRUNCATED AT 250 WORDS)


Structure of the N- and O-glycans of the A-chain of human plasma alpha 2HS-glycoprotein as deduced from the chemical compositions of the derivatives prepared by stepwise degradation with exoglycosidases. The structure of the glycans of the A-chain of human plasma alpha 2HS-glycoprotein was established from the chemical compositions of its derivatives prepared by sequential enzymatic degradation of the carbohydrate moiety, from the determination of the kind and amount of the monosaccharides liberated after each step of the enzymatic digestion, and from the distinct specificity of the highly purified exoglycosidases. The exoglycosidases were three sialidases (Vibrio cholerae, fowl plague virus, and Arthrobacter ureafaciens), two beta-galactosidases (Streptococcus pneumoniae and bovine testis), one alpha-N-acetylgalactosaminidase, one beta-N-acetylglucosaminidase, and one alpha-mannosidase. Utilizing sialidases with different cleavage specificities, the number of alpha 2-3- and alpha 2-6-linked sialic acid residues could be separately determined. As to the beta-galactosidases, the enzyme isolated from S. pneumoniae cleaves only beta 1-4-linked galactose residues, whereas the bovine testes enzyme acts on both the beta 1-4- and beta 1-3-linked galactose residues. Jack bean beta-N-acetylglucosaminidase cleaves beta 1-2, beta 1-4, and beta 1-6 GlcNAc with higher activity for the beta 1-2. Jack bean alpha-mannosidase cleaves alpha 1-2, alpha 1-6, and alpha 1-3 Man with greater activity for alpha 1-2 and alpha 1-6. Bovine liver alpha-N-acetylgalactosaminidase cleaves O-linked GalNAc. On the basis of these results, the A-chain of alpha 2 HS-glycoprotein was found to possess two biantennary N-glycans and two O-linked trisaccharides.


The oxygen-evolving complex requires chloride to prevent hydrogen peroxide formation. Illumination of PSII core preparations can cause the production of H2O2 at rates which approach 60 mumol of H2O2 (mg of Chl.h)-1. The rate of peroxide production is maximal at pH 7.2 at low sucrose concentrations and at concentrations of Cl- (1.5-3.0 mM) that limit the rate of the oxidation of water to O2. The rate of H2O2 production increased with pH from pH 6.8 to 7.2 and was inversely proportional to the oxidation of water to O2 from pH 6.8 to 7.5. While EDTA does not inhibit H2O2 production, this reaction is abolished by 5 mM NH2OH and inhibited by the same concentrations of NH3 that affect water oxidation which indicates that the oxygen-evolving complex is responsible for the production of peroxide generated upon illumination of PSII core preparations. These results support a mechanism in which bound Cl- in the S2 state is displaced by OH- ions which are then oxidized by the OEC to form H2O2. Thus, the OEC requires Cl- to prevent access to the active site of the OEC until four oxidizing equivalents can be generated to allow the oxidation of water to O2.


Anomeric specificity of D-xylose isomerase. Crystal structures of complexes of D-xylose isomerase with deoxysugars have been determined. Deoxynojirimycin is a structural analogue of alpha-pyranose and mimics the binding of these aldose substrates. The structure of this complex supports the hypothesis that an imidazole group catalyzes ring opening of the pyranose. The steric restrictions in the active site of the enzyme prevent a beta-pyranose from binding in the same way. For the reverse reaction with ketoses, the anomeric specificity is less certain. Dideoxyimino-D-glucitol is a structural analogue of the ketose alpha-D-furanose. The binding of the inhibitor dideoxyimino-D-glucitol to the crystals of the enzyme does not mimic the binding of the reactive alpha-D-fructofuranose. Superposition of the nonphysiological substrate alpha-D-fructofuranose onto the atomic positions of dideoxyimino-D-glucitol is not possible due to the steric restrictions of the active site. However, by utilizing the approximate 2-fold symmetry of the sugar, a stereochemically sensible model is produced which is consistent with other data. In addition to reaction with alpha-D-furanose, the enzyme probably reacts with open ring keto sugars which are present at significant concentrations. Other sugars which resemble furanoses either do not inhibit significantly or are not observed in the crystals bound in a single conformation.


Molecular movements in the actomyosin complex: F-actin-promoted internal cross-linking of the 25- and 20-kDa heavy chain fragments of skeletal myosin subfragment. We describe, for the first time, the F-actin-promoted changes in the spatial relationship of strands in the NH2-terminal 25-kDa and COOH-terminal 20-kDa heavy chain fragments of the skeletal myosin subfragment 1 (S-1), detected by their exclusive chemical cross-linking in the rigor F-actin-S-1 complex with m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS). Quantitative electrophoretic analysis of the reaction products showed extensive conversion of the 95-kDa heavy chain of the actin-bound S-1 into a new species with an apparent mass of 135 kDa (yield = 50-60%), whereas the heavy chain mobility remained unaffected when actin was omitted. The 135-kDa entity retained the fluorescence of AEDANS-S-1 but not of AEDANS-actin, indicating that it was not a cross-linked acto-heavy chain adduct. Its extent of production depended markedly on the S-1: actin molar ratio and was maximum near a ratio of 1:4. The MBS treatment of acto-S-1 led also to some covalent actin-actin oligomers which could be suppressed by using trypsin-truncated F-actin lacking Cys-374, without altering the generation of the 135-kDa heavy chain derivative.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of 2,3-butanedione monoxime on myosin and myofibrillar ATPases. An example of an uncompetitive inhibitor. 2,3-Butanedione monoxime (BDM) reversibly inhibits force production in muscle. At least part of its action appears to be directly on the contractile apparatus. To understand better its mechanism of action, we studied the effect of BDM on the steps of myosin subfragment 1 Mg(2+)-ATPase in 0.1 M potassium acetate, pH 7.4. Because of the rapidity of certain processes, we experimented at 4 degrees C and our main technique was the rapid flow quench method. By varying the experimental conditions (relative concentrations of reagents, time scale, quenching agent), it was possible to study selectively the different steps of the S1 Mg(2+)-ATPase: [formula: see text] At saturation (20 mM), BDM had two major effects on the ATPase. First, it increased the equilibrium constant of the cleavage step (K3) from 2 to > 10. Second, it slowed the kinetics of the release of Pi by an order of magnitude (k4; from 0.054 to 0.004 s-1). By contrast, the kinetics of the binding of ATP (k) and the release of ADP (k6) were little affected by BDM. Thus, the oxime appears to interact specifically with M**.ADP.Pi, and it is a rare example of an uncompetitive inhibitor. Its effect is to reduce the steady-state concentration of the "strong" actin binding state M*.ADP and to increase that of the "weak" binding state, M**.ADP.Pi. The effect of BDM on the initial ATPase of Ca2+ activated myofibrils was very similar to that on S1 ATPase. Thus, with myofibrils too BDM seems to exert its main effect subsequent to the initial binding and cleavage steps.(ABSTRACT TRUNCATED AT 250 WORDS)


Conformation of tachyplesin I from Tachypleus tridentatus when interacting with lipid matrices. The mode of action of tachyplesin I, an antimicrobial cationic heptadecapeptide amide isolated from the hemocyte debris of a horseshoe crab, Tachypleus tridentatus, toward lipid matrices was studied with synthetic tachyplesin I, its analogs with Phe in place of Trp or Tyr, a linear analog with no disulfide bonds, and two linear short fragments. Circular dichroism spectra showed that tachyplesin I took an antiparallel beta-structure in buffer solution and a certain less ordered structure in acidic liposomes composed of egg phosphatidylcholine and egg phosphatidylglycerol (3:1). Spectrophotometric titration of the peptides with laurylphosphorylcholine revealed that both Trp and Tyr residues orient toward the inside of lipid matrices, suggesting that they are on the same side of the peptide backbone. The carboxyfluorescein leakage experiment and fluorescence data indicated that tachyplesin I interacted strongly with neutral and acidic lipid bilayers and an aromaticity-rich hydrophobic part of the peptide was embedded in lipid membranes. All the peptides except for the short fragments were almost equally active in lipopolysaccharide binding. The energy-transfer experiment showed that a conformational change occurred such that the Tyr and Trp residues are positioned more closely to each other in acidic liposomes than in buffer solution. The present study strongly suggested that amphipathic lipid bilayers induced a conformational change of tachyplesin I from an energetically stable beta-structure to a less ordered, probably more amphipathic structure.


Molten globule monomer to condensed dimer: role of disulfide bonds in platelet factor-4 folding and subunit association. Platelet factor 4 (PF4) exhibits high affinity for heparin and exists as a tetramer in solution under physiologic conditions. Reduction of the two disulfide bridges in PF4 increases the protein's dissociation constant for heparin approximately 20-fold and shifts the highest apparent aggregation state from tetramer to dimer as evidenced by gel filtration, chemical cross-linking, and 1H-NMR studies. 1H-NMR spectra of reduced PF4 monomers generally show narrower, less dispersed, upfield-shifted NH and alpha H resonances, suggesting the presence of an unfolded monomer state. Reduced PF4 monomer folding, however, is evidenced by the presence of about 12 relatively long-lived backbone NHs and by CD spectra that indicate conservation of overall secondary structure. These data suggest the presence of a molten globule-type state. Urea denaturation shifts this apparent molten globule to a fully unfolded state characterized by more random coil-like resonance shifts. The reduced PF4 dimer state yields NMR and CD data consistent with preservation of tertiary structural folds found for the native species. In this regard, the reduced PF4 folding transition is thermodynamically linked with dimer formation which stabilizes tertiary structure. Monomer-dimer association equilibria for reduced PF4 essentially follow the same pH and salt titration trends as reported previously for native PF4 dimers [Mayo, K. H., & Chen, M. J. (1989) Biochemistry 28, 9469-9478], indicating that that dimer interface is generally conserved in the absence of disulfide constraints. Reduced PF4 tetramers are not apparent under any conditions investigated, suggesting that disulfides are necessary for efficient antiparallel beta-sheet alignment between dimer pairs.


Structure of the proteolytic fragment F34 of calmodulin in the absence and presence of mastoparan as revealed by solution X-ray scattering. The solution X-ray scattering technique has been applied to examine the conformations of the proteolytic fragment F34 (78Asp-148Lys) of calmodulin in the absence of both Ca2+ and mastoparan, in the presence of Ca2+ only, and in the presence of both Ca2+ and mastoparan. The radius of gyration and the molecular weight for the F34 fragment increased by 1.1 +/- 0.3 A and 19%, respectively, upon binding of both 2 mol of Ca2+/mol to the F34 fragment and mastoparan to form the tertiary complex. A smaller change was found for the Ca(2+)-saturated F34 fragment in the absence of mastoparan (0.3 +/- 0.3 A) without any change of the molecular weight. The analysis based on the small-angle scattering data showed that the F34 fragment in the presence of Ca2+ alone preserved the tertiary structure of the globular domain in the crystal to a great extent. Further analyses based on a two-domain model showed that the center-to-center distance between F34 and mastoparan is about 12.7 A, if the structure of the F34 fragment in the presence of mastoparan resembles that in the absence of mastoparan and if mastoparan in the complex retains an alpha-helical conformation. The modeling studies using their crystal structure coordinates have been made on the basis of the solution X-ray scattering data. The combined results support a model proposed by Persechini and Kretsinger [Persechini, A., & Kretsinger, R. H. (1988) J. Cardiovasc. Pharmacol. 12 (Suppl. 5), S1-S12], although the center-to-center distance between mastoparan and the F34 fragment is shorter by about 5 A than that in their model.(ABSTRACT TRUNCATED AT 250 WORDS)


Trimerization of the heat shock transcription factor by a triple-stranded alpha-helical coiled-coil. We have isolated and characterized a 91 amino acid fragment of the heat shock transcription factor from both Saccharomyces cerevisiae and Kluyveromyces lactis. The two protein fragments behave similarly: they form homotrimers, as indicated by sedimentation equilibrium and cross-linking, and contain approximately 80% alpha-helix, as indicated by circular dichroism. Sedimentation velocity and diffusion coefficients indicate that they have an elongated, nonspherical shape. We conclude the following: these fragments contain a domain which forms a trimer via a triple-stranded alpha-helical coiled-coil, similar to that found in influenza hemagglutinin.


Interleukin-6, a marker of preservation injury in clinical lung transplantation. Interleukin-6 (IL-6) is one of the cytokines produced by human alveolar macrophages, lung parenchyma, and other cells in response to injury and infection. We hypothesized that IL-6 is released from poorly preserved lung grafts and may serve as a marker of preservation injury. Sixteen patients who received lung allografts were enrolled in this study. The average ischemic time was 284 +/- 78 minutes. Serum IL-6 level was measured before and at 4 and 24 hours after reperfusion of the grafts by an enzyme-linked immunosorbent assay. Preservation injury was assessed by (1) the need for prolonged intubation (> 7 days), (2) the arterial/alveolar oxygen tension ratio (PaO2/PAO2 ratio) at 4 hours after graft reperfusion (only in heart-lung or double lung recipients), (3) the presence of diffuse alveolar damage on first lung biopsy, and (4) the 30-day graft survival rate. IL-6 level peaked at 4 hours after reperfusion and returned to baseline at 24 hours. The patients were divided into group I (n = 6) and group II (n = 10), depending on whether the 4-hour IL-6 level was more than 1000 pg/ml or less than 500 pg/ml, respectively. Group I patients required longer intubation (p < 0.01) and had a lower PaO2/PAO2 ratio (p < 0.001), more diffuse alveolar damage (p < 0.01), and a lower graft survival rate (p < 0.01) than those of group II. No bacterial, fungal, or viral infection was found during postoperative week 1 in either group.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of allopurinol pretreatment with pulmonary flush on lung preservation. This study was designed to test whether use of allopurinol could improve lung preservation after 6 hours of cold storage. Thirty-two rabbits were divided into four groups (n = 8 each group): (1) the control group received no flush or storage, (2) the EC group received Euro-Collins (EC) solution for both flush and storage, (3) the Allo-F group received Euro-Collins solution with allopurinol (1 mmol/L) for both flush and storage, and (4) the Allo-R group received Euro-Collins solution to which allopurinol (1 mmol/L) was added only to the reperfused blood. For groups 2 through 4, the lungs were flushed (40 ml/kg) in situ, excised, and then stored at 4 degrees C. After storage, the lungs were reperfused for 1 hour with an in vitro blood-perfused ventilated model. Lung function was measured during reperfusion with mean pulmonary arterial pressure, end-inspiratory airway pressure, and blood gas data. The lung wet/dry weight ratio was used to measure lung edema. The lungs in the EC group had a significant increase in mean pulmonary arterial pressure, airway pressure, and wet/dry weight ratio when compared with the control group. The mean pulmonary arterial pressure in either of the groups receiving allopurinol was consistently lower than that in the EC group. The airway pressure in the Allo-R group also significantly decreased compared with the EC group.(ABSTRACT TRUNCATED AT 250 WORDS)


Graft cytokine mRNA activity in rat single lung transplants by reverse transcription-polymerase chain reaction: effect of cyclosporine. Intragraft levels of cytokine mRNA were studied in an orthotopic rat left lung transplant model. Three groups of rats were compared at 7 days after transplantation. Isogeneic (Lewis to Lewis), allogeneic (Brown-Norway to Lewis) untreated, and cyclosporine-treated (25 mg/kg/day, intramuscularly) allogeneic animals underwent analysis of cytokine mRNA isolated from total RNA in freshly excised grafts. Reverse transcription-polymerase chain reaction amplification of interleukin (IL)-2, IL-4, and actin (control) mRNA was performed with custom-synthesized oligonucleotide amplimers targeted to known sequences of rat IL-2 and IL-4 cDNA. Semiquantitative analysis was performed by radioanalytic scanning of gel preparations. Sample specimens from the retrieved grafts were also graded histologically for rejection on a five-point scale. Rejection was most severe in the untreated allografts (p < 0.003). IL-2 mRNA was significantly greater in the untreated allografts when compared with isografts (p < 0.05) and cyclosporine-treated allografts (p < 0.05). No significant differences in IL-4 mRNA between groups were observed. We conclude that semiquantitative analysis of cytokine mRNA by reverse transcription-polymerase chain reaction is a useful and sensitive method for the study of acute rejection in lung grafts and that this technique may become an important tool in future studies of cytokine-mediated responses in cyclosporine-treated allografts.


Leukocyte-depleted reperfusion of transplanted human hearts: a randomized, double-blind clinical trial. Standard methods of myocardial preservation for heart transplantation have generally provided good results. Preservation times beyond 3 hours, however, have been associated with decreased survival. Leukocyte-mediated reperfusion injury is partly responsible for decreased graft function after prolonged graft ischemia. Leukocyte-depleted reperfusion has been shown experimentally to improve cardiac function after cold ischemic arrest. To determine the efficacy and safety of leukocyte-depleted reperfusion, 20 patients were enrolled in a randomized, double-blind clinical trial to be treated with either warm whole blood reperfusion (group I; n = 9) or warm leukocyte-depleted blood reperfusion (group II; n = 11). Reperfusion with leukocyte-depleted blood or whole blood was carried out for 10 minutes, with enriched cardioplegic solution added for the first 3 minutes of reperfusion. The mean donor and recipient age and the ischemic time (142 versus 153 minutes) were not significantly different between the two groups. Coronary sinus release of creatinine phosphokinase-MB 5 minutes after reperfusion was significantly less in group II (1.65 EU/min) than in group I (3.83 units/min; p = 0.05). Thromboxane B2 release was also significantly less (p = 0.05) in group II (33.6 pg/min) than in group I (67.0 pg/min). All hearts functioned adequately in both groups. The duration of inotropic support was shorter in group II than in group I, but the difference was not statistically significant. Postoperative hemodynamics, rejection episodes, and infectious complications were also not significantly different between groups in a mean follow-up of 9 months. Mean ejection fraction 1 month after operation was 65% in both groups. One early death occurred at 66 days secondary to infection; two late deaths occurred in group II, both from rejection. Leukocyte-depleted reperfusion is safe and easily applied in the operating room. Furthermore, leukocyte-depleted reperfusion decreases biochemical evidence of reperfusion injury. Although not influencing postoperative cardiac function when the ischemic time is short, less than 3 hours, leukocyte-depleted reperfusion may prevent significant reperfusion injury and improve posttransplantation graft function when ischemic times are long. Safe extension of the ischemic time would expand the donor pool and allow for better crossmatching.


Remnant atrial function in human and animal recipients of a total artificial heart. The native atria remains intact after total artificial heart (TAH) implantation. The electrical activity of the recipient's atria can be recorded from wires placed during TAH implantation. Regulating TAH heart rate by coupling it with native atrial activity has the potential for a more physiologically responsive TAH. The reactivity of the atrial impulse rate is a critical component of this link, but little is known about atrial responsiveness after TAH placement. Two human and three animal TAH recipients had recordable atrial electrical activity. Human atrial impulse rate after TAH was relatively constant at rest but unresponsive to physiologic stimuli. Analysis of human atrial contraction provided no discernable effect on ventricular filling. Animal atrial impulse rate at rest was more rapid than calves without a TAH. The bovine TAH recipients had an atrial impulse rate that responded to catecholamine stimulation and blockade. Isoproterenol caused a significant rise in atrial impulse rate (152 +/- 16 impulses per minute to 216 +/- 24 impulses per minute; p < 0.05) and propranolol caused a decrease in atrial impulse rate (142 +/- 20 impulses per minute to 122 +/- 19 impulses per minute; p > 0.05). Despite beta blockade, the atrial impulse rate remained abnormally elevated secondary to unknown factors. Animal atrial contraction did appear to intermittently augment TAH ventricular filling. These data indicate that the atria remains electrically intact after TAH implantation. The human atrial impulse rate was unresponsive to physiologic stimuli although the animal atrial impulse rate was affected by exogenous catecholamine administration, but the rate remained abnormally rapid.(ABSTRACT TRUNCATED AT 250 WORDS)


Changes in serum catecholamine levels in patients who are brain dead. Prospective blood samplings from 15 patients admitted with a Glasgow Coma Score of less than 7 were obtained to observe and compare epinephrine, norepinephrine, and dopamine serum levels in patients with brain injury before, after, and in the absence of brain death. Nine of the patients developed or were admitted after brain death. Wide variations in catecholamine blood levels over time were documented, and subgroup analysis precluded useful statistical comparison or inference of the data. The data are presented therefore as descriptive observations only. No apparent differences were noted between similarly injured patients in whom brain death did not develop and patients before brain death or between patients with penetrating versus nonpenetrating brain injury. Brain death was preceded by hypertension and corresponding elevations in serum catecholamine levels in one patient with complete data. Catecholamine levels appeared to fall after brain death in most patients. Only minimal changes in myocardial histology were present in three donor hearts, and the two transplanted hearts functioned satisfactorily. Serum catecholamine measurement or monitoring does not provide a precise method of determining potential injury to the donor heart before or after brain death. Other experimental data and clinical observation indicate that some hearts may be injured in the donor during the evolution of brain death. Pharmacologic intervention may prevent such injury in experimental animals but must be used before brain death is induced. Such interventions should be studied in selected human donors before brain death to determine whether cardiac function is improved in the donor or recipient.


Methotrexate pulse therapy in the treatment of recurrent acute heart rejection. Despite cytolytic induction therapy and triple-drug immunosuppression, acute allograft rejection continues to cause important morbidity and occasional death after heart transplantation. Between November 1, 1988, and May 1, 1990, 24 patients received methotrexate pulse therapy for recurrent or persistent acute rejection despite methylprednisolone, OKT3, or antithymocyte globulin therapy. Methotrexate was administered as a daily oral dose of 2.5 to 15 mg on 1 day/week over 3 weeks (longer in 15 patients because of either severe leukopenia with temporary interruption of therapy or recurrent rejection during methotrexate therapy) with reduction or discontinuation of azathioprine. Rejection incidence was reduced from 1.1 episodes/patient month before methotrexate therapy to 0.2 episodes/patient month after completion of therapy (p = 0.0001). Two patients died within 3 months after treatment, one of cytomegalovirus pneumonia and one of lymphoma. Mean white blood count (WBC) fell from 6900 per ml before methotrexate therapy to 3700 during the first month of methotrexate therapy (p = 0.0005). The lowest WBC typically occurred about 3 weeks after starting methotrexate therapy, and a transient WBC of less than 1000/ml developed in seven patients. By multivariable analysis, the WBC 1 month after starting methotrexate therapy was significantly related to greater bone marrow suppression (lower WBC), immediately before methotrexate therapy, greater overall immunosuppression (more rejection episodes) during the 3 months before methotrexate therapy, and a higher total dose of methotrexate. The following conclusions can be drawn: (1) Methotrexate is a useful adjunct in the treatment of recurrent or persistent rejection.(ABSTRACT TRUNCATED AT 250 WORDS)


Sinoatrial node dysfunction in recipients of domino heart transplants: complication of a surgical harvesting technique. Two of six domino transplantations performed at our institution required permanent pacemaker implantation as a result of persistent sinoatrial node dysfunction and symptomatic nodal rhythm. Retrospective analysis of several potential etiologic factors showed that the only obvious difference between these two patients and the remaining four domino transplant recipients without this complication was the technique used during harvesting of the cardiac graft. The hearts from the two patients with sinoatrial node dysfunction were harvested using a "right atrial cuff preservation" technique, and the hearts of the remaining four patients were harvested with a standard bicaval division technique. Based on this experience, we think that this right atrial cuff preservation harvesting technique carries a potential risk for sinoatrial node damage, and we do not recommend its use for domino transplantation.


Clinical heart transplantation without routine endomyocardial biopsy. In a prospective clinical study, routine endomyocardial biopsy (EMB) for early detection of cardiac allograft rejection was replaced by two noninvasive diagnostic techniques. In 32 patients who underwent transplantation during a 6-month period, intramyocardial electrogram (IMEG) amplitude was recorded with the telemetry function of a dual-chamber pacemaker system. An amplitude decrease of more than 8% below the individual range of variability in overnight long-term recordings served as an indicator of rejection. A prolongation of the time interval Te--the time span between maximal posterior wall contraction and peak retraction velocity--of more than 20 msec was used as an additional indicator of rejection-related left ventricular dysfunction. For patient safety, routine EMBs were still performed but remained undisclosed to the transplantation team. The pathologist was asked to intervene in cases of discrepancies between biopsy results and medical decisions, but no such intervention was necessary. Twenty-seven rejection episodes were treated medically, of which 22 were diagnosed noninvasively. In four patients, EMB, ordered because noninvasive parameters remained inconclusive, led to medical treatment. No false negative IMEG results were observed. Retrospective correlation with rejection gave a 100% negative predictive value for IMEG and a 96.9% negative predictive value for echocardiographic diagnosis. We conclude that omission of a schedule of routine biopsies is justified, if the described techniques of IMEG and echocardiography are meticulously applied and indicate normal cardiac function. EMB remains indicated if noninvasive parameters are not conclusive or if false-positive IMEG results, which were observed in the presence of infection or arrhythmias, are suspected. The frequency of EMB can be reduced by 90%, compared to conventional routine biopsy schedules. Diagnostic safety is increased because the additional information gained from IMEG and echocardiography is helpful in the interpretation of EMBs.


Cyclosporine interactions with miconazole and other azole-antimycotics: a case report and review of the literature. Several antimicrobial drugs have been shown to pharmacokinetically interact with cyclosporine. On two separate occasions, we observed increases in cyclosporine plasma concentrations during concomitant miconazole therapy in a heart transplant patient with an infection secondary to Pseudallescheria boydii. To our knowledge, no interaction between cyclosporine and miconazole has previously been reported. In addition, drug interactions were observed between cyclosporine and ketoconazole and possibly between cyclosporine and SCH 39304, an investigational azole-antifungal agent. No interaction was noted between cyclosporine and fluconazole. In general, clinicians should anticipate drug interactions between cyclosporine and azole-antimycotic agents.


Early diagnosis and follow-up by echocardiography of acquired cor triatriatum after orthotopic heart transplantation. Acquired cor triatriatum in a heart transplant recipient, secondary to a prominent left atrial suture line and torsion of the atria, was diagnosed immediately after cardiopulmonary bypass by transesophageal echocardiography. The patient was followed with serial echocardiograms and right heart catheterizations, showing resolution of the obstruction.


Hemodynamic performance in a heterotopically transplanted dog heart: proposal of techniques for working left heart model of heterotopic (abdominal) heart transplantation. To study hemodynamics together with various aspects of rejection after experimental heart transplantation, we developed a technique to produce a working left heart model of heterotopic (abdominal) heart transplantation. The interatrial septum and tricuspid valve of the donor heart are removed. The pulmonary arterial trunk, pulmonary veins, and inferior vena cava are ligated, and the stumps of the donor aorta and superior vena cava are anastomosed in an end-to-side fashion to the recipient abdominal aorta and inferior vena cava, respectively. Arterial blood from the recipient abdominal aorta thus perfuses the donor myocardium through the coronary artery, and the donor left ventricle receives venous blood from the recipient inferior vena cava as preload. In this model, the donor left ventricle does not pump out enough venous blood to desaturate the recipient femoral arterial blood but does generate approximately the same pressure as the recipient's heart. This model is reproducible, easy to manage, and can be applied to heterotopic heart transplantation in various experimental animals including rats.


Acute septal myocardial infarction diagnosed by endomyocardial biopsy in a heart transplant recipient. We describe an unusual case of acute septal myocardial infarction in a heart transplant recipient. The clinical presentation was most suggestive of acute rejection; the correct diagnosis was first made by endomyocardial biopsy and was then verified by coronary angiography. Acute myocardial infarction should be included in the differential diagnosis of acute rejection after heart transplantation and included among the possible diagnoses made by endomyocardial biopsy in these patients.


Efficacy of dobutamine in the failing transplanted heart. Acute rejection often leads to severe myocardial failure and death. Surprisingly, no systematic study on the efficacy of beta-adrenergic pharmacologic agents have been reported to the present. Because of all the pathophysiologic alterations documented during rejection, we expected an inappropriate response to inotropic drugs, so we have questioned the value of dobutamine during those circumstances. Twelve dogs underwent orthotopic transplantation and were prepared with implantable devices for serial hemodynamic studies to be performed on the resting unanesthetized subject. Of this number, six dogs were studied while they were in immediate postoperative heart failure (3 hours after operation), and the same study was performed when myocardial failure secondary to rejection occurred (5 to 7 days). After basal state measurement, 5 and 10 micrograms.kg-1.min-1 of dobutamine were infused continuously, and the hemodynamic response during the two phases was compared. The baseline cardiac index in the immediate postoperative period was 1.4 +/- 0.4 L.min-1.m2 and 1.8 +/- 1.0 L.min-1.m2 during rejection, showing a similar degree of heart failure. Dobutamine (5 micrograms.kg-1.min-1) increased cardiac index by 97% 3 hours after transplantation and by 35% during rejection (p < 0.05). With 10 micrograms.kg-1.min-1 of dobutamine, the difference between increments was not significant (99% versus 79%). Raising the infusion rate of the drug to 15 and 20 micrograms.kg-1.min-1 during rejection increased cardiac index by 97% and 118%, respectively. Interestingly, no detrimental tachycardia occurred with this increased dosage. Heart failure secondary to acute rejection can therefore be improved by dobutamine.(ABSTRACT TRUNCATED AT 250 WORDS)


Living with a donor heart: feelings and attitudes of patients toward the donor and the donor organ. For the patient, heart transplantation means more than an operation; adjustment to its rigors requires a high degree of personal strength and adequacy of coping skills. The goal of our study was to gain insight into how heart transplant patients cope with the fact that their own heart has been replaced by a donor organ from an unknown dead donor who was the target of disease, accident, or even suicide. Over a period of 2 years 44 transplant patients were interviewed after rehabilitation in a semi-structured interview regarding their feelings about and reactions to the graft and the donor. Their answers were recorded, transcribed, and analyzed as to content. Three groups of patients were identified: (1) the complete deniers (N = 15), who denied thinking about the donor; (2) the partial deniers (N = 17), who were aware of avoiding thinking about the donor; and those who coped (N = 12), who accepted the death of the donor as reality and also reported having more or less close connections with the donor. Eighty-two percent of the patients interviewed accepted the donor heart immediately as their own, whereas the remaining 18% avoided talking and thinking about the graft and donor. The findings are supported by verbatim statements of patients. The role of defense mechanisms in heart transplant patients is discussed.


The psychosocial impact of pediatric heart transplantation. Children with terminal heart disease experience a dramatic improvement in functional status after heart transplantation but may be at increased risk for problems in psychosocial adaptation. Selected psychosocial outcomes were assessed in 49 pediatric heart transplant recipients and their families from five heart transplantation centers. Heart transplant recipients did not appear significantly different from their peers on self-report measures of self-concept and anxiety, but they showed significantly less social competence and more behavior problems than a normative population. Behavior problems observed were most frequently suggestive of depression and were significantly associated with greater family stress and diminished family resources for managing stress. The study findings further suggest that the heart transplant recipients' ability to verbalize or ventilate their feelings and concerns to others seems to facilitate psychosocial adaptation. Assessment of stress, resources, and coping is imperative to enable health professionals to promote the psychosocial adaptation of pediatric heart transplant recipients and their families.


Clinical results of heart transplantation in recipients over 55 years of age with donors over 40 years of age. Between November 1985 and November 1991, 180 patients underwent heart transplantation at our institution; 55 of these patients (31%) were over 55 years of age. Eighteen patients (10%) received hearts from donors who were over 40 years of age (mean age, 47 years; range, 40 to 55 years); (group 1); 37 hearts (21%) were from donors who were under 40 years of age (mean age, 23 years; range, 8 to 38 years). Mean recipient age was 59 years (range, 55 to 64 years) and 57 years (range, 55 to 68 years) in groups 1 and 2, respectively. The main indication for transplantation was ischemic heart disease in group 1 and dilated cardiomyopathy in group 2. Perioperative mortality and intensive care assistance were similar in the two groups. Survival was 88% versus 84% at 1 year and 81% versus 80% at 4 years in groups 1 and 2, respectively. Although infections were more frequent in group 1 (0.27 versus 0.11 episode/patient), the incidence of acute rejection was comparable in the two groups (1.50 versus 1.65 episode/patient). Angiographic and echocardiographic controls showed normal graft function up to 4 years, with low incidence of chronic rejection in both groups. We conclude that heart transplantation in patients over 55 years of age with donors over 40 years of age offers excellent short-term and mid-term results. The consideration of older donors makes heart transplantation a valid therapeutic option for selected patients in the sixth and seventh decade of life, in spite of chronic donor shortage.


Adult heart transplantation: adverse role of chronic alcoholism in donors on early graft function. Because of the increasing shortage of heart donors, selection criteria have been gradually extended. The purpose of this study was to determine the donor-related factors implied in early graft dysfunction and to define new selection criteria. The 70 consecutive adult patients who underwent heart transplantation in our institution between January 1988 and February 1992 were retrospectively studied. Mean donor age was 38 +/- 11 years (10 donors were more than 50 years of age; two donors were more than 60 years of age). Mean ischemic time was 130 +/- 39 minutes. An important proportion of donors (20%) had a history of chronic alcoholism. Thirteen patients experienced immediate graft dysfunction; five of them died within the first operative month. The different parameters studied, which were found to have no significant influence on the early graft function, were the age of the donor, the duration of inotropic support and the dose administered, a relative hemodynamic instability, resuscitation maneuvers, chest trauma, and weight mismatch between donor and recipient. Ischemic time was significantly longer in patients who died of cardiac dysfunction (p < 0.05). Chronic alcoholism in the donor was a very detrimental factor: 54% of patients who had early graft dysfunction versus only 12% of patients who had immediate normal graft function had received a graft from an alcoholic donor (p = 0.003). Excluding such alcoholic donors or reserving them for critically-ill recipients, with an increased risk of early graft dysfunction would be preferable.


Relationship of cardiac allograft size and pulmonary vascular resistance to long-term cardiopulmonary function. The purpose of this study was to evaluate the long-term cardiopulmonary function of heart transplant patients who received disproportionately sized allografts for varying levels of pulmonary vascular resistance. Resting hemodynamics and oxygen uptake during exercise were recorded at 1 year after transplantation in 52 patients. No differences in resting heart rate, cardiac output, stroke volume, peak oxygen uptake during exercise, and exercise duration were found in recipients of undersized hearts (donor:recipient weight ratio [D:R] < 0.75), sized-matched hearts (D:R = 0.75 to 1.25), and oversized (D:R > 1.25) hearts. In a further analysis according to preoperative pulmonary vascular resistance, resting cardiac output (5.8 +/- 1.3 L/min) was normal, and peak exercise oxygen uptake (22.7 +/- 8.0 ml/kg/min) was mildly decreased in recipients of size-matched allografts with a pulmonary vascular resistance of less than 3 Wood units (size-matched hearts, with mild or no pulmonary vascular resistance). Of patients with moderate pulmonary hypertension (pulmonary vascular resistance > or = 3 Wood units), resting cardiac output was normal (5.1 +/- 0.6 L/min) in recipients of oversized hearts and was reduced (4.7 +/- 1.0 L/min) in recipients of sized-matched hearts (p < 0.05 versus recipients of size-matched hearts with pulmonary vascular resistance less than 3 Wood units).(ABSTRACT TRUNCATED AT 250 WORDS)


Cefaclor uptake by the proton-dependent dipeptide transport carrier of human intestinal Caco-2 cells and comparison to cephalexin uptake. The human Caco-2 cell line spontaneously differentiates in culture to epithelial cells possessing intestinal enterocytic-like properties. These cells possess a proton-dependent dipeptide transport carrier that mediates the uptake of the cephalosporin antibiotic cephalexin (Dantzig, A.H. and Bergin, L. (1990) Biochim. Biophys. Acta 1027, 211-217). In the present study, the uptake of cefaclor was examined and found to be sodium-independent, proton-dependent, and energy-dependent. The initial rate of D-[3-phenyl-3H]cefaclor uptake was measured over a wide concentration range; uptake was mediated by a single saturable transport carrier with a Km of 7.6 mM and a Vmax of 7.6 nmol/min per mg protein and by a non-saturable component. Uptake was inhibited by dipeptides but not amino acids. The carrier showed a preference for the L-isomer. The effect of the presence of a 5-fold excess of other beta-lactam antibiotics was examined on the initial rates of 1 mM cefaclor and 1 mM cephalexin uptake. Uptake rates were inhibited by the orally absorbed antibiotics, cefadroxil, cefaclor, loracarbef, and cephradine and less so by the parenteral agents tested. The initial uptake rates of both D-[9-14C]cephalexin and D-[3-phenyl-3H]cefaclor were competitively inhibited by cephalexin, cefaclor, and loracarbef with Ki values of 9.2-13.2, 10.7-6.2, and 7.7-6.4 mM, respectively. Taken together, these data suggest that a single proton-dependent dipeptide transport carrier mediates the uptake of these orally absorbed antibiotics into Caco-2 cells, and provide further support for the use of Caco-2 cells as a cellular model for the study of the intestinal proton-dependent dipeptide transporter.


Effect of estradiol on accelerated atherosclerosis in rabbit heterotopic aortic allografts. Migration and proliferation of vascular smooth muscle cells are early and major events in the formation of atherosclerotic lesions. We report on an aorta transplant model in rabbits wherein myointimal proliferation is inhibited by 17-beta-estradiol. The abdominal aortas of outbred white New Zealand rabbits were harvested and allografted to the carotid artery of the recipient. The animals, which were fed either a normal or a high-cholesterol (0.5%) diet, were killed 3 weeks later. The degree of myointimal proliferation was measured with a digitized system attached to a light microscope. The myointimal hyperplasia was expressed as the cross section area of the intima/the area of the intima + the area of the media x 100. Transmission electron micrographs were obtained for all vessels. Intimal thickening was shown mainly to consist of proliferating smooth muscle cells. The cholesterol diet resulted in significantly higher serum total cholesterol levels compared to animals on a normal diet (p < 0.0001) but did not affect serum high-density lipoprotein-cholesterol or serum triglyceride levels. The cholesterol diet was also associated with a greater but not significant amount of intimal thickening. Treatment with 17-beta-estradiol significantly decreased both serum triglyceride concentration (p < 0.05) and myointimal thickening (p < 0.01) in cholesterol-fed animals. Transmission electron microscopy showed that the endothelial cells appeared structurally normal in the estradiol-treated animals. Further, estradiol prevented the appearance of vacuolized macrophages. Thus estradiol may decrease myointimal thickening by preserving the endothelium and preventing macrophage appearance in the intima.(ABSTRACT TRUNCATED AT 250 WORDS)


The conformational behaviour of phosphatidylinositol in model membranes: 2H-NMR studies. Dimyristoylphosphatidylinositol (DMPI) has been synthesized with the appropriate natural stereochemistry and labelled with deuterium at specific sites in the D-myo-inositol headgroup. 2H-NMR spectroscopy of DMPI in its lamellar phase at a molar ratio of water-to-lipid RW/L of 129 and at 70 degrees C reveals quadrupolar splittings delta v of 3.83 and 2.17 kHz, respectively, for the five axially oriented C-D bonds and the single equatorially oriented C-D bond of the D-myo-inositol headgroup. Between RW/L ratios of 129 and 210 and between 30 degrees C and 80 degrees C the value of the ratio of these splittings delta nu ax/delta nu eq varies significantly (between 1.17 and 4.38). If it is assumed that, at a particular temperature, there is a single preferred orientation of the inositol headgroup, and that motion of the DPMI molecule establishes axial symmetry with respect to the bilayer normal then the ratio of these quadrupolar splittings can be used to impose constraints on that orientation. For example, the data are inconsistent with a situation in which the inositol ring lies parallel to the membrane surface and are difficult to reconcile with an arrangement where the inositol ring lies perpendicular to the surface. Computational modelling identifies four possible 'tilted' orientations, all of which are consistent with the data, and two of these allow good intramolecular hydrogen bonds to be formed. In one there is hydrogen bonding between the inositol C2-OH and the phosphate pro-R oxygen. This is close to the conformation previously identified as being dominant in DMSO solution (Bushby, R.J., Byard, S.J., Hansbro, P.M. and Reid, D.G. (1990) Biochim. Biophys. Acta 1044, 231-236).


Effects of double-filtration plasmapheresis and a platelet-activating factor antagonist on the prolongation of xenograft survival. Xenotransplantation may be considered a possible solution to the clinical donor-organ shortage. We studied the effects of the removal of preformed natural antibodies against donor tissue by double-filtration plasmapheresis (DFPP) and the administration of a platelet-activating factor antagonist on the prolongation of xenograft survival using the pig-to-dog model. The porcine hearts were perfused with the blood of mongrel dogs. Pairs of animals were divided into four groups: group I (n = 3) was given no pretreatment before perfusion; group II (n = 6) was pretreated by DFPP; group III (n = 3) was given a platelet-activating factor antagonist intravenously at a dose of 0.1 mg/kg 5 minutes before and after perfusion; group IV (n = 5) was pretreated by DFPP and then given a platelet-activating factor antagonist. The titer of natural antibodies was measured by lymphocytotoxicity and hemagglutination. Heart specimens were examined by immunohistochemical staining for deposits of dog antibodies. No immunosuppressive drugs were given in this study. The porcine hearts resumed beating immediately after perfusion in all groups. The mean duration of the graft beating was 17.0 +/- 2.0 minutes for group I, 215.2 +/- 22.6 minutes for group II (p < 0.001 compared with group I), 18.3 +/- 2.1 minutes for group III, and 317.0 +/- 26.5 minutes for group IV (p < 0.001 compared with group II). Immunoglobulin (Ig) M, IgG, complements (C3, C4), and fibrinogen were removed significantly from the perfusing blood, and natural antibody titer was scarcely detectable in groups II and IV after DFPP.(ABSTRACT TRUNCATED AT 250 WORDS)