The C-terminal domain of Escherichia coli ribosomal protein L7/L12 can occupy a location near the factor-binding domain of the 50S subunit as shown by cross-linking with N-[4-(p-azidosalicylamido)butyl]-3-(2'-pyridyldithio)propionamide.

All large ribosomal subunits contain two dimers composed of small acidic proteins that are involved in binding elongation factors during protein synthesis. The ribosomal location of the C-terminal globular domain of the Escherichia coli ribosomal acidic protein L7/L12 has been determined by protein cross-linking with a new heterobifunctional, reversible, photoactivatable reagent, N-[4-(p-azidosalicylamido)-butyl]-3-(2'-pyridyldithio)propionamide . Properties of this reagent are described. It was first radiolabeled with 125I and then attached through the formation of a disulfide bond to a unique cysteine of L7/L12, introduced by site-directed mutagenesis at residue 89. Intact 50S ribosomal subunits were reconstituted from L7/L12-depleted cores and the radiolabeled L7/L12Cys89. Irradiation of the reconstituted subunits resulted in photo-cross-linking between residue 89 and other ribosomal components. Reductive cleavage of the disulfide cross-link resulted in transfer of the 125I label from L7/L12Cys89 to the other cross-linked components. Two radiolabeled proteins were identified, L11 and L10. The location of both of these proteins is well established to be at the base of the L7/L12 stalk near the binding sites for the N-terminal domain of both L7/L12 dimers, and for elongation factors. The result indicates that L7/L12 can have a bent conformation bringing the C-terminal domain of at least one of the L7/L12 dimers at or near the factor-binding domain. The cross-linking method with radiolabeled N-[4-(p-azidosalicylamido)butyl]-3-(2'-pyridyldithio)propionamide should be applicable for studies of other multicomponent complexes that can be reconstituted.


Genomic cloning of the gene for an IgE-binding lectin reveals unusual utilization of 5' untranslated regions.

epsilon BP (for epsilon binding protein) is a M(r) 31,000 S-type animal lectin that binds to IgE and has been identified as the homologue of Mac-2, a macrophage cell-surface marker, as well as the lectins RL-29, CBP35, and L-34. The protein is composed of two domains with the amino-terminal portion containing tandem repeats of nine amino acids and the carboxyl-terminal half containing consensus sequences shared by S-type animal lectins. We determined the genomic map in both rat and mouse and isolated overlapping genomic clones that contain the 5' two-thirds of the murine gene. The remaining portion of the gene was obtained by polymerase chain reaction (PCR) amplification of genomic murine DNA followed by subcloning into plasmid vectors. The epsilon BP gene is composed of six exons separated by five introns. The entire amino-terminal repetitive sequence is contained in exon III, and the carboxyl-terminal domain is encoded by the three succeeding exons (IV, V, VI). The latter three exons correspond well in size and share sequence homology with three exons coding for 14-kDa S-type lectins. The sequence in exon I offers an explanation for the generation of two mRNAs differing only in their 5' untranslated sequences, previously reported in Mac-2 cDNA clones. Using cDNA synthesis and PCR amplification, we determined that two alternative splice sites are used in many different types of cells. This alternative splicing results in different 5' untranslated regions of the murine epsilon BP mRNA.


Multidomain binding of transforming growth factor alpha to the epidermal growth factor receptor.

Solubilized epidermal growth factor receptor (EGF-R) has been used in an extension of the Geysen epitope mapping protocol in order to provide additional insight into the amino acid residues in human transforming growth factor alpha (hTGF alpha) which are critical to recognition and binding. Overlapping heptapeptides which encompassed the 50 amino acid primary sequence of hTGF alpha were synthesized on a polyethylene solid phase, and the amount of detergent-solubilized EGF-R bound to each peptide was measured using ELISA. EGF-R appeared to bind reproducibly to four heptapeptides cognate to sequences in both the N- and C-domains of hTGF alpha (residues 22-28, 28-34, 36-42, and 44-50). Visualization of these four regions on three-dimensional solution phase structures of hTGF alpha, derived from 1H NMR measurements [Kline, T.-P., Brown, F.K., Brown, S.C., Jeffs, P.W., Kopple, K.D., & Mueller, L. (1990) Biochemistry 29, 7805-7813], indicated that the peptide segments are located on a single face of the protein and suggested the presence of a potential receptor binding cavity. If peptide segments within both the N- and C-domains of hTGF alpha are involved in binding to EGF-R, then this has direct consequences for possible molecular mechanisms by which receptor activation might take place. For example, the observed conformational flexibility in the six NMR-derived hTGF alpha structures due to variations in the main-chain torsion angles of Val-33, in combination with the involvement of residues from both domains in the proposed binding cavity, may imply that receptor activation results from interdomain reorientation in the protein ligand.(ABSTRACT TRUNCATED AT 250 WORDS)


Determination of the solution structure of a synthetic two-site calcium-binding homodimeric protein domain by NMR spectroscopy.

The solution structure of a 34-residue synthetic calcium-binding peptide from site III of chicken troponin-C has been determined by 1H NMR spectroscopy. In solution and in the presence of calcium this peptide forms a symmetric two-site homodimeric calcium-binding domain (Shaw et al., 1990). The solution structure of this dimer was determined from the measurement of 470 NOEs from a 75-ms NOESY data set. For the dimer structure determination, the constraint list included 868 distance restraints, 44 phi angles, and 24 chi 1 and 2 chi 2 angles. Seven structures were calculated by restrained molecular dynamics using a procedure in which intramonomer distances were used first and then all distances, intra- and intermonomer, were input during further dynamics. The structures exhibited a fold very similar to the C-terminal domain of troponin-C comprised of a pair of helix-loop-helix calcium-binding sites. The rms deviation of these structures for backbone atoms between residues 97-122 and 97'-122' for the dimer was 0.82 A. The dimer structure was also calculated to be more symmetric than sites III and IV in troponin-C.


Crystal and molecular structure of a DNA fragment containing a 2-aminoadenine modification: the relationship between conformation, packing, and hydration in Z-DNA hexamers.

The crystal and molecular structure of d(CGUA'CG)2 (where A' is 2-aminoadenine) has been determined and refined to an R factor of 13.8% for data 8.0-1.3 A. The structure is very similar to the original Z-DNA structures with the sequence d(CGCGCG)2 [Gessner, R. V., Frederick, C. A., Quigley, G. J., Rich, A., & Wang, A. H.-J. (1989) J. Biol. Chem. 264, 7921] and shows that the substitution of 2-aminoadenine-uracil base pairs in the two central steps is consistent with Z-DNA formation. In addition, we show how waters mediating intermolecular interactions may help to explain the ZI-ZII conformational pattern found in many Z-DNA structures.


A sequence element necessary for self-cleavage of the antigenomic hepatitis delta RNA in 20 M formamide.

The genomic and antigenomic RNAs of hepatitis delta virus are capable of self-cleavage and show no significant sequence similarities to other known self-cleaving RNAs. We have derived an antigenomic delta RNA which cleaves to completion in 15 s in 9 mM magnesium at 37 degrees C and is capable of efficient self-cleavage in concentrations of formamide as high as 20 M. Cleavage in high concentrations of denaturant is dependent upon the presence of a polypurine sequence element, GGAGA, located between 81 and 85 nucleotides downstream of the cleavage site. Mutation of the initial G81G82 to C81C82, or removal of the sequence element, results in a loss of the ability to cleave in high formamide concentrations. Changing the final U-2C-1 of a pyrimidine-rich region, UCUUC, just upstream of the cleavage site, to G-2G-1 severely affects the self-cleavage, but introducing the two mutations, GG to CC and UC to GG, into the same molecule, restoring potential base pairing, partially restores the formamide stability. Relocating the GGAGA sequence upstream of the cleavage site also results in partial restoration of the formamide cleavage. Although the GGAGA sequence is important for self-cleavage under denaturing conditions, it does not appear to be necessary for HDV RNA cleavage in normal buffer conditions.


2'-Fluoro- and 2'-amino-2'-deoxynucleoside 5'-triphosphates as substrates for T7 RNA polymerase.

2'-Fluoro- and 2'-amino-2'-deoxynucleoside 5'-triphosphates have been investigated as substrates for T7 RNA polymerase. Michaelis-Menten kinetic parameters are reported for the incorporation of 2'-fluoro-2'-deoxyuridine, 2'-fluoro-2'-deoxycytidine, and 2'-amino-2'-deoxyuridine into runoff transcripts. The 2'-amino derivative of uridine is a better substrate than the 2'-fluoro derivative. Gel electrophoretic analysis shows that full-length transcripts with a length of 2500 nucleotides can be obtained with the analogues, although a considerable amount of shorter fragments accompanies the full-length product. In keeping with the kinetic analysis, the 2'-aminouridine triphosphate gives a cleaner product than the 2'-fluoro analogue. Transcription of two tRNA genes shows that such shorter templates can be transcribed to full-length products essentially without premature termination with any of the analogues.


Nonidentical DNA-binding sites of endonuclease NaeI recognize different families of sequences flanking the recognition site.

NaeI endonuclease uses a two-site binding mechanism to cleave substrate DNA: reaction-rate studies imply that occupancy of the second DNA site causes an allosteric change in the protein that enables DNA cleavage at the first site [Conrad, M., & Topal, M. D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9707-9711]. Measurements of relative binding affinities for 14-base-pair DNA fragments containing the NaeI recognition sequence GCCGGC and various flanking sequences showed that the two DNA-binding sites are not identical. G.C-rich flanking sequences were preferred by the activator binding site, whereas A.T-rich flanking sequences were preferred by the substrate binding site: GGGTGCCGGCAGGG was preferred 8-fold more by the activator site but 14-fold less by the substrate site than TTTCGCCGGCGTTT. Substitution of pyrimidine or 7-deazapurine for purine immediately 3' to GCCGGC reduced DNA affinity for only the activator site by up to 26-fold, implying that the activator DNA-binding site requires N-7 base contacts immediately flanking GCCGGC. The implications of nonidentical DNA-binding sites, one of which binds a specific DNA site to allosterically activate the other, are discussed.


Three-dimensional solution structure of the B domain of staphylococcal protein A: comparisons of the solution and crystal structures.

The three-dimensional solution structure of the recombinant B domain (FB) of staphylococcal protein A, which specifically binds to the Fc portion of immunoglobulin G, was determined by NMR spectroscopy and hybrid distance geometry-dynamical simulated annealing calculations. On the basis of 692 experimental constraints including 587 distance constraints obtained from the nuclear Overhauser effect (NOE), 57 torsion angle (phi, chi 1) constraints, and 48 constraints associated with 24 hydrogen bonds, a total of 10 converged structures of FB were obtained. The atomic root mean square difference among the 10 converged structures is 0.52 +/- 0.10 A for the backbone atoms and 0.98 +/- 0.08 A for all heavy atoms (excluding the N-terminal segment from Thr1 to Glu9 and the C-terminal segment from Gln56 to Ala60, which are partially disordered). FB is composed of a bundle of three alpha-helices, i.e., helix I (Gln10-His19), helix II (Glu25-Asp37), and helix III (Ser42-Ala55). Helix II and helix III are antiparallel to each other, whereas the long axis of helix I is tilted at an angle of about 30 degrees with respect to those of helix II and helix III. Most of the hydrophobic residues of FB are buried in the interior of the bundle of the three helices. It is suggested that the buried hydrophobic residues form a hydrophobic core, contributing to the stability of FB.(ABSTRACT TRUNCATED AT 250 WORDS)


A possible role for the conserved trimer interface of ferritin in iron incorporation.

Ferritin is a large protein, highly conserved among higher eukaryotes, which reversibly stores iron as a mineral of hydrated ferric oxide. Twenty-four polypeptides assemble to form a hollow coat with the mineral inside. Multiple steps occur in iron core formation. First, Fe2+ enters the protein. Then, several alternate paths may be followed which include oxidation at site(s) on the protein, oxidation on the core surface, and mineralization. Sequence variations occur among ferritin subunits which are classified as H or L; Fe2+ oxidation at sites on the protein appears to be H-subunit-specific or protein-specific. Other steps of ferritin core formation are likely to involve conserved sites in ferritins. Since incorporation of Fe2+ into the protein must precede any of the other steps in core formation, it may involve sites conserved among the various ferritin proteins. In this study, accessibility of Fe2+ to 1,10-phenanthroline, previously shown to be inaccessible to Fe2+ inside ferritin, was used to measure Fe2+ incorporation in two different ferritins under various conditions. Horse spleen ferritin (L/H = 10-20:1) and sheep spleen ferritin (L/H = 1:1.6) were compared. The results showed that iron incorporation measured as inaccessibility of Fe2+ to 1,10-phenanthroline increased with pH. The effect was the same for both proteins, indicating that a step in iron core formation common among ferritins was being measured. Conserved sites previously proposed for different steps in ferritin core formation are at the interfaces of pairs and trios of subunits. Dinitrophenol cross-links, which modify pairs of subunits and affect iron oxidation, had no effect on Fe2+ incorporation.(ABSTRACT TRUNCATED AT 250 WORDS)


Kinetic resolution of peptide bond and side chain far-UV circular dichroism during the folding of hen egg white lysozyme.

The kinetics of regain of the native ellipticity in the far- and near-UV spectra have been investigated during the refolding at pH 7.8 and 20 degrees C of guanidine-unfolded, nonreduced hen egg white lysozyme. Stopped-flow studies showed that the ellipticities at 260 and 289.5 nm exhibit biphasic kinetics with rate constants of about 50 s-1 and 2.5 s-1 for the rapid and slow phase, respectively. The ellipticity in the far-UV obeyed triphasic kinetics. In addition to a rapid and a slow phase with rate constants similar to those observed in the near-UV, a "burst" of ellipticity was shown to occur in the dead time of the experiments. The effects of low pH and of concentrations of guanidine ranging from 0.075 to 1.5 M on the rapid and slow rate constants were studied. Under all conditions investigated, the rate constants observed in the far- and near-UV for a given phase were the same, thus suggesting that the molecular events observed in the two regions of the UV spectrum are either identical or strongly coupled. Continuous-flow experiments at different wavelengths between 214 and 240 nm under conditions where the dead time for the observation was only 4 ms, followed by a detailed analysis of the kinetics of ellipticity change at each wavelength, provided the spectrum of the molecular species formed at the end of the burst phase. This spectrum was found to closely fit that predicted from the secondary structure of native lysozyme.(ABSTRACT TRUNCATED AT 250 WORDS)


Spectroscopic analysis of a methionine-48 to tyrosine mutant of chicken troponin C.

A mutant (M48Y) of chicken skeletal muscle troponin C was prepared in which Tyr replaced Met-48 of the recombinant protein (rTnC). Since Tyr and Trp are normally absent, spectral properties could be unambiguously assigned to the site of substitution. In the crystal structure, this residue lies at the COOH-terminal end of the B-helix of the N domain in a region postulated to undergo a significant conformational change to a more polar environment upon Ca2+ binding [Herzberg et al. (1986) J. Biol. Chem. 261, 2638-2644]. Comparison of the far-UV CD spectra of M48Y and rTnC in the absence and presence of Ca2+ indicated no overall structural alteration due to the mutation. However, Ca2+ titration of the ellipticity change showed a reduction in Ca2+ affinity and cooperativity of sites I and II. A Ca(2+)-induced increase in the near-UV ellipticity of M48Y at pH 7.12 and a red shift in its UV absorbance spectrum occurred over a range of free [Ca2+] attributable to the N-domain transition only. This was largely abolished at pH 5.3 where Ca2+ no longer binds to sites I and II. That region of the 1H NMR spectrum attributable to Tyr was broadened upon Ca2+ binding. These Ca(2+)-induced changes are consistent with the environment of the Tyr side chain becoming chiral, less polar, and more immobile, all in a direction opposite to that predicted. These observations indicate that while the general features of the postulated model are valid, it is unlikely to be correct in detail.


Reversible dimer dissociation of tubulin S and tubulin detected by fluorescence anisotropy.

Concentration-dependent dissociation of dimers of goat brain tubulin S and tubulin was studied by fluorescence anisotropy. Upon dilution, assembly-competent fluorescein 5'-maleimide labeled dimers of tubulin S and tubulin show a progressive decrease in fluorescence anisotropy. That this lowering of anisotropy results from the dissociation of tubulin S dimers into monomers was shown by dilution experiments with unlabeled homologous and heterologous proteins. A nonlinear least-squares fit of the data gave a dissociation constant of 7.1 x 10(-8) M for tubulin S compared to 7.2 x 10(-7) M for tubulin at 25 degrees C in 0.1 M PEM buffer, pH 7.0. van't Hoff plots of dimer-monomer dissociation of tubulin S and tubulin also show considerable differences in delta H and delta S. Effects of ionic strength and colchicine on the equilibrium constants are also substantially different for tubulin and tubulin S. The implications of these observations on the influence of C-terminal tails on tubulin structure are discussed.


Differential scanning calorimetry of thermal unfolding of the methionine repressor protein (MetJ) from Escherichia coli.

The thermal stability of the methionine repressor protein from Escherichia coli (MetJ) has been examined over a wide range of pH (pH 3.5-10) and ionic strength conditions using differential scanning calorimetry. Under reducing conditions, the transitions are fully reversible, and thermograms are characteristic of the cooperative unfolding of a globular protein with a molecular weight corresponding to the MetJ dimer, indicating that no dissociation of this dimeric protein occurs before unfolding of the polypeptide chains under most conditions. In the absence of reducing agent, repeated scans in the calorimeter show only partial reversibility, though the thermodynamic parameters derived from the first scans are comparable to those obtained under fully reversible conditions. The protein is maximally stable (Tm 58.5 degrees C) at about pH 6, close to the estimated isoelectric point, and stability is enhanced by increasing ionic strength in the range I = 0.01-0.4 M. The average calorimetric transition enthalpy (delta Hm) for the dimer is 505 +/- 28 kJ mol-1 under physiological conditions (pH 7, I = 0.125, Tm = 53.2 degrees C) and shows a small temperature dependence which is consistent with an apparent denaturational heat capacity change (delta Cp) of about +8.9 kJ K-1 mol-1. The effects of both pH and ionic strength on the transition temperature and free energy of MetJ unfolding are inconsistent with any single amino acid contribution and are more likely the result of more general electrostatic interactions, possibly including significant contributions from electrostatic repulsion between the like-charged monomers which can be modeled by a Debye-Hückel screened potential.


Synergistic inhibition of Escherichia coli aspartate transcarbamylase by CTP and UTP: binding studies using continuous-flow dialysis.

The allosteric control of Escherichia coli aspartate transcarbamylase (ATCase) involves feedback inhibition by both CTP and UTP, although it is only in the presence of CTP that UTP appears to inhibit the activity of the enzyme. In order to better understand the parts played by both pyrimidine nucleotides in this synergistic inhibition, binding studies were performed by continuous-flow dialysis and ultracentrifugation methods. The results obtained show that UTP binds to ATCase in the absence of CTP. Nevertheless, this binding does not induce any inhibition unless CTP is present. The mutual influence of CTP and UTP on their respective binding constants suggests that they bind to the same regulatory sites. However, the results obtained cannot be satisfactorily explained by a simple competition between the nucleotides, and it is shown that reciprocal affinity enhancements play a fundamental role. CTP enhances the affinity of UTP for the regulatory sites 80-fold, and conversely, UTP enhances the affinity of CTP 5-fold. Interestingly, the isolated regulatory subunits bind the two pyrimidine nucleotides following the same pattern as the entire enzyme. These observations indicate that the synergistic inhibition mechanism relies entirely on interactions between the two adjacent allosteric sites which belong to the same regulatory dimer.


Interaction of C225SR1 mutant subunit of ribonucleotide reductase with R2 and nucleoside diphosphates: tales of a suicidal enzyme.

Ribonucleotide reductase (RDPR) from Escherichia coli is composed of two subunits, R1 and R2, both of which are required to catalyze the conversion of nucleotides to deoxynucleotides. This reduction process is accompanied by oxidation of two cysteines within the active site to a disulfide. One of these putative active site cysteines, C225, has been mutated to a serine, and the properties of this mutant (C225SR1) have been investigated in detail. Incubation of C225SR1 and R2 with [3'-3H,U-14C]UDP results in time-dependent inactivation of the enzyme! This inactivation is accompanied by production of 2.4 uracils, 3H2O, and 3H,14C-labeled protein with an absorbance change at 320 nm. There is an isotope effect (kH/k3H) on uracil production of 3.2. In addition, the tyrosyl radical on R2 is reduced. The observation of 3H2O, indicative of 3' carbon-hydrogen bond cleavage and loss of the tyrosyl radical, provides a direct test of our mechanistic hypothesis that cleavage of this bond occurs concomitantly with tyrosyl radical reduction. Incubation of [3'-2H]UDP with C225SR1 and R2 resulted in a V and V/K isotope effect on loss of the radical of 2.0 and 2.0, respectively. These studies provide the first direct evidence for protein radical involvement in catalysis. Reduction of the tyrosyl radical on R2 is accompanied by a stoichiometric cleavage of the R1 polypeptide into two new polypeptides of 26 and 61 kDa. The 26-kDa polypeptide is the N-terminus of R1, and hence cleavage of the polypeptide is occurring in the region of the mutation. The N-terminus of the 61-kDa polypeptide is blocked.(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of C439SR1, a mutant of Escherichia coli ribonucleotide diphosphate reductase: evidence that C439 is a residue essential for nucleotide reduction and C439SR1 is a protein possessing novel thioredoxin-like activity.

Ribonucleotide reductase from Escherichia coli catalyzes the conversion of nucleotides to deoxynucleotides. Cysteine 439 is proposed to be the protein radical on R1 which initiates the reduction reaction by cleavage of the 3' carbon-hydrogen bond of the nucleotide (Mao et al., 1992a,b). C439 is thus proposed to be essential for catalysis. The C439S mutant of R1 (C439SR1) was prepared. The structure of this mutant was determined to be similar to wt-R1, based on identical CD spectra, isolation via an affinity column specific for the allosteric binding domain, binding of the substrate GDP, and competition with R1 for binding to R2. Preparations of C439SR1 are contaminated with low levels of wt-R1 due to the expression system. The wt-R1 in these preparations can be specifically inactivated by the stoichiometric mechanism-based inhibitor, 2'-azido-2'-deoxyuridine 5'-diphosphate. The activity of the resulting C439SR1 was shown to be less than 0.03% that of the corresponding wt-R1. This is the lower limit of detection with the present assay method. Thus C439 appears to be essential for catalysis. During these studies an unexpected activity of the C439SR1 was uncovered. Its additional cysteines, presumably C754 and C759, appear to function as a thioredoxin with the wt-R1, even though it is incapacitated with respect to nucleotide reduction.


3-Carboxy-cis,cis-muconate lactonizing enzyme from Pseudomonas putida is homologous to the class II fumarase family: a new reaction in the evolution of a mechanistic motif.

The gene (pcaB) for 3-carboxymuconate lactonizing enzyme (CMLE; 3-carboxymuconate cycloisomerase; EC 5.5.1.2) from Pseudomonas putida has been cloned into pMG27NS, a temperature-sensitive expression vector, and expressed in Escherichia coli N4830. The specific activity and kinetic parameters of the recombinant CMLE were comparable to those previously reported. A comparison of the deduced amino acid sequence of CMLE with sequences available in the PIR and Genbank databases revealed that CMLE has highly significant sequence homology to the class II fumarase family, particularly to adenylosuccinate lyase from Bacillus subtilis. CMLE has no significant homology to muconate lactonizing enzyme (MLE) from P. putida, its sister enzyme in the beta-ketoadipate pathway. These findings fully corroborate a prediction made by us on the basis of mechanistic and stereochemical analyses of CMLE and MLE [Chari, R. V. J., Whitman, C. P., Kozarich, J. W., Ngai, K.-L., & Ornston, L. N. (1987) J. Am. Chem. Soc. 109, 5514-5519] and suggest that CMLE and MLE were recruited into this specialized pathway from two different enzyme families.


Conferral of malonyl coenzyme A sensitivity to purified rat heart mitochondrial carnitine palmitoyltransferase.

An immunoaffinity column against the 86-kDa malonyl-CoA-binding protein of beef heart mitochondria was prepared, and the properties of the eluates were compared to those of eluates of an anti-carnitine palmitoyltransferase immunoaffinity column. Both eluates contain seven to eight major proteins with a malonyl-CoA-binding capacity of approximately 5 nmol/mg of protein; in contrast, the eluates from a preimmune IgG column did not contain any of the major proteins. The eluates from both immunoaffinity columns conferred malonyl-CoA sensitivity to purified rat heart mitochondrial carnitine palmitoyltransferase (CPTi/CPT-II). Addition of phospholipids increased the degree of malonyl-CoA inhibition. Doubling the amount of column eluate approximately doubled the malonyl-CoA sensitivity when added to a fixed amount of CPT; i.e., the inhibition increased from 32 to 67%. These results show that CPTi/CPT-II is capable of exhibiting malonyl-CoA sensitivity in the presence of malonyl-CoA-binding proteins. The results do not support the concept that the 86-kDa malonyl-CoA-binding protein is detergent-inactivated carnitine palmitoyltransferase I;rather, they suggest that it is a regulatory subunit of a carnitine palmitoyltransferase complex.


Active site structure of methylamine dehydrogenase: hydrazines identify C6 as the reactive site of the tryptophan-derived quinone cofactor.

To identify the reactive part of the orthoquinone function of the tryptophan-derived cofactor found in methylamine dehydrogenase (MADH), we have determined the crystal structures of MADH from Thiobacillus versutus inhibited by methylhydrazine and (2,2,2-trifluoroethyl)hydrazine. Extra electron density attached to C6 of the tryptophyl tryptophanquinone cofactor shows that this atom and not C7 is the reactive part of the ortho-quinone moiety. The density retained after hydrazine inhibition is much less extensive than expected, however, suggesting that partial breakdown of the inhibitors after reaction with the cofactor may take place. A detailed description is presented of the cofactor environment in an improved model of MADH which now includes information from the recently determined gene sequence of the cofactor-containing subunit [Ubbink, M., van Kleef, M.A.G., Kleinjan, D., Hoitink, C.W.G., Huitema, F., Beintema, J.J., Duine, J.A., & Canters, G.W. (1991) Eur. J. Biochem. 202, 1003-1012]. We hypothesize that Asp76 is responsible for proton abstraction from the alpha-carbon of the substrate during catalysis.


Refined atomic model of wheat serine carboxypeptidase II at 2.2-A resolution.

The crystal structure of the homodimeric serine carboxypeptidase II from wheat (CPDW-II, M(r) 120K) has been determined and fully refined at 2.2-A resolution to a standard crystallographic R factor of 16.9% using synchrotron data collected at the Brookhaven National Laboratory. The model has an rms deviation from ideal bond lengths of 0.018 A and from bond angles of 2.8 degrees. The model supports the general conclusions of an earlier study at 3.5-A resolution and will form the basis for investigation into substrate binding and mechanistic studies. The enzyme has an alpha + beta fold, consisting of a central 11-stranded beta-sheet with a total of 15 helices on either side. The enzyme, like other serine proteinases, contains a "catalytic triad" Ser146-His397-Asp338 and a presumed "oxyanion hole" consisting of the backbone amides of Tyr147 and Gly53. The carboxylate of Asp338 and imidazole of His397 are not coplanar in contrast to the other serine proteinases. A comparison of the active site features of the three families of serine proteinases suggests that the "catalytic triad" should actually be regarded as two diads, a His-Asp diad and a His-Ser diad, and that the relative orientation of one diad with respect to the other is not particularly important. Four active site residues (52, 53, 65, and 146) have unfavorable backbone conformations but have well-defined electron density, suggesting that there is some strain in the active site region. The binding of the free amino acid arginine has been analyzed by difference Fourier methods, locating the binding site for the C-terminal carboxylate of the leaving group. The carboxylate makes hydrogen bonds to Glu145, Asn51, and the amide of Gly52. The carboxylate of Glu145 also makes a hydrogen bond with that of Glu65, suggesting that one or both may be protonated. Thus, the loss of peptidase activity at pH > 7 may in part be due to deprotonation of Glu145. The active site does not reveal exposed peptide amides and carbonyl oxygen atoms that could interact with substrate in an extended beta-sheet fashion. The fold of the polypeptide backbone is completely different than that of trypsin or subtilisin, suggesting that this is a third example of convergent molecular evolution to a common enzymatic activity. Furthermore, it is suggested that the active site sequence motif "G-X-S-X-G/A", often considered the hallmark of serine peptidase or esterase activity, is fortuitous and not the result of divergent evolution.(ABSTRACT TRUNCATED AT 400 WORDS)


Oligosaccharide structures of the major exoglucanase secreted by Saccharomyces cerevisiae.

We have determined the structures of the N-linked carbohydrate chains, released by endo H, of exoglucanase II that are secreted by wild-type Saccharomyces cerevisiae and by the mnn1 mnn9 and mnn1 glycosylation mutants. The mnn9 mutation does not significantly affect N-linked oligosaccharides of exoglucanase II since we found almost identical structures in both mutant strains consisting of a slightly enlarged core with the basic structure shown in A (where M = mannose). Most of the molecules (77%) were phosphorylated on one of the starred mannoses (34%) or on both (43%) with a diesterified (alpha M-->P-->) or monoesterified phosphate group. In addition, some of the molecules apparently escape normal processing and retain the alpha-(1-->2)-linked mannose (italicized) and/or the three glucoses that are characteristic of the lipid-linked precursor (structure B). In the wild type, we found the same basic structure but more [formula; see text] than 90% of the molecules were modified with one to four alpha-(1-->3)-linked mannoses, which were absent in the strains bearing the mnn1 mutation (structure C). The proportion of acidic components was similar to that found in the mutants (78%), although, in this case, the monophosphorylated forms were more abundant (50%) than the diphosphorylated ones (28%). Most of the phosphate groups (69%) were diesterified by a disaccharide (alpha M-->3 alpha M-->P-->) instead of the single mannose found when the mnn1 mutation was present. In both mnn1 and wild type 10-15% of the oligosaccharides had an extra alpha-(1-->6)-linked mannose in the outer chain, a structure described in the recently isolated vrg1 mutant [Ballou, L., Hitzeman, R.A., Lewis, M. S., & Ballou, C. E. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 3209-3212].


Structural elucidation of a novel family of acyltrehaloses from Mycobacterium tuberculosis.

Analysis of the lipids of Mycobacterium tuberculosis H37Rv, by both normal- and reverse-phase thin-layer chromatography, revealed a series of novel glycolipids based on 2,3-di-O-acyltrehalose. The structures of these acylated trehaloses were elucidated by a combination of gas chromatography-mass spectrometry, 1H, 13C, two-dimensional 1H-1H, and 1H-13C nuclear magnetic resonance spectrometry. The fatty acyl substituents were mainly of three types: saturated straight-chain C16-C19 acids; C21-C25 "mycosanoic acids"; and C24-C28 "mycolipanolic acids." Analysis of one of the major 2,3-di-O-acyltrehaloses by two-dimensional 1H-chemical shift correlated and 1H-detected heteronuclear multiple-bond correlation spectroscopy established that the C18 saturated straight-chain acyl group was located at the 2 position and that the C24 mycosanoyl substituent was at the 3 position of the same "right-hand" glucosyl residue. At least six molecular species differing only in their fatty acid content comprised this family of di-O-acylated trehaloses. We regard these acyltrehaloses as elemental forms of the multiglycosylated acyltrehaloses (the lipooligosaccharides) perhaps due to an inability of the majority of isolates of virulent tubercle bacilli to glycosylate core acyltrehaloses. The acyltrehaloses are minor but consistent components of virulent M. tuberculosis and apparently the basis of the specific serological activity long associated with its lipid fractions.


Effect of membrane fluidity and fatty acid composition on the prothrombin-converting activity of phospholipid vesicles.

Vesicles composed of phospholipids with different fatty acyl side chains have been utilized to examine the importance of the nonpolar membrane region for the prothrombin-converting activity of procoagulant phospholipid vesicles. Membranes composed of phosphatidylserine (PS) and phosphatidylcholine (PC) with unsaturated fatty acyl side chains were more active in prothrombin activation than membranes composed of phospholipids with saturated fatty acyl chains. This phenomenon was observed above the phase transition temperature, i.e., on membranes in the liquid-crystalline state. The prothrombin-converting activity of saturated phospholipids approached the activity of unsaturated phospholipids at high factor Va concentrations, which is indicative for a less favorable equilibrium constant for prothrombinase assembly on membrane surfaces composed of saturated phospholipids. The difference between saturated and unsaturated phospholipids was annulled on membranes with high mole percentages of PS. This may result from a compensating contribution of electrostatic forces to the binding equilibria involved in prothrombinase assembly. Additional effects on the prothrombin-converting activity were observed when membranes containing saturated phospholipids were studied below their phase transition temperature. In agreement with Higgins et al. [(1985) J. Biol. Chem. 260, 3604-3612], we found that the time required for the assembly of prothrombinase from membrane-bound factors Xa and Va is considerably prolonged on solid membranes. However, we also observed an effect of membrane fluidity on the steady-state rate of prothrombin activation. Kinetic experiments at saturating factor Va concentrations showed that the transition from the liquid-crystalline to the gel state caused a more than 9-fold decrease of the kcat of prothrombin activation without affecting the Km for prothrombin.(ABSTRACT TRUNCATED AT 250 WORDS)


Detection of internal motions in oligosaccharides by 1H relaxation measurements at different magnetic fields.

The effect of internal motions on proton relaxation data in oligosaccharides has been investigated experimentally. 1H steady-state and transient NOEs together with 13C T1's have been measured at two magnetic field strengths. The existence of internal motions leads to additional modulations of the dipolar interaction between proton pairs, thus producing a range of spectral density functions for these interactions. As a result, it is possible to show that protons relaxing through fixed distances have a different ratio of relaxation parameters, acquired at 500 and 300 MHz, compared to those relaxing through fluctuating distances. This approach has been used to unequivocally establish for two disaccharides the existence of internal motions on the time scale of the overall tumbling.


Conformational changes in phospholipase A2 upon binding to micellar interfaces in the absence and presence of competitive inhibitors. A 1H and 15N NMR study.

An NMR study has been made of porcine pancreatic phospholipase A2 (PLA) in three environments: free in solution, in a binary complex with dodecylphosphocholine micelles, and in a ternary complex with a micelle and the substrate-like inhibitor (R)-1-octyl-2-(N-dodecanoylamino)-2-deoxyglycero-3-phosph oglycol. 1H and 15N chemical shifts, amide exchange rates, and NOE intensities are compared for the enzyme in different environments. From these data, structural differences are found for the N-terminal part, the end of the surface loop at residues Tyr69 and Thr70, and the active site residue His48, and also for the Ca-binding loop (residues 28-32). Specifically, when binding to a micelle, the side chains of residues Ala1, Trp3, and Tyr69, as well as all protons of Thr70, are found to be closer together. After subsequent introduction of the competitive inhibitor, further changes are found for these residues. The N-terminus is flexible in PLA free in solution, in contrast with the crystal structures where it adopts an alpha-helical conformation. According to the NMR data, this helix is rigidly formed only in the ternary complex. Furthermore, in the ternary complex, the N-terminal amino group and the exchangeable hydrogen at N3 of the ring of His48 are observed. We propose that PLA is activated in two steps. An initial conformational change occurs upon binding to a micellar interface. The catalytically active conformation of the enzyme, which has an extensive network of hydrogen bonds, is formed only when binding a substrate or competitive inhibitor at a lipid-water interface.


Response of the phosphatidylcholine headgroup to membrane surface charge in ternary mixtures of neutral, cationic, and anionic lipids: a deuterium NMR study.

Deuterium nuclear magnetic resonance (2H NMR) spectroscopy was used to investigate the response of the phosphatidylcholine headgroup of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) to changes in surface electrostatic charge in membranes consisting of ternary mixtures of lipids. DMPC was deuterated at the choline alpha- and beta-methylene segments. The membrane surface charge was manipulated by the simultaneous addition of cationic didodecyldimethylammonium bromide (DDAB) and anionic 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) to neutral DMPC. Addition of increasing amounts of DDAB caused a progressive decrease (increase) in the 2H NMR quadrupole splitting from DMPC-alpha-d2 (DMPC-beta-d2). Addition of increasing amounts of DMPG caused a progressive increase (decrease) in the quadrupole splitting from DMPC-alpha-d2 (DMPC-beta-d2). Qualitatively, the 2H NMR quadrupole splitting charge response exhibited the same main features for ternary mixtures of DDAB/DMPG/DMPC and binary mixtures of DDAB/DMPC or DMPG/DMPC. Quantitatively, however, the 2H NMR quadrupole splittings obtained from ternary mixtures did not coincide with those obtained from binary mixtures of nominally identical surface charge densities. Hence, the quadrupole splitting did not respond directly to the net membrane surface charge. Instead, the quadrupole splitting measured for a given ternary lipid composition could be reproduced by summing the individual effects of the charged lipids in binary mixtures, weighted according to their appropriate mole fractions.


Influence of lipid composition on the orientational order in Acholeplasma laidlawii strain B membranes: a deuterium NMR study.

2H NMR techniques have recently been developed to determine the complete orientational order profile of lipid bilayers employing lipids containing perdeuteriated palmitic acid [Lafleur, M., Fine, B., Sternin, E., Cullis, P.R., & Bloom, M. (1989) Biophys. J. 56, 1037-1041]. In this work, these techniques have been applied to study order profiles in intact membranes derived from Acholeplasma laidlawii strain B. It is shown that complete orientational order profiles can be readily obtained from the intact membranes of A. laidlawii B grown on equimolar amounts of perdeuteriated palmitic acid and a nondeuteriated fatty acid of varying length and unsaturation. By variation of the fatty acid composition employing mixtures of perdeuteriated palmitic acid with myristic, elaidic, oleic, or linoleic acid, a range of hydrocarbon order compatible with high rates and extents of cell growth has been obtained where the average order parameter, mean value of S, varies over the range 0.140-0.176. This same variation in order is seen for liposomes derived from total lipids extracted from these intact membranes. 2H NMR studies on liposomes composed of individual species of the extracted lipids indicate that modulation of the membrane lipid headgroup composition has the potential to play an important role in maintaining the membrane order within this range.


Peptide binding to lipid bilayers. Nonclassical hydrophobic effect and membrane-induced pK shifts.

The binding of the cyclic peptide (+)-D-Phe1-Cys2-Phe3-D-Trp4-(+)-Lys5-Thr6- Cys7-Thr(ol)8, a somatostatin analogue (SMS 201-995), and the potential-sensitive dye 2-(p-toluidinyl)naphthalene-6-sulfonate (TNS) to lipid membranes was investigated with high-sensitivity titration calorimetry. The binding enthalpy of the peptide was found to vary dramatically with the vesicle size. For highly curved vesicles with a diameter of d congruent to 30 nm, the binding reaction was enthalpy-driven with delta H congruent to -7.0 +/- 0.3 kcal/mol; for large vesicles with more tightly packed lipids, the binding reaction became endothermic with delta H congruent to +1.0 +/- 0.3 kcal/mol and was entropy-driven. In contrast, the free energy of binding was almost independent of the vesicle size. The thermodynamic analysis suggests that the observed enthalpy-entropy compensation of about 8 kcal/mol can be related to a change in the internal tension of the bilayer and is brought about by an entropy increase of the lipid matrix. The "entropy potential" of the membrane may have its molecular origin in the excitation of the hydrocarbon chains to a more disordered configuration and may play a more important role in membrane partition equilibria than the classical hydrophobic effect. The binding of the peptide to the membrane surface induced a pK shift of the peptide terminal amino group. Neutral membranes were found to destabilize the NH3+ group, leading to a decrease in pK; negatively charged membranes, generated an apparent increase in pK due to the increase in proton concentration near the membrane surface. No pK shifts were seen for TNS. Titration calorimetry combined with the Gouy-Chapman theory can be used to determine both the reaction enthalpy and the binding constant of the membrane-binding equilibrium.


Binding between maleimidobenzoyl-G-actin and myosin subfragment 1.

It is well known that the structural interactions between S-1 moieties of myosin molecules ("cross bridges") and actin molecules in polymerized ("F") form are thought to underlie muscle contraction. It is surmised that such interactions are unitary (actin:S-1 = 1:1), but actual demonstration thereof is handicapped by intrinsic properties of the proteins. Recently, it has been reported that chemically modified [with m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)] actin maintains its monomeric ("G") form and makes a stable unitary complex with S-1 but does not activate the S-1 ATPase [Bettache, N., Bertrand, R., & Kassab, R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6028-6032]. However, we recently showed that when MBS-G-actin and S-1 are covalently cross-linked by 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC), ATPase activity is restored [Hozumi, T. (1991) Biochem. Int. 23, 835-843]. Here we investigated the interface between MBS-G-actin and S-1 using the techniques of tryptic digestion and EDC-cross-linking. MBS-G-actin specifically protected the N-terminal region of S-1 heavy chain against tryptic cleavage at the 25 kDa/50 kDa junction, which is different from the effect that a protomer within F-actin has on the protection of the 25 kDa/50 kDa junction. In addition, the cross-linking pattern between MBS-G-actin and S-1 was different from that between F-actin and S-1. When MBS-G actin was cross-linked to trypsin-treated S-1, no cross-linked product was observed.(ABSTRACT TRUNCATED AT 250 WORDS)


Oxygen binding constants and stepwise enthalpies for human and bovine hemoglobin at pH 7.6.

A high-precision thin-layer gas-solution microcalorimeter has been developed to study the binding reactions of gaseous ligands with ligand-binding macromolecules in a manner analogous to that of the Gill thin-layer optical apparatus [Doleman & Gill (1976) Anal. Biochem. 87, 127]. We have generated differential heat-binding curves of oxygen binding to human and bovine hemoglobin in phosphate buffer at pH 7.6, with the enzyme-reducing system of Hayashi et al. [(1973) Biochim. Biophys. Acta 310, 309]. Experiments were conducted at a number of different temperatures in order to expand the data field, allowing for separation of enthalpy and free energy parameters. This type of experimental analysis makes no assumptions of optical linearity between the various heme groups and reveals that the triply ligated species is measurably significant for both human and bovine hemoglobin. It was also determined that the concentration of doubly ligated species of bovine hemoglobin is relatively low. The experiments indicate that the reactions for both hemoglobins are enthalpy-driven for oxygen stepwise additions 1, 2, and 4 while being entropy-driven for step 3. Human hemoglobin oxygen-binding experiments were also performed with the Gill thin-layer optical apparatus under solution conditions identical to those used in the calorimeter. The experiments revealed that if optical linearity is assumed, the overall third equilibrium constant is negative or near zero. This indicated that either the optical cell's performance is much poorer than the thin-layer calorimeter or there is an appreciable nonlinear optical effect.


Synthetic analogs of the carboxyl-terminus of beta-thyrotropin: the importance of basic amino acids in receptor binding activity.

Previously, using a synthetic peptide strategy, we determined that four distinct regions of human beta-thyrotropin (beta TSH) were responsible for interaction of TSH with the TSH receptor. The most potent of these four regions was the carboxyl-terminus of the subunit, represented by the peptide sequence beta 101-112, which inhibited binding of radiolabeled beta TSH to receptor in radioreceptor assay with an IC50 of approximately 100 microM. In the current studies, we systematically substituted the native amino acids in region beta 101-112 with alanine, and we have determined which residues within this span are important to the binding activity of TSH to its receptor. Substitution of Lys101, Asn103, Tyr104, Cys105, Lys107, and Lys110 with alanine each caused a significant fall in activity as compared to the native sequence, whereas substitution at the remaining positions had little or no effect. Because three of these residues are positively charged at physiologic pH, we hypothesized that this charge may be important to the binding activity of the sequence. We modified the charge characteristics of the region by synthesizing two series of analogs in which the residues identified in the alanine substitution studies were substituted with Arg, D-Lys, and D-Arg at each position. In addition, a series of analogs containing basic residues, either added to or substituted for nonbasic residues in the sequence beta 101-112, was synthesized. Substitution of Arg, D-Lys, and D-Arg for Lys101, Lys107, and Lys110 had little effect on activity; however, inclusion of additional basic residues in the beta 101-112 sequence significantly enhanced the inhibitory activity of the region.(ABSTRACT TRUNCATED AT 250 WORDS)


Acetyladenylate or its derivative acetylates the chemotaxis protein CheY in vitro and increases its activity at the flagellar switch.

CheY, a key protein in the mechanism of bacterial chemotaxis, is known to interact with the flagellar switch and thereby cause clockwise rotation. This activity of CheY was significantly increased by producing acetyladenylate (AcAMP) within cytoplasm-free bacterial envelopes containing purified CheY. This was achieved by including in the envelopes the enzyme acetyl-CoA synthetase (ACS) and ATP, and adding acetate externally. The fraction of clockwise-rotating envelopes, tethered to glass by their flagella, increased from 14% to 58% by the presence of AcAMP (or its derivative). In parallel experiments carried out with [14C]acetate under similar conditions, CheY became acetylated: [1-14C]acetate was as effective as [2-14C]acetate in labeling CheY, and ACS-dependent labeling of CheY by [alpha-32P]ATP was not detected. The switch proteins, FliG, FliM, and FliN, isolated to purity, were not acetylated. The acetylation was specific for CheY and dependent on its native conformation. The acetylated form the CheY was estimated to be more active than its nonacetylated form by 4-5 orders of magnitude. Acetylated CheY was stable in the presence of the strong nucleophiles hydroxylamine or ethanolamine, indicative of N-acetylation. There was a correlation between the activity of CheY in vivo and its ability to be acetylated in vitro. Thus, proteins with a single substitution at their active site, CheY57DE and CheY109KR, are not active in vivo and accordingly were not acetylated in vitro; in contrast, the protein CheY13DK is active in vivo and was normally acetylated in vitro. The possibility that CheY acetylation plays a role in bacterial chemotaxis is discussed.


31P NMR and X-ray diffraction study of the effect of photopolymerization on lipid polymorphism.

It was recently shown that oligolamellar vesicles of 3:1 mixtures of dioleoylphosphatidylethanolamine (DOPE) and the photopolymerizable lipid 1,2-bis[10-(2',4'-hexadienoyloxy)decanoyl]-sn-glycero-3-phosphocho line (SorbPC) are destabilized by polymerization of the SorbPC [Lamparski, H., Liman, U., Frankel, D.A., Barry, J.A., Ramaswami, V., Brown, M.F., & O'Brien, D.F. (1992) Biochemistry 31, 685-694]. The current work describes the polymorphic phase behavior of these mixtures in extended bilayers, as studied by 31P NMR spectroscopy and X-ray diffraction. In the NMR experiments, samples with varying degrees of polymerization were slowly raised in temperature, with spectra acquired every 2.5-10 degrees C. In the unpolymerized mixiture, and in those photopolymerized samples where the monomeric SorbPC was decreased by 33% and 51%, an isotropic signal grew progressively until no signal from the lamellar liquid-crystalline (L alpha) phase remained. In the highly polymerized sample with a 90% loss of monomeric SorbPC, less than 20% of the lipids underwent this transition. In none of the samples was an inverted hexagonal phase (HII) observed, under conditions of slow heating to almost 100 degrees C. The X-ray diffraction studies indicated that samples which exhibit the isotropic NMR signal corresponded to a structure exhibiting no well-defined crystalline order, which upon thermal cycling became an inverted cubic phase belonging to either the Pn3m or Pn3 space groups. The temperature of the transition to the cubic precursor decreased as the extent of polymerization increased, demonstrating that photopolymerization of these lipid bilayers can significantly alter the composition and thermotropic phase behavior of the mixture.


Role of glycosylation on the secretion and biological activity of erythropoietin.

The erythropoietin (EPO) molecule contains four carbohydrate chains. Three contain N-linkages to asparagines at positions 24, 38, and 83, and one contains an O-linkage to a serine at position 126. We constructed human EPO variants that eliminated the three N-glycosylation sites by replacing the asparagines with glutamines singly or in combination. The O-linked carbohydrate chain was removed by replacing the serine with glutamine, valine, histidine, or alanine. A variant with a double mutation (Gln38,83) and another with a triple mutation (Gln24,38,83) were secreted poorly from COS1 and CHO cells even though RNA encoding these variants was present. All other variants with mutations in N-linked glycosylation sites were secreted normally. Removal of any of the N-glycosylation sites reduced the in vivo but not the in vitro biological activity of the EPO molecule. All the mutations at Ser126, the O-glycosylation site, were secreted normally. In vitro activity was also unaffected except for Ala126 which had a 50-fold decrease. The Val126 variant was tested in vivo, and its specific activity was only slightly less than that of the native EPO, which indicates that the O-linked carbohydrate is not essential for activity.


Structural basis for recognition of polyglutamyl folates by thymidylate synthase.

Thymidylate synthase (TS) catalyzes the final step in the de novo synthesis of thymidine. In vivo TS binds a polyglutamyl cofactor, polyglutamyl methylenetetrahydrofolate (CH2-H4folate), which serves as a carbon donor. Glutamate residues on the cofactor contribute as much as 3.7 kcal to the interaction between the cofactor, substrate, and enzyme. Because many ligand/receptor interactions appear to be driven largely by hydrophobic forces, it is surprising that the addition of hydrophilic, soluble groups such as glutamates increases the affinity of the cofactor for TS. The structure of a polyglutamyl cofactor analog bound in ternary complex with deoxyuridine monophosphate (dUMP) and Escherichia coli TS reveals how the polyglutamyl moiety is positioned in TS and accounts in a qualitative way for the binding contributions of the different individual glutamate residues. The polyglutamyl moiety is not rigidly fixed by its interaction with the protein except for the first glutamate residue nearest the p-aminobenzoic acid ring of folate. Each additional glutamate is progressively more disordered than the previous one in the chain. The position of the second and third glutamate residues on the protein surface suggests that the polyglutamyl binding site could be utilized by a new family of inhibitors that might fill the binding area more effectively than polyglutamate.


Oligomerization of the cytoplasmic fragment from the aspartate receptor of Escherichia coli.

We have observed that a 31-kDa cloned fragment from the Escherichia coli aspartate receptor exhibits a reversible monomer-oligomer reaction. The fragment, derived from the cytoplasmic region of the receptor (c-fragment), contains the signaling functions of the receptor. The wild-type and nine missense mutant fragments were analyzed. The latter were selected by the effect of the mutations on the signaling properties of the intact receptor, which induced either persistent smooth swimming or tumbling in bacteria [Mutoh, N., Oosawa, K., & Simon, M. I. (1986) J. Bacteriol. 167, 992-998]. In pH 7.0 buffer, the mutations caused five out of the six smooth mutant c-fragments to form oligomers, while neither the three tumble mutant nor wild-type fragments exhibited significant oligomer formation. At a lower pH (5.5), all of the fragments displayed some tendency to form oligomers. The equilibria between the monomer and the oligomers were monitored by gel permeation chromatography (GPC) which resolved two to three forms with apparent molecular weights between 110,000 and 270,000. The proportions of the different forms depended on concentration, indicating an association-dissociation reaction. Static light scattering (SLS) was used to demonstrate that the solution molecular mass of the wild-type c-fragment was 31 kDa and not 110 kDa as indicated by chromatography. One oligomer-forming c-fragment (S461L) eluted as the monomer and one other form, which was determined to be a dimer by SLS. The weight-average molecular weights, calculated from GPC data as a function of protein concentration, agreed well with the weight-average molecular weights obtained by SLS for this mutant.(ABSTRACT TRUNCATED AT 250 WORDS)


Mechanism of magainin 2a induced permeabilization of phospholipid vesicles.

The magainins, peptide antibiotics secreted by the frog Xenopus laevis, have previously been shown to permeabilize phospholipid vesicles. To elucidate the mechanism of permeabilization, we have conducted detailed kinetic studies of magainin 2 amide (mgn2a)-induced release of 6-carboxyfluorescein from vesicles of phosphatidylserine. The results show that dye release occurs in (at least) two stages--an initial rapid phase, with t1/2 approximately 3 s, followed by a much slower phase that approaches zero leakage rate before all the dye is released. Light-scattering studies showed that mgn2a does not cause gross changes in vesicle structure. The peptide was found to rapidly equilibrate between vesicles; this was demonstrated by determining a binding isotherm for the peptide-lipid interaction, and by showing that addition of unloaded vesicles rapidly quenches peptide-induced leakage from loaded vesicles. Transient dye release in the presence of an equilibrating peptide can be explained in two ways: (1) the peptide exists only transiently in an active form; (2) the vesicles are only transiently leaky. Preincubation of mgn2a at assay concentrations in buffer alone or with unloaded vesicles did not inactivate the peptide. Therefore, rapid leakage is probably due to transient destabilization of the vesicle upon addition of mgn2a.


Novel alpha- and omega-conotoxins from Conus striatus venom.

Three neurotoxic peptides from the venom of Conus striatus have been purified, biochemically characterized, and chemically synthesized. One of these, an acetylcholine receptor blocker designated alpha-conotoxin SII, has the sequence GCCCNPACGPNYGCGTSCS. In contrast to all other alpha-conotoxins, SII has three disulfide bonds (instead of two), has no net positive charge, and has a free C-terminus. The other two paralytic peptides are Ca channel-targeted omega-conotoxins, SVIA and SVIB. omega-SVIA is the smallest natural omega-conotoxin so far characterized and has the sequence CRSSGSPCGVTSICCGRCYRGKCT-NH2. Although omega-conotoxin SVIA is a potent paralytic toxic in lower vertebrate species, it was much less effective in mammals. The third toxin, omega-conotoxin SVIB, has the sequence CKLKGQSCRKTSYDCCSGSCGRSGKC-NH2. This peptide has a different pharmacological specificity from other omega-conotoxins previously purified from Conus venoms; only omega-conotoxin SVIB has proven to be lethal to mice upon ic injection. Binding competition experiments with rat brain synaptosomal membranes indicate that the high-affinity binding site for omega-conotoxin SVIB is distinct from the high-affinity omega-conotoxin GVIA or MVIIA site.


The two polypeptide chains in fibronectin are joined in antiparallel fashion: NMR structural characterization.

The fibronectin C-terminal interchain disulfide-linked heptapeptide dimer (Val-Asn-Cys-Pro-Ile-Glu-Cys)2 has been investigated via 1H NMR spectroscopy in both water and dimethyl sulfoxide (DMSO) solutions. Proton Overhauser experiments in DMSO indicate unambiguously that the two fibronectin polypeptide chains are linked head-to-tail (N-terminus to C-terminus), in an antiparallel fashion. It is found that the structure of the peptide is extended. From the 1H NMR interproton distance and angle constraints, the preferred mean (time-averaged) conformations in both H2O and DMSO were derived using distance geometry and molecular mechanics algorithms. The two conformations, although significantly dissimilar, exhibit the common feature of a structurally parallel (as opposed to chemically antiparallel) fibronectin alpha/beta chain array.


Regulation of the chromaffin granule aggregating activity of annexin I by phosphorylation.

Annexin I (lipocortin I) binds to secretory granule membranes and promotes their aggregation in a Ca(2+)-dependent manner [Creutz, C. E., et al. (1987) J. Biol. Chem. 262, 1860-1868; Drust, D. S., & Creutz, C. E. (1988) Nature 331, 88-91]. It is also phosphorylated on serine residues when bovine chromaffin cells are stimulated to secrete [Michener, M. L., et al. (1986) J. Biol. Chem. 261, 6548-6555], suggesting phosphorylation may be involved in modulating the function of annexin I. We report here that phosphorylation of the N-terminal tail by protein kinase C strongly inhibits the ability of annexin I to aggregate chromaffin granules by increasing the calcium requirement 4-fold. This inhibition was readily reversed when the protein was dephosphorylated by protein phosphatase 2A. The inhibition was not due to inability of phosphorylated annexin I to bind to chromaffin granules, since the phosphorylated form bound to the granule membrane at slightly lower levels of calcium than the native form. The phosphorylated annexin I also bound to 20% phosphatidylserine/80% phosphatidylcholine vesicles at lower Ca2+ levels than the native form. The inhibitory effect of phosphorylation on the granule aggregating activity of annexin I was found to be amplified by an unusual mechanism: The phosphorylated form inhibited the activity of the unphosphorylated form. The possible importance of the regulation of annexin I activity by phosphorylation in exocytosis is discussed.


Insulin induces the phosphorylation of DNA-binding nuclear proteins including lamins in 3T3-F442A.

Insulin binding to its plasma membrane receptor stimulates a cascade of protein kinases and phosphatases which ultimately affects multiple processes in the membrane, cytosol, and nucleus of the cell, including transcription of specific genes. To gain insight into the relationship between the kinase cascade and the mechanism of insulin-induced nuclear events, we have studied the effect of insulin on the phosphorylation of DNA-binding nuclear proteins in differentiated NIH-3T3-F442A adipocytes. Insulin induced the phosphorylation of seven DNA-binding proteins: pp34, pp40, pp48, pp62, pp64, pp66, and pp72. The half-maximal response was observed at 10-30 min and reached its maximum at 60 min. The insulin-induced phosphorylation of each of these proteins was dose-dependent with ED50S of 2-10 nM. The phosphorylation of pp62, pp64, and pp72 took place on serine residues. On the basis of immunoprecipitation and immunoblotting experiments with anti-lamin antibodies, we found that the insulin-induced DNA-binding phosphoproteins pp62, pp64, pp66, and possibly pp48 were related to lamins A and C. The ED50 for insulin-stimulated lamin phosphorylation was approximately 10 nM, and phosphorylation was half-maximal at 30 min. The insulin-dependent phosphorylation of lamins and other DNA-binding proteins (pp34, pp40, and pp72) may play a mediatory role in the long-term effects of insulin.


Postbinding characterization of five naturally occurring mutations in the human insulin receptor gene: impaired insulin-stimulated c-jun expression and thymidine incorporation despite normal receptor autophosphorylation.

Some patients with extreme insulin resistance have mutations in their insulin receptor gene. We previously identified five such mutations located in the extracellular domain of the insulin receptor (Asn-->Lys15, His-->Arg209, Phe-->Val382, Lys-->Glu460, and Asn-->Ser462) and studied the effects of these mutations upon posttranslational processing, insulin binding, and tyrosine autophosphorylation. We now characterize the ability of these mutant receptors to mediate biological actions of insulin in transfected NIH-3T3 fibroblasts. All cell lines expressing mutant receptors showed marked impairment in insulin-stimulated c-jun expression and thymidine incorporation when compared with cells expressing wild-type human insulin receptors. The most severe impairment was seen in cells expressing the Val382 mutant (a mutation which causes an intrinsic defect in receptor autophosphorylation). These cells had insulin responses similar to the untransfected cells (used as a negative control). In contrast, cells expressing the Lys15 mutant have the ability to achieve a normal level of maximal autophosphorylation but require an abnormally high concentration of insulin to do so (as the result of decreased insulin binding affinity). These cells show a higher basal rate and much lower insulin stimulation of both c-jun expression and thymidine incorporation when compared with the cells expressing the wild-type human insulin receptors. This pattern is also seen in the cells expressing the other mutants with normal autophosphorylation (Arg209, Glu460, and Ser462). Although the most severe defects in insulin action are seen with the mutation which has an intrinsic defect in receptor autophosphorylation, the ability to undergo normal autophosphorylation does not seem to preclude mutations from impairing the ability of receptors to mediate some of the actions of insulin.


Mechanism of Agrobacterium beta-glucosidase: kinetic studies.

The beta-glucosidase from Agrobacterium faecalis (previously Alcaligenes faecalis) has been subjected to a detailed kinetic investigation with a range of substrates to probe its specificity and mechanism. It has a relatively broad specificity for the substrate sugar moiety and exhibits a classical pH dependence for its kinetic parameters with three different substrates and an identical pH dependence for its inactivation by a mechanism-based inactivator, cyclophellitol. Measurement of kcat and Km values for a series of aryl glucoside substrates has allowed construction of a Bronsted plot, the concave-downward shape of which is consistent with the anticipated two-step mechanism involving a glucosyl-enzyme intermediate which is formed and hydrolyzed via oxocarbonium ion-like transition states. The slope of the leaving group-dependent portion of the Bronsted plot (beta 1g = -0.7) indicates a large degree of bond cleavage at the transition state. Secondary deuterium kinetic isotope effects measured for five different aryl glucosides are also consistent with this mechanism and further suggest that the transition state for formation of the glucosyl-enzyme intermediate, probed with the slower substrates for which kH/kD = 1.06, is more SN2-like than that for its hydrolysis (for which kH/kD = 1.11). Reasons for this difference are proposed, and values of Ki for several ground-state and transition-state analogue inhibitors are presented which support the concept of sp2-hybridized transition states.


Isopentenyl-diphosphate isomerase: irreversible inhibition by 3-methyl-3,4-epoxybutyl diphosphate.

Isopentenyl-diphosphate:dimethylallyl-diphosphate isomerase (EC 5.3.3.2) catalyzes the 1,3-allylic rearrangement of the homoallylic substrate isopentenyl diphosphate (IPP) to its allylic isomer, dimethylallyl diphosphate (DMAPP). Incubation of yeast IPP isomerase with 3-methyl-3,4-epoxybutyl diphosphate (EIPP) resulted in a time-dependent first-order loss of activity characteristic of an active-site-directed irreversible process, where k2 = 0.63 +/- 0.10 min-1 and KI = 0.37 +/- 0.11 microM. A 1:1 covalent E-I complex was formed upon incubation with [1-14C]EIPP. The inhibited enzyme was treated with trypsin to give two radioactive fragments, which were purified by reversed-phase HPLC on a C18 column. The modified amino acid in each fragment was identified as C139 by sequencing the radiolabeled peptides. Incubation of IPP isomerase with [2,4,5-13C3]EIPP gave a 13C-labeled E-I complex. A 1H-13C heteronuclear multiquantum correlation spectrum had strong cross-peaks at 1.2/28 and 2.9/48 ppm, which we assigned to the labeled methyl group and C(4) methylene, respectively, of the inhibitor. In addition, a weak signal at 2.17/42 ppm may be from the C(2) methylene. Comparison of these chemical shifts with those of a synthetic adduct isolated from treatment of EIPP with cysteine indicates C139 attacks C(4) of EIPP to generate a thioether linkage between the enzyme and the inhibitor.


Inactivation of a beta-glucosidase through the accumulation of a stable 2-deoxy-2-fluoro-alpha-D-glucopyranosyl-enzyme intermediate: a detailed investigation.

The inactivation of glycosidases by 2-deoxy-2-fluoroglycosides has been shown previously to occur via the accumulation of a covalent 2-deoxy-2-fluoro-alpha-D-glucopyranosyl enzyme intermediate [Withers, S. G., & Street, I. P. (1988) J. Am. Chem. Soc. 110, 8551]. Further characterization of this process with Agrobacterium beta-glucosidase is described, and the range of glycosides engaging in this behavior is examined. Inactivation is shown to be accompanied by the release of a stoichiometric "burst" of aglycon, thereby providing a new class of active site titration agents for glycosidases. The rate of inactivation is shown to be very strongly dependent on the leaving group ability of the aglycon, the slowest inactivator studied (p-nitrophenyl2-deoxy-2-fluoro-beta-D-glucopyranoside) yielding only partial inactivation due to turnover of the intermediate becoming competitive with its formation. Such turnover of the intermediate is shown to be greatly accelerated by transglycosylation to a suitable glycoside bound in the aglycon site, resulting in the release of a disaccharide product which was isolated and characterized. The pH dependences of both the formation and the hydrolysis of the 2-deoxy-2-fluoroglycosyl-enzyme closely resemble those of each step for normal catalysis, indicating that the same catalytic groups are involved in both processes. A model system for the partial "steady-state" inactivation observed previously [Withers, S. G., Rupitz, K., & Street, I. P. (1988) J. Biol. Chem. 263, 7929] with certain other glycosidases was established by incubating the enzyme with an inactivator known to undergo relatively rapid transglycosylation in the presence of various concentrations of a suitable reactivator.(ABSTRACT TRUNCATED AT 250 WORDS)


Changing patterns of adolescent sexual behavior: consequences for health and development.

Sexuality is a fundamental quality of human life, important for health, happiness, individual development, and indeed for the preservation of the human race. During the dynamic period of adolescence in which the passage from childhood to maturity takes place, sexuality takes on new dimensions; feelings become more intense, relationships become more complex, and the consequences of sexual behavior are radically altered. This not only affects the behavior of young people but also of those who interact with them, their families and peers, and those who work in the health, education, youth, social welfare, and other sectors. In the contemporary world the conditions of life for many young people have also changed, and with it patterns of sexual behavior. In general, earlier puberty, later marriage, a decline in the family leading to less control and more autonomy, and intense exposure to sexual stimuli via the mass media and travel across cultural boundaries have made pre-marital adolescent sexual activity more common. This has added to traditional problems of early marriage, newer problems of early pregnancy, childbirth, and induced abortion outside of marriage, sexually transmitted diseases, and human immunodeficiency syndrome infection leading to acquired immunodeficiency syndrome. But the work of the World Health Organization (WHO), along with many others in the field, strongly suggests that given appropriate information and services, trust and equity between the sexes, young people will behave responsibly and well. In this paper some of the findings from methods developed by WHO for research, training, advocacy, and evaluation, and findings in relation to patterns and determinants of sexual and reproductive health and development will be described, and future directions suggested.


Models for effective prevention.

The social influence models do provide some optimism for primary prevention efforts. Prevention programs appear most effective when 1) the target behavior of the intervention has received increasing societal disapproval (such as cigarette smoking), 2) multiple years of behavioral health education are planned, and 3) community-wide involvement or mass media complement a school-based peer-led program (45,46). Short-term programs and those involving alcohol use have had less favorable outcomes. Future research in primary prevention should address concerns of high-risk groups and high-risk countries, such as lower income populations in the United States or countries that have large adolescent homeless populations. The utilization of adolescent leaders for program dissemination might be particularly critical in these settings. A second major and global concern should focus upon alcohol use and alcohol-related problems. In many communities adolescent alcohol use is normative and even adult supported. Thus, young people are getting quite inconsistent messages on alcohol from their schools, from TV, from peers, and from parents. This inconsistency may translate into many tragic and avoidable deaths for young people. Clearly, in the area of alcohol-related problems, community-wide involvement may be necessary. A third direction for prevention research should involve issues of norms, access, and enforcement including policy interventions, such as involve the availability of cigarette vending machines or the ease of under-age buying or levels of taxation. These methods affect adolescents more acutely since their financial resources, for the most part, are more limited. These policy level methods also signify to adolescents what adults consider appropriate.(ABSTRACT TRUNCATED AT 250 WORDS)


Dietary habits, food consumption, and nutrient intake during adolescence.

Adolescents and young adults adopt eating patterns which may well form the basis of their dietary habits for much of their lives. At the same time, this section of the population can come under considerable pressure from the world at large, to conform to the current trends in fashion, language, behavior, or foods. Recent developments in Western dietary practice have tended to leave adolescents vulnerable to low intakes of energy and of some nutrients, as snacking accounts for an increasing proportion of dietary intake. Achieving a balanced diet is more difficult when the most popular and widely available snack foods are high in sugar or in fat. Results from major British studies, together with a review of data from other developed countries, support the need for more effective dietary education for adolescents and for children generally.


Comparison of the self-image of teenagers in Finland, the United States, and Germany.

In this research, the self-image of Finnish adolescents was compared with the self-images of American and West German adolescents. Results show that there are statistically significant differences between Finnish adolescents and American adolescents in five areas. Finnish adolescents are emotionally healthier than American adolescents, and their mood is better. American adolescents have higher vocational and educational goals than Finnish adolescents, and their adaptation skills to immediate environment and their coping skills are better. Differences between Finnish and West German adolescents are found in four scales. The affective harmony of the psychic structure of Finnish adolescents and their object relationships and friendships are better developed than those of West German adolescents. West German adolescents have higher vocational and educational goals than Finnish adolescents and their coping skills with themselves, other people, and their own world are better. Emotional tone (mood) is an area where Finnish adolescents scored higher than the American and the West German adolescents. Vocational and educational goals and adjustment skills are well developed in adolescents from the United States and West Germany. Adolescents in these three countries are alike in Impulse Control, Body Image, Sexual Attitudes, and Family Relations.


Atypical eating disorders.

Some patients with eating disorders have neither anorexia nervosa (A.N.) nor bulimia. Cases which do not rigorously meet the DSM-III-R criteria for anorexia nervosa or for bulimia are usually defined as "eating disorders N.O.S." Among them are patients with pathological characteristics very closely related to the above-mentioned categories. Others, however, although affected by an eating disorder, present a quite different clinical picture from either A.N. or bulimia. In a study of 80 eating disorder cases, only 45 met the strict definition of A.N. or bulimia. The other 35 were diagnosed as atypical eating disorders and are the focus of this presentation. 29 were classified as Eating Disorders N.O.S. and 6 as obesity. Co-morbidity, gender and age data, and clinical vignettes are presented.


A tentative epidemiologic approach to suicide prevention in adolescence.

During adolescence (by convention, 10-19 years of age), the global mortality rates are relatively low in most countries. Nevertheless, suicidal mortality rates are usually second (after all accidents) or third (after accidents and homicides), depending on the country. Rates in males are regularly higher than those in females. Owing to the universal absence of a systematic recording of parasuicide cases, morbidity data are obviously much more difficult to get and to analyze properly. Data based on hospital admissions give only approximations. Nevertheless, in this age group, parasuicide is expected to be many times more frequent in females than in males. Epidemiologic studies on relapses as well as parasuicide follow-up are rare. Taking into account the number and heterogeneity of known risk factors, suicidal behavior prediction is based on probability statistics: valuable only for groups ("high-risk profiles"), never for individuals. This is particularly true in adolescence, when so many changes occur rapidly. Preventative measures are to be discussed according to their pertinence in each of the three classic levels of prevention.


Adolescence: the crossroads of violence.

We sought to distinguish between aggressivity and true violence. Violence is a vital force, a life instinct, or, more particularly, a survival instinct. Violence is neither good nor bad. Everything depends on the way in which this essential instinct is used in the succession of crises that mark the affective development of each and every one of us, and, in particular, during the crisis of adolescence. The measure of prevention could be based on the elements of pure violence and not on established aggressivity. The primary prevention of the deviations toward natural violence fits into the framework of the education for health. That is only possible if we today prepare future parents and educators of the children of tomorrow.


Self-reported anger in black high school adolescents.

The purpose of this study was to explore the recognition and expression of anger in black high school adolescents. A total of 56 teens, aged 14-19 years, responded to questions about their recognition of anger, how and to whom they express anger, and to whom they refrain from expressing anger. They also stated their opinions about acceptable and unacceptable expressions of anger and its relationship to depression or suicide. Data were analyzed using frequency tabulations for all questions on the survey instrument. Specific variables of age, grade in school, gender, and family composition were analyzed by one-sample chi 2 tests (alpha set at 0.05). The study demonstrated 1) all the teens surveyed could recognize when they were angry; 2) most teens expressed anger to their friends, to their siblings, and to their mothers; 3) younger teens (ages 14-15 years) when compared to older teens (ages 18-19 years), identified mother as the one who made them angry; 4) females were more likely to feel like crying when angry; 5) females were more likely to feel like being silent when angry; 6) students from one- and two-parent homes did not differ in their expression of anger. Implications of this study include the recognition that anger is a natural, human emotion. Adolescents need to observe adults who can effectively manage behavior associated with anger. Problem solving skills, stress management techniques, and role play situations can be utilized as effective tools in the recognition and expression of anger in acceptable ways and in attempts at the prevention of dysfunctional anger.


Prevalence of physical and sexual assault in pregnant adolescents.

Few studies have addressed the prevalence of violence among pregnant adolescents. We interviewed 342 pregnant teenagers 17 years of age or younger for a history of assault; 9% reported physical assault, 8% sexual assault, and 8% both physical and sexual assault. Of those physically abused, 40% had been hit during pregnancy. The most common perpetrator of physical assault was a member of their family of origin as compared to a mate (46% versus 33%), although a boyfriend or spouse was the attacker in 80% of cases in which abuse had increased during pregnancy. The face or neck was the most common site of contact. A total of 14% reported being hit in the abdomen, one-third of them while pregnant. We conclude that a significant proportion of pregnant teenagers have experienced violence and therefore should be screened routinely for a history of abuse.


Violence and its relationship to substance use in adolescent pregnancy.

We surveyed 342 pregnant adolescents 17 years old and younger for a history of physical or sexual assault and substance use to investigate whether victims of childhood violence are at increased risk of smoking and using alcohol or drugs. A total of 95% of new patients who attended the university's teen pregnancy clinic between May 8, 1989, and December 8, 1990, were interviewed. Substance use was reported seven times more often in those with a history of combined physical and sexual assault, five times more frequently by those who had been sexually assaulted, and three times more often in those who had been physically assaulted than adolescents without a history of assault. Violence was associated with substance use in all ethnic groups although this relationship was modified by ethnicity. Among Hispanics, an association was observed between physical assault and tobacco use. Sexual and combined physical and sexual assault were strongly associated with use of alcohol among blacks. All categories of violence were associated with drug use among all ethnic groups. When use of each substance was analyzed by the adolescent's relationship to the perpetrator, drug use was most strongly associated with assault by a mate, whereas tobacco or alcohol use was more often associated with assault by a member of the victim's family of origin.


Factors related to substance use by pregnant, school-age adolescents.

This study provides information on substance use among pregnant adolescents, and examines social influence, intrapersonal, and environmental factors associated with substance use during pregnancy in adolescence. The sample consists of premaritally pregnant adolescents (N = 241), who were interviewed as part of a longitudinal study of patterns of drug use among pregnant and parenting school-age adolescents. The findings indicate that, although the sample demonstrated a high rate of prepregnancy substance use, a significant drop in use occurred during pregnancy. Logistic regression analysis indicated that perceived harm of using substances while pregnant, best friend's substance use, boyfriend's substance use, and school status were related to substance use during pregnancy, even after controlling for the effects of prepregnancy substance use. The findings have implications for substance use prevention and intervention programs for pregnant and parenting adolescents.


Substance abuse and syphilis in urban adolescents: a new risk factor for an old disease.

The incidence of syphilis is increasing. To identify the epidemiologic characteristics of adolescents with syphilis in an urban adolescent clinic, we performed a retrospective review and case-control study of 59 patients with syphilis and 111 sex-matched controls seen at our institution between 1985 and 1988. Only 24% of cases occurred in males, in contrast with 61% in a 1977-1981 study. Almost one-half (46%) of cases occurred in asymptomatic individuals, and only 24% were diagnosed at the initial visit. Patients with syphilis were more likely to have a history of substance abuse (odds ratio, 4.94; 95% confidence interval, 1.83, 13.3). This study suggests that the epidemiology of syphilis in adolescents may be changing. The high percentage of asymptomatic syphilis cases underscores the need for a high index of suspicion, particularly in sexually active adolescents who abuse drugs.


Problem drinking among college freshmen.

Entering college freshmen (n = 308) completed a questionnaire which assessed drinking behaviors and identified students at risk for problem drinking as defined by the CAGE (focuses on Cutting down on drinking, Annoyance by criticism by others about drinking, Guilty feelings about drinking, and the use of an Eye opener) questionnaire and Perceived-Benefit-of-Drinking Scale (PBDS). Students were 50% male with a mean age of 17.9 years. In the past month, 17% had drunk on 10 or more occasions, and 18% had binged on 6 or more occasions. CAGE scores of 2 or greater were obtained by 21% and PBDS scores of 3 or greater by 29%, reflecting high risk for problem drinking. High-risk CAGE and PBDS scores were associated with frequent drinking and binging. Student reports of parental problem drinking were not associated with high risk for problem drinking. Intent to join a fraternity or sorority (the Greek system) was associated with frequent drinking, binging, and high-risk CAGE and PBDS scores. Approaches to screening for problem drinking which emphasize attitudes and beliefs may be useful. The Greek system appears to be attractive to high-risk students and should be a focus of prevention programming.


Relationship of AIDS-related attitudes to sexual behavior changes in adolescents.

The impact of the acquired immunodeficiency syndrome (AIDS) epidemic on a group of adolescents was investigated by surveying 197 sexually active, predominantly African-American, urban high school students. Reported sexual behavior changes were evaluated in relation to AIDS-related knowledge and attitudes. Over 50% of the students decreased their frequency of sexual activity, increased their condom use, and/or decreased their number of partners. These students had significantly higher scores on a measure of worry about vulnerability to human immunodeficiency virus (HIV) infection than those whose behavior had not changed. AIDS knowledge, AIDS beliefs, and AIDS-related anxiety interacted with gender to affect sexual behavior change. Male students reporting decreased frequency of sexual activity, for example, had more accurate beliefs about AIDS than males reporting no decrease. Among female students, however, those reporting decreased frequency had less accurate beliefs than those reporting no decrease. These results highlight the importance of considering gender and specific sexual behaviors when designing AIDS education interventions.


AIDS/HIV knowledge level and perceived chance of having HIV among rural adolescents.

Behaviors that increase the risk of acquired immunodeficiency syndrome (AIDS) and human immunodeficiency virus (HIV) among adolescents living in rural areas have been reported to be as frequent as those of lower socioeconomic minority youth living in large urban areas. Little is known, however, about whether rural adolescents possess adequate knowledge upon which to make responsible decisions to avoid exposure to HIV. In order to address this deficit, we administered the Centers for Disease Control (CDC) 1989 Secondary School Health Risk Survey to 294 sixth, seventh, and eighth grade students (30.2% sample) from a rural county with significant social problems including epidemic sexually transmitted diseases STDs, sex-for-drugs, poverty, and drug abuse. The sample was 65% African-American, 50% female, with a mean age of 12.9 +/- 1.3 years. Although 68% reported having received school-based AIDS education, a lower proportion (greater than or equal to 10%) the students were found to correctly answer 8 of 17 AIDS/HIV knowledge questions than those from a national comparison group. The mean was 12.8 +/- 3.1 of 17 items answered correct. Lower AIDS/HIV knowledge was associated with lower school grade (rho = 0.46, p less than or equal to 0.0001); being African-American, Hispanic, or Native American (p less than or equal to 0.043); and never receiving school-based AIDS/HIV education (p less than or equal to 0.0001). Based on multivariate analysis of variance (ANOVA), only school-based AIDS/HIV education was a significant predictor (p less than or equal to 0.0001) of knowledge.(ABSTRACT TRUNCATED AT 250 WORDS)


Partner-specific condom use among adolescent women clients of a family planning clinic.

Because inconsistent condom use could put adolescent women at an increased risk for sexually transmitted diseases, it is important to understand when and with whom they use condoms. This study examined partner-specific condom use over time among adolescent women. The data were from a clinic-based, prospective study of 308 adolescent women who had at least one sex partner during a 6-month follow-up. Their condom use was examined with three types of partners: exclusive, nonexclusive primary, and nonexclusive secondary. Predictors of consistent condom use (using condoms 100% of the time with a specific partner) were explored in a multiple logistic regression analysis. Consistent condom use was more likely to occur in shorter relationships (less than 3 months) and with partners who preferred condoms for contraception. It was no more likely to occur with nonexclusive partners than with exclusive partners, and it was somewhat less likely to occur among consistent oral contraceptive users. These findings emphasize the importance of educating adolescent women to introduce and maintain condom use with all partners.


Self-efficacy, hedonistic expectancies, and condom-use intentions among inner-city black adolescent women: a social cognitive approach to AIDS risk behavior.

The effects on condom-use intentions of an acquired immune deficiency syndrome (AIDS) prevention intervention based on social cognitive theory were investigated among 19 sexually active black adolescent women recruited from an inner-city family planning clinic. The women received the social cognitive intervention designed to increase perceived self-efficacy and favorable outcome expectancies about the hedonistic consequences of using condoms or one of two control interventions: An information-alone intervention designed to increase AIDS knowledge or a general health-promotion intervention designed to provide information about important health problems other than AIDS. All interventions lasted 105 min and involved films and small-group exercises. Participants' evaluations did not differ among conditions. As hypothesized, analysis of covariance indicated that participants in the social cognitive condition reported greater intentions to use condoms than did those in the two control conditions. In addition, participants in the social cognitive condition scored higher in perceived self-efficacy and favorable hedonistic expectancies--the two hypothesized mediators of the intervention effect. Although participants who received the information-alone intervention subsequently had greater AIDS knowledge than did those in the health promotion condition, they did not express greater intentions to use condoms. These results highlight the value of a social cognitive approach to AIDS risk behavior: outcome expectancies regarding the effects of precautionary practices on sexual enjoyment and perceived self-efficacy to implement such practices play an important role in decisions about condom use.


Comparison of cytobrush with Cervex-Brush for endocervical cytologic sampling.

This study was designed to compare the quality of the Papanicolaou (Pap) smear and side effects associated with the Ayres spatula/cytobrush combination and the Cervex-Brush. We evaluated 165 Pap smears, of which 84 (51%) were cytobrush/spatula specimens, and 81 (49%) were from Cervex-Brush specimens. The cytobrush/Ayres spatula combination and the Cervex-Brush alone were equally successful in detecting squamous cells, however, the cytobrush/Ayres spatula combination was significantly better in picking up endocervical cells than the Cervex-Brush (p less than 0.01). There were no significant differences between the two techniques in degree of bleeding and pain in adolescents. The combination of the cytobrush and spatula appears to be superior to the Cervex-Brush alone in producing adequate Pap smears.


Smoking and apolipoproteins in adolescents. The Niño Jesus Group.

The relation between smoking and blood lipids and apolipoproteins (A1,B100) were studied in a group of 1024 12- to 18-year-old school children in the Comunidad de Madrid. The percentage of smokers was 19% (17% for girls and 21% for boys). The average consumption of cigarettes per day was 7.83 +/- 5.06 in boys and 6.04 +/- 3.49 in girls (p less than 0.05). As compared with male nonsmokers, male smokers showed a higher mean level of low-density lipoprotein (LDL) cholesterol (112 versus 100 mg/dL, p less than 0.05), a higher LDL cholesterol to HDL-cholesterol ratio (2.27 versus 1.94, p less than 0.001), a higher mean level of apolipoprotein B100 (59 versus 53 mg/dL, p less than 0.05), and a higher apolipoprotein B100 to apolipoprotein A1 ratio (0.45 versus 0.40, p less than 0.01). Female smokers tended to show the same results, although significant differences were only found for LDL cholesterol to HDL cholesterol ratio and apolipoprotein B100 to apolipoprotein A1 ratio (1.8 versus 1.59 and 0.41 versus 0.38 respectively, both p less than 0.05). This work provides new data about the effects of smoking on apolipoproteins in adolescents and emphasizes on the need for preventive programs.


Self-reported weight and height in adolescents and their parents.

Self-reported and measured weight and height were compared in a sample of adolescents aged 15 years (109 boys; 95 girls) and their parents (135 fathers; 190 mothers) recruited from secondary schools in the urban area of Geelong, Victoria, Australia. On average the adolescents' self-reported weight and height did not differ to a greater extent from the measured values than did that of their parents for their own weight and height but differences for individuals were much more variable. Self-reported weight was significantly underestimated and height over-estimated by both adolescents and parents. Body size had little effect on the extent of underestimation of weight and overestimation of height. The precision of reporting varied both with age and sex, while reporting bias in the parents, but not the adolescents', was influenced by father's occupation score. The educational level of the parents, however, had no statistically significant effect on reporting bias. The extent to which weight was underestimated and height overestimated was no greater than that observed in adults and suggests that group means reported for weight and height are likely to be as valid a measure of actual weight and height as in adults.