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PubMed2005

Isolation of TAK-779-resistant HIV-1 from an R5 HIV-1 GP120 V3 loop library.

The human immunodeficiency virus (HIV-1) envelope glycoprotein (GP) 120 interacts with CD4 and the CCR5 coreceptor for viral entry. The V3 loop in GP120 is a crucial region for determining coreceptor usage during viral entry, and a variety of amino acid substitutions has been observed in clinical isolates. To construct an HIV-1 V3 loop library, we chose 10 amino acid positions in the V3 loop and incorporated random combinations (27,648 possibilities) of the amino acid substitutions derived from 31 R5 viruses into the V3 loop of HIV-1(JR-FL) proviral DNA. The constructed HIV-1 library contained 6.6 x 10(6) independent clones containing a set of 0-10 amino acid substitutions in the V3 loop. To address whether restricted steric alteration in the V3 loop could confer resistance to an entry inhibitor, TAK-779, we selected entry inhibitor-resistant HIV-1 by increasing the concentration of TAK-779 from 0.10 to 0.30 microM in PM1-CCR5 cells with high expression of CCR5. The selected viruses at passage 8 contained five amino acid substitutions in the V3 loop without any other mutations in GP120 and showed 15-fold resistance compared with the parental virus. These results indicated that a certain structure of the V3 loop containing amino acid substitutions derived from 31 R5 viruses can contribute to the acquisition of resistance to entry inhibitors binding to CCR5. Taken together, this type of HIV-1 V3 loop library is useful for isolating and analyzing the specific biological features of HIV-1 with respect to alterations of the V3 loop structure.

Amides↗
PubMed2002

Molecular modeling and dynamics studies of HIV-1 kissing loop structures.

Recognition of an RNA loop by another RNA loop is involved in several biological functions. The dimerization of two copies of the HIV-1 genomic RNA is thought to be involved in several steps of the retroviral life cycle. It has been shown that the dimerization of the two HIV-1 RNA genomes is initiated by the so called kissing loop. The 9nt kissing loop consists of a palindromic 6nt sequence that forms Watson-Crick base-pairs at the kissing site in HIV-1. We report the results of our molecular modeling and dynamics studies on two major subtype isolates (MAL and LAI) of HIV-1 kissing loop structures. From our modeling studies, we conclude that the conformation of the loop in the monomer might be closer to the A-RNA-like conformation in order to form an initial kissing structure. This is achieved by the stacking interactions of the bases at the 3' end of the loop and by the intramolecular tertiary interactions of a single linker nucleotide. We discuss the effect of the loop size and the structural limitations on the formation of kissing loop structures. Also, we propose a possible mechanism to convert the kissing loop structure to a stable extended duplex structure without unwinding the stems.

Base Composition↗
PubMed2026

Chiron3D: an interpretable deep learning framework for understanding the DNA code of chromatin looping.

MOTIVATION: Three-dimensional folding of the genome into structures such as chromatin loops is essential for gene regulation. Current experimental methods for mapping these structures, like Hi-C and HiChIP, are labor-intensive and require repeated assays to test hypothesized mutation effects. This motivates the need for predictive approaches that reveal the sequence determinants of chromatin loops. RESULTS: In this work, we present a novel and interpretable computational pipeline for predicting CTCF-mediated chromatin loops. We propose Chiron3D, a DNA-only model trained in a cell-type specific manner to predict CTCF HiChIP contact maps. By leveraging pre-trained embeddings from a foundation model, our approach is competitive with baselines that take CTCF ChIP-seq as additional input, while enabling nucleotide-level attribution to the input DNA sequence. Using our framework, we provide likely mechanistic insights into the physical control of loop dynamics. Specifically, we find that the strength of the loop extrusion anchorage site is largely governed by the amount and binding affinity of CTCF sites at the boundaries. Furthermore, we reveal that loop stability is regulated by the amount of intra-loop CTCF binding sites, where fewer intra-loop sites are associated with greater loop stability. Using targeted, single-nucleotide edit simulations with Chiron3D, we show that both loop strength and stability can be precisely controlled. Together, these results provide novel mechanistic insights into the physical control of genome organization and highlight the potential of decoding the DNA sequence logic in silico. AVAILABILITY: The Chiron3D pipeline is made available at https://github.com/BoevaLab/Chiron3D.

Chromatin↗
PubMed2001

t-loops at trypanosome telomeres.

Mammalian telomeres form large duplex loops (t-loops) that may sequester chromosome ends by invasion of the 3' TTAGGG overhang into the duplex TTAGGG repeat array. Here we document t-loops in Trypanosoma brucei, a kinetoplastid protozoan with abundant telomeres due to the presence of many minichromosomes. These telomeres contained 10-20 kb duplex TTAGGG repeats and a 3' TTAGGG overhang. Electron microscopy of psoralen/UV cross-linked DNA revealed t-loops in enriched telomeric restriction fragments and at the ends of isolated minichromosomes. In mammals, t-loops are large (up to 25 kb), often comprising most of the telomere. Despite similar telomere lengths, trypanosome t-loops were much smaller (approximately 1 kb), indicating that t-loop sizes are regulated. Coating of non-cross-linked minichromosomes with Escherichia coli single-strand binding protein (SSB) often revealed 3' overhangs at both telomeres and several cross-linked minichromosomes had t-loops at both ends. These results suggest that t-loops and their prerequisite 3' tails can be formed on the products of both leading and lagging strand synthesis. We conclude that t-loops are a conserved feature of eukaryotic telomeres.

Animals↗
PubMed1998

Biased short tract repair of palindromic loop mismatches in mammalian cells.

Mismatch repair of palindromic loops in the presence or absence of single-base mismatches was investigated in wild-type and mismatch-binding defective mutant Chinese hamster ovary cells. Recombination intermediates with a maximum heteroduplex DNA (hDNA) region of 697 bp contained a centrally located, phenotypically silent 12-base palindromic loop mismatch, and/or five single-base mismatches. In wild-type cells, both loops and single-base mismatches were efficiently repaired (80-100%). When no other mismatches were present in hDNA, loops were retained with a 1.6-1.9:1 bias. However, this bias was eliminated when single-base mismatches were present, perhaps because single-base mismatches signal nick-directed repair. In the multiple marker crosses, most repair tracts were long and continuous, with preferential loss of markers in cis to proximal nicks, consistent with nicks directing most repair in this situation. However, approximately 25% of repair tracts were discontinuous as a result of loop-specific repair, or from segregation or short tract repair of single-base mismatches. In mutant cells, single-base mismatches were repaired less frequently, but the loop was still repaired efficiently and with bias toward loop retention, indicating that the defect in these cells does not affect loop-specific repair. Repair tracts in products from mutant cells showed a wide variety of mosaic patterns reflecting short regions of repair and segregation consistent with reduced nick-directed repair. In mutant cells, single-base mismatches were repaired more efficiently in the presence of the loop than in its absence, a likely consequence of corepair initiated at the loop.

Animals↗
PubMed1995

Structural characterization of d(CAACCCGTTG) and d(CAACGGGTTG) mini-hairpin loops by heteronuclear NMR: the effects of purines versus pyrimidines in DNA hairpins.

The DNA decamers, d(CAACCCGTTG) and d(CAACGGGTTG) were studied in solution by proton and heteronuclear NMR. Under appropriate conditions of pH, temperature, salt concentration and DNA concentration, both decamers form hairpin conformations with similar stabilities [Avizonis and Kearns (1995) Biopolymers, 35, 187-200]. Both decamers adopt mini-hairpin loops, where the first and last four nucleotides are involved in Watson-Crick hydrogen bonding and the central two nucleotides, CC or GG respectively, form the loop. Through the use of proton-proton, proton-phosphorus and natural abundance proton-carbon NMR experiments, backbone torsion angles (beta, gamma and epsilon), sugar puckers and interproton distances were measured. The nucleotides forming the loops of these decamers were found to stack upon one another in an L1 type of loop conformation. Both show gamma tr and unusual beta torsion angles in the loop-closing nucleotide G7, as expected for mini-hairpin loop formation. Our results indicate that the beta and epsilon torsion angles of the fifth and sixth nucleotides that form the loop and the loop-closing nucleotide G7 are not in the standard trans conformation as found in B-DNA. Although the loop structures calculated from NMR-derived constraints are not well defined, the stacking of the bases in the two different hairpins is different. This difference in the base stacking of the loop may provide an explanation as to why the cytosine-containing hairpin is thermodynamically more stable than the guanine-containing hairpin.

Base Sequence↗
PubMed1999

Conformation of the primary binding loop folded through an intramolecular interaction contributes to the strong chymotrypsin inhibitory activity of the chymotrypsin inhibitor from Erythrina variegata seeds.

We previously demonstrated that amino acid residues Gln62 (P3), Phe63 (P2), Leu64 (P1), and Phe67 (P3') in the primary binding loop of Erythrina variegata chymotrypsin inhibitor (ECI), a member of the Kunitz inhibitor family, are involved in its strong inhibitory activity toward chymotrypsin [Iwanaga et al. (1998) J. Biochem. 124, 663-669]. To determine whether or not these four amino acid residues predominantly contribute to the strong inhibitory activity of ECI, they were simultaneously replaced by Ala. The results showed that a quadruple mutant, Q62A/F63A/L64A/F67A, retained considerable inhibitory activity (Ki, 5.6 x 10(-7) M), indicating that in addition to the side chains of these four amino acid residues, the backbone structure of the primary binding loop in ECI is essential for the inhibitory activity toward chymotrypsin. Two chimeric proteins, in which the primary binding loops of ECI and ETIa were exchanged: an isoinhibitor from E. variegata with lower chymotrypsin inhibitory activity, were constructed to determine whether the backbone structure of the primary binding loop of ECI was formed by the amino acid residues therein, or through an interaction between the primary binding loop and the residual structure designated as the "scaffold." A chimeric protein, ECI/ETIa, composed of the primary binding loop of ECI and the scaffold of ETIa showed weaker inhibitory activity (Ki, 1.3 x 10(-6) M) than ECI (Ki, 9.8 x 10(-8) M). In contrast, a chimera, ETIa/ECI, comprising the primary binding loop of ETIa and the scaffold of ECI inhibited chymotrypsin more strongly (Ki, 5.7 x 10(-7) M) than ETIa (Ki, 1.3 x 10(-6) M). These results indicate that the intramolecular interaction between the primary binding loop and the scaffold of ECI plays an important role in the strong inhibitory activity toward chymotrypsin. Furthermore, surface plasmon resonance analysis revealed that the side chains on the primary binding loop of ECI contribute to both an increase in the association rate constant (kon) and a decrease in the dissociation rate constant (koff) for the ECI-chymotrypsin interaction, whereas the backbone structure of the primary binding loop mainly contributes to a decrease in the dissociation rate constant.

Alanine↗
PubMed2004

Accurate and efficient loop selections by the DFIRE-based all-atom statistical potential.

The conformations of loops are determined by the water-mediated interactions between amino acid residues. Energy functions that describe the interactions can be derived either from physical principles (physical-based energy function) or statistical analysis of known protein structures (knowledge-based statistical potentials). It is commonly believed that statistical potentials are appropriate for coarse-grained representation of proteins but are not as accurate as physical-based potentials when atomic resolution is required. Several recent applications of physical-based energy functions to loop selections appear to support this view. In this article, we apply a recently developed DFIRE-based statistical potential to three different loop decoy sets (RAPPER, Jacobson, and Forrest-Woolf sets). Together with a rotamer library for side-chain optimization, the performance of DFIRE-based potential in the RAPPER decoy set (385 loop targets) is comparable to that of AMBER/GBSA for short loops (two to eight residues). The DFIRE is more accurate for longer loops (9 to 12 residues). Similar trend is observed when comparing DFIRE with another physical-based OPLS/SGB-NP energy function in the large Jacobson decoy set (788 loop targets). In the Forrest-Woolf decoy set for the loops of membrane proteins, the DFIRE potential performs substantially better than the combination of the CHARMM force field with several solvation models. The results suggest that a single-term DFIRE-statistical energy function can provide an accurate loop prediction at a fraction of computing cost required for more complicate physical-based energy functions. A Web server for academic users is established for loop selection at the softwares/services section of the Web site http://theory.med.buffalo.edu/.

Membrane Proteins↗
PubMedDate not supplied

Biomechanical and clinical evaluation of a modified 3-loop pulley suture pattern for reattachment of canine tendons to bone.

OBJECTIVE: To describe a modified 3-loop pulley suture pattern for the reattachment of canine tendons to bone along with a biomechanical comparison with the locking-loop suture. STUDY DESIGN: In vitro biomechanical study and clinical case report. ANIMALS OR SAMPLE POPULATION: Biomechanical study: 10 paired gastrocnemius tendons and calcaneii harvested from 5 canine cadavers. CASE REPORT: a Doberman with avulsion of the gastrocnemius tendon of insertion. METHODS: Biomechanical study: paired tendons were reattached to the calcaneus with either a modified 3-loop pulley pattern or a locking-loop pattern. Tensile loading to failure was performed. A direct, non-contact, method of gap measurement, using digital video, was used to measure gap formation. Load required to initiate gap formation (defined as load at a 1 mm gap) and to produce a 3 mm gap was evaluated in addition to maximum load and gap at failure. RESULTS: Mean (+/-SEM) 1 mm gap loads were 31.0+/-4.2 and 17.2+/-2.5 N, mean 3 mm gap loads were 49.1+/-2.4 and 28.9+/-3.2 N, and mean maximum loads were 72.9+/-4.3 and 55.8+/-2.2 N for the modified 3-loop pulley suture and the locking-loop suture, respectively. These differences were statistically significant (P<.05). The gap at failure was similar for both repairs. The clinical case remained sound 7 months postoperatively. CONCLUSIONS: A modified 3-loop pulley pattern is biomechanically superior to a locking-loop pattern for reattachment of the canine gastrocnemius tendon to bone and may be suitable for clinical use. CLINICAL RELEVANCE: Tendon repairs with a gap >3 mm are reported to be at increased risk of rupture during the first 6 weeks postoperatively. A modified 3-loop pulley pattern resists gap formation better than a locking-loop pattern.

Animals↗
PubMed2000

Cysteine-scanning mutagenesis of the periplasmic loop regions of PomA, a putative channel component of the sodium-driven flagellar motor in Vibrio alginolyticus.

The sodium-driven motor consists of the products of at least four genes, pomA, pomB, motX, and motY, in Vibrio alginolyticus. PomA and PomB, which are homologous to the MotA and MotB components of proton-driven motors, have four transmembrane segments and one transmembrane segment, respectively, and are thought to form an ion channel. In PomA, two periplasmic loops were predicted at positions 21 to 36 between membrane segments 1 and 2 (loop(1-2)) and at positions 167 to 180 between membrane segments 3 and 4 (loop(3-4)). To characterize the two periplasmic loop regions, which may have a role as an ion entrance for the channel, we carried out cysteine-scanning mutagenesis. The T186 residue in the fourth transmembrane segment and the D71, D148, and D202 residues in the predicted cytoplasmic portion of PomA were also replaced with Cys. Only two mutations, M179C and T186C, conferred a nonmotile phenotype. Many mutations in the periplasmic loops and all of the cytoplasmic mutations did not abolish motility, though the five successive substitutions from M169C to K173C of loop(3-4) impaired motility. In some mutants that retained substantial motility, motility was inhibited by the thiol-modifying reagents dithionitrobenzoic acid and N-ethylmaleimide. The profiles of inhibition by the reagents were consistent with the membrane topology predicted from the hydrophobicity profiles. Furthermore, from the profiles of labeling by biotin maleimide, we predicted more directly the membrane topology of loop(3-4). None of the loop(1-2) residues were labeled, suggesting that the environments around the two loops are very different. A few of the mutations were characterized further. The structure and function of the loop regions are discussed.

Amino Acid Sequence↗
PubMed2000

Impact of human immunodeficiency virus type 1 RNA dimerization on viral infectivity and of stem-loop B on RNA dimerization and reverse transcription and dissociation of dimerization from packaging.

The kissing-loop domain (KLD) encompasses a stem-loop, named kissing-loop or dimerization initiation site (DIS) hairpin (nucleotides [nt] 248 to 270 in the human immunodeficiency virus type 1 strains HIV-1(Lai) and HIV-1(Hxb2)), seated on top of a 12-nt stem-internal loop called stem-loop B (nt 243 to 247 and 271 to 277). Destroying stem-loop B reduced genome dimerization by approximately 50% and proviral DNA synthesis by approximately 85% and left unchanged the dissociation temperature of dimeric genomic RNA. The most affected step of reverse transcription was plus-strand DNA transfer, which was reduced by approximately 80%. Deleting nt 241 to 256 or 200 to 256 did not reduce genome dimerization significantly more than the destruction of stem-loop B or the DIS hairpin. We conclude that the KLD is nonmodular: mutations in stem-loop B and in the DIS hairpin have similar effects on genome dimerization, reverse transcription, and encapsidation and are also "nonadditive"; i.e., a larger deletion spanning both of these structures has the same effects on genome dimerization and encapsidation as if stem-loop B strongly impacted DIS hairpin function and vice versa. A C258G transversion in the palindrome of the kissing-loop reduced genome dimerization by approximately 50% and viral infectivity by approximately 1.4 log. Two mutations, CGCG261-->UUAA261 (creating a weaker palindrome) and a Delta241-256 suppressor mutation, were each able to reduce genome dimerization but leave genome packaging unaffected.

Animals↗
PubMed1998

Reverse transcription of a self-primed retrotransposon requires an RNA structure similar to the U5-IR stem-loop of retroviruses.

An inverted repeat (IR) within the U5 region of the Rous sarcoma virus (RSV) mRNA forms a structure composed of a 7-bp stem and a 5-nucleotide (nt) loop. This U5-IR structure has been shown to be required for the initiation of reverse transcription. The mRNA of Tf1, long terminal repeat-containing retrotransposon from fission yeast (Schizosaccharomyces pombe) contains nucleotides with the potential to form a U5-IR stem-loop that is strikingly similar to that of RSV. The putative U5-IR stem-loop of Tf1 consists of a 7-bp stem and a 25-nt loop. Results from mutagenesis studies indicate that the U5-IR stem-loop in the mRNA of Tf1 does form and that it is required for Tf1 transposition. Although the loop is required for transposition, we were surprised that the specific sequence of the nucleotides within the loop was unimportant for function. Additional investigation indicates that the loss of transposition activity due to a reduction in the loop size to 6 nt could be rescued by increasing the GC content of the stem. This result indicates that the large loop in the Tf1 mRNA relative to that of the RSV allows the formation of the relatively weak U5-IR stem. The levels of Tf1 proteins expressed and the amounts of Tf1 RNA packaged into the virus-like particles were not affected by mutations in the U5-IR structure. However, all of the mutations in the U5-IR structure that caused defects in transposition produced low amounts of reverse transcripts. A unique feature in the initiation of Tf1 reverse transcription is that, instead of a tRNA, the first 11 nt of the Tf1 mRNA serve as the minus-strand primer. Analysis of the 5' end of Tf1 mRNA revealed that the mutations in the U5-IR stem-loop that resulted in defects in reverse transcription caused a reduction in the cleavage activity required to generate the Tf1 primer. Our results indicate that the U5-IR stems of Tf1 and RSV are conserved in size, position, and function.

Avian Sarcoma Viruses↗
PubMedDate not supplied

Automatic selection of loop breakers for genetic linkage analysis.

Pedigree loops pose a difficult computational challenge in genetic linkage analysis. The most popular linkage analysis package, LINKAGE, uses an algorithm that converts a looped pedigree into a loopless pedigree, which is traversed many times. The conversion is controlled by user selection of individuals to act as loop breakers. The selection of loop breakers has significant impact on the running time of the subsequent linkage analysis. We have automated the process of selecting loop breakers, implemented a hybrid algorithm for it in the FASTLINK version of LINKAGE, and tested it on many real pedigrees with excellent performance. We point out that there is no need to break each loop by a distinct individual because, with minor modification to the algorithms in LINKAGE/FASTLINK, a single individual that participates in multiple marriages can serve as a loop breaker for several loops. Our algorithm for finding loop breakers, called LOOPBREAKER, is a combination of: (1) a new algorithm that is guaranteed to be optimal in the special case of pedigrees with no multiple marriages and (2) an adaptation of a known algorithm for breaking loops in general graphs. The contribution of this work is the adaptation of abstract methods from computer science to a challenging problem in genetics.

Algorithms↗
PubMed2006

Dynamics of the WPD loop of the Yersinia protein tyrosine phosphatase.

The bacterial protein tyrosine phosphatase YopH is an essential virulence determinant in Yersinia spp., causing gastrointestinal diseases and the plague. Like eukaryotic PTPases, YopH catalyzes the hydrolysis of the phosphate moiety of phosphotyrosine within a highly conserved binding pocket, which is also characterized by the closure of the so-called "WPD loop" upon ligand binding. In this study, we investigate the conformational changes and dynamics of the WPD loop by molecular dynamics simulations. Consistent with experimental observations, our simulations show that the WPD loop of YopH is intrinsically flexible and fluctuates between the open and closed conformation with a frequency of approximately 4 ns for the apo, native protein. The region of helix alpha4 spanning loop 384-392, which has been revealed experimentally as a second substrate-binding site in YopH, is found to be highly associated with the WPD loop, stabilizing it in the closed, active conformation, and providing a structural basis for the cooperation of the second-substrate binding site in substrate recognition. Loop L4 (residues 323-327) is shown to be involved in a parallel, correlated motion mode with the WPD loop that contributes the stabilization of a more extended open conformation. In addition, we have simulated the loop reopening in the ligand-bound protein complex by applying the locally enhanced sampling method. Finally, the dynamic behavior of the WPD loop for the C403S mutant differs from the wild-type YopH remarkably. These results shed light on the role of the WPD loop in PTPase-mediated catalysis, and are useful in structure-based design for novel, selective YopH inhibitors as antibacterial drugs.

Bacterial Outer Membrane Proteins↗
PubMed2005

Force systems yielded by different designs of T-loop.

OBJECTIVES: To determine the mechanical characteristics of beta-titanium T-loops with and without helices, with 0 and 180 degree gable bends and constructed from 0.017 inch x 0.025 inch and 0.019 inch x 0.025 inch wire. METHODS: Forty beta-titanium T-loops were centrally positioned in a universal testing machine. The horizontal force and the moment/force ratios during activation were recorded at 1 mm intervals, up to a maximum of 7 mm. The data were analysed with analysis of variance complemented by the Tukey test for multiple comparisons. RESULTS: The results demonstrated that the transverse section of the wire had the greatest effect on the horizontal force produced by the loops. Significantly lower levels of horizontal force were obtained with loops made with the smaller 0.017 inch x 0.025 inch wire. Loops with gable bends yielded high moment/force ratios, whereas loops without gable bends had low moment/force ratios. In general, T-loops with helices yielded lower magnitudes of horizontal force and moment/force ratios than plain T-loops. CONCLUSIONS: The horizontal forces and moment/force ratios generated by plain T-loops with 180 degree gable bends yielded more adequate force systems. Incorporation of helices in the design of T-loops seems to be unnecessary.

Biomechanical Phenomena↗
PubMed1996

Endotoxin-induced enteric hypomotility in jaundiced loops in vitro.

Biliary obstruction may be accompanied by systemic endotoxemia due to increased growth of enteric microbiota and failure of hepatic clearance mechanisms. This endotoxemia is related to increased postoperative morbidity and mortality. An increased growth of the aerobic flora has been demonstrated experimentally in the presence of biliary obstruction, and in previous studies we observed intestinal hypomotility of jaundiced loops in vitro. To determine the ileal motor response in the presence of jaundice caused by biliary obstruction and in the presence of endotoxemia, an in vitro study was carried out on ileal segments from 10 female Holtzman rats, 2-3 months old, weighing 200 to 300 g, divided into two groups (N = 5); A, washed loops of jaundiced rats, and B, washed loops of jaundiced rats to which endotoxin was added. On the seventh postoperative day, we evaluated the effect of exogenous endotoxin (E. coli 0111:B4, Sigma) on the motor response to acetylcholine of distal ileal segments isolated from both animal groups. A 4-cm ileal segment, located 10 cm from the ileal papilla, was removed and studied in an organ chamber in order to assess dose-response curves to acetylcholine. There was an increase in threshold dose in jaundiced loops with intraluminally injected endotoxin when compared with the loops without intraluminal endotoxin (291 +/- 188 vs 8.5 +/- 6.7 microM, P < 0.05). The maximum contraction was reduced in jaundiced loops with intraluminal endotoxin in relation to control loops (5.3 +/- 1.7 vs 18.7 +/- 4.8 mm, P < 0.05), and pD2 was also reduced in jaundiced loops with intraluminal endotoxin in relation to control loops (2.4 +/- 0.6 vs 3.7 +/- 0.5, P < 0.05). There was no statistical difference between jaundiced loops with and without intraluminal endotoxin when the maximal contraction doses were compared (86 +/- 66 vs 48 +/- 22 mM, P > 0.05). These results demonstrate that intraluminal endotoxin depressed enteric motility in jaundiced rats.

Animals↗
PubMed1987

Impaired intestinal sodium and chloride transport in the blind loop syndrome of the rat.

Self-filling blind loops of rat jejunum were used as a model for the blind loop syndrome in humans. Electrical resistance, short circuit current, and unidirectional sodium and chloride fluxes were measured using the Ussing technique. Whereas net fluxes for sodium and chloride did not differ significantly from zero in the blind loop or in the control, unidirectional fluxes of either direction were decreased and electrical resistance was increased, indicating an increase in the tightness of the intestinal wall. Measurements of alternating current impedance and micropuncture experiments revealed that this was due to an increase in epithelial resistance from 9 +/- 1 omega X cm2 (n = 15, results of both methods) to 27 +/- 4 omega X cm2 (n = 15) and in subepithelial resistance from 40 +/- 2 omega X cm2 (n = 15) to 76 +/- 7 omega X cm2 (n = 15). As the ratio of epithelial to subepithelial resistance was similar in the blind loop and in the control, lower transport rates in the blind loop are indicative of impaired epithelial transport function. Subsequently, two different transport systems were characterized. First, the 3-o-methyl-glucose-induced, phlorizin-reversible increase in short circuit current, representing glucose-coupled sodium absorption, showed a 77% decrease in maximum velocity in the blind loop and no change in Km. Second, the chloride-induced, bumetanide-reversible increase in short circuit current in tissues stimulated simultaneously by prostaglandin E1 and theophylline, representing rheogenic chloride secretion, also showed a decrease in maximum velocity (of 83%) and no change in Km. A morphometric analysis revealed that the crypt surface area increased by 100% in the blind loop, whereas the villous surface area was not significantly different between blind loops and controls. We conclude that the jejunal self-filling blind loop is characterized by impaired active ion transport processes and an increase in epithelial and subepithelial resistance.

Animals↗
PubMed2001

Prediction of a 12-residue loop in bovine pancreatic trypsin inhibitor: effects of buried water.

The x-ray conformations of 5-, 7-, 9-, and 12-residue loops in bovine pancreatic trypsin inhibitor (BPTI) were predicted by the use of multiple independent Monte Carlo simulating annealing (MCSA) runs starting from random conformations. Four buried water molecules interacted with a 12-residue loop that started at residue 8 and ended at residue 19, and that included the binding region. The final conformation at the end of an MCSA run was characterized. Solvation free energy based on the solvent accessible surface area was included in the energy function at low simulated annealing temperatures. Conformational states were interactively separated by a recently developed algorithm. Computed loops were characterized in terms of total energy, and backbone and side chain root mean square deviations (RMSDs) between computed native loop conformations and the x-ray conformation. The 12-residue loop was computed with and without buried water [called WL12(8-19) and L12(8-19), respectively]. The backbone was reliably and reproducibly computed to within 1.1 A in L12(8-19) and 0.9 A in WL12(8-19). L12(8-19) required significantly more MCSA runs to achieve the same level of reproducibility as WL12(8-19). Based on the size of the cluster of low energy native loop conformations, and the computational effort, WL12(8-19) had greater entropy. In calculations of 7-, 9-, and 12-residue loops without buried water, the effects of buried water became obvious in the 12-residue loop calculation, which interacted with all four buried water molecules. Nearly all conformations of the native loop conformer had a hydrogen bond between the Lys 15 side chain and the backbone of Gly 12, Pro 13, and Cys 14, which may have implications in the rate of exchange of buried water with bulk solvent and in protein folding. The present version of MCSA program was more efficient than earlier versions.

Animals↗