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In the human DNA mismatch repair (MMR) system, hMSH2 forms the hMutSalpha and hMutSbeta complexes with hMSH6 and hMSH3, respectively, whereas hMLH1 and hPMS2 form the hMutLalpha heterodimer. These complexes, together with other components in the MMR system, correct single-base mismatches and small insertion/deletion loops that occur during DNA replication. Microsatellite instability (MSI) occurs when the loops in DNA microsatellites are not corrected because of a malfunctioning MMR system. Low-frequency MSI (MSI-L) is seen in some chronically inflamed tissues in the absence of genetic inactivation of the MMR system. We hypothesize that oxidative stress associated with chronic inflammation might damage protein components of the MMR system, leading to its functional inactivation. In this study, we demonstrate that noncytotoxic levels of H2O2 inactivate both single-base mismatch and loop repair activities of the MMR system in a dose-dependent fashion. On the basis of in vitro complementation assays using recombinant MMR proteins, we show that this inactivation is most likely due to oxidative damage to hMutSalpha, hMutSbeta, and hMutLalpha protein complexes. We speculate that inactivation of the MMR function in response to oxidative stress may be responsible for the MSI-L seen in nonneoplastic and cancer tissues associated with chronic inflammation.

Infection by enveloped viruses initially involves membrane fusion between viral and host cell membranes. The fusion peptide plays a crucial role in triggering this reaction. To clarify how the fusion peptide exerts this specific function, we carried out biophysical studies of three fusion peptide analogs of influenza virus hemagglutinin HA2, namely E5, G13L, and L17A. E5 exhibits an activity similar to the native fusion peptide, whereas G13L and L17A, which are two point mutants of the E5 analog, possess much less fusion activity. Our CD data showed that the conformations of these three analogs in SDS micelles are pH-dependent, with higher alpha-helical contents at acidic pH. Tryptophan fluorescence emission experiments indicated that these three analogs insert deeper into lipid bilayers at acidic pH. The three-dimensional structure of the E5 analog in SDS micelles at pH 4.0 revealed that two segments, Leu(2)-Glu(11) and Trp(14)-Ile(18), form amphipathic helical conformations, with Gly(12)-Gly(13) forming a hinge. The hydrophobic residues in the N- and C-terminal helices form a hydrophobic cluster. At neutral pH, however, the C-terminal helix of Trp(14)-Ile(18) reduces dramatically, and the hydrophobic core observed at acidic pH is severely disrupted. We suggest that the disruption of the C-terminal helix renders the E5 analog fusion-inactive at neutral pH. Furthermore, the decrease of the hinge and the reduction of fusion activity in G13L reveal the importance of the hinge in fusion activity. Also, the decrease in the C-terminal helix and the reduction of fusion activity in L17A demonstrates the importance of the C-terminal helix in fusion activity. Based on these biophysical studies, we propose a model that illustrates the structural change of the HA2 fusion peptide analog and explains how the analog interacts with the lipid bilayer at different pH values.

Conformations of a 48-mer peptide corresponding to the amino-terminal region of influenza HA2 in aqueous and membranous environments were studied. In aqueous solution the peptide was found to be oligomeric and its helicity was enhanced at higher concentrations. The conformation in phospholipid bilayer and insertion depth into the sodium dodecyl sulfate (SDS) micelle for the fusion peptide were in line with those determined for the amino-terminal 25-mer analog. The turn of residues 28-31 found in the crystal structure of hemagglutinin at neutral pH persisted in the presence of SDS at pH 5.0. Except for the turn, conformational lability of the amino portion of HA2 is suggested by comparison of the secondary structure determined herein with that obtained with the influenza fusion protein crystallized in the aqueous phase at neutral pH. The backbone amide proton exchange experiment suggested an interaction with the micellar surface for the segment carboxy-terminal to the fusion peptide domain., ((c)2001 Elsevier Science.)

A 43-mer peptide derived from the coiled coil domain of the transmembrane glycoprotein, gp41, of human immunodeficiency virus type 1, was synthesized. Light scattering measurements suggested that the peptide molecules likely exist in the aqueous solution in trimeric form. Circular dichroism experiments showed a moderate helix population enhancement for the peptide in 80% methanol solution relative to helicity in sodium dodecyl sulfate micellar suspension. NMR spectroscopy indicated that the N-terminal section of the peptide was conformationally more sensitive to the medium. The conformationally labile regions contain residues implicated in gp41-gp120 association. Our data support the idea that the coiled coil region is responsible for oligomerization of the gp41 ectodomain and suggest a site of conformational isomerization following receptor binding-induced gp120 dissociation from gp41.

JC virus (JCV), along with other members of the polyomavirus family, encodes a class of highly conserved proteins, T antigens, that are capable of inducing aneuploidy in cultured cells. We have previously isolated T-antigen DNA variants of JCV from both colon cancer tissues and the corresponding nonneoplastic gastrointestinal tissues, raising new questions about the role of JCV in the development of chromosomal instability of the colon. Based on the sequence of the transcriptional control region (TCR), JCV can be classified as archetype or tandem repeat variants. Among the latter, Mad-1, the prototype virus first isolated from a patient with progressive multifocal leukoencephalopathy, is characterized by lacking the 23- and 66-bp sequences that are present in the archetype and by duplication of a 98-bp sequence. In this study, we evaluated differences in the TCR of JCV isolated from colon cancer tissues and nonneoplastic epithelium. To characterize JCV variants, we first treated eight pairs of DNA samples from colon cancers and noncancerous tissue with topoisomerase I and then amplified and cloned the JCV TCR. We obtained 285 recombinant clones from the JCV TCR, 157 from nonneoplastic samples, and 128 from colon cancer tissues. Of these clones, 262 spanned the length of the JCV Mad-1 TCR: 99.3% from nonneoplastic samples and 82.8% from colon cancer tissues. In sequencing 54 clones in both directions, we did not find archetype JCV either in the nonneoplastic tissue or in the cancer samples. From all colon cancer tissues, 18 clones had a deletion of one 98-bp tandem repeat. This deleted strain was not detected in any of the nonneoplastic tissues (14 versus 0% [chi(2) = 23.6; P < 0.001]). Our study demonstrates that the only JCV strain present in the human colon is Mad-1, and the variant with a single 98-bp sequence is found exclusively in the cancer tissues. This strain may be involved in the development of chromosomal instability.

New clinical scales for semiquantitating disease activity in systemic lupus erythematosus (SLE) are widely used in research. They are reliable and valid measures. One of the original scales, the Systemic Lupus Activity Measure (SLAM), has been modified based on experience with it in multi-observer studies and training of individuals in its use. We tested the psychometric properties of the revised SLAM (SLAM-R). SLAM-R was tested on 30 SLE patients, who fulfilled 1997 revised ACR criteria and were selected to represent a range of disease activity. The patients were evaluated independently by two physicians, who studied the instruction booklet and who had never used SLAM-R, on two occasions 2-4 weeks apart. At the first visit, the physician's global assessment of activity using visual analog scale, anti-dsDNA Ab, C3 and C4 were checked for construct validity. The psychometric properties were analyzed with nested analysis of variance and Pearson's correlation coefficient using SAS. All patients were female, the median age was 31 (15-52) y, and the mean score of SLAM-R was 10.5 +/- 5.3 (3-26). Estimates of reliability were 0.78 of inter-rater, 0.61 of inter-visit, 0.76 of physician 1 between visits, and 0.56 of physician 2 between visits. Among subcategories except 'Eye,' the 'Gastrointestinal' category had the highest (0.96) and the 'Neuromotor' category had the lowest inter-rater reliability (0.50). With respect to construct validity, the correlation of SLAM-R scores with the disease activity variables except C4 was high and statistically significant. In conclusion, the SLAM-R is reliable and valid for measuring clinical disease activity in SLE.

Evans' syndrome is characterised by the simultaneous or sequential occurrence of Coombs'-positive haemolytic anaemia (AIHA) and immune thrombocytopenia without underlying aetiology. It has been found to be associated with collagen vascular diseases, especially systemic lupus erythematosus (SLE) and scleroderma. However, Evans' syndrome with dermatomyositis is very rare. A 59-year-old woman, who had been taking high-dose prednisolone for a month and cyclosporin for 10 days for dermatomyositis, developed purpura on the left popliteal fossa. The platelet and haemoglobin levels decreased to 77,000/mm3 and 9.8 g/dl, respectively. Antiplatelet antibody was positive. Thrombocytopenia responded to intravenous immunoglobulin (IVIG) for a short time, but further decreased in a week. Her blood film showed features of haemolytic anaemia. Laboratory findings showed reticulocytosis and a positive direct Coombs' test. Bone marrow examination showed a mild hyperplasia of erythroid precursors and megakaryocytes. The patient was successfully treated with cyclophosphamide in addition to oral prednisolone. AIHA in connective tissue disease may develop gradually and show a benign clinical course in most patients. Therefore, we suggest that patients with dermatomyositis and anaemia should always be checked for haemolysis if there is no other explanation.

Background & Aims: JC virus (JCV), a human polyomavirus, has been found in a limited number of normal human tissues and cancers. The oncogenic potential of this virus is mediated by a transforming protein, the T antigen (TAg). We have previously demonstrated the presence of JCV-TAg in colorectal cancers, in adjacent normal colonic mucosa from these patients, and in the human colon cancer cell line SW480. The mode of transmission of this virus is unclear, and we hypothesized that the gastrointestinal (GI) tract may be a reservoir for the virus., Methods: DNA was extracted from 129 normal GI tissue samples collected from 33 patients. Topoisomerase I-assisted polymerase chain reaction (PCR) was used to detect the virus using exact and degenerate primers. Nested PCR and Southern blot analysis confirmed the identity of the PCR products. Single-stranded conformation polymorphism (SSCP) analysis and sequencing were used to evaluate the presence of viral quasispecies., Results: JCV sequences were found in 75.8% of patients (70.6% of upper GI and 81.2% of colonic samples); no significant differences in rates of infection were found by site. The use of degenerate primers combined with topoisomerase I treatment led to viral detection in 58.9% of samples, compared with 27.9% of samples using exact primers and topoisomerase I (P < 0.01). SSCP and sequencing analysis confirmed the amplification of viral quasispecies and the authenticity of TAg sequences., Conclusions: The results show that JCV DNA sequences are highly prevalent in the human upper and lower gastrointestinal tract of immunocompetent individuals.

Membrane fusion is a key step in the virus mediated cell fusion. The vesicular dispersion serves as a model system to study the membrane fusion. We employed dynamic and static light scattering to study the fusion of phosphatidylcholine vesicles in the presence of model fusion peptide fragments from the hemagglutinin HA2 protein. The fusion-induced aggregation under the present experimental setup exhibited strong pH dependence, similar to the parental viral protein. Replacement of the glycine residue at the extreme amino terminus by glutamic acid (G1E) abolished fusion activity. The average molecular mass and diameter of vesicular dispersion obtained from static and dynamic light scattering measurements respectively at neutral and acidic pH showed about three fold increase in acidic solution containing wild type fusion peptide. The light scattering data are consistent with lipid mixing results. The present work demonstrates the utility of light scattering as a facile means to monitor the fusion process.

Background and Aims: Ulcerative colitis (UC) is regarded as a rare disease in developing countries, but accurate data are generally lacking. We performed the present study to evaluate the incidence and prevalence of UC in Korea., Methods: A retrospective study was performed from 1986 to 1997 in the Songpa-Kangdong district of Seoul, Korea. To recruit UC patients as completely as possible, multiple information sources including all medical facilities in the study area and three referral centres located nearby, but outside the study area were used. The incidence and prevalence rates were adjusted using the 1997 Korean population statistics., Results: During the study period, a total of 94 incident cases were identified, for an adjusted mean annual incidence rate of 0.68 per 100,000 inhabitants. On 31 December 1997, 91 patients with UC lived in the study area, giving an adjusted prevalence rate of 7.57 per 100,000 inhabitants. By using the Poisson regression analysis, the annual incidence rate increased significantly from 0.20 per 100,000 inhabitants in 1986-1988 to 1.23 per 100,000 inhabitants in 1995-1997 (P < 0.005). Patient age at diagnosis, the interval from onset of symptoms to diagnosis, and the disease extent at diagnosis were fairly constant throughout the study period., Conclusions: The incidence and prevalence of UC in our study area are still low compared with those of Western countries, but the incidence rate is steadily increasing.

The conformation and interactions with membrane mimics of the NH(2)-terminal fragment 1-25 of HA2, HA2-(1-25), of influenza virus were studied by spectroscopic methods. Secondary structure analysis of circular dichroism data revealed 45% helix for the peptide at pH 5.0. Tryptophan fluorescence quenching by acrylamide and NMR experiments established that the Trp(14) is inside the vesicular interior and residues 16-18 are at the micellar aqueous boundary. NBD fluorescence enhancement of the NH(2)-terminal labeled fluorophore on the vesicle-bound peptide indicated that the NH(2) terminus of the fusion peptide was located in the hydrophobic region of the lipid bilayer. No significant change in insertion depth was observed between pH 5.0 and 7.4. Collectively, these spectroscopic measurements pointed to an equilibrium between helix and non-helix conformations, with helix being the dominant form, for the segment in the micellar interior. The conformational transition may be facilitated by the high content of glycine, a conformationally flexible amino acid, within the fusion peptide sequence. Self-association of the 25-mer peptide was observed in the N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine SDS-gel electrophoresis experiments. Incorporating the NMR signal attenuation, fluorescence, and gel electrophoresis data, a working model for the organization of the fusion peptide in membrane bilayers was proposed.

Steady-state levels of human DNA mismatch repair (MMR) transcripts and proteins were measured in MMR-proficient and -deficient cell lines by the newly developed competitive quantitative reverse transcription- polymerase chain reaction and Western analysis normalized with purified proteins. In MMR-proficient cells, hMSH2 is the most abundant MMR protein and is expressed 3 to 5 times more than hMLH1. The hMLH1 protein was expressed 1.5 to 2.5 times more than hPMS2. Steady-state levels of mRNA expression correlated well with protein expression. hMSH2-mutated LoVo cells did not express detectable hMSH3 or hMSH6 proteins. Similarly, hMLH1-mutated HCT116 cells did not express detectable hMLH1 or hPMS2 protein, whereas in hMLH1-restored HCT116+ch3 cells, hPMS2 protein was re-expressed. In hMSH6-mutated HCT15 cells, both hMSH3 protein and mRNA were increased. In SV40-transformed lung fibroblasts, all MMR mRNAs and proteins examined were expressed at levels 1.5-5-fold higher than in their nontransformed counterpart. The steady-state levels of MMR proteins indicate that substantially more hMutS proteins, which are involved in DNA mismatch recognition, are present in comparison with the hMutL proteins. Stability of hMSH3 and hMSH6 proteins appears to depend upon the presence of the hMSH2 protein, and, similarly, the stability of the hPMS2 protein depends upon hMLH1. When the hMSH6 is mutationally inactivated, hMSH3 increases by both transcriptional up-regulation and enhanced protein stability. A balanced up-regulation of all of the components was seen after viral transformation in a fibroblast model. Quantitative changes of the MMR components are a potential mechanism to modify the DNA MMR capabilities of a cell.

Five partially overlapping synthetic peptides containing the N-terminal portion of the leucine zipper (LZ)-like domain of human immunodeficiency virus envelope glycoprotein gp41 were used to deduce the helix initiation site. Circular dichroism (CD) data suggested a strong helix-inducing motif, LLRA. The coupling constant and nuclear Overhauser effect (NOE) results obtained from nuclear magnetic resonance experiments in 20% trifluoroethanol aqueous solution at 280 K for the four decapeptides under study suggested that the motif XLLRA, where X is a group or an amino acid residue capable of forming hydrogen bond to arginine, constitutes a helix nucleation core. A similar conclusion was reached for a pentadecapeptide in water, suggesting that the result was not dependent on both chain length and the helix promoting medium. Detailed analysis of NOE and CD data from the four decapeptides indicated that the acetyl group and asparagine had a strong tendency to be helix N-capping, in confirmation of previous studies. Molecular modeling using restraints derived from NOE data showed that van der Waals, hydrophobic interactions and hydrogen bonds contribute synergetically to the stability of the core structure. The concept of nucleation core consisting of a few amino acids may be generally applied in proton design and folding studies.

The in vitro refolding of hen egg-white lysozyme is studied in the presence of various osmolytes. Proline is found to prevent aggregation during protein refolding. However, other osmolytes used in this study fail to exhibit a similar property. Experimental evidence suggests that proline inhibits protein aggregation by binding to folding intermediate(s) and trapping the folding intermediate(s) into enzymatically inactive, "aggregation-insensitive" state(s). However, elimination of proline from the refolded protein mixture results in significant recovery of the bacteriolytic activity. At higher concentrations (>1.5 M), proline is shown to form loose, higher-order molecular aggregate(s). The supramolecular assembly of proline is found to possess an amphipathic character. Formation of higher-order aggregates is believed to be crucial for proline to function as a protein folding aid. In addition to its role in osmoregulation under water stress conditions, the results of this study hint at the possibility of proline behaving as a protein folding chaperone.

The structural effect of a proline in a helix in trifluoroethanol (TFE)/water medium was examined on a 29-mer peptide and its proline analog derived from the leucine zipper (LZ)-like motif of gp41 (the transmembrane glycoprotein of HIV-1) by NMR and circular dichroism (CD) spectroscopies. Lower helical content was found for the proline mutant from the CD study. NMR data show that distortion of the helix by proline is local and occurs mainly on the N-terminal side of the substitution site. Molecular dynamics computation exhibits a bending of the helical axis of 30 degrees +/- 10 degrees, in agreement with X-ray diffraction results. Light-scattering experiments indicated that the average aggregation number of the proline-substituted mutant is substantially lower than that of the wild-type peptide. From the ratio of dissociation constants of the wild-type and the proline mutant peptides, the difference in free energy of trimeric formation is calculated to be 2.1 kcal/mol. Thermal stability, helicity, and the average aggregation number for the helix oligomers were found to be correlated. The structural alteration and the reduced coiled coil stability may account for the deficiency in the biological functions of the proline mutants of gp41 and in the inhibitory action of proline-substituted peptides. These effects may also be important in unraveling the roles played by proline in transmembrane proteins., (Copyright 1999 Academic Press.)

When the Y chromosomes from certain populations of Mus musculus domesticus are introduced into the mouse strain C57BL/6 (B6), testis determination can fail, resulting in gonads developing either as ovotestes (with both ovarian and testicular components) or as ovaries. Not all Y(DOM) chromosomes cause sex reversal. Y(DOM) chromosomes are divided into three classes based upon their ability to induce testes in B6. The molecular basis underlying the three Y(DOM) classes is an enigma. The simplest explanation is that they harbor different alleles of the testis-determining gene, Sry. Sequencing of Sry(DOM) genes has indeed identified polymorphisms. However, none were unequivocally linked to the sex-reversal trait. It was concluded that all SRY(DOM) proteins are functionally equivalent. Using a semiquantitative RT-PCR assay, we now show that representatives of the three Y(DOM) classes have variant Sry expression patterns, that severity of sex reversal correlates with Sry mRNA titers, and that genetic correction of the sex reversal results in the upregulation of Sry expression. We propose that the variant Sry expression patterns result from polymorphisms at the site of a putative Sry enhancer., (Copyright 1999 Academic Press.)

A technique of arthroscopic anterior cruciate ligament reconstruction that does not disturb the epiphyseal plate in the young patient with open physis is presented. A cryopreserved bone-Achilles tendon allograft was incorporated by an interference screw fixation to the bone plug in the tibia and an over-the-top positioning of the tendon on the femoral side. For this procedure, the minimal patient age that the thickness of the epiphysis can accept an interference screw greater than 15 mm in length is 8 years. An intra-articular reconstruction of anterior cruciate ligament with the cryopreserved Achilles allograft using our technique is safe and recommendable for young patients with open physis.

Background/aims: Anti-Helicobacter pylori (H. pylori) treatment for low-grade gastric mucosa-associated lymphoid tissue (MALT) lymphoma has been the subject of attention. The aim of this study was to determine the proportion of such cases which could be suitable candidates for H. pylori eradication for the purpose of cure; we focused on gross morphology and lymph node metastasis., Methodology: We retrospectively reviewed the medical records of 53 patients diagnosed and treated for gastric MALT lymphoma at Seoul National University Hospital between 1992 and 1996., Results: According to Isaacson's classification, 60% of cases were low-grade, and H. pylori was detected in 88% of them. In low-grade disease, gastroscopy revealed superficial lesions in 56% of cases, ulcerofungating lesions were found in as much as 19%, and ulceroinfiltrating in 25%. Even in low-grade disease, invasion of proper muscle, or deeper, was seen in 28% of patients, and lymph node involvement in 36%; even in low-grade disease confined to mucosa and submucosa, the rate of lymph node involvement was 40%. All cases which, on gastroscopy, appeared to be gastritis or benign ulcer-like lesions were free of lymph node metastasis, but in low-grade disease, this proportion was only 16%. In 33% of cases, pre-operative clinical stage I--as shown by abdominal CT--was found post-operatively to be stage II. The negative predictive value of lymph node detection by CT was 68%., Conclusions: In low-grade gastric MALT lymphoma, the lymph node involvement rate was too high to be neglected. In detecting lymph node metastasis, the diagnostic accuracy of CT was too low. The proportion of suitable candidates for anti-H. pylori treatment for low-grade gastric MALT lymphoma was not high, and in clinical practice, anti-H. pylori treatment in such cases should at present be very carefully applied.

A peptide of 51 amino acids corresponding to the NH2-terminal region (5-55) of the glycoprotein gp41 of human immunodeficiency virus type 1 was synthesized to study its conformation and assembly. Nuclear magnetic resonance experiments indicated the sequence NH2-terminal to the leucine zipper-like domain of gp41 was induced into helix in the micellar solution, in agreement with circular dichroism data. Light scattering experiment showed that the peptide molecules self-assembled in water into trimeric structure on average. That the peptide molecules oligomerize in aqueous solution was supported by gel filtration and diffusion coefficient experiments. Molecular dynamics simulation based on the NMR data revealed a flexible region adjacent to the hydrophobic NH2 terminus of gp41. The biological significance of the present findings on the conformational flexibility and the propensity of oligomerization of the peptide may be envisioned by a proposed model for the interaction of gp41 with membranes during fusion process.

Patients with systemic lupus erythematosus (SLE) are reported to have an increased risk of malignancy, especially lymphoproliferative disorders. We decribe the occurrence of ileocaecal intussusception secondary to Burkitt's lymphoma in a patient with SLE. A 23-year-old woman, who had been diagnosed with SLE 2 years ago, developed intermittent abdominal pain with a palpable mass. Computed tomography and a double-contrast barium enema showed a lobulated mass with intussusception at the ileocaecal junction. Right hemicolectomy and splenectomy was performed after histopathological examinations on colonoscopic biopsy revealed Burkitt's lymphoma. Fourteen months after chemotherapy, there is no evidence of recurrence of the Burkitt's lymphoma. When a patient with SLE has abdominal complaints, besides serositis, lupus enteritis such as peptic ulcer disease, mesenteric vasculitis with or without complications and pancreatitis, we have to consider intussusception secondary to gastrointestinal lymphoma as one of the differential diagnoses. Therefore, we should thoroughly investigate patients with SLE presenting with abdominal pain and not simply consider it afeature of lupus enteritis until other causes have been ruled out.

The binding affinity of a protein kinase C substrate, neurogranin peptide NG(28-43), to a sodium dodecyl sulfate micelle was analyzed quantitatively by the diffusion coefficient (Da) of the peptide determined by pulsed field gradient NMR. By use of a two-state model, the fraction of the peptide in the bound state, and hence the binding constant, can be estimated. The obtained binding constant is within the same order of magnitude as those reported for similar systems using other techniques. The present method may be generalized to measure the formation constants of other peptide:micelle complexes.

The exchange broadening of backbone amide proton resonances of a 23-mer fusion peptide of the transmembrane subunit of HIV-1 envelope glycoprotein gp41, gp41-FP, was investigated at pH 5 and 7 at room temperature in perdeuterated sodium dodecyl sulfate (SDS) micellar solution. Comparison of resonance peaks for these pHs revealed an insignificant change in exchange rate between pH 5 and 7 for amide protons of residues 4 through 14, while the exchange rate increase at neutral pH was more prominent for amide protons of the remaining residues, with peaks from some protons becoming undetectable. The relative insensitivity to pH of the exchange for the amide protons of residues 4 through 14 is attributable to the drastic reduction in [OH-] in the micellar interior, leading to a decreased exchange rate. The A15-G16 segment represents a transition between these two regimes. The data are thus consistent with the notion that the peptide inserts into the hydrophobic core of a membrane-like structure and the A15-G16 dipeptide is located at the micellar-aqueous boundary.

The folding of cardiotoxin analogue III (CTX III), a small (60 amino acids), all beta-sheet protein from the venom of the Taiwan Cobra (Naja naja atra) is here investigated. The folding kinetics is monitored by using a variety of techniques such as NMR, fluorescence, and circular dichroism spectroscopy. The folding of the protein is complete within a time scale of 200 ms. The earliest detectable event in the folding pathway of CTX III is the formation of a hydrophobic cluster, which possess strong affinity to bind to nonpolar dye such as 1-anilino-8-napthalene-sulfonic acid. Quenched-flow deuterium-hydrogen exchange experiments indicate that the segment spanning residues 51-55 along with Lys23, Ile39, Val49, Tyr51 and Val52 could constitute the "hydrophobic cluster." Folding kinetics of CTX III based on the amide-protection data reveals that the triple-stranded, antiparallel beta-sheet segment, which is located in the central core of the molecule, appears to fold faster than the double-stranded beta-sheet segment.

A peptide based on the N-terminal fusion domain of gp41 of human immunodeficiency virus type 1 (HIV-1) and its tryptophan analog were synthesized to examine the secondary structure in the micellar environment. Nuclear magnetic resonance (NMR), circular dichroism and electron paramagnetic resonance experiments indicated that the gp41 fusion peptide inserted into the micelle primarily as a helix (59%), with substantial beta-structure (26.7%). Deep penetration of the peptide into the apolar hydrocarbon core was supported by the results of fluorescence experiments in which the tryptophan analog exhibited a blue shift of about 30 nm in the presence of a sodium dodecyl sulfate micelle, in 1,2-dimyristoyl-rac-glycero-3-phosphocholine, and in 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine vesicular solutions. The results of spin label-attenuated 1H resonance experiments show that the region C-terminal to G16, which contains a turn structure, exhibited substantial interaction with the micelle, suggesting that it lies on the surface of micelle. Molecular simulation based on data from NMR experiments revealed a flexible hinge at residues 15 and 16 (alanine and glycine, respectively) from the N terminus of the peptide located at the micelle-solution interface. The highly conserved A15-G16 dipeptide may play a role in the function of fusion domain of HIV-1 envelope glycoprotein.

NMR and CD studies were carried out on a peptide representing the hydrophobic N-terminal domain of envelope glycoprotein of human immunodeficiency virus type-1 in solutions of varying polarity. It was found that in aquaeous solution the amide proton of glycine in the FLG motif resonated at a considerably high field and its chemical shift, within the limit of experimental precision, had a temperature coefficient of zero in the range studied. The upfield shift of NH of the glycine could be largely attributed to the ring-current effect of phenylalanine in the FLG motif that participated in a type-1 beta turn with a short Cbeta(i)-NH(i+2) distance. The slower proton-deuterion exchange for the glycine amide proton relative to that of other glycines was consistent with a folded structure for the motif in aquaeous solution. Results of the molecular simulation showed that this proton was shielded from the solvent by non-polar side chains of the amino acid residues surrounding the turn stabilized by hydrophobic interactions, thus explaining the zero temperature coefficient of the proton chemical shift. The structural stabilizing effect of the hydrophobic interaction was supported by the behavior of the proton in less polar Me2SO solution, in which the anomaly in the chemical shift and its temperature coefficient was less prominent. Detailed secondary-structure analysis suggested that the beta turn of the FLG motif may act as an initiation core for helix formation, probably because the turn readily transforms into helical form.

Two-dimensional NMR, circular dichroism (CD) experiments and molecular modeling were performed to study the secondary structure of a 44-mer peptide fragment derived from the C4 region of gp120 of human immunodeficiency virus in aqueous solution. It was found a nascent helical structure exists following a type I turn near the N-terminus of the peptide. The proline residue in the turn appears to serve as a helix initiator. The helical structure was in fast dynamic equilibrium with beta- or random coil form on the NMR scale. A reverse turn was identified at a section containing two consecutive proline residues. A nascent helical structure has been detected for the region near the C-terminus of the 44-mer peptide. Higher helical content for the peptide is also indicated by CD studies on TFE titration. Thus it is proposed that, in more apolar medium, the Pro-Pro turn and the segment amino-terminal to it, spanning about 20 amino acids, may be converted into helix structure. Moreover, the region near the C-terminus of the peptide may also be induced into helix, so that a helix-turn-helix structure may be formed in the C4 domain of gp120. A helical wheel representation of this stretch shows amphipathicity of the helix. The biological implication of the conformational adaptibility of the peptide was discussed.

A peptide corresponding to the neuronal protein neurogranin (NG) residues 28-43, NG(28-43), and its analog, [A35]NG(28-43), have been investigated by NMR, electron paramagnetic resonance (EPR), and circular dichroism (CD) spectroscopies. The peptides existed in aqueous solution predominantly in random form. However, a nascent helical structure was detected in the central region of the parent peptide from NMR data. Furthermore, a helical structure can be detected for both peptides with greater induced secondary structure for the parent peptide in the presence of sodium dodecyl sulfate (SDS) micelle. The formation of micelles for SDS was confirmed by results from EPR as well as 13C NMR. As shown by CD experiments, helical conformer was induced for NG(28-43) in vesicular solution containing phosphatidyl serine (PS), whereas no helix can be discerned for the peptide in phosphatidyl choline (PC)-containing vesicular solution. Together with the induction of the peptide into helix in SDS micellar solution as suggested by both NMR and CD data, these results underscored the electrostatic contribution to the interaction of the PKC substrate peptides and proteins with membrane. According to NMR and CD data, a dynamic equilibrium existed between free and micelle-bound states for the peptide. Moreover, proton-deuterium exchange results and SDS-induced linewidth broadening of proton resonances allowed delineation of the orientation of the amphipathic helix on the surface of SDS micelle. The result was supported by spin label experiments that indicated F35 of NG(28-43) interacted strongly with the hydrocarbon interior of micelle. Based on the experimental findings, a working model was proposed that attempted to partly explain the roles played by the nonpolar amino acid near the phosphorylation site, by the negatively charged phospholipids, and by the basic amino acids of the substrate.

Objective: Because psychiatrists do not have a consistent way to classify and define the forms of child abuse that may be mistaken for ritual abuse, the objective of this paper is to create a comprehensive differential diagnosis of allegations of ritual abuse., Method: The authors reviewed 60 articles, chapters, and books that contained allegations of ritual abuse or behaviors that might be mistaken for ritual abuse, that were made by patients or caretakers., Results: This paper clarifies the behaviors that represent or may be mistaken for ritual abuse: Cult-based ritual abuse, pseudoritualistic abuse, activities by organized satanic groups, repetitive psychopathological abuse, sexual abuse by pedophiles, child pornography portraying ritual abuse, distorted memory, false memory, false report due to a severe mental disorder, pseudologia phantastica, adolescent behavior simulating ritual abuse, epidemic hysteria, deliberate lying, and hoaxes., Conclusions: The differential diagnosis of allegations of ritual abuse is important in both clinical and forensic psychiatry. In some cases, it will not be possible to tell whether a particular allegation is factual or what the underlying mental processes are. It is important to separate the role of the mental health professional as therapist from the role as an expert witness in court.

Comparison of interaction energy between an oligonucleotide and a DNA-binding ligand in the minor and major groove modes was made by use of restrained molecular dynamics. Distortion in DNA was found for the major groove mode whereas less significant changes for both ligand and DNA were detected for the minor groove binding after molecular dynamics simulation. The conformation of the ligand obtained from the major groove modes resembles that computed with the ligand soaked in water. The van der Waals contact energy was found to be as significant as electrostatic energy and more important for difference in binding energy between these two binding modes. The importance of van der Waals force in groove binding was supported by computations on the complex formed by the repressor peptide fragment from the bacteriophage 434 and its operator oligonucleotide.

The interaction between the cobalt(III) complex of a bleomycin functional model (AMPHIS-NET) and the oligonucleotide d(CGCAATTGCG)2 and the structural features of the 1:1 ligand-DNA complex have been determined by high-resolution two-dimensional nuclear magnetic resonance methods and restrained molecular dynamics calculations. The intermolecular nuclear Overhauser effect (NOE) cross-peaks between ligand protons and the DNA minor groove protons suggest that the cobalt(III) complex of AMPHIS-NET binds in the minor groove of DNA at the central AATT site. The NOE connectivities also clearly indicate that the H8 pyridine proton and the H2 imidazole proton in the metal-binding domain interact with the H4' sugar proton of C19 and the H4' sugar proton of A5, respectively, which defines a structure where the metal binding moiety of Co(III).AMPHIS-NET participates in binding to the DNA and extends into the region two base pairs beyond the central AATT site in the minor groove. This binding model is in accord with the consistently observed nondiffusion DNA cleavage in locations two to three residues beyond the end of AT-rich binding sites induced by the corresponding iron(II) complexes of AMPHIS-NET and other AMPHIS-lexitropsin hybrids of the bleomycin functional model compounds.

The thermal unfolding of an all beta-sheet protein, cardiotoxin analogue III, from the Taiwan Cobra (Naja naja atra) is studied at pH 2.0, 4.0 and 6.0. At pH 4.0, using circular dichroism and 1-anilino naphthalene-8-sulphonic acid (ANS) fluorescence binding studies, a stable partially structured intermediate is detected at 90 degrees C.

Two-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy experiments were carried out to determine the conformation of a CD4 fragment (81-92) TYICEVEDQKEE and its di-benzylated analogue in aqueous solution. The refined structures obtained by use of distance geometry and simulated annealing for both peptides revealed a marked departure from that of the corresponding sequence within the D1D2 fragment of CD4 determined by x-ray crystal studies. Near the amino terminus of both peptides, a distinct loop exists that is stabilized by the hydrophobic interaction between the side chains of the amino acids in the region. For the parent peptide and Cys84 and Glu85 dibenzylated analogues, the orientation of the loop is different with respect to the reverse turn formed by Val86 through Gln89. In particular, the side chains of Val86 point in different directions for these two peptide analogues. The C- terminus of both peptide analogues exhibits a faster motional characteristic. The deuterium-proton exchange experiment showed a very slowly exchanged Gln89 backbone NH for the di-benzylated peptide, suggesting its participation in the hydrogen bond to Val86 C=O. The negatively charged side chains of Glu87 and Asp88 are found to jut out, similar to the crystal structure. The difference between the two peptides in inhibiting the syncytium formation is postulated to arise from the more defined Val86-Gln89 turn and the orientation of the apolar side chains of the residues in the stretch thought to participate in the cell-fusion process.

The alpha 1 subunit of the gamma-aminobutyric acid (GABA)A receptor is highly expressed in a subgroup of neurons in the hippocampal formation. The distribution and chemical identities of these neurons in the dentate gyrus have been studied with double-labeling in situ hybridization and immunohistochemical methods. Double labeling for the alpha 1 subunit and glutamate decarboxylase 65 (GAD65) mRNAs indicated that virtually all neurons in the dentate gyrus that are heavily labeled for the alpha 1 subunit are GABA neurons. However, many GAD65 mRNA-labeled neurons in the hilus do not contain high levels of the alpha 1 subunit mRNA and protein. Studies were thus conducted to determine if the somatostatin neurons of the hilus were part of the alpha 1 subunit-labeled group. Double labeling for the alpha 1 subunit and pre-prosomatostatin mRNAs demonstrated virtually no co-localization of these mRNAs in hilar neurons. Thus, the strongly labeled alpha 1 mRNA-containing neurons and the somatostatin neurons constitute two distinct populations of hilar GABA neurons. Double labeling for the alpha 1 subunit polypeptide and its mRNA with immunohistochemical and in situ hybridization methods demonstrated directly that neurons of the dentate gyrus that express high levels of the alpha 1 subunit mRNA are the same neurons that show extensive labeling for the alpha 1 subunit along their somal and dendritic surfaces. The high levels of alpha 1 subunit expression in some populations of GABA neurons could be related to prominent disinhibitory functions of these neurons.

The three-dimensional structure in solution of cardiotoxin II, a membrane toxin from the venom of Taiwan cobra, Naja naja atra, was determined using 1H nuclear magnetic resonance spectroscopy and molecular modeling based on the hybrid distance geometry/dynamic simulated annealing technique. A complete sequence-specific proton assignment was obtained, and the secondary structures of the protein were determined from information on nuclear Overhauser effect connectivities, coupling constants, and hydrogen exchange were confirmed using the main-chain-directed strategy. Twelve simulated annealing structures found to be within a single family were selected based on the condition of distance constraint violation less than 0.02 nm and the dihedral angle violation less than 4 degrees. The average atomic root mean square deviation between the selected structures and their geometric average are 0.079 nm for the backbone atoms and 0.137 nm for all heavy atoms; they are 0.044 nm and 0.117 nm, respectively, when considering the secondary structural residues only. The molecule adopts a compact structure consisting of three major loops emerging from a globular head. These loops contain five strands to form double- and a triple-stranded antiparallel beta sheets. Comparisons are made between this structure and those of its homologous cardiotoxins in order to derive further information on their structural variations.

Solution conformation of CD4 fragment 81-92 TYICEVEDQKEE and two of its benzylated analogues was determined by two-dimensional 1H NMR spectroscopy, distance geometry and simulated annealing techniques. The structures of both benzylated derivatives are similar but are distinct from that of wild-type dodecapeptide. It is concluded from structural analysis that bulky side chain(s) of amino acid(s) at an appropriate position can have a marked effect on the conformation and thus the functions of a peptide.

The structure in solution of cardiotoxin III, a membrane toxin purified from the venom of the Taiwan cobra, Naja naja atra, is reported. Sequence-specific assignment of 1H-NMR lines was completed and the NMR data show the presence of a triple and a double-stranded antiparallel beta-sheet. Many NOE cross peaks identified in NOESY spectra were applied as distance constraints based on a hybrid distance geometry/dynamical simulated annealing technique; 20 structures were found within a single family. The average value of atomic RMS differences between the 20 structures and their geometric mean is 0.087 nm for the backbone atoms and 0.152 nm for all heavy atoms; they are 0.055 nm and 0.12 nm, respectively for the segments of secondary structure. In these selected structures the backbone of the polypeptide chain folds such that five strands emerge from a globular head. Three major loops link these strands to form a double and a triple-stranded antiparallel beta-sheet. Comparison of the structures of the toxin in solution with the X-ray crystal structure of its homologous protein, cardiotoxin V4II from Naja mossambica mossambica, showed good agreement between the structures except at segments of the turns. As the functions of short neurotoxins and cardiotoxins are distinct, despite their similar secondary structural patterns and tertiary folding, a comparative analysis has been carried out between cardiotoxin III and short neurotoxins of known structures. We discuss their structural features in order to clarify relationships between their structure and function.

A 49-year-old man with liver cirrhosis and hypertension was found to have hyperkalemia out of a degree of renal insufficiency and metabolic acidosis with low to normal anion gap, aggravated by volume contraction with diarrhea and medications (captopril, spironolactone and atenolol) interfering with potassium homeostasis. Plasma renin activity and serum aldosterone levels of this patient on a regular diet after discontinuation of medications were very low compared to those of five other cirrhotic patients with normokalemia as controls. Also, the renin-aldosterone stimulation testing on this patient performed by sodium restricted diet and furosemide, upright position and by angiotensin converting enzyme inhibition (captopril, 50 mg) showed the blunted renin and aldosterone responses to each of these stimuli, almost no changes from baseline renin and aldosterone levels, it was concluded that the underlying defect responsible for hyperkalemia in this case was hyporeninemic hypoaldosteronism and this was aggravated by other factors or drugs affecting potassium homeostasis.

The conformation of two substance P (SP) related hexapeptides. Glp-Phe-Phe-(L-Pro)-Leu-Met.NH2 (I) and Glp-Phe-Phe-(D-Pro)-Leu-Met.NH2 (II), in two solvents, chloroform-d and trifluoroethanol(TFE)-d3/H2O, was studied by two-dimensional NMR methods, including COSY, TOCSY, ROESY and HMQC. The study shows that these two peptides exist predominantly in the extended form in TFE/H2O, but in general exhibit a reverse-turn structure in chloroform. I is clearly less ordered than II in both solvents. Furthermore, extensive Phe3-Pro4 cis<==>trans isomerization was found in I but not in II. The differences in the conformational behavior of these two peptides, which are selective agonists for neurokinin NK1 and NK2 receptors, respectively, are discussed.

The characterization of mineral-associated polyanions from the unicellular alga Pleurochrysis carterae is described. This species is useful for the study of mineralization, because it produces calcified scales known as coccoliths in homogeneous cell culture. Three acidic polysaccharides (PS-1, PS-2, and PS-3) were extracted from the coccoliths with EDTA and were separated and purified by differential precipitation with magnesium ions and chromatography on DEAE-cellulose. PS-1 and PS-3 are predominantly polymers of galacturonic acid containing lesser amounts of other monosaccharides. PS-2 has an unusual structure. Chemical, enzymatic, and two-dimensional NMR analyses demonstrate that the repeating unit of PS-2 is [----4)D-glucuronate(beta 1----2)meso-tartrate(3----1)glyoxylate(1-]n. Thus PS-2 has a high density of negatively charged groups available for calcium ion binding, similar to the phosphoprotein polyanions of other species. Polysaccharides containing tartrate and/or glyoxylate have not been previously described; these residues may be introduced into PS-2 by a postpolymerization process involving oxidative cleavage of glucuronate or mannuronate residues.

The novel deoxyribonucleotide alpha-[d(CpCpTpTpCpC)] and its complement beta-[d(GpGpApApGpG)] were synthesized by the phosphotriester method. 1H-NMR-NOE examination of the alpha-hexamer revealed that the cytosine and thymine bases appear to adopt anti conformations in this strand. In addition the deoxyribose of the thymidine moieties may adopt average conformations approximating to C3'-endo while the cytidine furanose groups are close to C2'-endo conformations. Both hyperchromicity in thermal melting and detection of base paired imino protons in 1H-NMR studies in H2O provide evidence for the annealing of alpha-d[CCTTCC] with its complement beta-d[GGAAGG] in potassium phosphate buffer pH 7.1 containing 10 mM magnesium chloride. Under these conditions thermal melting begins at 38 degrees C and its complete at approximately 45 degrees C. NOE experiments do not permit a decision on the polarity of annealing (predicted to be parallel) for this particular pair of sequences.

The complementary consensus acceptor exon:intron junction d(ApCpCpTpGpTpApG) has been synthesized by a modified phosphotriester method. The non self-complementary octamer exists in the random coil form in aqueous buffer at 20 degrees C as evidenced by temperature variable 1H-NMR and NOE measurements. The non-exchangeable proton assignments were secured using a combination of techniques including two-dimensional COSY, NOESY and 1H-1H-INADEQUATE. The octamer was annealed with the primary consensus sequence d(CpTpApCpApGpGpT). Confirmation of complete duplex formation was confirmed by detection and assignment of imino protons in D2O:H2O mixtures. Assignment of the non-exchangeable proton signals in the duplex consensus junction was then secured by a combination of two-dimensional COSY correlations, NOESY and NOE experiments. Determination of individual vicinal coupling constants in the component deoxyribose moieties permitted deduction of the population of S conformations in this sequence. It is concluded that the consensus acceptor junction exists in solution in a conformation belonging to the B family, and that the bases are oriented anti. In addition the deoxyribose moieties in the 5' regions exist predominantly in the S form (2'endo-3'exo) whereas those residues on or adjacent to the junction on the primary strand show more N character (2'exo-3'endo). The contiguous bases A5-G6 (adjacent to the junction) and A15-G16 are stacked more closely than the other neighbor bases in this duplex sequence. These subtle structural and conformational differences in the exon:intron junction may serve as recognition signals for these critical sites in the genome.

The consensus acceptor exon:intron junction d(CpTpApCpApGpGpT) has been synthesized by a modified phosphotriester method. The non-self complementary octamer exists in the single strand form in aqueous buffer at 20 degrees C as evidenced by temperature variable 1H-NMR and NOE measurements. The non-exchangeable proton assignments were secured using a combination of techniques including two-dimensional COSY, NOESY and the double quantum technique 1H-1H-INADEQUATE as well as inversion recovery T1 experiments. The new technique of 31P-1H shift correlation is particularly valuable in removing certain ambiguities in the sugar proton assignments. Characteristic chemical shifts for the base protons which are determined by their immediate molecular environments are also useful in assignments. The consensus acceptor exon:intron junction adopts a random coil conformation in solution under the experimental conditions employed.

Structural and dynamic properties of the self-complementary decadeoxyribonucleotide d(CGCAATTGCG)2 and the interaction between a prototype lexitropsin, or information-reading oligopeptide, and the decadeoxyribonucleotide are deduced by using high-resolution 1H NMR techniques. The nonexchangeable and imino proton resonances of d(CGCAATTGCG)2 have been completely assigned by two-dimensional NMR studies. The decadeoxyribonucleotide exists as a right-handed B-DNA. In the 1H NMR spectrum of the 1:1 complex, the selective chemical shifts and removal of degeneracy of AH2(4), AH2(5), T-CH3(6), and T-CH3(7) due to the anisotropy effects of the heterocyclic moieties of the ligand, and with lesser effects at the flanking base sites C(3) and G(8), locate the drug centrally in the decadeoxyribonucleotide. This conclusion is supported by plots of individual chemical shift changes across the decadeoxyribonucleotide. Similarly, imino protons IV and V experience larger shifts and II and III smaller shifts in accord with this conclusion while drug complexation permits the detection of imino proton I. Strong nuclear Overhauser effects (NOEs) between pyrrole H5 and AH2(5), and weaker NOEs to AH1'(5), TH3'(6), and AH2'(5), firmly locate the ligand in the minor groove. Intraligand NOEs between the adjacent heterocyclic moieties indicate that the lexitropsin is subject to propeller twisting about the N6-C9 bond in both the bound and free forms. Nuclear Overhauser effect spectroscopy (NOESY) and correlated spectroscopy (COSY) experiments also indicate that the removal of degeneracy of the C16 methylene protons upon complexation may arise from restricted rotation about the C15-N9, C15-C16, and C16-C17 bonds. Specific hydrogen bonds between amide NH groups on the concave face of the ligand (N4H, N6H, N9H) and adenine N3 or thymine O2 on the floor of the minor groove are in accord with displacement of the hydration shell by the drug. NOE measurements on the decadeoxyribonucleotide in the 1:1 complex confirm it exists as a right-handed helix and belongs to the B family. Exchange NMR effects permit an estimate of a rate of approximately equal to 44 s-1 for the two-site exchange of the lexitropsin between two equivalent sites on the decamer with delta G++ approximately equal to 70 +/- 5 kJ mol-1 at 294 K. Alternative mechanisms for this exchange process are considered.

The beta-complementary hexamer, beta-d[GTACGC], to the alpha-sequence, alpha-d[CATGCG], was synthesized by the phosphotriester method. The non-exchangeable proton assignments were obtained using 1D- and 2D-NMR techniques, including NOE, COSY and NOESY. The beta-strand exists as a random coil at 21 degrees C; however, at 4 degrees C, it forms an antiparallel self-recognition duplex annealing at positions 1-4. The beta-strand was annealed to the alpha-strand, and confirmation of complete annealing was obtained by detection and assignment of the six base pair imino protons in H2O/D2O solution at 21 degrees C. 1D-NOE experiments of the alpha, beta duplex d[alpha-(CATGCG) X beta-(GTACGC)] reveal that (i) it exists in aqueous solution in a conformation that belongs to the B family, (ii) it is 70 +/- 10% right-handed, (iii) the sugar-base orientations of the beta-strand are anti, and the deoxyribose units exist predominantly in the 2'-endo-3'-exo conformation. NOE measurements of the imino proton signals in the alpha, beta duplex reveal that the duplex exhibits parallel polarity.