The effect of amino acid substitution on protein-folding and -unfolding transition studied by computer simulation

Protein-folding and -unfolding transitions were studied by the method of computer simulation. The protein was modeled as a two-dimensional lattice polymer. Various energy terms were assumed to be operative between units composing the polymer. But hydrophobic interactions were neglected explicitly. Both thermodynamic and kinetic quantities were obtained from the simulation, and from their temperature dependence in the transition zone characteristics of the conformational transition of proteins were discussed. Two amino acid substituted models, differing in the location of substitution, were studied and compared with the original in order to clarify the effect of substitution on conformational transition of proteins. The following conclusions were reached in this study: (1) The relaxation time of the slow mode, which reflects the overall folding and unfolding processes, shows a peak near the transition temperature, while that of the fast mode is almost independent of temperature. The peak of the slow mode occurs at a slightly lower temperature than the transition temperature. (2) The dependence of the logarithm of the rate constants on the inverse of temperature (Arrhenius plot) is linear. Therefore, the plot of the free energy of activation vs temperature is linear. (3) The values of kinetic parameters obtained suggest that in the activated state the intramolecular interactions are half broken, while the state is close to the native state on the entropy axis. (4) The amino acid substitution, which is modeled as having slightly unfavorable short-range interactions, causes the substituted ones to be slightly unstable. Moreover, it causes the folding transition to slow. From the analysis of the way slowing down is observed in the two substituted models, we conclude that a structure, designed to model a β-sheet, is formed before it gets assembled with other structures, which are designed to model α-helices. The process of assembly occurs nearly at the activated state of the folding and unfolding transition. (5) It is suggested from this study that the maximum of folding rate constant in the Arrhenius plot that has been observed experimentally in real proteins is likely due to hydrophobic interactions.     

Effect of an inhibitor of glucosylceramide synthesis on cultured rabbit skin fibroblasts

1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an effective inhibitor of UDP-glucose:ceramide glucosyltransferase, caused growth inhibition of cultured rabbit skin fibroblasts in a dose-dependent manner. At 50 microM both threo and erythro isomers of PDMP completely suppressed the cell growth. Major gangliosides of the fibroblasts, GM3 and GD3, were greatly reduced in amounts in the presence of threo-PDMP and accumulation of ceramides was observed. Surface labeling with galactose oxidase and [3H]NaBH4 demonstrated that neural glycosphingolipids with four or more sugars present on the surface of control cells were not detectable when the fibroblasts were grown in medium containing threo-PDMP. Metabolic labeling of cellular glycosphingolipids with [14C]-galactose showed reduced incorporation of radioactivity into gangliosides and neutral glycosphingolipids when threo-PDMP was present in the medium. In contrast, the erythro isomer of PDMP did not affect the biosynthesis of glycosphingolipids, a result suggesting that the inhibitory effect of erythro-PDMP on cell growth was due to a mechanism other than the inhibition of glucosyltransferase.     

Mucin-hypersecreting bile duct neoplasm characterized by clinicopathological resemblance to intraductal papillary mucinous neoplasm (IPMN) of the pancreas

Background

Ultrasound-guided vacuum-assisted breast biopsy technology is extremely useful for diagnostic biopsy of suspicious breast lesions and for attempted complete excision of appropriately selected presumed benign breast lesions.

Case presentation

A female patient presented with 16 breast lesions (eight within each breast), documented on ultrasound and all presumed to be fibroadenomas. Over a ten and one-half month period of time, 14 of these 16 breast lesions were removed under ultrasound guidance during a total of 11 separate 8-gauge Mammotome^® excision procedures performed during seven separate sessions. Additionally, two of these 16 breast lesions were removed by open surgical excision. A histopathologic diagnosis of fibroadenoma and/or fibroadenomatous changes was confirmed at all lesion excision sites. Interval follow-up ultrasound imaging revealed no evidence of a residual lesion at the site of any of the 16 original breast lesions.

Conclusion

This report describes an innovative approach of utilizing ultrasound-guided 8-gauge vacuum-assisted breast biopsy technology for assisting in achieving complete eradication of multiple bilateral fibroadenomas in a patient who presented with 16 documented breast lesions. As such, this innovative approach is highly recommended in similar appropriately selected patients.     

Reduced snow cover changes nitrogen use in canopy and understory vegetation during the subsequent growing season

Plant and Soil - We examined how mechanical management of invasive macrophyte, Typha × glauca alters plant-soil interactions underlying carbon processes and nutrient cycling, which are...     

Suppression of lysophosphatidic acid and lysophosphatidylcholine formation in the plasma in vitro: proposal of a plasma sample preparation method for laboratory testing of these lipids

It is now established that lysophosphatidic acid (LPA) and lysophosphatidylcholine (LPC) play important roles in a variety of biological responses, especially in the area of vascular biology, and determination of their concentrations in the plasma is believed to be clinically relevant. Preparation of the measurement samples is a difficult task, however, because the blood levels of these lipids can be easily increased by in vitro manipulation after venepuncture. In this study, we examined the optimal conditions for the preparation of plasma samples for the measurement of LPA and LPC. It appears that regulation of platelet activation and the enzymatic activity of lysophospholipase D/autotaxin and lecithin-cholesterol acyltransferase is important to suppress the undesirable formation of LPA and LPC after venepuncture. We found that in vitro formation of LPA and LPC was negligible when whole blood samples were mixed with 7.5 mM EDTA plus 10% (v/v) citrate-theophylline-adenosine-dipyridamole (CTAD) and when all of the procedures, including the plasma preparation and preservation until measurement, were performed at 4 degrees C. Thus, although the plasma levels of LPA and LPC can be easily altered, laboratory testing of these important bioactive lipids for clinical purposes may be conducted reliably if the samples are prepared under stringent conditions.     

Development of internal standard for lipoprotein subclass analysis using dual detection gel-permeation high-performance liquid chromatography system

The LipoSEARCH® System is an innovative lipoprotein class analysis method based on gel-permeation high-performance liquid chromatography (HPLC). This system uses a gel permeation column to separate the major lipoprotein subclasses (chylomicron, very low-density lipoprotein, low-density lipoprotein, and high-density lipoprotein) in serum according to particle size and splits them into two pathways to measure total cholesterol (TC; esterified + unesterified cholesterol) and triglyceride (TG) concentrations simultaneously to obtain chromatograms for each.These chromatograms were analyzed based on the results of the calibration serum by fitting Gaussian curves to profile the 20 lipoprotein subclasses defined in detail. An important assumption of this HPLC system is its simultaneous detection of two pathways to guarantee the accuracy of each analysis. Therefore, in the present study, we investigated the development of an internal standard that can guarantee the simultaneous detection of this system by adding a pigment to the serum. We focused on quinone pigments with absorption at 550 nm, which is the wavelength used for the enzymatic assay of TC and TG concentrations in the system. As a result, we succeeded in producing overlapping pigment peaks that appeared after the analytical chromatograms in two pathways. It is also suggested that the pigment solution as an internal standard is stable in freezing storage and has little effect on the analysis. The developed internal standard is expected to contribute to the accuracy assurance of lipoprotein analysis by this dual-detection HPLC system.     

Autotaxin–lysophosphatidic acid–LPA3 signaling at the embryo‐epithelial boundary controls decidualization pathways

During pregnancy, up‐regulation of heparin‐binding (HB‐) EGF and cyclooxygenase‐2 (COX‐2) in the uterine epithelium contributes to decidualization, a series of uterine morphological changes required for placental formation and fetal development. Here, we report a key role for the lipid mediator lysophosphatidic acid (LPA) in decidualization, acting through its G‐protein‐coupled receptor LPA₃ in the uterine epithelium. Knockout of Lpar3 or inhibition of the LPA‐producing enzyme autotaxin (ATX) in pregnant mice leads to HB‐EGF and COX‐2 down‐regulation near embryos and attenuates decidual reactions. Conversely, selective pharmacological activation of LPA₃ induces decidualization via up‐regulation of HB‐EGF and COX‐2. ATX and its substrate lysophosphatidylcholine can be detected in the uterine epithelium and in pre‐implantation‐stage embryos, respectively. Our results indicate that ATX–LPA–LPA₃ signaling at the embryo‐epithelial boundary induces decidualization via the canonical HB‐EGF and COX‐2 pathways.     

A short-term,simple method for detection of N-nitrosocompounds produced from sodium nitrite and morpholine in stomach

Syrian hamsters in the 11th or 12th day of pregnancy were given sodium nitrite and morpholine simultaneously by stomach tube. The embryonic cells were cultured for 72 h in normal MEM medium plus 10% fetal calf serum and then transferred into medium containing 8-azaguanine. After cultivation in the selection medium, number of 8 azaguanine-resistant colonies was scored. As the results, this oral concurrent transplacental application of sodium nitrite and morpholine can cause 8 azaguanine-resistant mutants on the cultured embryonic cells from mothers that received these chemicals. Nitrosomorpholine was only detected in stomach of animals treated with sodium nitrite and morpholine.