Characterization of a trifluoroethanol-induced partially folded state of alpha-lactalbumin

The protein alpha-lactalbumin exists in a partially folded molten globule state at pH 2.0, the A state. This state is believed to be compact, possessing a similar amount of secondary structure to the native state but having a flexible tertiary structure comprised mainly of non-specific hydrophobic clustering of residues. Addition of trifluoroethanol (TFE) to bovine, human and guinea pig alpha-lactalbumin at pH 2.0 has been found in each case to induce a conformational transition in the A state as monitored by circular dichroism, nuclear magnetic resonance chemical shifts, and 1-anilinonaphthalene-8-sulphonate binding. The mid-point of this transition is near 15% (v/v) TFE and is effectively complete by 50% (v/v) TFE at 315 K. Far ultraviolet circular dichroism ellipticities at 208 nm and 220 nm, usually taken as a measure of the degree of helical character, are substantially more negative in the TFE state than in the A state. Furthermore, backbone amide protons protected from solvent exchange in the A state are generally at least as strongly protected in the TFE state; patterns of protection appear similar in the two states and include at least part of both the B and C alpha-helices. One major difference from the A state is nevertheless evident: the ability to bind the fluorescent probe 1-anilinonaphthalene-8-sulphonate, characteristic of molten globule states, is lost in the TFE state. Like the A state, the TFE state of alpha-lactalbumin shows little chemical shift dispersion of side-chain resonances. Extensive line broadening in the nuclear magnetic resonance spectra, characteristic of slow conformational averaging in the A state, is, however, much reduced in the TFE state. The line narrowing observed in the TFE state has made it possible to obtain directly sequence-specific assignments for about 25% of the 123 residues of bovine alpha-lactalbumin in 50% (v/v) TFE. Two helices are amongst regions of structure so far identified from short-range backbone nuclear Overhauser enhancement (NOE) connectivities in two-dimensional spectra of the TFE state. One of the helices (residues 86 to 96) corresponds to the C-helix in the native structure. The other (residues 35 to 41) corresponds, however, to a region of the sequence that is not helical in the native state. The partially folded state of alpha-lactalbumin formed in TFE, therefore, supports both native and non-native secondary structure in the absence of persistent long-range tertiary structure.     

(R)-(-)- and (S)-(+)-Synadenol: synthesis, absolute configuration, and enantioselectivity of antiviral effect

Synthesis of (R)-(-)- and (S)-(+)-synadenol (1a and 2a, 95-96% ee) is described. Racemic synadenol (1a + 2a) was deaminated with adenosine deaminase to give (R)-(-)-synadenol (1a) and (S)-(+)-hypoxanthine derivative 5. Acetylation of the latter compound gave acetate 6. Reaction with N, N-dimethylchloromethyleneammonium chloride led to 6-chloropurine derivative 7. Ammonolysis furnished (S)-(+)-synadenol (2a). Absolute configuration of 1a was established by two methods: (i) synthesis from (R)-methylenecyclopropanecarboxylic acid (8) and (ii) X-ray diffraction of a single crystal of (-)-synadenol hydrochloride. Racemic methylenecyclopropanecarboxylic acid (10) was resolved by a modification of the described procedure. The R-enantiomer 8 was converted to ethyl ester 13 which was brominated to give vicinal dibromides 14. Reduction with diisobutylaluminum hydride then furnished alcohol 15 which was acetylated to the corresponding acetate 16. Alkylation-elimination procedure of adenine with 16 yielded acetates 17 and 18. Deprotection with ammonia afforded a mixture of Z- and E-isomers 1a and 19 of the R-configuration. Comparison with products 1a and 2a by chiral HPLC established the R-configuration of (-)-synadenol (1a). These results were confirmed by X-ray diffraction of a single crystal of (-)-synadenol hydrochloride. The latter forms a pseudosymmetric dimer with adenine-adenine base pairing in the lattice with the nucleobase in an anti-like conformation. Enantiomers 1a and 2a exhibit varied enantioselectivity toward different viruses. Both enantiomers are equipotent against human cytomegalovirus (HCMV) and varicella zoster virus (VZV). The S-enantiomer 2a is somewhat more effective than R-enantiomer 1a in herpes simplex virus 1 and 2 (HSV-1 and HSV-2) assays. By contrast, enantioselectivity of antiviral effect is reversed in Epstein-Barr virus (EBV) and human immunodeficiency virus type 1 (HIV-1) assays where the R-enantiomer 1a is preferred. In these assays, the S-enantiomer 2a is less effective (EBV) or devoid of activity (HIV-1).     

alpha-Methylene-gamma-butyrolactones: synthesis and vasorelaxing activity assay of coumarin, naphthalene, and quinoline derivatives

Certain alpha-methylene-gamma-butyrolactone derivatives of coumarin, naphthalene, and quinoline were synthesized and evaluated for vasorelaxing effects on isolated rat thoracic aorta. The 7-[(2,3,4,5-tetrahydro-2-methyl-4-methylene-5-oxo-2-furanyl)methoxy]-2H- 1- benzopyran-2-ones, which have an aliphatic methyl substituent at the lactone C2, were more active than their C2-phenyl counterparts against high-K+ (80 mM) medium, Ca2+ (1.9 mM)-induced vasoconstriction and the norepinephrine (NE, 3 microM)-induced phasic and tonic constrictions (2a vs. 2b; 2c vs. 2d; 2e vs. 2f; 2g vs. 2h). Although 3-chloro-7-[(2,3,4,5-tetrahydro-2-methyl-4-methylene-5-oxo-2- furanyl)methoxy]-4-methyl-2H-1-benzopyran-2-one (2g) demonstrated the most potent inhibitory activities on the NE-induced phasic and tonic constrictions at concentrations of as low as 10 micrograms/ml, it possesses both affinity for NE-receptor and intrinsic activity to trigger the vasoconstriction. However, 8-[(2,3,4,5-tetrahydro-2-methyl-4-methylene-5-oxo-2- furanyl)methoxy]quinoline (10a) and other quinoline derivatives (11a, 12a) are pure irreversible non-competitive blockers of NE-receptor with no intrinsic activity. The aromatic ring played an important role in the vasorelaxing effects of alpha-methylene-gamma-butyrolactones; naphthalene was inactive, quinolines exhibited only affinity to the alpha-receptor, and coumarins possessed both affinity and intrinsic activity.     

Establishment of an optimal hypo-osmotic swelling test by examining single spermatozoa in four different hypo-osmotic solutions

In order to find an optimal hypo-osmotic swelling test (HOST) and to identify viable sperm cells from patients with asthenozoospermia for intracytoplasmic sperm injection (ICSI), we tested single motile and non-motile spermatozoa in four hypo-osmotic solutions by micromanipulation. The four solutions were: A, H2O; B, 50 mOsm NaCl; C, 150 mOsm NaCl and D, 150 mOsm sodium citrate and fructose. Eosin Y staining was then carried out for evaluation of viability of the spermatozoa after HOST. Using motile spermatozoa, no statistical difference was found in HOST-positive spermatozoa between these four solutions. There were more viable sperm cells after HOST in solutions C and D, as noted by Eosin Y staining. After non-motile spermatozoa were incubated for 1 min in the four solutions, HOST with solution C gave the best results for identification of viable sperm cells compared to the other three solutions. When motile spermatozoa were incubated in solution C or solution D for 30 min, the result of HOST with solution C (10.8% dead spermatozoa) was superior to that of solution D (49.1% dead spermatozoa). In conclusion, the HOST protocol using 150 mOsm NaCl (solution C) for 1 min yielded the best results for selection of viable spermatozoa. This procedure should be used for selection of viable spermatozoa for ICSI in patients with 100% non-motile spermatozoa.     

Tissue-engineered nipple reconstruction

We describe a simple, effective approach to the creation of autologous tissue-engineered cartilage in the shape of a human nipple by injecting a reverse thermosensitive polymer seeded with autologous chondrocytes in an immunocompetent porcine animal model. A biodegradable, biocompatible copolymer of polyethylene oxide and polypropylene oxide (Pluronic F-127), which exists as a liquid below 4 degrees C and polymerizes to a thick gel when it is exposed to physiologic temperatures (body temperatures), was used as a vehicle for chondrocyte delivery and as a scaffold to guide growth. Autologous chondrocytes isolated from porcine auricular elastic cartilage and suspended in 30% (weight/volume) Pluronic F-127 were injected on the ventral surface of the pigs from which the cells had been isolated. A circumferential subdermal suture was used to support the contour of the implant and assist in its projection in the form of a human nipple. After 3 weeks, the skin over and surrounding the implant was tattooed to create the appearance of a human nipple-areolar complex. As controls, an equal number of injections were made using either cells alone (not suspended in hydrogel), or hydrogel alone. After 10 weeks, all specimens were excised and examined both grossly and histologically. Before harvesting, visual inspection of the tattooed chondrocyte-Pluronic F-127 hydrogel implant sites revealed that they closely resembled a human female nipple-areolar complex. Nodules were similar in size, shape, and texture to a human nipple at each injection site. Glistening opalescent tissue was surgically isolated from each implant site. Hematoxylin and eosin, safranine o, trichrome blue, and Verhoeff's stains of the experimental implants showed nodules with the characteristic histologic signs of elastic cartilage. Control injections of copolymer hydrogel alone exhibited no evidence of cartilage formation. Control injections of chondrocytes alone showed evidence of dissociated microscopic nodules of elastic cartilage.     

Relationship between endothelial cell turnover and permeability to horseradish peroxidase

1. The atrioventricular node (AVN) is an important part of the conduction system in the heart and is a significant site of antiarrhythmic drug action. The class 1 antiarrhythmic propafenone is effective in treating a variety of arrhythmias, including those involving the AVN. In this study, we have investigated the effects of propafenone on ionic currents in single rabbit AVN cells, focusing in particular on those on L-type calcium current (ICa,L). 2. With a standard K-based internal dialysis solution, exposure to 5 microM propafenone reduced significantly the amplitude of ICa,L. In spontaneously active AVN myocytes, action potential upstroke velocity was decreased by propafenone exposure, consistent with the observed change in ICa,L. 3. By use of a Cs-based internal dialysis solution to record ICa,L selectively, voltage clamp test pulses were applied from a holding potential of -40 mV to +10 mV (stimulation frequency 0.33 Hz). Propafenone 5 microM reduced mean ICa,L density at +10 mV from -9.58 +/- 1.05 pA/pF to -4.19 +/- 0.60 pA/pF (P < 0.002). A range of propafenone concentrations were applied which reduced ICa,L in a dose-dependent manner (IC50 1.7 microM). When test pulses were applied to a range of potentials, propafenone reduced ICa,L at each potential without significantly affecting the activation curve for this current. Thus, propafenone reduced ICa,L conductance, without affecting the voltage-dependent activation properties of the current. 4. ICa,L block by propafenone exhibited tonic-, use- and frequency-dependent characteristics. 5. In the presence of propafenone, the voltage-dependence of inactivation of ICa,L was shifted 8 mV in the hyperpolarizing direction. Also, the recovery of ICa,L from inactivation was slowed by propafenone. 6. The ICa,L blocking properties of propafenone may mediate some of the antiarrhythmic properties of this agent, particularly in regions of the heart such as the AVN in which ICa,L contributes significantly to the action potential upstroke.     

The effect of education on mortality among older Taiwanese and its pathways

The present research examines the impact of education on the mortality of older Taiwanese during a 4-year interval from April 1989 to April 1993. Data used for this study come from the Taiwan Survey of Health & Living Status of the Elderly (1989). The research decomposes the effect of education into the direct effect and the indirect effects by means of health status, health behaviors, and social relationships. We have shown that, of the total effect of educational attainment on the mortality of older Taiwanese, about 83% represents indirect influences by means of the 3 mediating factors, particularly health status. On the other hand, the magnitude of the direct effect, which might reflect influences of additional intervening variables on old-age mortality, is low and not statistically significant. The results demonstrate that the apparent strong effect of education on mortality among older Taiwanese can be accounted for parsimoniously through 3 major pathways.     

Effect of electroacupuncture on gastric acid secretion and gut hormones

21 patients with mild type of chronic superficial gastritis were selected in this study. The effect of electroacupuncture in Zhongwan (RM12), Neiguan (P6) and Sanyinjiao (Sp6) on gastric acid secretion, serum gastrin, plasma somatostatin, plasma motilin concentration and erythrocyte acetylcholinesterase (AchE) activity were observed. The results were as follows: There were significant decreases in gastric acid output, serum gastrin concentration and AchE activity (P < 0.05), but no significant changes in plasma somatostatin and motilin concentration (P > 0.05) after simultaneous electroacupuncture in Zhongwan, Neiguan and Sanyinjiao.     

Influence of panel-reactive antibody on survival and rejection after lung transplantation

BACKGROUND: Panel-reactive antibody (PRA) is commonly used before thoracic organ transplantation to estimate a potential recipient's degree of humoral sensitization. METHODS: To assess the influence of an elevated PRA on survival and the incidence of rejection in pulmonary transplantation, the records of 247 patients that underwent single or double lung transplantation were reviewed. RESULTS: Twenty-one of 247 patients (8.5%) had PRA values greater than 10%. Survival of this population was not significantly different from that of patients with low PRA levels: 74% (low PRA) vs 65% (elevated PRA) at 1 year and 58% in both groups at 3 years. The acute rejection rates (episodes/first 100 days) for the elevated and low PRA groups were 2.1 and 1.9, respectively (p = NS). Obliterative bronchiolitis developed in 38.9% of the high and 31.2% of the low PRA groups (p = NS). Six of 247 patients had a retrospective positive lymphocytotoxic cross-match result; three had PRA values greater than 10%. Patients with a positive cross-match result experienced similar survival and incidence of rejection as the remainder of the population. Among 957 patients evaluated for lung transplantation, 16 (1.7%) had a PRA (with dithiothreitol) greater than 15%. All had a history of pregnancy, blood transfusion, connective tissue disease, or previous transplantation. CONCLUSIONS: Humoral sensitization is uncommon in the lung transplantation population. A modestly elevated PRA does not predict survival or the development of acute rejection or bronchiolitis obliterans. PRA testing before lung transplantation should be reserved for those patients with specific risk factors for humoral sensitization.     

Re-expression of the alpha 2 beta 1 integrin abrogates the malignant phenotype of breast carcinoma cells

To assess the role of altered alpha 2 beta 1 integrin expression in breast cancer, we expressed the alpha 2 beta 1 integrin de novo in a poorly differentiated mammary carcinoma that expressed no detectable alpha 2-integrin subunit. Expression of the alpha 2 beta 1 integrin resulted in a dramatic phenotypic alteration from a fibroblastoid, spindle-shaped, non-contact-inhibited, motile, and invasive cell to an epithelioid, polygonal-shaped, contact-inhibited, less motile, and less invasive cell. Although expression of the alpha 2 subunit did not alter adhesion to collagen, it profoundly altered cell spreading. Re-expression of the alpha 2 beta 1 integrin restored the ability to differentiate into gland-like structures in three-dimensional matrices and markedly reduced the in vivo tumorigenicity of the cells. These results indicate that the consequences of diminished alpha 2 beta 1-integrin expression in the development of breast cancer and, presumably, of other epithelial malignancies are increased tumorigenicity and loss of the differentiated epithelial phenotype.