Dynamic light scattering and circular dichroism studies on heat-induced gelation of hard-keratin protein aqueous solutions

Animal hairs consist of aggregates of dead cells filled with keratin protein gel. We succeeded in preparing water-soluble hard-keratin proteins and reconstructing the keratin gels by heat-induced disulfide linkages in vitro. Here, the roles of intermolecular hydrophobic interaction and disulfide bonding between the proteins in the gel were discussed. Water-soluble keratin proteins consisting of mixtures of type I ( approximately 48 kDa) and type II ( approximately 61 kDa) were prepared from wool fibers as S-carboxymethyl alanyl disulfide keratin (CMADK). The gelation was achieved by heating an aqueous solution containing at least 0.8 wt % CMADK at 100 degrees C. CMADK solutions with different urea or N-ethylmaleimide concentrations or pH were exposed to dynamic light scattering (DLS) and circular dichroism (CD). DLS clarified the gelation point of CMADK solutions and provided information on the changes in keratin cluster size. DLS suggested two types of gelation mechanism. One was the regenerated chemical disulfide bonding between keratins from CMAD parts of chains. After the gel formed, this bond became important to maintain the gel structure. The other was the physical assembly due to hydrophobic interaction between alpha-helix parts of keratin chains. This hydrophobic assembly also played an important role during gelation. CD confirmed a conformational change in the keratin protein, resulting heat-induced gelation. CD clarified the relationship between keratin protein conformation and gelation, i.e., a rodlike conformation with many alpha-helix structures was necessary to associate keratin chains and form a gel network.     

Reducing salt intake for prevention of cardiovascular diseases in high-risk patients by advanced health education intervention (RESIP-CVD study), Northern Thailand: study protocol for a cluster randomized trial

ABSTRACT: BACKGROUND: Decreasing salt consumption can prevent cardiovascular diseases (CVD). Practically, it is difficult to promote people's awareness of daily salt intake and to change their eating habits in terms of reducing salt intake for better cardiovascular health. Health education programs visualizing daily dietary salt content and intake may promote lifestyle changes in patients at high risk of cardiovascular disease. METHODS: This is a cluster randomized trial. A total of 800 high-CVD-risk patients attending diabetes and hypertension clinics at health centers in Muang District, Chiang Rai province, Thailand, will be studied with informed consent. A health center recruiting 100 participants is a cluster, the unit of randomization. Eight clusters will be randomized into intervention and control arms and followed up for 1 year. Within the intervention clusters the following will be undertaken: (1) salt content in the daily diet will be measured and shown to study participants; (2) 24-hour salt intake will be estimated in overnight-collected urine and the results shown to the participants; (3) a dietician will assist small group health education classes in cooking meals with less salt. The primary outcome is blood pressure change at the 1-year follow-up. Secondary outcomes at the 1-year follow-up are estimated 24-hoursalt intake, incidence of CVD events and CVD death. The intention-to-treat analysis will be followed.Blood pressure and estimated 24-hour salt intake will be compared between intervention and control groups at the cluster and individual level at the 1-year follow-up. Clinical CVD events and deaths will be analyzed by time-event analysis. Retinal blood vessel calibers of CVD-risk patients will be assessed cross-sectionally. Behavioral change to reduce salt intake and the influencing factors will be determined by structured equation model (SEM). Multilevel regression analyses will be applied. Finally, the cost effectiveness of the intervention will be analyzed. DISCUSSION: This study is unique as it will recruit the individuals most vulnerable to CVD morbidity and mortality by applying the general Framingham CVD risk scoring system. Dietary salt reduction will be applied as a prioritized, community level intervention for the prevention of CVD in a developing country.Trial registrationISRCTN39416277.     

Immobilization ofβ-fructofuranosidase fromAureobasidium on DEAE-cellulose

Summary -Fructofuranosidase, which produces fructo-oligosaccharides (1-kestose and nystose) from sucrose, was purified fromAureobasidium and immobilized on DEAE-cellulose at especially high efficiency (95%). The enzymatic profiles of the immobilized enzyme were almost identical to those of the native form except that the stability was slightly improved. The immobilized enzyme was stable during long-term continuous reaction for up to 360 h.     

Exhaustive crystal structure search and crystal modeling of beta-chitin

An exhaustive search of the crystal structure of beta-chitin was carried out by simultaneously optimizing all the structural parameters based on published X-ray diffraction data and stereochemical criteria. The most probable structure was characterized by a parallel-up chain polarity, a gg orientation of hydroxymethyl groups and an intermolecular hydrogen bond along the a-axis, which essentially reproduced the original structure proposed by Gardner and Blackwell. The proposed crystal structure was subsequently subjected to crystal modeling using the AMBER force field. The probable orientation of hydroxyl groups and their motional behaviors is proposed based on calculations for the crystal models identified. Solvated crystal models exhibited a slightly deformed structure with the formation of appreciable numbers of hydrogen bonds along the b-axis.     

Effects of Candesartan on Electrical Remodeling in the Hearts of Inherited Dilated Cardiomyopathy Model Mice

Inherited dilated cardiomyopathy (DCM) is characterized by dilatation and dysfunction of the ventricles, and often results in sudden death or heart failure (HF). Although angiotensin receptor blockers (ARBs) have been used for the treatment of HF, little is known about the effects on postulated electrical remodeling that occurs in inherited DCM. The aim of this study was to examine the effects of candesartan, one of the ARBs, on cardiac function and electrical remodeling in the hearts of inherited DCM model mice (TNNT2 ΔK210). DCM mice were treated with candesartan in drinking water for 2 months from 1 month of age. Control, non-treated DCM mice showed an enlargement of the heart with prolongation of QRS and QT intervals, and died at t_1/2 of 70 days. Candesartan dramatically extended the lifespan of DCM mice, suppressed cardiac dilatation, and improved the functional parameters of the myocardium. It also greatly suppressed prolongation of QRS and QT intervals and action potential duration (APD) in the left ventricular myocardium and occurrence of ventricular arrhythmia. Expression analysis revealed that down-regulation of Kv4.2 (I_to channel protein), KChIP2 (auxiliary subunit of Kv4.2), and Kv1.5 (I_Kur channel protein) in DCM was partially reversed by candesartan administration. Interestingly, non-treated DCM heart had both normal-sized myocytes with moderately decreased I_to and I_Kur and enlarged cells with greatly reduced K⁺ currents (I_to, I_Kur I_K1 and I_ss). Treatment with candesartan completely abrogated the emergence of the enlarged cells but did not reverse the I_to, and I_Kur in normal-sized cells in DCM hearts. Our results indicate that candesartan treatment suppresses structural remodeling to prevent severe electrical remodeling in inherited DCM.     

TRPC3-mediated Ca2+ influx contributes to Rac1-mediated production of reactive oxygen species in MLP-deficient mouse hearts

Obesity-related metabolic abnormalities, including chronic inflammation and oxidative stress, increase the risk of colorectal cancer. Dysregulation of the renin–angiotensin system (RAS) also plays a critical role in obesity-related metabolic disorders and in several types of carcinogenesis. In the present study, we examined the effects of an angiotensin-converting enzyme (ACE) inhibitor and angiotensin-II type 1 receptor blocker (ARB), both of which inhibit the RAS, on the development of azoxymethane (AOM)-initiated colonic premalignant lesions in C57BL/KsJ-db/db (db/db) obese mice. Male db/db mice were given 4 weekly subcutaneous injections of AOM (15 mg/kg body weight), and then, they received drinking water containing captopril (ACE inhibitor, 5 mg/kg/day) or telmisartan (ARB, 5 mg/kg/day) for 7 weeks. At sacrifice, administration of either captopril or telmisartan significantly reduced the total number of colonic premalignant lesions, i.e., aberrant crypt foci and β-catenin accumulated crypts, compared to that observed in the control group. The expression levels of TNF-α mRNA in the colonic mucosa of AOM-treated db/db mice were decreased by captopril and telmisartan. Captopril lowered the expression levels of TNF-α, IL-1β, IL-6, and PAI-1 mRNAs, while telmisartan lowered the expression levels of COX-2, IL-1β, IL-6, and PAI-1 mRNAs in the white adipose tissues of these mice. In addition, these agents significantly reduced the levels of urinary 8-OHdG, a surrogate marker of oxidative damage to DNA, in the experimental mice. These findings suggested that both ACE inhibitor and ARB suppress chemically-induced colon carcinogenesis by attenuating chronic inflammation and reducing oxidative stress in obese mice. Therefore, targeting dysregulation of the RAS might be an effective strategy for chemoprevention of colorectal carcinogenesis in obese individuals.     

Drastic Ca2+ sensitization of myofilament associated with a small structural change in troponin I in inherited restrictive cardiomyopathy

Six missense mutations in human cardiac troponin I (cTnI) were recently found to cause restrictive cardiomyopathy (RCM). We have bacterially expressed and purified these human cTnI mutants and examined their functional and structural consequences. Inserting the human cTnI into skinned cardiac muscle fibers showed that these mutations had much greater Ca2+-sensitizing effects on force generation than the cTnI mutations in hypertrophic cardiomyopathy (HCM). The mutation K178E in the second actin-tropomyosin (Tm) binding region showed a particularly potent Ca2+-sensitizing effect among the six RCM-causing mutations. Circular dichroism and nuclear magnetic resonance spectroscopy revealed that this mutation does not extensively affect the structure of the whole cTnI molecule, but induces an unexpectedly subtle change in the structure of a region around the mutated residue. The results indicate that the K178E mutation has a localized effect on a structure that is critical to the regulatory function of the second actin-Tm binding region of cTnI. The present study also suggests that both HCM and RCM involving cTnI mutations share a common feature of increased Ca2+ sensitivity of cardiac myofilament, but more severe change in Ca2+ sensitivity is associated with the clinical phenotype of RCM.     

Synthetic studies on oligosaccharides composed of 5-thioglucopyranose units

Glycosylation reactions of 5-thioglucopyranosyl trichloroacetimidates bearing ethereal protective groups at the 2-O-position 14-15, and 37 proceed smoothly to give alpha-glycosides stereoselectively by using a catalytic amount of silyl triflate. This methodology allowed us to achieve syntheses of sulfur-substituted isomaltotetraoside 2 and maltotetraoside 3. These studies also revealed that benzoyl-protected 5-thioglucopyranosyl trichloroacetimidate 12 underwent beta-selective glycosylation with C6-OH glucopyranosyl acceptors upon activation by BF3OEt2. This was applied for preparation of sulfur-substituted gentiobiosides 1 and 46.