We applied a new method, "protein-ligand interaction using mass spectrometry, titration, and H/D exchange" (PLIMSTEX) [Zhu, M. M. (2003) J. Am. Chem. Soc. 125, 5252-5253], to determine the conformational changes, binding stoichiometry, and binding constants for Ca(2+) interactions with calmodulin (CaM) under varying conditions of electrolyte identity and ionic strength. The outcome shows that CaM becomes less solvent-accessible and more compact upon Ca(2+)-binding, as revealed by the PLIMSTEX curve. The formation of CaM-4Ca species is the biggest contributor to the shape of the titration curve, indicating that the formation of this species accounts for the largest conformational change in the stepwise Ca(2+) binding. The Ca(2+)-binding constants, when comparisons permit, agree with those in the literature within a factor of 3. The binding is influenced by ionic strength and the presence of other cations, although many of these cations do not cause conformational change in apo-CaM. Furthermore, Ca(2+)-saturated CaM exhibits larger protection and higher Ca(2+) affinity in media of low rather than high ionic strength. Both Ca(2+) and Mg(2+) bind to CaM with different affinities, causing different conformational changes. K(+), if it does bind, causes no detectable conformational change, and interactions of Ca(2+) with CaM in the presence of Li(+), Na(+), and K(+) occur with similar affinities and associated changes in solvent accessibility. These metal ion effects point to nonspecific rather than competitive binding of alkali-metal ions. The rates of deuterium uptake by the various CaM-xCa species follow a three-group (fast, intermediate, slow), pseudo-first-order kinetics model. Calcium binding causes the number of amide hydrogens to shift from the fast to the slow group. The results taken together not only provide new insight into CaM but also indicate that both PLIMSTEX and kinetic modeling of H/D exchange data may become general methods for probing protein conformations and quantifying protein-ligand interactions.

Formation of depurinating adducts by reaction of catechol estrogen-3,4-quinones with DNA was proposed to be a tumor initiating event by estrogens [E.L. Cavalieri et al. (1997) Proc. Natl Acad. Sci. USA, 94, 10937-10942]. Under estrogenic imbalance, oxidation of catechol estrogens to quinones may compete with their detoxification by protective enzymes. The quinones formed can be detoxified by reaction with glutathione (GSH) or can covalently bind to DNA. To provide more support for this hypothesis, we developed a method to identify and quantify GSH, cysteine (Cys) and N-acetylCys conjugates of 4-hydroxyestrogens (4-OHE) in the kidneys of male Syrian hamsters treated with 4-hydroxyestradiol (4-OHE2) by intraperitoneal injection. The highest level of conjugates was observed 1 h after treatment, and almost none was detected after 24 h. Dose-response studies indicated conjugate formation after treatment with 0.5 micromol of 4-OHE2/100 g body weight, and formation increased up to a treatment level of 12 micromol/100 g body weight. GSH, Cys and N-acetylCys conjugates of 4-OHE were identified in the picomole range by high-performance liquid chromatography (HPLC) with multichannel electrochemical detection and confirmed by HPLC/tandem mass spectrometry. Treatment of tissue homogenates with beta-glucuronidase/sulfatase at 37 degrees C for 6 h before extraction resulted in a 12- to 20-fold increase in Cys conjugates from picomole to nanomole levels. Similar enhancement was observed by just incubating the tissue at 37 degrees C for 6 h. Evidence for the 4-OHE-1-N7Gua depurinating adducts was obtained by mass spectrometry. We conclude that GSH and Cys conjugates of the 4-OHE and the 4-OHE-N7Gua adducts can be utilized as biomarkers to detect estrogenic imbalance and potential susceptibility to tumor initiation.

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Chronic diarrhea is one of the most common complaints in clinical practice for both adults and children. The purpose of this study was to assess the commonly used Chinese herbal medicine navel therapy for the treatment of chronic diarrhea (traditional Chinese medicine syndrome of spleen deficiency). The literature search was up to June 2018. Four types of studies (clinical trials, case series, case reports, and experts' experience) researched on Chinese medicine navel therapy used alone or combined with other therapies for the treatment of chronic diarrhea all included. Information on prescriptions, effectiveness, and safety of intervention was collected. Traditional Chinese Medicine Inheritance Support System V2.5 was used to do data analysis. We included 416 studies. All these studies carried out in 194 cities of China. We obtained the most commonly used single herbs, herbal medicine combination, and Chinese patent medicine for both adults and children. The top 3 single herbs were Caryophylli Flos (Dingxiang), Cinnamomi Cortex (Rougui), and Euodiae Fructus (Wuzhuyu). The most frequently used Chinese patent medicines were Ding Gui Infantile Navel Paste and Huoxiang Zhengqi Liquid. The effectiveness assessment was based on clinical trials, but we did not perform a meta‐analysis because of different study design and unsatisfactory methodological quality. No serious adverse reaction happened in original studies. The application of Chinese medicine navel therapy could be one of the ideal treatments for chronic diarrhea in the future, especially for children. However, high‐quality studies are very needed to provide clear evidence.

Rheumatoid arthritis (RA) is an autoimmune disorder that affects the joint synovium. Anserine is a functional dipeptide containing methylhistidine and β-alanine, and is present in the brain and skeletal muscle of birds and mammals. Glucosamine is an amino sugar used in the synthesis of glycosylated proteins and lipids. We evaluated the effects of anserine and glucosamine on RA. Rats were assigned into the control group, RA group, anserine group (1 mg/kg), glucosamine group (200 mg/kg), or anserine plus glucosamine group (anserine, 1 mg/kg + glucosamine, 200 mg/ kg). Treatment was continued for 45 consecutive days and was administered orally. The serum levels of catalase, glutathione peroxidase (Gpx), superoxide dismutase (SOD), reduced glutathione (GSH), lipid peroxidation, uric acid, nitric oxide, ceruloplasmin, zinc, copper, prostaglandin E 2 (PGE 2 ), matrix metalloproteinase (MMP)-3, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 were assayed. The mRNA and protein levels of nuclear factor (NF)-κB and inducible nitric oxide synthase (iNOS) in synovial tissue were also determined. Anserine plus glucosamine significantly increased the catalase, SOD, Gpx, GSH, and zinc levels compared to the control, anserine, and glucosamine groups. Also, anserine plus glucosamine significantly reduced the PGE 2 , MMP-3, TNF-α, IL-1β, and IL-6 levels compared to the control, anserine, and glucosamine groups. Furthermore, anserine plus glucosamine significantly reduced the mRNA and protein levels of NF-κB and iNOS compared to the control, anserine, and glucosamine groups. Therefore, supplementation of anserine plus glucosamine shows therapeutic potential for RA.