The mechanisms of reversible immobilization of fowl spermatozoa at body temperature

Intact fowl spermatozoa became almost immotile at 40 degrees C, but motility increased significantly at 30 degrees C. The oxygen consumption at both temperatures was 8-11 microliters O2/10(10) spermatozoa.min-1. The ATP concentration at 40 degrees C was higher than that at 30 degrees C but ADP concentration at 30 degrees C was higher than that at 40 degrees C. Consequently, the ATP/ADP ratio at 30 degrees C (1.9-2.2) increased to 3.5-3.7 at 40 degrees C. The motility of intact spermatozoa at 40 degrees C was effectively restored by 2 mM-Ca2+, 10% seminal plasma and 10% peritoneal fluid taken at the time of ovulation. In contrast, these effectors did not restore the motility of demembranated spermatozoa at 40 degrees C. Motility of demembranated spermatozoa was restored at 30 degrees C. These results suggest that the immobilization of fowl spermatozoa at 40 degrees C occurs due to a decrease in flagellar dynein ATPase activity. Furthermore, the action of effectors for motility such as Ca2+ may not be directly on the axoneme, but mediated by solubilized substances which have been removed by demembranation of the spermatozoa.     

Target Binding to S100B Reduces Dynamic Properties and Increases Ca2 +-Binding Affinity for Wild Type and EF-Hand Mutant Proteins

Mutations in the second EF-hand (D61N, D63N, D65N, and E72A) of S100B were used to study its Ca^2 + binding and dynamic properties in the absence and presence of a bound target, TRTK-12. With ^D63NS100B as an exception (^D63NK_D = 50 ± 9 μM), Ca^2 + binding to EF2-hand mutants were reduced by more than 8-fold in the absence of TRTK-12 (^D61NK_D = 412 ± 67 μM, ^D65NK_D = 968 ± 171 μM, and ^E72AK_D = 471 ± 133 μM), when compared to wild-type protein (^WTK_D = 56 ± 9 μM). For the TRTK-12 complexes, the Ca^2 +-binding affinity to wild type (^WT + TRTKK_D = 12 ± 10 μM) and the EF2 mutants was increased by 5- to 14-fold versus in the absence of target (^{D61N + TRTK}K_D = 29 ± 1.2 μM, ^{D63N + TRTK}K_D = 10 ± 2.2 μM, ^{D65N + TRTK}K_D = 73 ± 4.4 μM, and ^{E72A + TRTK}K_D = 18 ± 3.7 μM). In addition, R_ex, as measured using relaxation dispersion for side‐chain ¹⁵N resonances of Asn63 (^D63NS100B), was reduced upon TRTK-12 binding when measured by NMR. Likewise, backbone motions on multiple timescales (picoseconds to milliseconds) throughout wild type, ^D61NS100B, ^D63NS100B, and ^D65NS100B were lowered upon binding TRTK-12. However, the X-ray structures of Ca^2 +-bound (2.0 Å) and TRTK-bound (1.2 Å) ^D63NS100B showed no change in Ca^2 + coordination; thus, these and analogous structural data for the wild-type protein could not be used to explain how target binding increased Ca^2 +-binding affinity in solution. Therefore, a model for how S100B–TRTK‐12 complex formation increases Ca^2 + binding is discussed, which considers changes in protein dynamics upon binding the target TRTK-12.     

Long-term hydrochemical monitoring in an Oyasan Experimental Forest Watershed comprised of two small forested watersheds of Japanese cedar and Japanese cypress

Forest ecosystems are self-fertilizing systems, and development of forest stands depends on nutrient supply via biogeochemical cycling within the ecosystem. Therefore, it is important to clarify the nutrient cycle mediating growth and development. In addition, long-term hydrochemical monitoring is needed to understand the influence of environmental changes on biogeochemical cycling in forest ecosystems. The Oyasan Experimental Forest Watershed (OEFW) is located in the Field Museum Oyasan, the university forest of Tokyo University of Agriculture and Technology, in Gunma prefecture, Japan. OEFW comprises two small adjacent forested watersheds—A-watershed and B-watershed—with respective areas of 1.3 and 1.8 ha. A-watershed is a reestablished forest planted with sugi (Japanese cedar; Cryptomeria japonica) and hinoki (Japanese cypress; Chamaecyparis obtusa) in 1976, and has been managed intensively with fertilizer application. By contrast, B-watershed is an established forest planted with sugi and hinoki in 1907. No forest practices have been carried out except for thinning of suppressed trees in 1983. However, the sugi plantation on the lowest slope (18% of the watershed area) was cut in 2000, and sugi was replanted the following year. In this data paper, we present data on the daily precipitation, discharge, pH, and concentrations of major nutrients (Ca²⁺, Mg²⁺, K⁺, Na⁺, NH₄ ⁺, Cl⁻, NO₃ ⁻, and SO₄ ²⁻) in rainwater and stream water since November 1978. The arithmetical mean pH of precipitation, stream water in A- and B-watershed from the beginning of the monitoring to the present were 4.77 ± 0.67, 6.85 ± 0.41 and 6.88 ± 0.36 (average ± SD), respectively. The arithmetical mean concentrations in precipitation in mmol_c L⁻¹ were 0.030 ± 0.030 for Ca²⁺, 0.010 ± 0.011 for Mg²⁺, 0.009 ± 0.013 for K⁺, 0.020 ± 0.024 for Na⁺, 0.035 ± 0.041 for NH₄ ⁺, 0.026 ± 0.029 for Cl⁻, 0.033 ± 0.038 for NO₃ ⁻, and 0.046 ± 0.043 for SO₄ ²⁻. The mean concentrations in stream water in A-watershed were 0.180 ± 0.032 for Ca²⁺, 0.073 ± 0.013 for Mg²⁺, 0.018 ± 0.009 for K⁺, 0.182 ± 0.024 for Na⁺, 0.010 ± 0.010 for NH₄ ⁺, 0.060 ± 0.008 for Cl⁻, 0.111 ± 0.038 for NO₃ ⁻, and 0.074 ± 0.012 for SO₄ ²⁻; whereas for B-watershed the mean concentrations were 0.169 ± 0.025 for Ca²⁺, 0.079 ± 0.016 for Mg²⁺, 0.018 ± 0.005 for K⁺, 0.192 ± 0.026 for Na⁺, 0.010 ± 0.010 for NH₄ ⁺, 0.065 ± 0.010 for Cl⁻, 0.093 ± 0.025 for NO₃ ⁻, and 0.087 ± 0.011 for SO₄ ²⁻.     

Accuracy of Optimized Chemical-exchange Parameters Derived by Fitting CPMG R 2 Dispersion Profiles when R 2 0a ≠ R 2 0b

The transverse relaxation rate, R₂, measured as a function of the effective field (R₂ dispersion) using a Carr-Purcell-Meiboom-Gill (CPMG) pulse train, is well suited to detect conformational exchange in proteins. The dispersion data are commonly fitted by a two-site (sites a and b) exchange model with four parameters: the relative population, p_a, the difference in chemical shifts of the two sites, δω, the correlation time for exchange, τ_ex, and the intrinsic relaxation rate (i.e., transverse relaxation rate in the absence of chemical exchange), R₂⁰. Although the intrinsic relaxation rates of the two sites, R₂^0a and R₂^0b, can differ, they are normally assumed to be the same (i.e., R₂^0a = R₂^0b = R₂⁰) when fitting dispersion data. The purpose of this investigation is to determine the magnitudes of the errors in the optimized exchange parameters that are introduced by the assumption that R₂^0a = R₂^0b. In order to accomplish this goal, we first generated synthetic constant-time CPMG R₂ dispersion data assuming two-site exchange with R₂^0a ≠ R₂^0b, and then fitted the synthetic data assuming two-site exchange with R₂⁰ = R₂^0a = R₂^0b. Although all the synthetic data generated assuming R₂^0a ≠ R₂^0b were well fitted (assuming R₂^0a = R₂^0b), the optimized values of p_a and τ _ex differed from their true values, whereas the optimized values of δω values did not. A theoretical analysis using the Carver–Richards equation explains these results, and yields simple, general equations for estimating the magnitudes of the errors in the optimized parameters, as a function of ( R₂^0a − R₂^0b).     

Chemical exchange effects during refocusing pulses in constant-time CPMG relaxation dispersion experiments

In the analysis of the constant-time Carr-Purcell-Meiboom-Gill (CT-CPMG) relaxation dispersion experiment, chemical exchange parameters, such as rate of exchange and population of the exchanging species, are typically optimized using equations that predict experimental relaxation rates recorded as a function of effective field strength. In this process, the effect of chemical exchange during the CPMG pulses is typically assumed to be the same as during the free-precession. This approximation may introduce systematic errors into the analysis of data because the number of CPMG pulses is incremented during the constant-time relaxation period, and the total pulse duration therefore varies as a function of the effective field strength. In order to estimate the size of such errors, we simulate the time-dependence of magnetization during the entire constant time period, explicitly taking into account the effect of the CPMG pulses on the spin relaxation rate. We show that in general the difference in the relaxation dispersion profile calculated using a practical pulse width from that calculated using an extremely short pulse width is small, but under certain circumstances can exceed 1 s^?1. The difference increases significantly when CPMG pulses are miscalibrated.     

A murine model of neonatal diabetes mellitus in Glis3-deficient mice

Glis3 is a member of the Gli-similar subfamily. GLIS3 mutations in humans lead to neonatal diabetes, hypothyroidism, and cystic kidney disease. We generated Glis3-deficient mice by gene-targeting. The Glis3^−/− mice had significant increases in the basal blood sugar level during the first few days after birth. The high levels of blood sugar are attributed to a decrease in the Insulin mRNA level in the pancreas that is caused by impaired islet development and the subsequent impairment of Insulin-producing cell formation. The pancreatic phenotypes indicate that the Glis3-deficient mice are a model for GLIS3 mutation and diabetes mellitus in humans.     

Engineered Human Antibody Constant Domains with Increased Stability

The immunoglobulin (Ig) constant CH2 domain is critical for antibody effector functions. Isolated CH2 domains are promising as scaffolds for construction of libraries containing diverse binders that could also confer some effector functions. However, previous work has shown that an isolated murine CH2 domain is relatively unstable to thermally induced unfolding. To explore unfolding mechanisms of isolated human CH2 and increase its stability γ1 CH2 was cloned and a panel of cysteine mutants was constructed. Human γ1 CH2 unfolded at a higher temperature (T_m = 54.1 °C, as measured by circular dichroism) than that previously reported for a mouse CH2 (41 °C). One mutant (m01) was remarkably stable (T_m = 73.8 °C). Similar results were obtained by differential scanning calorimetry. This mutant was also significantly more stable than the wild-type CH2 against urea induced unfolding (50% unfolding at urea concentration of 6.8 m versus 4.2 m). The m01 was highly soluble and monomeric. The existence of the second disulfide bond in m01 and its correct position were demonstrated by mass spectrometry and nuclear magnetic resonance spectroscopy, respectively. The loops were on average more flexible than the framework in both CH2 and m01, and the overall secondary structure was not affected by the additional disulfide bond. These data suggest that a human CH2 domain is relatively stable to unfolding at physiological temperature, and that both CH2 and the highly stable mutant m01 are promising new scaffolds for the development of therapeutics against human diseases.Monoclonal antibodies (mAbs)² with high affinity and specificity are now well established therapeutics and invaluable tools for biological research. It appears that their use will continue to expand in both targets and disease indications. However, a fundamental problem for full-size mAbs that limits their applications is their poor penetration into tissues (e.g. solid tumors) and poor or absent binding to regions on the surface of some molecules (e.g. on the HIV envelope glycoprotein) that are accessible by molecules of smaller size. Antibody fragments, e.g. Fabs (∼60 kDa) or single chain Fv fragments (scFvs) (20∼30 kDa), are significantly smaller than full-size antibodies (∼150 kDa), and have been used as imaging reagents and candidate therapeutics. Even smaller fragments of antibodies are of great interest and advantageous for pharmaceutical applications including cancer targeting and imaging.During the last decade a large amount of work has been aimed at developing of small size binders with scaffolds based on various highly stable human and non-human molecules (1–8). A promising direction is the development of binders based on the heavy or light chain variable region of an antibody; these fragments ranging in size from 11 kDa to 15 kDa were called “domain antibodies” or “dAbs” (7, 9). A unique kind of antibodies composed only of heavy chains are naturally formed in camels, dromedaries, and llamas, and their variable regions can also recognize antigens as single domain fragments (10). Not only is the overall size of the dAbs much smaller than that of full-size antibodies but also their paratopes are concentrated over a smaller area so that the dAbs provide the capability of interacting with novel epitopes that are inaccessible to conventional antibodies or antibody fragments with paired light and heavy chain variable domains.The structure of the constant antibody domains is similar to that of the variable domains consisting of β-strands connected mostly with loops or short helices. The second domain of the α, δ, and γ heavy chain constant regions, CH2, is unique in that it exhibits very weak carbohydrate-mediated interchain protein-protein interactions in contrast to the extensive interchain interactions that occur between the other domains. The expression of murine CH2 in bacteria, which does not support glycosylation, results in a monomeric domain (11). It has been hypothesized that the CH2 domain (CH2 of IgG, IgA, and IgD, and CH3 of IgE and IgM) could be used as a scaffold and could offer additional advantages compared with those of dAbs because it contains binding sites or portions of binding sites conferring effector and stability functions (12).It was found previously that an isolated murine CH2 is relatively unstable at physiological temperature with a temperature of 50% unfolding (T_m) slightly higher than 37 °C (11). We have hypothesized that human CH2 would exhibit different stability because of significant differences in the sequence compared with its murine counterpart. Therefore, we have extensively characterized the stability of an isolated unglycosylated single CH2 domain. We found that its stability is significantly higher than the previously reported for the murine CH2. We further increased the stability of the human CH2 by engineering an additional disulfide bond between the A and G strands. One of the newly developed mutants, denoted as m01, exhibited significantly higher stability (T_m = 73.8 °C) than that of wild type CH2. We suggest that both the wild type CH2 and the newly developed mutant, m01, could be used as scaffolds for binders. These results also demonstrate for the first time that the stability of constant antibody domains can be dramatically increased by engineering of an additional disulfide bond. The increase in stability of isolated domains may result in an increase in stability of larger antibody fragments, e.g. Fc, and therefore could have implications as a general method for increasing antibody stability. Thus, it appears that further development of CH2 and its more stable variants as scaffolds could provide new opportunities for identification of potentially useful therapeutics.     

Polycystic Kidney Disease in the Medaka (Oryzias latipes) pc Mutant Caused by a Mutation in the Gli-Similar3 (glis3) Gene

Polycystic kidney disease (PKD) is a common hereditary disease in humans. Recent studies have shown an increasing number of ciliary genes that are involved in the pathogenesis of PKD. In this study, the Gli-similar3 (glis3) gene was identified as the causal gene of the medaka pc mutant, a model of PKD. In the pc mutant, a transposon was found to be inserted into the fourth intron of the pc/glis3 gene, causing aberrant splicing of the pc/glis3 mRNA and thus a putatively truncated protein with a defective zinc finger domain. pc/glis3 mRNA is expressed in the epithelial cells of the renal tubules and ducts of the pronephros and mesonephros, and also in the pancreas. Antisense oligonucleotide-mediated knockdown of pc/glis3 resulted in cyst formation in the pronephric tubules of medaka fry. Although three other glis family members, glis1a, glis1b and glis2, were found in the medaka genome, none were expressed in the embryonic or larval kidney. In the pc mutant, the urine flow rate in the pronephros was significantly reduced, which was considered to be a direct cause of renal cyst formation. The cilia on the surface of the renal tubular epithelium were significantly shorter in the pc mutant than in wild-type, suggesting that shortened cilia resulted in a decrease in driving force and, in turn, a reduction in urine flow rate. Most importantly, EGFP-tagged pc/glis3 protein localized in primary cilia as well as in the nucleus when expressed in mouse renal epithelial cells, indicating a strong connection between pc/glis3 and ciliary function. Unlike human patients with GLIS3 mutations, the medaka pc mutant shows none of the symptoms of a pancreatic phenotype, such as impaired insulin expression and/or diabetes, suggesting that the pc mutant may be suitable for use as a kidney-specific model for human GLIS3 patients.     

Emergence of the circadian sleep-wake rhythm might depend on conception not on birth timing

Sleep and Biological Rhythms - Developmental changes in the sleep-wake rhythm of preterm infants were compared with those of full-term infants, to clarify the timing of the developmental change of...