Effects of freezing and activation on membrane quality and DNA damage in Xenopus tropicalis and Xenopus laevis spermatozoa.

There is growing concern over the effect of sperm cryopreservation on DNA integrity and the subsequent development of offspring generated from this cryopreserved material. In the present study, membrane integrity and DNA stability of Xenopus laevis and Xenopus tropicalis spermatozoa were evaluated in response to cryopreservation with or without activation, a process that happens upon exposure to water to spermatozoa of some aquatic species. A dye exclusion assay revealed that sperm plasma membrane integrity in both species decreased after freezing, more so for X. laevis than X. tropicalis spermatozoa. The sperm chromatin dispersion (SCD) test showed that for both X. tropicalis and X. laevis, activated frozen spermatozoa produced the highest levels of DNA fragmentation compared with all fresh samples and frozen non-activated samples (P<0.05). Understanding the nature of DNA and membrane damage that occurs in cryopreserved spermatozoa from Xenopus species represents the first step in exploiting these powerful model organisms to understand the developmental consequences of fertilising with cryopreservation-damaged spermatozoa.

Validation of the sperm chromatin dispersion (SCD) test in the amphibian Xenopus laevis using in situ nick translation and comet assay.

The integrity of sperm DNA is becoming increasingly recognised as an important parameter of semen quality, but there are no published reports of this procedure for any amphibian. The primary aim of this study was to apply a modified sperm chromatin dispersion (SCD) test (Halomax) to an amphibian sperm model (African clawed frog; Xenopus laevis) and to validate the assay against in situ nick translation (ISNT) and the double-comet assay procedure. Inactivated spermatozoa were collected from fresh testes (n=3). Sperm DNA fragmentation (SDF) for each sperm sample was conducted immediately following activation (T0) and again after 1h (T1) and 24h (T24) of incubation at room temperature in order to produce a range of spermatozoa with differing levels of DNA damage. The SCD procedure resulted in the production of three nuclear morphotypes; amphibian sperm morphotype 1 (ASM-1) and ASM-2 showed no evidence of DNA damage, whereas ASM-3 spermatozoa were highly fragmented with large halos of dispersed DNA fragments and a reduced nuclear core. ISNT confirmed that ASM-3 nuclei contained damaged DNA. There was a significant correlation (r=0.9613) between the levels of ASM-3 detected by the SCD test and SDF revealed by the double-comet assay.

Fibrillogenesis in dense collagen solutions: a physicochemical study.

Fibrillogenesis, the formation of collagen fibrils, is a key factor in connective tissue morphogenesis. To understand to what extent cells influence this process, we systematically studied the physicochemistry of the self-assembly of type I collagen molecules into fibrils in vitro. We report that fibrillogenesis in solutions of type I collagen, in a high concentration range close to that of living tissues (40-300 mg/ml), yields strong gels over wide pH and ionic strength ranges. Structures of gels were described by combining microscopic observations (transmission electron microscopy) with small- and wide-angle X-ray scattering analysis, and the influence of concentration, pH, and ionic strength on the fibril size and organization was evaluated. The typical cross-striated pattern and the corresponding small-angle X-ray scattering 67-nm diffraction peaks were visible in all conditions in the pH 6 to pH 12 range. In reference conditions (pH 7.4, ionic strength=150 mM, 20 degrees C), collagen concentration greatly influences the overall macroscopic structure of the resultant fibrillar gels, as well as the morphology and structure of the fibrils themselves. At a given collagen concentration, increasing the ionic strength from 24 to 261 mM produces larger fibrils until the system becomes biphasic. We also show that fibrils can form in acidic medium (pH approximately 2.5) at very high collagen concentrations, beyond 150 mg/ml, which suggests a possible cholesteric-to-smectic phase transition. This set of data demonstrates how simple physicochemical parameters determine the molecular organization of collagen. Such an in vitro model allows us to study the intricate process of fibrillogenesis in conditions of molecular packing close to that which occurs in biological tissue morphogenesis.

Cooperative ordering of collagen triple helices in the dense state.

Extracellular matrixes such as bone, skin, cornea, and tendon have ordered structures comprised for the most part of collagen, an elongated protein of well-defined dimensions and composition. Here we show how the cooperative ordering of collagen triple helices in the dense fluid state is exploited to produce dense ordered collagen matrixes. The spontaneous formation of a birefringent phase occurs at critical concentrations that increase from 50-60 to 80-85 mg/mL as the acetic acid concentration of the solvent increases from 5 to 500 mM. We studied by small-angle X-ray scattering (SAXS) the local liquidlike positional order across the isotropic/anisotropic phase transition by unwinding the cholesteric phase with moderate shearing stress. Interparticle scattering gives rise to a broad interference peak. The average distance between triple helices, dav, is thus estimated and decreases linearly as a function of phi-1/2 from 12.7 +/- 0.9 nm (22.5 mg/mL) to 5.0 +/- 0.6 nm (166.4 mg/mL). Equilibrium concentrations and the order parameter of the nematic phase agree reasonably well with theoretical predictions for semiflexible macromolecules. Striated fibrils with a high degree of alignment were obtained by fine-tuning the delicately balanced electrostatic interactions, which yielded strong elastic gels with a hierarchical organization very similar to that of major biological tissues. Typical Bragg reflections corresponding to the 67 nm period characteristic of collagen fibrils in biological tissues were recorded by SAXS with ordered collagen matrixes reconstituted in vitro.

Whole linted cottonseed as a forage substitute: fiber effectiveness and digestion kinetics.

Six ruminally and duodenally cannulated Holstein cows were used in a 6 x 6 Latin square design to 1) evaluate the potential interaction in effectiveness of neutral detergent fiber (NDF) from whole cottonseed (WCS) when it was substituted for forage NDF (FNDF) and fed with ground (G) or steam-flaked (SF) corn and 2) to determine whether the kinetic properties of NDF digestion further clarify the effectiveness of WCS. The six dietary treatments were: forage control with G corn (21% FNDF), 5% WCS with G or SF corn (18% FNDF), 10% WCS with G or SF corn (15% FNDF), and 15% WCS with G corn (12% FNDF). Based on chewing activity, the NDF from WCS was estimated to be 84% (SE = 36%) as effective as alfalfa silage NDF. Decreasing passage and digestion rates of potentially digestible NDF with increasing WCS increased the evacuated pool size of ruminal DM, apparently explaining the similar ruminal mat consistency among treatments. Measures of effectiveness ofWCS treatments did not interact with corn source. Fluid dilution rate was estimated based on a two-compartment model describing Co dilution, but no treatment differences were detected. There was a strong linear bias for estimates of ruminal NDF digestibility based on a single compartment model using the digestion rate of potentially digestible NDF and the passage rate of either indigestible NDF or digestible NDF when compared with NDF digestibility calculated using duodenal flows. Although further verification is needed, these digestion and passage kinetics help explain why WCS are effective at stimulating chewing during eating and rumination.

Liquid crystalline ordering of procollagen as a determinant of three-dimensional extracellular matrix architecture.

The precise molecular mechanisms that determine the three-dimensional architectures of tissues remain largely unknown. Within tissues rich in extracellular matrix, collagen fibrils are frequently arranged in a tissue-specific manner, as in certain liquid crystals. For example, the continuous twist between fibrils in compact bone osteons resembles a cholesteric mesophase, while in tendon, the regular, planar undulation, or "crimp", is akin to a precholesteric mesophase. Such analogies suggest that liquid crystalline organisation plays a role in the determination of tissue form, but it is hard to see how insoluble fibrils could spontaneously and specifically rearrange in this way. Collagen molecules, in dilute acid solution, are known to form nematic, precholesteric and cholesteric phases, but the relevance to physiological assembly mechanisms is unclear. In vivo, fibrillar collagens are synthesised in soluble precursor form, procollagens, with terminal propeptide extensions. Here, we show, by polarized light microscopy of highly concentrated (5-30 mg/ml) viscous drops, that procollagen molecules in physiological buffer conditions can also develop long-range nematic and precholesteric liquid crystalline ordering extending over 100 microm(2) domains, while remaining in true solution. These observations suggest the novel concept that supra-fibrillar tissue architecture is determined by the ability of soluble precursor molecules to form liquid crystalline arrays, prior to fibril assembly.

Relationship between clinical and biologic variables and chloramphenicol pharmacokinetic parameters in pediatric patients with sepsis.

OBJECTIVE: To evaluate the influence of several clinical and biologic factors on the disposition kinetics of oral chloramphenicol in pediatric patients and to determine the usefulness of this information to predict chloramphenicol serum concentrations. STUDY DESIGN: The clinical, biologic, and pharmacokinetic data of 30 consecutive pediatric patients diagnosed with sepsis and admitted to a tertiary care center were analyzed retrospectively. The patients were randomly assigned to a study group and a validation group. The model was developed by a three-step approach involving Bayesian estimation of pharmacokinetic parameters, selection of covariates by principal component analysis, and final selection by stepwise multiple linear regression. The model was tested in the study group and compared with a general population model using a prediction error analysis. RESULTS: Regression analysis revealed that weight, albumin, and white blood cell (WBC) count were the most important determinants for chloramphenicol distribution volume, whereas age, WBC count, and serum creatinine were the most important determinants for chloramphenicol clearance. The performance of the constructed population model improved significantly in terms of both bias and precision compared with the general model when tested in the validation group. CONCLUSIONS: Clinical and biologic factors may significantly influence chloramphenicol's disposition in pediatric patients with sepsis and therefore should be considered in programming dosage regimens.

RNA-dependent cytoplasmic anchoring of a transcription factor subunit during Xenopus development.

The CCAAT box transcription factor (CBTF) is a multimeric transcription factor that activates expression of the haematopoietic regulatory factor, GATA-2. The 122 kDa subunit of this complex, CBTF(122), is cytoplasmic in fertilized Xenopus eggs and subsequently translocates to the nucleus prior to activation of zygotic GATA-2 transcription at gastrulation. Here we present data suggesting both a role for CBTF(122) prior to its nuclear translocation and the mechanism that retains it in the cytoplasm before the midblastula transition (MBT). CBTF(122) and its variant CBTF(98) are associated with translationally quiescent mRNP complexes. We show that CBTF(122) RNA binding activity is both necessary and sufficient for its cytoplasmic retention during early development. The introduction of an additional nuclear localization signal to CBTF(122) is insufficient to overcome this retention, suggesting that RNA binding acts as a cytoplasmic anchor for CBTF(122). Destruction of endogenous RNA by microinjection of RNase promotes premature nuclear translocation of CBTF(122). Thus, the nuclear translocation of CBTF(122) at the MBT is likely to be coupled to the degradation of maternal mRNA that occurs at that stage.

Structural aspects of fish skin collagen which forms ordered arrays via liquid crystalline states.

The ability of acid-soluble type I collagen extracts from Soleidae flat fish to form ordered arrays in condensed phases has been compared with data for calf skin collagen. Liquid crystalline assemblies in vitro are optimized by preliminary treatment of the molecular population with ultrasounds. This treatment requires the stability of the fish collagen triple helicity to be controlled by X-ray diffraction and differential scanning calorimetry and the effect of sonication to be evaluated by viscosity measurements and gel electrophoresis. The collagen solution in concentrations of at least 40 mg ml(-1) showed in polarized light microscopy birefringent patterns typical of precholesteric phases indicating long-range order within the fluid collagen phase. Ultrastructural data, obtained after stabilization of the liquid crystalline collagen into a gelated matrix, showed that neutralized acid-soluble fish collagen forms cross-striated fibrils, typical of type I collagen, following sine wave-like undulations in precholesteric domains. These ordered geometries, approximating in vivo situations, give interesting mechanical properties to the material.

[Bayesian prediction of chloramphenicol blood levels in children with sepsis and malnutrition]. / Predicción bayesiana de las concentraciones séricas de cloranfenicol en niños con sepsis y desnutrición.

OBJECTIVE: To validate the population pharmacokinetic parameters of chloramphenicol in pediatric patients with sepsis and malnutrition (PPSM) using a bayesian forecasting program. DESIGN: Retrospective evaluation of predictive performance of a bayesian program in PPSM. SETTING: Tertiary care center. PATIENTS: Fifteen MPSP and ten NMPSP that receiving treatment with chloramphenicol. METHODS AND MAIN RESULTS: In the first part of the study, the medical records of 10 MPSP and 10 NMPSP who had received treatment with chloramphenicol were reviewed. The population pharmacokinetic parameter values for each group were estimated using a nonparametric expectation maximization algorithm (NPEM). In the second part, data gathered from five other MPSP receiving chloramphenicol were entered into a bayesian program. Chloramphenicol pharmacokinetic values for each of these five patients were estimated, first using the values of NMPSP as a priori distribution and then repeating the analysis using the MPSP values. The bayesian serum chloramphenicol concentrations predicted for each population model were compared with the actual peaks and troughs. The specific model for MPSP permitted forecasting the peak and trough serum chloramphenicol concentrations with less bias and a better precision compared with the NMPSP population model. CONCLUSIONS: These data indicate that chloramphenicol pharmacokinetics in PPSM can be predicted with minimal bias and good precision using a bayesian forecasting program, allowing a better control of the chloramphenicol serum concentrations. In addition, the limited number of samples required by the bayesian method may represent an important economical benefit for the patient.

Banded patterns in liquid crystalline phases of type I collagen: relationship with crimp morphology in connective tissue architecture.

Solutions of type I acid soluble collagen were studied in light and electron microscopy at concentrations over 40 mg/ml. Banded patterns spontaneously emerge in samples observed between crossed polars between slide and coverslip. The textures are interpreted as precholesteric, appearing at the transition between the isotropic phases, due to random molecular order, and the cholesteric phase corresponding to a highly organized three-dimensional structure. Type I collagen banded patterns correspond to regular undulations of the molecular directions with an observed periodicity in the range of 1 to 10 microm. This interpretation is verified by ultrastructural analysis of precholesteric samples gelled under ammonium vapors. Results are discussed in regard to banded patterns described either within synthetic polymer systems or within collagen extracellular matrices. Self-assembled liquid crystalline phases of collagen generate crimp morphologies. Their possible relationship with early secretion steps in the development of connective tissues is discussed.

[Cefuroxime Bayesian pharmacokinetics in severely ill septic children]. / Farmacocinética bayesiana de cefuroxima en niños infectados gravemente enfermos.

OBJECTIVE: To estimate the cefuroxime pharmacokinetic parameters in critically ill pediatric septic patients using a Bayesian pharmacokinetic method and three serum drug assays per patient. DESIGN: Cross-sectional study of critically ill pediatric patients undergoing therapeutic monitoring of cefuroxime serum concentrations. SETTING: Tertiary care center. PATIENTS: Nine critically ill pediatric patients with sepsis and septic shock treated with cefuroxime. METHODS: Timed serum concentrations of cefuroxime were obtained in each patient. The Vd (volume of distribution) and the Kel (constant of elimination) were estimated by means of a Bayesian iterative two-stage algorithm, using the information of three serum drug concentrations per patient at optimal times. The parameters were also estimated by the traditional method of non linear least square regression in eight samples. RESULTS: The Bayesian Vd was very similar to the traditional Vd with a correlation coefficient of 0.99 and a small bias of -0.04 L/kg whereas the Kel had a correlation of 0.90 and bias of -0.29 h-1. The mean Bayesian Vd was 0.68 L/kg, a larger value than that reported in non critically ill patients. CONCLUSIONS: We offer a tentative cefuroxime pharmacokinetic model for critically ill pediatric septic patients which may be useful for the control of cefuroxime serum concentrations. Also, our study underscored the potential usefulness of a Bayesian pharmacokinetic approach as a tool for therapeutic drug monitoring in critically ill patients.

The maternal CCAAT box transcription factor which controls GATA-2 expression is novel and developmentally regulated and contains a double-stranded-RNA-binding subunit.

The transcription factor GATA-2 is expressed at high levels in the nonneural ectoderm of the Xenopus embryo at neurula stages, with lower amounts of RNA present in the ventral mesoderm and endoderm. The promoter of the GATA-2 gene contains an inverted CCAAT box conserved among Xenopus laevis, humans, chickens, and mice. We have shown that this sequence is essential for GATA-2 transcription during early development and that the factor binding it is maternal. The DNA-binding activity of this factor is detectable in nuclei and chromatin bound only when zygotic GATA-2 transcription starts. Here we report the characterization of this factor, which we call CBTF (CCAAT box transcription factor). CBTF activity mainly appears late in oogenesis, when it is nuclear, and the complex has multiple subunits. We have identified one subunit of the factor as p122, a Xenopus double-stranded-RNA-binding protein. The p122 protein is perinuclear during early embryonic development but moves from the cytoplasm into the nuclei of embryonic cells at stage 9, prior to the detection of CBTF activity in the nucleus. Thus, the accumulation of CBTF activity in the nucleus is a multistep process. We show that the p122 protein is expressed mainly in the ectoderm. Expression of p122 mRNA is more restricted, mainly to the anterior ectoderm and mesoderm and to the neural tube. Two properties of CBTF, its dual role and its cytoplasm-to-nucleus translocation, are shared with other vertebrate maternal transcription factors and may be general properties of these proteins.

Methods for the analysis of DNA-protein interactions.

The interaction of proteins with DNA is a central theme of molecular biology. In this article, we review some of the principal techniques currently used for the identification and characterization of DNA binding proteins, and for investigation of the molecular interactions that are responsible for the recognition of specific DNA sequences.