Subzero water transport characteristics of boar spermatozoa confirm observed optimal cooling rates

Incomplete understanding of the water transport parameters (reference membrane permeability, L(pg), and activation energy, E(Lp)) during freezing in the presence of extracellular ice and cryoprotective agents (CPAs) is one of the main limiting factors in reconciling the difference between the numerically predicted value and the experimentally determined optimal rates of freezing in boar (and in general mammalian) gametes. In the present study, a shape-independent differential scanning calorimeter (DSC) technique was used to measure the water transport during freezing of boar spermatozoa. Water transport data during freezing of boar sperm cell suspensions were obtained at cooling rates of 5 and 20 degrees C/min in the presence of extracellular ice and 6% (v/v) glycerol. Using previously published values, the boar sperm cell was modeled as a cylinder of length 80.1 microm and a radius of 0.31 microm with an osmotically inactive cell volume, V(b), of 0.6 V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained data, the best-fit water transport parameters (L(pg) and E(Lp)) were determined. The "combined-best-fit" parameters at 5 and 20 degrees C/min for boar spermatozoa in the presence of extracellular ice are: L(pg) = 3.6 x 10(-15) m(3)/N. s (0.02 microm/min-atm) and E(Lp) = 122.5 kJ/mole (29.3 kcal/mole) (R(2) = 0.99); and the corresponding parameters in the presence of extracellular ice and glycerol are: L(pg)[cpa] = 0.90 x 10(-15) m(3)/N. s (0.005 microm/min-atm) and E(Lp)[cpa] = 75.7 kJ/mole (18.1 kcal/mole) (R(2) = 0.99). The water transport parameters obtained in the present study are significantly different from previously published parameters for boar and other mammalian spermatozoa obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The theoretically predicted optimal rates of freezing using the new parameters ( approximately 30 degrees C/min) are in close agreement with previously published but experimentally determined optimal cooling rates. This analysis reconciles a long-standing difference between theoretically predicted and experimentally determined optimal cooling rates for boar spermatozoa.     

Frankia KB5 Possesses a Hydrogenase Immunologically Related to Membrane-Bound [NiFe]-Hydrogenases

The immunological relationship of the hydrogenase in Frankia KB5 to hydrogenases in other microorganisms was investigated using antisera raised against holo-[NiFe]-hydrogenases isolated from Alcaligenes latus, Azotobacter vinelandii, Ralstonia eutropha, and the small and large hydrogenase subunits from Bradyrhizobium japonicum. The antisera raised against the A. latus, R. eutropha, and B. japonicum (large subunit) polypeptides were found to recognize two polypeptides, corresponding to the unprocessed and processed forms of the hydrogenase subunit in Frankia KB5. None of the antisera, including the antibodies produced against the small hydrogenase subunit isolated from B. japonicum, recognized any polypeptide related to the small hydrogenase subunit in Frankia KB5. An immunogold localization study of the intracellular distribution of hydrogenase in Frankia KB5, with the cryo-section technique, showed that labeling in the membrane of both hyphae and vesicles was positively correlated with hydrogenase activity. Received: 6 November 2000 / Accepted: 18 December 2000     

Genetic Linkage and Cotransfer of a Novel, vanB-Containing Transposon (Tn5382) and a Low-Affinity Penicillin-Binding Protein 5 Gene in a Clinical Vancomycin-Resistant Enterococcus faecium Isolate

Mechanisms for the intercellular transfer of VanB-type vancomycin resistance determinants and for the almost universal association of these determinants with those for high-level ampicillin resistance remain poorly defined. We report the discovery of Tn5382, a ca. 27-kb putative transposon encoding VanB-type glycopeptide resistance in Enterococcus faecium. Open reading frames internal to the right end of Tn5382 and downstream of the vanX_B dipeptidase gene exhibit significant homology to genes encoding the excisase and integrase of conjugative transposon Tn916. The ends of Tn5382 are also homologous to the ends of Tn916, especially in regions bound by the integrase enzyme. PCR amplification experiments indicate that Tn5382 excises to form a circular intermediate in E. faecium. Integration of Tn5382 in the chromosome of E. faecium C68 has occurred 113 bp downstream of the stop codon for the pbp5 gene, which encodes high-level ampicillin resistance in this clinical isolate. Transfer of vancomycin, ampicillin, and tetracycline resistance from C68 to an E. faecium recipient strain occurs at low frequency in vitro and is associated with acquisition of a 130- to 160-kb segment of DNA that contains Tn5382, the pbp5 gene, and its putative repressor gene, psr. The interenterococcal transfer of this large chromosomal element appears to be the primary mechanism for vanB operon spread in northeast Ohio. These results expand the known family of Tn916-related transposons, suggest a mechanism for vanB operon entry into and dissemination among enterococci, and provide an explanation for the nearly universal association of vancomycin and high-level ampicillin resistance in clinical E. faecium strains.     

Evidence that induction and suppression of mutations and recombinations by chemical mutagens in S. cerevisiae during mitosis are jointly correlated.

Mutagen-induced intergenic and interallelic recombination as well as forward mutation were studied in one and the same strain of S. cerevisiae. In nontoxic dose ranges, the induction of mutants and recombinants was parallel after treatment with ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-methyl-N'-nitro-M-nitrosoguanidine (MNNG), triethylene melamine (TEM), 4-nitroquinoline 1-oxide (4-NQO), sodium nitrite (NaNO2), and 1-fluoro-2,4-dinitrobenzene (2,4-DNFB). Acridine orange (AO) after treatment without light induced recombinants, but reduced the frequency of spontaneous mutations. In combination with TEM, AO exerted the same effect, i.e., reduced its mutagenic effect and enhanced its recombinogenic effect. 4,5,6-Trichloro-2-(2,4-dichlorophenoxy) phenol (Cl5-predioxin) induced mutants and intergenic recombinants, but specifically reduced the spontaneous frequency of interallelic recombinants. In combination with TEM, it enhanced its mutagenic and intergenic recombinogenic effects but reduced its interallelic recombinogenic effect. The main conclusions of the present study, that is 1. Essentially similar lesions can lead to different genetic consequences, and 2. Induction of mutation and recombination are jointly correlated, i.e., suppression of mutations leads to an enhancement of recombinations, while suppression of recombinations leads to an enhancement of mutations, are used to set up a speculative concept for mutation and recombination induction in the diploid yeast cell during mitosis.     

Vascular Factors and Multiple Measures of Early Brain Health: CARDIA Brain MRI Study

Objective

To identify early changes in brain structure and function that are associated with cardiovascular risk factors (CVRF).

Design

Cross-sectional brain Magnetic Resonance I (MRI) study.

Setting

Community based cohort in three U.S. sites.

Participants

A Caucasian and African-American sub-sample (n= 680; mean age 50.3 yrs) attending the 25 year follow-up exam of the Coronary Artery Risk Development in Young Adults Study.

Primary and Secondary Outcomes

3T brain MR images processed for quantitative estimates of: total brain (TBV) and abnormal white matter (AWM) volume; white matter fractional anisotropy (WM-FA); and gray matter cerebral blood flow (GM-CBF). Total intracranial volume is TBV plus cerebral spinal fluid (TICV). A Global Cognitive Function (GCF) score was derived from tests of speed, memory and executive function.

Results

Adjusting for TICV and demographic factors, current smoking was significantly associated with lower GM-CBF and TBV, and more AWM (all <0.05); SA with lower GM-CBF, WM-FA and TBV (p=0.01); increasing BMI with decreasing GM-CBF (p<0003); hypertension with lower GM-CBF, WM-FA, and TBV and higher AWM (all <0.05); and diabetes with lower TBV (p=0.007). The GCS was lower as TBV decreased, AWM increased, and WM-FA (all p<0.01).

Conclusion

In middle age adults, CVRF are associated with brain health, reflected in MRI measures of structure and perfusion, and cognitive functioning. These findings suggest markers of mid-life cardiovascular and brain health should be considered as indication for early intervention and future risk of late-life cerebrovascular disease and dementia.     

Matrix quality and disturbance frequency drive evolution of species behavior at habitat boundaries

Previous theoretical studies suggest that a species' landscape should influence the evolution of its dispersal characteristics, because landscape structure affects the costs and benefits of dispersal. However, these studies have not considered the evolution of boundary crossing, that is, the tendency of animals to cross from habitat to nonhabitat (“matrix”). It is important to understand this dispersal behavior, because of its effects on the probability of population persistence. Boundary‐crossing behavior drives the rate of interaction with matrix, and thus, it influences the rate of movement among populations and the risk of dispersal mortality. We used an individual‐based, spatially explicit model to simulate the evolution of boundary crossing in response to landscape structure. Our simulations predict higher evolved probabilities of boundary crossing in landscapes with more habitat, less fragmented habitat, higher‐quality matrix, and more frequent disturbances (i.e., fewer generations between local population extinction events). Unexpectedly, our simulations also suggest that matrix quality and disturbance frequency have much stronger effects on the evolution of boundary crossing than either habitat amount or habitat fragmentation. Our results suggest that boundary‐crossing responses are most affected by the costs of dispersal through matrix and the benefits of escaping local extinction events. Evolution of optimal behavior at habitat boundaries in response to the landscape may have implications for species in human‐altered landscapes, because this behavior may become suboptimal if the landscape changes faster than the species' evolutionary response to that change. Understanding how matrix quality and habitat disturbance drive evolution of behavior at boundaries, and how this in turn influences the extinction risk of species in human‐altered landscapes should help us identify species of conservation concern and target them for management.     

Association between Plasma 25-Hydroxyvitamin D,Ancestry and Aggressive Prostate Cancer among African Americans and European Americans in PCaP

Background

African Americans (AAs) have lower circulating 25-hydroxyvitamin D3 [25(OH)D3] concentrations and higher prostate cancer (CaP) aggressiveness than other racial/ethnic groups. The purpose of the current study was to examine the relationship between plasma 25(OH)D3, African ancestry and CaP aggressiveness among AAs and European Americans (EAs).

Methods

Plasma 25(OH)D3 was measured using LC-MS/MS (Liquid Chromatography Tandem Mass Spectrometry) in 537 AA and 663 EA newly-diagnosed CaP patients from the North Carolina-Louisiana Prostate Cancer Project (PCaP) classified as having either ‘high’ or ‘low’ aggressive disease based on clinical stage, Gleason grade and prostate specific antigen at diagnosis. Mean plasma 25(OH)D3 concentrations were compared by proportion of African ancestry. Logistic regression was used to calculate multivariable adjusted odds ratios (OR) and 95% confidence intervals (95%CI) for high aggressive CaP by tertile of plasma 25(OH)D3.

Results

AAs with highest percent African ancestry (>95%) had the lowest mean plasma 25(OH)D3 concentrations. Overall, plasma 25(OH)D3 was associated positively with aggressiveness among AA men, an association that was modified by calcium intake (OR_T3vs.T1: 2.23, 95%CI: 1.26–3.95 among men with low calcium intake, and OR_T3vs.T1: 0.19, 95%CI: 0.05–0.70 among men with high calcium intake). Among EAs, the point estimates of the ORs were <1.0 for the upper tertiles with CIs that included the null.

Conclusions

Among AAs, plasma 25(OH)D3 was associated positively with CaP aggressiveness among men with low calcium intake and inversely among men with high calcium intake. The clinical significance of circulating concentrations of 25(OH)D3 and interactions with calcium intake in the AA population warrants further study.     

Quality by design approach for viral clearance by protein a chromatography

Protein A chromatography is widely used as a capture step in monoclonal antibody (mAb) purification processes. Antibodies and Fc fusion proteins can be efficiently purified from the majority of other complex components in harvested cell culture fluid (HCCF). Protein A chromatography is also capable of removing modest levels of viruses and is often validated for viral clearance. Historical data mining of Genentech and FDA/CDER databases systematically evaluated the removal of model viruses by Protein A chromatography. First, we found that for each model virus, removal by Protein A chromatography varies significantly across mAbs, while remains consistent within a specific mAb product, even across the acceptable ranges of the process parameters. In addition, our analysis revealed a correlation between retrovirus and parvovirus removal, with retrovirus data generally possessing a greater clearance factor. Finally, we describe a multivariate approach used to evaluate process parameter impacts on viral clearance, based on the levels of retrovirus‐like particles (RVLP) present among process characterization study samples. It was shown that RVLP removal by Protein A is robust, that is, parameter effects were not observed across the ranges tested. Robustness of RVLP removal by Protein A also correlates with that for other model viruses such as X‐MuLV, MMV, and SV40. The data supports that evaluating RVLP removal using process characterization study samples can establish multivariate acceptable ranges for virus removal by the protein A step for QbD. By measuring RVLP instead of a model retrovirus, it may alleviate some of the technical and economic challenges associated with performing large, design‐of‐experiment (DoE)—type virus spiking studies. This approach could also serve to provide useful insight when designing strategies to ensure viral safety in the manufacturing of a biopharmaceutical product. Biotechnol. Bioeng. 2014;111: 95–103. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Alterations in the red blood cell membrane proteome in alzheimer's subjects reflect disease-related changes and provide insight into altered cell morphology

Background  

Our earlier studies have shown that red blood cell (RBC) morphology in Alzheimer's disease (AD) subjects was altered (> 15% of the RBCs were elongated as compared to 5.9% in normal controls (p < 0.0001)). These results suggested alterations in the RBC membrane architecture in AD subjects, possibly due to RBC-β-amyloid interactions and/or changes in the expression of membrane proteins. We hypothesized that the observed changes could be due to changes in the level of the protein components of the cytoskeleton and those linked to the RBC membrane. To examine this, we performed a proteomic analysis of RBC membrane proteins of AD subjects, and their age-matched controls using one pool of samples from each group, following their separation by SDS-PAGE, in-gel Tryptic digestion, LC-MS-MS of peptides generated, and a label-free approach of semi-quantitative analysis of their relative MS spectral intensities.     

Adaptive resistance of Saccharomyces cerevisiae to chronic treatment with mutagens being due to a dominant mutation

The exposure of mammalian cells or tumors for weeks or months to low non-lethal doses of cytostatic drugs may induce multi-drug resistance, which can be enhanced by a variety of DNA-damaging agents. In yeast multi-drug resistance to a variety of drugs has been observed. DNA-damaging agents have not yet been tested. As the appearance of resistance is the result of long-term exposure, we decided to extend the application of test substances to a period of up to 400 days. In such long-term experiments S. cerevisiae MP1 adapted to treatment with low doses of mutagens. Consistent results were obtained for both alkylating and non-alkylating mutagenic substances. Furthermore, the adaptive resistance to the alkylating agent also adapted cells to the non-alkylating agent, which implies that there may be a single pathway for mutagens with different modes of action. Random spore analysis of adapted yeast cells and the back-cross to the parental wild type indicates that a single dominant mutation is responsible for the adaptive resistance.