Radiation inactivation target size of rat adipocyte glucose transporters in the plasma membrane and intracellular pools

The in situ assembly states of the glucose transport carrier protein in the plasma membrane and in the intracellular (microsomal) storage pool of rat adipocytes were assessed by studying radiation-induced inactivation of the D-glucose-sensitive cytochalasin B binding activities. High energy radiation inactivated the glucose-sensitive cytochalasin B binding of each of these membrane preparations by reducing the total number of the binding sites without affecting the dissociation constant. The reduction in total number of binding sites was analyzed as a function of radiation dose based on target theory, from which a radiation-sensitive mass (target size) was calculated. When the plasma membranes of insulin-treated adipocytes were used, a target size of approximately 58,000 daltons was obtained. For adipocyte microsomal membranes, we obtained target sizes of approximately 112,000 and 109,000 daltons prior to and after insulin treatment, respectively. In the case of microsomal membranes, however, inactivation data showed anomalously low radiation sensitivities at low radiation doses, which may be interpreted as indicating the presence of a radiation-sensitive inhibitor. These results suggest that the adipocyte glucose transporter occurs as a monomer in the plasma membrane while existing in the intracellular reserve pool either as a homodimer or as a stoichiometric complex with a protein of an approximately equal size.     

Artifacts following gold staining of Western-blotted membranes

Protein samples were separated by sodium dodecyl-sulfate-polyacrylamide gel electrophoresis, electrophoretically transferred to nitrocellulose membranes, and stained with colloidal gold. Three artifact bands appeared demonstrating apparent molecular weights of 58,000, 63,000, and 65,000. The appearance of these bands was due to oxidized dithiothreitol used to denature the proteins prior to electrophoresis. The appearance of the artifact bands could be avoided by the addition of excess iodoacetamide to the denatured protein sample and by limiting staining time to 1.5 h at 13 V/cm.     

Addition of Cryoprotectant Significantly Alters the Epididymal Sperm Proteome

Although cryopreservation has been developed and optimized over the past decades, it causes various stresses, including cold shock, osmotic stress, and ice crystal formation, thereby reducing fertility. During cryopreservation, addition of cryoprotective agent (CPA) is crucial for protecting spermatozoa from freezing damage. However, the intrinsic toxicity and osmotic stress induced by CPA cause damage to spermatozoa. To identify the effects of CPA addition during cryopreservation, we assessed the motility (%), motion kinematics, capacitation status, and viability of epididymal spermatozoa using computer-assisted sperm analysis and Hoechst 33258/chlortetracycline fluorescence staining. Moreover, the effects of CPA addition were also demonstrated at the proteome level using two-dimensional electrophoresis. Our results demonstrated that CPA addition significantly reduced sperm motility (%), curvilinear velocity, viability (%), and non-capacitated spermatozoa, whereas straightness and acrosome-reacted spermatozoa increased significantly (p < 0.05). Ten proteins were differentially expressed (two decreased and eight increased) (>3 fold, p < 0.05) after CPA, whereas NADH dehydrogenase flavoprotein 2, f-actin-capping protein subunit beta, superoxide dismutase 2, and outer dense fiber protein 2 were associated with several important signaling pathways (p < 0.05). The present study provides a mechanistic basis for specific cryostresses and potential markers of CPA-induced stress. Therefore, these might provide information about the development of safe biomaterials for cryopreservation and basic ground for sperm cryopreservation.     

Molecular Reclassification of Crohn’s Disease: A Cautionary Note on Population Stratification

Complex human diseases commonly differ in their phenotypic characteristics, e.g., Crohn’s disease (CD) patients are heterogeneous with regard to disease location and disease extent. The genetic susceptibility to Crohn’s disease is widely acknowledged and has been demonstrated by identification of over 100 CD associated genetic loci. However, relating CD subphenotypes to disease susceptible loci has proven to be a difficult task. In this paper we discuss the use of cluster analysis on genetic markers to identify genetic-based subgroups while taking into account possible confounding by population stratification. We show that it is highly relevant to consider the confounding nature of population stratification in order to avoid that detected clusters are strongly related to population groups instead of disease-specific groups. Therefore, we explain the use of principal components to correct for population stratification while clustering affected individuals into genetic-based subgroups. The principal components are obtained using 30 ancestry informative markers (AIM), and the first two PCs are determined to discriminate between continental origins of the affected individuals. Genotypes on 51 CD associated single nucleotide polymorphisms (SNPs) are used to perform latent class analysis, hierarchical and Partitioning Around Medoids (PAM) cluster analysis within a sample of affected individuals with and without the use of principal components to adjust for population stratification. It is seen that without correction for population stratification clusters seem to be influenced by population stratification while with correction clusters are unrelated to continental origin of individuals.     

Drought mildly reduces plant dominance in a temperate prairie ecosystem across years

1. Shifts in dominance and species reordering can occur in response to global change. However, it is not clear how altered precipitation and disturbance regimes interact to affect species composition and dominance.
2. We explored community‐level diversity and compositional similarity responses, both across and within years, to a manipulated precipitation gradient and annual clipping in a mixed‐grass prairie in Oklahoma, USA. We imposed seven precipitation treatments (five water exclusion levels [?20%, ?40%, ?60%, ?80%, and ?100%], water addition [+50%], and control [0% change in precipitation]) year‐round from 2016 to 2018 using fixed interception shelters. These treatments were crossed with annual clipping to mimic hay harvest.
3. We found that community‐level responses were influenced by precipitation across time. For instance, plant evenness was enhanced by extreme drought treatments, while plant richness was marginally promoted under increased precipitation.
4. Clipping promoted species gain resulting in greater richness within each experimental year. Across years, clipping effects further reduced the precipitation effects on community‐level responses (richness and evenness) at both extreme drought and added precipitation treatments.
5. Synthesis: Our results highlight the importance of studying interactive drivers of change both within versus across time. For instance, clipping attenuated community‐level responses to a gradient in precipitation, suggesting that management could buffer community‐level responses to drought. However, precipitation effects were mild and likely to accentuate over time to produce further community change.

     

Failed Stabilization for Long-Term Potentiation in the Auditory Cortex of Fmr1 Knockout Mice

Fragile X syndrome is a developmental disorder that affects sensory systems. A null mutation of the Fragile X Mental Retardation protein 1 (Fmr1) gene in mice has varied effects on developmental plasticity in different sensory systems, including normal barrel cortical plasticity, altered ocular dominance plasticity and grossly impaired auditory frequency map plasticity. The mutation also has different effects on long-term synaptic plasticity in somatosensory and visual cortical neurons, providing insights on how it may differentially affect the sensory systems. Here we present evidence that long-term potentiation (LTP) is impaired in the developing auditory cortex of the Fmr1 knockout (KO) mice. This impairment of synaptic plasticity is consistent with impaired frequency map plasticity in the Fmr1 KO mouse. Together, these results suggest a potential role of LTP in sensory map plasticity during early sensory development.