Pyruvate metabolism in mitochondria from cucumber cotyledons during early seedling development

Mitochondria were isolated from cucumber cotyledons during earlyseedling growth, and their capacity for pyruvate metabolisminvestigated. The rate of pyruvate oxidation was low. Evidenceis presented that suggests that this is due to low activitiesof the pyruvate transporter. Key words: Cotyledon, cucumber, germination, pyruvate oxidation     

The cytology of apocrine sweat glands

Summary The apocrine sweat glands of cat and monkey have been studied by light and electron microscopy. The apocrine secretory cells of the cat are columnar cells with prominent apical cytoplasmic caps extending into the gland lumen beyond the zone of terminal bars (zonulae occludentes). Many secretory vacuoles are present in the cytoplasm, and they contain acid mucopolysaccharide demonstrable by light microscopy. These secretory vacuoles arise from prosecretory vacuoles in the region of the Golgi apparatus and are liberated from the apical cell surface as in other merocrine cells. The apocrine duct is short and the cells have scant mitochondria. The apocrine secretory cells of the monkey have secretory vacuoles similar to those of the cat but are fewer in number. The monkey apocrine cells also contain unidentified bodies similar to those seen in Langerhans cells of the epidermis. These cells liberate secretory vacuoles in a merocrine manner. Apocrine or decapitation secretion is regarded as an artifact.This investigation was supported in part by United States Public Health Service research grants GM-03784 and GM-10102 from the Institute of General Medical Sciences.     

Evaluation of the influence of anisotropic indirect nuclear spin-spin coupling tensors on effective residual dipolar couplings for model peptides

Residual dipolar couplings (RDCs) observed between nuclear spins in molecules in partially oriented media have become a valuable source of information for NMR spectroscopists seeking to structurally characterize biological macromolecules. Examination of the form of the direct (D) and indirect (J) nuclear spin-spin coupling Hamiltonians indicates that all observed RDCs contain an unknown contribution from the anisotropic part of J (J) in addition to the direct dipolar contribution, D _PQ. Here, we evaluate the influence of J on RDCs through a series of DFT calculations on model peptides. Very small corrections to one-bond RDCs measured between heavy atoms in peptides and proteins are recommended: +0.51% for N-C spin pairs, and +0.45% for C-C spin pairs. The corrections to RDCs involving at least one proton are negligible. This latter point is likely to be equally applicable to nucleic acids and oligosaccharides in addition to peptides and proteins. Finally, the orientations of the J(N, C) and J(C, C) tensors in the molecular framework are reported for glycylglycine.     

The role of changing pH on olfactory success of predator–prey interactions in green shore crabs,Carcinus maenas

Aquatic Ecology - Arguably climate change is one of the biggest challenges faced by many organisms. One of the more significant of these is the decreasing pH level of the ocean, a consequence of...     

CTCF cis-Regulates Trinucleotide Repeat Instability in an Epigenetic Manner: A Novel Basis for Mutational Hot Spot Determination

At least 25 inherited disorders in humans result from microsatellite repeat expansion. Dramatic variation in repeat instability occurs at different disease loci and between different tissues; however, cis-elements and trans-factors regulating the instability process remain undefined. Genomic fragments from the human spinocerebellar ataxia type 7 (SCA7) locus, containing a highly unstable CAG tract, were previously introduced into mice to localize cis-acting “instability elements,” and revealed that genomic context is required for repeat instability. The critical instability-inducing region contained binding sites for CTCF—a regulatory factor implicated in genomic imprinting, chromatin remodeling, and DNA conformation change. To evaluate the role of CTCF in repeat instability, we derived transgenic mice carrying SCA7 genomic fragments with CTCF binding-site mutations. We found that CTCF binding-site mutation promotes triplet repeat instability both in the germ line and in somatic tissues, and that CpG methylation of CTCF binding sites can further destabilize triplet repeat expansions. As CTCF binding sites are associated with a number of highly unstable repeat loci, our findings suggest a novel basis for demarcation and regulation of mutational hot spots and implicate CTCF in the modulation of genetic repeat instability.     

Genetic stability of bone marrow-derived human mesenchymal stromal cells in the Quantum System

Background aimsThe Quantum® Cell Expansion System (Quantum; Terumo BCT, Inc, Lakewood, CO, USA) is a novel hollow fiber-based device that automates and closes the cell culture process, reducing labor intensive tasks such as manual cell culture feeding and harvesting. The manual cell selection and expansion processes for the production of clinical-scale quantities of bone marrow-derived human mesenchymal stromal cells (BM-hMSCs) have been successfully translated onto the Quantum platform previously. The formerly static, manual, in vitro process performed primarily on tissue culture polystyrene substrates may raise the question of whether BM-hMSCs cultured on a hollow fiber platform yields comparable cell quality.MethodsA rigorous battery of assays was used to determine the genetic stability of BM-hMSCs selected and produced with the Quantum. In this study, genetic stability was determined by assessing spectral karyotype, micronucleus formation and tumorigenicity to resolve chromosomal aberrations in the stem cell population. Cell phenotype, adherent growth kinetics and tri-lineage differentiation were also evaluated. HMSC bone marrow aspirates, obtained from three approved donors, were expanded in parallel using T225 culture flasks and the Quantum.ResultsBM-hMSCs harvested from the Quantum demonstrated immunophenotype, morphology and tri-lineage differentiation capacity characteristics consistent with the International Society of Cell Therapy standard for hMSCs. Cell populations showed no malignant neoplastic formation in athymic mice 60 days post-transplant, no clonal chromosomal aberrations were observed and no DNA damage was found as measured by micronucleus formation.ConclusionsQuantum-produced BM-hMSCs are of comparable quality and demonstrate analogous genetic stability to BM-hMSCs cultured on tissue culture polystyrene substrates.     

Role and location of NAD malic enzyme in thermogenic tissues of Araceae

This work was done to discover how those nonphotosynthetic tissues of the Araceae that become thermogenic release, as CO2, carbon recently fixed by phosphoenolpyruvate carboxylase. Extracts of clubs of the spadix of Arum maculatum showed no activity for phosphoenolpyruvate carboxykinase and low activities of NADP malic enzyme. NAD malic enzyme activity in the above extracts and in those of thermogenic tissues of other Araceae was appreciable. Analysis of homogenates of clubs of Typhonium giraldii by differential centrifugation and sucrose gradients showed that NAD malic enzyme was confined to mitochondria. Centrifugation of mitochondria after freezing and thawing left all the NAD malic enzyme in the supernatant. NAD malic enzyme in isolated, intact mitochondria was completely latent, and was completely protected from exogenous trypsin. The responses of this latency and protection to different concentrations of Triton X-100 suggested that none of the NAD malic enzyme was accessible from either the outside or the intermembrane space of the mitochondria. Treatment of excised clubs of A. maculatum with 2-N-butylmalonate largely prevented the development of the rapid respiration responsible for thermogenesis, and severely inhibited dark fixation of 14CO2. The conclusion is that in mature clubs of the Araceae phosphoenolpyruvate is converted to malate in the cytosol by phosphoenolpyruvate carboxylase and NAD malate dehydrogenase, and that this malate then enters the mitochondrial matrix where it is converted to pyruvate by NAD malic enzyme.     

DnaJ/Hsc70 chaperone complexes control the extracellular release of neurodegenerative‐associated proteins

It is now known that proteins associated with neurodegenerative disease can spread throughout the brain in a prionlike manner. However, the mechanisms regulating the trans‐synaptic spread propagation, including the neuronal release of these proteins, remain unknown. The interaction of neurodegenerative disease‐associated proteins with the molecular chaperone Hsc70 is well known, and we hypothesized that much like disaggregation, refolding, degradation, and even normal function, Hsc70 may dictate the extracellular fate of these proteins. Here, we show that several proteins, including TDP‐43, α‐synuclein, and the microtubule‐associated protein tau, can be driven out of the cell by an Hsc70 co‐chaperone, DnaJC5. In fact, DnaJC5 overexpression induced tau release in cells, neurons, and brain tissue, but only when activity of the chaperone Hsc70 was intact and when tau was able to associate with this chaperone. Moreover, release of tau from neurons was reduced in mice lacking the DnaJC5 gene and when the complement of DnaJs in the cell was altered. These results demonstrate that the dynamics of DnaJ/Hsc70 complexes are critically involved in the release of neurodegenerative disease proteins.     

Strengthening Community-Based Vital Events Reporting for Real-Time Monitoring of Under-Five Mortality: Lessons Learned from the Balaka and Salima Districts in Malawi

Background

Malawi ratified a compulsory birth and death registration system in 2009. Until it captures complete coverage of vital events, Malawi relies on other data sources to calculate mortality estimates. We tested a community-based method to estimate annual under-five mortality rates (U5MR) through the Real-Time Monitoring of Under-Five Mortality (RMM) project in Malawi. We implemented RMM in two phases, and conducted an independent evaluation of phase one after 21 months of implementation. We present results of the phase two validation that covers the full project time span, and compare the results to those of the phase one validation.

Methods and Findings

We assessed the completeness of the counts of births and deaths and the accuracy of disaggregated U5MR from the community-based method against a retrospective full pregnancy history for rolling twelve-month periods after the independent evaluation. We used full pregnancy histories collected through household interviews carried out between November 2013 and January 2014 as the validation data source. Health Surveillance Agents (HSAs) across the 160 catchment areas submitted routine reports on pregnancies, births, and deaths consistently. However, for the 15-month implementation period post-evaluation, average completeness of birth event reporting was 76%, whereas average completeness of death event reporting was 67% relative to that expected from a comparable pregnancy history. HSAs underestimated the U5MR by an average of 21% relative to that estimated from a comparable pregnancy history.

Conclusions

On a medium scale, the community-based RMM method in Malawi produced substantial underestimates of annualized U5MR relative to those obtained from a full pregnancy history, despite the additional incentives and quality-control activities. We were not able to achieve an optimum level of incentive and support to make the system work while ensuring sustainability. Lessons learned from the implementation of RMM can inform programs supporting community-based interventions through HSAs in Malawi.