Hydration of biological molecules: lipids versus nucleic acids

We used FTIR spectroscopy to comparatively study the hydration of films prepared from nucleic acids (DNA and double-stranded RNA) and lipids (phosphatidylcholines and phosphatidylethanolamines chosen as the most abundant ones) at room temperature by varying the ambient relative humidity in terms of solvent-induced structural changes. The nucleic acids and phospholipids both display examples of polymorphism on the one hand and structural conservatism on the other; even closely related representatives behave differently in this respect. DNA undergoes a hydration-driven A-B conformational transition, but RNA maintains an A-like structure independently of the water activity. Similarly, a main transition between the solid and liquid-crystalline phases can be induced lyotropically in certain phosphatidylcholines, while their phosphatidylethanolamine counterparts do not exhibit chain melting under the same conditions. A principal difference concerning the structural changes that occur in the studied biomolecules is given by the relevant water-substrate stoichiometries. These are rather high in DNA and often low in phospholipids, suggesting different mechanisms of action of the hydration water that appears to induce structural changes on global- and local-mode levels, respectively.     

Development of EGF-conjugated liposomes for targeted delivery of boronated DNA-binding agents

Liposomes are of interest as drug delivery tools for therapy of cancer and infectious diseases. We investigated conjugation of epidermal growth factor, EGF, to liposomes using the micelle-transfer method. EGF was conjugated to the distal end of PEG-DSPE lipid molecules in a micellar solution and the EGF-PEG-DSPE lipids were then transferred to preformed liposomes, either empty or containing the DNA-binding compound, water soluble acridine, WSA. We found that the optimal transfer conditions were a 1-h incubation at 60 degrees C. The final conjugate, (125)I-EGF-liposome-WSA, contained approximately 5 mol % PEG, 10-15 EGF molecules at the liposome surface, and 10(4) to 10(5) encapsulated WSA molecules could be loaded. The conjugate was shown to have EGF-receptor-specific cellular binding in cultured human glioma cells.     

Adaptation of a South American malaria vector to laboratory colonization suggests faster-male evolution for mating ability

Background  

Anopheles (Nyssorhynchus) albitarsis (Diptera: Culicidae) is one of the very few South American mosquito vectors of malaria successfully colonized in the laboratory. These vectors are very hard to breed because they rarely mate in artificial conditions. A few years ago a free-mating laboratory colony of An. albitarsis sensu stricto was established after about 30 generations of artificial-mating. To begin to understand the process of adaptation of these malaria vectors to the laboratory we have compared the insemination rates of colony mosquitoes to those from the original population in both artificial and free-mating crosses. We also carried out crossing experiments between the two types of mosquitoes for a preliminary analysis of the genetic basis of such adaptation.     

The Association of the Vanin-1 N131S Variant with Blood Pressure Is Mediated by Endoplasmic Reticulum-Associated Degradation and Loss of Function

High blood pressure (BP) is the most common cardiovascular risk factor worldwide and a major contributor to heart disease and stroke. We previously discovered a BP-associated missense SNP (single nucleotide polymorphism)–rs2272996–in the gene encoding vanin-1, a glycosylphosphatidylinositol (GPI)-anchored membrane pantetheinase. In the present study, we first replicated the association of rs2272996 and BP traits with a total sample size of nearly 30,000 individuals from the Continental Origins and Genetic Epidemiology Network (COGENT) of African Americans (P = 0.01). This association was further validated using patient plasma samples; we observed that the N131S mutation is associated with significantly lower plasma vanin-1 protein levels. We observed that the N131S vanin-1 is subjected to rapid endoplasmic reticulum-associated degradation (ERAD) as the underlying mechanism for its reduction. Using HEK293 cells stably expressing vanin-1 variants, we showed that N131S vanin-1 was degraded significantly faster than wild type (WT) vanin-1. Consequently, there were only minimal quantities of variant vanin-1 present on the plasma membrane and greatly reduced pantetheinase activity. Application of MG-132, a proteasome inhibitor, resulted in accumulation of ubiquitinated variant protein. A further experiment demonstrated that atenolol and diltiazem, two current drugs for treating hypertension, reduce the vanin-1 protein level. Our study provides strong biological evidence for the association of the identified SNP with BP and suggests that vanin-1 misfolding and degradation are the underlying molecular mechanism.     

The Impact of Stressful Life Events on Excessive Alcohol Consumption in the French Population: Findings from the GAZEL Cohort Study

Background

Major life changes may play a causative role in health through lifestyle factors, such as alcohol. The objective was to examine the impact of stressful life events on heavy alcohol consumption among French adults.

Methods

Trajectories of excessive alcohol consumption in 20,625 employees of the French national gas and electricity company for up to 5 years before and 5 years after an event, with annual measurements from 1992. We used repeated measures analysis of time series data indexed to events, employing generalized estimating equations.

Results

For women, excessive alcohol use increased before important purchase (p = 0.021), children leaving home (p<0.001), and death of loved ones (p = 0.03), and decreased before widowhood (p = 0.015); in the year straddling the event, increased consumption was observed for important purchase (p = 0.018) and retirement (p = 0.002); at the time of the event, consumption decreased for marriage (p = 0.002), divorce, widowhood, and death of loved one (all p<0.001), and increased for retirement (p = 0.035). For men, heavy alcohol consumption increased in the years up to and surrounding the death of loved ones, retirement, and important purchase (all p<0.001), and decreased after (all p<0.001, except death of loved one: p = 0.006); at the time of the event, consumption decreased for all events except for children leaving home and retirement, where we observed an increase (all p<0.001). For women and men, heavy alcohol consumption decreased prior to marriage and divorce and increased after (all p<0.001, except for women and marriage: p = 0.01).

Conclusion

Stressful life events promote healthy and unhealthy alcohol consumption. Certain events impact alcohol intake temporarily while others have longer-term implications. Research should disentangle women''s and men''s distinct perceptions of events over time.     

Ultrastructural localization of amiloride-sensitive sodium channels and Na+,K(+)-ATPase in the rat's olfactory epithelial surface

Several studies have indicated that olfactory responses are impeded by amiloride. Therefore, it was of interest to see whether, and if so which, olfactory epithelial cellular compartments have amiloride- sensitive structures. Using ultrastructural methods that involved rapid freezing, freeze-substitution and low temperature embedding of olfactory epithelia, this study shows that, in the rat, this tissue is immunoreactive to antibodies against amiloride sensitive Na(+)- channels. However, microvilli of olfactory supporting cells, as opposed to receptor cilia, contained most of the immunoreactive sites. Apices from which the microvilli sprout and receptor cell dendritic knobs had much less if any of the amiloride-antibody binding sites. Using a direct ligand-binding cytochemical method, this study also confirms earlier ones that showed that olfactory receptor cell cilia have Na+, K(+)-ATPase. It is proposed that supporting cell microvilli and the receptor cilia themselves have mechanisms, different but likely complementary, that participate in regulating the salt concentration around the receptor cell cilia. In this way, both structures help to provide the ambient mucous environment for receptor cells to function properly. This regulation of the salt concentration of an ambient fluid environment is a function that the olfactory epithelium shares with cells of transporting epithelia, such as those of kidney.      

Sodium chloride impacts glycosylation and N- and O-glycan site occupancy of an Fc-fusion protein

Fc-fusion proteins are highly complex molecules, difficult to manufacture at scale. In this work, undesired proteoforms were detected during the manufacture of a therapeutic fusion protein produced in CHO cells. These species were characterized using gel electrophoresis, size exclusion chromatography and liquid chromatography-mass spectrometry leading to the identification of low molecular weight proteoforms presenting low N- and O-glycan site occupancy, as well as a low sialylation content. Upstream process parameters were investigated, and fusion protein quality was shown to be linked to the sodium chloride content of the medium. A mitigation strategy was developed to avoid formation of unwanted glyco-variants, resulting in an increased yield of highly glycosylated Fc-fusion protein. The effect of sodium chloride was shown to be independent of the osmolality increase and was hypothesized to be linked to a modulation of Golgi acidity, which is required for the correct localization and function of glycosyltransferases. Altogether, this study highlights the importance of the salt balance in cell culture media used to produce highly sialylated and occupied glycoproteins, helping to maximize the yield and increase robustness of processes aiming at producing biopharmaceutical complex therapeutic molecules.     

Silver Impregnation of Alzheimer's Neurofibrillary Changes Counterstained for Basophilic Material and Lipofuscin Pigment

A method is described in which selective silver staining of Alzheimer's neurofibrillary changes is combined with staining of cell nuclei, Nissl material, and lipofuscin granules. Formalin fixed, paraffin embedded sections of human autopsy tissue are silver stained according to a method proposed by Gallyas. Lipofuscin is stained by crotonaldehyde fuchsin following performic acid oxidation. Nissl substance is visualized by either Darrow red or gallocyanin-chrome alum staining. Architectonic units showing the specific pathology and the neuronal types prone to develop the neurofibrillary changes can be recognized using this technique.