The Effect of Magnesium and Calcium Ions on Adenosine Triphosphatases from Wheat and Oat Roots at Different pH

Microsomal fractions from wheat (Triticum vulgare) and oat (Avena sativa) roots were used to study Mg²⁺ and Ca²⁺ activated adenosine triphosphatases, their dependence of pH, and how Mg²⁺ and Ca²⁺ compete or add in stimulation and inhibition. Wheat gives a high proportion of Ca²⁺ stimulated ATPase. Less effect is obtained with Mg²⁺. The characteristics of oar ATPase are the reverse. The ATPase from the wheat roots depends on the mineral nutrition. A kinetïc analysis shows one site, where Mg²⁺ and Ca²⁺ at low concentrations (or complexes between the di-valents and ATP) cooperate in the activation of the ATPase. The action of this site is more dearly expressed at pH 6.0 than at 6.8, and more clearly in the preparations from low salt roots than in those from high salt conditions. In another site, which is particularly evident in preparations from high salt roots tested at pH 6.8, high concentrations of Mg²⁺ inhibit the ATPase; this inhibition is competitively relieved by Ca²⁺. The specific activity of the ATPase from high salt roots of wheat is higher than that from low salt roots, although the amount of protein of the fraction studied remains the same, when calculated per g fresh weight of the roots.     

A 15-kilobase-pair region of the human cytomegalovirus genome which includes US1 through US13 is dispensable for growth in cell culture.

The genome of a temperature-sensitive, DNA-negative mutant of human cytomegalovirus was cloned in cosmids and analyzed by restriction endonuclease mapping and Southern blotting. The data presented show that in the mutant genome, nearly half of the short segment was deleted (14.3 to 15.1 kb; map position, 0.83 to 0.9), including the genes for a potential immediate early protein (US3) and a structural glycoprotein of 47 to 52 kDa (US6 through US11). The deleted DNA region was replaced by a 20.8- to 21.6-kb fragment that represented an inverted repetition of the retained portion of the short segment (map position, 0.92 to 1.0), suggesting that US20 through US36 were duplicated in the mutant. Northern (RNA) blots with appropriate probes of total cell RNA extracted from mutant-infected cells confirmed the absence of mRNAs originating from US3 or from US8 through US11. It is concluded that the deleted genes are dispensable for human cytomegalovirus replication in cell culture.     

Isolation of prolyl-tRNA synthetase as a free form and as a form associated with glutamyl-tRNA synthetase.

Rat liver prolyl-tRNA synthetase was purified as a dimer of M(r) 60,000 subunits not associated with other aminoacyl-tRNA synthetases and as a form associated with glutamyl-tRNA synthetase. Proteolysis of the dimeric enzyme generated a less active form with M(r) 52,000 subunits and an inactive form with M(r) 40,000 subunits. A second species was isolated with polypeptides of M(r) 60,000 and 150,000. This form dissociated during gel filtration chromatography being partially resolved into the M(r) 150,000 and 60,000 components; glutamyl-tRNA synthetase was associated with the larger polypeptide and prolyl-tRNA synthetase with the smaller component. Antibodies against the M(r) 60,000 polypeptide reacted with the M(r) 60,000 and 150,000 polypeptides. Gel filtration of extracts revealed multiple forms of prolyl- and glutamyl-tRNA synthetase. Antibody against the M(r) 60,000 component detected the M(r) 60,000 and 150,000 polypeptides throughout the chromatogram; these forms could be partially separated by polyethylene glycol fractionation. The M(r) 150,000 and 60,000 polypeptides were detected by Western blot analysis of crude extracts prepared under several conditions. Antibody to prolyl-tRNA synthetase reacted with a M(r) 150,000 polypeptide of the aminoacyl-tRNA synthetase core complex identified previously as glutamyl-tRNA synthetase.     

Impact of Natural Variations in Freeze-Drying Parameters on Product Temperature History: Application of Quasi Steady-State Heat and Mass Transfer and Simple Statistics

Inter- and intra-batch variability in heat and mass transfer during the drying phase of lyophilization is well recognized. Heat transfer variability between individual vials in the same batch arise from both different positions in the vial array and from variations in the bottom contour of the vials, both effects contributing roughly equally to variations in the effective heat transfer coefficient of the vials, K_v. Both effects can be measured in the laboratory, and variations in average K_v values as a function of vial position in the array for lab and production can be calculated by use of the simple steady-state heat and mass transfer theory. Typically, in the laboratory dryer, vials on the edge of the array, “edge vials,” run 2–4°C warmer than “center vials,” but differences between laboratory and manufacturing temperatures are modest. The variability in mass transfer can be assigned to major variations in ice nucleation temperature (both intra-batch and inter-batch), including major differences between laboratory and manufacturing. The net effect of all random variations, for each class of vial, can be evaluated by a simple statistical model-propagation of error, which then allows prediction of the distribution in product temperatures and drying times, and therefore prediction of percent of vials dry and percent of vials collapsed and proximity to the edge of failure for a given process. Good agreement between theoretical and experimentally determined maximum temperatures in primary drying and percent collapsed product demonstrates the calculations have useful accuracy.     

Molecular phylogenetic species delimitation in the aquatic genus Ottelia (Hydrocharitaceae) reveals cryptic diversity within a widespread species

Journal of Plant Research - Ottelia, a pantropical genus of aquatic plants belonging to the family Hydrocharitaceae, includes several narrowly distributed taxa in Asia. Although the Asian species...     

Adaptation of CD8 T Cell Responses to Changing HIV-1 Sequences in a Cohort of HIV-1 Infected Individuals Not Selected for a Certain HLA Allele

HIV evades CD8 T cell mediated pressure by viral escape mutations in targeted CD8 T cell epitopes. A viral escape mutation can lead to a decline of the respective CD8 T cell response. Our question was what happened after the decline of a CD8 T cell response and - in the case of viral escape – if a new CD8 T cell response towards the mutated antigen could be generated in a population not selected for certain HLA alleles. We studied 19 antiretroviral-naïve HIV-1 infected individuals with different disease courses longitudinally. A median number of 12 (range 2-24) CD8 T cell responses towards Gag and Nef were detected per study subject. A total of 30 declining CD8 T cell responses were studied in detail and viral sequence analyses showed amino acid changes in 25 (83%) of these. Peptide titration assays and definition of optimal CD8 T cell epitopes revealed 12 viral escape mutations with one de-novo response (8%). The de-novo response, however, showed less effector functions than the original CD8 T cell response. In addition we identified 4 shifts in immunodominance. For one further shift in immunodominance, the mutations occurred outside the optimal epitope and might represent processing changes. Interestingly, four adaptations to the virus (the de-novo response and 3 shifts in immunodominance) occurred in the group of chronically infected progressors. None of the subjects with adaptation to the changing virus carried the HLA alleles B57, B*58:01 or B27. Our results show that CD8 T cell responses adapt to the mutations of HIV. However it was limited to only 20% (5 out of 25) of the epitopes with viral sequence changes in a cohort not expressing protective HLA alleles.     

Capsid Mutated Adeno-Associated Virus Delivered to the Anterior Chamber Results in Efficient Transduction of Trabecular Meshwork in Mouse and Rat

Background

Adeno associated virus (AAV) is well known for its ability to deliver transgenes to retina and to mediate improvements in animal models and patients with inherited retinal disease. Although the field is less advanced, there is growing interest in AAV’s ability to target cells of the anterior segment. The purpose of our study was to fully articulate a reliable and reproducible method for injecting the anterior chamber (AC) of mice and rats and to investigate the transduction profiles of AAV2- and AAV8-based capsid mutants containing self-complementary (sc) genomes in the anterior segment of the eye.

Methodology/Principle Findings

AC injections were performed in C57BL/6 mice and Sprague Dawley rats. The cornea was punctured anterior of the iridocorneal angle. To seal the puncture site and to prevent reflux an air bubble was created in the AC. scAAVs expressing GFP were injected and transduction was evaluated by immunohistochemistry. Both parent serotype and capsid modifications affected expression. scAAV2- based vectors mediated efficient GFP-signal in the corneal endothelium, ciliary non-pigmented epithelium (NPE), iris and chamber angle including trabecular meshwork, with scAAV2(Y444F) and scAAV2(triple) being the most efficient.

Conclusions/Significance

This is the first study to semi quantitatively evaluate transduction of anterior segment tissues following injection of capsid-mutated AAV vectors. scAAV2- based vectors transduced corneal endothelium, ciliary NPE, iris and trabecular meshwork more effectively than scAAV8-based vectors. Mutagenesis of surface-exposed tyrosine residues greatly enhanced transduction efficiency of scAAV2 in these tissues. The number of Y-F mutations was not directly proportional to transduction efficiency, however, suggesting that proteosomal avoidance alone may not be sufficient. These results are applicable to the development of targeted, gene-based strategies to investigate pathological processes of the anterior segment and may be applied toward the development of gene-based therapies for glaucoma and acquired or inherited corneal anomalies.     

Low levels of taurine introgression in the current Brazilian Nelore and Gir indicine cattle populations

Background

Nelore and Gir are the two most important indicine cattle breeds for production of beef and milk in Brazil. Historical records state that these breeds were introduced in Brazil from the Indian subcontinent, crossed to local taurine cattle in order to quickly increase the population size, and then backcrossed to the original breeds to recover indicine adaptive and productive traits. Previous investigations based on sparse DNA markers detected taurine admixture in these breeds. High-density genome-wide analyses can provide high-resolution information on the genetic composition of current Nelore and Gir populations, estimate more precisely the levels and nature of taurine introgression, and shed light on their history and the strategies that were used to expand these breeds.

Results

We used the high-density Illumina BovineHD BeadChip with more than 777 K single nucleotide polymorphisms (SNPs) that were reduced to 697 115 after quality control filtering to investigate the structure of Nelore and Gir populations and seven other worldwide populations for comparison. Multidimensional scaling and model-based ancestry estimation clearly separated the indicine, European taurine and African taurine ancestries. The average level of taurine introgression in the autosomal genome of Nelore and Gir breeds was less than 1% but was 9% for the Brahman breed. Analyses based on the mitochondrial SNPs present in the Illumina BovineHD BeadChip did not clearly differentiate taurine and indicine haplotype groupings.

Conclusions

The low level of taurine ancestry observed for both Nelore and Gir breeds confirms the historical records of crossbreeding and supports a strong directional selection against taurine haplotypes via backcrossing. Random sampling in production herds across the country and subsequent genotyping would be useful for a more complete view of the admixture levels in the commercial Nelore and Gir populations.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0109-5) contains supplementary material, which is available to authorized users.     

Solution-Mediated Phase Transformation of Haloperidol Mesylate in the Presence of Sodium Lauryl Sulfate

Forming a salt is a common way to increase the solubility of a poorly soluble compound. However, the solubility enhancement gained by salt formation may be lost due to solution-mediated phase transformation (SMPT) during dissolution. The SMPT of a salt can occur due to a supersaturated solution near the dissolving surface caused by pH or other solution conditions. In addition to changes in pH, surfactants are also known to affect SMPT. In this study, SMPT of a highly soluble salt, haloperidol mesylate, at pH 7 in the presence of a commonly used surfactant, sodium lauryl sulfate (SLS), was investigated. Dissolution experiments were performed using a flow-through dissolution apparatus with solutions containing various concentrations of SLS. Compacts of haloperidol mesylate were observed during dissolution in the flow-through apparatus using a stereomicroscope. Raman microscopy was used to characterize solids. The dissolution of haloperidol mesylate was significantly influenced by the addition of sodium lauryl sulfate. In conditions where SMPT was expected, the addition of SLS at low concentrations (0.1–0.2 mM) reduced the dissolution of haloperidol mesylate. In solutions containing concentrations of SLS above the critical micelle concentration (CMC) (10–15 mM), the dissolution of haloperidol mesylate increased compared to below the CMC. The solids recovered from solubility experiments of haloperidol mesylate indicated that haloperidol free base precipitated at all concentrations of SLS. Above 5 mM of SLS, Raman microscopy suggested a new form, perhaps the estolate salt. The addition of surfactant in solids that undergo solution-mediated phase transformation can add complexity to the dissolution profiles and conversion.     

Structural basis for the stereospecific inhibition of the dual proline/hydroxyproline catabolic enzyme ALDH4A1 by trans‐4‐hydroxy‐L‐proline

Aldehyde dehydrogenase 4A1 (ALDH4A1) catalyzes the final steps of both proline and hydroxyproline catabolism. It is a dual substrate enzyme that catalyzes the NAD⁺‐dependent oxidations of L‐glutamate‐γ‐semialdehyde to L‐glutamate (proline metabolism), and 4‐hydroxy‐L‐glutamate‐γ‐semialdehyde to 4‐erythro‐hydroxy‐L‐glutamate (hydroxyproline metabolism). Here we investigated the inhibition of mouse ALDH4A1 by the six stereoisomers of proline and 4‐hydroxyproline using steady‐state kinetics and X‐ray crystallography. Trans‐4‐hydroxy‐L‐proline is the strongest of the inhibitors studied, characterized by a competitive inhibition constant of 0.7 mM, followed by L‐proline (1.9 mM). The other compounds are very weak inhibitors (approximately 10 mM or greater). Insight into the selectivity for L‐stereoisomers was obtained by solving crystal structures of ALDH4A1 complexed with trans‐4‐hydroxy‐L‐proline and trans‐4‐hydroxy‐D‐proline. The structures suggest that the 10‐fold greater preference for the L‐stereoisomer is due to a serine residue that hydrogen bonds to the amine group of trans‐4‐hydroxy‐L‐proline. In contrast, the amine group of the D‐stereoisomer lacks a direct interaction with the enzyme due to a different orientation of the pyrrolidine ring. These results suggest that hydroxyproline catabolism is subject to substrate inhibition by trans‐4‐hydroxy‐L‐proline, analogous to the known inhibition of proline catabolism by L‐proline. Also, drugs targeting the first enzyme of hydroxyproline catabolism, by elevating the level of trans‐4‐hydroxy‐L‐proline, may inadvertently impair proline catabolism by the inhibition of ALDH4A1.