Rapid separation of low molecular weight solutes from liposomes without dilution

Liposomes can be separated from low molecular weight solutes on minicolumns of Sephadex G-50 made from the barrels of 1- or 5-ml plastic syringes. Excess fluid is first removed from the Sephadex beads by centrifugation and a mixture of liposomal entrapped and free solute is applied to the column bed. The centrifugation is repeated forcing the liposomal material through the column into a test tube while the free solute is quantitatively retained in the Sephadex. The procedure is applicable to a variety of solutes and 92 to 100% recovery is achieved for both charged and neutral liposomes. This technique has advantages over other methods for separating extraliposomal solutes from liposomes. Numerous samples can be processed simultaneously within minutes with no dilution of the liposomal preparation. Nonentrapped solute within the Sephadex can be easily recovered in a small volume of water or buffer.     

Functional Effects of a Restrictive-Cardiomyopathy-Linked Cardiac Troponin I Mutation (R145W) in Transgenic Mice

The human cardiac troponin I (hcTnI) mutation R145W has been associated with restrictive cardiomyopathy. In this study, simultaneous measurements of ATPase activity and force in skinned papillary fibers from hcTnI R145W transgenic mice (Tg-R145W) were explored. Tg-R145W fibers showed an ∼ 13-16% increase in maximal Ca²⁺-activated force and ATPase activity compared to hcTnI wild-type transgenic mice. The force-generating cross-bridge turnover rate (g) and the energy cost (ATPase/force) were the same in all groups of fibers. Also, the Tg-R145W fibers showed a large increase in the Ca²⁺ sensitivity of both force development and ATPase. In intact fibers, the mutation caused prolonged force and intracellular [Ca²⁺] transients and increased time to peak force. Analysis of force and Ca²⁺ transients showed that there was a 40% increase in peak force in Tg-R145W muscles, which was likely due to the increased Ca²⁺ transient duration. The above cited results suggest that: (1) there would be an increase in resistance to ventricular filling during diastole resulting from the prolonged force and Ca²⁺ transients that would result in a decrease in ventricular filling (diastolic dysfunction); and (2) there would be a large (approximately 53%) increase in force during systole, which may help to partly compensate for diastolic dysfunction. These functional results help to explain the mechanisms by which these mutations give rise to a restrictive phenotype.     

DNA methylation changes in photoperiod-thermo-sensitive male sterile rice PA64S under two different conditions

Epigenetic modification can occur at a high frequency in crop plants and might generate phenotypic variation without changes in DNA sequences. DNA methylation is an important epigenetic modification that may contribute to environmentally-induced phenotypic variations by regulating gene expression. Rice Photoperiod-Thermo-Sensitive Genic Male Sterile (PTGMS) lines can transform from sterility to fertility under lower temperatures and short-day (SD) conditions during anther development. So far, little is known about the DNA methylation variation of PTGMS throughout the genome in rice. In this study, we investigated DNA cytosine methylation alterations in the young panicles of PTGMS line PA64S under two different conditions using methylation sensitive amplified polymorphism (MSAP) method. Compared with the DNA methylation level of PA64S under lower temperatures and SD conditions (fertility), higher methylation was observed in PA64S (sterility). The sequences of 25 differentially amplified fragments were successfully obtained and annotated. Three methylated fragments, which are homologous to D2, NAD7 and psaA, were confirmed by bisulfite sequencing and their expression levels were also evaluated by qPCR. Real time quantitative PCR analysis revealed that five of the six selected methylated genes were downregulated in PA64S (sterility). These results suggested that DNA methylation may be involved in the sterility–fertility transition of PA64S under two different environmental conditions.     

Isolation and characterization of cDNA clones encoding a 18.8 kDa polypeptide,the product of the gene psaL,associated with photosystem I reaction center from spinach

Several cDNA clones encoding subunit XI of photosystem I reaction center (PSI-L) have been isolated from two INE" BORDER="0">gt11 expression libraries based on polyadenylated RNA of spinach seedlings illuminated for 4 and 16 h, respectively. The precursor polypeptide made from these recombinant DNAs in vitro can be efficiently imported into isolated spinach chloroplasts. It is correctly processed to the size of the authentic polypeptide and integrates into the photosystem I assembly. The 834 nucleotide sequence of the longest cDNA insert encodes a precursor polypeptide of 24 kDa (216 residues) and a mature protein of probably 18.8 kDa (169 residues). Hydropathy analysis suggests that the polypeptide contains two transmembrane segments. The protein appears to originate in a single-copy gene in spinach and to be decoded from RNA species of ca. 900 bases.     

MHC Class I Immunopeptidome: Past,Present, and Future

In the 35 years since the revelation that short peptides bound to major histocompatibility complex class I and II molecules are the secret of the major histocompatibility complex–restricted nature of T-cell recognition, there has been enormous progress in characterizing the immunopeptidome, the repertoire of peptide presented for immunosurveillance. Here, the major milestones in the journey are marked, the contribution of proteasome-mediated splicing to the immunopeptidome is discussed, and exciting recent findings relating the immunopeptidome to the translatome revealed by ribosome profiling (RiboSeq) is detailed. Finally, what is needed for continued progress is opined about, which includes the infusion of talented young scientists into the antigen-processing field, currently undergoing a renaissance; thanks in part to the astounding success of T-cell–based cancer immunotherapy.     

CHANGES IN PHOTOSYNTHESIS IN RESPONSE TO COMBINED IRRADIANCE AND TEMPERATURE STRESS IN CYANOBACTERIAL SURFACE WATERBLOOMS1

Buoyant cyanobacteria, previously mixed throughout the water column, float to the lake surface and form a surface waterbloom when mixing subsides. At the surface, the cells are exposed to full sunlight, and this abrupt change in photon irradiance may induce photoinhibition; at the same time, temperature rises as well. This study investigated the damaging effects of this increase in temperature as well as the ecologically more relevant combination of both an increased temperature and a high photon irradiance. Analysis of surface blooms with oxygen microelectrodes showed that integrated oxygen contents that are dependent on the balance of photosynthetic oxygen evolution and respiratory oxygen uptake decreased when temperature was raised above the lake temperature. Gross rates of photosynthesis were unaffected by temperatures up to of 35°C; hence, a moderate increase in temperature mainly stimulated oxygen uptake. Preincubation of cells of the cyanobacterium Anabaena flos-aquae (Lyngb.) de Brébisson at temperatures up to 35°C did not affect the subsequent measurement of rates of net photosynthesis. Another 5°C rise in temperature severely damaged the photosynthetic apparatus. Failure to restore net rates of photosynthesis was coupled to a strong quenching of the ratio of variable to maximum fluorescence, F_v/F_m, that was the result of a rise in F_o. A combination of high temperature and high photon irradiance was more damaging than high temperature alone. In contrast, low photon irradiances offered substantial protection against heat injury of the photosynthetic apparatus. I conclude from this study that because cyanobacteria usually are acclimated to low average irradiance prior to bloom formation, there is a reasonable risk of chronic photoinhibition. The increase in temperature will enhance the photodamage of cells in the top layer of the bloom. Low photon irradiances in subsurface layers will offer protection against heat injury. If the high temperatures extend to the deepest, dark layers of the bloom, damage in those layers is likely to occur.     

Ultrastructure of retrocerebral complex of the earwig, Euborellia annulipes, and rôle of aorta as a neurohaemal organ

The ultrastructure of the retrocerebral endocrine-aortal complex of the earwig, Euborellia annulipes has been studied. The space between the inner and outer stromal layers of the aorta is occupied by numerous axon terminals and pre-terminals containing large electron dense granules (NS-I) of approximately 100 to 220 nm and a few axon terminals having small granules (NS-II) of approximately 40 to 90 nm; the former appear to belong to medial neurosecretory A-cells, and the latter to the B-cells of the brain. The corpora cardiaca consist of intrinsic cells with mitochondria and multivesicular bodies. Granules of type NS-II and NS-III are observed in the axon terminals and pre-terminals in the corpora cardiaca. The NS-II are identical to those found in the aorta and are probably the secretions of the lateral B-cells. Granules of type NS-III are 40 to 120 nm and electron dense, and are intrinsic in origin. Similar granules occur in the intrinsic cells of the corpora cardiaca. E M studies have confirmed the rôle of the aorta as a neurohaemal organ for the medial neurosecretory cells, and the corpora cardiaca for the lateral neurosecretory cells of the brain. The corpora cardiaca also act as a reservoir for the intrinsic secretion. The corpus allatum is a solid body consisting of parenchymal cells with prominent nuclei, mitochondria, and endoplasmic reticulum. In between its cells are occasional glial cells and also neurosecretory as well as non-neurosecretory axons. The gland is devoid of A-cell NSM.     

Non-innocent ligand reservoirs for reducing or oxidizing equivalents in carbonylrhenium(I) complexes: 1,1′-Bis(diphenylphosphino)ferrocene (dppf) and bis-triazinyl-pyridine (BTP)

Organometallic complexes of Re(I) with ligands having opposite redox properties have been synthesized and structurally characterized. X-ray crystal structures of the complexes show typical fac-Re^I(CO)₃ coordination to the redox active ligands. Complete electrochemical and spectroelectrochemical studies on the ligands and the metal complexes were performed. The IR-spectroelectrochemical responses were monitored using the fac-Re(CO)₃ unit as a probe. The 15-20 cm⁻¹ hypsochromic or bathochromic shift of the ν_CO bands upon reduction or oxidation is attributed to ligand-centered processes.     

Characterization of <Emphasis Type="Italic">twist</Emphasis> and <Emphasis Type="Italic">snail</Emphasis> gene expression during mesoderm and nervous system development in the polychaete annelid <Emphasis Type="Italic">Capitella</Emphasis> sp. I

To investigate the evolutionary history of mesoderm in the bilaterian lineage, we are studying mesoderm development in the polychaete annelid, Capitella sp. I, a representative lophotrochozoan. In this study, we focus on the Twist and Snail families as candidate mesodermal patterning genes and report the isolation and in situ expression patterns of two twist homologs (CapI-twt1 and CapI-twt2) and two snail homologs (CapI-sna1 and CapI-sna2) in Capitella sp. I. CapI-twt1 is expressed in a subset of mesoderm derivatives during larval development, while CapI-twt2 shows more general mesoderm expression at the same stages. Neither twist gene is detected before the completion of gastrulation. The two snail genes have very distinct expression patterns. At cleavage and early gastrula stages, CapI-sna1 is broadly expressed in precursors of all three germ layers and becomes restricted to cells around the closing blastopore during late gastrulation; CapI-sna2 expression is not detected at these stages. After gastrulation, both snail genes are expressed in the developing central nervous system (CNS) at stages when neural precursor cells are internalized, and CapI-sna1 is also expressed laterally within the segmental mesoderm. Based on the expression patterns in this study, we suggest a putative function for Capitella sp. I twist genes in mesoderm differentiation and for snail genes in regulating CNS development and general cell migration during gastrulation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.