Pouring and Running a Protein Gel by reusing Commercial Cassettes

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry and molecular biology researchers^1-4. For laboratories that perform daily analyses of proteins, the cost of commercially available polyacrylamide gels (˜$10/gel) can be considerable over time. To mitigate this cost, some researchers prepare their own polyacrylamide gels. Traditional methods of pouring these gels typically utilize specialized equipment and glass gel plates that can be expensive and preclude pouring many gels and storing them for future use. Furthermore, handling of glass plates during cleaning or gel pouring can result in accidental breakage creating a safety hazard, which may preclude their use in undergraduate laboratory classes. Our protocol demonstrates how to pour multiple protein gels simultaneously by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4°C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment. In addition, plastic gel cassettes are extremely resistant to breakage, which makes them ideal for undergraduate laboratory classrooms.     

Selective accumulation of Th2-skewing immature erythroid cells in developing neonatal mouse spleen

Environmental factors likely regulate neonatal immunity and self-tolerance. However, evidence that the neonatal immune system is suppressed or deviated is varied depending on the antigen and the timing of antigen exposure relative to birth. These disparate findings may be related to the availability of the appropriate antigen presenting cells but also point to the possibility of homeostatic changes in non-lymphoid cells in the relevant lymphoid tissues. Here we show that, while leukocytes are the most abundant cell population present in spleen during the first 4-5 days after birth, a massive accumulation of nucleated immature erythroid population in the spleen takes places on day 6 after birth. Although the relative frequency of these immature erythorid cells slowly decreases during the development of neonates, they remain one of the most predominant populations up to three weeks of age. Importantly, we show that the immature erythroid cells from neonate spleen have the capacity to modulate the differentiation of CD4 T cells into effector cells and provide a bias towards a Th2 type instead of Th1 type. These nucleated erythroid cells can produce cytokines that participate in the Th2/Th1 balance, an important one being IL-6. Thus, the selective accumulation of immature erythroid cells in the spleen during a specific period of neonatal development may explain the apparent differences observed in the type(s) of immune responses generated in infants and neonates. These findings are potentially relevant to the better management of immune deficiency in and to the design of vaccination strategies for the young.     

Crystal structures and small-angle x-ray scattering analysis of UDP-galactopyranose mutase from the pathogenic fungus Aspergillus fumigatus

UDP-galactopyranose mutase (UGM) is a flavoenzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, which is a central reaction in galactofuranose biosynthesis. Galactofuranose has never been found in humans but is an essential building block of the cell wall and extracellular matrix of many bacteria, fungi, and protozoa. The importance of UGM for the viability of many pathogens and its absence in humans make UGM a potential drug target. Here we report the first crystal structures and small-angle x-ray scattering data for UGM from the fungus Aspergillus fumigatus, the causative agent of aspergillosis. The structures reveal that Aspergillus UGM has several extra secondary and tertiary structural elements that are not found in bacterial UGMs yet are important for substrate recognition and oligomerization. Small-angle x-ray scattering data show that Aspergillus UGM forms a tetramer in solution, which is unprecedented for UGMs. The binding of UDP or the substrate induces profound conformational changes in the enzyme. Two loops on opposite sides of the active site move toward each other by over 10 Å to cover the substrate and create a closed active site. The degree of substrate-induced conformational change exceeds that of bacterial UGMs and is a direct consequence of the unique quaternary structure of Aspergillus UGM. Galactopyranose binds at the re face of the FAD isoalloxazine with the anomeric carbon atom poised for nucleophilic attack by the FAD N5 atom. The structural data provide new insight into substrate recognition and the catalytic mechanism and thus will aid inhibitor design.     

Changes in mitochondrial membrane potential and accumulation of reactive oxygen species precede ultrastructural changes during ovule abortion

In many species, environmental stress reduces plant fertility. In Arabidopsis thaliana, a significant fraction of this reduction in plant fertility results from ovule abortion and embryo senescence. In this species, environmental conditions were identified that induced 94% of the developing ovules to either undergo stress-induced ovule abortion or embryo senescence (Sun et al. Plant Physiol 135:2358–2367, 2004). Following salt stress, physiological and anatomical changes were first detected in the female gametophyte of an aborting ovule. Two to four hours after a period of salt stress that induces most ovules to abort, the mitochondrial membrane potential dissipated. Subsequently, cells in the gametophyte accumulated reactive oxygen species, which are known to be molecules that promote programmed cell death (PCD). Because mitochondria often play an important role in PCD, these organelles were closely examined for changes in structure. Although the anatomy of mitochondria varied, reproducible changes in mitochondria structure were not observed. Nonetheless, other changes in ultrastructure were found. In some aborting gametophytes, concentric rings of endoplasmic reticulum were formed. In a fraction of the aborting ovules, cytoplasmic contents and organelles were invaginated into the vacuole. Even in cryofixed sections, many of these bodies appeared indistinct, which is consistent with the degradation of their contents.     

Exogenous hyalin and sea urchin gastrulation. Part III: biological activity of hyalin isolated from Lytechinus pictus embryos

Hyalin is a large glycoprotein, consisting of the hyalin repeat domain and non-repeated regions, and is the major component of the hyaline layer in the early sea urchin embryo of Strongylocentrotus purpuratus. The hyalin repeat domain has been identified in proteins from organisms as diverse as bacteria, sea urchins, worms, flies, mice and humans. While the specific function of hyalin and the hyalin repeat domain is incompletely understood, many studies suggest that it has a functional role in adhesive interactions. In part I of this series, we showed that hyalin isolated from the sea urchin S. purpuratus blocked archenteron elongation and attachment to the blastocoel roof occurring during gastrulation in S. purpuratus embryos, (Razinia et al., 2007). The cellular interactions that occur in the sea urchin, recognized by the U.S. National Institutes of Health as a model system, may provide insights into adhesive interactions that occur in human health and disease. In part II of this series, we showed that S. purpuratus hyalin heterospecifically blocked archenteron-ectoderm interaction in Lytechinus pictus embryos (Alvarez et al., 2007). In the current study, we have isolated hyalin from the sea urchin L. pictus and demonstrated that L. pictus hyalin homospecifically blocks archenteron-ectoderm interaction, suggesting a general role for this glycoprotein in mediating a specific set of adhesive interactions. We also found one major difference in hyalin activity in the two sea urchin species involving hyalin influence on gastrulation invagination.     

Climate change and postglacial human dispersals in southeast Asia

Modern humans have been living in Island Southeast Asia (ISEA) for at least 50,000 years. Largely because of the influence of linguistic studies, however, which have a shallow time depth, the attention of archaeologists and geneticists has usually been focused on the last 6,000 years--in particular, on a proposed Neolithic dispersal from China and Taiwan. Here we use complete mitochondrial DNA (mtDNA) genome sequencing to spotlight some earlier processes that clearly had a major role in the demographic history of the region but have hitherto been unrecognized. We show that haplogroup E, an important component of mtDNA diversity in the region, evolved in situ over the last 35,000 years and expanded dramatically throughout ISEA around the beginning of the Holocene, at the time when the ancient continent of Sundaland was being broken up into the present-day archipelago by rising sea levels. It reached Taiwan and Near Oceania more recently, within the last approximately 8,000 years. This suggests that global warming and sea-level rises at the end of the Ice Age, 15,000-7,000 years ago, were the main forces shaping modern human diversity in the region.     

Mannose-binding lectin deficiency is associated with early onset of polyarticular juvenile rheumatoid arthritis: a cohort study

Background  

Mannose-binding lectin (MBL) is an innate immune protein. The aim of our study was to determine whether genetically determined MBL deficiency is associated with susceptibility to juvenile rheumatoid arthritis (JRA) and whether MBL2 genotypes are associated with JRA severity.     

Fluorometric studies of ligand-induced conformational changes of CD38

The lymphoid surface antigen CD38 is a NAD⁺-glycohydrolase that also catalyzes the transformation of NAD⁺ into cyclic ADP-ribose, a calcium mobilizing second messenger. In addition, ligation of CD38 by antibodies triggers signaling in lymphoid cells. Since the cytoplasmic tail of CD38 is dispensable for this latter property, we have previously proposed that CD38-mediated receptor signal transduction might be regulated by its conformational state. We have now examined the molecular changes of this protein during its interaction with NAD⁺ by measuring the intrinsic fluorescence of CD38. We have shown that addition of the substrate produced a dramatic decrease in the fluorescence of the catalytically active recombinant soluble ectodomain of murine CD38. Analysis of this event revealed that the catalytic cycle involves a state of the enzyme that is characterized by a low fluorescence which, upon substrate turnover, reverts to the initial high intrinsic fluorescence level. In contrast, non-hydrolyzable substrates trap CD38 in its altered low fluorescence state. Studies with the hydrophilic quencher potassium iodide revealed that the tryptophan residues that are mainly involved in the observed changes in fluorescence, are remote from the active site. Similar data were also obtained with human CD38, indicating that studies of intrinsic fluorescence will be useful in monitoring the transconformation of CD38 from different species. Together, these data demonstrate that CD38 undergoes a reversible conformational change after substrate binding, and suggest a mechanism by which this change could alter interactions with different cell-surface partners.     

Thymidylate synthetase-catalyzed conversions of E-5-(2-bromovinyl)-2'-deoxyuridylate

E-5-(2-Bromovinyl)-2'-deoxyuridylate (BrvdUMP), the first metabolite in the processing of the antiviral agent E-5-(2-bromovinyl)-2'-deoxyuridine (BrvdUrd), is an excellent alternate substrate for dTMP synthetase. The nucleophilic catalyst of the enzyme adds to the 6-position of the heterocycle and converts the normally inert 5-bromovinyl group of BrvdUMP to a reactive allylic bromide in which both carbons of the side chain are susceptible to nucleophilic attack. These centers react with nucleophiles in the reaction mixture, 2-mercaptoethanol and water, to give three diastereomeric products which have been isolated and characterized. Possible implications of these findings as related to the mechanism and selectivity of BrvdUrd as an antiviral agent are discussed.     

Isolation of species-specific and stage-specific adhesion promoting component by disaggregation of intact sea urchin embryo cells

The species-specific and developmental stage-specific aggregation-enhancing supernatant isolated from intact sea urchin (Strongylocentrotus purpuratus) blastula cells incubated in Ca²⁺---Mg²⁺-free sea water is a hemagglutinin. This material agglutinated trypsinized, fixed human type O and B (inhibited by -galactose) erythrocytes, whereas control erythrocytes in Millipore-filtered sea water did not agglutinate. The blastula supernatant agglutinates both live and fixed S. purpuratus blastula cells. Fixed cells were chosen in these experiments so that a standardized, highly reproducible system could be produced by pooling batches of blastula cells. Dissociation supernatant (DS)-mediated agglutination of S. purpuratus blastula cells was blocked by -galactose and N-acetyl- -galactosamine by 10 min of incubation, but not by -glucose, -fucose, -mannose, -glucosamine, -mannosamine or N-acetyl- -mannosamine (all at 0.1 M concentration, the concentration chosen as a result of preliminary experiments). The results were consistently observed in scores of experiments and suggest that DS binds cells together via -galactose-like and N-acetyl- -galactosamine-like residues. We also found that aggregates of live blastula cells formed in the presence of DS gave rise, after 24 h incubation, to viable, swimming embryoids, suggesting that DS-mediated adhesion is physiologically meaningful.