Frequency and epitope recognition of anti-ribosome P antibodies from humans with systemic lupus erythematosus and MRL/lpr mice are similar

Autoantibodies reactive against a shared, conserved epitope on the ribosomal phosphoproteins P0, P1, and P2 occur in approximately 15% of patients with SLE and are relatively specific for this disease. To determine whether anti-P antibodies occur in murine lupus, serum from MRL/lpr and NZB/W F1 mice were analyzed by immunoblotting as well as by ELISA using a synthetic peptide Ag. Of those analyzed, 4 of 35 (11%) MRL/lpr, 0 of 25 NZB/W F1 and 0 of 13 control NIH/Swiss mice had anti-P antibodies. Anti-P specificity was confirmed by immunoblotting of ribosomal proteins separated by two-dimensional gel electrophoresis and by inhibition of anti-P reactivity on immunoblots with the synthetic peptide Ag. These findings indicate a striking similarity in the frequency and fine epitope specificity of anti-P antibodies in humans and MRL/lpr mice with SLE.     

Elements within the beta-lactoglobulin gene inhibit expression of human serum albumin cDNA and minigenes in transfected cells but rescue their expression in the mammary gland of transgenic mice.

Two new beta-lactoglobulin (BLG)/human serum albumin (HSA) hybrid gene vectors were constructed and tested for expression in COS-7 cells and in transgenic mice. The HSA sequences were inserted between the second and sixth BLG exons. Transient transfection experiments with these vectors as well as a series of additional vectors with either the BLG 5'- or 3'- intragenic sequences revealed that sequences within BLG exon 1/intron 1/exon 2 abrogated BLG- directed HSA expression in vitro, regardless of the presence of HSA introns or the origin of the 3' polyadenylation signal. In contrast, the same BLG expression cassette enabled the efficient expression of HSA cDNA or minigene in the mammary gland of transgenic mice with subsequent secretion of the corresponding protein into the milk of 56 and 82%, respectively of the mouse strains at levels up to 0.3 mg/ml. Previous attempts to express HSA cDNA inserted into exon 1 of the BLG gene had failed [Shani,M., Barash,I., Nathan,M., Ricca,G., Searfoss,G.H., Dekel,I., Faerman,A., Givol,D. and Hurwitz,D.R. (1992) Transgenic Res. 1, 195- 208]. The new BLG expression cassette conferred more stringent tissue specific expression than previously described BLG/HSA constructs [Barash,I, Faerman,A., Ratovitsky,T, Puzis,R., Nathan,M., Hurwitz,D.R. and Shani, M. (1994) Transgenic Res. 3, 141-151]. However, it was not able to insulate the transgenes from the surrounding host DNA sequences and did not result in copy number dependent expression in transgenics. Together, the in vitro and in vivo results suggest both positive and negative regulatory elements within the BLG intragenic sequences evaluated. The new BLG construct represents an extremely valuable vector for the efficient expression of cDNAs in the mammary gland of transgenic animals.     

The plant glycosyltransferase clone collection for functional genomics

The glycosyltransferases (GTs) are an important and functionally diverse family of enzymes involved in glycan and glycoside biosynthesis. Plants have evolved large families of GTs which undertake the array of glycosylation reactions that occur during plant development and growth. Based on the Carbohydrate‐Active enZymes (CAZy) database, the genome of the reference plant Arabidopsis thaliana codes for over 450 GTs, while the rice genome (Oryza sativa) contains over 600 members. Collectively, GTs from these reference plants can be classified into over 40 distinct GT families. Although these enzymes are involved in many important plant specific processes such as cell‐wall and secondary metabolite biosynthesis, few have been functionally characterized. We have sought to develop a plant GTs clone resource that will enable functional genomic approaches to be undertaken by the plant research community. In total, 403 (88%) of CAZy defined Arabidopsis GTs have been cloned, while 96 (15%) of the GTs coded by rice have been cloned. The collection resulted in the update of a number of Arabidopsis GT gene models. The clones represent full‐length coding sequences without termination codons and are Gateway^® compatible. To demonstrate the utility of this JBEI GT Collection, a set of efficient particle bombardment plasmids (pBullet) was also constructed with markers for the endomembrane. The utility of the pBullet collection was demonstrated by localizing all members of the Arabidopsis GT14 family to the Golgi apparatus or the endoplasmic reticulum (ER). Updates to these resources are available at the JBEI GT Collection website http://www.addgene.org/ .     

The role of farm structure on bird assemblages around a Kenyan tropical rainforest

Although it is clear that the farmlands neighbouring fragmented forests are utilized by some forest birds, it is not clear how birds in general respond to farmland habitat mosaic. An effort was made to determine how bird density and foraging assemblages were influenced by farm structural characteristics and distance from forest edge. Thirty farms up to a distance of 12 km around Kakamega forest in western Kenya were studied. Farm structure entailed size, hedge volume, habitat heterogeneity, woody plant density, plant diversity and crop cover. Birds were surveyed using line transects and DISTANCE analyses and classified into six feeding guilds and three habitat associations. Size of farms increased away from the forest, as woody plant density, plant diversity, indigenous trees and subsistence crop cover declined. The most important farm structure variable was hedge volume, which enhanced bird species richness, richness of shrub‐land bird species and insectivorous bird density (R = 0.58, P < 0.01). Bird density increased with tree density while indigenous trees were suitable for insectivores and nectarivores. There were very few forest bird encounters. Agricultural practices incorporating maintenance of hedges and sound selection of agroforestry trees can enhance conservation of birds on farmland, though, not significantly for forest species.     

A Journey from Mammals to Yeast with Vacuolar H+-ATPase (V-ATPase)

The vacuolar H⁺-ATPase (V-ATPase) is one of the most fundamental enzymes in nature. It functions in almost every eukaryotic cell and energizes a wide variety of organelles and membranes. V-ATPase has a structure and mechanism of action similar to F-ATPase and several of their subunits probably evolved from common ancestors. In eukaryotic cells, F-ATPase is confined to the semiautonomous organelles, chloroplasts and mitochondria, which contain their own genes that encode some of the F-ATPase subunits. In contrast to F-ATPases, whose primary function in eukaryotic cells is to form ATP at the expense of the protonmotive force (pmf), V-ATPases function exclusively as ATP-dependent proton pumps. The pmf generated by V-ATPases in organelles and membranes of eukaryotic cells is utilized as a driving force for numerous secondary transport processes. It was the survival of the yeast mutant without the active enzyme and yeast genetics that allowed the identification of genuine subunits of the V-ATPase. It also revealed special properties of individual subunits, factors that are involved in the enzyme's biogenesis and assembly, as well as the involvement of V-ATPase in the secretory pathway, endocytosis, and respiration. It may be the insect V-ATPase that unconventionally resides in the plasma membrane of their midgut, that will give the first structure resolution of this complex.     

The maize gene maternal derepression of r1 encodes a DNA glycosylase that demethylates DNA and reduces siRNA expression in the endosperm

Demethylation of transposons can activate the expression of nearby genes and cause imprinted gene expression in the endosperm; this demethylation is hypothesized to lead to expression of transposon small interfering RNAs (siRNAs) that reinforce silencing in the next generation through transfer either into egg or embryo. Here we describe maize (Zea mays) maternal derepression of r1 (mdr1), which encodes a DNA glycosylase with homology to Arabidopsis thaliana DEMETER and which is partially responsible for demethylation of thousands of regions in endosperm. Instead of promoting siRNA expression in endosperm, MDR1 activity inhibits it. Methylation of most repetitive DNA elements in endosperm is not significantly affected by MDR1, with an exception of Helitrons. While maternally-expressed imprinted genes preferentially overlap with MDR1 demethylated regions, the majority of genes that overlap demethylated regions are not imprinted. Double mutant megagametophytes lacking both MDR1 and its close homolog DNG102 result in early seed failure, and double mutant microgametophytes fail pre-fertilization. These data establish DNA demethylation by glycosylases as essential in maize endosperm and pollen and suggest that neither transposon repression nor genomic imprinting is its main function in endosperm.

Demethylation by DNA glycosylases is important for endosperm development, but only a subset of the affected loci are imprinted, suggesting demethylation may have additional functions.

IN A NUTSHELL Background: In 1970, Jerry Kermicle reported that maize kernels could have dramatically different pigmentation depending on which parent the r1 gene is inherited from. This was the first discovery of many genomically imprinted genes that are selectively expressed from the maternal genome in endosperm. Later, Kermicle also discovered a mutant with poor maternal r1 expression. He hypothesized that the normal function of the mutated gene would be to derepress maternal r1; hence the name maternal depression of r1 (mdr1). The identify of mdr1 has remained unknown since then, but studies using Arabidopsis thaliana have revealed that DNA demethylation by enzymes called DNA glycosylases is important for expression of some maternally inherited genes in endosperm. Question: We wanted to identify the mdr1 gene. We hypothesized that mdr1 would reveal insights into molecular mechanisms of genomic imprinting in maize. Findings: We discovered that mdr1 encodes one of two DNA glycosylases with high expression in endosperm. We found that at least one of the two must be functional for endosperm to develop normally, but the one encoded by mdr1 is expressed higher. Surprisingly, most of the genes the mdr1 DNA glycosylase demethylates do not appear to be genomically imprinted, and about half the DNA it demethylates is not even near genes. These findings suggest that DNA glycosylases also have an undiscovered function unrelated to genomic imprinting in endosperm. Next steps: We want to know how specific regions in the genome are targeted for demethylation. What distinguishes these regions from other regions in endosperm? And what keeps them from being demethylated in other tissues? On the flip side, little is known about the effect of demethylation in endosperm, other than genomic imprinting. We want to know what effect DNA demethylation by DNA glycosylases has on chromatin structure and why it is important.     

Greater subchondral vBMD at the tibia is observed between 1 and 5 years of anterior cruciate ligament injury

Objectives:This study aimed to determine if differences exist in tibial subchondral bone and muscle imbalances between individuals with and without an Anterior Cruciate Ligament (ACL) repair within the past 1 to 5 years (median 3 years).Methods:Fifteen individuals (ages 18-23 years) that had a unilateral ACL repair with no contralateral knee injuries and 15 age- and sex-matched controls (no prior knee injuries) were recruited to participate. Subchondral bone was measured using peripheral quantitative computed tomography (pQCT) distal to the tibial plateau. Muscle force, power, and force efficiency were measured using single leg jumps performed on a force platform.Results:Within subject analysis showed a greater subchondral vBMD in the injured versus uninjured legs of cases (278±11 mg/cm³ and 258±6 mg/cm³, respectively, mean±SD, p=0.01). Subchondral vBMD was greater on the injured leg of cases than controls (267±8 mg/cm³ and 237±8 mg/cm³, respectively, marginal mean±SE, p=0.01). No differences were observed between cases and controls for muscle force, power, or force efficiency.Conclusions:Greater subchondral bone mineral density was observed in participants between 1- and 5-years post-op. Given the results of this study and the known long-term effects of ACL injuries, future research must continue to focus on the prevention of these injuries.     

The evolving redox chemistry and bioavailability of vanadium in deep time

The incorporation of metal cofactors into protein active sites and/or active regions expanded the network of microbial metabolism during the Archean eon. The bioavailability of crucial metal cofactors is largely influenced by earth surface redox state, which impacted the timing of metabolic evolution. Vanadium (V) is a unique element in geo–bio‐coevolution due to its complex redox chemistry and specific biological functions. Thus, the extent of microbial V utilization potentially represents an important link between the geo‐ and biospheres in deep time. In this study, we used geochemical modeling and network analysis to investigate the availability and chemical speciation of V in the environment, and the emergence and changing chemistry of V‐containing minerals throughout earth history. The redox state of V shifted from a more reduced V(III) state in Archean aqueous geochemistry and mineralogy to more oxidized V(IV) and V(V) states in the Proterozoic and Phanerozoic. The weathering of vanadium sulfides, vanadium alkali metal minerals, and vanadium alkaline earth metal minerals were potential sources of V to the environment and microbial utilization. Community detection analysis of the expanding V mineral network indicates tectonic and redox influence on the distribution of V mineral‐forming elements. In reducing environments, energetic drivers existed for V to potentially be involved in early nitrogen fixation, while in oxidizing environments vanadate () could have acted as a metabolic electron acceptor and phosphate mimicking enzyme inhibitor. The coevolving chemical speciation and biological functions of V due to earth's changing surface redox conditions demonstrate the crucial links between the geosphere and biosphere in the evolution of metabolic electron transfer pathways and biogeochemical cycles from the Archean to Phanerozoic.     

Heterogeneous Effects of Calorie Restriction on In Vivo Glucose Uptake and Insulin Signaling of Individual Rat Skeletal Muscles

Calorie restriction (CR) (consuming ∼60% of ad libitum, AL, intake) improves whole body insulin sensitivity and enhances insulin-stimulated glucose uptake by isolated skeletal muscles. However, little is known about CR-effects on in vivo glucose uptake and insulin signaling in muscle. Accordingly, 9-month-old male AL and CR (initiated when 3-months-old) Fischer 344xBrown Norway rats were studied using a euglycemic-hyperinsulinemic clamp with plasma insulin elevated to a similar level (∼140 µU/ml) in each diet group. Glucose uptake (assessed by infusion of [¹⁴C]-2-deoxyglucose, 2-DG), phosphorylation of key insulin signaling proteins (insulin receptor, Akt and Akt substrate of 160kDa, AS160), abundance of GLUT4 and hexokinase proteins, and muscle fiber type composition (myosin heavy chain, MHC, isoform percentages) were determined in four predominantly fast-twitch (epitrochlearis, gastrocnemius, tibialis anterior, plantaris) and two predominantly slow-twitch (soleus, adductor longus) muscles. CR did not result in greater GLUT4 or hexokinase abundance in any of the muscles, and there were no significant diet-related effects on percentages of MHC isoforms. Glucose infusion was greater for CR versus AL rats (P<0.05) concomitant with significantly (P<0.05) elevated 2-DG uptake in 3 of the 4 fast-twitch muscles (epitrochlearis, gastrocnemius, tibialis anterior), without a significant diet-effect on 2-DG uptake by the plantaris or either slow-twitch muscle. Each of the muscles with a CR-related increase in 2-DG uptake was also characterized by significant (P<0.05) increases in phosphorylation of both Akt and AS160. Among the 3 muscles without a CR-related increase in glucose uptake, only the soleus had significant (P<0.05) CR-related increases in Akt and AS160 phosphorylation. The current data revealed that CR leads to greater whole body glucose disposal in part attributable to elevated in vivo insulin-stimulated glucose uptake by fast-twitch muscles. The results also demonstrated that CR does not uniformly enhance either insulin signaling or insulin-stimulated glucose uptake in all muscles in vivo.