Goodpasture antigen-binding protein and its spliced variant, ceramide transfer protein, have different functions in the modulation of apoptosis during zebrafish development

Human Goodpasture antigen-binding protein (GPBP) is an atypical protein kinase that phosphorylates the Goodpasture auto-antigen, the alpha3 chain of collagen IV. The COL4A3BP gene is alternatively spliced producing two protein isoforms: GPBP and GPBPDelta26. The latter lacks a serine-rich domain composed of 26 amino acid residues. Both isoforms also function as ceramide transfer proteins (CERT). Here, we explored the function of Gpbp and GpbpDelta26/CERT during embryogenesis in zebrafish. We cloned both splice variants of the zebrafish gene and found that they are differentially expressed during development. We used antisense oligonucleotide-mediated loss-of-function and synthetic mRNA-based gain-of-function approaches. Our results show that the loss-of-function phenotype is linked to cell death, evident primarily in the muscle of the somites, extensive loss of myelinated tracks, and brain edema. These results indicate that disruption of the nonvesicular ceramide transport is detrimental to normal embryonic development of somites and brain because of increased apoptosis. Moreover, this phenotype is mediated by Gpbp but not GpbpDelta26/CERT, suggesting that Gpbp is an important factor for normal skeletal muscle and brain development.     

Cre/<Emphasis Type="Italic">lox</Emphasis> system to develop selectable marker free transgenic tobacco plants conferring resistance against sap sucking homopteran insect

A binary expression vector was constructed containing the insecticidal gene Allium sativum leaf agglutinin (ASAL), and a selectable nptII marker gene cassette, flanked by lox sites. Similarly, another binary vector was developed with the chimeric cre gene construct. Transformed tobacco plants were generated with these two independent vectors. Each of the T(0) lox plants was crossed with T(0) Cre plants. PCR analyses followed by the sequencing of the target T-DNA part of the hybrid T(1) plants demonstrated the excision of the nptII gene in highly precised manner in certain percentage of the T(1) hybrid lines. The frequency of such marker gene excision was calculated to be 19.2% in the hybrids. Marker free plants were able to express ASAL efficiently and reduce the survivability of Myzus persiceae, the deadly pest of tobacco significantly, compared to the control tobacco plants. Results of PCR and Southern blot analyses of some of the T(2) plants detected the absence of cre as well as nptII genes. Thus, the crossing strategy involving Cre/lox system for the excision of marker genes appears to be very effective and easy to execute. Documentation of such marker excision phenomenon in the transgenic plants expressing the important insecticidal protein for the first time has a great significance from agricultural and biotechnological points of view.     

Ecological factors affect the level and scaling of avian BMR

The basal rate of metabolism (BMR) in 533 species of birds, when examined with ANCOVA, principally correlates with body mass, most of the residual variation correlating with food habits, climate, habitat, a volant or flightless condition, use or not of torpor, and a highland or lowland distribution. Avian BMR also correlates with migratory habits, if climate and a montane distribution is excluded from the analysis, and with an occurrence on small islands if a flightless condition and migration are excluded. Residual variation correlates with membership in avian orders and families principally because these groups are behaviorally and ecologically distinctive. However, the distinction between passerines and other birds remains a significant correlate of avian BMR, even after six ecological factors are included, with other birds having BMRs that averaged 74% of the passerine mean. This combination of factors accounts for 97.7% of the variation in avian BMR. Yet, migratory species that belong to Anseriformes, Charadriiformes, Pelecaniformes, and Procellariiformes and breed in temperate or polar environments have mass-independent basal rates equal to those found in passerines. In contrast, penguins belong to an order of polar, aquatic birds that have basal rates lower than passerines because their flightless condition depresses basal rate. Passerines dominate temperate, terrestrial environments and the four orders of aquatic birds dominate temperate and polar aquatic environments because their high BMRs facilitate reproduction and migration. The low BMRs of tropical passerines may reflect a sedentary lifestyle as much as a life in a tropical climate. Birds have BMRs that are 30-40% greater than mammals because of the commitment of birds to an expensive and expansive form of flight.     

Dual Functions for the Schizosaccharomyces pombe Inositol Kinase Ipk1 in Nuclear mRNA Export and Polarized Cell Growth

The inositol 1,3,4,5,6-pentakisphosphate (IP₅) 2-kinase (Ipk1) catalyzes the production of inositol hexakisphosphate (IP₆) in eukaryotic cells. Previous studies have shown that IP₆ is required for efficient nuclear mRNA export in the budding yeast Saccharomyces cerevisiae. Here, we report the first functional analysis of ipk1⁺ in Schizosaccharomyces pombe. S. pombe Ipk1 (SpIpk1) is unique among Ipk1 orthologues in that it harbors a novel amino (N)-terminal domain with coiled-coil structural motifs similar to those of BAR (Bin-amphiphysin-Rvs) domain proteins. Mutants with ipk1⁺ deleted (ipk1Δ) had mRNA export defects as well as pleiotropic defects in polarized growth, cell morphology, endocytosis, and cell separation. The SpIpk1 catalytic carboxy-terminal domain was required to rescue these defects, and the mRNA export block was genetically linked to SpDbp5 function and, likely, IP₆ production. However, the overexpression of the N-terminal domain alone also inhibited these functions in wild-type cells. This revealed a distinct noncatalytic function for the N-terminal domain. To test for connections with other inositol polyphosphates, we also analyzed whether the loss of asp1⁺ function, encoding an IP₆ kinase downstream of Ipk1, had an effect on ipk1Δ cells. The asp1Δ mutant alone did not block mRNA export, and its cell morphology, polarized growth, and endocytosis defects were less severe than those of ipk1Δ cells. Moreover, ipk1Δ asp1Δ double mutants had altered inositol polyphosphate levels distinct from those of the ipk1Δ mutant. This suggested novel roles for asp1⁺ upstream of ipk1⁺. We propose that IP₆ production is a key signaling linchpin for regulating multiple essential cellular processes.     

Transitional B cell fate is associated with developmental stage-specific regulation of diacylglycerol and calcium signaling upon B cell receptor engagement

Functional peripheral mature follicular B (FoB) lymphocytes are thought to develop from immature transitional cells in a BCR-dependent manner. We have previously shown that BCR cross-linking in vitro results in death of early transitional (T1) B cells, whereas late transitional (T2) B cells survive and display phenotypic characteristics of mature FoB cells. We now demonstrate that diacylglycerol (DAG), a lipid second messenger implicated in cell survival and differentiation, is produced preferentially in T2 compared with T1 B cells upon BCR cross-linking. Consistently, inositol 1,4,5-triphosphate is also produced preferentially in T2 compared with T1 B cells. Unexpectedly, the initial calcium peak appears similar in both T1 and T2 B cells, whereas sustained calcium levels are higher in T1 B cells. Pretreatment with 2-aminoethoxydiphenylborate, an inhibitor of inositol 1,4,5-triphosphate receptor-mediated calcium release, and verapamil, an inhibitor of L-type calcium channels, preferentially affects T1 B cells, suggesting that distinct mechanisms regulate calcium mobilization in each of the two transitional B cell subsets. Finally, BCR-mediated DAG production is dependent upon Bruton's tyrosine kinase and phospholipase C-gamma2, enzymes required for the development of FoB from T2 B cells. These results suggest that calcium signaling in the absence of DAG-mediated signals may lead to T1 B cell tolerance, whereas the combined action of DAG and calcium signaling is necessary for survival and differentiation of T2 into mature FoB lymphocytes.     

Pyruvate kinase M2 in chronic inflammations: a potpourri of crucial protein–protein interactions

Cell Biology and Toxicology - Chronic inflammation (CI) is a primary contributing factor involved in multiple diseases like cancer, stroke, diabetes, Alzheimer’s disease, allergy, asthma,...     

Enhanced expression of Arabidopsis rubisco small subunit gene promoter regulated Cry1Ac gene in chickpea conferred complete resistance to Helicoverpa armigera

A major pest of chickpea, Helicoverpa armigera, can be controlled by expressing genes from the bacterium Bacillus thuringiensis as an environmentally compatible option. Here we show that transgenic chickpeas containing a cry1Ac gene conferred a high degree of resistance to H. armigera. The Agrobacterium binary vector contained the nptII gene as the selectable marker and cry1Ac gene driven by the Arabidopsis rubisco small subunit gene (ats1A) promoter. We generated 54 and 47 independent transgenic lines using truncated (trcry1Ac) and full-length versions of the cry1Ac (flcry1Ac) gene, respectively. Of these lines, twelve transmitted the trcry1Ac transgene to the next generation at a 3:1 ratio, while only 8 flcry1Ac lines segregated in a 3:1 ratio. Five lines expressed trCry1Ac protein > 50 μg/g fresh weight, however, only one line accumulated about 30 μg/g flCry1Ac protein. Such high levels of trCry1Ac protein have not been reported before in chickpea. When trCry1Ac lines were challenged to whole plant bioassays in the greenhouse, lowest pod damage was observed in BS100B (1.4%) followed by BS81P (4.4%), and BS100E (6.2%) compared to the parental line (49.9%). The phenotypes of the lines expressing high levels of Cry1Ac protein were indistinguishable from their null segregants and controls. Thus, trCry1Ac lines could be suitable for crossing with our existing Cry2Aa lines for generation of a pyramided Bt chickpea for enhanced insect resistance management in the field.

     

Density functional studies on the electron affinities of DNA and RNA bases

Influence of basis sets on electron affinities (EAs) of DNA and RNA bases has been investigated using density functional method (B3LYP functional) with different basis sets (6-31G, TZVP and 6-311+ + G**). Effect of some PBE functionals namely, PBEOP, PBELYP and PBEVWN, on EA values of the nucleobases was studied using basis set which predicted the most reliable values with B3LYP functional. Observation of the trends in the values of EA and dipole moment of the molecules enable us to identify the features of a basis set that shows the presence of dipole-bound state of some of the nucleobases. The vertical electron affinities with B3LYP and PBEOP functionals are close to the experimental values. The adiabatic electron affinities of uracil and thymine were found to be positive for basis set with diffuse functions using B3LYP functional. Adenine does not have a stable covalently bound anion at all levels of basis sets and functionals. The sign of adiabatic electron affinity value of cytosine is inconsistent with that of experimental value but in agreement with previous theoretical results. For guanine the adiabatic electron affinity value with 6-311+ + G** basis set was found to be very high as comparison with other two basis sets confirming the formation of mixed covalent-dipole character.