Purification and Characterization of Recombinant Polymeric Hemoglobin P1 of Glycera dibranchiata

The apoprotein of component P1 of the polymeric fraction of the intracellular hemoglobin of the marine polychaete Glycera dibranchiata has been expressed at a high level in Escherichia coli. The expressed globin was reconstituted with heme and purified. The N-terminal sequence of the recombinant P1 is identical to the cDNA-derived sequence of cloned P1 (Zafar et al., Biochem. Biophys. Acta, 1041, 117-123, 1990). Gel filtration, SDS-PAGE, optical spectra over the range 200-650 nm, and circular dichroism over the range 200-250 nm of the purified recombinant P1 were very similar to the polymeric fraction of native Glycera hemoglobin. The molar ellipticity at 222 nm provided an estimate of 77% for the α-helical content of the recombinant P1, in excellent agreement with that calculated from the crystal structure of Glycera monomeric component M-II. Although the oxygen binding affinity of the recombinant P1 is higher than that of the polymeric fraction of Glycera hemoglobin (3-4 torr vs 7-13 torr), which consists of at least six different single-chain hemoglobins, the Hill coefficient is lower (1.0-1.2 vs 1.2-1.4).     

Deletion analysis of the essentiality of penicillin-binding proteins 1A, 2B and 2X of Streptococcus pneumoniae

Abstract An internal fragment from each of the penicillinebinding protein (PBP) 1A, 2B and 2X genes of Streptococcus pneumoniae , which included the region encoding the active-site serine residue, was replaced by a fragment encoding spectinomycin resistance. The resulting constructs were tested for their ability to transform S. pneumoniae strain R6 to spectinomycin resistance. Spectinomycin-resistant transformants could not be obtained using either the inactivated PBP 2X or 2B genes, suggesting that deletion of either of these genes was a lethal event, but they were readily obtained using the inactivated PBP 1A gene. Analysis using the polymerase chain reaction confirmed that the latter transformants had replaced their chromosomal copy of the PBP 1A gene with the inactivated copy of the gene. Deletion of the PBP 1A gene was therefore tolerated under laboratory conditions and appeared to have little effect on growth or susceptibility to benzylpenicillin.     

Identification of photo-inactive phytochrome A in etiolated seedlings and photo-active phytochrome B in green leaves of the aurea mutant of tomato

The contents of spectrophotometrically measurable phytochrome A (PhyA) and phytochrome B (PhyB) and the corresponding immunochemically detectable apoproteins (PHYA and PHYB) were examined in dark- and light-grown tissues of the aurea mutant of tomato and its wild-type (WT). The amount of PHYA in etiolated aurea seedlings was found to be about 20% of that in the WT; this PHYA showed no photoreversible changes in absorbance, no downregulation of the level of PHYA in light-grown seedlings, and no differential proteolysis of Pr and Pfr species in vitro which was seen in the case of the WT. By contrast, the amount of PHYB in aurea seedlings was not significantly different from that in WT seedlings. Phytochrome isolated from green leaves of the aurea mutant and purified by ion-exchange chromatography showed a red/far-red reversible spectral change, and its elution profile during chromatography was essentially similar to that of PHYB. The results indicate that aurea is a mutant that is deficient in photoactive PhyA at the etiolated stage, when it contains a spectrally inactive PHYA. However, the mutant contains spectrally active PhyB in its green tissue as does the WT.     

Facile technique of protein precipitation by application of electric current

Summary Precipitation of proteins has been achieved following passage of direct electric current in various protein solutions. Application of as low as 3 V of electric current showed precipitation but the rate increased with increase in electric current. With 9 V there was more than 85% precipitation of protein within 15 min. Precipitation occurred at a wide range of pH and temperature. Electrophoretic analysis of precipitated proteins show that they are not denatured by application of electric current. Proteins thus precipitated can be easily recovered by centrifugation.     

ABC transporters mined through comparative transcriptomics associate with organ-specific accumulation of picrosides in a medicinal herb,Picrorhiza kurroa

Protoplasma - Picrorhiza kurroa Royle ex Benth is a valuable medicinal herb of North-Western Himalayas due to presence of two major bioactive compounds, picroside-I and picroside-II used in the...     

Speed Breeding Opportunities and Challenges for Crop Improvement

Journal of Plant Growth Regulation - Crop improvement in light of the rapidly changing climate and the increasing human population continues to be one of the primary concerns for researchers across...     

Fecundity of two hillstream fishes, Garra lamta (Ham.) and Garra gotyla gotyla (gray)

The fecundity of two hillstream fishes, Garra lamta and G. gotyla gotyla, is related to their total body length (L), total body weight (W), ovary length (X) and ovary weight (V). The relationships to L, W, X and V were all linear. The correlation coefficient values (r) showed that total body length and total body weight were the best indices in fecundity estimates in both fishes.     

Variability of milk fat globule membrane protein gene between cattle and riverine buffalo.

A study on butyrophilin (BTN) gene was conducted to detect variability at nucleotide level between cattle and buffalo. Hae III PCR-RFLP was carried out in crossbred cattle and it revealed polymorphism at this locus. Three genotypes namely, AA, BB and AB and two alleles were observed with frequencies 0.78, 0.17, 0.04 and 0.87, 0.13, respectively. The sequences of different cattle, buffalo and sheep breeds have been reported in the EMBL gene bank with accession numbers: AY491468 to AY491475. The nucleotides, which have been substituted from allele A to B, were found to be C to G (71st nucleotide), C to T (86th nucleotide), A to T (217th nucleotide), G to A (258th nucleotide), A to C (371st nucleotide) and C to T (377th nucleotide). The nucleotide substitution at 71st, 86th and 377th position of the fragment were expected to be a silent mutation where as nucleotide changes at 217th, 258th and 371st positions were expected to be substituted by lysine with arginine, valine with isoleucine and leucine with proline in allele B. The differences of nucleotides and amino acids between cattle, buffalo and sheep breeds have been revealed and on the basis of nucleotide as well as protein variability the phylogenetic diagram have been developed indicating closeness between cattle and buffalo.     

1α,25-dihydroxy-24-oxo-16-ene vitamin D3, a metabolite of a synthetic vitamin D3 analog, 1α,25-dihydroxy-16-ene vitamin D3, is equipotent to its parent in modulating growth and differentiation of human leukemic cells

1α,25(OH)₂-16-ene-D₃, a synthetic analog of the steroid hormone, 1α,25(OH)₂D₃, has great potential to become a drug in the treatment of leukemia and other proliferative disorders, because of its minimal in vivo calcemic activity associated with a potent inhibitory effect on cell growth. However, at present, the mechanisms through which 1α,25(OH)₂-16-ene-D₃ expresses its biological activities are still not completely understood. Our previous in vitro study in a perfused rat kidney indicated for the first time that 1α,25(OH)₂-16-ene-D₃ and 1α,25(OH)₂D₃ are metabolized differently. 1α,25(OH)₂-24-oxo-16-ene-D₃, an intermediary metabolite of 1α,25(OH)₂-16-ene-D₃ formed through the C-24 oxidation pathway, accumulated significantly in the perfusate when compared to 1α,25(OH)₂-24-oxo-D₃, the corresponding intermediary metabolite of 1α,25(OH)₂D₃. In a subsequent in vivo study, we also reported that 1α,25(OH)₂-24-oxo-16-ene-D₃ exerted immunosuppressive activity equal to its parent, without causing significant hypercalcemia. In order to establish further the critical role of 1α,25(OH)₂-24-oxo-16-ene-D₃, in generating some of the key biological activities ascribed to its parent, we performed the present in vitro study using a human myeloid leukemic cell line (RWLeu-4) as a model. Comparative target tissue metabolism studies indicated that 1α,25(OH)₂-16-ene-D₃ and 1α,25(OH)₂D₃ are metabolized differently in RWLeu-4 cells, and the differences were similar to the ones we previously observed in the rat kidney. The significant finding was the accumulation of 1α,25(OH)₂-24-oxo-16-ene-D₃ in RWLeu-4 cells because of its resistance to further metabolism. Biological activity studies indicated that both 1α,25(OH)₂-16-ene-D₃ and its 24-oxo metabolite produced growth inhibition and promoted differentiation of RWLeu-4 cells to the same extent, and these activities were several fold higher than those exerted by 1α,25(OH)₂D₃. In addition, the genomic action of each vitamin D compound was assessed in a rat osteosarcoma cell line (ROS 17/2.8) by measuring its ability to transactivate a gene construct containing the vitamin D response element of the osteocalcin gene linked to the growth hormone reporter gene. In these studies, both 1α,25(OH)₂-16-ene-D₃ and its 24-oxo metabolite exerted similar but potent transactivation activity which was several fold greater than that exerted by 1α,25(OH)₂D₃ itself. In summary, our results indicate that the production and slow clearance of the bioactive intermediary metabolite, 1α,25(OH)₂-24-oxo-16-ene-D₃, in RWLeu-4 cells contributes significantly to the final expression of the enhanced biological activities ascribed to its parent analog, 1α,25(OH)₂-16-ene-D₃.