Isolation of α-tubulin genes from the human malaria parasite, Plasmodium falciparum: sequence analysis of α-tubulin

As a step towards identifying exploitable differences between host and parasite at the molecular level, we have isolated and sequenced genomic clones encompassing an entire alpha-tubulin gene (designated alpha-tubulin I) from the human malaria parasite, Plasmodium falciparum. The gene, which contains two introns, encodes a product with a predicted length of 453 amino acid residues (50.3 kD). The protein sequence shows a high degree of homology to other alpha-tubulins, particularly that of the coccidian parasite, Toxoplasma gondii (94%), whose gene carries introns in identical positions. Only one copy of the alpha-tubulin I gene itself was found, although a second gene designated alpha-II was also identified which is closely related but which differs at both the nucleotide and amino acid sequence levels. The alpha-I and beta-tubulin genes were found to reside on different chromosomes.     

A Thermostable Acid α-Glucosidase from Tetrahymena thermophila: Purification and Characterization

An acid α-glucosidase (EC 3.2.1.20) was purified to homogeneity from the culture medium of Tetrahymena thermophila CU 399. Its general molecular, catalytic and immunological properties were compared to those of the T. pyriformis W enzyme. The enzyme from T. thermophila was a 105-kD monomer and the N-terminus (25 amino acid residues) displayed some homology with that of T. pyriformis enzyme. The purified enzyme was most active at 56° C and showed resistance to thermal inactivation. The acid α-glucosidase appears to have α-1,6-glucosidase as well as α-1,4-glucosidase activity. The Km values determined with p-nitrophenyl-α-glucopyranoside, maltose, isomaltose and glycogen were 0.7 mM, 2.5 mM, 28.5 mM and 18.5 mg/ml, respectively. The enzyme was antigenically distinct from T. pyriformis acid α-glucosidase.     

The extension of α-d-1,3-branch linkages by 1,3-α-d-glucan synthase from Streptococcus sobrinus

In the presence of an acceptor, 1,3-alpha-D-glucan synthase of Streptococcus sobrinus synthesizes water-insoluble glucans from sucrose. Under such conditions, 1,3-alpha-D-glucoside linkages were extended without any change in the glucose-residue number between the 1,3,6-branch points on the acceptor. From these results, the mechanisms of water-insoluble-glucan formation were proposed as follows: (i) the attachment of an acceptor to the glucan binding sites of 1,3-alpha-D-glucan synthase occurs during the initiation of the reaction, and concurrently determines the positions of the branched portions of 1,3,6 on the acceptor, and (ii) the 1,3-alpha-D-glucoside linkage extends from these positions.     

A subset of newly synthesized polypeptides in mitochondria from human endothelial cells exposed to hydroperoxide stress.

The synthesis of 40 polypeptides in mitochondria was found to be stimulated after transient exposure of human endothelial cells to sublethal levels of hydroperoxides, such as H(2)O(2), using comparative two-dimensional polyacrylamide gel electrophoresis. Eleven proteins were identified; these include 60 kDa heat shock protein (HSP60), a mitochondrial type of 70 kDa HSP (mtHSP70), manganese-dependent superoxide dismutase (MnSOD), three metabolic enzymes in citric acid cycle, two components for respiratory chain complexes, a ribosomal protein for translation in mitochondria (RM12), and an unnamed protein. These proteins are involved in reduction-oxidation and protein biogenesis, suggesting that their synthesis, which is triggered under oxidative stress conditions, is aimed at playing a defensive role in mitochondria. Moreover, mtHSP70, HSP60, MnSOD, and RM12 were revealed as their respective precursor proteins with mitochondrial targeting sequences. The preproteins of HSP60 and mtHSP70 were transiently accumulated in mitochondria after the removal of H(2)O(2) in a processing competent state, while the accumulated preprotein of MnSOD localized inside mitochondria and remained unchanged. Membrane potential of mitochondria and cellular ATP levels were unchanged under these conditions. Taken together, these results suggest that hydroperoxide stress leads to preprotein accumulation, possibly due to the impairment of the protein-processing system in mitochondria, independent of membrane potential dissipation and ATP depletion.     

A potent anti-oxidant property: fluorescent recombinant α-phycocyanin of Spirulina

Aims:  To express and product a fluorescent antioxidant holo-α-phycocyanin (PC) of Spirulina platensis ( Sp ) with His-tag (rHHPC; recombinant holo-α-phycocyaninof Spirulina platensis with His-tag) in 5-l bench scale.
Methods and Results:  A vector harbouring two cassettes was constructed: cpcA along with cpcE - cpcF in one cassette; ho1 - pcyA in the other cassette. Lyases CpcE/F of Synechocystis sp. PCC6803 ( S6 ) could catalyse the 82 site Cys in apo-α-PC of Sp linking with bilin chromophores, and rHHPC was biosynthesized in Escherichia coli BL21. The constant feeding mode was adopted, and transformant reached the biomass of rHHPC up to 0·55 g l⁻¹ broth in 5-litre bench scale. rHHPC was purified by Ni²⁺ affinity column conveniently. The absorbance and the fluorescence emission spectra of rHHPC had λ_max at 621 and 650 nm, respectively. The IC₅₀ values of rHHPC were 277·5 ± 25·8 μ g ml⁻¹ against hydroxyl radicals and 20·8 ± 2·2  μ g ml⁻¹ against peroxyl radicals.
Conclusions:  Combinational biosynthesis of rHHPC was feasible, and the constant feeding mode was adopted to produce good yields of rHHPC. Fluorescent rHHPC with several unique qualitative and quantitative features was effective on scavenging hydroxyl and peroxyl radicals.
Significance and impact of the study:  A potent antioxidant rHHPC was co-expressed, produced and characterized for nutritional and pharmacological values, which would help to develop phycobiliproteins' applications in their fluorescent and biological activities.     

Dα3, a New Functional α Subunit of Nicotinic Acetylcholine Receptors from Drosophila

Abstract: Nicotinic acetylcholine (ACh) receptors (nAChRs) are important excitatory neurotransmitter receptors in the insect CNS. We have isolated and characterized the gene and the cDNA of a new nAChR subunit from Drosophila . The predicted mature nAChR protein consists of 773 amino acid residues and has the structural features of an ACh-binding α subunit. It was therefore named Dα3, for D rosophila α -subunit 3 . The dα3 gene maps to the X chromosome at position 7E. The properties of the Dα3 protein were assessed by expression in Xenopus oocytes. Dα3 did not form functional receptors on its own or in combination with any Drosophila β-type nAChR subunit. Nondesensitizing ACh-evoked inward currents were observed when Dα3 was coexpressed with the chick β2 subunit. Half-maximal responses were at ∼0.15 µ M ACh with a Hill coefficient of ∼1.5. The snake venom component α-bungarotoxin (100 n M ) efficiently but reversibly blocked Dα3/β2 receptors, suggesting that Dα3 may be a component of one of the previously described two classes of toxin binding sites in the Drosophila CNS.     

The appearance of α, β and γ crystallins in an anophthalmic strain of mice

Abstract. A certain percentage of congenitally anophthalmic mouse embryos have the ability to generate small lens vesicles that have previously been shown to produce alpha crystallin at 13-day gestation. Further immunohistological analysis of 13- and 15-day-gestation anophthalmia embryos indicates that beta crystallin is present in those 13-day embryos which have lens vesicles with lens-fiber formation. Also, 15-day embryos with lenses demonstrating fiber elongation can produce both beta and gamma crystallins. The conclusion is drawn that the genetic potential to produce at least three characteristic biochemical markers of normal lens differentiation is present in the anophthalmia mutant. The spatial distribution patterns of the crystallins in normal and anophthalmia embryos were similar. However, there appeared to be a transposition in the temporal appearance of beta and gamma crystallins in the anophthalmia mutant. Optic cups and associated lenses in 15-day anophthalmia specimens were much smaller than those in controls. The optic and lens rudiments in these anophthalmia embryos were fairly proportional in size, which indicates that some degree of allometric growth compensation had occurred during the course of development. This ability for differential growth compensation in the mouse eye appears to be restricted to the predifferentiative stages of eye formation.     

The nucleotide sequence of an α-amylase gene from an alkalopsychrotrophic Micrococcus sp

An alpha-amylase gene from Micrococcus sp. 207 was cloned into Escherichia coli JM101 using the vector pHSG399. The constructed recombinant plasmid pYK63 contained a 4.8 kb chromosomal DNA fragment derived from strain 207 DNA. The cloned amylase isolated from E. coli JM101 (pYK63) produced mainly maltotetraose from starch, and exhibited temperature and pH activity profiles closely similar to those of the enzyme from the original strain. Nucleotide sequence analysis of the cloned DNA fragment revealed one open reading frame containing the gene which consisted of 3312 bp (1104 amino acids). When compared with several other alpha-amylases, three consensus sequences were identified in the region of the active site. About 300 amino acid residues were present both upstream and downstream of the active site region.     

The role of carbonic anhydrase α-CA4 in the adaptive reactions of photosynthetic apparatus: the study with α-CA4 knockout plants

Protoplasma - The role of α-carbonic anhydrase 4 (α-CA4) in photosynthetic machinery functioning in thylakoid membranes was studied, using Arabidopsis thaliana wild type plants (WT) and...     

Neuropeptide-γ: A Peptide Isolated from Rabbit Intestine that Is Derived from γ-Preprotachykinin

The neurokinin A-like immunoreactivity in an extract of rabbit small intestine was resolved into two molecular forms by gel permeation chromatography. These components were purified to apparent homogeneity by reverse-phase HPLC. The primary structure of the larger component was established as the following: Asp-Ala-Gly-His-Gly-Gln-Ile-Ser-His-Lys-Arg-His-Lys-Thr-Asp-Ser-Phe-Val- Gly-Leu - Met.NH2. This amino acid sequence represents residues (72-92) of gamma-preprotachykinin, as predicted from the nucleotide sequence of a cloned cDNA from the rat. The peptide, termed neuropeptide-gamma, lacks residues (3-17) of neuropeptide K, and this segment is specified exactly by exon 4 in the preprotachykinin gene. The smaller form of neurokinin A-like immunoreactivity was identical to neurokinin A. Neuropeptide K was not present in the extract, demonstrating that the pathways of post-translational processing of beta- and gamma-preprotachykinins in the rabbit gut are different.     

Organization of the genes coding for the reaction-centre L and M subunits and B870 antenna polypeptides α and β from the aerobic photosynthetic bacterium Erythrobacter species OCH114

In the aerobic photosynthetic bacterium Erythrobacter species OCH114 the structural genes coding for the light-harvesting (LH) complex B870 and the reaction-centre (RC) polypeptides (the gene products of the pufB, pufA, pufL and pufM genes) are mapped on a 2.728 kbp EcoRI fragment. Sequencing of this fragment revealed that the deduced amino acid sequences contain 50 (B870 beta), 52 (B850 alpha), 283 (RCL) and 331 (RCM) residues with the corresponding molecular weights of 5592, 5814, 31364, and 37671, respectively. In the corresponding mRNA a 'hairpin' structure (delta G degrees = -26.6 kcal) is predicted to be located immediately downstream of pufA. The RC and LH polypeptides are highly homologous to those of the purple photosynthetic bacteria Rhodobacter capsulatus, Rhodobacter sphaeroides and Rhodopseudomonas viridis. Directly downstream of pufM there is an open reading frame (ORF) of unknown size. Partial sequencing indicates that this ORF is highly homologous to the cytochrome subunit of the photosynthetic reaction centre from R. viridis. In the puf operon no pufQ or pufX genes could be found, but the bchA gene is located upstream of that operon. Plasmid pESS8.9 containing the 2.728 kbp EcoRI fragment reconstituted a photoinactive mutant of Erythrobacter species OCH114. Comparative analysis of the DNA region upstream of the puf operon and of bacteriochlorophyll (Bchl) synthesis indicated that Bchl synthesis and puf gene expression are regulated differently in Erythrobacter and purple bacteria, respectively.     

Phosphorylation of the α and β polypeptides of the light-harvesting complex I (B870) of Rhodobacter capsulatus in an in vitro translation system

Abstract Sporozoites and unsporulated oocysts of Eimeria tenella were shown to contain a pyrophosphate-dependent phosphofructokinase (PP_i-PFK) but apparently lack an ATP-specific activity. The PP_i-PFK resembles those that occur in a number of other protists in being reversible and not subject to metabolic control. In contrast, the ADP-utilising pyruvate kinase, present in two developmental stages of the parasite, exhibited strong positive cooperativity with respect to its substrate, phosphoenolpyruvate, and was shown to be allostetically activated by glucose 6-phosphate, fructose 6-phosphate and AMP. It is suggested that the PP_i-PFK represents an adaptation of the parasite towards life in an environment containing only low concentrations of oxygen and that the unusual allosteric regulation of pyruvate kinase evolved to compensate for glycolysis not being controlled at the PP_i-PFK step.     

Rapid Communication: A cis-Acting Element, Tα-1, in the Upstream Region of Rod α-Transducin Gene that Binds a Developmentally Regulated Retina-Specific Nuclear Factor

Abstract: The G protein transducin (T) is an integral component of the signal transduction pathway in photoreceptors. We have identified a cis -acting element, Ta-1, in the upstream region of the mouse rod a-T (T_rα) gene that may be important for tissue-specific expression. Tα-1 binds a retina-specific nuclear factor of apparent molecular mass of 90 kDa. Binding to the Tα-1 site is developmentally regulated and peaks between postnatal days 6 and 9. This corresponds to the time of rod photoreceptor maturation and the rise in T_rα gene expression. The sequence of Tα-1 shows homology with RET-1, a cis -acting element in the proximal promoter of opsin gene that binds a distinct retina-specific factor. Tα-1 and RET-1 sequences may have been derived from a prototype Tα-1/RET-1 sequence, evolved to confer photoreceptor specificity on retina-specific genes.     

Characterization of a Thermostable α-Amylase from Bacillus licheniformis NCIB 6346

With one exception (NCIB 9668), the extracellular amylases from 10 strains of Bacillus licheniformis were thermostable and retained more than 98% of their original activity after incubation at 85°C for 60 min. The enzyme from B. licheniformis NCIB 6346 was purified 30-fold by ion-exchange chromatography and was characterized. It had an endo-action on starch yielding maltopentaose as the major product, and was identified as an α-amylase. The purified enzyme had a molecular weight of 62 650, was stable between pH 7 and 10 and was maximally active at 70-90°C at pH 7.0. It closely resembled commercial thermostable α-amylases in its general properties and it is concluded that B. licheniformis provides a good source of these enzymes.