Photosynthesis research in India: transition from yield physiology into molecular biology

Photosynthesis research in India can be traced back several thousand years, with the mention of the Sun energizing the plants, which form food for all living creatures on the earth (from the Mahabharata, the great epic, ca. 2600 B.C.) and the report of Sage Parasara (ca. 100 B.C.) on the ability of plants to make their own food, due to their pigments. With the pioneering studies by Sir Jagdish Chandra Bose, work on photosynthesis proceeded steadily during the first half of the 20th century. Some of the classic reports during this period are: malate metabolism in Hydrilla, spectrophotometric estimation of chlorophylls, importance of spectral quality for photosynthesis – an indication of two photosystems, photoinactivation of photosynthesis, and importance of flag leaf photosynthesis to grain yield. After the 1960s, there was a burst of research in the areas of physiology and biochemistry of carbon assimilation and photochemistry. A significant transition occurred, before the beginning of new millennium, into the area of molecular biology of chloroplasts, regulation of photosynthesis and stress tolerance. Future research work in India is geared to focus on the following aspects of photosynthesis: elucidation/analysis of genes, molecular biology/evolution of enzymes, development/use of transgenics and modeling. This revised version was published online in August 2006 with corrections to the Cover Date.     

The discovery of the nature of ferredoxin in photosystems: A recollection

I describe here my recollection of the story of the discovery of the nature of ferredoxin in photosystems that began in 1965: this story involved the EPR measurements by a young physicist J.H.M. Thornley, using samples provided by J.F. Gibson and D. Hall, and in collaboration with F.R. Whatley.     

The use of region-specific antibodies in the characterization and localization of vasoactive intestinal polypeptide-like substances in the rat gastrointestinal tract

Antisera specific for different regions of porcine VIP have been used in radioimmunoassay and immunohistochemical studies of immunoreactive VIP in rat small and large intestine. Cation exchange chromatography of intestinal extracts separated two major and one minor peak of immunoreactivity. One major peak eluted in a similar position to natural porcine VIP and was read equally by NH₂-terminal-specific, and mid- and COOH-terminal-specific antisera. A second major peak, and the minor peak, eluted earlier than porcine VIP, and were read significantly less well with mid- and COOH-terminal antisera compared with NH₂-terminal-specific antisera. All forms of VIP occurred mainly in extracts of muscle layers of the gut, and no antiserum revealed more than trace amounts of immunoreactivity in mucosal extracts. In immunohistochemical studies all antisera demonstrated fluorescent nerve fibres in the enteric plexuses, circular smooth muscle and lamina propria; some antisera demonstrated nerve cell bodies predominantly in the submucous plexus. NH₂-terminal-specific antisera also demonstrated a sparse population of mucosal endocrine-like cells in the ileum and colon that were not seen with other antisera. It is concluded that VIPergic neurons of the rat gut contain a peptide closely resembling porcine VIP and at least two less basic variants with similar NH₂-terminal antigenic determinants. VIP-like peptides may also occur in endocrine cells, but since these peptides appearto fact that the majority of neuronal VIP in rat gut exists in a form that is both chromatographically and immunochemically distinct from porcine VIP, and may well possess different biological properties.     

Gene 67, a new,essential bacteriophage T4 head gene codes for a prehead core component,PIP: II. The construction in vitro of unconditionally lethal mutants and their maintenance

We have obtained frameshift mutations of the bacteriophage T4 gene 67 by manipulating restriction cleavage sites within the gene cloned onto small plasmids. When these mutated genes were recombined back into the T4 genome the resulting phages were inviable. They could only be propagated by complementation in strains carrying a cloned, non-mutated copy of the gene on a plasmid. These experiments demonstrate that gene 67 is essential for T4 growth. Electron microscopy of bacteria infected with 67^? phages revealed that phage head morphogenesis was blocked at an early stage and particles resembling abnormal preheads were found in large numbers. The gene 67 product, PIP, is therefore essential for correct prehead assembly.     

William Bateson from <Emphasis Type="Italic">Balanoglossus</Emphasis> to <Emphasis Type="Italic">Materials for the Study of Variation</Emphasis>: The Transatlantic Roots of Discontinuity and the (un)naturalness of Selection

William Bateson (1861–1926) has long occupied a controversial role in the history of biology at the turn of the twentieth century. For the most part, Bateson has been situated as the British translator of Mendel or as the outspoken antagonist of W.␣F. R. Weldon and Karl Pearson’s biometrics program. Less has been made of Bateson’s transition from embryologist to advocate for discontinuous variation, and the precise role of British and American influences in that transition, in the years leading up to the publication of his massive Materials for the Study of Variation (1894). In this paper, I first attempt to trace Bateson’s development in his early career before turning to search for the development of the moniker “anti-Darwinist” that has been attached to Bateson in well-known histories of the neo-Darwinian Synthesis.     

Chlorophyll isolation,structure and function: major landmarks of the early history of research in the Russian Empire and the Soviet Union

This paper covers major events of the early history of chlorophyll research in the Russian Empire and the Soviet Union from 1771 until 1952, when the modern period of studies on photosynthesis began in full swing. Short biographical sketches of key scientists, reviews of their major research contributions and some selected photographs are included. This revised version was published online in August 2006 with corrections to the Cover Date.     

Relationship between the "phospholipid effect" and calcium in the thyroid. I. - Effects of calcium ions, E.G.T.A., ionophore A 23187, verapamil and chlorpromazine on resting and stimulate thyroid slices.

The "phospholipid effect" which is the enhanced turnover of the phosphorylinositol group of phosphatidylinositol (PI) occurs in the thyroid of response to thyreostimulin (TSH). The possibility that Ca2+ ions are involved in this stimulation has been investigated with pig thyroid slices. Experiments performed in media without Ca2+ or containing E.G.T.A. (2 mM), indicate that it is not the extracellular Ca2+ which is implied, but rather the intracellular Ca2+. The ionophore A23187 (6.10(-6) M) increases the specific radioactivity of the acid soluble precursors, but has also a specific effect on the PI turnover, which is additive with the effect of a high concentration of TSH (50 mU/ml). Washing and loading of slices with various Ca2+ concentrations show that 0.9 mM restores the TSH phospholipid effect. Verapamil (10(-3) M) and Chlorpromazine (10(-3) M) redirect glycerolipid metabolism by increasing PI and phosphatidic acid (PA) synthesis at the expense of other glycerolipids, as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). These results suggest that the "phospholipid effect" is not a result of Ca2+ entry into the thyroid cells. On the contrary, it seems that this increased turnover of PI in "long term" incubations (3 hr). An additive and acute effect of TSH effect is more pronounced when Ca2+ movements     

On the integrated frameworks of species concepts: Mayden’s hierarchy of species concepts and de Queiroz’s unified concept of species

Richard L. Mayden and Kevin de Queiroz have devised and developed ‘a hierarchy of species concepts’ and ‘a unified species concept’, respectively. Although their integrated frameworks of species concepts are rather different as to how to integrate the diverse modern concepts of species, the end result is that they are likely to agree on species recognition in nature, because they virtually share the same major components (i.e. evolutionary or lineage concept of species; same way of delimiting species), and have the same important consequences. Both the hierarchical and unified frameworks, however, are interpreted to have shortcoming regarding the way of integrating the modern species concepts. I reformulate these ideas into a framework of species concepts as follows: It treats the idea of species as population‐level evolutionary lineages (sensu Wiley 1978 ) as the concept for species category, and it adopts the contingent biological properties of species (e.g. internal reproductive isolation, diagnosability, monophyly) as operational criteria in delimiting species. I also suggest that existing and revised versions of the integrated framework of species concepts all are not new species concepts, but versions of the evolutionary species concept, because they treat the evolutionary (or lineage) species concept as the concept for species category.     

Magnetic circular dichroism studies of hemoglobin: The reduction of ferrihemoglobin by ferrocytochrome b5 and characterization of the high-spin hydroxy species of mixed-valence hemoglobin

The final step in the erythrocyte methemoglobin reduction pathway, the transfer of an electron from cytochrome b₅, to methemoglobin, has been studied using magnetic circular dichroism spectroscopy. Spectral analysis allowed us to determine accurately the concentration of each redox species in mixtures of the two heme-proteins and to follow simultaneously the kinetics of the appearance or disappearance of each of these species during reduction reactions. Our analysis detected a substantial increase in the high-spin hydroxymethemoglobin species in the partially reduced bovine hemoglobin tetramer. This species was sensitive to the degree of reduction and pH, and was spectrally similar to fluoride methemoglobin. At pH 7.8. 100% of the hydroxide component of methemoglobin was in the high-spin form when two or more subunits were in the ferrous form. Kinetic analysis of bovine methemoglobin reduction yielded values for the apparent first-order rates for the tetrameric species possessing four, three, two, and one ferric subunit. Further analysis showed that the reduction kinetics can also be described by an equilibrium state, pure competitive inhibition model for enzyme catalysis in which ferrous and ferric subunits of hemoglobin compete for cytochrome b₅ This analysis generated a K_D that depends on ionic strength and hemoglobin tetramer conformation, a V_max that was independent of these factors, and an inhibition constant that was equal to K_d. This model is consistent with the hypothesis that the reduction of methemoglobin can be separated into two steps, the ionic interaction between cytochrome b₅ and hemoglobin and the electron transfer.     

Prevalence of known mutations and a novel missense mutation (M694K) in the MEFV gene in a population from the Eastern Anatolia Region of Turkey

Familial Mediterranean fever (FMF) is an autosomal recessive disorder characterized by recurrent attacks of fever and serositis. Mutations in the Mediterranean fever gene (MEFV) localized on the short arm of chromosome 16 cause FMF. Over 90 MEFV missense/nonsense mutations have been identified so far in FMF patients, mostly in the 10th exon of the gene.     

Apolar distal pocket mutants of yeast cytochrome c peroxidase: Hydrogen peroxide reactivity and cyanide binding of the TriAla,TriVal, and TriLeu variants

Three yeast cytochrome c peroxidase (CcP) variants with apolar distal heme pockets have been constructed. The CcP variants have Arg48, Trp51, and His52 mutated to either all alanines, CcP(triAla), all valines, CcP(triVal), or all leucines, CcP(triLeu). The triple mutants have detectable enzymatic activity at pH 6 but the activity is less than 0.02% that of wild-type CcP. The activity loss is primarily due to the decreased rate of reaction between the triple mutants and H₂O₂ compared to wild-type CcP. Spectroscopic properties and cyanide binding characteristics of the triple mutants have been investigated over the pH stability region of CcP, pH 4 to 8. The absorption spectra indicate that the CcP triple mutants have hemes that are predominantly five-coordinate, high-spin at pH 5 and six-coordinate, low-spin at pH 8. Cyanide binding to the triple mutants is biphasic indicating that the triple mutants have two slowly-exchanging conformational states with different cyanide affinities. The binding affinity for cyanide is reduced at least two orders of magnitude in the triple mutants compared to wild-type CcP and the rate of cyanide binding is reduced by four to five orders of magnitude. Correlation of the reaction rates of CcP and 12 distal pocket mutants with H₂O₂ and HCN suggests that both reactions require ionization of the reactants within the distal heme pocket allowing the anion to bind the heme iron. Distal pocket features that promote substrate ionization (basic residues involved in base-catalyzed substrate ionization or polar residues that can stabilize substrate anions) increase the overall rate of reaction with H₂O₂ and HCN while features that inhibit substrate ionization slow the reactions.     

Regulation and function of avian selenogenome

Background

Selenium (Se) is an essential micronutrient required by avian species. Dietary Se/vitamin E deficiency induces three classical diseases in chicks: exudative diathesis, nutritional pancreatic atrophy, and nutritional muscular dystrophy.

The validation of housekeeping genes as a reference in quantitative Real Time PCR analysis : Application in the milk somatic cells and frozen whole blood of goats infected with caprine arthritis encephalitis virus

The validation of housekeeping genes (HKGs) for normalization of RNA expression in Real-Time PCR is crucial to obtain the most reliable results. There is limited information on reference genes used in the study of gene expression in milk somatic cells and the frozen whole blood of goats. Thus, the aim of this study was to propose the most stable housekeeping genes that can be used as a reference in Real-Time PCR analysis of milk somatic cells and whole blood of goats infected with caprine arthritis encephalitis virus (CAEV). Animals were divided into two groups: non-infected (N = 13) and infected with CAEV (N = 13). Biological material (milk somatic cells and whole blood) was collected 4 times during the lactation period (7, 30, 100 and 240 days post-partum). The expression levels of candidate reference genes were analyzed using geNorm and NormFinder software. The stability of candidates for reference gene expression was analyzed for CAEV-free (control) and CAEV-infected groups, and also for both groups together (combined group). The stability of expression of β-actin (ACTB), glyceraldehyde-3P-dehydrogenase (GAPDH), cyclophilin A (PPIA), RNA18S1, ubiquilin (UBQLN1) and ribosomal protein large subunit P0 (RPLP0) was determined in milk somatic cells, while ACTB, PPIA, RPLP0, succinate dehydrogenase complex subunit A (SDHA), zeta polypeptide (YWHAZ), battenin (CLN3), eukaryotic translation initiation factor 3K (EIF3K) and TATA box-binding protein (TBP) were measured in frozen whole blood of goats. PPIA and RPLP0 were considered as the most suitable internal controls as they were stably expressed in milk somatic cells regardless of disease status, according to NormFinder software. Furthermore, geNorm results indicated the expression of PPIA/RPLP0 genes as the best combination under these experimental conditions. The results of frozen whole blood analysis using NormFinder software revealed that the most stable reference gene in control, CAEV-infected and combined groups is YWHAZ, and – according to the geNorm results – the combined expression of PPM/YWHAZ genes is the best reference in the presented experiment. The usefulness in gene expression analysis of whole blood samples frozen immediately in liquid nitrogen and stored at -80 °C was also proved.     

A new protein from human plasma lipoproteins: isolation and partial characterization of apolipoprotein G]

A new apolipoprotein has been identified in VHDL1 and in HDL. This protein is immunologically distinct from already isolated apoproteins. It was isolated by column chromatography on hydroxylapatite. In polyacrylamide gel electrophoresis, its mobility is very close to that of apo D. The amino acid composition differs from those of the well characterized polypeptides of the human plasma lipoproteins. It contains glucosamine. The apparent molecular weight is 72 000 +/- 2 000 in the presence and absence of reducing agent. According to the ABCDEF nomenclature, this protein can be named apolipoprotein G (apo G). It is present in a lipoprotein distinct from the lipoproteins A and D among the VHDL1 : this new lipoprotein can be named lipoprotein G (LPG).     

In silico analysis of Single Nucleotide Polymorphisms (SNPs) in human BRAF gene

BRAF gene mutations are frequently seen in both inherited and somatic diseases. However, the harmful mutations for BRAF gene have not been predicted in silico. Owing to the importance of BRAF gene in cell division, differentiation and secretion processes, the functional analysis was carried out to explore the possible association between genetic mutations and phenotypic variations. Genomic analysis of BRAF was initiated with SIFT followed by PolyPhen and SNPs&GO servers to retrieve the 85 deleterious non-synonymous SNPs (nsSNPs) from dbSNP. A total of 5 mutations i.e. c.406T>G (S136A), c.1446G>T (R462I), c.1556 A>G (K499E), c.1860 T>A (V600E) and c.2352 C>T (P764L) that are found to exert benign effects on the BRAF protein structure and function were chosen for further analysis. Protein structural analysis with these amino acid variants was performed by using I-Mutant, FOLD-X, HOPE, NetSurfP, Swiss PDB viewer, Chimera and NOMAD-Ref servers to check their solvent accessibility, molecular dynamics and energy minimization calculations. Our in silico analysis suggested that S136A and P764L variants of BRAF could directly or indirectly destabilize the amino acid interactions and hydrogen bond networks thus explain the functional deviations of protein to some extent. Screening for BRAF, S136A and P764Lvariants may be useful for disease molecular diagnosis and also to design the molecular inhibitors of BRAF pathways.