Functional evolution of C(4) pyruvate, orthophosphate dikinase

Pyruvate,orthophosphate dikinase (PPDK) plays a controlling role in the PEP-regeneration phase of the C(4) photosynthetic pathway. Earlier studies have fully documented its biochemical properties and its post-translational regulation by the PPDK regulatory protein (PDRP). However, the question of its evolution into the C(4) pathway has, until recently, received little attention. One assumption concerning this evolution is that changes in catalytic and regulatory properties of PPDK were necessary for the enzyme to fulfil its role in the C(4) pathway. In this study, the functional evolution of PPDK from its ancient origins in the Archaea to its ascension as a photosynthetic enzyme in modern C(4) angiosperms is reviewed. This analysis is accompanied by a comparative investigation into key catalytic and regulatory properties of a C(3) PPDK isoform from Arabidopsis and the C(4) PPDK isoform from Zea mays. From these analyses, it is proposed that PPDK first became functionally seated in C(3) plants as an ancillary glycolytic enzyme and that its transition into a C(4) pathway enzyme involved only minor changes in enzyme properties per se.     

The mechanism of action of virginiamycin M on the binding of aminoacyl-tRNA to ribosomes directed by elongation factor Tu

Mitochondrial DNA from Ustilago cynodontis has been investigated in several of its properties. Its dG + dC content is equal to 33.5%; its buoyant density (1.698 g/cm3) is higher, by 5 mg/cm3, and its melting temperature (82.5 degrees C) is lower than expected for a bacterial DNA having the same base composition; the first derivative of its melting curve indicates a large compositional heterogeneity, its molarity of elution from hydroxyapatite is high, 0.28 M phosphate, and allows its partial separation from nuclear DNA. Degradation by micrococcal nuclease indicates that about 25% of the DNA is formed by stretches having no more than 15% dG + dC. Finally, the unit size of mitochondrial genome is about 50 X 10(6). In most of its properties, the mitochondrial genome of U. cynodontis presents strong analogies with that of Saccharomyces cerevisiae. A parallel investigation on mitochondrial DNA from Acanthamoeba castellanii which has as genome unit size of only 27 X 10(6), has shown that this shares with the former the dG + dC content (32.9%), the melting temperature (82.5 degrees C), a large compositional heterogeneity and a very similar pattern of micrococcal nuclease degradation; its buoyant density (1.692 g/cm3) and its molarity of elution from hydroxyapatite (0.25 M phosphate) are, however, normal, probably because of a different short-sequence pattern and the fact that its dA + dT-rich stretches are shorter, on the average.     

Development and in vitro evaluation of thiolated chitosan--Poly(methacrylic acid) nanoparticles as a local mucoadhesive delivery system

The main objective of this study was to develop a local, oral mucoadhesive metronidazole benzoate (MET) delivery system that can be applied and removed by the patient for the treatment of periodontal diseases. The results of present study revealed that the retention time of MET at its absorption site could be increased by formulating it into nanoparticles using thiolated chitosan (TCS)-poly(methacrylic acid) (PMAA). The nanoparticles of MET prepared from TCS-PMAA may represent a useful approach for targeting its release at its site of absorption, sustaining its release and improving its oral availability.     

Antitumor action of seminal ribonuclease, its dimeric structure, and its resistance to the cytosolic ribonuclease inhibitor

Bovine seminal RNase (BS-RNase) is a homodimeric enzyme with a cytotoxic activity selective for tumor cells. In this study, the relationships of its cytotoxic activity to its dimeric structure and its resistance to the cytosolic RNase inhibitor (cRI) are investigated systematically by site-directed mutagenesis. The results show that (1) the dimericity of BS-RNase is essential for its full cytotoxic action; (2) the role of the dimeric structure in the antitumor activity is that of making the enzyme insensitive to the cytosolic RNase inhibitor; (3) a RNase may not be completely insensitive to cRI to exploit a full cytotoxic potential.     

Male‐female differences in family size preferences among college students

Abstract

Three dimensions of isolated spousal separation largely determine its fertility impact: (1) length, (2) “completeness” (degree to which it is eliminating fecundability during fecundable months), and (3) its timing as defined by the reproductive state of the wife at its start. While it is obvious that by starting when the wife is in the fecundable state its net delaying of next conception is maximized, it is much less clear with which reproductive state its start should coincide in order to minimize its fertility impact. By means of a simple model, it is shown that for minimum impact a separation should start later in a pregnancy the longer is post‐partum anovulation, the higher is the level of intrauterine mortality, and the shorter is the period of separation. Conditions of natural fertility are assumed.     

PLP-dependent H(2)S biogenesis

The role of endogenously produced H(2)S in mediating varied physiological effects in mammals has spurred enormous recent interest in understanding its biology and in exploiting its pharmacological potential. In these early days in the field of H(2)S signaling, large gaps exist in our understanding of its biological targets, its mechanisms of action and the regulation of its biogenesis and its clearance. Two branches within the sulfur metabolic pathway contribute to H(2)S production: (i) the reverse transsulfuration pathway in which two pyridoxal 5'-phosphate-dependent (PLP) enzymes, cystathionine β-synthase and cystathionine γ-lyase convert homocysteine successively to cystathionine and cysteine and (ii) a branch of the cysteine catabolic pathway which converts cysteine to mercaptopyruvate via a PLP-dependent cysteine aminotransferase and subsequently, to mercaptopyruvate sulfur transferase-bound persulfide from which H(2)S can be liberated. In this review, we present an overview of the kinetics of the H(2)S-generating reactions, compare the structures of the PLP-enzymes involved in its biogenesis and discuss strategies for their regulation. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.     

On the equivalence point for ammonium (de)protonation during its transport through the AmtB channel

Structural characterization of the bacterial channel, AmtB, provides a glimpse of how members of its family might control the protonated state of permeant ammonium to allow for its selective passage across the membrane. In a recent study, we employed a combination of simulation techniques that suggested ammonium is deprotonated and reprotonated near dehydrative phenylalanine landmarks (F107 and F31, respectively) during its passage from the periplasm to the cytoplasm. At these landmarks, ammonium is forced to maintain a critical number ( approximately 3) of hydrogen bonds, suggesting that the channel controls ammonium (de)protonation by controlling its coordination/hydration. In the work presented here, a free energy-based analysis of ammonium hydration in dilute aqueous solution indicates, explicitly, that at biological pH, the transition from ammonium (NH(4)(+)) to ammonia (NH(3)) occurs when these species are constrained to donate three hydrogen bonds or less. This result demonstrates the viability of the proposal that AmtB indirectly controls ammonium (de)protonation by directly controlling its hydration.     

Phorbol ester-treated human acute myeloid leukemia cells secrete G-CSF, GM-CSF and erythroid differentiation factor into serum-free media in primary culture.

Upon treatment with the phorbol ester, tetradecanoylphorbol 13-acetate (PMA), peripheral mononuclear blood cells from patients with acute myeloid leukemia secrete into serum-free cell-conditioned media (PMA-CCM) at least three distinct nondialysable 'hematopoietic' factors: granulocyte-colony-stimulating factor (G-CSF), granulocyte/macrophage-colony-stimulating factor (GM-CSF) and erythroid differentiation factor (EDF, activin A). G-CSF was identified by its stimulation of [3H]thymidine incorporation into a G-CSF-responsive cell line, NSF-60, and the inhibition of its stimulation by a G-CSF-specific monoclonal antibody (MAB). GM-CSF was identified by its stimulation of [3H]thymidine incorporation into a GM-CSF-responsive line, TALL-101, and the inhibition of its stimulation by a GM-CSF-specific MAB. EDF was identified by its ability to stimulate erythroid differentiation in mouse erythroleukemia cell lines, its identical retention times to those of authentic EDF on three successive reverse-phase HPLC columns and characterization of its penultimate N-terminal residue as leucine which is the same as that of authentic EDF. Both authentic EDF and the erythroid-stimulating activity in PMA-CCM were found to act synergistically with a suboptimal inducing concentration of a well-studied inducing agent, dimethyl sulfoxide, in inducing erythroid differentiation. In addition, a fourth activity was observed in PMA-CCM: normal human fetal bone marrow cell-proliferation stimulating activity (FBMC-PSA). FBMC-PSA was identified by its ability to stimulate the growth of granulocytes and macrophages in FBMC suspension cultures, which neither recombinant G-CSF or GM-CSF were found to do.     

Three Distinct Types of Monoclonal Antibodies After Long-Term Immunization of Rats with Mouse Nerve Growth Factor

This article reports the results of a systematic investigation of the different types of antibodies produced in the course of a long-term immunization of rats with mouse nerve growth factor (NGF). We have characterized three types of monoclonal antibodies, namely: (1) antibodies that bind to NGF and inhibit its binding to target cells and its biological activity in culture (type A); (2) antibodies that bind to and precipitate NGF but do not inhibit its binding to target cells or its biological activity (type B); (3) antibodies that fail to recognize NGF itself, but inhibit nonetheless its binding to target cells (type C). These antibodies bind to an antigen present on NGF target cells and not on rat fibroblasts lacking NGF receptor. They appear thus to be antiidiotypic antibodies directed against the NGF receptor, developed as a consequence of the long-term immunization with NGF.     

Subunit A of the E. coli ATP synthase: reconstitution and high resolution NMR with protein purified in a mixed polarity solvent

p23 is a regulatory co-chaperone of heat shock protein (Hsp) 90, but can also act as a general molecular chaperone by itself. Using novel point mutations of p23 that disrupt its interaction with Hsp90 we found its co-chaperone function to be required for its inhibitory effect on glucocorticoid receptor (GR). The C-terminal region of p23, which is required for its chaperone activity, is dispensable for inhibition of GR. Importantly, similar results were obtained with a constitutively active GR. Thus, the action of p23 on the nuclear stage of GR regulation requires its Hsp90 co-chaperone function, but not its chaperone activity.     

Metal binding and structure-activity relationship of the metalloantibiotic peptide bacitracin

Bacitracin is a widely used metallopeptide antibiotic produced by Bacillus subtilis and Bacillus licheniformis with a potent bactericidal activity directed primarily against Gram-positive organisms. This antibiotic requires a divalent metal ion such as Zn(2+) for its biological activity, and has been reported to bind several other transition metal ions, including Mn(2+), Co(2+), Ni(2+), and Cu(2+). Despite the widespread use of bacitracin since its discovery in the early 1940s, the structure-activity relationship of this drug has not been established and the coordination chemistry of its metal complexes was not fully determined until recently. This antibiotic has been suggested to influence cell functioning through more than one route. Since bacterial resistance against bacitracin is still rare despite several decades of widespread use, this antibiotic can serve as an ideal lead for the design of potent peptidyl antibiotics lacking bacterial resistance. In this review, the results of physical (including NMR, EPR, and EXAFS) and molecular biological studies regarding the synthesis and structure of bacitracin, the coordination chemistry of its metal derivatives, the mechanism of its antibiotic actions, its influence on membrane function, and its structure and function relationship are discussed.     

Focal adhesion kinase regulation of N-WASP subcellular localization and function

N-WASP is a member of the WASP family of proteins that regulate actin cytoskeleton remodeling. FAK is a cytoplasmic tyrosine kinase implicated in integrin signaling during cell migration. Here we identify a direct interaction between N-WASP and FAK and show that N-WASP is phosphorylated by FAK at a conserved tyrosine residue, Tyr(256). We found that phosphorylation of Tyr(256) affected N-WASP nuclear localization, suggesting that phosphorylation of N-WASP by FAK may regulate its activity in vivo by altering its subcellular localization. We also showed that the nuclear localization of N-WASP is dependent on its being in the open conformation either after its activation by Cdc42 or the truncation of the C-terminal VCA domain. Phosphorylation of Tyr(256) of N-WASP could reduce its interaction with nuclear importin NPI-1, which might be responsible for its decreased nuclear localization. Lastly, we show that phosphorylation of Tyr(256) plays an important role in promoting cell migration. Together, these results suggest a novel regulatory mechanism of N-WASP by tyrosine phosphorylation and subcellular localization and its potential role in the regulation of cell migration.     

The role of posttranslational modification in moonlighting glyceraldehyde-3-phosphate dehydrogenase structure and function

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a moonlighting protein exhibiting distinct activities apart from its classical role in glycolysis. Regulation of its moonlighting functions and its subcellular localization may be dependent on its posttranslational modification (PTM). The latter include its phosphorylation, which is required for its role in intermembrane trafficking, synaptic transmission and cancer survival; nitrosylation, which is required for its function in apoptosis, heme metabolism and the immune response; acetylation which is necessary for its modulation of apoptotic gene regulation; and N-acetylglucosamine modification which may induce changes in GAPDH oligomeric structure. These findings suggest a structure function relationship between GAPDH posttranslational modification and its diverse moonlighting activities.

     

大蒜不同品种蒜薹发育的解剖学研究

通过形态观测和石蜡切片法,比较了2个大蒜品种的蒜薹发育和解剖结构。结果表明：（1）“陇县火蒜”比“改良蒜”蒜薹的表皮细胞形状规则,排列致密;角质层较薄;（2）“陇县火蒜”比“改良蒜”蒜薹表面的气孔数量少,但开张度大;分泌细胞出现早、体积大、数量多;维管束数量少、直径小;（3）“陇县火蒜”蒜薹髓细胞卫多边形,髓细胞间隙率小,而“改良蒜”蒜薹的髓细胞呈椭圆形,髓细胞间隙率大。     

Phosphatidylinositol-4-phosphate 5-kinase regulates fission yeast cell integrity through a phospholipase C-mediated protein kinase C-independent pathway

Fission yeast its3-1 mutant is an allele of the essential gene its3+ that encodes a phosphatidylinositol-4-phosphate 5-kinase (PIP5K) that produces phosphatidylinositol 4,5-bisphosphate. We found that the its3-1 mutant is sensitive to micafungin, a (1,3)-beta-D-glucan synthase inhibitor, suggesting a cell wall integrity defect. Consistently, its3-1 mutation caused synthetic lethality with a (1,3)-beta-D-glucan synthase mutant, bgs1-i2, and its3-1 mutant cells showed aberrant localization of green fluorescent protein-Bgs1. Similar aberrant localization of green fluorescent protein-tagged Rgf1, a putative phosphatidylinositol 4,5-bisphosphate-binding guanine nucleotide exchange factor for Rho protein, in its3-1 mutants was observed, suggesting a defective Rgf1/Rho pathway. To unravel the molecular mechanism(s), putative downstream components of PIP5K signaling were analyzed. Unexpectedly, overexpression of phospholipase C (Plc1), but not that of protein kinase C (PKC; Pck1 and Pck2), suppressed the phenotypes of the its3-1 mutant. These findings indicate that PKCs are not involved in the suppression, and further analysis revealed that PKCs are not downstream of Plc1 in fission yeast. Also, the enzymatic activity of Plc1 is essential for the suppression of the phenotypes and for the viability of the its3-1 mutant. These findings suggest that Its3 PIP5K regulates cell integrity through a Plc1-mediated PKC-independent pathway, in addition to the Rho/PKC pathway.     

Biophoton emission

The phenomenon of ultraweak photon emission from living systems was further investigated in order to elucidate the physical properties of this radiation and its possible source. We obtained evidence that the light has a high degree of coherence because of (1) its photon count statistics, (2) its spectral distribution, (3) its decay behavior after exposure to light illumination, and (4) its transparency through optically thick materials. Moroever, DNA is apparently at least an important source, since conformational changes induced with ethidium bromide in vivo are clearly reflected by changes of the photon emission of cells. The physical properties of the radiation are described, taking DNA as an exciplex laser system, where a stable state can be reached far from thermal equilibrium at threshold.     

Regulation of protein synthesis by estradiol-17beta in cultured hepatocytes

Estradiol-17beta added to cultured chick embryo hepatocytes induced the appearance in the medium of a phosphoprotein, identified as phosvitin on the basis of: (i) its behaviour on ionic exchange columns; (ii) its SDS-acrylamide gel electrophoretic mobility; (iii) its amino acid composition. The hormone treatment was also followed by a decreased synthesis of other proteins secreted by the hepatocytes.     

Homology and structure-function correlations between alpha 1-acid glycoprotein and serum retinol-binding protein and its relatives

Although the serum protein alpha 1-acid glycoprotein (AGP) or orosomucoid has been extensively studied, its relationships with other proteins have been controversial and its precise physiological function has remained unclear. It is shown here that AGP is significantly similar in amino acid sequence and in the locations of introns in its structural gene to members of a protein superfamily that includes serum retinol-binding protein (RBP), beta-lactoglobulin (LG), alpha 2u-globulin, and protein HC (alpha 1-microglobulin). The view that the three-dimensional structure of AGP is closely similar to the published structures of RBP and LG is supported by its homology with these proteins, similarities in disulfide bond arrangements, and its secondary structure profile, predicted from the amino acid sequence. The relationship of AGP with this particular protein family indicates that its well-characterized ability to bind lipophilic drugs and certain steroids is a reflection of its true biological role. It is proposed that AGP and the other members of this extensive group of proteins should be designated lipocalins to reflect a common ability to bind lipophiles by enclosure within their structures in a manner that minimizes solvent contact.     

Physiology and Nutrition of Lampropedia hyalina

A detailed study of the physiology and nutrition of Lampropedia hyalina revealed that it is an aerobic, cytochrome-containing chemoheterotroph which is limited in its energy sources to a few intermediates (and close derivatives) of the Krebs cycle. Reducing compounds at low levels are potent growth inhibitors. The microbe has no photosynthetic ability (despite its previous taxonomic position with the sulfur purple bacteria). The results of a general investigation of its physiology are reported. Added biotin and thiamine are needed for growth in defined media; pantothenate is strongly stimulatory. Alanine, arginine, and tyrosine, as well as NH(4)Cl, serve as sole nitrogen sources. A unique motion exhibited by cells of a rapidly growing culture is described. Aspects of its metabolism of poly-beta-hydroxybutyrate and limiting aspects of its physiology as related to its ecology are discussed.