bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of osmiophilic bodies and Pfg377 expression in female gametocyte emergence and mosquito infectivity in the human malaria parasite Plasmodium falciparum
Authors: De Koning‐Ward Tania F. , Olivieri Anna , Bertuccini Lucia , Hood Andrew , Silvestrini Francesco , Charvalias Konstantinos , Berzosa Díaz Pedro , Camarda Grazia , McElwain Terry F. , Papenfuss Tony , Healer Julie , Baldassarri Lucilla , Crabb Brendan S. , Alano Pietro , Ranford‐Cartwright Lisa C.
Published: August 2010

Osmiophilic bodies are membrane‐bound vesicles, found predominantly in Plasmodium female gametocytes, that become progressively more abundant as the gametocyte reaches full maturity. These vesicles lie beneath the subpellicular membrane of the gametocyte, and the release of their contents into the parasitophorous vacuole has been postulated to aid in the escape of gametocytes from the erythrocyte after ingestion by the mosquito. Currently, the only protein known to be associated with osmiophilic bodies in Plasmodium falciparum is Pfg377, a gametocyte‐specific protein expressed at the onset of osmiophilic body development. Here we show by targeted gene disruption that Pfg377 plays a fundamental role in the formation of these organelles, and that female gametocytes lacking the full complement of osmiophilic bodies are significantly less efficient both in vitro and in vivo in their emergence from the erythrocytes upon induction of gametogenesis, a process whose timing is critical for fertilization with the short‐lived male gamete. This reduced efficiency of emergence explains the significant defect in oocyst formation in mosquitoes fed blood meals containing Pfg377‐negative gametocytes, resulting in an almost complete blockade of infection.
Reciprocal oxylipin‐mediated cross‐talk in the Aspergillus–seed pathosystem
Authors: Brodhagen Marion , Tsitsigiannis Dimitrios I. , Hornung Ellen , Goebel Cornelia , Feussner Ivo , Keller Nancy P.
Published: August 2010

In Aspergilli, mycotoxin production and sporulation are governed, in part, by endogenous oxylipins (oxygenated, polyunsaturated fatty acids and metabolites derived therefrom). In Aspergillus nidulans, oxylipins are synthesized by the dioxygenase enzymes PpoA, PpoB and PpoC. Structurally similar oxylipins are synthesized in seeds via the action of lipoxygenase (LOX) enzymes. Previous reports have shown that exogenous application of seed oxylipins to Aspergillus cultures alters sporulation and mycotoxin production. Herein, we explored whether a plant oxylipin biosynthetic gene (ZmLOX3) could substitute functionally for A. nidulans ppo genes. We engineered ZmLOX3 into wild‐type A. nidulans, and into a ΔppoAC strain that was reduced in production of oxylipins, conidia and the mycotoxin sterigmatocystin. ZmLOX3 expression increased production of conidia and sterigmatocystin in both backgrounds. We additionally explored whether A. nidulans oxylipins affect seed LOX gene expression during Aspergillus colonization. We observed that peanut seed pnlox2–3 expression was decreased when infected by A. nidulansΔppo mutants compared with infection by wild type. This result provides genetic evidence that fungal oxylipins are involved in plant LOX gene expression changes, leading to possible alterations in the fungal/host interaction. This report provides the first genetic evidence for reciprocal oxylipin cross‐talk in the Aspergillus–seed pathosystem.
Intracellular levels and activity of PvdS, the major iron starvation sigma factor of Pseudomonas aeruginosa
Authors: Tiburzi Federica , Imperi Francesco , Visca Paolo
Published: August 2010

In Pseudomonas aeruginosa the iron starvation sigma factor PvdS directs the transcription of pyoverdine and virulence genes under iron limitation. PvdS activity is modulated by pyoverdine through the surface signalling cascade involving the FpvA receptor and the inner membrane‐spanning sensor FpvR. To gain insight into the molecular mechanisms enabling PvdS to compete with the major sigma RpoD for RNA polymerase (RNAP) binding, we determined the intracellular levels of RNAP, RpoD and PvdS in P. aeruginosa PAO1, and the effect of pyoverdine signalling on PvdS activity. Under iron limitation, P. aeruginosa contains 2221 and 933 molecules of RNAP and RpoD per cell respectively. PvdS attains 62% of RpoD levels. The high PvdS content is partly offset by retention of 30% of PvdS on the membrane, lowering the concentration of cytosolic PvdS to 45% of RpoD levels. RNAP purification from iron‐starved P. aeruginosa cells demonstrated that PvdS–RNAP is poorly represented compared with RpoD–RNAP (1 and 27% of total RNAP respectively). Pyoverdine signalling does not affect the PvdS cellular content but facilitates PvdS release from the membrane, increasing its cytosolic concentration from 35% in both pvdF and fpvA signalling mutants to 70% in the wild type and 83% in the fpvR mutant.
Inadequate inhibition of host RNA polymerase restricts T7 bacteriophage growth on hosts overexpressing udk
Authors: Qimron Udi , Kulczyk Arkadiusz W. , Hamdan Samir M. , Tabor Stanley , Richardson Charles C.
Published: August 2010

Overexpression of udk, an Escherichia coli gene encoding a uridine/cytidine kinase, interferes with T7 bacteriophage growth. We show here that inhibition of T7 phage growth by udk overexpression can be overcome by inhibition of host RNA polymerase. Overexpression of gene 2, whose product inhibits host RNA polymerase, restores T7 phage growth on hosts overexpressing udk. In addition, rifampicin, an inhibitor of host RNA polymerase, restores the burst size of T7 phage on udk‐overexpressing hosts to normal. In agreement with these findings, suppressor mutants that overcome the inhibition arising from udk overexpression gain the ability to grow on hosts that are resistant to inhibition of RNA polymerase by gene 2 protein, and suppressor mutants that overcome a lack of gene 2 protein gain the ability to grow on hosts that overexpress udk. Mutations that eliminate or weaken strong promoters for host RNA polymerase in T7 DNA, and mutations in T7 gene 3.5 that affect its interaction with T7 RNA polymerase, also reduce the interference with T7 growth by host RNA polymerase. We propose a general model for the requirement of host RNA polymerase inhibition.
Osmiophilic bodies and the odd organelles of alveolates
Authors: Hayton Karen , Templeton Thomas J.
Published: August 2010

The apicomplexa are parasitic protozoa that are responsible for important human and animal diseases, including malaria, toxoplasmosis, cryptosporidiosis, coccidiosis and babesiosis. Like other members of the superphylum Alveolata, apicomplexans have regulated exocytosis of specialized secretory organelles, such as the apicomplexan‐specific rhoptries and micronemes that are required for host cell invasion. The secretions of another class of organelles, the dense granules and osmiophilic bodies, are proposed to be required for maintenance of the parasitophorous vacuole and host cell egress. Little is known about the osmiophilic bodies and to date only one protein, P377, has been localized to this organelle. In this issue, de Koning‐Ward et al. describe the disruption of pfg377 in the virulent human malaria parasite, Plasmodium falciparum, which results in reduced osmiophilic body formation, a marked decrease in female fitness, and dramatically impaired infectivity to mosquitoes. These findings suggest that targeting PFG377 may be a strategy to block parasite transmission.
Electron tomography of the Maurer's cleft organelles of Plasmodium falciparum‐infected erythrocytes reveals novel structural features
Authors: Hanssen Eric , Sougrat Rachid , Frankland Sarah , Deed Samantha , Klonis Nectarios , Lippincott‐Schwartz Jennifer , Tilley Leann
Published: August 2010

During intraerythrocytic development, the human malaria parasite, Plasmodium falciparum, establishes membrane‐bound compartments, known as Maurer's clefts, outside the confines of its own plasma membrane. The Maurer's compartments are thought to be a crucial component of the machinery for protein sorting and trafficking; however, their ultrastructure is only partly defined. We have used electron tomography to image Maurer's clefts of 3D7 strain parasites. The compartments are revealed as flattened structures with a translucent lumen and a more electron‐dense coat. They display a complex and convoluted morphology, and some regions are modified with surface nodules, each with a circular cross‐section of ∼25 nm. Individual 25 nm vesicle‐like structures are also seen in the erythrocyte cytoplasm and associated with the red blood cell membrane. The Maurer's clefts are connected to the red blood cell membrane by regions with extended stalk‐like profiles. Immunogold labelling with specific antibodies confirms differential labelling of the Maurer's clefts and the parasitophorous vacuole and erythrocyte membranes. Spot fluorescence photobleaching was used to demonstrate the absence of a lipid continuum between the Maurer's clefts and parasite membranes and the host plasma membrane.
Trigger enzymes: bifunctional proteins active in metabolism and in controlling gene expression
Authors: Commichau Fabian M. , Stülke Jörg
Published: August 2010

All regulatory processes require components that sense the environmental or metabolic conditions of the cell, and sophisticated sensory proteins have been studied in great detail. During the last few years, it turned out that enzymes can control gene expression in response to the availability of their substrates. Here, we review four different mechanisms by which these enzymes interfere with regulation in bacteria. First, some enzymes have acquired a DNA‐binding domain and act as direct transcription repressors by binding DNA in the absence of their substrates. A second class is represented by aconitase, which can bind iron responsive elements in the absence of iron to control the expression of genes involved in iron homoeostasis. The third class of these enzymes is sugar permeases of the phosphotransferase system that control the activity of transcription regulators by phosphorylating them in the absence of the specific substrate. Finally, a fourth class of regulatory enzymes controls the activity of transcription factors by inhibitory protein–protein interactions. We suggest that the enzymes that are active in the control of gene expression should be designated as trigger enzymes. An analysis of the occurrence of trigger enzymes suggests that the duplication and subsequent functional specialization is a major pattern in their evolution.
Vitamin B12 partners the CarH repressor to downregulate a photoinducible promoter in Myxococcus xanthus
Authors: Pérez‐Marín Mari Cruz , Padmanabhan S. , Polanco María Carmen , Murillo Francisco José , Elías‐Arnanz Montserrat
Published: August 2010

A light‐inducible promoter, PB, drives expression of the carB operon in Myxococcus xanthus. Repressed by CarA in the dark, PB is activated when CarS, produced in the light, sequesters CarA to prevent operator‐CarA binding. The MerR‐type, N‐terminal domain of CarA, which mediates interactions with both operator and CarS, is linked to a C‐terminal oligomerization module with a predicted cobalamin‐binding motif. Here, we show that although CarA does bind vitamin B12, mutating the motif involved has no effect on its ability to repress PB. Intriguingly, PB could be repressed in the dark even with no CarA, so long as B12 and an intact CarA operator were present. We have discovered that this effect of B12 depends on the gene immediately downstream of carA. Its product, CarH, also consists of a MerR‐type, N‐terminal domain that specifically recognizes the CarA operator and CarS, linked to a predicted B12‐binding C‐terminal oligomerization module. The B12‐mediated repression of PB in the dark is relieved by deleting carH, by mutating the DNA‐ or B12‐binding residues of CarH, or by illumination. Our findings unveil parallel regulatory circuits that control a light‐inducible promoter using a transcriptional factor repertoire that includes a paralogous gene pair and vitamin B12.
The Bacillus subtilis	σM regulon and its contribution to cell envelope stress responses
Authors: Eiamphungporn Warawan , Helmann John D.
Published: August 2010

The Bacillus subtilis extracytoplasmic function (ECF) σM factor is activated by cell envelope stress elicited by antibiotics, and by acid, heat, ethanol and superoxide stresses. Here, we have used several complementary approaches to identify genes controlled by σM. In many cases, expression is only partially dependent on σM because of both overlapping promoter recognition with other ECF σ factors and the presence of additional promoter elements. Genes regulated by σM have a characteristic pattern of induction in response to cell envelope‐acting antibiotics as evidenced by hierarchical clustering analysis. σM also contributes to the expression of the Spx transcription factor and thereby indirectly regulates genes of the Spx regulon. Cell envelope stress responses also include regulons controlled by σW, σB and several two‐component regulatory systems (e.g. LiaRS, YycFG, BceRS). Activation of the σM regulon increases expression of proteins functioning in transcriptional control, cell wall synthesis and shape determination, cell division, DNA damage monitoring, recombinational repair and detoxification.
Pneumocystis PCINT1, a molecule with integrin‐like features that mediates organism adhesion to fibronectin
Authors: Kottom Theodore J. , Kennedy Cassie C. , Limper Andrew H.
Published: August 2010

Pneumocystis species cause severe pneumonia during chronic immunosuppression, especially in patients with AIDS or malignancy. Adhesion of Pneumocystis to extracellular matrix proteins, particularly fibronectin, associated with alveolar epithelial cell surfaces, triggers organism proliferative pathways. Herein, we report the characterization of a novel Pneumocystis molecule with considerable structural features of an integrin‐like extracellular matrix adhesion receptor. A PCINT1115 bp probe was initially identified from partial sequence present within the Pneumocystis genome project database. A full‐length 3018 bp cDNA was subsequently obtained with extensive homology to the C‐terminal region of Candida albicans INT1 (31% blastx), a gene originally described as encoding an integrin‐like molecule implicated in adhesion, growth, and virulence. Sequence analysis of PCINT1 indicated that the Pneumocystis molecule contained both a putative internal RGD motif and four Metal Ion‐Dependent Attachment Sites (MIDAS) motifs required for coordination of divalent cations, as well as a specific tyrosine residue found in the cytoplasmic tails of some integrin receptors and C. albicans INT1. Northern, Western and immunofluorescence studies demonstrated that the trophic forms of Pneumocystis, known to be the life cycle forms that tightly adhere to lung epithelium, expressed the molecule to a substantially greater degree than cystic forms. Heterologous expression of PCINT1 in yeast followed by application to human fibronectin‐coated surfaces demonstrated these yeast display PCINT1 on their surfaces and subsequently gain the ability to bind fibronectin in a cation dependent fashion. Taken together, these results indicate that Pneumocystis expresses a novel integrin‐like PCINT1 molecule sufficient to mediate interactions with extracellular matrix fibronectin, an integral component of host–cell organism interactions during this infection.
GTP‐dependent polymerization of the tubulin‐like RepX replication protein encoded by the pXO1 plasmid of Bacillus anthracis
Authors: Anand Syam P. , Akhtar Parvez , Tinsley Eowyn , Watkins Simon C. , Khan Saleem A.
Published: August 2010

RepX protein encoded by the pXO1 plasmid of Bacillus anthracis is required for plasmid replication. RepX harbours the tubulin signature motif and contains limited amino acid sequence homology to the bacterial cell division protein FtsZ. Although replication proteins are not known to polymerize, here we show by electron microscopy that RepX undergoes GTP‐dependent polymerization into long filaments. RepX filaments assembled in the presence of GTPγS were more stable than those assembled in the presence of GTP, suggesting a role for GTP hydrolysis in the depolymerization of the filaments. Light scattering studies showed that RepX underwent rapid polymerization, and substitution of GTP with GTPγS stabilized the filaments. RepX exhibited GTPase activity and a mutation in the tubulin signature motif severely impaired its GTPase activity and its polymerization in vitro. Unlike FtsZ homologues, RepX harbours a highly basic carboxyl‐terminal region and exhibits GTP‐dependent, non‐specific DNA binding activity. We speculate that RepX may be involved in both the replication and segregation of the pXO1 plasmid.
An aniline blue staining procedure for confocal microscopy and 3D imaging of normal and perturbed cellular phenotypes in mature Arabidopsis embryos
Authors: Bougourd Sue , Marrison Jo , Haseloff Jim
Published: August 2010

A new method is described for fluorescent imaging of mature Arabidopsis embryos that enables their cellular architecture to be visualized without the need for histological sectioning. Mature embryos are stained with aniline blue and cleared with chloral hydrate to allow high‐resolution confocal imaging of individual cells within the embryo prior to germination. The technique allows the collection of longitudinal optical sections throughout the cotyledon, hypocotyl and root of wild‐type Arabidopsis C24 embryos. Every cell within the mature embryo can be visualized with sufficient clarity and resolution to allow three‐dimensional analysis of cellular architecture. Optical sectioning of mutant gnom, short‐root and scarecrow embryos, and through root meristems disrupted as a consequence of targeted misexpression of diphtheria toxin, demonstrate the potential of this technique for visualizing the cellular organization of mutant and perturbed embryonic phenotypes.
Arabidopsis ARR1 and ARR2 response regulators operate as transcriptional activators
Authors: Sakai Hiroe , Aoyama Takashi , Oka Atsuhiro
Published: August 2010

The genes coding for the response regulators ARR1 and ARR2 have previously been identified by in silico screening of an expression sequence tag database and subsequent cloning from both Arabidopsis cDNA and genomic libraries. Their structures, in which the N‐terminal signal receiver domain is followed by the output domain, are characteristic of typical bacterial response regulators of the two‐component regulatory systems that control responses to a variety of environmental stimuli. Here we present evidence that these response regulators actually work as transcription factors. ARR1 and ARR2 were localized in the nuclei of plant cells regardless of the presence or absence of their signal receiver domain. Their middle segments, which faintly resemble the mammalian oncogene product Myb, were capable of binding double‐stranded DNA in a sequence‐specific manner in vitro. Their C‐terminal halves functioned as transactivation domains in plant cells when combined with the DNA‐binding domain of yeast GAL4. They thus possess all the essential components of a transcriptional activator. Both ARR1 and ARR2 promoted expression of a reporter gene in plant cells through their own target sequence. Truncation of their N‐terminal signal receiver domain led to an increase in transactivation. An as yet unidentified phospho‐relay signal may modulate the capability for transactivation and/or DNA binding through the signal receiver domain.
Temperature‐dependent translational regulation of the ER ω‐3 fatty acid desaturase gene in wheat root tips
Authors: Horiguchi Gorou , Fuse Takuichi , Kawakami Naoto , Kodama Hiroaki , Iba Koh
Published: August 2010

The proportion of α‐linolenic acid (18:3) among the total fatty acids in root tissue increases as the growth temperature decreases. Endoplasmic reticulum ω‐3 fatty acid desaturase is responsible for the production of most of the 18:3 in root tissue. In this study, the effect of temperature on expression of the TaFAD3 gene, which encodes endoplasmic reticulum ω‐3 desaturase in wheat (Triticum aestivum L. cv. Chihoku), was analysed at the mRNA and protein levels. In wheat root tips grown at 30 and 10°C, the proportions of 18:3 among total fatty acids were 22% and 55%, respectively. The level of TaFAD3 protein in microsomal preparations of the root tips grown at 10°C was approximately 7.5 times higher than that of the root tips grown at 30°C. The increased level of TaFAD3 protein at the lower temperature was accompanied by enhanced association of TaFAD3 mRNA with polyribosomes. In contrast, the level of TaFAD3 mRNA in root tips grown at 10°C was slightly higher than in those grown at 30°C. These results suggest that, in root tips, the increase in the 18:3 level at low temperature is achieved directly by an increase in the amount of TaFAD3 protein, and that temperature‐dependent translational regulation of the TaFAD3 gene, rather than its transcriptional regulation, contributes to modulation of the TaFAD3 protein accumulation.
The MuDR transposon terminal inverted repeat contains a complex plant promoter directing distinct somatic and germinal programs
Authors: Raizada Manish N. , Benito Maria‐Ines , Walbot Virginia
Published: August 2010

The Mu transposons of maize are under stringent developmental control. Elements excise at high frequencies in terminally dividing somatic cells, but not in meristems. Mu elements in germinal cells amplify, without excision, and insert throughout the genome. All activities require MuDR, which encodes two genes, mudrA and mudrB, whose near‐identical promoters are located in the transposon terminal inverted repeats (TIR). We have fused the 216 bp TIR of the mudrB gene to GUS and luciferase reporters. We demonstrate that TIRB programs reporter expression in diverse, meristematic somatic cells, paradoxically in those cells in which Mu excisions are repressed. In germinal cells, immature tassel and mature pollen, reporter expression increases up to 20‐fold compared to leaf. By RNA blot hybridization, we demonstrate that endogenous mudrB and mudrA transcripts increase significantly in mature pollen; sequence comparisons demonstrate that the MuDR TIRs contain plant cell‐cycle enhancer motifs and functionally defined pollen enhancers. Therefore, the MuDR TIR promoters are developmentally regulated in both somatic and germinal tissues. Because database sequence analysis suggests that the MuDR TIR enhancers should be functional in both monocots and dicots, we suggest that the native MuDR promoter be used in attempts to transfer the unique behavior of Mu transposition to heterologous hosts.
Probenazole induces systemic acquired resistance in Arabidopsis with a novel type of action
Authors: Yoshioka Keiko , Nakashita Hideo , Klessig Daniel F. , Yamaguchi Isamu
Published: August 2010

Probenazole (PBZ; 3‐allyloxy‐1,2‐benzisothiazole‐1,1‐dioxide), which is the active ingredient in Oryzemate, has been used widely in Asia to protect rice plants against the rice blast fungus Magnaporthe grisea. To study PBZ's mode of action, we analyzed its ability, as well as that of its active metabolite 1, 2‐benzisothiazol‐3 (2H)‐one 1,1‐dioxide (BIT) to induce defense gene expression and resistance in Arabidopsis mutants that are defective in various defense signaling pathways. Wild‐type Arabidopsis treated with PBZ or BIT exhibited increased expression of several pathogenesis‐related genes, increased levels of total salicylic acid (SA), and enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC 3000 and the oomycete pathogen Peronospora parasitica Emco5. The role of several defense signaling hormones, such as SA, ethylene and jasmonic acid (JA), in activating resistance following PBZ or BIT treatment was analyzed using NahG transgenic plants and etr1‐1 and coi1‐1 mutant plants, respectively. In addition, the involvement of NPR1, a key component in the SA signaling pathway leading to defense responses, was assessed. PBZ or BIT treatment did not induce disease resistance or PR‐1 expression in NahG transgenic or npr1 mutant plants, but it did activate these phenomena in etr1‐1 and coi 1‐1 mutant plants. Thus SA and NPR1 appear to be required for PBZ‐ and BIT‐mediated activation of defense responses, while ethylene and JA are not. Furthermore, our data suggest that PBZ and BIT comprise a novel class of defense activators that stimulate the SA/NPR1‐mediated defense signaling pathway upstream of SA.
Substrate preferences of O‐methyltransferases in alfalfa suggest new pathways for 3‐O‐methylation of monolignols
Authors: Parvathi Kota , Chen Fang , Guo Dianjing , Blount Jack W. , Dixon Richard A.
Published: August 2010

Measurement of relative O‐methyltransferase activities against all potential substrates in the monolignol pathway in developing alfalfa stem extracts revealed activities in the order: caffeoyl CoA > caffeoyl alcohol > 5‐hydroxyferulic acid > caffeoyl aldehyde > 5‐hydroxyconiferyl alcohol > 5‐hydroxyferuloyl CoA > 5‐hydroxyconiferaldehyde > caffeic acid. Maxima for all activities occurred in the seventh internode. In stem extracts from transgenic alfalfa with antisense downregulated caffeoyl CoA O‐methyltransferase (CCoAOMT), activities with all substrates except for the two coenzyme A esters were unaffected. In contrast, downregulation of caffeic acid O‐methyltransferase (COMT) reduced activities against the non‐esterifed substrates in the order: 5‐hydroxyconiferyl alcohol > 5‐hydroxyferulic acid and caffeoyl alcohol > caffeoyl aldehyde > caffeic acid > 5‐hydroxyconiferaldehyde. Recombinant COMT expressed in Escherichia coli exhibited the highest Vmax/Km values with 5‐hydroxyconiferaldehyde and caffeoyl aldehyde, and the lowest with caffeic acid. These results indicate that COMT is unlikely to methylate caffeic acid during lignin biosynthesis in vivo, and provide enzymatic evidence for an alternative pathway to monolignols involving methylation of caffeoyl aldehyde and/or caffeoyl alcohol by COMT. The concept of independent pathways to guaiacyl and syringyl monolignols is discussed.
REVOLUTA regulates meristem initiation at lateral positions
Authors: Otsuga Denichiro , DeGuzman Bernadette , Prigge Michael J. , Drews Gary N. , Clark Steven E.
Published: August 2010

While the shoot apical meristem (SAM) is indirectly responsible for the initiation of all above‐ground postembryonic organs, in most plants the vast majority of these organs are directly initiated by lateral meristems. In Arabidopsis thaliana, the lateral meristems include flower meristems (FMs), which form on the flanks of the SAM, and lateral shoot meristems (LSMs), which develop in leaf axils. While significant progress has been made on the molecular genetic basis of SAM initiation during embryo development, relatively little is known about the initiation of meristems at lateral positions. Here we have characterized the phenotypic consequences and genetic interactions of mutations in the REVOLUTA (REV) gene, with an emphasis on the role of REV in lateral meristem initiation. Our observations indicate that REV is required for initiation of both LSMs and FMs, and likely acts in the same pathway as, and upstream of, known meristem regulators. We identified the REV gene and found it encodes a predicted homeodomain/leucine zipper transcription factor that also contains a START sterol‐lipid binding domain. REV is the same as the IFL gene. REV was expressed at the earliest stages of LSM and FM formation. Within the inflorescence shoot meristem, REV expression appeared to predict 3–5 incipient flower primordia on the flanks of the SAM, and REV expression at stage 1 and stage 2 matched that of WUS and STM, respectively. We propose that REV acts at lateral positions to activate the expression of known meristem regulators.
MOLECULAR FORCES IN PROTEIN STRUCTURE AND CRYSTALLOGRAPHY
Authors: Ramachandran G. N.
Published: August 2010

The paper deals with the nature of the intermolecular forces and the laws governing these, which play an important part in the stability of molecular crystals and in the conformation of biological macromolecules. The method of contact criteria which give the disallowed ranges of conformations is briefly discussed and then the energy criteria are considered. Intermolecular potential functions for non‐bonded interactions (both van der Waals and repulsive), electrostatic forces, hydrogen bonds, and hydrophobic interactions are discussed, followed by a treatment of intramolecular distortions of bond lengths, bond angles, and torsional angles. The last of these are important for the study of conformations. The possibility of non‐planar distortion of peptide units is specially discussed. Application of these ideas to the calculation of the structures of molecular crystals is indicated.
A STUDY OF THE THERMAL DENATURATION OF RIBONUCLEASE BY DIFFERENTIAL SCANNING CALORIMETRY
Authors: Delben F. , Crescenzi V. , Quadrifoglio F.
Published: August 2010

A study by differential scanning calorimetry (DSC) of ribonuclease thermal denaturation in water, 1.5, 2.5, and 4M aqueous urea, and in 1M and 2M aqueous hexame‐thylenetetramine has been carried out. Application of the DSC technique to this type of investigation appears a useful one, in our experience. Our results indicate that whereas urea only slightly changes enthalpy variation accompanying denaturation but lowers the melting temperature of ribonuclease, hexamethylenetetramine does not effect either factor. According to these results, which are beyond experimental errors, the ability of hexamethylenetetramine in influencing hydrophobic‐forces‐driven phenomena (Barone, Crescenzi & Quadrifoglio 1967) has thus apparently no counterpart in any important action of this compound on the stability of a partially hydrophobic protein in aqueous solution.
A FORTRAN IV E AMINO ACID ANALYZER COMPUTER PROGRAM
Authors: Gerding J. J. T.
Published: August 2010

A computer program has been written in basic Fortran IV (E level subset) which can be used on many computers and which enables calculation of amino acid analyser data in a short time. Special care has been taken to simplify the data entry. For every analysis (long or short column) only two data cards are required. Surveyable data entry forms, including typing instructions for the key punch operator, have been printed. These data entry forms can also be used for the standard calibrations runs.

The program is specially suited for laboratories where many routine amino acid analyses have to be performed on a two‐column system and where the apparatus is not yet fully automated. The data input can be given in integrator units or in H X W units. The output is rendered as surveyable as possible in order to avoid human errors in the steps that are not yet automated. The data input is reprinted together with the calculated results on the data output sheet. The correctness of the results can then easily be checked.
THE OXIDATION WITH N‐BROMOSUCCINIMIDE OF THE SINGLE TRYPTOPHAN RESIDUE IN RIBONUCLEASE T1
Authors: Kawashima Seiichi , Ando Toshio
Published: August 2010

1. The single tryptophan residue‐59 present in RNase T1 was oxidized almost specifically by NBS in 8 M urea. The enzymatic activity of RNase T1 decreased with the increase in the amount of NBS used, and complete inactivation took place within 10 minutes when 2 equivalents of the reagent were used.

2. During the oxidation with NBS of RNase T1 no cleavage of peptide bonds was found and none of the amino acid residues other than tryptophan was affected except for oxidation of cystine to cysteic acid in a small amount (up to 20 %). The complete inactivation of the enzyme occurred when the tryptophan residue was completely oxidized.

3. The oxidized RNase T1 possessed a lower solubility than the native enzyme. Measurements of the optical rotatory dispersion and the circular dichroism indicated the occurrence, of some but not so great conformational changes in the enzyme by the oxidation.

4. These residts clearly demonstrated the importance of the single tryptophan residue in RNase T1 for the proper function of the enzyme.
THE INCORPORATION OF AMINO ACIDS BY FREE POLYRIBOSOMES AND ROUGH ENDOPLASMIC RETICULUM IN A CELL‐FREE SYSTEM
Authors: Bont W. S. , Geels J. , Rezelman G.
Published: August 2010

The incorporation of amino acids into TCA * precipitable polypeptides has been studied in a cell‐free system isolated from rat liver. A new procedure is described for the isolation of all amino‐acyl‐tRNA synthetases necessary for optimal amino acid incorporation. Polyribosomes were present either as membrane‐bound or as free polyribosomes. Differences in incorporation between bound and free polyribosomes were observed. In contrast to free polyribosomes the incorporation of (14C)leucine in bound polyribosomes was not stimulated by addition of all the other non‐radioactive amino acids. The plateau for incorporation of (14C)leucine into bound polyribosomes was reached after 5 min and for free polyribosomes after 60 min. From these findings it was concluded that in vitro, apart from a limited translation, further translation of bound polyribosomes was blocked.
THE CONFORMATION OF THE HIGH‐SULPHUR PROTEINS OF WOOL. I. THE PREPARATION AND PROPERTIES OF A WATER‐SOLUBLE METAKERATIN
Authors: Bhatnagar G. M. , Crewther W. G.
Published: August 2010

Whereas the S‐cyanoethyl derivatives of the high‐sulphur kerateines extracted from Merino wool were virtually insoluble in aqueous buffer solutions, the disulphide form of a high‐sulphur fraction designated Metakeratin‐B(5,0‐8+4) (M‐B(5.0‐8+4)), obtained by dialysing the corresponding kerateine fraction in the presence of oxygen, was soluble in aqueous buffer solutions at pH 8.0. The material was highly aggregated in solution and could be partially disaggregated by 8 M urea. Gel filtration of the meta‐keratin fraction in 8 M urea on Sephadex G‐200 indicated that the molecular weights ranged from values exceeding 100,000 (excluded from column) to about 10,000. The latter value was confirmed by sedimentation measurements. When reduced and alkylated with iodoacetate the S‐carboxymethylkerateine fraction so formed (SCMK‐5(5.o 8.4)) contained chiefly components with molecular weights of about 10,000 as determined by gel filtration.

M‐B(5,0‐8.4) showed a minimum solubility at about pH 4.4 in salt solutions, the pH of minimum solubility decreasing to about 3.0 with progressive S‐carboxymethylation of the protein. No evidence could be obtained for the presence in M‐B(5.0.8.4) of asymmetrical disulphide groups of the form CyS‐S‐CH2COOH. The incorporation of 8 M urea in solutions of the metakeratin at pH 8 gave rise to a positive difference spectrum, suggesting that few if any additional tyrosine residues become accessible in this solvent. Optical rotatory dispersion measurements gave a value of–50 for the parameter b0 in the Moffitt‐Yang equation. In the presence of 8 M urea the value of b0 became zero. M‐B(5,0‐8.4) was digested relatively slowly by Pronase P but the initial rate of digestion increased greatly as the disulphide bonds were converted progressively to S‐carboxy‐methyl groups, the greatest change in rate occurring over the range 10 to 20% conversion of the cystine residues to S‐carboxymethylcysteine residues.

The wavelength of maximum fluorescence of M‐B(5.0‐8.4) was unaffected by the S‐carboxymethylation but the intensity of the fluorescence was greatly increased by this modification. The ultraviolet absorption curve for the metakeratin showed absorption maxima at about 276 and 330 nm. With progressive S‐carboxymethylation the ab‐sorbance over the whole range of wavelengths decreased, the absorbance peak at about 330 nm disappeared, and an additional peak at about 280 nm appeared.

The results suggest that the reduced chains of the high‐sulphur protein fraction from wool, when allowed to oxidize to the disulphide form, tend to fold in a manner resembling native globular proteins. The unit structures so formed appear to be stabilized by intrachain disulphide bonds.
CORRELATIONS BETWEEN AMINO ACID SEQUENCE AND CONFORMATTON OF IMMUNOGLOBULIN LIGHT CHAINS
Authors: Welscher H. D.
Published: August 2010

The significance of nonpolar and of charged residues for the corrformation of light chains is discussed, based on the amino acid sequence of 15 light chains and on the amino acid composition of light chains from I1 species.

The nonpolar residues contribute to the conformational stability by hydrophobic interactions with an average hydrophobicity of about 1000 caliresictue; this value is essentially independent of isotypic and ictiotypic sequence variability, and is similar to that for other globular proteins. While ihe total h.vdrophobicity of many globular proteins containing no SS bridges compensates afniost exclusively for their cotiforma‐ tional entropy, a significant discrepancy is observed in the case of light chains. This rnav rejlect the loss of conformational entropy clue to ihe two SS bridges forming independent covalent loops. The ioni7cd residue;, give a fractional charge close to 0.20 unitsiresirlue. This relatively low value, together with the relatively high average hydrophobicity, cxplains qualitatively many of the a5sociation and thermal properties of light chains. Variable and constant regions of light chains exhibit nearly identical average hydrophobicities and fracfional charges, suggesting that the molecules are folded into two distinct structural subunits of compact conformation.

Some preliirrirrary results on heavy chains are also considered: The fructional charge of the Fdjragments are much lowcr than that of the Fc fragments. This may fucilitate the intimate contact oj the light chain with the former fragment in the intact antibody molecule.
DYE‐SENSITIZED SELECTIVE PHOTO‐OXIDATION OF CYSTEINE
Authors: Jori Giulio , Galiazzo Guido , Scoffone Ernesto
Published: August 2010

The phtalein derivative cresol red and the fuchsin dye crystal violet have been found to sensitize the selective photo‐oxidation of cysteine, both free and incorporated into a protein. The specificity for cysteine of these dyes was checked in aqueous solution over the pH range 2.5‐9, as well as in aqueous acetic acid. In acidic solutions, cysteine is quantitatively converted to cysteic acid; on the contrary, in neutral or alkaline media, the photo‐oxidative process is competed by a dark reaction, which causes the oxidation of cysteine to cystine. The irradiation of lysozyme, after reduction of a single disulphide bridge, in 16%acetic acid solution and in the presence of either sensitizer yielded a disulphonic derivative, which displayed about 40% enzymic activity. Moreover, fully reduced lysozyme, after photo‐oxidation under the same conditions, appeared to contain eight cysteic acid residues per protein molecule and was devoid of enzymic activity. Therefore, this method appears to be suitable for the selective and quantitative modification of the cysteinyl residues in biologically active polypeptides and for investigating their importance for the biological function.
L‐5‐HYDROXYLYSINE AS A CONSTITUENT OF THE SHELL MEMBRANES OF THE HEN'S EGG
Authors: Candlish J. K. , Scougall R. K.
Published: August 2010

Hydroxylysine was detected in hydrolysates of the inner and outer membranes of the shell of the hen's egg by ion‐exchange and paper chromatography. N‐ɛ‐DNP‐hydroxy‐lysine was detected by paper chromatography of hydrolysates of dinitrophenylated membranes. Complete amino acid analysis showed hydroxylysine to be the least abundant of the known amino acids in the membranes (1‐2 moles/1(fig protein).
AMINO ACID COMPOSITIONS and N‐TERMINAL AMINO ACIDS OF ALL THE TRYPTIC PEPTIDES FROM THE α and β POLYPEPTIDE CHAINS OF ADULT HEMOGLOBIN OF THE RHESUS MONKEY (Macaca mulatto)
Authors: Matsuda Genji , Maita Tetsuo , Take Hiroshi
Published: August 2010

Globin prepared from adult hemoglobin of the rhesus monkey was separated into α and β polypeptide chains by the countercurrent distribution method. The α and β polypeptide chains were digested with trypsin. All the tryptic peptides were isolated and purified by column and paper chromatography. The amino acid compositions and the N‐terminal amino acid of these tryptic peptides were analyzed.

According to the results thus obtained, the α and β polypeptide chains of adult hemoglobin of the rhesus monkey are respectively composed of 141 and 146 amino acid residues just as those of human hemoglobin. However, it is presumed that there exist at least four amino acid substitutions in the a polypeptide chain and eight in the β polypeptide chain.
STUDIES ON THE CONFORMATION OF AMINO ACIDS*
Authors: Ponnuswamy P. K. , Sasisekharan V.
Published: August 2010

In this paper, the results of the potential energy calculations on the conformations of some amino acids are given. The non‐bonded, electrostatic, and torsional contributions to the total energy of the molecule were calculated using appropriate functions and their role in predicting the conformation of amino acids is discussed. It is shown that electrostatic interaction between the amino group and the carboxyl group is mainly responsible for the plane of the carboxyl group to be coplanar with the plane through N, Cα, and C1atoms. It is also shown that the presence of a γ‐atom in position II necessiates a large deviation from the coplanarity between the carboxyl plane and the N, Cα, C plane.

The results indicate that the backbone conformations of all the amino acids are nearly the same and could be shown to be the same as those of glycine and alanine. Also, the theoretically predicted conformations about the Cα‐Cβ bond in aminobutyric acid could explain the observed γ‐atom positions in all the amino acids.
STUDIES ON BACTERIAL PROTEASES
Authors: Tsuru D. , Yoshimoto T. , Yoshida H. , Kira H. , Fukumoto J.
Published: August 2010

Amino acid composition of neutral protease obtained from the culture broth o/Bacillus subtilis var. amylosacchariticus was dertermined. The enzyme was composed of 308 residues of amino acids: Lys15 His5, Arg9 Asp43, Thr31, Ser30, Glu23, Pro11, Gly27, Ala27, Cys0, Val16, Met2, He12, Leu22., Tyr23, Phe9, Trp3, and amide‐ammonia32 on a basis of molecular weight of 34,000. Of 23 residues of tyrosine, 15 groups were found to be normally ionized with the apparent pK value of 10.0, whereas the remainder was ionized only after irreversible unmasking of the tyrosyi residues at higher pH with the pK value of about 12.2. The optical rotatory dispersion measurements showed that [a]D, a0, b0, and Lc were –28, –129, –173, and 276 m/μ, respectively, suggesting the enzyme to contain less than 30 % of α‐helix structure. The negative minimum of the Cotton effect lay at 233 m/μ with the [m] value of 3,700.
THE PRIMARY STRUCTURE OF THE α and β POLYPEPTIDE CHAINS OF ADULT HEMOGLOBIN OF THE RHESUS MONKEY (Macaca mulatto)
Authors: Matsuda Genji , Maita Tktsuo , Ota Hisahiro , Takei Hiroshi
Published: August 2010

Countercurrent distribution separated rhesus monkey hemoglobin into α and β polypeptide chains, which were respectively oxidized with performic acid, dissolved in 8 M urea to be denatured at 60° for 45 min, and then digested with pepsin at 37° for 2 hours. Peptic peptides thus obtained from each polypeptide chain were isolated and purified by column‐ and by paper chromatography, and the amino acid composition of each peptide was analyzed. Sequence analysis was carried out by using PTC and DNP methods, if necessary.

From the results thus obtained, the present authors established the primary structure of rhesus monkey hemoglobin, taking account of the amino acid sequence in all the tryptic peptides from the α and β polypeptide chains previously reported.
ON THE CONFORMATION OF HISTONES: CD STUDIES ON THE LYSINE‐ and SERINE‐RICH FRACTIONS FROM AVIAN ERYTHROCYTES
Authors: Tamburro A. M. , Scatturin A. , Vidau G.
Published: August 2010

Circular dichroism studies of avian erythrocytes lysine‐rich and serine‐rich histone fractions are reported. Both histones are essentially disordered in aqueous solutions. In aqueous methanol the lysine‐rich fraction undergoes a coil‐to‐helix transition.

The same property is enhanced in the serine‐rich histone, and at high methanol percentage the fraction is partially in β form.
IDENTITY OF TRYPSIN INHIBITORS ISOLATED FROM BOVINE LIVER and PANCREAS
Authors: Chauvet Jacqueline , Acher Roger
Published: August 2010

A trypsin inhibitor, isolated from bovine liver, has been compared to the cat ionic trypsin inhibitor produced by bovine pancreas (Kunitz’inhibitor). The electrophoretic properties and the amino acid compositions of both inhibitors are identical. The tryptic peptides of the inhibitor isolated from liver have been purified by paper chromato‐electrophoresis. The peptide map is similar to that of pancreatic inhibitor and the amino acid compositions of the 9 fragments are virtually the same in both cases.
THERMOPHILIC AMINOPEPTIDASES FROM BAC.STEAROTHERMOPHILUS
Authors: Moser P. , Roncari G. , Zuber H.
Published: August 2010

The thermophilic (thermostable) aminopeptidase I (AP I) can be converted into a stable apoenzyme by dialysis at pH 5.6 against EDTA. Presumably during the formation of the apoenzyme (pH 5.6) the removal of the Co2+ and the alteration of the pH (from 7.2 to 5.6) increase the negative charge of the enzyme molecule (gel electrophoresis), which presumably causes a change in conformation and an increase in the number of accessible carboxyl groups. The ORD remains unaltered during this process. The AP I in Co2‐‐free buffer and the apoenzyme are thermolabile at pH 5.6 and pH 8.1, respectively, which reveals the importance of the role played by the Co2+ ions bound at the active site or by additional Co2‐ ions bound at other points of the molecule with regard to the thermostability of the native AP I. Upon recombination with Co2+, an active aminopeptidase (AP I*) is obtained from the apoenzyme (pH 5.6). The recombined AP I*, however, can no longer be stained to the same extent as the native AP I in gel electrophoresis with Coomassie Brilliant Blue R 250 or with Amido Black 10B. AP I has an M̄app of 400,000 = 45,000 (sedimentation equilibrium) and an MS,D, of 395,000 ± 30,000 (S20,W, C O=‐ 16.5 and D20,W,C=O= 3.9‐10‐7). The inactive apoenzyme displays a slightly reduced S20,W, of 13.8, while the ORD is the same as for active AP I. In 8M urea and 10‐2M EDTA at pH 5.6, AP I decomposes into subunits (MS,D‐36,500 ± 4,000; S20,W ‐ 1.95 ± 0.1; D20,W= 5.0 ± 0.25‐10‐7). The “helical” content of 22% in native AP I decreases to 6% in the process. AP I is largely stable in 8M urea (peptide hydrolysis activity and ORD are unchanged; slight decrease in S20,W= 14.4; the inhibition of LNA hydrolysis by 8M urea is fully reversible). The activity of the AP I preparations depends on the conditions of the procedures for isolating the enzyme. The activation of AP I at higher temperature is dependent on Co2‐ ion concentration and on temperature. Recombined AP I* (from apoenzyme) is not activatable. The activation of AP I at 80° reaches a maximum after 30 min.
STUDIES ON THE CONFORMATION OF AMINO ACIDS
Authors: Chanhrasekaran R. , Ramachandran G. N.
Published: August 2010

The distribution of the values of the dihedral angles (χj) in the side group conformations of various amino acid residues in the three proteins myoglobin, lysozyme and tosyl‐α.‐chomotrypsin has been analysed. The observations in these protein structures are found to agree well with the theoretical distribution worked out on the basis of contact criteria and the number of allowed conformations of each category. The nature of the conformations are, in general, similar to those observed in simple amino acid structures. Hydrogen‐bonding interactions between side group and backbone seem to play a significant role in determining the conformations of certain polar side groups like serine, threonine, and aspartic acid.