bluuebunny/crossref_metadata_2025 huggingface dataset:
Endocytosis, membrane recycling and sorting of GPI‐anchored proteins: Trypanosoma brucei as a model system
Authors: Overath Peter , Engstler Markus
Published: July 2004

In the flagellated protozoon Trypanosoma brucei, endo‐ and exocytosis are restricted to a small area of the plasma membrane, the flagellar pocket. All endosomal compartments and the single Golgi complex are located within the posterior part of the cell between the flagellar pocket and the nucleus. The use of reverse genetic tools, including RNA interference, in combination with quantitative 3D‐fluorescence and electron microscopic techniques has provided an insight into endosomal membrane traffic, which occurs at a very high rate and appears to exhibit a lower level of complexity than in mammalian cells. The flagellate is an excellent model system for studies on endocytosis, sorting and recycling of glycosylphosphatidylinositol‐anchored glycoproteins, because 107 molecules of the variant surface glycoprotein form a dense coat at the cell's surface. Because the endocytic rate varies widely at different stages in the parasite's life cycle, trypanosomes may be used for investigating developmental aspects of their endocytic system.
A prospective association between cocoon use in infancy and childhood asthma
Authors: Trevillian L. F. , Ponsonby A. L. , Dwyer T. , Lim L. L. Y. , Kemp A. , Cochrane J. , Carmichael A.
Published: July 2004

There is increasing evidence for a role for bedding items in the development of asthma. The use of some forms of synthetic bedding, such as foam mattresses and pillows, is associated with a significantly increased risk of childhood wheeze. Our aim was to examine prospectively whether the use of synthetic cocoon/baby nests in infancy is associated with the subsequent development of wheeze in childhood. Data collected in 1988 as part of the Tasmanian Infant Health Survey were linked to the cross‐sectional Childhood Asthma Survey conducted in 1995 in Tasmania, Australia.

We were able to match 863 records out of the 1111 in the 1988 survey. Information including parental, child‐care, and the infant's sleeping environment was collected at home interview in 1988 when the infant was 1 month of age. Data including sleep environment and asthma symptoms were available for each child at age 7 years. A generalised linear model was used to calculate the adjusted relative risk (RR) estimates for symptoms of wheeze and infant cocoon use. For children who were placed in a cocoon in infancy, there was an increased risk of recent wheeze (adjusted RR = 4.33 [95% CI 2.08, 9.02]) and night wheeze (adjusted RR = 3.35 [95% CI 1.52, 7.39]) at age 7 years.

In view of the increasing prevalence of childhood asthma, the identification of potentially modifiable environmental factors which might operate in infancy is of importance. The present findings implicate infant bedding choice as a significant factor and further studies on the infant sleeping environment are indicated.
Transition from Birth to Ten to Birth to Twenty: the South African cohort reaches 13 years of age
Authors: Richter Linda M. , Norris Shane A. , De Wet Thea
Published: July 2004

Birth to Ten now Birth to Twenty (BT20), is the largest and longest running longitudinal birth cohort study in Africa. In this paper, the methods, magnitude and significance of recruitment, follow‐up and attrition are described. Although more than 5000 births were notified in the area in the 7‐week enrolment period in early 1990, only 3275 children were established to have been born to women who were residents in the Greater Johannesburg Metropolitan area for at least the first 6 months of the child's life. Seventy per cent of these children and their families have been followed up for more than 12 years, indicating an average attrition rate of less than 3% per annum, with most attrition occurring in the first 2 years of the study. The most common reason for attrition was movement out of the study area, although detailed follow‐up, and the extent of contact re‐established at later points, indicate very high levels of circular migration among women and young children between urban and rural areas, as well as very high levels of residential mobility within urban areas. There has been no differential loss of vulnerable families and children. African women living in Soweto are the most consistent participants in the study. A bias, by population group membership and residential area, was introduced in the recruitment phases of the project by the difficulty of enrolling the small proportion of people in the metropolis, largely Whites, who used private delivery services in 1989–90.
The ACR4 receptor‐like kinase is required for surface formation of epidermis‐related tissues in Arabidopsis thaliana
Authors: Watanabe Masaru , Tanaka Hirokazu , Watanabe Daisuke , Machida Chiyoko , Machida Yasunori
Published: July 2004

In higher plants, an outer layer of meristematic cells, the protoderm, forms early in embryogenesis and this layer gives rise to the epidermis in differentiating tissues. We proposed previously that an Arabidopsis thaliana homolog of crinkly4 (ACR4), a gene for a receptor‐like protein kinase, would be involved in differentiation and/or maintenance of epidermis‐related tissues. In the present study, we isolated loss‐of‐function acr4 mutants by a reverse genetic approach. Our extensive analyses using the transmission electron microscopy and the toluidine blue test – a method that has recently been developed for the rapid visualization of defects in the leaf cuticle – showed that the acr4 mutations significantly affected the differentiation of leaf epidermal cells, suggesting similar roles for ACR4 and CR4 in the differentiation of leaf epidermis. Our acr4 mutants also had various abnormalities related to epidermal differentiation, which included disorganized cell layers in the integument and endothelium of ovules. In addition, the green fluorescent protein fused to ACR4 was localized preferentially on the lateral and basal plasma membranes in the epidermis of the leaf primordia, suggesting a role for ACR4 in epidermal differentiation at cell surfaces that make contact with adjacent cells. Furthermore, the loss‐of‐function mutations in the ACR4 and ABNORMAL LEAF SHAPE1 (ALE1) genes, which encode a putative subtilisin‐like serine protease, synergistically affected the function of the epidermis such that most leaves fused. Thus, ACR4 seems to play an essential role in the differentiation of proper epidermal cells in both vegetative and reproductive tissues.
Site‐directed mutagenesis and protein 3D‐homology modelling suggest a catalytic mechanism for UDP‐glucose‐dependent betanidin 5‐O‐glucosyltransferase from Dorotheanthus bellidiformis
Authors: Hans Judith , Brandt Wolfgang , Vogt Thomas
Published: July 2004

In livingstone daisy (Dorotheanthus bellidiformis), betanidin 5‐O‐glucosyltransferase (UGT73A5) is involved in the regiospecific glucosylation of betanidin and various flavonols. Based on sequence alignments several amino acid candidates which might be essential for catalysis were identified. The selected amino acids of the functionally expressed protein, suggested to be involved in substrate binding and turnover, were substituted via site‐directed mutagenesis. The substitution of two highly conserved amino acids, Glu378, located in the proposed UDP‐glucose binding site, and His22, located close to the N‐terminus, led to the complete loss of enzyme activity. A 3D model of this regiospecific betanidin and flavonoid glucosyltransferase was constructed and the active site modelled. This model was based on the crystallographic structure of a bacterial UDP‐glucose‐dependent glucosyltransferase from Amycolatopsis orientalis used as a template and the generated null mutations. To explain the observed inversion in the configuration of the bound sugar, semiempirical calculations favour an SN‐1 reaction, as one plausible alternative to the generally proposed SN‐2 mechanism discussed for plant natural product glucosyltransferases. The calculated structural data do not only explain the abstraction of a proton from the acceptor betanidin, but further imply that the reaction mechanism might also involve a catalytic triad, with similarities described for the serine protease family.
The fusarium wilt resistance locus Fom‐2 of melon contains a single resistance gene with complex features
Authors: Joobeur Tarek , King Joseph J. , Nolin Shelly J. , Thomas Claude E. , Dean Ralph A.
Published: July 2004

The soil‐borne fungus Fusarium oxysporum f.sp. melonis causes significant losses in the cultivated melon, a key member of the economically important family, the Cucurbitaceae. Here, we report the map‐based cloning and characterization of the resistance gene Fom‐2 that confers resistance to race 0 and 1 of this plant pathogen. Two recombination events, 75 kb apart, were found to bracket Fom‐2 after screening approximately 1324 gametes with PCR‐based markers. Sequence analysis of the Fom‐2 interval revealed the presence of two candidate genes. One candidate gene showed significant similarity to previously characterized resistance genes. Sequence analysis of this gene revealed clear polymorphisms between resistant and susceptible materials and was therefore designated as Fom‐2. Analysis of susceptible breeding lines (BL) presenting a haplotype very similar to the resistant cultivar MR‐1 indicated that a gene conversion had occurred in Fom‐2, resulting in a significant rearrangement of this gene. The second candidate gene which shared high similarity to an essential gene in Arabidopsis, presented an almost identical sequence in MR‐1 and BL, further supporting Fom‐2 identity. The gene conversion in Fom‐2 produced a truncated R gene, revealing new insights into R gene evolution. Fom‐2 was predicted to encode an NBS‐LRR type R protein of the non‐TIR subfamily. In contrast to most members of this class a coiled‐coil structure was predicted within the LRR region rather than in the N‐terminal. The Fom‐2 physical region contained retroelement‐like sequences and truncated genes, suggesting that this locus is complex.
LeSTIG1, an extracellular binding partner for the pollen receptor kinases LePRK1 and LePRK2, promotes pollen tube growth in vitro
Authors: Tang Weihua , Kelley Dior , Ezcurra Inés , Cotter Robyn , McCormick Sheila
Published: July 2004

As pollen tubes grow through the pistil they are thought to perceive and respond to diverse signals. The tomato pollen‐specific receptor kinases LePRK1 and LePRK2 might participate in signaling during pollen tube growth. We previously showed that the extracellular domain of LePRK2 interacts with a pollen protein, LAT52, before but not after pollen germination. To determine whether LePRK2 might have different binding partner(s) after pollen germination, we characterized two more proteins that, like LAT52, were identified in yeast two‐hybrid screens using the extracellular domains of LePRK1 and LePRK2 as baits. We show that LeSHY, a leucine‐rich repeat protein from pollen, and LeSTIG1, a small cysteine‐rich protein from pistil, can bind the extracellular domains of both LePRK1 and LePRK2 in vitro. In vitro binding assays with the extracellular domain of LePRK2 suggested that LeSTIG1 could displace binding of LAT52, consistent with the idea that LePRK1 and LePRK2 might interact with different ligands at different stages of pollen tube growth. Exogenous LeSTIG1 promotes pollen tube growth in vitro. The interaction of these pollen kinases with LeSTIG1 supports the notion that LePRK1 and LePRK2 are involved in mediating pollen–pistil interactions.
Endoplasmic reticulum‐resident proteins are constitutively transported to vacuoles for degradation
Authors: Tamura Kentaro , Yamada Kenji , Shimada Tomoo , Hara‐Nishimura Ikuko
Published: July 2004

Soluble endoplasmic reticulum (ER)‐resident proteins have very long lives because of their ER residency. This residency depends largely on ER‐retrieval signals at their C‐terminus. We examined the long‐term destiny of endogenous ER‐resident proteins, a lumenal binding protein (BiP) and a protein disulfide isomerase (PDI), with cultured cells of Arabidopsis. ER residents, in contrast to vacuolar proteinases, were considerably degraded in cells at the stationary phase. A subcellular fractionation analysis suggested that ER residents were transported into the vacuoles, which accumulated the residents lacking the ER‐retrieval signals. We showed that the PDI located in the vacuoles had high mannose glycans, but not complex glycans, which suggested that the ER resident was transported to the vacuoles independent of the medial/trans‐Golgi complex. To visualize the pathway of transport of ER‐resident proteins, tobacco BY‐2 cells were transformed with a chimeric gene encoding an ER‐targeted green fluorescent protein (30 kDa GFP‐HDEL). In the transformed cells at the stationary phase, GFP fluorescence was observed in the vacuoles. A subcellular fractionation revealed that a trimmed form of 27 kDa GFP was localized in the vacuoles. Treatment with E‐64d, an inhibitor of papain‐type cysteine proteinases that inhibits the degradation of GFP in the vacuoles, resulted in a stable accumulation of 27 kDa GFP in the vacuoles, even in the logarithmic phase. Our results suggest that endogenous ER residents are transported constitutively to the vacuoles by bypassing the Golgi complex and are then degraded.
Targeting of Proteins Derived from Self‐Processing Polyproteins Containing Multiple Signal Sequences
Authors: de Felipe Pablo , Ryan Martin D.
Published: July 2004

The 18aa 2A self‐cleaving oligopeptide from foot‐and‐mouth disease virus can be used for co‐expression of multiple, discrete proteins from a single ORF. 2A mediates a co‐translational cleavage at its own C‐terminus and is proposed to manipulate the ribosome into skipping the synthesis of a specific peptide bond (producing a discontinuity in the peptide backbone), rather than being involved in proteolysis. To explore the utility of the system to target discrete processing products, self‐processing polyproteins comprising fluorescent proteins flanking 2A were constructed, permutating both the type of signal sequence and the location within the polyprotein. A polyprotein comprising a protein bearing an N‐terminal signal sequence, 2A, then a protein lacking any signal sequence, was constructed. Interestingly, both proteins were translocated into the endoplasmic reticulum. Despite the discontinuity in the peptide backbone, the mammalian ribosome:translocon complex did not disassemble – the second protein (lacking any signal) ‘slipstreamed’ through the translocon formed by the first (signal‐bearing) protein. These polyprotein systems provide a novel method of targeting proteins to different subcellular sites by transfection with a plasmid encoding a single ORF. The inclusion of a fluorescent reporter enables visualisation of expression levels, whilst inclusion of a selectable marker enables stable cell‐lines to be established rapidly.
Chlamydia– Host Cell Interactions: Recent Advances on Bacterial Entry and Intracellular Development
Authors: Dautry‐Varsat Alice , Balañá María Eugenia , Wyplosz Benjamin
Published: July 2004

Bacteria of the Chlamydiales order are very successful intracellular organisms that grow in human and animal cells, and even in amoebae. They fulfill several essential functions to enter their host cells, establish an intracellular environment favorable for their multiplication and exit the host cell. They multiply in a unique organelle called the inclusion, which is isolated from the endocytic but not the exocytic pathway. A combination of host cell factors and of proteins secreted by the bacteria, from within the inclusion, contribute to the establishment and development of this inclusion. Here we review recent data on the entry mechanisms and maturation of the inclusion.
Increased prevalence of latex‐sensitization among children with chronic renal failure
Authors: Dehlink E. , Prandstetter C. , Eiwegger T. , Putschögl B. , Urbanek R. , Szépfalusi Z.
Published: July 2004

Background:  Type‐I‐allergy to natural rubber latex (NRL) has been shown to be more prevalent among certain groups of patients. Children suffering from chronic renal failure (CRF) could be a suspected risk group because of their intense exposure to latex through catheters, gloves and anesthetic equipment during frequent hospitalizations from early life on. We investigated the prevalence of latex‐sensitization among this group of patients and sought to identify risk factors.

Methods:  Ninety‐three patients (mean age 10.5 years) suffering from CRF were assessed by questionnaire‐based history (details on renal disease, number and kind of surgical procedures, family and personal history of atopic diseases, allergic reactions to NRL, and the use of pacifiers) and by measurement of total and latex‐specific serum immunoglobulin (Ig)E.

Results:  Ten of 93 (10.8%) patients showed elevated latex‐specific IgE‐levels. One of 10 patients reported clinical symptoms to latex‐allergen, but no allergic reactions to NRL during medical care were reported. Sensitized patients were significantly more likely to be atopic, reflected by a positive history of other allergies as well as elevated total serum IgE‐levels, and had a significantly higher number of urogenital surgeries (P = 0.02 in all cases, Fisher's exact and Wilcoxon test, respectively).

Conclusion:  This study demonstrates that children with CRF are at increased risk of latex‐hypersensitivity. Significant associations with atopy and repeated surgeries were observed. Larger studies are required to elucidate whether these children are also at increased risk of anaphylaxis and therefore deserve preventive measures.
Gene Expression Evidence Indicates that Nucleotides 507–513 and 1434–1440 in 16s rRNA are Organized in Close Proximity on the Escherichia Coli 30s Ribosomal Subunit
Authors: Kaloyanova Dora , Xu Jianhua , Ivanov Ivan G. , Abouhaidar Mounir G.
Published: July 2004

A non‐Shine‐Dalgarno translational initiator is identified in Escherichia coli. The nucleotide sequence ACCUACUCGAGUUAG, designated as PL, is capable of initiating translation of pokeweed antiviral protein (PAP) and human calcitonin (hCT) mRNAs in E. coli cells. The yield of recombinant protein was double that obtained with the consensus Shine‐Dalgarno‐sequence‐(SD)‐driven translation. The PL sequence is composed of two heptanucleotides (ACCUACU, box I and GAGUUAG, box 11) which are complementary to nucleotides 1434–1440 and 507–513, respectively, in 16s rRNA. Mutational analysis shows that the translation initiation efficiency with either box alone is much lower than that obtained with the entire PL sequence, indicating that the boxes interact simultaneously with both complementary regions in 16s rRNA during the translation initiation step. Based on these results, we propose that the two widely separated regions 507–513 (part of helical domain 18) and 1434–1440 (belonging to helical domain 44) are organized in close proximity to each other and to the ribosome decoding center on the surface of the E. coli 30s ribosomal subunit.
Overexpression of Human poly(ADP‐Ribose) Polymerase in Transfected Hamster Cells Leads to Increased Poly(ADP‐Ribosyl)ation and Cellular Sensitization to γ irradiation
Authors: Van Gool Léon , Meyer Ralph , Tobiasch Edda , Cziepluch Celina , Jauniaux Jean‐Claude , Mincheva Antoaneta , Lichter Peter , Poirier Guy G. , Bürkle Alexander , Küpper Jan‐Heiner
Published: July 2004

Poly(ADP‐ribosyl)ation is a posttranslational modification of nuclear proteins catalyzed by poly(ADP‐ribose) polymerase (PARP), an enzyme which uses NAD+ as substrate. Binding of PARP to DNA single‐strand or double‐strand breaks leads to enzyme activation. Inhibition of poly(ADP‐ribose) formation impairs the cellular recovery from DNA damage. Here we describe stable transfectants of the Chinese hamster cell line CO60 that constitutively overexpress human PARP (COCF clones). Immunofluorescence analysis of γ‐irradiation‐stimulated poly(ADP‐ribose) synthesis revealed consistently larger fractions of cells positive for this polymer in the COCF clones than in control clones, which failed to express human PARP. HPLC‐based quantitative determination of in vivo levels of poly(ADP‐ribose) confirmed this result and revealed that the basal polymer levels of undamaged ceils were significantly higher in the COCF clones. The COCF clones were sensitized to the cytotoxic effects of γ irradiation compared with control transfectants and parental cells. This effect could not be explained by depletion of cellular NAD+ or ATP pools. Together with the well‐known cellular sensitization by inhibition of poly(ADP‐ribosyl)ation, our data lead us to hypothesize that an optimal level of cellular poly(ADP‐ribose) accumulation exists for the cellular recovery from DNA damage.
Characterization of Two Nuclear Proteins that Interact with Cytochrome P‐450 1A2 mRNA
Authors: Raffalli‐Mathieu Françoise , Geneste Olivier , Lang Matti A.
Published: July 2004

Regulation of the expression of the cytochrome P‐450 1a2 gene (cyp1a2) occurs mainly at the transcriptional level, but the molecular events involved in the induction process are partly unknown. Some reports have proposed involvement of post‐transcriptional mechanisms [Adesnik, M. & Atchison, M. (1986)Crit. Rev. Biochem. 19, 247–305; Silver, G. & Krauter, K. S. (1990)Mol. Cell. Biol. 10, 6765–6768], Here we report the identification of two proteins in the nuclear fraction of mouse liver, with specific binding characteristics towards CYP1A2 mRNA. The proteins have apparent molecular masses of 37 kDa and 46 kDa and exhibit a high affinity for a poly(U) motif in the 3′ untranslated region of CYP1A2 mRNA. This motif seems to be important for their specific and apparently competitive binding to CYP1A2 mRNA. Treatment of mice with an inducer of CYP1A2, 3‐methylcholanthrene, increases the binding of the 46‐kDa protein and decreases the binding of the 37‐kDa protein to the mRNA, suggesting that changes in the binding of the proteins to the mRNA could play a role in the upregulation of CYP1A2 mRNA by 3‐methylcholanthrene. Phosphorylation of the 46‐kDa protein, or of an intermediary factor, may play a role in its binding activity. Furthermore, the 46‐kDa but not the 37‐kDa protein is recognized by a monoclonal antibody against the heterogeneous nuclear ribonucleoprotein C, a nuclear protein probably involved in pre‐mRNA processing. While more work is needed to understand the function of the proteins that bind to the 3′ untranslated region of CYP1A2, it is possible that the 37‐kDa protein has a role in the maintenance of uninduced levels of CYP1A2 mRNA, while the 46‐kDa protein could be important in the maturation of elevated levels of CYP1A2 pre‐mRNA, during induction.
Molecular Cloning and Characterization of ConBr, the Lectin of Canavalia Brasiliensis Seeds
Authors: Grangeiro Thalles B. , Schriefer Albert , Calvete Juan J. , Raida Manfred , Urbanke Claus , Barral‐Netto Manoel , Cavada Benildo S.
Published: July 2004

ConBr, a lectin isolated from Canavalia brusiliensis seeds, shares with other legume plant lectins from the genus Canavalia (Diocleinae subtribe) primary carbohydrate recognition specificity for D‐mannose and D‐glucose. However, ConBr exerts different biological effects than concanavalin A, the lectin of Canavalia ensiformis seeds, regarding induction of rat paw edema, peritoneal macrophage spreading in mouse, and in vitro human lymphocyte stimulation. The primary structure of ConBr was established by cDNA cloning, amino acid sequencing, and mass spectrometry. The 237‐amino‐acid sequence of ConBr displays Ser/Thr heterogeneity at position 96, indicating the existence of two isoforms. The mature Canavalia brasiliensis lectin monomer consists of a mixture of predominantly full‐length polypeptide (α‐chain) and a small proportion of fragments 1–118 (β‐chain) and 119–237 (γ‐chain). Although ConBr isolectins and concanavalia A differ only in residues at positions 58, 70, and 96, ConBr monomers associate into dimers and tetramers in a different pH‐dependent manner than those of concanavalin A. The occurrence of glycine at position 58 does not allow formation of the hydrogen bond that in the concanavalin A tetramer exists between Asp58 of subunit A and Sec62 of subunit C. The consequence is that the α carbons of the corresponding residues in ConBr are 1.5 Å closer that in concanavalin A, and ConBr adopts a more open quaternary structure than concanavalin A. Our data support the hypothesis that substitution of amino acids located at the subunit interface of structurally related lectins of the same protein family can lead to different quaternary conformations that may account for their different biological activities.
Multitryptophan‐Fluorescence‐Emission Decay of β‐Glycosidase From the Extremely Thermophilic Archaeon Sulfolobus Solfataricus
Authors: Bismuto Ettore , Irace Gaetano , D'Auria Sabalo , Rossi Mosè , Nucci Roberto
Published: July 2004

The emission decay of intrinsic fluorescence of the extremely thermophilic β‐glycosidase from Sulfolobus solfataricus has been investigated as functions of temperature and of iodide‐quencher concentration by frequency‐domain fluorometry. This protein contains 68 tryptophans and provides a matrix for correlation of the average spectroscopic behaviour with solvent exposure and local dynamics. At each temperature, the emission is very heterogeneous and interpretable in terms of quasicontinuous bimodal distribution of fluorescence lifetimes. We associate the component of the bimodal distribution to two distinct classes of tryptophanyl residues that differ in microenvironmental characteristics. Temperature and quenching experiments show that the long‐lived component includes tryptophanyl residues located in buried regions with high rigidity; the short distributional component corresponds to tryptophans embedded in more flexible and exposed regions. This proposal has been confirmed by examination of the crystallographic structure. The data suggest that, at least for this protein, there is a good correlation between residue exposure and lifetime distributional components. The conformational dynamics of the two classes of tryptophanyl residues is affected differently by temperature, suggesting that the protein regions in which they are located give different contributions to enzyme properties, such as flexibility, stability and function.
Purification and Characterization of an Aspergillus Oryzae‐Produced Carboxylesterase that Catalyzes O‐Deacetylation of a Fully Acetylated O‐Glucoside of N‐Phenylacetohydroxamic Acid
Authors: Yoshioka Tadao , Ohno Hiroaki , Uematsu Takayoshi
Published: July 2004

A carboxylesterase {23,4,6‐tetra‐O‐acetyl‐1‐[N‐acetyl‐N‐phenylamino)oxy]‐1‐deoxy‐β‐d‐glucopyranoside (GPA) O‐deacetylase} from a culture product of Aspergillus oryzae (Taka diastase) was purified 8500‐fold with a yield of 3%. The molecular mass of the purified enzyme was shown to be 35 ± 1 kDa by SDS/PAGE. The enzyme shows a selective O‐deacetylation activity of GPA to give the fully O‐deacetylated glucoside. Among the substrates tested, the enzyme did not hydrolyze benzoyl and phenylacetyl esters and acetamides. In the hydrolysis of p‐nitrophenyl esters, the acyl preference is acetyl > propionyl > butyryl, judging from the Vmax/Km values. A good correlation between log(Vmax/Am) and the Taft's Es constant of the alkyl group of the acyl moiety was obtained. The optimum pH was around 7.3 at 37°C, and the enzyme was inhibited by mercuric chloride, p‐chloromercuribenzoate and diisopropyl fluorophosphate. This enzyme should be useful for the selective removal of acetyl groups that serve to protect hydroxyl groups during carbohydrate synthesis.
Oxidative Injury of Synapse and Alteration of Antioxidative Defense Systems in Rats, and Its Prevention by Vitamin E
Authors: Urano Shiro , Asai Yuki , Makabe Shinobu , Matsuo Mitsuyoshi , Izumiyama Naotaka , Ohtsubo Koichiro , Endo Toyoshige
Published: July 2004

In order to define whether active oxygen species actually induce oxidative damage to the nervous system, and how antioxidative defense systems are changed by oxidative stress, morphological and functional changes in the nervous system and antioxidant status were investigated. When rats were exposed to 100% oxygen in a chamber, many morphological changes, e.g. swollen astrocytes around vessels, deformed nuclei in nerve cells, pigmentation, swollen mitochondria, and abnormal accumulation of synaptic vesicles in swollen nerve terminals, were observed by electron microscopy. When synaptosomes isolated from oxygen‐exposed rats were stimulated by KCl, acetylcholine release from the terminal was decreased more significantly than in synaptosomes from unexposed rats (P <0.01). Synaptic plasma membrane fluidity decreased in response to oxygen exposure, and plasma membrane permeability to sucrose was increased significantly (P<0.05). The cholesterol/phospholipid ratio of the plasma membranes was increased by oxidative stress and the content of unsaturated fatty acids, especially arachidonic acid and docosahexaenoic acid, decreased. The levels of thiobarbituric‐acid‐reactive substances in the plasma membranes of oxygen‐exposed rats were significantly higher than in unexposed rats (P <0.01). These results suggest that free radicals derived from oxygen may attack nerve terminals and peroxidize the plasma membrane. It was found that in response to the oxidative stress, the status of the defense system in synapse, i.e. the concentration of vitamin E, activities of superoxide dismutase and glutathione peroxidase changed, and that many of the changes observed were reduced remarkably by the intraperitoneal administration of vitamin E prior to stress. Data support the idea that vitamin E contributes to the protection against nerve dysfunction caused by oxidative stress.
Structural Organization of A Multifunctional Polyketide Synthase Involved in the Biosynthesis of the Macrolide Immunosuppressant FK506
Authors: Motamedi Haideh , Cai Sheng‐Jian , Shafiee Ali , Elliston Keith O.
Published: July 2004

The immunosuppressant FK506 is a 23‐membered macrocyclic polyketide produced by several Streptomyces species. Sequencing of a 19.5‐kb contiguous segment of DNA from the FK506 gene cluster of Streptomyces sp. MA6548 revealed the presence of a single 19.3‐kb open reading frame designated fkbA. fkbA encodes a component of the FK506 polyketide synthase, a complex enzyme system which catalyzes synthesis of the polyketide portion of FK506. The predicted product of gene fkbA is a 630660‐Da protein (6420 amino acids) that contains 19 independent domains with a high degree of amino acid sequence similarity to the catalytic activities of known fatty acid synthases. The identified domains are arranged into four repeated modules with a linear organization precisely as that of animal fatty acid synthase and type I polyketide synthase. Each module participates in one round of chain extension and subsequent processing and thus FkbA polypeptide catalyzes four of the ten condensation steps required for synthesis of the FK506 macrolactone ring. Disruption of fkbA results in the generation of an FK506 non‐producing mutant demonstrating direct involvement of fkbA in the biosynthesis of FK506.
Dynamic Structure/Function Relationships in the α‐Chymotrypsin Deactivation Process by Heat and pH
Authors: Lozano Pedro , De Diego Teresa , Iborra José Luis
Published: July 2004

The activity decay of α‐chymotrypsin due to temperature and pH has been related to the associated structural changes of the protein that could be studied with the help of melting temperature which was determined by using ultraviolet absorption spectroscopy, and fluorescence spectra measurements. The kinetic behaviour in activity loss of α‐chymotrypsin followed a two‐step deactivation model, involving an intermediate state. The increase in temperature and pH showed a clear deactivation effect, reducing exponentially the half‐life of the enzyme. At pH 7.0, this two‐step deactivation process was also observed with both the maximum fluorescence intensity (Imax) and emission wavelength (λmax). The series‐type kinetic model allowed to establish a clear correlation between the activity and the fluorescence spectral normalized data: the intermediate state of the enzyme occurred at an identical deactivation denaturation level (α1), and a proportionality between the decay rate constants was observed. As a function of the incubation temperature, another correlation was observed between the α1 profile, initial λmax and thermal unfolding transition, allowing to identify the intermediate state of the kinetic model as that obtained at the melting temperature (43.9°C).
Identification of an N‐Glycosylated Region of Pig Zona Pellucida Glycoprotein ZPB that is involved in Sperm Binding
Authors: Yonezawa Naoto , Mitsui Sachiko , Kudo Katsuyasu , Nakano Minoru
Published: July 2004

ZPB, one of the pig zona pellucida glycoproteins, can be purified after removal of sialylated and/or sulfated N‐acetylpolylactosamine from the nonreducing region of its carbohydrate chains by digestion with endo‐β‐galactosidase. Among the components produced, only ZPB shows sperm‐binding activity after the digestion. Recently, we have shown that N‐linked carbohydrate chains of endo‐β‐galactosidase‐digested ZPB (EβG‐ZPB) are predominantly involved in sperm binding [Yonezawa, N., Aoki, H., Hatanaka, Y. & Nakano, M. (1995) Eur. J. Biochem. 233, 35–41]. In this study, to define the sperm‐binding region in EβG‐ZPB, glycopeptides were purified from lysyl endopeptidase digests of EβG‐ZPB and analyzed for sperm‐binding activity by an in vitro competition assay. The locations of the glycopeptides were determined from partial amino acid sequences, amino acid and sugar composition analyses, and apparent molecular masses after SDS/PAGE. The N‐terminal fragment (amino acid residues 137–247), which contains two N‐linked carbohydrate chains, showed a significant inhibition of sperm‐egg binding. However, the fragment that had one N‐linked carbohydrate chain (residues 325–341) and the fragment that had two or three O‐linked carbohydrate chains (residues 248–324) did not inhibit sperm‐egg binding. Thus, the two N‐linked carbohydrate chains in the N‐terminal fragment of EβG‐ZPB are important for sperm binding of pig zona pellucida.
The Gap‐Junction Protein Connexin 56 is Phosphorylated in the Intracellular Loop and the Carboxy‐Terminal Region
Authors: Berthoud Viviana M. , Beyer Eric C. , Kurata Wendy E. , Lau Alan F. , Lampe Paul D.
Published: July 2004

The lens gap‐junction protein, connexin 56, is modified by phosphorylation. Two‐dimensional mapping of tryptic phosphopeptides of 32P‐labeled connexin 56 from primary chicken‐lens cultures showed that treatment with 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) induced an increase in phosphorylation of connexin 56 at specific constitutively phosphorylated sites. Treatment with 8‐Br‐cAMP or forskolin did not induce substantial changes in connexin 56 phosphorylation. Two phosphorylation sites within connexin 56, S493 and S118, were identified after HPLC purification and peptide sequencing of tryptic phosphopeptides from bacterially expressed connexin 56 fusion proteins phosphorylated by protein kinase C or protein kinase A in vitro. Comparisons of the two‐dimensional maps of tryptic phosphopeptides from in vitro phosphorylated connexin 56 fusion proteins and in vivo phosphorylated connexin 56 showed that S493 and S118 were constitutively phosphorylated in lentoid‐containing cultures, and that treatment with TPA induced an increase in phosphorylation of the peptides containing S118. It is suggested that phosphorylation of connexin 56 at S118 is involved in the TPA‐induced decrease in intercellular communication and acceleration of connexin 56 degradation.
Up‐Regulation of the Ubiquitin‐Conjugating and Proteolytic Systems in Murine Acquired Immunodeficiency Syndrome
Authors: Crinelli Rita , Fraternale Alessandra , Casabianca Anna , Magnani Mauro
Published: July 2004

Lymphoproliferation, chronic B‐cell activation resulting in hypergammaglobulinemia, and profound immunodeficiency are prominent features of retro virus‐induced murine acquired immunodeficiency syndrome (murine AIDS). Here we demonstrate that in murine AIDS the ATP‐dependent and ubiquitin‐dependent proteolytic system is strongly affected, at least in the lymph nodes of infected mice. Solid‐phase immunochemical assays show that the ubiquitin–conjugate pools increase by about threefold 10 weeks after infection, then decline slightly 15 weeks after infection to a twofold increase. Accumulation of ubiquitin conjugates is accompanied by induction of the ubiquitin‐conjugating pathway, involving several carrier‐protein isozymes (E2), mainly 14‐kDa E2 and 17‐kDa E2. Furthermore, accumulation of ubiquitin conjugates and induction of the conjugating system are coincident with an increase in the proteolytic activity supported by the 26S proteolytic complex. However, 15 weeks after infection, when the conjugation rate and levels of ubiquitin conjugates decrease, proteasome activity returns to values similar to those of the control, suggesting that a higher proteosomal activity is no longer needed. The concerted induction of the ubiquitin‐conjugating and proteolytic systems in murine AIDS apparently does not involve the breakdown of viral products nor is it supported by virus‐coded events, but probably arises as a cellular response to viral infection.
Direct Electrochemistry of Megasphaera Elsdenii Iron Hydrogenase
Authors: Butt Julea N. , Filipiak Marian , Hagen Wilfred R.
Published: July 2004

The Fe‐hydrogenase from Megasphaera elsdenii undergoes direct electron exchange with glassy carbon electrodes. Cyclic voltammetry defines the catalytic performance of the enzyme over a continuous but precisely defined range of potentials. In the presence of H2 and protons the bias of the enzyme towards H2 production is readily visualised. Variation of the response with pH indicates that protein ionisations with pK of approximately 6.7 and 8.3 regulate the catalytic activity. Possible origins for these observations in the chemistry of the H2‐activating site are discussed. The mid‐wave potential of the catalytic response, Emid, is defined as the catalytic operating potential of the enzyme. Under an atmosphere of hydrogen Emid, =−421 ± 10 mV, pH 7 with a variation of −21 ± 4 mV pH−1, 22°C. Deviation of Emid from the thermodynamic potential of the hydrogen/proton couple reflects the enzyme's influence over the catalysed reaction. Emid is the reduction potential of the H2‐activating centre (H‐cluster) in the absence of kinetic bottle‐necks at other steps in the reaction mechanism.
Dimeric Binding of The Mouse Germ Cell Nuclear Factor
Authors: Boromeyer Uwe
Published: July 2004

The mouse germ cell nuclear factor (GCNF), a member of the nuclear receptor superfamily, is highly expressed during spermatogenesis, oogenesis, and during neuronal embryonic differentiation. The in vitro translated receptor binds autonomously to the direct repeat of the sequence 5′‐AGGTCA‐3′. To gain insights into the determinants necessary for DNA binding, I have generated truncated GCNF molecules by introducing carboxy‐terminal deletions into expression constructs. An electrophoretic mobility‐shift assay with these polypeptides shows that amino acids in addition to the core DNA‐binding domain are important for specific binding. To address the question of whether the protein binds as monomer, homodimer, or heterodimer, I used different approaches. Analysis of the full‐length protein was possible with GCNF polypeptides that contain epitopes of six consecutive histidines. Using a monoclonal antibody directed against these epitopes, I demonstrate that two GCNF molecules bind to a direct repeat. Dimerization between wild‐type and truncated GCNF is shown by an electrophoretic mobility‐shift analysis with a mixture of the proteins. In addition, I show that there is no in vitro interaction of GCNF with the retinoid X receptor, a promiscous partner of many nuclear receptors. The data suggest that GCNF may excert its in vivo function independently of other nuclear receptors.
High‐Molecular‐Mass Complexes of Human Minichromosome‐Maintenance Proteins in Mitotic Cells
Authors: Richter Andreas , Knippers Rolf
Published: July 2004

Minichromosome‐maintenance (Mcm) proteins perform essential functions regulating the replication of eukaryotic genomes. In interphase cells they are either bound to a nuclear structure, most probably chromatin, or occur as free multiprotein complexes in the nucleoplasm. Mcm proteins are displaced from their chromatin sites during S phase, and several become highly phosphorylated during mitosis. We investigated whether phosphorylation affects the ability of mitotic Mcm proteins to form multiprotein complexes. Our results clearly show that phosphorylated mitotic Mcm proteins form a 14–15‐S complex, probably consisting of one molecule each of the six known human Mcm proteins.
Characterization of Human Endothelin B Receptor and Mutant Receptors Expressed in Insect Cells
Authors: Doi Tomoko , Hiroaki Yoko , Arimoto Ikuyo , Fujiyoshi Yoshinori , Okamoto Tomoyuki , Satoh Misako , Furuichi Yasuhiro
Published: July 2004

Endothelin type‐B receptor (ETBR) forms a stable complex with its ligand, endothelin‐1. To facilitate biochemical and biophysical studies of human ETBR, several ETBR mutants carrying a hexahistidine tag sequence at the N or C terminus were expressed in Sf9 cells and were purified by a combination of biotinylated endothelin‐1‐ligand‐affinity and nickel‐affinity chromatographies. The ligand‐free receptor was purified by dissociating the ligand · receptor complex with 2 M NaSCN, whereas the ligand‐bound ETBR was purified by the use of thiol‐sensitive biotinylated endothelin‐1. While the wild‐type ETBR was expressed at about 100 pmol 125I‐endothelin‐1‐binding activity/mg membrane protein, the deletion of 36 residues from the N‐terminus reduced the expressed activity to about 30%. On the other hand, the lack of glycosylation and the replacement of 2–9 residues in the N‐terminal tail resulted in a 20–40% reduction in the expressed activity. Among the mutant proteins, [H57–H62, G63–G65]ETBR, carrying six His residues in the N‐terminal tail, was studied extensively because it was purified most effectively. Ligand‐free [H57–H62, G63–G65]ETBR, purified in digitonin, retained full ligand‐binding activity, while other detergents led to partial denaturation of the receptor after solubilization or after elution with NaSCN. On the other hand, ligand‐bound [H57–H62, G63–G65]ETBR could be purified in various detergents, such as n‐octyl‐β‐d‐glucopyranoside or n‐decyl‐β‐d‐maltopyranoside. Ligand‐free [H57–H62, G63–G65]ETBR reconstituted in phospholipid vesicles stimulated the binding of guanosine 5'‐3‐O‐(thio)triphosphate by Gq in the presence of endothelin‐1. Ligand‐bound [H57–H62, G63–G65]ETBR showed similar catalytic activity in nucleotide exchange by Gq. These results indicate that the ligand receptor complex in a detergent‐micellar solution retained the biologically active structure, and that the presence of ligand, endothelin‐1, in the receptor molecule reinforces the stable assembly of a helical bundle and therefore the active structure.
Structures of Polymeric Products Isolated From the Lipopolysaccharides of Reference Strains for Acinetobacter Baumannii O23 and O12
Authors: Haseley Simon R. , Traub Walter H. , Wilkinson Stephen G.
Published: July 2004

A polysaccharide containing D‐galactose (Gal), 2‐acetamido‐2‐deoxy‐D‐galactose (GalNAc), 2‐acet‐amido‐2‐deoxy‐D‐glucose (GlcNAc), and 3‐deoxy‐3‐(D‐3‐hydroxybutyramido)‐D‐quinovose (Qui3NR) was isolated from lipopolysaccharide (LPS) obtained from cells walls of the reference strain for Acinetobacter baumannii O23. By means of NMR studies, methylation analysis, and chemical degradations, the repeating unit of the polymer was identified as a branched pentasaccharide with the structure 1. The same polymer was apparently also present in LPS of the reference strain for serogroup O12, together with a second polymer based on a branched tetrasaccharide with the structure 2. This second polymer has previously been isolated as the O16 antigen of A. baumannii [Haseley, S. R., Diggle, H. J. & Wilkinson, S. G. (1996) Carbohydr. Res. 293, 259–265] and is probably present as a minor component of the LPS of A. baumannii O11 [Haseley, S. R. & Wilkinson, S. G. (1996) Eur. J. Biochem. 237, 266–271].
Structural and functional analysis of the promoter of the hepatic lipase gene
Authors: Chang Shau‐Feng , Scharf Jens‐Gerd , Will Hans
Published: July 2004

Hepatic lipase (HL) gene transcription is almost exclusively limited to hepatocytes. Here we have studied sequences and transcription factors regulating basal and hepatocyte‐restricted HL promoter activity. Sequencing of a cloned 3.4‐kb HL promoter fragment revealed three Alu repeat sequences and a consensus hepatocyte‐enriched nuclear transcription factor 1 (HNF1) binding site located upstream of one major and one minor transcription initiation site. By transfection of cell lines of hepatic and non‐hepatic origin and of primary hepatocyte cultures, sequences controlling basic HL promoter activity and negative elements located downstream and upstream thereof which extinguish or enhance this activity were defined. Some HL‐promoter fragments with internal deletions were active only in primary hepatocyte cultures. Human HNF1 protein was shown to bind to the HL‐specific HNF1 response element and the activity of a heterologous promoter was enhanced by HL‐HNF1 in rat primary hepatocyte cultures but not in the context of the authentic 3.4‐kb HL promoter sequences. In cell lines the presence of HNF4 but not of HNF1 and vHNF1 mRNA was found to correlate with HL gene expression although no perfect consensus HNF4 binding motif was detected in the promoter region tested. Taken together, these data indicate that hepatocyte‐specific HL gene transcription is controlled by positive and negative transcription regulatory proteins which bind to sequence motifs within and outside of the proximal 3.4‐kb promoter fragment studied. For the elucidation of the control of HL promoter activity in vivo the use of primary hepatocyte cultures is essential.
Differential Expression of three Arabidopsis Genes Encoding the B′ Regulatory Subunit of Protein Phosphatase 2A
Authors: Latorre Keith A. , Harris Darby M. , Rundle Sabine J.
Published: July 2004

Numerous plant processes ranging from signal transduction to metabolism appear to be mediated, in part, by type 2 A protein serine/threonine phosphatases (PP2A). In an effort to identify factors that control the activity of this enzyme in plants, we have isolated and characterized DNA sequences encoding the B′ regulatory subunit of PP2A from Arabidopsis thaliana. Specifically, we used PCR to amplify a segment of Arabidopsis cDNA that encodes a conserved section of the B′ polypeptide. This PCR fragment was subsequently used as a probe to screen an Arabidopsis cDNA library and cDNA clones derived from three distinct genes were identified. The AtB'α and AtBβgenes encode highly similar 57‐kDa B′ regulatory subunits while the third gene, AtBγ, encodes a more divergent 59‐kDa B′ protein. A comparison of the three Arabidopsis B′ polypeptides to those of yeast and animals shows the core region of this protein to be the most conserved while the amino and carboxy termini vary both in length and sequence. Genomic Southern blots indicate that at most the Arabidopsis genome contains five genes encoding the B′ regulatory subunit. The three genes identified in this study are expressed in all Arabidopsis organs, albeit at varying levels. In addition, mRNAs derived from the three genes accumulate differentially in response to heat shock. Our results indicate that the activity of plant PP2A might be regulated by a B′ type regulatory subunit similar to those found in animals and yeast, and suggest possible roles for B′‐containing PP2A complexes within plant cells.