bluuebunny/crossref_metadata_2025 huggingface dataset:
Coordinate repression of regulators of embryonic identity by PICKLE during germination in Arabidopsis
Authors: Dean Rider Stanley , Henderson James T. , Jerome Ronald E. , Edenberg Howard J. , Romero‐Severson Jeanne , Ogas Joe
Published: July 2003

In angiosperms, germination represents an important developmental transition during which embryonic identity is repressed and vegetative identity emerges. PICKLE (PKL) encodes a CHD3‐chromatin‐remodeling factor necessary for the repression of expression of LEAFY COTYLEDON1 (LEC1), a central regulator of embryogenesis. A candidate gene approach and microarray analysis identified nine additional genes that exhibit PKL‐dependent repression of expression during germination. Transcripts for all three LEAFY COTYLEDON genes, LEC1, LEC2, and FUS3, exhibit PKL‐dependent repression, and all three transcripts are elevated more than 100‐fold in pkl primary roots that inappropriately express embryonic traits (pickle roots). Three other genes that exhibit PKL‐dependent regulation have expression patterns correlated with zygotic or somatic embryogenesis, and one gene encodes a putative Lin‐11, Isl‐1, MEC‐3 (LIM) domain transcriptional regulator that is preferentially expressed in siliques. Genes that exhibit PKL‐dependent repression during germination are not necessarily regulated by PKL at other points in development. Our data suggest that PKL selectively regulates a suite of genes during germination to repress embryonic identity. In particular, we propose that PKL acts as a master regulator of the LEAFY COTYLEDON genes, and that joint derepression of these genes is likely to contribute substantially to expression of embryonic identity in pkl seedlings.
Synergistic interaction of the two paralogous Arabidopsis genes LRX1 and LRX2 in cell wall formation during root hair development
Authors: Baumberger N. , Steiner M. , Ryser U. , Keller B. , Ringli C.
Published: July 2003

LRR‐extensins (LRX) form a family of structural cell wall proteins containing a receptor‐like domain. The functional analysis of Arabidopsis LRX1 has shown that it is involved in cell morphogenesis of root hairs. In this work, we have studied LRX2, a paralog of LRX1. LRX2 expression is mainly found in roots and is responsive to factors promoting or repressing root hair formation. The function of LRX1 and LRX2 was tested by the expression of a truncated LRX2 and different LRX1/LRX2 chimaeric proteins. Using complementation of the lrx1 phenotype as the parameter for protein function, our experiments indicate that LRX1 and LRX2 are functionally similar but show differences in their activity. Genetic analysis revealed that single lrx2 mutants do not show any defect in root hair morphogenesis, but synergistically interact with the lrx1 mutation. lrx1/lrx2 double mutants have a significantly enhanced lrx1 phenotype, resulting in frequent rupture of the root hairs soon after their initiation. Analysis of the root hair cell wall ultrastructure by transmission electron microscopy (TEM) revealed the formation of osmophilic aggregates within the wall, as well as local disintegration of the wall structure in the double mutant, but not in wild‐type plants. Our results indicate that LRX1 and LRX2 have overlapping functions in root hair formation, and that they likely regulate cell morphogenesis by promoting proper development of the cell wall.
The capacity of transgenic tobacco to send a systemic RNA silencing signal depends on the nature of the inducing transgene locus
Authors: Mallory Allison C. , Mlotshwa Sizolwenkosi , Bowman Lewis H. , Vance Vicki B.
Published: July 2003

RNA silencing is a conserved eukaryotic pathway in which double‐stranded RNA (dsRNA) triggers destruction of homologous target RNA via production of short‐interfering RNA (siRNA). In plants, at least some cases of RNA silencing can spread systemically. The signal responsible for systemic spread is expected to include an RNA component to account for the sequence specificity of the process, and transient silencing assays have shown that the capacity for systemic silencing correlates with the accumulation of a particular class of small RNA. Here, we report the results of grafting experiments to study transmission of silencing from stably transformed tobacco lines in the presence or absence of helper component‐proteinase (HC‐Pro), a viral suppressor of silencing. The studied lines carry either a tail‐to‐tail inverted repeat, the T4‐IR transgene locus, or one of two different amplicon transgene loci encoding replication‐competent viral RNA. We find that the T4‐IR locus, like many sense‐transgene‐silenced loci, can send a systemic silencing signal, and this ability is not detectably altered by HC‐Pro. Paradoxically, neither amplicon locus effectively triggers systemic silencing except when suppressed for silencing by HC‐Pro. In contrast to results from transient assays, these grafting experiments reveal no consistent correlation between capacity for systemic silencing and accumulation of any particular class of small RNA. In addition, although all transgenic lines used to transmit systemic silencing signals were methylated at specific sites within the transgene locus, silencing in grafted scions occurred without detectable methylation at those sites in the target locus of the scion.
Molecular characterization of the Arabidopsis 9‐cis epoxycarotenoid dioxygenase gene family
Authors: Tan Bao‐Cai , Joseph Leina M. , Deng Wen‐Tao , Liu Lijuan , Li Qin‐Bao , Cline Kenneth , McCarty Donald R.
Published: July 2003

A key regulated step in abscisic acid (ABA) biosynthesis in plants is catalyzed by 9‐cis epoxycarotenoid dioxygenase (NCED), which cleaves 9‐cis xanthophylls to xanthoxin, a precursor of ABA. In Arabidopsis, ABA biosynthesis is controlled by a small family of NCED genes. Nine carotenoid cleavage dioxygenase (CCD) genes have been identified in the complete genome sequence. Of these, five AtNCEDs (2, 3, 5, 6, and 9) have been cloned and studied for expression and subcellular localization. Although all five AtNCEDs are targeted to plastids, they differ in binding activity of the thylakoid membrane. AtNCED2, AtNCED3, and AtNCED6 are found in both stroma and thylakoid membrane‐bound compartments. AtNCED5 is exclusively bound to thylakoids, whereas AtNCED9 remains soluble in stroma. A quantitative real‐time PCR analysis and histochemical staining of promoter::GUS activity in transgenic Arabidopsis revealed a complex pattern of localized NCED expression in well‐watered plants during development. AtNCED2 and AtNCED3 account for the NCED activity in roots, with localized expression in root tips, pericycle, and cortex cells at the base of lateral roots. Localized AtNCED2 and AtNCED3 expression in pericycle cells is an early marker of lateral initiation sites. AtNCED5, AtNCED6, AtNCED3, and AtNCED2 are expressed in flowers with very high AtNCED6::GUS activity occurring in pollen. AtNCED5::GUS, and to lesser degrees, AtNCED2::GUS and AtNCED3::GUS are expressed in developing anthers. AtNCED5, AtNCED6, AtNCED9, and AtNCED3 contribute to expression in developing seeds with high levels of AtNCED6 present at an early stage. GUS analysis indicates that AtNCED3 expression is confined to the base of the seed, whereas AtNCED5 and AtNCED6 are expressed throughout the seed. Consistent with the studies conducted by Iuchi and his colleagues in 2001, AtNCED3 is the major stress‐induced NCED in leaves. Our results indicate that developmental control of ABA synthesis involves localized patterns of AtNCED gene expression. In addition, differential membrane‐binding capacity of AtNCEDs is a potential means of post‐translational regulation of NCED activity.
An Arabidopsis protein closely related to Synechocystis cryptochrome is targeted to organelles
Authors: Kleine Tatjana , Lockhart Peter , Batschauer Alfred
Published: July 2003

Cryptochromes (CRYs) are blue/UV‐A photoreceptors related to the DNA repair enzyme DNA photolyase. They have been found in plants, animals and most recently in the cyanobacterium Synechocystis. Closely related to the Synechocystis cryptochrome is the Arabidopsis gene At5g24850. Here, we show that the encoded protein of At5g24850 binds flavin adenine dinucleotide (FAD). It has no photolyase activity, and is likely to function as a photoreceptor. We have named it At‐cry3 to distinguish it from the other Arbabidopsis cryptochrome homologues At‐cry1 and At‐cry2. At‐cry3 carries an N‐terminal sequence, which mediates import into chloroplasts and mitochondria. Furthermore, we show that At‐cry3 binds DNA. DNA binding was also demonstrated for the Synechocystis cryptochrome, indicating that both photoreceptors could have similar modes of action. Based on the finding of a new cryptochrome class in bacteria and plants, it has been suggested that cryptochromes evolved before the divergence of eukaryotes and prokaryotes. However, our phylogenetic analyses are also consistent with an alternative explanation that the presence of cryptochromes in the plant nuclear genome is the result of dual horizontal gene transfer. That is, CRY1 and CRY2 genes may originate from an endosymbiotic ancestor of modern‐day α‐proteobacteria, while the CRY3 gene may originate from an endosymbiotic ancestor of modern‐day cyanobacteria.
Urban malaria: primary caregivers’ knowledge, attitudes, practices and predictors of malaria incidence in a cohort of Ugandan children
Authors: Njama Denise , Dorsey Grant , Guwatudde David , Kigonya Kate , Greenhouse Bryan , Musisi Stephen , Kamya Moses R.
Published: July 2003

Objectives To assess malaria‐related knowledge, attitude and practices (KAP) among primary caregivers, to identify associations between primary caregivers’ characteristics and positive KAP towards malaria, and to identify independent predictors of childhood malaria incidence in an urban setting.

Methods Children aged 6 months to 5 years living in Kampala, Uganda were enrolled as part of a longitudinal study on antimalarial therapy. Primary caregivers of 307 children were interviewed and information was collected on demographics, malaria‐related KAP, environmental and household factors. Malaria incidence was measured prospectively using passive surveillance.

Results A total of 90% of respondents reported mosquitoes and/or malaria as the cause of fever. Caregivers reported that if their child had fever, 63% would go to a clinic or hospital as their first action and 97% as their first or second action. Only 38% knew that chloroquine was the recommended first‐line treatment for malaria and 29% knew the correct dose. Preventive measures for malaria were reported in 45% of households but only 25% reported using bednets. Higher levels of education for the caregiver were associated with positive malaria‐related KAP. Malaria incidence varied widely. The following were independent predictors of malaria incidence: (1) Children aged 24–41 months at enrolment had a higher incidence of malaria. (2) Reported bednet or chemoprophylaxis use reduced the incidence of malaria. (3) A child's place of residence was associated with incidence. (4) Children from households using open water sources had a higher incidence than those using closed sources.

Conclusion Primary caregivers were knowledgeable about malaria and used modern health care facilities but knew less about the proper administration of antimalarials and had limited use of preventive measures. Malaria incidence was associated with child's age at enrolment, geography, source of water and the use of preventive measures.
Clinical presentation and diagnostic sensitivity of laboratory tests for Strongyloides stercoralis in travellers compared with immigrants in a non‐endemic country
Authors: Sudarshi Sonali , Stümpfle Richard , Armstrong Margaret , Ellman Thomas , Parton Simon , Krishnan Prabha , Chiodini Peter L. , Whitty Christopher J. M.
Published: July 2003

Objectives To assess whether the clinical and laboratory methods for diagnosing Strongyloides stercoralis infection in non‐endemic countries is different between those who are chronically exposed and those who travel.

Methods Analysis of laboratory and clinical data from 204 patients having S. stercoralis infection at the Hospital for Tropical Diseases, London.

Results Sixty‐four travellers and 128 immigrants from endemic countries had laboratory‐proven strongyloides. In those with microscopically proven disease, serology was 73% sensitive in travellers and 98% sensitive in immigrants (P < 0.001). There was no difference in the eosinophil count between the two groups with 19% having a normal count. Patterns of symptoms varied between the groups, and around one‐third were asymptomatic in both groups. Serology was of limited use in follow‐up.

Conclusions Eosinophil count and stool microscopy are insufficiently sensitive to be used alone for screening strongyloides. The sensitivity of serology is good in immigrants with chronic infection, but lower in travellers.
A fatal case of human babesiosis in Portugal: molecular and phylogenetic analysis
Authors: Centeno‐Lima S. , Do Rosário V. , Parreira R. , Maia A. J. , Freudenthal A. M. , Nijhof A. M. , Jongejan F.
Published: July 2003

We report the first case of human babesiosis in Portugal. A 66‐year‐old splenectomized man was admitted to a Lisbon hospital after 1 week of fever, abdominal pain, anorexia and nausea. A high parasitaemia (30%) of Babesia parasites was found in Giemsa‐stained blood smears and, despite treatment, the patient died several weeks later of renal failure. Ethylenediaminetetraacetic acid blood samples were processed for polymerase chain reaction (PCR) and reverse line blot hybridization to confirm and characterize the Babesia infection. The amplified PCR product was cloned and subsequently sequenced. Molecular analysis showed that the infection was caused by Babesia divergens and that other blood parasites were not involved. Phylogenetic analysis showed that the 18 S ribosomal RNA gene sequence was similar to three other European isolates of B. divergens. In view of the high risk for splenectomized individuals, strict measures should be taken to avoid tick bites.
Low awareness of intestinal schistosomiasis in northern Senegal after 7 years of health education as part of intense control and research activities
Authors: Sow Seydou , de Vlas Sake J. , Mbaye Amadou , Polman Katja , Gryseels Bruno
Published: July 2003

We evaluated the awareness of and knowledge about intestinal schistosomiasis in a highly infected rural community of northern Senegal where a variety of health information and education activities had taken place for 7 years as a component of different research and control programmes. As the infection had been introduced only recently, an initial ‘zero’ knowledge can be assumed. Most of the health education activities had been performed with adapted messages through local health and community workers. By a questionnaire, 566 individuals were asked simple questions on symptoms, mode of transmission, the sources of information and health‐seeking behaviour. About 86% of the respondents stated that they knew what schistosomiasis was, and 92% that in case of illness they would seek treatment at the health centre. However, only half of the people accurately quoted symptoms associated with intestinal schistosomiasis: diarrhoea, abdominal pain and bloody stools. The majority of respondents realized that the disease was somehow linked with water and (lack of) hygiene, but only 44% of respondents reported water contact as the source of infection. Ultimately, only 30% of the respondents gave adequate answers about both symptoms and mode of transmission. We conclude that even intense and long‐lasting education efforts for a specific and straightforward problem as schistosomiasis are not enough to have profound impact on the knowledge of rural traditional communities.
Performance of the acetic acid test when used in field conditions as a screening test for cervical cancer
Authors: Claeys P. , De Vuyst H. , Gonzalez C. , Garcia A. , Bello R. E. , Temmerman M.
Published: July 2003

Objective To assess if visual inspection with acetic acid (VIA) is a useful alternative screening test for cervical cancer, when used in a resource‐poor setting with an existing cytology‐based screening programme.

Methods Women living in Rivas district (Nicaragua), who attended the programme, were concurrently screened with VIA and Papanicolau (PAP) smear. Screening was performed by health providers who had received training in VIA and a refresher course in cytology. Women testing positive for either of the results were referred for colposcopy and biopsy when indicated. The performance of VIA was compared with PAP smear, calculating the relative true and false positive rate (RELTPR and RELFPR) and for a high threshold on biopsy (cervical intraepithelial neoplasia grade 2 or a higher grade). We determined the trade‐off between both tests by calculating the ratio of extra false positives detected through extra true positives (EFP:ETP ratio).

Results A total of 1076 patients were screened. Nearly 33% had a positive screening test. On biopsy, 7.6% had a low‐grade intraepithelial lesion, 4.5% a high‐grade intraepithelial lesion (HSIL) and 0.5% invasive cancer. The RELTPR (VIA to PAP) was 1.96, the RELFPR 5.02 and the EFP:ETP ratio 8.04. VIA detected twice as much HSIL and invasive cancers as the PAP smear. Yet, for every extra diagnosis, eight extra false positives had to be examined at the referral level.

Conclusions The VIA spectacularly increases the number of HSIL and invasive cancers detected. The high FPR is a concern for the organization of the referral level. There is a need to establish uniform criteria on test positivity and to further improve the performance in field conditions.
Which health services reduce maternal mortality? Evidence from ratings of maternal health services
Authors: Bulatao Rodolfo A. , Ross John A.
Published: July 2003

We report cross‐national regressions for maternal mortality in 49 developing countries, using indices of the adequacy of maternal health services derived from ratings by at least 10 experts per country. As in previous such regressions, a socioeconomic factor – in this case per capita income – has a significant effect, but having a trained attendant at delivery does not. Instead, the ratings index for access to services has a consistent, significant effect regardless of which estimates of maternal mortality ratios are predicted. Further analysis suggests that access to treatment for pregnancy complications and to services that help avoid pregnancy and birth are most closely related to lower mortality. Service ratings are interdependent, however, so that focusing only on individual services may not be productive.
Monitoring mosquito net coverage for malaria control in Africa: possession vs. use by children under 5 years
Authors: Korenromp Eline L. , Miller John , Cibulskis Richard E. , Kabir Cham M. , Alnwick David , Dye Christopher
Published: July 2003

Objectives To investigate the strengths and weaknesses of the indicators ‘proportion of households possessing mosquito net(s)’ and ‘proportion of children under 5 years of age who slept under a net the preceding night’ for monitoring malaria control.

Methods Review of data from household surveys including demographic and health surveys in sub‐Saharan African countries.

Results Net possession ranged among 14 surveyed regions from 0.1% to 28.5% for insecticide‐treated nets (ITNs) and among 69 regions from 3.6% to 79.7% for any net. Reported use during the preceding night by children under 5 years of age was between 0% and 16% for ITNs and between 0.7% and 74.5% for any net. On average, in households owning ITN(s), 55% of children slept under it (R2 = 0.97, P < 0.001). For any net, use was −4.2% + 0.875 × possession (R2 = 0.89, P < 0.001); the use of nets, however, also varied somewhat among the surveyed countries (P = 0.003). In‐depth surveys suggested that use was lower than possession because: (i) nets were scarce (mean 1.8 per possessing household); (ii) nets were not always used for children and (iii) use was lower during hot, dry months than during cool rainy months, and many surveys had been conducted in the dry season.

Conclusions Not all mosquito nets owned by African households are being used for young children. Household education on the consistent use of nets for this vulnerable group is called for in malaria control programmes. Regular, district‐level rapid assessments of household possession of nets should complement ongoing in‐depth surveys. Data on ‘use during the preceding night’ must be interpreted taking the survey season into account.
Evaluation of the seroprevalence of rubella in the region of Dakar (Senegal)
Authors: Dromigny J. A. , Nabeth P. , Perrier Gros Claude J. D.
Published: July 2003

Data on the seroepidemiology of rubella in tropical African countries are still scarce. To determine the seroprevalence in Senegal, we conducted a 6 year retrospective survey among women of child bearing age in the urban region of Dakar, Senegal. The global seroprevalence determined with a commercial enzyme immunoassay among 3471 serological results was 90.1%. The distribution of this prevalence appeared stable with no significant difference between the years of study, age groups, and the socio‐economic level of the districts of residence of the patients. Compared to seroepidemiological surveys performed in other western African countries, our data suggest an important and stable circulation of the virus in the region of Dakar. The lack of data on rubella and congenital rubella syndrome (CRS) in Senegal should encourage medical authorities to establish a national rubella surveillance network in order to develop a strategy to survey and control CRS in the country.
The effect of soil type and climate on hookworm (Necator americanus) distribution in KwaZulu‐Natal, South Africa
Authors: Mabaso M. L. H. , Appleton C. C. , Hughes J. C. , Gouws E.
Published: July 2003

We investigated environmental factors influencing the distribution of hookworm infection in KwaZulu‐Natal, South Africa. Prevalence data were sourced from previous studies and additional surveys carried out to supplement the database. When geo‐referenced the data revealed that higher prevalences are limited to areas below 150 m above sea level, and low prevalences to areas above this altitude. Using univariate analysis we investigated the differences in environmental factors in the two areas. The relationship between hookworm prevalence, altitude and climate‐derived variables was assessed using Pearson correlation coefficient, and that of soil type using the t‐test. Multivariate analysis was used to determine environmental factors that combine best to provide favourable conditions for hookworm distribution. The results revealed that areas ≤150 m above sea level, i.e. the coastal plain, supported high prevalences (x̄ = 45, n = 51), and were characterized by sandy soils with a clay content of less than 15%, warm temperatures and relatively high rainfall. Areas >150 m above sea level, i.e. inland, supported low mean hookworm prevalences (x̄ = 6, n = 21), and were characterized by soils with a clay content of more than 45%, variable temperatures and moderate rainfall. Hookworm prevalence also decreased southwards as temperatures became slightly cooler, rainfall remained more‐or‐less constant and the coastal plain narrowed. In the multivariate model prevalence was most significantly correlated with the mean daily minimum temperature for January followed by the mean number of rainy days for January. This indicates the importance of summer conditions in the transmission of hookworm infection in KwaZulu‐Natal and suggests that transmission may be seasonal.
Pentoxifylline adjunct improves prognosis of human cerebral malaria in adults
Authors: Das B. K. , Mishra S. , Padhi P. K. , Manish R. , Tripathy R. , Sahoo P. K. , Ravindran B.
Published: July 2003

Fifty‐two adult patients with cerebral malaria were randomly categorized into two groups to receive either quinine dihydrochloride (Qn) alone or a combination of Qn and pentoxifylline (Px). Thirty‐two of them received intravenous (i.v.) Qn (group I), and 20 patients (group II) received i.v. Qn along with parenteral Px support (10 mg/kg/day) for the initial 3 days. There was significant improvement in coma resolution time in group II (21.6 ± 13.9 h) in comparison with group I (63.5 ± 19.7 h) (P < 0.001), and mortality was 25% of patients in group I against 10% patients receiving Px adjunct (P > 0.05). Three days post‐therapy, serum tumour necrosis factor‐α (TNF‐α) levels decreased significantly in patients on Px support (day 0 TNF = 415.62 ± 477.80 pg/ml; day 3 TNF = 47.92 ± 27.9 pg/ml; P = 0.0029). There was no significant change in TNF levels in those on quinine alone (day 0 TNF = 477.08 ± 933.90 pg/ml; day 3 TNF = 589 ± 602.3 pg/ml; P > 0.05). There were no serious side‐effects necessitating withdrawal of patients receiving Px therapy.
Visceral leishmaniasis and HIV in Tigray, Ethiopia
Authors: Lyons Suzi , Veeken Hans , Long Jean
Published: July 2003

Objectives To identify characteristics that increased the risk of mortality in Ethiopian visceral leishmaniasis patients in a treatment programme managed by Médecins sans Frontières, in Tigray, Northern Ethiopia.

Methods Retrospective review of a cohort of 791 patients treated for visceral leishmaniasis.

Results The cohort displayed all the classical signs and symptoms of the disease. The case fatality rate was 18.5% (146) (95% CI: 15.8–21.3%). Logistic regression showed that individuals who experienced at least one episode of vomiting or haemorrhage were more likely to die than those who did not. A subcohort of individuals who tested human immunodeficiency virus (HIV)‐positive were more than four times more likely to die than those who tested HIV‐negative (OR 4.5, 95% CI: 1.8–11.4).

Conclusion This study identifies characteristics associated with death in this population and highlights the devastating effect of co‐infection with visceral leishmaniasis and HIV in the African context.
Psychosocial aspects of health seeking behaviours of patients with Buruli ulcer in southern Benin
Authors: Aujoulat Isabelle , Johnson Christian , Zinsou Claude , Guédénon Augustin , Portaels Françoise
Published: July 2003

We investigated cultural beliefs and psychosocial factors associated with Buruli ulcer in southern Benin in order to elaborate and deliver appropriate health education messages. We conducted a qualitative study among 130 adults and 30 children in Zou province, a highly endemic region. Focus group interviews of inhabitants, patients and their assistants, health care professionals and traditional healers took place in Dasso, Ouinhi, Sagon and Zagnanado. Drawing sessions followed by individual interviews were organized among school children in Dasso and Sagon. We found that although Buruli ulcer is well known and recognized – even at a very early stage of the disease – and perceived as threatening, most people are reluctant to seek treatment at the health care centre. They are unclear about the origin of the disease (environmental factors or sorcery) and treatment is considered devastating, expensive and ineffective in some cases.
p53‐induced inhibition of protein synthesis is independent of apoptosis
Authors: Constantinou Constantina , Bushell Martin , Jeffrey Ian W. , Tilleray Vivienne , West Matthew , Frost Victoria , Hensold Jack , Clemens Michael J.
Published: July 2003

Activation of a temperature‐sensitive form of p53 in murine erythroleukaemia cells results in a rapid impairment of protein synthesis that precedes inhibition of cell proliferation and loss of cell viability by several hours. The inhibition of translation is associated with specific cleavages of polypeptide chain initiation factors eIF4GI and eIF4B, a phenomenon previously observed in cells induced to undergo apoptosis in response to other stimuli. Although caspase activity is enhanced in the cells in which p53 is activated, both the effects on translation and the cleavages of the initiation factors are completely resistant to inhibition of caspase activity. Moreover, exposure of the cells to a combination of the caspase inhibitor z‐VAD.FMK and the survival factor erythropoietin prevents p53‐induced cell death but does not reverse the inhibition of protein synthesis. We conclude that the p53‐regulated cleavages of eIF4GI and eIF4B, as well as the overall inhibition of protein synthesis, are caspase‐independent events that can be dissociated from the induction of apoptosis per se.
Subcellular localization of yeast Sec14 homologues and their involvement in regulation of phospholipid turnover
Authors: Schnabl Martina , Oskolkova Olga V. , Holič Roman , Brežná Barbara , Pichler Harald , Zágoršek Miloš , Kohlwein Sepp D. , Paltauf Fritz , Daum Günther , Griač Peter
Published: July 2003

Sec14p of the yeast Saccharomyces cerevisiae is involved in protein secretion and regulation of lipid synthesis and turnover in vivo, but acts as a phosphatidylinositol–phosphatidylcholine transfer protein in vitro. In this work, the five homologues of Sec14p, Sfh1p–Sfh5p, were subjected to biochemical and cell biological analysis to get a better view of their physiological role. We show that overexpression of SFH2 and SFH4 suppressed the sec14 growth defect in a more and SFH1 in a less efficient way, whereas overexpression of SFH3 and SFH5 did not complement sec14. Using C‐terminal yEGFP fusions, Sfh2p, Sfh4p and Sfh5p are mainly localized to the cytosol and microsomes similar to Sec14p. Sfh1p was detected in the nucleus and Sfh3p in lipid particles and in microsomes. In contrast to Sec14p, which inhibits phospholipase D1 (Pld1p), overproduction of Sfh2p and Sfh4p resulted in the activation of Pld1p‐mediated phosphatidylcholine turnover. Interestingly, Sec14p and the two homologues Sfh2p and Sfh4p downregulate phospholipase B1 (Plb1p)‐mediated turnover of phosphatidylcholine in vivo. In summary, Sfh2p and Sfh4p are the Sec14p homologues with the most pronounced functional similarity to Sec14p, whereas the other Sfh proteins appear to be functionally less related to Sec14p.
A novel mass spectrometric approach to the analysis of hormonal peptides in extracts of mouse pancreatic islets
Authors: Ramström Margareta , Hagman Charlotte , Tsybin Youri O. , Markides Karin E. , Håkansson Per , Salehi Albert , Lundquist Ingmar , Håkanson Rolf , Bergquist Jonas
Published: July 2003

Liquid chromatography mass spectrometry (LC‐MS) is a valuable tool in the analysis of proteins and peptides. The combination of LC‐MS with different fragmentation methods provides sequence information on components in complex mixtures. In this work, on‐line packed capillary LC electrospray ionization Fourier transform ion cyclotron resonance MS was combined with two complementary fragmentation techniques, i.e. nozzle‐skimmer fragmentation and electron capture dissociation, for the determination of hormonal peptides in an acid ethanol extract of mouse pancreatic islets. The most abundant peptides, those derived from proinsulin and proglucagon, were identified by their masses and additional sequence‐tag information established their identities. Interestingly, the experiments demonstrated the presence of truncated C‐peptides, des‐(25–29)‐C‐peptide and des‐(27–31)‐C‐peptide. These novel findings clearly illustrate the potential usefulness of the described technique for on‐line sequencing and characterization of peptides in tissue extracts.
Kinetic characterization, structure modelling studies and crystallization of Trypanosoma brucei enolase
Authors: Hannaert Véronique , Albert Marie‐Astrid , Rigden Daniel J. , Theresa da Silva Giotto M. , Thiemann Otavio , Garratt Richard C. , Van Roy Joris , Opperdoes Fred R. , Michels Paul A. M.
Published: July 2003

In this article, we report the results of an analysis of the glycolytic enzyme enolase (2‐phospho‐d‐glycerate hydrolase) of Trypanosoma brucei. Enolase activity was detected in both bloodstream‐form and procyclic insect‐stage trypanosomes, although a 4.5‐fold lower specific activity was found in the cultured procyclic homogenate. Subcellular localization analysis showed that the enzyme is only present in the cytosol. The T. brucei enolase was expressed in Escherichia coli and purified to homogeneity. The kinetic properties of the bacterially expressed enzyme showed strong similarity to those values found for the natural T. brucei enolase present in a cytosolic cell fraction, indicating a proper folding of the enzyme in E. coli. The kinetic properties of T. brucei enolase were also studied in comparison with enolase from rabbit muscle and Saccharomyces cerevisiae. Functionally, similarities were found to exist between the three enzymes: the Michaelis constant (Km) and KA values for the substrates and Mg2+ are very similar. Differences in pH optima for activity, inhibition by excess Mg2+ and susceptibilities to monovalent ions showed that the T. brucei enolase behaves more like the yeast enzyme. Alignment of the amino acid sequences of T. brucei enolase and other eukaryotic and prokaryotic enolases showed that most residues involved in the binding of its ligands are well conserved. Structure modelling of the T. brucei enzyme using the available S. cerevisiae structures as templates indicated that there are some atypical residues (one Lys and two Cys) close to the T. brucei active site. As these residues are absent from the human host enolase and are therefore potentially interesting for drug design, we initiated attempts to determine the three‐dimensional structure. T. brucei enolase crystals diffracting at 2.3 Å resolution were obtained and will permit us to pursue the determination of structure.
Structural analysis of lipopolysaccharides from Haemophilus influenzae serotype f
Authors: Yildirim Håkan H. , Hood Derek W. , Moxon E. Richard , Schweda Elke K. H.
Published: July 2003

Structural elucidation of the lipopolysaccharide (LPS) from three serotype f Haemophilus influenzae clinical isolates RM6255, RM7290 and RM6252 has been achieved using NMR spectroscopy techniques and ESI‐MS on O‐deacylated LPS and core oligosaccharide material (OS) as well as ESI‐MSn on permethylated dephosphorylated OS. This is the first study to report structural details on LPS from serotype f strains. We found that the LPSs of all strains were highly heterogeneous mixtures of glycoforms expressing the common H. influenzae structural element l‐α‐d‐Hepp‐(1→2)‐[PEtn→6]‐l‐α‐d‐Hepp‐(1→3)‐[β‐d‐Glcp‐(1→4)]‐l‐α‐d‐Hepp‐(1→5)‐[PPEtn→4]‐α‐Kdo‐(2→6)‐lipid A with variable length of OS chains linked to each of the heptoses. The terminal heptose (HepIII) in RM6255 is substituted at the O‐3 position by a β‐d‐Glcp residue whereas HepIII in strains RM7290 and RM6252 is substituted at O‐2 by the globoside unit (α‐d‐Galp‐(1→4)‐β‐d‐Galp‐(1→4)‐β‐d‐Glc) or truncated versions thereof. The central heptose (HepII) is substituted by an α‐d‐Galp‐(1→4)‐β‐d‐Galp‐(1→4)‐β‐d‐Glcp‐(1→4)‐α‐d‐Glcp unit in RM7290 and RM6252 or truncated versions thereof. Strain RM6255 does not express galactose in its LPS and only shows a cellobiose unit elongating from HepII (β‐d‐Glcp‐(1→4)‐α‐d‐Glcp). ESI‐MSn on dephosphorylated and permethylated OS provided information on the existence of additional minor isomeric glycoforms.
Gene regulation by tetracyclines
Authors: Berens Christian , Hillen Wolfgang
Published: July 2003

The Tet repressor protein (TetR) regulates transcription of a family of tetracycline (tc) resistance determinants in Gram‐negative bacteria. The resistance protein TetA, a membrane‐spanning H+‐[tc·M]+ antiporter, must be sensitively regulated because its expression is harmful in the absence of tc, yet it has to be expressed before the drugs' concentration reaches cytoplasmic levels inhibitory for protein synthesis. Consequently, TetR shows highly specific tetO binding to reduce basal expression and high affinity to tc to ensure sensitive induction. Tc can cross biological membranes by diffusion enabling this inducer to penetrate the majority of cells. These regulatory and pharmacological properties are the basis for application of TetR to selectively control the expression of single genes in lower and higher eukaryotes. TetR can be used for that purpose in some organisms without further modifications. In mammals and in a large variety of other organisms, however, eukaryotic transcriptional activator or repressor domains are fused to TetR to turn it into an efficient regulator. Mechanistic understanding and the ability to engineer and screen for mutants with specific properties allow tailoring of the DNA recognition specificity, the response to inducer tc and the dimerization specificity of TetR‐based eukaryotic regulators. This review provides an overview of the TetR properties as they evolved in bacteria, the functional modifications necessary to transform it into a convenient, specific and efficient regulator for use in eukaryotes and how the interplay between structure − function studies in bacteria and specific requirements of particular applications in eukaryotes have made it a versatile and highly adaptable regulatory system.
Purification and characterization of novel salt‐active acharan sulfate lyase from Bacteroides stercoris HJ‐15
Authors: Hong Sung‐Woon , Shin Ho‐Young , Kim Yeong Shik , Kim Dong‐Hyun
Published: July 2003

Salt‐active acharan sulfate lyase (no EC number) has been purified from Bacteroides stercoris HJ‐15, which was isolated from human intestinal bacteria with GAG degrading enzymes. The enzyme was purified to apparent homogeneity by a combination of QAE‐cellulose, diethylaminoethyl (DEAE)‐cellulose, CM‐Sephadex C‐50, HA ultrogel and phosphocellulose column chromatography with the final specific activity of 81.33 µmol·min−1·mg−1. The purified salt‐active acharan sulfate lyase was activated to 5.3‐fold by salts (KCl and NaCl). The molecular weight of salt‐active acharan sulfate lyase was 94 kDa by SDS/PAGE and gel filtration. The salt‐active acharan sulfate lyase showed optimal activity at pH 7.2 and 40 °C. Salt‐active acharan sulfate lyase activity was potently inhibited by Cu2+, Ni2+ and Zn2+. This enzyme was inhibited by some agents, butanediol and p‐chloromercuric sulfonic acid, which modify arginine and cysteine residues. The purified Bacteroidal salt‐active acharan sulfate lyase acted to the greatest extent on acharan sulfate, to a lesser extent on heparan sulfate and heparin. The biochemical properties of the purified salt‐active acharan sulfate lyase are different from those of the previously purified heparin lyases. However, these findings suggest that the purified salt‐active acharan sulfate lyase may belong to heparin lyase II.
Alternative binding modes of an inhibitor to two different kinases
Authors: De Moliner Erika , Brown Nick R. , Johnson Louise N.
Published: July 2003

Protein kinases are targets for therapeutic agents designed to intervene in signaling processes in the diseased state. Most kinase inhibitors are directed towards the conserved ATP binding site. Because the essential features of this site are conserved in all eukaryotic protein kinases, it is generally assumed that the same compound will bind in a similar manner to different protein kinases. The inhibitor 4,5,6,7‐tetrabromobenzotriazole (TBB) is a selective inhibitor for the protein kinase CK2 (IC50 1.6 µm) (Sarno et al. (2001) FEBS Letts.496, 44–48). Three other kinases [cyclin‐dependent protein kinase 2 (CDK2), phosphorylase kinase and glycogen synthase kinase 3β] exhibit approximately 10‐fold weaker affinity for TBB than CK2. We report the crystal structure of TBB in complex with phospho‐CDK2–cyclin A at 2.2 Å resolution and compare the interactions with those observed for TBB bound to CK2. TBB binds at the ATP binding site of both kinases. In CDK2, each of the four bromine atoms makes polar contacts either to main chain oxygens in the hinge region of the kinase or to water molecules, in addition to several van der Waals contacts. The mode of binding of TBB to CDK2 is different from that to CK2. TBB in CDK2 is displaced more towards the hinge region between the N‐ and C‐terminal lobes and rotated relative to TBB in CK2. The ATP binding pocket is wider in CDK2 than in CK2 resulting in fewer van der Waals contacts but TBB in CK2 does not contact the hinge. The structures show that, despite the conservation of the ATP binding pocket, the inhibitor is able to exploit different recognition features so that the same compound can bind in different ways to the two different kinases.
Structure of the O‐polysaccharide from Proteus myxofaciens
Authors: Sidorczyk Zygmunt , Kondakova Anna N. , Zych Krystyna , Senchenkova Sof'ya N. , Shashkov Alexander S. , Drzewiecka Dominika , Knirel Yuriy A.
Published: July 2003

The O‐polysaccharide (O‐antigen) was obtained from the lipopolysaccharide of Proteus myxofaciens, a Proteus strain producing copious amounts of slime, which was isolated from the gypsy moth larvae. The structure of the polysaccharide was studied by chemical analysis and 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY and H‐detected 1H,13C HMQC experiments. It was found that the polysaccharide contains an amide of glucuronic acid (GlcA) with an unusual α‐linked amino acid, Nε‐[(R)‐1‐carboxyethyl]‐l‐lysine (2S,8R‐alaninolysine, 2S,8R‐AlaLys), and has a linear tetrasaccharide repeating unit of the following structure:

This structure is unique among known bacterial polysaccharide structures. On the basis of these and serological data, it is proposed that P. myxofaciens be classified into a new Proteus serogroup, O60, of which this strain is the single representative. Structural and serological relatedness of P. myxofaciens to other AlaLys‐containing O‐antigens of Proteus and Providencia is discussed.
Weak organic acid stress inhibits aromatic amino acid uptake by yeast, causing a strong influence of amino acid auxotrophies on the phenotypes of membrane transporter mutants
Authors: Bauer Bettina E. , Rossington Danielle , Mollapour Mehdi , Mamnun Yasmine , Kuchler Karl , Piper Peter W.
Published: July 2003

The ability of yeasts to grow in the presence of weak organic acid preservatives is an important cause of food spoilage. Many of the determinants of acetate resistance in Saccharomyces cerevisiae differ from the determinants of resistance to the more lipophilic sorbate and benzoate. Interestingly, we show in this study that hypersensitivity to both acetate and sorbate results when the cells have auxotrophic requirements for aromatic amino acids. In tryptophan biosynthetic pathway mutants, this weak acid hypersensitivity is suppressed by supplementing the medium with high levels of tryptophan or, in the case of sorbate sensitivity, by overexpressing the Tat2p high affinity tryptophan permease. Weak acid stress therefore inhibits uptake of aromatic amino acids from the medium. This allows auxotrophic requirements for these amino acids to strongly influence the resistance phenotypes of mutant strains. This property must be taken into consideration when using these phenotypes to attribute functional assignments to genes. We show that the acetate sensitivity phenotype previously ascribed to yeast mutants lacking the Pdr12p and Azr1p plasma membrane transporters is an artefact arising from the use of trp1 mutant strains. These transporters do not confer resistance to high acetate levels and, in prototrophs, their presence is actually detrimental for this resistance.
UMP kinase from the Gram‐positive bacterium Bacillus subtilis is strongly dependent on GTP for optimal activity
Authors: Gagyi Cristina , Bucurenci Nadia , Sîrbu Ovidiu , Labesse Gilles , Ionescu Mihaela , Ofiteru Augustin , Assairi Liliane , Landais Stéphanie , Danchin Antoine , Bârzu Octavian , Gilles Anne‐Marie
Published: July 2003

The gene encoding Bacillus subtilis UMP kinase (pyrH/smbA) is transcribed in vivo into a functional enzyme, which represents approximately 0.1% of total soluble proteins. The specific activity of the purified enzyme under optimal conditions is 25 units·mg−1 of protein. In the absence of GTP, the activity of B. subtilis enzyme is less than 10% of its maximum activity. Only dGTP and 3′‐anthraniloyl‐2′‐deoxyguanosine‐5′‐triphosphate (Ant‐dGTP) can increase catalysis significantly. Binding of Ant‐dGTP to B. subtilis UMP kinase increased the quantum yield of the fluorescent analogue by a factor of more than three. UTP and GTP completely displaced Ant‐dGTP, whereas GMP and UMP were ineffective. UTP inhibits UMP kinase of B. subtilis with a lower affinity than that shown towards the Escherichia coli enzyme. Among nucleoside monophosphates, 5‐fluoro‐UMP (5F‐UMP) and 6‐aza‐UMP were actively phosphorylated by B. subtilis UMP kinase, explaining the cytotoxicity of the corresponding nucleosides towards this bacterium. A structural model of UMP kinase, based on the conservation of the fold of carbamate kinase and N‐acetylglutamate kinase (whose crystals were recently resolved), was analysed in the light of physicochemical and kinetic differences between B. subtilis and E. coli enzymes.
Transglutaminase from Streptomyces mobaraensis is activated by an endogenous metalloprotease
Authors: Zotzel J. , Keller P. , Fuchsbauer H.‐L.
Published: July 2003

Streptomyces mobaraensis secretes a Ca2+‐independent transglutaminase (TGase) that is activated by removing an N‐terminal peptide from a precursor protein during submerged culture in a complex medium [Pasternack, R., Dorsch, S., Otterbach, J. T., Robenek, I. R., Wolf, S. & Fuchsbauer, H.‐L. (1998) Eur. J. Biochem. 257, 570–576]. However, an activating protease could not be identified, probably because of the presence of a 14‐kDa protein (P14) belonging to the Streptomyces subtilisin inhibitor family. In contrast, if the microorganism was allowed to grow on a minimal medium, several soluble proteases were extracted, among them the TGase‐activating protease (TAMEP). TAMEP was purified by sequential chromatography on DEAE‐ and Arg‐Sepharose and used to determine the cleavage site of TGase. It was clearly shown that the peptide bond between Phe(−4) and Ser(−5) was hydrolyzed, indicating that at least one additional peptidase is necessary to complete TGase processing, even if TAMEP cleavage was sufficient to obtain total activity. Sequence analysis from the N‐terminus of TAMEP revealed the close relationship to a zinc endo‐protease from S. griseus. The S. griseus protease differs from other members of the M4 protease family, such as thermolysin, in that it may be inhibited by the Streptomyces subtilisin inhibitor. P14 likewise inhibits TAMEP in approximately equimolar concentrations, suggesting its important role in regulating TGase activity.