bluuebunny/crossref_metadata_2025 huggingface dataset:
Healthcare Heroes and COVID-19: Understanding Vaccine Perception and Practice in a Nigerian Tertiary Care Centre
Authors: Umenweke Chukwuemeka Chibuzo , Edeh Gabriel Chidera , Onwuaghamba Chidimma Praise , Umenweke Chisom Amara , Eze Cynthia , Emetu Chisom Christine , Chukwujekwu Chimdalu Obialunamma , Diogu Chizoba Jessica
Published: February 2025

Background: The COVID-19 pandemic underscored the critical role of vaccination in mitigating transmission and severe outcomes, particularly among high-risk groups like healthcare workers (HCWs). Healthcare workers have an important role to play in the success of COVID-19 vaccination programs as their practice and recommending the vaccines to the public is dependent on their perceptions towards the vaccine.
Methodology: This cross-sectional study assessed the perceptions, practices, and determinants of COVID-19 vaccination among HCWs at Nnamdi Azikiwe University Teaching Hospital (NAUTH), Nigeria, a pivotal site for national vaccination efforts. A stratified sampling method was used to recruit 260 HCWs, including doctors, nurses, paramedics, and administrative staff. Data were collected via validated questionnaires and analyzed using descriptive and inferential statistics.
Results: This survey revealed high awareness (98.8%) of COVID-19 vaccines, with 66.9% demonstrating a good perception of vaccine safety and efficacy. However, only 39.6% had received at least one vaccine dose, highlighting a stark perception-practice gap. Vaccination rates varied by profession. Doctors had the highest uptake (54.5%), while nurses exhibited the lowest (26.8%). Key motivators included civic responsibility (55.3%) and fear of COVID-19 (48.5%), whereas barriers centered on safety concerns (53.1%), perceived inadequate clinical trials (48.8%), and mistrust in institutions (50.4%). Some socio-demographic disparities were significant (p ≤ 0.05), with older, male, and tertiary-educated HCWs more likely to vaccinate.
Conclusion: Despite accessibility at NAUTH (71.5% availability), hesitancy persisted due to misinformation and fear of adverse effects. The findings advocate for targeted interventions addressing profession-specific concerns, enhanced public health communication, and community-driven advocacy to bridge the uptake gap. Strengthening transparency in vaccine development and addressing systemic mistrust are vital for achieving global immunization targets in resource-limited settings.
Breve Panorama sobre Instrumentos para Rastreio do Transtorno do Espectro Autista (TEA) em Adultos: Resultados de uma Revisão Integrativa
Authors: F.  A. Ferreira Lucas , N. Marques Louise , Salustiano Pedro , C. Souza Emanuelle , B. Benevides Silvia , G. B. Soares Artur , C. Maselli João Felippe , L.-Fernandez Jesus , Murray Christopher , Anunciação Luis
Published: February 2025

Autism spectrum disorder (ASD) is a complex clinical condition. Although less common, ASD diagnosis in adults has gained more attention, bringing new theoretical and clinical challenges. Screening tools are widely used to assist clinicians but few are available for this population; some have weak psychometric and statistical support and poor focus on behaviors more characteristic of autism in women. Thus, this study conducted an integrative review on the PubMed and SciELO databases to fi nd tools that were published from 2017 to 2023 in Portuguese or English. We found 189 studies, of which 16 met our inclusion criteria. These were grouped into two main domains: literature review studies on available tools and those that adapted international tools to the Brazilian context. We concluded that (a) few tools are currently available, (b) their psychometric evidence varied from inadequate to adequate, and (c) tools more targeted to the female population, especially in Brazil, remain lacking. This study discussed these results and recommended that screening tools complement the diagnostic process due to the need for new assessment instruments with robust psychometric properties.Keywords: Autism spectrum disorder, tools, integrative review, adult
Characterization of sulphydryl groups of the mitochondrial phosphate translocator by a maleimide spin label
Authors: Houstek J. , Bertoli E. , Stipani I. , Pavelka S. , Megli f.M. , Palmieri F.
Published: July 2002

A maleimide spin label strongly inhibits the phosphate/H+ symporter of rat liver mitochondria. While inducing half‐maximal inhibition of transport, the spin label reacts preferentially with the SH groups of the carrier, which are at least of two types. One type of SH group is localized close to the surface of the membrane and its environment does not significantly influence the mobility of the probe. The second type of SH group is buried in the membrane, is not accessible to ascorbate or chromium oxalate and its environment greatly restricts the motion of the probe.
The complete amino acid sequence of the major mammalian neurofilament protein (NF‐L)
Authors: Geisler Norbert , Plessmann Uwe , Weber Klaus
Published: July 2002

The first complete amino acid sequence of a neurofilament protein has been established. Porcine NF‐L contains 548 residues corresponding to a molecular mass of ~62 kDa. This value is noticeably smaller than the 68–72 kDa estimates from gel electrophoresis. Sequence comparison among the 6 non‐epithelial intermediate filament (IF) proteins of warm‐blooded vertebrates shows that the three NF proteins are the most remote members. Additionally and unexpectedly they reveal among each other lower sequence identity than the three non‐neuronal IF proteins GFAP, desmin, and vimentin where the last two are particularly closely related. Certain schemes of IF protein evolution are discussed.
Cloning of two β‐tubulin related genes in tetrahymena pyriformis
Authors: Barahona Isabel , Galego Lisete , Jacquet Michel , Rodrigues-Pousada Claudina
Published: July 2002

Genomic clones containing β‐tubulin sequences were isolated from a pBR322 genomic library of Tetrahymena pyriformis. Each clone has been mapped and the identity of the tubulin‐related genes has been established by cross‐hybridization with a cDNA containing a complete β‐tubulin coding region from Chlamydomonas and by the positive hybrid‐selected translation assay. The respective tubulin‐related genes, βTT1 and βTT2, differ from each other although they contain a homologous region which is also homologous with a heterologous probe. Tetrahymena β‐tubulin‐related genes are physically linked and clustered in the genome.
Amino acid sequence of β‐galactoside‐binding bovine heart lectin
Authors: Southan Christopher , Aitken Alastair , Childs Robert A. , Abbott William M. , Feizi Ten
Published: July 2002

A variety of animal tissues contain β‐galactoside‐binding lectins with molecular masses in the range 13–17 kDa. There is evidence that these lectins may constitute a new protein family although their function in vivo is not yet clear. In this work the major part of the amino acid sequence of the 13 kDa lectin from bovine heart muscle has been determined. Comparison of this sequence with the cDNA‐deduced sequence published for the chick embryo skin lectin showed 58% homology. Comparison of the bovine lectin sequence with partial sequences from two cDNA clones from a human hepatoma library and partial amino acid sequences of human lung lectin showed 70, 40 and 85% homology, respectively. The sequences of these vertebrate lectins are thus clearly related, supporting earlier results of immunological cross‐reactivity within this group of proteins. Computer searching of protein sequence databases did not detect significant homologies between the bovine lectin sequence and other known proteins.
Chronic exposure to dexamethasone induces hypomethylation of ornithine decarboxylase genes in a human myeloma cell line
Authors: Leinonen Pekka , Alhonen-Hongisto Leena , Laine Raija , Jänne Olli A. , Jänne Juhani
Published: July 2002

Chronic exposure of a human myeloma cell line to dexamethasone resulted in a selection of cells resistant to the growth‐inhibitory action of the glucocorticoid. Upon acute exposure of the parental myeloma cells to dexamethasone growth inhibition was associated with depression of ornithine decarboxylase (ODC, EC 4.1.1.17) activity. However, in cells adapted to grow in the presence of micromolar concentrations of dexamethasone, ODC activity was fully comparable to that in the parental cells. Restriction enzyme analyses with the two isoschizomers HpaII and MspI as well as with the methylation‐sensitive C⨍oI, indicated that the otherwise heavily methylated ODC gene(s) were rendered hypomethylated in the myeloma cells resistant to dexamethasone. This hypomethylation within and/or around ODC genes was associated with a 2–4‐fold enhancement of accumulation of ODC mRNA.
Glucagon‐like peptides activate hepatic gluconeogenesis
Authors: Mommsen Thomas P. , Andrews P.C. , Plisetskaya Erika M.
Published: July 2002

Piscine (anglerfish, catfish, coho salmon) glucagon‐like peptides (GLPs), applied at 3.5 nM, stimulate (1.1–1.9‐fold) flux through gluconeogenesis above control levels in isolated trout and salmon hepatocytes. Human GLP‐1 and GLP‐2 also activate gluconeogenesis, but to a lesser degree than their piscine counterparts. Minor increases of substrate oxidation are noticed at times of peak gluconeogenic activation through GLPs. These hormones, which are derived from the same precursor peptide as glucagon are more potent activators of gluconeogenesis than glucagon when applied at equimolar concentrations, and do not appear to employ cAMP or cGMP as the intracellular messenger in hepatic tissue.
Involvement of tryptase‐related cellular protease(s) in human immunodeficiency virus type 1 infection
Authors: Hattori Toshio , Koito Atsushi , Takatsuki Kiyoshi , Kido Hiroshi , Katunuma Nobuhiko
Published: July 2002

Trypstatin, a new cellular Kunitz‐type protease inhibitor purified from rat mast cells, inhibited syncytium formation in human immunodeficiency virus type 1 (HIV‐1)‐infected CCRF‐CEM and uninfected Molt‐4 clone 8 at a concentration of 1 μM. Anti‐rat tongue mast cell tryptase antibodies reacted with Molt‐4 clone 8 cells, as determined by Western blot and by immunofluorescence. In addition, the antibody inhibited syncytium formation. These findings along with homologous sequences with trypstatin and a neutralizing epitope of gp120 of HIV‐1 suggest that a tryptase‐like cellular enzyme(s) is involved in HIV‐1 infection.
Digital fluorescence imaging of fusion of influenza virus with erythrocytes
Authors: Georgiou George N. , Morrison Ian E.G. , Cherry Richard J.
Published: July 2002

Fusion of influenza virus with human erythrocytes at pH 5.2 was followed by fluorescence microscopy using a cooled slow‐scan CCD camera. The high sensitivity of the CCD permits repetitive digital imaging of the same cells with minimal photobleaching. The experimental conditions were such that only a small number of virus particles were adsorbed per cell. Quantitative analysis of the data indicated that for most cells only a single fusion event took place. This was, however, sufficient to cause haemolysis within 30 min at 20–22°C for about 60% of cells. There was a highly variable time lag between fusion and haemolysis. The lateral diffusion coefficient of virus particles on the cell surface when bound at pH 7.4 was < 2 × 10−13 cm2·s−1. The technique should be of value for more detailed studies of the dynamics of viral and other membrane fusion events.
Voltage‐dependent transient calcium currents in freshly dissociated capillary endothelial cells
Authors: Bossu J.-L. , Feltz A. , Rodeau J.-L. , Tanzi F.
Published: July 2002

Dissociated capillary endothelial cells display a voltage‐dependent Ca current activating around the resting potential. The initial transient component of the current corresponds to a Ca channel of the T type. Some cells also display a plateau component corresponding to a distinct dihydropyridine‐sensitive Ca channel. Depolarization induced by high external K+ elicits an increase in cytoplasmic Ca concentration. Confluent cells have been found to express the same Ca permeabilities.
The cold‐induced denaturation of lactate dehydrogenase at sub‐zero temperatures in the absence of perturbants
Authors: Hatley Ross H.M. , Franks Felix
Published: July 2002

Lactate dehydrogenase; Enzyme denaturation, cold; pH; Temperature; Cryosolvent
Fluorescence‐based continuous assay for the aspartyl protease of human immunodeficiency virus‐1
Authors: Geoghegan Kieran F. , Spencer Robin W. , Danley Dennis E. , Contillo Leonard G. , Andrews Glenn C.
Published: July 2002

5‐Dimethylaminonaphthalene‐1‐sulfonyl‐Ser‐Gln‐Asn‐Tyr‐Pro‐Ile‐Val‐Trp (Dns‐SQNYPIVW) is a fluorescent substrate for the aspartyl protease of human immunodeficiency virus‐1. In intact substrate, fluorescence of Trp (λex 290 nm, λem 360 nm) was 60% quenched by energy transfer to the dansyl group. Protease‐catalyzed cleavage at the Tyr‐Pro bond abolished the energy transfer, and the consequent increase in Trp fluorescence was used to follow the enzymatic reaction. At substrate concentrations <60 μM, initial reaction velocity increased as a linear function of substrate concentration, with k
cat/K
M= 9700 M−1s−1. Limited solubility and internal fluorescence quenching precluded a determination of K
M for Dns‐SQNYPIVW, but this was clearly > 100 μM.
The reactivity of hydrazine with photosystem II strongly depends on the redox state of the water oxidizing system
Authors: Messinger J. , Renger G.
Published: July 2002

The decay kinetics of the redox states S2 and S3 of the water‐oxidizing enzyme have been analyzed in isolated spinach thylakoids in the absence and presence of the exogenous reductant hydrazine. In control samples without NH2NH2 a biphasic decay is observed. The rapid decline of S2 and S3 with yD as reductant exhibits practically the same kinetics with t
1/2 = 6‐7 s at pH = 7.2 and 7°C. The slow reduction (order of 5‐10 min at 7°C) of S2 and S3 with endogenous electron donors other than yD is about twice as fast for S2 as for S3 under these conditions. In contrast, the hydrazine‐induced reductive shifts of the formal redox states Si (i = 0⋯3) are characterized by a totally different kinetic pattern: (a) at 1 mMNH2NH2 and incubation on ice the decay of S2 is estimated to be at least 25 times faster (t
1/2⩽0.4 min) than the corresponding reaction of S3 (t
1/2≈13 min); (b) the NH2NH2‐induced decay of S2 is even slower (about twice) than the transformation of S1 into the formal redox state ‘S−1’ (t
1/2≈6 min), which gives rise to the two‐digit phase shift of the oxygen‐yield pattern induced by a flash train in dark adapted thylakoids. (c) the NH2NH2‐induced transformation S0→‘S−2’ [Renger, Messinger and Hanssum (1990) in: Curr. Res. Photosynth. (Baltscheffsky, M., ed). Vol. 1, pp. 845‐848, Kluwer, Dordrecht] is about three times faster (t
1/2≈ min) than the reaction. Based on these results, the following dependence on the redox state Si of the reactivity towards NH2NH2 is obtained: S3 < S1 < S0 ⪡ S2. The implications of this surprising order of reactivity are discussed.
Ultraweak photoemission from dark‐adapted leaves and isolated chloroplasts
Authors: Hideg Èva , Kobayashi Masaki , Inaba Humio
Published: July 2002

Dark‐adapted isolated spinach chloroplasts and leaves, unlike sub‐chloroplast fractions, are capable of emitting ultraweak light spontaneously (50–125 counts per cm2). The emission of leaves is due to two processes with activation energies of 97 and 25 kJ/mol while in isolated chloroplasts, it is the result of a single process (98 kJ/mol), as indicated by the Arrhenius plots of the intensity. Emission spectra demonstrate that the terminal step of these reactions is the excitation of chlorophyll in both samples. We suggest that the additional component in the ultraweak light emission of leaves may be related to mitochondria
Identification of an additional gene belonging to the α2adrenergic receptor family in the human genome by PCR
Authors: Chhajlani Vijay , Rangel Ninfa , Uhlén Staffan , Wikberg Jarl E.S.
Published: July 2002

We here describe the cloning of an additional gene, called α2‐1.8, which is similar to the previously cloned human α2‐adrenergic receptor located on chromosome 4. The α2‐1.8 gene was identified by using the polymerase chain reaction with primers specific for sequences in transmembrane regions 2 and 5 of the previously isolated human α2‐C4 and α2‐C10 adrenoceptor genes, which are localized on chromosomes 4 and 10, respectively. The new gene was identified by amplifying the 1.8 kb size fractionated region ofPstI restriction cut human genomic DNA. The previously cloned α2‐C10 and α2‐C4 genes were recovered at their expected locations, 0.96 and 5.9 kb, respectively. We have identified 387 bases of the new α2‐1.8 gene, and its sequence is identical to the previously described α2‐C4 gene, but is distinct from the α2‐C10 and α2‐C2 genes. Our results demonstrate that the α2‐C4 adrenergic receptor exists in more than one copy in the human genome.
Evidence for the involvement of type II domains in collagen binding by 72 kDa type IV procollagenase
Authors: Bányai László , Patthy László
Published: July 2002

The fibronectin‐related region of the 72 kDa type IV procollagenase has been expressed in E. coli as a β‐galactosidase fusion product. The fragment containing the three type II units of the protein was found to have affinity for denatured collagen, suggesting that these domains may be responsible for the collagen‐affinity of type IV collagenase. We have also shown that segment Ala‐Ala‐His‐Glu of type IV collagenase (residues 372–375), which is similar to a fibronectin‐segment previously implicated in collagen‐binding, is not essential for binding activity.
Identification of the polypeptides of the major light‐harvesting complex of photosystem II (LHCII) with their genes in tomato
Authors: Green Beverley R. , Shen Dingren , Aebersold Ruedi , Pichersky Eran
Published: July 2002

Using an improved SDS‐PAGE system, the polypeptides of the major chlorophylla/blight‐harvesting complex of PSII (LHCII) from tomato leaves were resolved into five polypeptide bands. All the polypeptides were matched with the genes encoding them by comparing amino acid sequences of tryptic peptides with gene sequences. The two major LHCII bands (usually comigrating as a ‘27 kDa’ polypeptide) were encoded bycab1 andcab3 (Type I LHCII) genes. A third strong band or about 25 kDa was encoded bycab4 (Type II) genes. Polypeptides from two minor bands of 23–24 kDa were not N‐teminally blocked; their N‐terminal sequences showed they were Type III LHCII proteins. One complete cDNA clone and several incomplete clones for Type III polypeptides were sequenced. Combined with the peptide sequences, the results indicate that there are at least four different Type III genes in tomato, encoding four almost identical polypeptides. Thus, all the LHCII CAB polypeptides have been identified, and each type of LHCII polypeptide is encoded by distinct gene or genes in tomato.
Artifactual detection of ADP‐dependent sucrose synthase in crude plant extracts
Authors: Perata P. , Pozueta-Romero J. , Yamaguchi J. , Akazawa T.
Published: July 2002

Results presented in a previous report from this laboratory indicated the presence, in crude extracts from sycamore (Acer pseudoplatanus) and spinach (Spinacea oleracea), of a sucrose synthase (EC 2.4.1.13) showing high affinity for ADP as the glucose acceptor in the sucrose‐cleaving reaction. In the present paper we report that the modified enzymatic method previously used to measure sucrose synthase activities leads to the detection of artifactual ADP‐dependent sucrose synthase, which in fact arises from the combined action of invertase (EC 3.2.1.26) and nucleoside diphosphate kinase (EC 2.7.4.6) activities. We also present data on the partial purification of nucleoside diphosphate kinase from sycamore cells.
Identification of the mitochondrial receptor complex in Saccharomyces cerevisiae
Authors: Moczko Martin , Dietmeier Klaus , Söllner Thomas , Segui Bartolome , Steger Heinrich F. , Neupert Walter , Pfanner Nikolaus
Published: July 2002

Mitochondrial protein import involves the recognition of preproteins by receptors and their subsequent translocation across the outer membrane. In Neurospora crassa, the two import receptors, MOM19 and MOM72, were found in a complex with the general insertion protein, GIP (formed by MOM7, MOM8, MOM30 and MOM38) and MOM22. We isolated a complex out of S. cerevisiae mitochondria consisting of MOM38/ISP42, the receptor MOM72, and five new yeast proteins, the putative equivalents of N. crassa MOM7, MOM8, MOM19, MOM22 and MOM30. A receptor complex isolated out of yeast cells transformed with N. crassa MOM19 contained the N. crassa master receptor in addition to the yeast proteins. This demonstrates that the yeast complex is functional, and provides strong evidence that we also have identified the yeast MOM19.
Primary structure determination and cloning of the cDNA encoding toxin 4 of the scorpion Centruroides noxius Hoffmann
Authors: Vázquez A. , Becerril B. , Martin B.M. , Zamudio F. , Bolívar F. , Possani L.D.
Published: July 2002

A peptide (toxin II‐10), shown to be a Na+ channel blocker, was purified from the venom of the scorpion Centruroides noxius Hoffmann and sequenced by Edman degradation. It has 66 amino acid residues with the C‐terminal residue (asparagine) amidated, as demonstrated by mass spectrometry. In addition, we report the cloning and the nucleotide sequence of the cDNA (CngtV) that codes for this toxin. We discuss the mechanism for processing the precursor peptide to its final form and compare the primary structure to that of other Na− channel toxins. Two distinct groups of toxins seem to emerge from this comparison, suggesting a structure—function relationship of these peptides towards the recognition of either mammalian or insect tissues.
The characterisation of the shikimate pathway enzyme dehydroquinase fromPisum sativum
Authors: Deka Ranjit K. , Anton Ian A. , Dunbar Bryan , Coggins John R.
Published: July 2002

Peptides accounting for 157 residues of the bifunctional shikimate pathway enzyme, dehydroquinase/shikimate dehydrogenase, ofPisum sativumwere sequenced. Three of the peptides were homologous to regions inEscherichia colidehydroquinase and two toE. colishikimate dehydrogenase. The pea dehydroquinase activity was inhibited by treatment with dehydroquinate plus sodium borohydride, establishing it as a type I dehydroquinase. Synthetic oligonucleotides designed from the amino acid sequence were used as PCR primers to amplify fragments ofP. sativumcDNA. DNA sequence analysis showed that these amplified products were derived from dehydroquinase/shikimate dehydrogenase CDNA. The complete amino acid sequence of the dehydroquinase domain has been defined; it is homologous to all other type I dehydroquinases and is N‐terminal.
On the presence of phosphorylated sphingoid bases in rat tissues A mass‐spectrometric approach
Authors: Van Veldhoven Paul P. , De Ceuster Patrick , Rozenberg Raoul , Mannaerts Guy P. , de Hoffmann Edmond
Published: July 2002

A simple and straightforward procedure to analyze phosphorylated sphingoid bases has been developed. After phase separation of lipid extracts under alkaline conditions, the compounds were quantitatively recovered in the aqueous upper phase. Following a clean‐up of the aqueous phase on C18‐solid phase extraction columns, the amino‐group of the bases was derivatized by means of phenylisothiocyanate addition. FAB‐MS of the phenylthiocarbamate derivatives of sphingenine‐ and sphinganine‐phosphate in the negative mode revealed the expected pseudo‐molecular ions (M‐1) at 513 m/z and 515 m/z, respectively. Moreover, a typical fragmentation pattern, characterized by the loss of the phenylthiocarbamate moiety (m/z = 135), was observed. When applied to rat tissues, the presence of sphingenine‐phosphate in brain, kidney and liver could easily be demonstrated. Highest levels, amounting to 5 nmol/g of wet weight, were present in brain.
Expression of GATA‐binding transcription factors in rat hepatocytes
Authors: Matsuda Koichi , Kobune Yoshiko , Noda Chiseko , Ichihara Akira
Published: July 2002

Recently, we demonstrated that a DNA‐binding protein(s) is involved in transcriptional repression of the rat serine dehydratase gene in fetal liver. Here, we report that a GAT(A/T) motif is the target sequence for the DNA‐binding protein. By screening a fetal liver cDNA library, we isolated a rat homolog of GATA‐1. Rat GATA‐1 expressed as a GST‐fusion protein in E. coli bound to the GAT(A/T) motif in the serine dehydratase gene. Northern analysis show that GATA‐1 and GATA‐4 mRNAs are expressed in fetal hepatocytes.
Red edge excitation shifts of crystallins and intact lenses
Authors: Rao S.Chenchal , Rao Ch.Mohan
Published: July 2002

The shift that occurs in the fluorescence emission wavelength upon changing the excitation wavelength towards the red edge of the absorption band is termed red edge excitation shift (REES). We have monitored the REES of intrinsic protein fluorescence of freshly isolated intact lenses, of individual crystallins in their native, denatured and photodamaged states and also of crystallin mixtures. The observed REES values for the lenses from different species are different suggesting that the mobilities and packing of the crystallins may vary with the species. Lens photodamage in all the cases resulted in an increase of REES. Denaturation of crystallins in solution reduces REES and renaturation restores it. Mixtures of α‐ and β‐crystallins prepared either by directly mixing equimolar solutions or mixing them in 4 M urea followed by dialysis (reconstituting) gave similar REES values indicating the absence of any specific interactions in dilute solutions. Possible existence of induced alterations facilitating inter‐crystallin interactions at high protein concentration is suggested.
Regulation of the cloned L‐type cardiac calcium channel by cyclic‐AMP‐dependent protein kinase
Authors: Perez-Reyes Edward , Yuan Weilong , Wei Xiangyang , Bers Donald M.
Published: July 2002

Hormones can regulate cardiac L‐type Ca2+ channels via cAMP‐dependent protein kinase (PKA) phosphorylation. However, regulation of the cloned L‐type Ca2+ channel has been difficult to demonstrate conclusively. We stably transfected a human embryonic kidney (HEK‐293) cell with the cardiac α1 andβ2 subunits, then examined PKA modulation of the α2+ current. Although forskolin did not increase basal Ca2+ current, the PKA inhibitors, H‐89 and Rp‐cAMPS, could inhibit basal current. We reversed H‐89 inhibition with either forskolin or okadaic acid. We conclude that the channel was phosphorylated under basal conditions, and that inhibition of PKA allowed dephosphorylation. These studies demonstrate that reversible PKA regulation of cloned Ca2+ channels can be studied in HEK‐293 cells.
The Saccharomyces cerevisiae OXA1 gene is required for the correct assembly of cytochrome c oxidase and oligomycin‐sensitive ATP synthase
Authors: Altamura Nicola , Capitanio Nazareno , Bonnefoy Nathalie , Papa Sergio , Dujardin Geneviève
Published: July 2002

The nuclear gene OXA1 was first isolated in Saccharomyces cerevisiae and found to be required at a post‐translational step in cytochrome c oxidase biogenesis, probably at the level of assembly. Mutations in OXA1 lead to a complete respiratory deficiency. The protein Oxa1p is conserved through evolution and a human homolog has been isolated by functional complementation of a yeast oxa1−
mutant. In order to further our understanding of the role of Oxa1p, we have constructed two yeast strains in which the OXA1 open reading frame was almost totally deleted. Cytochrome spectra and enzymatic activity measurements show the absence of heme aa
3 and of a cytochrome c oxido‐reductase activity and dramatic decrease of the oligomycin sensitive ATPase activity. Analysis of the respiratory complexes in non‐denaturing gels reveals that Oxa1p is necessary for the correct assembly of the cytochrome c oxidase and the ATP synthase complex.
The outcome of placenta accreta in Kuwait (1981–1993)
Authors: Makhseed M. , Moussa M.A.
Published: July 2002

Objectives: To study the demographic characteristics of patients with placenta accreta and to identify the clinical features, maternal and neonatal complications of this condition. Methods: Sixteen cases of placenta accreta were identified in the Maternity Hospital of Kuwait during the period January 1981 to July 1993. Medical records were reviewed regarding past obstetric history, type of placenta, clinical presentation, maternal and fetal outcome. Results: The rate of placenta accreta was found to be 98 per 1 000 000 deliveries. Emergency hysterectomy was needed in 87.5% of cases. There was one maternal death (6.25%) and three perinatal deaths (18.75%). Hemorrhage was the major presenting symptom either externally and antenatally in previa accreta or postpartum in accreta of the upper segment or internally in the same cases. The major postoperative complications were coagulopathy, urinary injury, pelvic hematoma and abscess, in addition to cardiac arrest. These complications, when considered separately, were not affected by a previous history of uterine scarring. Conclusions: Placenta accreta is a major cause of obstetric hemorrhage and has an adverse effect on maternal and neonatal outcome. It ought to be considered a possibility in patients with previous uterine scarring, placenta previa or retained placenta. It will remain a growing problem in the developing countries due to the rising incidence of previous multiple cesarean sections. Unfortunately the latter do not deter women in this part of the world from insisting upon having big families and refusing tubal ligation.
Two carbohydrate recognition domains of Hyphantria cunea lectin bind to bacterial lipopolysaccharides through O‐specific chain
Authors: Shin Sang Woon , Park Doo-Sang , Kim Sun Chang , Park Ho-Yong
Published: July 2002

We previously identified a novel lectin cDNA from the fall webworm [Shin et al. (1998) Insect Biochem. Mol. Biol. 28, 827–837], which encodes two carbohydrate recognition domains (CRD‐N and CRD‐C) and is up‐regulated following bacterial challenge. The lipopolysaccharide (LPS) binding activities of the recombinant CRD‐N and CRD‐C (rCRD‐N and rCRD‐C) were investigated by enzyme‐linked immunosorbent assay. The LPS binding of rCRD‐N and rCRD‐C was pH‐dependent: at pH below 6.0, they show a higher binding ability to LPS. The binding of the rCRD‐N was inhibited by both D‐mannose and N‐acetyl‐D‐glucosamine, whereas the binding of the rCRD‐C was inhibited only by D‐mannose. The binding of both rCRD‐N and rCRD‐C to Escherichia coli was mainly mediated through the O‐specific chain.
The roles of Glu93 and Tyr149 in astacin‐like zinc peptidases
Authors: Yiallouros Irene , Große Berkhoff Eva , Stöcker Walter
Published: July 2002

The catalytic zinc of astacin, a prototype of the astacin family and the metzincin superfamily of metalloproteinases is coordinated by three histidines, a glutamate bound water and a tyrosine. In order to assess the roles of active site key residues, two mutants, Glu93Ala‐astacin and Tyr149Phe‐astacin, were expressed in Escherichia coli, affinity‐purified and renatured. While the Glu93Ala mutant was inactive, the Tyr149Phe mutant retained about 2.5% residual activity toward Dns‐Pro‐Lys‐Arg*Ala‐Pro‐Trp‐Val, based on the k
cat/K
m value for recombinant wild‐type astacin. These results support a model in which Glu93 is the general base in substrate hydrolysis, whereas Tyr149 contributes to transition state binding.
Characterization of two genes, Sig1 and Sig2, encoding distinct plastid σ factors1 in the moss Physcomitrella patens: phylogenetic relationships to plastid σ factors in higher plants
Authors: Hara Keishi , Morita Masakatsu , Takahashi Rieko , Sugita Mamoru , Kato Seiji , Aoki Setsuyuki
Published: July 2002

We isolated the cDNA for a σ factor from the moss Physcomitrella patens, which possesses unusually large N‐terminal extension and the conserved subdomains 1.2–4.2. Phylogenetic analyses indicated that this novel σ factor and PpSIG1*
2, a plastid σ factor previously identified from Physcomitrella, were classified into SigA and SigB groups, two major classes of higher plant plastid σ factors, respectively. According to the nomenclature recently proposed, we renamed PpSIG1* into PpSIG2, and named the novel σ factor PpSIG1. A transient expression assay using a green fluorescent protein showed that the N‐terminal region of PpSIG1 acts as a chloroplast‐targeting signal. Reverse transcription‐PCR experiments showed that light induces the expression of the Sig1 and Sig2 genes encoding PpSIG1 and PpSIG2, respectively. Thus, PpSIG1 and PpSIG2 are likely plastid σ factors regulating plastid gene expression in response to light signals.
The p80 TNF receptor‐associated kinase (p80TRAK) associates with residues 354–397 of the p80 cytoplasmic domain: similarity to casein kinase
Authors: Darnay Bryant G. , Singh Sanjaya , Aggarwal Bharat B.
Published: July 2002

The cytoplasmic domain of the p80 TNF receptor associates with a protein kinase, termed p80TRAK, that phosphorylates both the p60 and p80 TNF receptors. To determine the region of the cytoplasmic domain that is necessary for binding of p80TRAK and the region that it phosphorylates, a series of deletions of the p80 cytoplasmic domain were constructed and expressed as glutathione‐S‐transferase fusion proteins. These fusions were then used to examine the binding of p80TRAK derived from cellular extracts. We found that out of 174 residues (266–439) in the cytoplasmic domain of p80 receptor, 44 residues (354–397) were sufficient for binding of p80TRAK. Interestingly, this was also the region that contained the phosphorylation site for p80TRAK. Phosphoamino acid analysis of this region revealed phosphorylation primarily on serine residues. Furthermore, we found that, like p80TRAK, purified casein kinase 1 (CK1) also binds to residues 354–397 of the p80 TNF receptor and causes its phosphorylation. Additionally, the activity of p80TRAK was inhibited by CK1‐7, the CK1‐specific inhibitor. Thus, our results indicate that p80TRAK associates with a short stretch of ∼44 residues located in the cytoplasmic domain of the p80 TNF receptor and that this kinase is similar to CK1.
Production of formyl‐CoA during peroxisomal α‐oxidation of 3‐methyl‐branched fatty acids
Authors: Croes Kathleen , Van Veldhoven Paul P , Mannaerts Guy P , Casteels Minne
Published: July 2002

α‐Oxidation of 3‐methyl‐substituted fatty acids was studied in purified rat liver peroxisomes. The experiments revealed that formyl‐CoA is formed during the α‐oxidation process. The amount of formyl‐CoA found constituted 2–5% of the amount of formate formed. Under the conditions used, no activation of exogenously added formate occurred in purified peroxisomes, whereas 95.5% of added synthetic formyl‐CoA was converted to formate. These data indicate that during α‐oxidation first formyl‐CoA is formed, which is then hydrolysed to formate.
Activation of mitogen‐activated protein kinase by the nociceptin receptor expressed in Chinese hamster ovary cells
Authors: Fukuda Kazuhiko , Shoda Takehiro , Morikawa Hitoshi , Kato Shigehisa , Mori Kenjiro
Published: July 2002

Activation of the nociceptin receptor stably expressed in Chinese hamster ovary cells induced a transient mitogen‐activated protein kinase (MAPK) activation, via pertussis toxin‐sensitive G‐proteins. The nociceptin receptor‐mediated MAPK activation was partially blocked by down‐regulation or inhibition of protein kinase C, and suppressed by pretreatment with a phosphatidylcholine‐specific phospholipase C inhibitor, D609. Furthermore, a tyrosine protein kinase inhibitor, genistein, and phosphatidylinositol 3‐kinase inhibitors, wortmannin and LY294002, affected the nociceptin‐induced MAPK activity. The nociceptin‐induced MAPK activation may lead to activation of phospholipase A2 and induce changes in gene expression.
Expression of arachidonate platelet‐type 12‐lipoxygenase in human rheumatoid arthritis type B synoviocytes
Authors: Liagre Bertrand , Vergne Pascale , Rigaud Michel , Beneytout Jean L
Published: July 2002

In the present study, we have demonstrated platelet‐type 12‐lipoxygenase (12‐LOX) expression in human rheumatoid arthritis (RA) type B synoviocytes by reverse‐transcription polymerase chain reaction (RT‐PCR). The presence of 12‐LOX mRNA in these cells was revealed by classical RT‐PCR analysis using platelet‐type 12‐LOX cDNA primers and the PCR fragment (246 bp) was purified, amplified and sequenced. By sequence analysis, this fragment was determined to be 100% identical to that from platelet‐type 12‐LOX cDNA. Immunofluorescence data demonstrate that interleukin‐1β (IL‐1β) increases cellular 12‐LOX protein. Other results associate specific inflammatory cytokines with the activity of 12‐LOX in human RA type B synoviocytes. IL‐1β increased 12S‐hydroxy‐5,8,10,14‐eicosatetraenoic acid (12‐HETE) production (4‐fold) and we also observed an increase in 12‐HETE production (2.5‐fold) after incubation of human RA type B synoviocytes with TNFα. In contrast to the action of IL‐1β on 12‐HETE synthesis, IL‐4 and IL‐6 did not enhance 12‐HETE production. This is the first demonstration of platelet‐type 12‐LOX cDNA derived from the mRNA of cultured human RA type B synoviocytes.
Generation and characterization of transgenic mice expressing a human mutant α‐galactosidase with an R301Q substitution causing a variant form of Fabry disease
Authors: Shimmoto Michie , Kase Ryoichi , Itoh Kohji , Utsumi Kouichi , Ishii Satoshi , Taya Choji , Yonekawa Hiromichi , Sakuraba Hitoshi
Published: July 2002

Transgenic mice expressing a human mutant α‐galactosidase with an R301Q substitution, which was found in a patient with a variant form of Fabry disease, were established. The mice transcribed a sufficient amount of α‐galactosidase mRNA, but the steady‐state levels of the enzyme protein were decreased in liver, kidney and heart, only residual activity being detected in these tissues. The mice will be useful for the clarification of the defective regulation of the structurally altered enzyme protein expressed by the mutant gene at the organ or individual level as well as for the evaluation of drugs that stabilize and/or activate the mutant α‐galactosidase.
Developmental regulation of the plant mitochondrial matrix located HSP70 chaperone and its role in protein import
Authors: Dudley P , Wood C.K , Pratt J.R , Moore A.L
Published: July 2002

Changes in the level of the mitochondrial chaperone mtHSP70 have been investigated in pea (Pisum sativum) leaf mitochondria by Western blot analysis and quantified by scanning densitometry. As pea leaves develop (from 6 days to 30 days of age) the levels of mtHSP70 decrease. Analysis of the levels of the α subunit of the F1ATPase show that the levels of this protein remain constant throughout the same developmental period, whereas the levels of the alternative oxidase increase. In vitro import of the alternative oxidase precursor protein into pea leaf mitochondria from day 6 to day 30 leaves and quantification by scanning densitometry indicates that protein import efficiency decreases with increasing maturity of the plant cell. Results are discussed in terms of how changing levels of the mtHSP70 chaperone, as a result of plant cell development, influence the efficiency of protein import.
The promyelocytic leukemia protein PML has a pro‐apoptotic activity mediated through its RING domain
Authors: Borden Katherine L.B , CampbellDwyer Elizabeth J , Salvato Maria S
Published: July 2002

The promyelocytic leukemia protein PML is known to form nuclear multiprotein complexes which are compromised in several pathogenic conditions including acute promyelocytic leukemia. We show that in cells infected with a single stranded RNA virus, which relocates PML bodies to the cytoplasm, the infected cells are more resistant to serum starvation induced apoptosis than their uninfected counterparts. Antisense PML oligonucleotides increase cell survival under serum deprivation conditions indicating that PML is directly involved in the apoptotic activity. Transient transfection studies have indicated that this pro‐apoptotic activity of PML is mediated through the zinc binding region known as the RING finger. Viral attack of PML nuclear bodies appears to allow the virus to deregulate host cell apoptotic machinery in order to establish chronic infection.
Copolymers of N‐isopropylacrylamide can trigger pH sensitivity to stable liposomes
Authors: Meyer Olivier , Papahadjopoulos Demetrios , Leroux Jean-Christophe
Published: July 2002

Stable liposomes were rendered pH‐sensitive by complexation to a polymer that undergoes marked temperature‐ and pH‐dependent water solubility changes. The N‐isopropylacrylamide‐methacrylic acid copolymer was prepared with or without octadecyl acrylate. At pH below the phase transition of the polymer, egg phosphatidylcholine liposomes quickly released a part of their contents only when associated with the octadecyl aliphatic chain grafted polymer at 37°C. Similarly, sterically stabilized liposomes also quickly released a significant part of the entrapped fluorescent markers at pH 5.5–4.9, values corresponding to those of endosomes/lysosomes. This new pH‐sensitive liposome‐polymer system may further improve the efficiency of liposomal drug delivery.
Site directed mutants of Noxiustoxin reveal specific interactions with potassium channels
Authors: Martı́nez F. , Muñoz-Garay C. , Gurrola G. , Darszon A. , Possani L.D. , Becerril B.
Published: July 2002

Several site directed mutations were introduced into a synthetic Noxiustoxin (NTX) gene. Alanine scanning of the nonapeptide at the N‐terminal segment of NTX (threonine 1 (T1) to serine 9 (S9)) was constructed and the recombinant products were obtained in pure form. Additionally, lysine 28 (K28) was changed to arginine (R) or glutamic acid (E), cysteine 29 was changed to alanine, and residues 37–39 (Tyr‐Asn‐Asn) of the carboxyl end were deleted. The recombinant mutants were tested for their ability to displace 125I‐NTX from rat brain synaptosome membranes, as well as for their efficiency in blocking the activity of Kv1.1 K+ channels expressed in Xenopus laevis oocytes. The main results indicate that residues K6, T8 at the amino end, and K28 and the tripeptide YNN at the carboxyl end are involved in specific interactions of NTX with rat brain and/or Kv1.1 K+ channels.
How do histone acetyltransferases select lysine residues in core histones?
Authors: Kimura Akatsuki , Horikoshi Masami
Published: July 2002

Acetylation of specific lysines of core histone N‐terminal tails correlates with chromatin assembly and specific regulation of gene expression. Core histones acetylated at particular lysines mediate effects on chromatin function; however, the manner in which different histone acetyltransferases (HATs) discriminate lysines is unknown. Here we propose a putative rule for lysine selection by HATs based on the primary sequence in the vicinity of lysines in the core histone N‐terminal tails and in flanking sequence. This provides insight into the molecular basis of site selection of core histones by HATs.
Conformational analysis of the endogenous μ‐opioid agonist endomorphin‐1 using NMR spectroscopy and molecular modeling
Authors: Podlogar Brent L. , Paterlini M.Germana , Ferguson David M. , Leo Gregory C. , Demeter David A. , Brown Frank K. , Reitz Allen B.
Published: July 2002

Endomorphin‐1 (Tyr‐Pro‐Trp‐Phe‐NH2) is a highly selective and potent agonist of the μ‐opioid receptor. To identify structural attributes unique to this opioid peptide and potential sites of recognition, a conformational analysis has been performed using multidimensional NMR and molecular modeling techniques. The spectroscopic results, derived from experiments in both DMSO and water, indicate that endomorphin‐1 exists in the cis‐ and trans‐configuration with respect to the Pro‐omega bond in approximately 25% and 75% populations, respectively. In DMSO, the cis‐configuration adopts a compact sandwich conformation in which the Tyr and Trp aromatic rings pack against the proline ring, whereas the trans‐configuration adopts an extended conformation. Although non‐random structure was not observed in water, condensed phase molecular dynamics calculations indicate that trans‐isomers dominate the population in this higher dielectric medium. Structural comparison of the cis‐ and trans‐configurations with morphine and selective μ‐peptide ligands PL‐017 and d‐TIPP, as well as the δ‐selective peptide ligands TIPP (δ‐antagonist, μ‐agonist) and DPDPE were also performed and suggest the trans‐isomer is likely the bioactive form. A hypothesis is proposed to explain μ‐ and δ‐selectivity based on the presence of spatially distinct selectivity pockets among these ligands.
The dermaseptin precursors: a protein family with a common preproregion and a variable C‐terminal antimicrobial domain
Authors: Amiche Mohamed , Seon Aurelia Anne , Pierre Thierry Nicolas , Nicolas Pierre
Published: July 2002

Preprodermaseptins are a group of antimicrobial peptide precursors found in the skin of a variety of frog species. Precursors of this family have very similar N‐terminal preprosequences followed by markedly different C‐terminal domains that correspond to mature antimicrobial peptides. Some of these peptides are 24–34 amino acids long and form well‐behaved amphipathic α‐helices, others are disulfide‐linked peptides of 20–46 residues, still others, highly hydrophobic, are the smallest antimicrobial peptides known so far being only 10–13 residues in length. All these peptides are broad‐spectrum microbicides that kill many bacteria, protozoa, yeasts and fungi by destroying or permeating the microbial membrane. In frogs belonging to the genus Phyllomedusinae, preprodermaseptins encoded peptides also include dermorphins and deltorphins, D‐amino acid‐containing heptapeptides which are very potent and specific agonists of the μ‐ or δ‐opioid receptors. The remarkable similarity between preproregions of precursors that give rise to peptides with very different primary structures, conformations and activities suggests that the corresponding genes originate from a common ancestor. The high conservation of the precursor prepropart indicates that this region must have an important function.
Arabidopsis knockout mutation of ADC2 gene reveals inducibility by osmotic stress
Authors: Soyka Stephan , Heyer Arnd G.
Published: July 2002

We isolated an Arabidopsis thaliana mutant line carrying an insertion of the En‐1 transposable element at the ADC2 locus. The insertion causes a knockout of the arginine decarboxylase 2 gene. We demonstrated that ADC2 is the gene responsible for induction of the polyamine biosynthetic pathway by osmotic stress. No induction of ADC activity by the osmolite sorbitol could be observed in the homozygous mutant, indicating a predominant role of ADC2 in stress response. ADC activity is reduced in the mutant by 44% under non‐stressed conditions and the mutant shows no obvious phenotype. This is the first report of a genetically mapped mutation in the polyamine biosynthetic pathway in plants.
CRTH2, an orphan receptor of T‐helper‐2‐cells, is expressed on basophils and eosinophils and responds to mast cell‐derived factor(s)
Authors: Nagata Kinya , Hirai Hiroyuki , Tanaka Kazuya , Ogawa Kazuyuki , Aso Takeshi , Sugamura Kazuo , Nakamura Masataka , Takano Shoichi
Published: July 2002

We have recently cloned a putative chemoattractant receptor, named CRTH2, which is preferentially expressed on human T‐helper‐ (Th) 2 but not Th1 cells. In this study, we demonstrated that CRTH2 is also highly expressed on peripheral blood basophils and eosinophils. Our search for a CRTH2 ligand identified mast cells as the possible producers of a ligand. When stimulated with an anti‐FcϵR1 antibody, cord blood‐derived mast cells secreted factor(s) that induced Ca2+ mobilization in CRTH2‐expressing K562 cells but not in mock transfected cells. These findings implied the involvement of CRTH2 in mast cell‐mediated immune responses such as allergic reactions.