bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Diagnostics of Adult Gliomas in Neuropathological Practice
Authors: Brandner Sebastian
Published: May 2021

This review focuses on adult gliomas, highlighting the most relevant biomarkers in the diagnosis of these tumours and the use of DNA methylation arrays to complement conventional molecular diagnostic techniques. The discovery and characterisation of diagnostic and prognostic biomarkers in brain tumours has significantly changed the neuropathological landscape over the last decade. These include mutations in the IDH1 and IDH2 genes in astrocytomas and oligodendrogliomas, histone H3 K27M mutations in midline gliomas, or BRAF mutations in a range of low-grade and high-grade glial and glioneuronal tumours. Other biomarkers of relevance are mutations in the TERT promoter, the ATRX gene, and genomic alterations such as 1p/19q codeletion, EGFR amplification, and chromosome 7 gain and 10 loss. The development of DNA methylation profiling and algorithmic classification of brain tumours has further enhanced the diagnostic abilities of neuropathologists. Methylation profiling is particularly useful for the diagnostic workup of biopsies with an inconclusive molecular test results, small samples, or samples with indistinctive low-grade or high-grade histology. This technology has become indispensable for the risk stratification of ependymal tumours, medulloblastomas and meningiomas.

Conclusion. This review highlights the importance of an integrated approach to brain tumour diagnostics and gives a balanced view of the relevance and choice of conventional and molecular techniques in the workup of adult gliomas in diagnostic neuropathology practice.

 
Genetics of Prostate Carcinoma
Authors: Krušlin Božo , Škara Lucija , Vodopić Tonći , Vrhovec Borna , Murgić Jure , Štimac Goran , Fröbe Ana , Lež Cvjetko , Ulamec Monika , Gall-Trošelj Koraljka
Published: May 2021

The aim of this review is to provide a brief overview of some current approaches regarding diagnostics, pathologic features, treatment, and genetics of prostate carcinoma (PCa). Prostate carcinoma is the most common visceral tumor and the second most common cancer-related cause of death in males. Clinical outcomes for patients with localized prostate cancer are excellent, but despite advances in prostate cancer treatments, castrate-resistant prostate cancer and metastatic prostate cancer patients have a poor prognosis. Advanced large-scale genomic studies revealed a large number of genetic alterations in prostate cancer. The meaning of these alterations needs to be validated in the specific prostate cancer molecular subtype context. Along these lines, there is a critical need for establishing genetically engineered mouse models, which would include speckle type BTB/POZ protein and isocitrate Dehydrogenase (NADP (+)) 1 mutant, as well as androgen receptor neuroendocrine subtypes of prostate cancer. Another urgent need is developing highly metastatic prostate cancer models, as only up to 17% of available models dis- play bone metastases and exhibit a less typical neuroendocrine prostate cancer or sarcomatoid carcinoma. Moreover, androgen deprivation and relapse should be mimicked in the genetically engineered mouse models, as androgen independence may yield a better model for metastatic castrate-resistant prostate cancer. The development of such refined animal models should be guid- ed by comparative genomics of primary versus corresponding metastatic tumors. Such an approach will have the potential to illuminate the key genetic events associated with specific molecular prostate cancer subsets and indicate directions for effective therapy.

Conclusion. Despite excellent results in the treatment of localized prostatic carcinoma, castrate-resistant prostate can- cer and metastatic prostate cancer have a poor prognosis. Advanced large-scale genomic studies revealed a large number of ge- netic alterations in PCa. Experimental models of prostate carcinoma in genetically modified mice could provide new data about the genetic changes in such cancers and help in developing better animal models for treatment resistant prostate carcinomas.
Classic Hodgkin Lymphoma – Old Disease, New Directions: An Update on Pathology, Molecular Features and Biological Prognostic Markers
Authors: Perry Anamarija M. , Smith Lauren B. , Bagg Adam
Published: May 2021

The aim of this paper is to review morphologic, immunophenotypic, and molecular features of classic Hodgkin lymphoma, as well as different prognostic markers in this neoplasm. Classic Hodgkin lymphoma (CHL) accounts for 15-25% of all lymphomas in the Western world. The hallmark of this disease is the neoplastic Hodgkin/Reed-Sternberg (HRS) cell, which is favored to be derived from germinal center B-cells but has lost many of the B-cell markers. HRS cells are scattered within a dense inflammatory infiltrate, and through a network of cytokines and chemokines they shape their microenvironment, evade immune response, survive, and grow. In the last two decades multiple prognostic markers related to HRS cells, the microenvironment or both, have been evaluated in patients with CHL. They include clinical, immunohistochemical, cytogenetic, and molecular markers that can predict survival and identify high-risk patients who will likely relapse after therapy. More recently, circulating tumor DNA analysis by next-generation sequencing has opened new avenues for diagnosis and disease monitoring after therapy. The increased understanding of molecular mechanisms underlying CHL pathogenesis has led to successful implementation of novel therapies, such as anti-PD-1 antibodies, which are becoming a mainstay of treatment in relapsed/refractory patients.

Conclusion. Currently, pathologic prognostic markers are not routinely assessed at initial diagnosis of CHL. However, as more therapies become available, it will be important to identify patients with high-risk disease who may benefit from more intense or targeted therapy upfront.
Kornelija Rakić: A Woman Doctor for Women and Children in Serbia and Bosnia and Herzegovina
Authors: Tahirović Husref , Fuchs Brigitte
Published: May 2021

This short biography focuses on the life and medical activities of Kornelija Rakić (1879–1952), a Serbian female pioneer of medicine from the then Hungarian province of Vojvodina, who acquired an MD from the University of Budapest in 1905. Rakić came from a humble background, and a Vojvodina Serbian women’s organization enabled her to become a physician and pursue her social medicine mission. After a futile attempt to open a private practice as a “woman doctor for women” in Novi Sad in 1906, she successfully applied to the Austro-Hungarian provincial government in Sarajevo for the position of an official female physician in occupied Bosnia. Rakić began her career as an Austro-Hungarian (AH) official female physician in Bihać (1908–1912) and was transferred to Banja Luka in 1912 and to Mostar in 1917–1918. Kornelija Rakić stayed in Mostar after the monarchy collapsed in 1918 and continued to work as a public health officer in the service of the Kingdom of Serbs, Croats and Slovenes, founded in 1918. Subsequently, she served as the head of the “dispensary for mothers and children” at the Public Health Centre in Mostar, founded in 1929, where she practiced until her retirement in 1949. After World War II, Rakić served as Vice President of the Red Cross Society in Mostar. She received numerous awards and medals from the Austro-Hungarian Empire, the Kingdom of Yugoslavia and the Federal People’s Republic of Yugoslavia. Kornelija Rakić died in Mostar in 1952 and was buried at the local Orthodox cemetery of Bjelušine.

 Conclusion. Kornelija Rakić (1879–1952) was the first Serbian female physician in Novi Sad, Vojvodina, and she was employed as an AH official female physician in Bihać (1908–1912), Banja Luka (1912–1917) and Mostar (1917–1918). After World War I, she participated in the establishment and expansion of public health institutions in Mostar and Herzegovina from 1918–1949 against the backdrop of the devastation of the two World Wars.
Staphylococcus saprophyticus and Escherichia coli: Tracking from sperm fertility potential to assisted reproductive outcomes
Authors: Ghasemian Fatemeh , Esmaeilnezhad Shahin , Mehdipour Moghaddam Mohammad Javad
Published: May 2021

Objective: Bacteriospermia and urogenital infections are common problems in male infertility. This study aimed to evaluate the effects of bacteriospermia on sperm parameters and clinical outcomes in semen samples infected with two common bacteria (Staphylococcus saprophyticus and Escherichia coli) in northern Iran.Methods: Microbiological tests were performed to isolate and identify organisms from 435 semen samples from infertile couples. Semen samples were assessed according to the World Health Organization criteria. The protamine status, chromatin structure, chromatin condensation, and acrosome reaction of sperm and assisted reproductive outcomes were determined in couples with different male infertility factors.Results: Among the total cases, the two most prevalent pathogens were considered: S. saprophyticus (38.2%) and E. coli (52.9%). In the semen samples infected with E. coli, the spontaneous acrosome reaction and abnormal chromatin condensation were more common (p<0.05). Significant increases in abnormal chromatin condensation and deprotamination were seen in the presence of S. saprophyticus. In washed semen, tight adhesion between the sperm midpiece and S. saprophyticus was observed. There was also a significant decrease in the fertilization rate using semen samples infected with S. saprophyticus and E. coli during in vitro fertilization cycles (p<0.001). In addition, the presence of S. saprophyticus and E. coli in semen samples was associated with a lower likelihood of clinical pregnancy in couples with various factors of male infertility.Conclusion: Poor results of assisted reproductive techniques may be correlated with semen samples infected with two common bacteria in northern Iran.
Der Ausschluss von Nachträgen in Pauschalpreisbauverträgen nach polnischem Recht
Authors: Barczewski Artur
Published: May 2021

Why go construction companies bankrupt on a mass scale in Poland? This question forms the starting point for an analysis of the phenomenon of construction insolvencies. The author sees the main cause for the undesirable developments in the specific design of Polish law. On the one hand there are only few requirements regarding construction design. As a consequence, construction projects are insufficiently planned. On the other hand, most construction contracts are lump sum contracts. The lump sum encompasses all necessary works. The author criticises the particular imbalance of the regulations, defines limits of permissible risk assumptions and prepares a proposal for a more balanced legislation.
¿Perciben los hablantes de español como lengua extranjera la lengua emocional igual que los nativos?
Authors: Masid Blanco Ocarina , Pérez Serrano Mercedes , Martín Leralta Susana
Published: May 2021

La valencia (cómo de positivo o negativo resulta un estímulo) y la activación (intensidad con la que se recibe un estímulo) se han reconocido como los dos ejes principales que articulan el componente afectivo. Por otro lado, se sabe que la lengua de la emoción es altamente figurativa y que la carga emocional que tienen las palabras en la L1 frente a la que tienen en las lenguas que se aprenden después no es la misma. Teniendo esto en cuenta, el objetivo de este estudio es comparar la forma en que perciben los hablantes nativos y no nativos la lengua emocional en términos de valencia y activación, de cara a determinar si existen diferencias entre la lengua figurativa y la literal. Para ello se encuestó a un total de 62 informantes (usuarios de español como L1 y sinohablantes usuarios de español como LX) acerca de su percepción de palabras en sentido literal y sus equivalentes figurados. Nuestros resultados muestran que los ítems figurados generan más activación que los literales en ambos grupos y que la activación ante los estímulos es más alta en L1. Los resultados contribuyen a la investigación abierta sobre la expresión de las emociones en relación con el aprendizaje de una nueva lengua y la aculturación emocional.
The pattern of nodal morphogen signaling is shaped by co-receptor expression
Authors: Lord Nathan D , Carte Adam N , Abitua Philip B , Schier Alexander F
Published: May 2021

Embryos must communicate instructions to their constituent cells over long distances. These instructions are often encoded in the concentration of signals called morphogens. In the textbook view, morphogen molecules diffuse from a localized source to form a concentration gradient, and target cells adopt fates by measuring the local morphogen concentration. However, natural patterning systems often incorporate numerous co-factors and extensive signaling feedback, suggesting that embryos require additional mechanisms to generate signaling patterns. Here, we examine the mechanisms of signaling pattern formation for the mesendoderm inducer Nodal during zebrafish embryogenesis. We find that Nodal signaling activity spans a normal range in the absence of signaling feedback and relay, suggesting that diffusion is sufficient for Nodal gradient formation. We further show that the range of endogenous Nodal ligands is set by the EGF-CFC co-receptor Oep: in the absence of Oep, Nodal activity spreads to form a nearly uniform distribution throughout the embryo. In turn, increasing Oep levels sensitizes cells to Nodal ligands. We recapitulate these experimental results with a computational model in which Oep regulates the diffusive spread of Nodal ligands by setting the rate of capture by target cells. This model predicts, and we confirm in vivo, the surprising observation that a failure to replenish Oep transforms the Nodal signaling gradient into a travelling wave. These results reveal that patterns of Nodal morphogen signaling are shaped by co-receptor-mediated restriction of ligand spread and sensitization of responding cells.
No relationship between frontal alpha asymmetry and depressive disorders in a multiverse analysis of five studies
Authors: Kołodziej Aleksandra , Magnuski Mikołaj , Ruban Anastasia , Brzezicka Aneta
Published: May 2021

For decades, the frontal alpha asymmetry (FAA) – a disproportion in EEG alpha oscillations power between right and left frontal channels – has been one of the most popular measures of depressive disorders (DD) in electrophysiology studies. Patients with DD often manifest a left-sided FAA: relatively higher alpha power in the left versus right frontal lobe. Recently, however, multiple studies failed to confirm this effect, questioning its reproducibility. Our purpose is to thoroughly test the validity of FAA in depression by conducting a multiverse analysis – running many related analyses and testing the sensitivity of the effect to changes in the analytical approach – on data from five independent studies. Only 13 of the 270 analyses revealed significant results. We conclude the paper by discussing theoretical assumptions underlying the FAA and suggest a list of guidelines for improving and expanding the EEG data analysis in future FAA studies.
Phenotypic plasticity underlies local invasion and distant metastasis in colon cancer
Authors: Sacchetti Andrea , Teeuwssen Miriam , Verhagen Mathijs , Joosten Rosalie , Xu Tong , Stabile Roberto , van der Steen Berdine , Watson Martin M , Gusinac Alem , Kim Won Kyu , Ubink Inge , Van de Werken Harmen JG , Fumagalli Arianna , Paauwe Madelon , Van Rheenen Jacco , Sansom Owen J , Kranenburg Onno , Fodde Riccardo
Published: May 2021

Phenotypic plasticity represents the most relevant hallmark of the carcinoma cell as it bestows it with the capacity of transiently altering its morphological and functional features while en route to the metastatic site. However, the study of phenotypic plasticity is hindered by the rarity of these events within primary lesions and by the lack of experimental models. Here, we identified a subpopulation of phenotypic plastic colon cancer cells: EpCAMlo cells are motile, invasive, chemo-resistant, and highly metastatic. EpCAMlo bulk and single-cell RNAseq analysis indicated (1) enhanced Wnt/β-catenin signaling, (2) a broad spectrum of degrees of epithelial to mesenchymal transition (EMT) activation including hybrid E/M states (partial EMT) with highly plastic features, and (3) high correlation with the CMS4 subtype, accounting for colon cancer cases with poor prognosis and a pronounced stromal component. Of note, a signature of genes specifically expressed in EpCAMlo cancer cells is highly predictive of overall survival in tumors other than CMS4, thus highlighting the relevance of quasi-mesenchymal tumor cells across the spectrum of colon cancers. Enhanced Wnt and the downstream EMT activation represent key events in eliciting phenotypic plasticity along the invasive front of primary colon carcinomas. Distinct sets of epithelial and mesenchymal genes define transcriptional trajectories through which state transitions arise. pEMT cells, often earmarked by the extracellular matrix glycoprotein SPARC together with nuclear ZEB1 and β-catenin along the invasive front of primary colon carcinomas, are predicted to represent the origin of these (de)differentiation routes through biologically distinct cellular states and to underlie the phenotypic plasticity of colon cancer cells.
The molecular coupling between substrate recognition and ATP turnover in a AAA+ hexameric helicase loader
Authors: Puri Neha , Fernandez Amy J , O'Shea Murray Valerie L , McMillan Sarah , Keck James L , Berger James M
Published: May 2021

In many bacteria and eukaryotes, replication fork establishment requires the controlled loading of hexameric, ring-shaped helicases around DNA by AAA+(ATPases Associated with various cellular Activities) ATPases. How loading factors use ATP to control helicase deposition is poorly understood. Here, we dissect how specific ATPase elements ofEscherichia coliDnaC, an archetypal loader for the bacterial DnaB helicase, play distinct roles in helicase loading and the activation of DNA unwinding. We have identified a new element, the arginine-coupler, which regulates the switch-like behavior of DnaC to prevent futile ATPase cycling and maintains loader responsiveness to replication restart systems. Our data help explain how the ATPase cycle of a AAA+-family helicase loader is channeled into productive action on its target; comparative studies indicate that elements analogous to the Arg-coupler are present in related, switch-like AAA+ proteins that control replicative helicase loading in eukaryotes, as well as in polymerase clamp loading and certain classes of DNA transposases.
An excitatory lateral hypothalamic circuit orchestrating pain behaviors in mice
Authors: Siemian Justin N , Arenivar Miguel A , Sarsfield Sarah , Borja Cara B , Erbaugh Lydia J , Eagle Andrew L , Robison Alfred J , Leinninger Gina , Aponte Yeka
Published: May 2021

Understanding how neuronal circuits control nociceptive processing will advance the search for novel analgesics. We use functional imaging to demonstrate that lateral hypothalamic parvalbumin-positive (LHPV) glutamatergic neurons respond to acute thermal stimuli and a persistent inflammatory irritant. Moreover, their chemogenetic modulation alters both pain-related behavioral adaptations and the unpleasantness of a noxious stimulus. In two models of persistent pain, optogenetic activation of LHPV neurons or their ventrolateral periaqueductal gray area (vlPAG) axonal projections attenuates nociception, and neuroanatomical tracing reveals that LHPV neurons preferentially target glutamatergic over GABAergic neurons in the vlPAG. By contrast, LHPV projections to the lateral habenula regulate aversion but not nociception. Finally, we find that LHPV activation evokes additive to synergistic antinociceptive interactions with morphine and restores morphine antinociception following the development of morphine tolerance. Our findings identify LHPV neurons as a lateral hypothalamic cell type involved in nociception and demonstrate their potential as a target for analgesia.
Beware the algorithm
Authors: van Endert Peter
Published: May 2021

Spliced peptides present on tumor cells can help to mount an immune response, but algorithms offer limited help in predicting which ones actually exist and perform this role in vivo.
dxpr: an R package for generating analysis-ready data from electronic health records—diagnoses and procedures
Authors: Tseng Yi-Ju , Chiu Hsiang-Ju , Chen Chun Ju
Published: May 2021

Background

Enriched electronic health records (EHRs) contain crucial information related to disease progression, and this information can help with decision-making in the health care field. Data analytics in health care is deemed as one of the essential processes that help accelerate the progress of clinical research. However, processing and analyzing EHR data are common bottlenecks in health care data analytics.

Methods

The dxpr R package provides mechanisms for integration, wrangling, and visualization of clinical data, including diagnosis and procedure records. First, the dxpr package helps users transform International Classification of Diseases (ICD) codes to a uniform format. After code format transformation, the dxpr package supports four strategies for grouping clinical diagnostic data. For clinical procedure data, two grouping methods can be chosen. After EHRs are integrated, users can employ a set of flexible built-in querying functions for dividing data into case and control groups by using specified criteria and splitting the data into before and after an event based on the record date. Subsequently, the structure of integrated long data can be converted into wide, analysis-ready data that are suitable for statistical analysis and visualization.

Results

We conducted comorbidity data processes based on a cohort of newborns from Medical Information Mart for Intensive Care-III (n = 7,833) by using the dxpr package. We first defined patent ductus arteriosus (PDA) cases as patients who had at least one PDA diagnosis (ICD, Ninth Revision, Clinical Modification [ICD-9-CM] 7470*). Controls were defined as patients who never had PDA diagnosis. In total, 381 and 7,452 patients with and without PDA, respectively, were included in our study population. Then, we grouped the diagnoses into defined comorbidities. Finally, we observed a statistically significant difference in 8 of the 16 comorbidities among patients with and without PDA, including fluid and electrolyte disorders, valvular disease, and others.

Conclusions

This dxpr package helps clinical data analysts address the common bottleneck caused by clinical data characteristics such as heterogeneity and sparseness.
New insight into the divergent responses of plants to warming in the context of root endophytic bacterial and fungal communities
Authors: Wei Xiaoting , Jiang Fengyan , Han Bing , Zhang Hui , Huang Ding , Shao Xinqing
Published: May 2021

Plant adaptation under climate changes is critical to the maintenance of terrestrial ecosystem structure and function. Studying the response of the endophytic community to climate warming is a novel way to reveal the mechanism of host environmental adaptability because of the prominent role endophytes play in host nutrient acquisition and stress tolerance. However, host performance was generally neglected in previous relevant research, which limits our understanding of the relationships between the endophytic community and host responses to climate warming. The present study selected two plants with different responses to climate warming.Elymus nutansis more suitable for growing in warm environments at low altitude compared toKobresia pygmaea.K. pygmaeaandE. nutanswere sampled along an altitude gradient in the natural grassland of Qinghai-Tibet Plateau, China. Root endophytic bacterial and fungal communities were analyzed using high throughput sequencing. The results revealed that hosts growing in more suitable habitats held higher endophytic fungal diversity. Elevation and host identity significantly affected the composition of the root endophytic bacterial and fungal community. 16S rRNA functional prediction demonstrated that hosts that adapted to lower temperatures recruited endophytic communities with higher abundance of genes related to cold resistance. Hosts that were more suitable for warmer and drier environments recruited endophytes with higher abundance of genes associated with nutrient absorption and oxidation resistance. We associated changes in the endophytic community with hosts adaptability to climate warming and suggested a synchronism of endophytic communities and hosts in environmental adaptation.
Integrative species delimitation based on COI, ITS, and morphological evidence illustrates a unique evolutionary history of the genus Paracercion (Odonata: Coenagrionidae)
Authors: Zhang Haiguang , Ning Xin , Yu Xin , Bu Wen-Jun
Published: May 2021

Paracercion are common ‘blue and black’ colored damselflies. We explore the species boundaries of Paracercion (Odonata: Coenagrionidae) using ABGD, bPTP, GMYC and Distance-based clustering. We finally got the molecular data of all nine species of Paracercion. P. hieroglyphicum and P. melanotum were combined into one putative species based on cytochrome c oxidase I (COI). However, they were separated into two putative species based on the nuclear segment including ITS1-5.8S-ITS2 (ITS). This suggests the introgression of mtDNA in Paracercion. Paracercion barbatum and Paracercion melanotum can be separated into two species based on COI, whereas they were combined into one putative species based on ITS, which suggests a hybridization event between them. The lower interspecific divergence (COI: 0.49%) between P. barbatum and Paracercion v-nigrum indicates a recent speciation event in Paracercion. Paracercion sieboldii and P. v-nigrum can be separated into two putative species based on COI, while they were frequently merged into the same putative species based on ITS. This can be explained by incomplete lineage sorting in nDNA. Besides, P. pendulum and P. malayanum were synonymized as junior synonyms of P. melanotum. P. luzonicum was confirmed not to belong to Paracercion. The possibility of introgression, hybridization, recent speciation and incomplete lineage sorting makes species delimitation, based on molecular data, difficult and complicates understanding of the evolutionary history of Paracercion. The discordance in COI and ITS also indicates the value of using markers from different sources in species delimitation studies.
The GRAS gene family in watermelons: identification, characterization and expression analysis of different tissues and root-knot nematode infestations
Authors: Lv Gongbo , Zheng Xing , Duan Yitian , Wen Yunyong , Zeng Bin , Ai Mingqiang , He Bin
Published: May 2021

The family of GRAS plant-specific transcription factor plays diverse roles in numerous biological processes. Despite the identification and characterization of GRAS genes family in dozens of plant species, until now, GRAS members in watermelon (Citrullus lanatus) have not been investigated comprehensively. In this study, using bioinformatic analysis, we identified 37 GRAS genes in the watermelon genome (ClGRAS). These genes are classified into 10 distinct subfamilies based on previous research, and unevenly distributed on 11 chromosomes. Furthermore, a complete analysis was conducted to characterize conserved motifs and gene structures, which revealed the members within same subfamily that have analogous conserved gene structure and motif composition. Additionally, the expression pattern of ClGRAS genes was characterized in fruit flesh and rind tissues during watermelon fruit development and under red light (RL) as well as root knot nematode infestation. Finally, for verification of the availability of public transcriptome data, we also evaluated the expression levels of randomly selected four ClGRAS genes under RL and nematode infection by using qRT-PCR. The qRT-PCR results indicated that several ClGRAS genes were differentially expressed, implying their vital role in RL induction of watermelon resistance against root-knot nematodes. The results obtained in this study could be useful in improving the quality of watermelon.
Molecular sizes of photolabeled GABA and benzodiazepine receptor proteins are identical
Authors: Asano Tomiko , Sakakibara Jinsaku , Ogasawara Nobuaki
Published: July 2002

[3H]Muscimol was irreversibly incorporated into rat cerebellar membranes upon irradiation with ultraviolet light. GABA agonists and antagonists inhibited this incorporation. The reversible muscimolbinding decreased after photoaffinity‐labeling of the membranes with muscimol. These results indicated that this irreversible incorporation is to the GABA receptor. Photolabeled GABA receptor protein showed its Mr
of 50000±1000 in SDS—polyacrylamide gel electrophoresis. This molecular size is identical to that of the benzodiazepine receptor which was photolabeled with 3H‐flunitrazepam.
Melphalan potently substitutes the N‐terminal Tyr of D‐Ala2‐Leu5‐enkephalin methyl ester
Authors: Szücs Mária , Di Gleria Katalin , Medzihradszky Kálmán
Published: July 2002

In search of an affinity label of the opioid receptor, the nitrogen mustard melphalan, Mel, was built into the peptide chain of D‐Ala2‐Leu5‐enkephalin (DALE) methyl ester in different positions. We report now that in contrast to the previous observations that an intact Tyr in position 1 is essential for opioid activity [(1980) Annu. Rev. Pharmacol. Toxicol. 20, 81‐110], substitution of Tyr by Mel did not result in a loss of the binding affinity. Mel1, Leu5‐enkephalin‐OMe competed for the binding sites of [3H]naloxone as potently as DALE did; IC50 values for both compounds were 50 nM. Mel substitution has led to one order potency decrease in binding to the δ‐sites. 0.5–1 μM of the compound irreversibly inactivates 50% of the binding sites of [3H]naloxone, and 5–10 μM of that of [3H]DALE. These results shed new light on the structural requirements established for opioid peptides. In addition, the new derivative can be used as an affinity label of the opioid receptor.
Circular dichroism spectra show abundance of β‐sheet structure in connectin, a muscle elastic protein
Authors: Maruyama Koscak , Itoh Yoshiharu , Arisaka Fumio
Published: July 2002

Circular dichroism spectra of native connectin from chicken breast muscle strongly suggested the abundant presence of β‐sheet structure, as much as 70% in 0.5 M KCl and 50 mM phosphate buffer, pH 7.5. α‐Helix was not detected. These results are in contradiction with the conclusion that native connectin from rabbit skeletal muscle consists entirely of random coil [(1984) J. Mol. Biol. 180, 331‐356].
Complete assignment of lysine resonances in 1H NMR spectra of proteins as probes of surface structure and dynamics
Authors: Chazin Walter J. , Rance Mark , Wright Peter E.
Published: July 2002

A strategy is presented for complete identification of 1H spin systems of lysine residues using sophisticated 2D NMR experiments. Relayed and remote connectivities within each spin system are determined for spin subsystems based at the backbone amide and Cϵ proton resonances. When complete spin system identification is combined with sequence‐specific assignment, protein surface structure and dynamics can be probed in a site‐specific manner. The interaction between the five lysine residues of French bean plastocyanin and a model redox partner Cr(CN)3−
6 has been examined using this approach.
Evidence for modulation of cell‐to‐cell electrical coupling by cAMP in mouse islets of Langerhans
Authors: Santos Rosa M. , Rojas Eduardo
Published: July 2002

The effects of forskolin on electrical coupling among pancreatic β‐cells were studied. Two microelectrodes were used to measure membrane potentials simultaneously in pairs of islet β‐cells. Intracellular injection of a current pulse (ΔI) elicited a membrane response ΔV
1 in the injected cell and also a response ΔV
2 in a nearby β‐cell confirming the existence of cell‐to‐cell electrical coupling among islet β‐cells. In the presence of glucose (7 mM), application of forskolin evoked a transient depolarization of the membrane and electrical activity suggesting that the drug induced a partial inhibition of the β‐cell membrane K+ conductance. Concomitant with this depolarization of the membrane there was a marked decrease in β‐cell input resistance (ΔV
2/ΔI) suggesting that exposure to forskolin enhanced intercellular coupling. Direct measurements of the coupling ratio ΔV
2/ΔV
1 provided further support to the idea that forskolin enhances electrical coupling among islet cells. Indeed, application of forskolin reversibly increased the coupling ratio. These results suggest that cAMP might be involved in the modulation of electrical coupling among islet β‐cells.
Immunogenic protein MPB57 from Mycobacterium bovis BCG: Molecular cloning, nucleotide sequence and expression
Authors: Yamaguchi Ryuji , Matsuo Kazuhiro , Yamazaki Akihiro , Nagai Sadamu , Terasaka Kunihiro , Yamada Takeshi
Published: July 2002

Using a single‐probe method, we have cloned the gene for an immunogenic MPB57 protein of Mycobacterium bovis BCG. The nucleotide sequence includes an ORF of 300 base pairs encoding a protein of 99 amino acids with an M
r of 10 818. This cloned gene was expressed in an Escherichia coli expression vector to give a mature protein which reacted with a polyclonal antibody raised against MPB57.
A highly selective ligand for brain δ opiate receptors, a ▿ EPhe4‐enkephalin analog, suppresses μ receptor‐mediated thermal analgesia by morphine
Authors: Shimohigashi Yasuyuki , Takano Yukio , Kamiya Hiro-o , Costa Tommaso , Herz Albert , Stammer Charles H.
Published: July 2002

[D‐Ala2,(2R,3S)‐▿
E
Phe4,Leu5]enkephalin (CP‐OH)[ ▿denoting cyclopropyl; superscript E indicating the E‐configuration about the cyclopropane ring], a highly selective opioid ligand for δ receptors in rat brain, but not for those in the mouse vas deferens, was examined for in vivo biological activities by intracerebroventricular administration. CP‐OH (5–20 μg) showed no analgesic activity in the hot plate (51°C) test using rats. However, it suppressed completely the analgesic effects of intraperitoneally administered morphine (3 mg/kg rat) in a dose‐dependent manner. CP‐OH showed no binding affinity for brain κ receptors to which dynorphin, an opioid peptide that inhibits morphine analgesia, binds predominantly. These results suggest that, besides the conventional δ receptors which mediate analgesia, the rat brain contains another δ‐like receptor which has a modulatory role to attenuate morphine‐induced analgesia mediated through the μ receptors, and that this modulatory receptor does not exist in the mouse vas deferens.
Interaction of a hydrophobic model peptide and its fatty acid derivative with lipid vesicles
Authors: Joseph M. , Nagaraj R.
Published: July 2002

The interaction of a peptide [Lys‐Phe‐Phe‐Phe‐Ile‐Ile‐Trp‐OCH3] and its fatty acid derivatives [Lys‐(ε‐palmitoyl)‐Phe‐Phe‐Phe‐Ile‐Ile‐Trp‐OCH3 and Lys‐{ε‐12‐(9‐anthroyloxy)stearic acid}‐Phe‐Phe‐Phe‐Ile‐Ile‐OCH3] with model membranes was investigated by fluorescence spectroscopy. The emission characteristics of the Trp fluorophore indicated that only the peptide with the fatty acid chain is associated with lipid vesicles. Quenching experiments with spin probes suggest an orientation for the fatty acylated peptide wherein the fatty acid chain is perpendicular to the bilayer surface and the peptide chain parallel to the bilayer surface.
Complete assignment of the 1H NMR spectrum and secondary structure of the DNA binding domain of GAL4
Authors: Gadhavi Paresh L. , Raine Andrew R.C. , Alefounder Peter R. , Laue Ernest D.
Published: July 2002

Complete 1H NMR resonance assignments are presented for the cysteine rich region of the DNA binding domain of the yeast transcriptional activator GAL4. The protein contains short helical regions between Asp‐12 and Leu‐19 and between Lys‐30 and Trp‐36. It is clearly distinct from the C2H2 class of zinc finger protein typified by the Xenopus laevis transcription factor (TF)IIIA. We also find that the first SP(X)(X) sequence, a recently proposed DNA binding motif (residues 41 to 44), appears to be tightly packed against the metal binding domain.
Diverse proteins homologous to inositol monophosphatase
Authors: Neuwald Andrew F. , York John D. , Majerus Philip W.
Published: July 2002

Bovine inositol monophosphatase (IMP) and several homologous proteins were found to share two sequence motifs with bovine inositol polyphosphate 1‐phosphatase (IPP). These motifs may correspond to binding sites within IMP and IPP for inositol phosphates or for lithium, since both substances are bound by these proteins. This suggests that the proteins homologous to IMP, which have diverse biological roles but whose function is not clear, may act by enhancing the synthesis or degradation of phosphorylated compounds.
Metabotropic responses to acetylcholine and serotonin of Xenopus oocytes injected with rat brain mRNA are transduced by different G‐protein subtypes
Authors: Kaneko Shuji , Takahashi Hideki , Satoh Masamichi
Published: July 2002

To assign the GTP‐binding protein (G‐protein) subtype involved in the signal transduction from exogenous receptors to phospholipase C in the Xenopus oocyte translation system, antisense DNA complementary to rat G‐protein α‐subunit MRNA was designed and injected together with rat brain poly(A)+ RNA. Current response of mRNA‐injected oocytes to acetylcholine (ACh) was suppressed dose‐dependently by a co‐injection of Gilα‐antisense DNA, but response of the same oocytes to serotonin (5‐HT) was not inhibited. In the oocytes co‐injected with Goα‐antisense DNA, the 5‐HT response was more effectively suppressed than the ACh response. These results suggest that Goα but not Gilα intermediates brain 5‐HTIC receptor function, and in contrast, muscarinic receptors derived from rat brain utilize Gilα rather than Goα to activate phospholipase C.
Is thioredoxin the physiological vitamin K epoxide reducing agent?
Authors: Preusch Peter C.
Published: July 2002

E. coli thioredoxin plus thioredoxin reductase have previously been shown to replace dithiothreitol as the electron donor for mammalian liver microsomal vitamin K epoxide reduction in vitro. Such activity is dependent on detergent disruption of the microsomal membrane integrity. A previously characterized salicylate‐inhibitable pathway for electron transfer from endogenous cylosolic reducing agents to the microsomal epoxide reducing warfarin‐inhibitable enzyme is not inhibited by known alternate substrates and inhibitors of the thioredoxin system nor by antibodies against thioredoxin.
Glycoprotein lib peptide 656–667 mimics the fibrinogen γ chain 402–411 binding site on platelet integrin GPIIb/IIIa
Authors: Calvete Juan J. , Rivas Germán , Schäfer Wolfram , McLane Mary Ann , Niewiarowski Stefan
Published: July 2002

The human integrin glycoprotein IIb/IIIa complex plays a central role in haemostasis as an inducible receptor for fibrinogen and other adhesive proteins at the platelet plasma membrane. Current evidence indicates that the ligand‐binding domain of GPIIb/IIIa is discontinuous and placed at the subunit interface. Here we show that a synthetic peptide containing the polypeptide stretch GPIIb 656–667, which is hidden within the resting platelet GPIIb/IIIa heterodimer but becomes exposed following platelet activation with thrombin, binds to soluble fibrinogen (n = 2.3 ± 1.3; K
d = 2 ± 0.8 × 10−5 M). This interaction is Ca2+‐independent and can be partially inhibited with synthetic fibrinogen γ‐chain peptide 400–411 but not with GRGDS. In addition, peptide GPIIb 656–667 inhibits in a dose‐dependent manner the aggregation of activated platelets (IC50 = 170μM). Altogether, our results indicate that the GPIIb 656–667 region may form part of the inducible fibrinogen binding site and may not overlap with the integrin RGD‐recognition domain.
Yeast coatomer contains a subunit homologous to mammalian β'‐COP
Authors: Harter Cordula , Draken Esther , Lottspeich Friedrich , Wieland Felix T.
Published: July 2002

The homologue of the mammalian coatomer complex was isolated from yeast cytosol and separated on a modified urea‐containing SDS‐polyacrylamide gel system. An additional band in the 100 kDa molecular weight range appeared when compared to the protein pattern obtained in conventional Laemmli gels, exactly as observed for mammalian coatomer. Cross‐reactivity with an anti‐peptide antibody raised against the C‐terminus of β‐COP from bovine, and N‐terminal sequence analysis, revealed that this protein from yeast is related to β‐COf from mammals.
Biochemical characterization of the presecretory protein translocation machinery of Escherichia coli
Authors: Tokuda Hajime
Published: July 2002

The protein translocation apparatus in Escherichia coli has been studied both genetically and biochemically. In vitro protein translocation systems involving everted membrane vesicles or reconstituted proteoliposomes have significantly contributed to biochemical clarification of the structure, mechanism and energetics of the apparatus. It is established that SecA, SecY and SecE are essential components, and play fundamental roles in the translocation reaction, and that both ATP and a proton motive force are required for the translocation. A new membrane factor, SecG, was found to participate in the formation of the apparatus, causing significant enhancement of the activity. SecD was found to play a role in the release of translocated proteins from the outer surface of the cytoplasmic membrane.
Refolding proteins by gel filtration chromatography
Authors: Werner Milton H. , Clore G.Marius , Gronenborn Angela M. , Kondoh Akiko , Fisher Robert J.
Published: July 2002

We have developed a facile means for the refolding of milligram quantities of purified proteins that employs gel filtration chromatography. We demonstrate by electrophoretic mobility shift and NMR spectroscopy that human ETS‐1 protein, bovine ribonucelase A and E. coli integration host factor can be refolded into the native conformation using this technique. We have extended this strategy to the preparation of miligram quantities of macromolecular complexes suitable for structural analysis by NMR spectroscopy or X‐ray crystallography. The diverse challenges to overcome in refolding these proteins illustrates the potential of this technique as a general approach for recovery of recombinant proteins produced as insoluble inclusion bodies.
Distinguishing between duplex and hairpin forms of RNA by 15N–1H heteronuclear NMR
Authors: Aboul-ela Fareed , Nikonowicz Edward P. , Pardi Arthur
Published: July 2002

A general method is described for distinguishing RNA hairpins from RNA duplexes by application of two‐dimensional filtered nuclear Overhauser enhancement spectra on a 1:1 mixture of unlabeled and 99% 15N‐labeled molecules. The method is applied to the RNA dodecamer rGGCGCUUGCGUC which can form an intramolecular hairpin under low salt conditions and a duplex in high salt. This procedure allows unambiguous identification of RNA hairpins or duplexes under the same conditions that are used in the NMR solution structure determination.
Use of a monoclonal antibody to detect DNA damage caused by the anticancer drug cis‐diamminedichloroplatinum (II) in vivo and in vitro
Authors: Chao Chuck C.-K. , Shieh Teh-Chien , Huang Haimei
Published: July 2002

A monoclonal antibody, MAb62‐5, was prepared and used to detect DNA damage due to the anticancer drug cis‐diamminedichloroplatinum (II) (or cisplatin). ELISA competition indicated that the binding of MAb62‐5 to cisplatin‐DNA was competitively inhibited (50% control) by 210 nM of cisplatin bound to DNA, cisplatin/nucleotide (DIN) = 0.2. Using a DNA mobility shift assay, MAb62‐5 binding activity was inhibited by 50% by ∼50‐fold molar excess of cisplatin‐DNA adducts (DIN = 0.08) whereas there was less than 5% inhibition by UV‐DNA adducts or mock‐treated DNA. In addition, MAb62‐5 showed a similar affinity to the cisplatin‐DNA adducts as compared to an endogenous cisplatin‐damaged DNA recognition protein. Using ELISA with this antibody, we have demonstrated a 2‐fold enhancement in excision repair of cisplatin‐DNA adducts in resistant HeLa cells. This is supported by the measurement of repair‐associated DNA strand breaks using alkaline elution and host cell reactivation of transfected plasmid DNA carrying cisplatin damage. These findings also provide a possible explanation for the complexicity of immunoassay in cells.
Effects of zopiclone on quality of life in insomnia
Authors: Hindmarch I
Published: July 2002

All of the components of quality of life can be altered in insomniacs because of sleep disorders themselves but theoretically also in consequence to side effects from hypnotic agents. The presented international study, carried out with a multicentre, randomised and double-blind design, investigated zopiclone effects on quality of life in insomniac patients as compared to placebo. A total of 458 patients completed the study, 231 receiving zopiclone 7.5 mg and 227 receiving placebo, with mean treatment durations of 48 and 44 days. Sleep parameters and quality of life, measured with extensive and validated scales, were assessed at baseline and at 14, 28 and 56 days after admission. Zopiclone showed hypnotic efficacy and significantly greater improvement of quality of life as compared to placebo, both at 14 days and at endpoint.
Suppression of GD1α ganglioside‐mediated tumor metastasis by liposomalized WHW‐peptide
Authors: Takikawa Masako , Kikkawa Hironori , Asai Tomohiro , Yamaguchi Naoto , Ishikawa Dai , Tanaka Michinori , Ogino Koichi , Taki Takao , Oku Naoto
Published: July 2002

GD1α ganglioside‐replica peptides were recently isolated from a phage‐displayed random pentadecapeptide library by assaying for inhibition of adhesion of RAW117‐H10 lymphosarcoma cells to hepatic sinusoidal microvessel endothelial (HSE) cells. We show here that the Trp‐His‐Trp (WHW) peptide was identified as a minimal sequence of the GD1α‐replica peptide WHWRHRIPLQLAAGR. The addition of WHW peptide‐attached liposomes displayed efficient inhibition of liver metastasis of RAW117‐H10 cells as well as of GD1α‐mediated adhesion of RAW117‐H10 cells to HSE cells in vitro. These results suggest that engineered liposomes for peptide delivery are applicable to treatment for metastasis.
Further studies on the reconstitution of glucosylceramidase activity by Sap C and anionic phospholipids
Authors: Salvioli Rosa , Tatti Massimo , Ciaffoni Fiorella , Vaccaro Anna Maria
Published: July 2002

The reconstitution of the activity of the lysosomal enzyme glucosylceramidase requires anionic phospholipids and, at least, a protein factor, saposin C (Sap C). We have previously proposed a mechanism for the glucosylceramidase activation [Vaccaro et al. (1993) FEBS Lett. 336, 159–162] which implies that Sap C promotes the association of the enzyme with anionic phospholipid‐containing membranes, thus favoring the contact between the enzyme and its lipid substrate, glucosylceramide. We have further investigated the properties of Sap C using a fluorescent hydrophobic probe such as 4,4′‐dianilino‐1,1′‐binaphthyl‐5,5′‐disulfonic acid (bis‐ANS). The binding between bis‐ANS and Sap C was pH‐dependent, indicating that protonation leads to increased exposure of hydrophobic surfaces of Sap C. The interaction of Sap C with membranes, triggered by the development of hydrophobic properties at low pH values, was affected by the content of anionic phospholipids, such as phosphatidylserine or phosphatidylinositol, suggesting that anionic phospholipids have the potential to modulate the insertion of Sap C in the hydrophobic environment of lysosomal membranes. We previously showed that Sap C and anionic phospholipids are both required for the binding of glucosylceramidase to large vesicles. We have presently observed that Sap C is able to promote the association of glucosylceramidase with the lipid surface only when anionic phospholipids exceed a concentration of 5–10%. This level can be reached by summing lower amounts of individual anionic phospholipids, since they have additive effects. The present data extend and refine our model of the mechanism of glucosylceramidase activation and stress the key role of pH, Sap C and anionic phospholipids in promoting the interaction of the enzyme with membranes.