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PubMed2006

Cryofixation methods for ultrastructural studies of Dictyostelium discoideum.

Cryopreservation methods, including rapid freezing, freeze-substitution, and low-temperature embedment, lead to superior ultrastructural preservation compared with traditional fixation procedures. This is particularly true for the multicellular stages of Dictyostelium discoideum, in which the hydrophobic sheath that surrounds the structures causes delayed penetration by the already slow-acting aqueous chemical fixatives, resulting in cell shape changes, loss of cell-cell contacts, and changes in cell-matrix interactions. The surface tension effects of traditional fixation methods can also result in disruption of the delicate structures. Depth of freezing is often greater than expected because of the relatively dehydrated state of the multicellular structures. Variations in freeze-substitution solvents and embedment media can be employed to allow for antigenic preservation. Commercial instruments exist for most of the procedures, but excellent results can be obtained using inexpensive hand-crafted apparatus.

Animals↗
PubMed1985

Metabolic phosphorylation and excretion of some nucleoside analogues in insects.

After oral administration to the hemipteran insect Pyrrhocoris apterus L. (Fireburg), the L-enantiomers and certain open-chain analogues of the nucleosides are rapidly converted into the corresponding monophosphates, which are then excreted. This metabolic phosphorylation is almost quantitative; it occurs at the primary as well as at the secondary hydroxylic groups. The process is abolished when the respective nucleoside analogues contain a free carboxylic group. By contrast, the phosphorylating capacity is unaffected by structural variations at the heterocyclic base. This phosphorylation and excretion may represent a part of detoxication mechanism for the above nucleoside analogues.

Animals↗
PubMed1987

Phenol-promoted structural transformation of insulin in solution.

Phenolic additives widely used for the preservation of insulin preparations can have a profound effect on the hormone's conformation in solution. m-Cresol, for instance, increases the circular dichroism in the far ultraviolet by 10-20%, corresponding to an increase in helix, and around 255 nm. The CD-spectral changes are strikingly similar to those brought about by halide ions which have been identified to reflect the 2 Zn----4 Zn insulin transition. Its most prominent element is the helix formation at the B-chain N-terminus. In both cases the changes fail to occur with dimeric insulin in the absence of Zn2 and with monomeric des-(B26-B30)-insulin. In the presence of Ni2 which is unable to replace Zn2 in 4 Zn insulin for coordinative reasons, the effect of m-cresol is impeded. m-Cresol thus induces a transition identical with or closely similar to the 2 Zn----4 Zn transformation. 2 Zn insulin crystals, when soaked in m-cresol containing solvents, are destroyed. Crystals grown in the presence of m-cresol, however, are monoclinic and containing symmetrical hexamers of, notably, 4 Zn conformation. Phenol, o- and p-cresol, m-nitrophenol, Nipagin M and benzene were further additives tested, all of them inducing largely the same spectral effects except for benzene. The results presented corroborate the close correspondence of insulin's structure in solution and in the crystal as well as insulin's capacity for structural variation.

Animals↗
PubMed1996

Purification and characterisation of the pyruvate decarboxylase from a haploid strain of Saccharomyces cerevisiae.

A novel purification procedure was developed for pyruvate decarboxylase (PDC, E.C. 1.1.1.4) from the haploid yeast strain YSH 4.127-1A expressing only one (PDC1) of the three structural genes for PDC. The purified enzyme is homotetrameric with a molecular mass of about 240,000 whereas PDC from brewer's yeast is a dimer of dimers composed of subunits of different size (alpha 2 beta 2) with the same molecular mass as the tetramer. Despite these structural variations there are no significant differences in the kinetic behaviour of the two enzyme species. PDC purified from the haploid yeast mutants shows a sigmoid dependence of the reaction rate from the substrate concentration due to the substrate activation. In the presence of the substrate surrogate pyruvamide the shape of the v/S plot is transformed into a hyperbolic one. As expected, polyclonal antibodies react with both the enzyme from haploid yeast strain mutants and that from brewer's yeast.

Ammonium Sulfate↗
PubMed2003

Malpighian tubule polytene chromosomes of Culex quinquefasciatus (Diptera, Culicinae).

Dipteran polytene chromosomes provide an excellent model for understanding in species complexes, as well as for structural and functional cytogenetics. The status of species in the Culex pipiens complex is controversial and the use of polytene chromosomes for cytogenetic analysis in the subfamily Culicinae has been difficult because of methodological problems. In this study, Malpighian tubule polytene chromosomes were obtained from young (0 to 12 h, 20 C) and old (20 to 42 h, 28 C) laboratory-bred C. pipiens quinquefasciatus pupae. The chromosome maps for this species were constructed and compared with published data for C. pipiens pipiens and C. p. quinquefasciatus. Although the banding patterns were conserved between subspecies, analysis of the structural variations in the bands and interbands revealed differences apparently related to the physiological stage and ecogeographical strain. The organization of the centromeric regions in larval and pupal chromosomes showed greater similarity to each other than did those of pupal and adult chromosomes. The use of pupal polytene chromosomes for in situ hybridization with vector competence probes is discussed.

Animals↗
PubMed2004

Use of molecular methods in identification of Candida species and evaluation of fluconazole resistance.

The aim of this study was to evaluate the use of one of the molecular typing methods such as PCR (polymerase chain reaction) following by RFLP (restriction fragment length polymorphism) analysis in the identification of Candida species and then to differentiate the identified azole susceptible and resistant Candida albicans strains by using AP-PCR (arbitrarily primed-polymerase chain reaction). The identification of Candida species by PCR and RFLP analysis was based on the size and primary structural variation of rDNA intergenic spacer regions (ITS). Forty-four clinical Candida isolates comprising 5 species were included to the study. The amplification products were digested individually with 3 different restriction enzymes: HaeIII, DdeI, and BfaI. All the isolates tested yielded the expected band patterns by PCR and RFLP analysis. The results obtained from this study demonstrate that Candida species can be differentiated as C. albicans and non-C. albicans strains only by using HaeIII restriction enzyme and BfaI maintains the differentiation of these non-C. albicans species. After identification Candida species with RFLP analysis, C. albicans strains were included to the AP-PCR test. By using AP-PCR, fluconazole susceptible and resistant strains were differentiated. Nine fluconazole susceptible and 24 fluconazole resistant C. albicans were included to the study. Fluconazole resistant strains had more bands when evaluating with the agarose gel electrophoresis but there were no specific discriminatory band patterns to warrant the differentiation of the resistance. The identification of Candida species with the amplification of intergenic spacer region and RFLP analysis is a practical, short, and a reliable method when comparing to the conventional time-consuming Candida species identification methods. The fluconazole susceptibility testing with AP-PCR seems to be a promising method but further studies must be performed for more specific results.

Antifungal Agents↗
PubMed2003

Effects of topoisomerases inhibitors protoberberine on Leishmania donovani growth, macrophage function, and infection.

DNA topoisomerases play a pivotal role in the regulation of cell division. Inhibition of Leishmania spp. topoisomerases represents an alternative to control parasite growth. Cancer research led to the development of several potent topoisomerase inhibitors such as topoisomerase I, topoisomerase II, or both (monobenzimidazole, terbenzimidazole, and protoberberine alkaloid-related compounds) that are effective antitumor agents. In the present study, we evaluated the efficacy of these compounds against Leishmania spp. growth in vitro. Some protoberberine compounds showed pronounced antileishmanial activity and were selected for further analysis in macrophages. These compounds did not affect macrophage viability and only slightly reduced macrophage nitric oxide generation in response to interferon-gamma. Moreover, exposure of infected macrophages to these compounds significantly reduced parasite loads. Collectively, our data suggest that protoberberine-related compounds have powerful antileishmania action and that minor structural variations among them can substantially improve their activity to restrict Leishmania spp. infection in vitro.

Animals↗
PubMed1995

Differences in carbohydrate composition of FSH preparations detected with lectin-ELISA systems.

FSH is a glycoprotein containing N-linked carbohydrates which exhibit a variety of forms ranging from mono- to multibranched structures. Variation in glycosylation, particularly the degree of terminal sialylation, determines the half-life of the hormone and hence its in vivo bioactivity. The glycoform content of FSH preparations can differ according to the source (e.g. pituitary, urine), cell line (for rDNA-derived material) and selectivity of purification procedures, and may create difficulties in the preparation and characterization of standards and therapeutic products. In order to develop a simple method to detect changes in glycocomposition, an FSH ELISA was modified by the incorporation of lectins of recognized sugar specificity, and used to examine the terminal sugar composition of ampouled preparations of pituitary, urinary and rDNA-derived FSH. FSH was captured with a specific monoclonal antibody (MAb) and detected with either biotinylated anti-FSH MAb (ELISA) or the sugar-specific lectins (L-ELISA) from Triticum vulgaris (sialic acid, SA), Sambucus nigra (alpha 2,6-linked SA), Maackia amurensis (alpha 2,3-linked SA) or Ricinus communis (free terminal galactose; GAL). Relative estimates of the amounts of terminal SA, its different forms and GAL were derived from the L-ELISA/ELISA data compared with the highly sialylated 1st International Standard for pituitary FSH (IS) 83/575. All the FSH preparations had less SA than the IS with the ratio of alpha 2,3- and alpha 2,6-linked SA varying between preparations. The amounts of alpha 2,6-linked SA relative to the IS were not significantly different in the urinary and pituitary preparations whereas alpha 2,3-linked SA in all preparations was generally less than that of the standard.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbohydrate Conformation↗
PubMed1979

Constituents of response rates.

Response rate and the proportion of time pigeons allocated to a key-pecking activity were measured on several basic types of reinforcement schedules. Reinforcement frequency was varied within each type of basic schedule, and the effects on two constituents of response rate were noted. Propensity, the proportion of time the birds spent on a platform in front of the key, showed very consistent effects as reinforcement frequency varied: in general, it decreased when reinforcement frequency markedly decreased and it increased when reinforcement frequency increased. Speed, key pecks per unit of time spent on the platform, showed inconsistent effects when reinforcement frequency varied. Consequently, response rate showed less consistent effects than did propensity. Cumulative response records demonstrated the existence of several different types of transitions or boundary states between the key-pecking activity and other activities. The types of transitions that occurred between activities depended on both the type of reinforcement schedule and the frequency of reinforcement. The propensity data support the position that general laws of behavior can be based on temporal measures of behavior. The speed data suggest that, if a complete assessment of the dynamic properties of behavior is to be achieved, measures of behavior must incorporate the structural variations in the operant unit.

Journal Article↗
PubMedDate not supplied

Spatial variability of nitrous oxide emissions and their soil-related determining factors in an agricultural field.

To evaluate spatial variability of nitrous oxide (N2O) emissions and to elucidate their determining factors on a field-scale basis, N2O fluxes and various soil properties were evaluated in a 100- x 100-m onion (Allium cepa L.) field. Nitrous oxide fluxes were determined by a closed chamber method from the one-hundred 10- x 10-m plots. Physical (e.g., bulk density and water content), chemical (e.g., total N and pH), and biological (e.g., microbial biomass C and N) properties were determined from surface soil samples (0-0.1 m) of each plot. Geostatistical analysis was performed to examine spatial variability of both N2O fluxes and soil properties. Multivariate analysis was also conducted to elucidate relationships between soil properties and observed fluxes. Nitrous oxide fluxes were highly variable (average 331 microg N m(-2) h(-1), CV 217%) and were log-normally distributed. Log-transformed N2O fluxes had moderate spatial dependence with a range of >75 m. High N2O fluxes were observed at sites with relatively low elevation. Multivariate analysis indicated that an organic matter factor and a pH factor of the principal component analysis were the main soil-related determining factors of log-transformed N2O fluxes. By combining multivariate analysis with geostatistics, a map of predicted N2O fluxes closely matched the spatial pattern of measured fluxes. The regression equation based on the soil properties explained 56% of the spatially structured variation of the log-transformed N2O fluxes. Site-specific management to regulate organic matter content and water status of a soil could be a promising means of reducing N2O emissions from agricultural fields.

Air Pollutants↗
PubMed2005

Pharmacoeconomic analyses using discrete event simulation.

To date, decision trees and Markov models have been the most common methods used in pharmacoeconomic evaluations. Both of these techniques lack the flexibility required to appropriately represent clinical reality. In this paper an alternative, more natural, way to model clinical reality--discrete event simulation--is presented and its application is illustrated with a real world example.A discrete event simulation represents the course of disease very naturally, with few restrictions. Neither mutually exclusive branches nor states are required, nor is a fixed cycle. All relevant aspects can be incorporated explicitly and efficiently. Flexibility in handling perspectives and carrying out sensitivity analyses, including structural variations, is incorporated and the entire model can be presented very transparently. The main limitations are imposed by lack of data to fit realistic models. Discrete event simulation, though rarely employed in pharmacoeconomics today, should be strongly considered when carrying out economic evaluations, particularly those aimed at informing policy makers and at estimating the budget impact of a pharmaceutical intervention.

Computer Simulation↗
PubMed2026

Multi-omics Investigations of Immune Microenvironment of Human Colorectal Cancer.

BACKGROUND/AIM: Colorectal cancer (CRC) remains a leading cause of cancer-related morbidity and mortality worldwide. Although immunotherapy has improved outcomes for a subset of patients, its limited efficacy in many cases highlights the need for a more comprehensive understanding of the CRC immune microenvironment. This study aimed to characterize the molecular landscape of the CRC immune microenvironment using an integrated multi-omics approach and to identify candidate regulatory molecules associated with immune remodelling. MATERIALS AND METHODS: We integrated structural variation, DNA methylation, chromatin accessibility, proteomic, and phosphoproteomic data generated from an in-house CRC cohort with transcriptomic data from The Cancer Genome Atlas (TCGA). Analyses focused on 1,539 immune-related genes (IRGs) associated with CD4+ T cells, B cells, and natural killer (NK) cells. Multi-layered genomic and proteomic analyses were performed to identify altered immune-related pathways, hub genes, candidate transcription factors, and upstream kinases. RESULTS: Higher infiltration of CD4+ T cells, B cells, and NK cells was associated with CRC. IRGs exhibited widespread alterations across genomic, epigenomic, transcriptomic, proteomic, and phosphoproteomic levels. IL10, LEP, ITGAM, and EGFR emerged as candidate hub genes. EGFR phosphorylation at S991 and T693 was significantly decreased in CRC. STAT2 and HSF1 were identified as candidate upstream transcription factors, while CDK2 emerged as a candidate upstream kinase associated with immune infiltration and immune checkpoint expression. CONCLUSION: This study provides a systematic multi-omics characterization of immune microenvironment remodelling in CRC and identifies candidate molecular regulators that may serve as potential targets for future immunotherapy research.

Humans↗
PubMed2000

A critical approach to the definition of Darwinian units of selection.

What are the biological units of selection? In fact, the notion of "unit of selection" (UOS) is blurred by ambiguity and controversy. To further evaluate the biological entities that are the objects of natural selection, three novel conceptual criteria (holism, minimalism, functionalism) are critically applied; they reveal, in addition to the self-evident case of the "individual," at least six distinct types of UOSs. These UOSs do not always have a defined structural organization; they can be parts of a living organism, a cohesive group of conspecifics, a multiunit entity, a totipotent cell, a DNA fragment, or a whole organism. UOS types diversify by amalgamation or parcelation processes of apparent entities. Therefore, previous attempts to characterize the UOSs solely on some morphological levels (gene, individual, group) without applying stringent criteria have failed to cope with the structural variations of natural phenomena and have led to the ambiguity of terms used.

Animals↗
PubMed1984

The characterization of hemoglobin Manitoba or alpha (2)102(G9)Ser----Arg beta 2 and hemoglobin Contaldo or alpha (2)103(G10)His----Arg beta 2 by high performance liquid chromatography.

Hb Contaldo with a His----Arg substitution at position 103(G10) of the alpha chain is a newly discovered unstable Hb variant observed in an Italian child. Its instability is probably due to the disruption of the hydrogen bond between alpha 103(G10)His and beta 108(G10)Asn. The structural variation in the core segment was determined through analysis of tryptic peptides from digests of the alpha X and oxidized alpha X (with performic acid) chains, which were separated by HPLC. Similar analyses were made for the alpha X chain of the rare Hb Manitoba in which alpha 102(G9) Ser is replaced by Arg. This variant was observed for the first time in an Italian patient, and was also studied in a member of a previously described Canadian family.

Amino Acids↗
PubMed1986

Cytochrome P-450scc a review of the specificity and properties of the cholesterol binding site.

Cytochrome P-450scc is unusual among members of this class of enzymes in showing a high degree of substrate specificity. Features of the cholesterol structure which are particularly important for binding include the 3 beta-hydroxyl, the delta 5-ring configuration, and the side-chain organization in the 20-22 region. Regarding the ring system, binding appears to require planarity and limited size at the 4-5-6 carbons (the A-B ring juncture). In the region of the 3 beta-hydroxyl, a "cleft" in the binding site extends about 4 A beyond the hydroxyl and can accommodate two additional ether-linked carbons. Evidence indicates that an enzyme residue hydrogen-bonds to the oxygen of the 3 beta hydroxyl, providing much of the energy for the initial enzyme-substrate interaction. The cytochrome shows less specificity for the side-chain structure, except in the region of carbons 20-22 where hydroxylation/side-chain cleavage takes place. The binding cleft for the side-chain is limited to approximately the length of the isocaproic group but can accommodate structural variations beyond the 22-position. Evidence indicates that the region near the 20-22 bond is more limited in size, and that an amino acid residue near the heme iron binds strongly and stereospecifically to the 22R-hydroxyl of the cleavage intermediates, 22R-hydroxycholesterol and 20 alpha, 22R-dihydroxycholesterol. The 22R-hydrogen of cholesterol is very close to the heme iron (approximately 3 A), while the 22S-hydrogen is slightly further (about 4 A). The size and bonding properties of the steroid binding/active site suggest a mechanism which accounts for the stereospecificity and sequence of reactions catalyzed by cytochrome P-450scc.

Adrenal Cortex↗
PubMed1975

Present status of the Duffy blood group system.

In 1950 a new blood group antibody was recognized in the serum of a multi-transfused hemophiliac patient. The reactive red-cell antigen was identified in 65% of random caucasians, and the systemic name Duffy was proposed. Two common antigens, Fy-a and Fy-b, were recognized and shown to be products of autosomal allelic genes, but the great majority of negro individuals were found to lack both antigens. In 1968 genetic studies showed Duffy to be linked to Un-1, which is an inherited structural variation of chromosome number 1. Duffy thus became the first autosomal blood group gene to be allocated to a specific chromosome. Recent studies have allowed recognition of three new antibodies (anti-Fy3, anti-Fy4, and anti-Fy5) in the Duffy system. The Rh blood group locus has also been assigned to chromosome 1, and there is evidence that the antigen defined by anti-Fy5 is a product of interaction between Duffy and Rh genes. The Duffy blood group appears to be one of importance in clinical blood transfusion practice, and possibly makes the greatest distinction of any of the red cell systems between different groups of the world population.

Anthropology↗
PubMed2006

Jugular tubercle: Morphometric analysis and surgical significance.

OBJECT: Maximizing intradural exposure via the extreme lateral infrajugular transcondylar-transtubercular exposure (ELITE) approach depends on understanding the fundamental anatomy of the jugular tubercle (JT). Drilling the JT can maximize the extent of exposure achieved with the ELITE approach. Removing the JT is critical for optimizing access to the inferior and midclival areas, vertebrobasilar artery junction, and ventral pons and medulla. METHODS: In this cadaveric study, the individual structural variations in the JT were evaluated in 100 split occipital bones. The mean length of the JT was 1.65 +/- 0.36 cm (range 1.2-3 cm); its mean width was 1.15 +/- 0.16 cm (range 0.7-1.7 cm); and its mean thickness was 0.61 +/- 0.15 cm (range 0.2-1 cm). The authors analyzed the difference in morphometric data with regard to right and left sides and found no statistically significant difference between the two sides. Furthermore, data from the morphometric study were compared with the results of 20 measurements obtained from three-dimensional computed tomography (3D CT) scans. Accordingly, the mean length of the JT was 1.35 +/- 0.15 cm (range 1-2.8 cm); the mean width, 1.10 +/- 0.12 cm (range 0.8-1.3 cm); and the mean thickness, 0.51 +/- 0.18 cm (range 0.2-1 cm). CONCLUSIONS: Morphometric data on the JT contribute significantly to the neurosurgeon's task of skull base drilling. The 3D CT scans were useful in the preoperative planning.

Body Weights and Measures↗
PubMed2026

Chromosome-Scale Genome of Zoonotic Eyeworm Thelazia callipaeda from China.

Thelazia callipaeda is a vector-borne zoonotic eyeworm infecting companion animals, wildlife, and humans, but chromosome-scale genomic resources from Chinese clinical material remain limited. We generated a genome supported by Pacific Biosciences (PacBio) high-fidelity (HiFi) sequencing and high-throughput chromosome conformation capture (Hi-C) from 100 adult worms recovered from naturally infected dogs in Beijing and compared its chromosome-scale organization with Portuguese assembly GCA_965194785.1. The final assembly spans 119.53 megabases (Mb) and comprises 115 top-level sequences, including four pseudomolecules totaling 91.26 Mb (76.34%) and 111 unanchored sequences. Genome-mode Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis recovered 98.5% complete chromadorean orthologues, and the representative 11,788-protein gene set recovered 92.6%. Sequence-level alignment resolved Chinese chromosomes 1-4 (chr1-chr4) to Portuguese chr1, chrX, chr3, and chr2, respectively, with retained alignments covering 95.9-99.2% of each Chinese pseudomolecule and estimated sequence identities of 99.75-99.91%. Strong chromosome-scale collinearity was accompanied by localized reverse-collinear regions, including 0.243 Mb and 0.115 Mb intervals on chr2-chrX and chr3-chr3. The anchored sequences contained 96.7% of predicted genes and were substantially more gene-dense than the unanchored sequences. These results establish a clinically sourced Chinese chromosome-scale reference and provide a validated framework for future individual-worm, population-genomic, structural-variation, and comparative genomic studies of this parasite.

Hi-C↗