Chain processes and their biophysical applications: Part I. General theory

A mathematical theory applicable to the biological effects of radiations as chain processes is developed. The theory may be interpreted substantially as a “hit theory” involving the concepts of “sensitive volume” or “target area”. The variability of the sensitivity of the organism to the radiation and its capacity of recovery between single hits is taken into account. It is shown that in a continuous irradiation of a biological aggregate in which the effect of each single hit cannot be observed, recovery and variation of sensitivity are formally equivalent to each other so that a discrimination between these two phenomena is possible only by discontinuous irradiation or by using different radiation intensities. Methods for the calculation of the “number of hits” and for the determination of the kinetics of the processes from “survival curves” or similar experimental data are given. The relation between the recovery and the Bunsen-Roscoe law is discussed. The case in which the injury of the organism is dependent on the destruction of more than one “sensitive volume” is also considered.     

Ecological time-series analysis through structural modelling with latent constructs: concepts, methods and applications

Time-series analyses in ecology usually involve the use of autoregressive modelling through direct and/or delayed difference equations, which severely restricts the ability of the modeler to structure complex causal relationships within a multivariate frame. This is especially problematic in the field of population regulation, where the proximate and ultimate causes of fluctuations in population size have been hotly debated for decades. Here it is shown that this debate can benefit from the implementation of structural modelling with latent constructs (SEM) to time-series analysis in ecology. A nonparametric bootstrap scheme illustrates how this modelling approach can circumvent some problems posed by the climate-ecology interface. Stochastic Monte Carlo simulation is further used to assess the effects of increasing time-series length and different parameter estimation methods on the performance of several model fit indexes. Throughout, the advantages and limitations of the SEM method are highlighted.     

Cryopreservation of articular cartilage. Part 1: conventional cryopreservation methods

There is increasing interest in the possibility of treating diseased or damaged areas of synovial joint surfaces by grafts of healthy allogeneic cartilage. Such grafts could be obtained from cadaver tissue donors or in the future they might be manufactured by 'tissue engineering' methods. Cartilage is an avascular tissue and hence is immunologically privileged but to take advantage of this is the graft must contain living cells. Preservation methods that achieve this are required to build up operational stocks of grafts, to provide a buffer between procurement and use, and to enable living grafts of a practical size to be provided at the right time for patient and surgeon. Review of the literature shows that it has been relatively straightforward to cryopreserve living isolated chondrocytes, but at the present time there is no satisfactory method to preserve cartilage between the time of procurement or manufacture and surgical use. In this paper, we review the relevant literature and we confirm that isolated ovine chondrocytes in suspension can be effectively cryopreserved by standard methods yet the survival of chondrocytes in situ in cartilage tissue is inadequate and extremely variable.     

Peptide-chelating agent conjugate for selective targeting of somatostatin receptor type 1: synthesis and characterization

Previously reported results suggest that the analogue of the somatostatin des-AA1,2,5[D-Trp8,IAmp9]-somatostatin (CH-275) peptide bearing chelating agents able to coordinate radioactive metals could be used for scintigraphic imaging of tumor lesions overexpressing sstr1. An efficient synthetic procedure for the preparation of the somatostatin analogue CH-275 and its conjugate DTPAGlu-Gly-CH-275, bearing the chelating agent DTPAGlu (DTPAGlu=N,N-bis[2-[bis(carboxy-ethyl)amino]ethyl]-L-glutamic acid) on the N-terminus, by solid-phase peptide synthesis and 9-fluorenylmethoxycarbonyl (Fmoc) chemistry, is here reported. Rapid and efficient labeling of DTPAGlu-Gly-CH-275 was achieved by addition of 111In(III) to the compound. Typical yields were greater than 97% as determined by reversed phase high performance liquid chromatography (HPLC) at specific activities in the range 4-9 GBq/micromol (100-250 Ci/mmol). A preliminary biological assay of the binding ability of 111In-DTPAGlu-Gly-CH-275 indicates, however, that the labeled compound does not display any specific interaction with somatostatin sstr1 receptors in the tested cell lines. To confirm this unexpected negative result, competition binding experiments were carried out, in which fixed tracer amounts of the 125I-labeled somatostatin-14 were incubated with the receptor-expressing cells in the presence of DTPAGlu-Gly-CH-275 or CH-275 at concentrations ranging from 10(-10) to 10(-3) M. While CH-275 shows a IC50 of 80 nM similar to that already found in displacement experiments on CHO-K1 sstr1-transfected cells, DTPAGlu-Gly-CH-275 displays instead very low or negligible affinity towards this receptor. The NMR solution characterization indicates that the presence of DTPAGlu does not influence the conformational and chemical features of the peptide moiety, thus suggesting that the loss in binding activity should be due to steric hindrance of either the chelating agent DTPAGlu or its indium complex.     

DNA binding ligands targeting drug-resistant Gram-positive bacteria. Part 1: Internal benzimidazole derivatives

Novel DNA minor-groove binding ligands with a promising antibacterial profile are described. Apart from excellent in vitro potency against multiple Gram-positive bacterial strains such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis (VRE), and penicillin-intermediate Streptococcus pneumoniae (PISP), a small subset of compounds was active against Gram-negative bacteria such as Escherichia coli (E. coli).     

Non-Ewald methods: theory and applications to molecular systems

Several non-Ewald methods for calculating electrostatic interactions have recently been developed, such as the Wolf method, the reaction field method, the pre-averaging method, and the zero-dipole summation method, for molecular dynamics simulations of various physical systems, including biomolecular systems. We review the theories of these approaches and their potential applications to molecular simulations, and discuss their relationships.     

Biological activity and receptor binding characteristics to various human tumors of acetylated somatostatin analogs.

Several somatostatin analogs with recently synthesized acetylated N terminus were assayed in vivo for their effects on sodium pentobarbital-stimulated growth hormone (GH) levels in fed male rats and gastrin-releasing peptide (14-27)-stimulated gastrin levels in fasted male rats. The binding characteristics of these analogs to somatostatin receptors were also examined in various human tumors and normal tissues. The analog RC-101-I, injected at a dose of 0.1 micrograms/100 g body wt, significantly suppressed GH release (P less than 0.01) for at least 2 hr. Analog RC-160-II caused the longest inhibition of GH release, greater than that induced by nonacetylated parent analog RC-160, with GH levels showing significant suppression (P less than 0.01) for more than 3 hr. Analogs RC-160-II and RC-101-I and RC-160, injected at a dose of 1.0 micrograms/100 g body wt, significantly (P less than 0.01) suppressed gastrin-releasing peptide (14-27)-stimulated serum gastrin. Analog RC-101-I was active in this test at a dose of 0.1 micrograms/100 g body wt. RC-160-II showed significant binding to somatostatin-14 receptors in all investigated tissues (human colon, human colon cancer, breast cancer, human pancreas and pancreatic cancer, human prostate and prostate cancer, and rat cerebral cortex), but there were marked variations in binding affinities among various normal and cancerous tissues. The highest affinity was found in membranes of colon cancer (Ka = 18.4 nM-1) and breast cancer (Ka = 12.46 nM-1). The binding affinity of RC-160-II to somatostatin receptors in membranes of the breast cancer was similar to that of RC-160. RC-101-I showed higher binding affinity to somatostatin-14 receptors than RC-160 in human breast, pancreatic, and prostate cancer. With the exception of breast cancer tissue, the binding affinity of RC-101-I was significantly lower than that of RC-160-II in membranes of all investigated tissues. It can be concluded that acetylated somatostatin analogs RC-101-I and RC-160-II possess prolonged and enhanced biological activities in suppressing serum GH and gastrin in rats. Significant variations in binding affinities for these analogs in different tissues and various tumors suggest that differences may exist between somatostatin receptors in normal versus malignant tissues. This raises the possibility that some of these analogs could be used more selectively in the treatment of various neoplasms.     

Automated docking of ligands to antibodies: methods and applications

Many approaches to studying protein-ligand interactions by computational docking are currently available. Given the structures of a protein and a ligand, the ultimate goal of all docking methods is to predict the structure of the resulting complex. This requires a suitable representation of molecular structures and properties, search algorithms to efficiently scan the configuration space for favorable interaction geometries, and accurate scoring functions to evaluate and rank the generated orientations. For many of the available methods, tests on experimentally known antibody-antigen or antibody-hapten complexes have appeared in the literature. In addition, some of them have been used in predictive studies on antibody-ligand interactions to provide structural insights where adequate experimental information is missing. The AutoDock program is presented as example of a method for flexibly docking ligands to antibodies. Applying parameters of the second-generation AMBER force field, three antibody-hapten complexes (AN02, DB3, NC6.8) are used as new test cases to analyze the ability of the method to reproduce experimental findings. The X-ray structures could be reconstituted and the corresponding solutions were ranked with best energy score in all cases. Docking to the free instead of the complexed NC6.8 structure indicated the limits of the rigid protein treatment, although fairly good guesses about the location of the binding site and the contact residues could still be obtained if conformational flexibility was allowed at least in the ligand.     

Minor and trace sterols in marine invertebrates. 1. General methods of analysis.

3beta-Hydroxy sterols occurring at a concentration of at least 0.001% of the sterol mixtures of Pseudoplexaura porosa and Plexaura homomalla have been fractionated using a series of refined techniques and subsequently analyzed using combined gas chromatography-mass spectrometry (GC-MS) in the development of a procedure for examining the minor and trace components of marine sterol mixtures. A total of 49 sterols were found which spanned a molecular weight range of 274 to 440. In addition delta4-3-keto analogs of cholesterol, 24-methylcholesterol and gorgosterol were found in the extracts of P. homomalla. Initial separation of various natural sterol-containing conjugates and free sterols was found to have a number of advantages. Fractional digitonin precipitation and alumina column chromatography were found to possess greater sterol separation abilities than previously recognized. Many of the minor sterols were found to possess novel structures including a series of short side chain sterols, 19-nor sterols, 5beta-stanols and 4-monomethyl sterols for which structure elucidation work is continuing.     

Metabolic pathway analysis: basic concepts and scientific applications in the post-genomic era.

This article reviews the relatively short history of metabolic pathway analysis. Computer-aided algorithms for the synthesis of metabolic pathways are discussed. Important algebraic concepts used in pathway analysis, such as null space and convex cone, are explained. It is demonstrated how these concepts can be translated into meaningful metabolic concepts. For example, it is shown that the simplest vectors spanning the region of all admissible fluxes in stationary states, for which the term elementary flux modes was coined, correspond to fundamental pathways in the system. The concepts are illustrated with the help of a reaction scheme representing the glyoxylate cycle and adjacent reactions of aspartate and glutamate synthesis. The interrelations between pathway analysis and metabolic control theory are outlined. Promising applications for genome annotation and for biotechnological purposes are discussed. Armed with a better understanding of the architecture of cellular metabolism and the enormous amount of genomic data available today, biochemists and biotechnologists will be able to draw the entire metabolic map of a cell and redesign it by rational and directed metabolic engineering.     

Differential expression of the human somatostatin receptor subtypes sst1 to sst5 in various adrenal tumors and normal adrenal gland.

Somatostatin (SRIF) is a widely distributed peptide with growth-inhibiting effects in various tumors. So far, five distinct human SRIF receptor subtypes (sst1-sst5) have been identified. We investigated expression of the five ssts in various adrenal tumors and in normal adrenal gland. Tissue was obtained from ten pheochromocytomas (PHEOs), nine cortisol-secreting adenomas (CPAs), eleven aldosterone secreting adenomas (APAs) and eight non-functional adenomas (NFAs) after retroperitoneoscopic surgery, and used for RNA extraction. Adrenal tissue surrounding the tumor was available for analysis in twenty-seven cases. Receptor expression was studied by RT-PCR using sst-specific primers and subsequently confirmed by Southern blotting. Expression of all five receptor subtypes was observed in RNA obtained from normal adrenal gland. Furthermore, each receptor subtype was expressed in more than 50 % of all tumors analyzed. No sst5 expression was found in PHEOs, while sst1 was present in nearly all of these tumors. Only a few of the CPAs expressed subtypes sst1 and sst4. Expression of all five subtypes was distributed equally in APAs. No sst4 was found in any of the NFAs. Differential expression of ssts in various adrenal tumors may point to new aspects in the pathogenesis of these adenomas. Furthermore, the presence of specific ssts could expand the diagnostic and therapeutic strategies during management. New subtype specific analogues of SRIF may be used in the future depending on the type of adrenal tumor and receptor subtype expressed.     

Large-scale mammalian cell culture: methods, applications and products.

Animal cell cultures are used to generate products of enormous biotechnological value. These systems rely on conventional manufacturing techniques using organisms that are the result of either cell fusions or genetic engineering. A wealth of new techniques has allowed improvements and developments to be made in culture medium composition, cell modification, and bioreactor design and operation. This progress is expected to be commercially exploited as new products reach the market place.     

Large-scale plant cell culture: methods, applications and products.

Recent significant contributions to the design of large-scale plant cell cultures for secondary metabolite production include: the development of a strategy to control the concentration of dissolved gases at constant shear; a surface immobilization technique to retain cell mass at high mixing rates; and a modified stirred-tank reactor with a mesh cage to prevent damage of hairy root cultures.     

Large-scale insect cell culture: methods, applications and products.

The primary development in large-scale insect cell culture over the past year has been the continuing accumulation of documented evidence (fundamental and applied) that conventional aerated stirred-tank and air-lift bioreactors may be employed for insect cell cultivation and recombinant protein production, provided that air sparging, agitation, and the addition to the medium of Pluronic F-68 and methyl cellulose polymers are carefully controlled.     

Approaches to Macroevolution: 1. General Concepts and Origin of Variation

Approaches to macroevolution require integration of its two fundamental components, i.e. the origin and the sorting of variation, in a hierarchical framework. Macroevolution occurs in multiple currencies that are only loosely correlated, notably taxonomic diversity, morphological disparity, and functional variety. The origin of variation within this conceptual framework is increasingly understood in developmental terms, with the semi-hierarchical structure of gene regulatory networks (GRNs, used here in a broad sense incorporating not just the genetic circuitry per se but the factors controlling the timing and location of gene expression and repression), the non-linear relation between magnitude of genetic change and the phenotypic results, the evolutionary potential of co-opting existing GRNs, and developmental responsiveness to nongenetic signals (i.e. epigenetics and plasticity), all requiring modification of standard microevolutionary models, and rendering difficult any simple definition of evolutionary novelty. The developmental factors underlying macroevolution create anisotropic probabilities—i.e., an uneven density distribution—of evolutionary change around any given phenotypic starting point, and the potential for coordinated changes among traits that can accommodate change via epigenetic mechanisms. From this standpoint, “punctuated equilibrium” and “phyletic gradualism” simply represent two cells in a matrix of evolutionary models of phenotypic change, and the origin of trends and evolutionary novelty are not simply functions of ecological opportunity. Over long timescales, contingency becomes especially important, and can be viewed in terms of macroevolutionary lags (the temporal separation between the origin of a trait or clade and subsequent diversification); such lags can arise by several mechanisms: as geological or phylogenetic artifacts, or when diversifications require synergistic interactions among traits, or between traits and external events. The temporal and spatial patterns of the origins of evolutionary novelties are a challenge to macroevolutionary theory; individual events can be described retrospectively, but a general model relating development, genetics, and ecology is needed. An accompanying paper (Jablonski in Evol Biol 2017) reviews diversity dynamics and the sorting of variation, with some general conclusions.