Commentary Polyamines to Target Drugs to Dna

In neutral solutions polyamines are fully protonated, and hence are really polyammonium cations (PAC). Spermine, for example, carries four positive charges in a linear system, H₃N⁺(CH₂)₃N⁺H₂(CH₂)₄N⁺H₂ (CH₂)₃N⁺H₃. There is a very powerful coulombic interaction between aqueous DNA and such cations, thus the cations are attracted to the DNA over large distances, and once close to the DNA normally remain there for long periods. A key issue is; are the cations mobile, or do they remain at one preferred site for significant periods? The latter is the currently preferred concept, but NMR and EPR evidence will be presented in favour of the former. If the former is correct, then PACs may be able to act as good drug delivery systems. In its simple form the concept is that any drug that acts directly on DNA can be chemically bound to a PAC. Once in the cell, this PAC-drug complex (PAC-D) will be carried to DNA and will move very rapidly along the exposed strands until it recognises the site of action. This may be some special base sequence region, a damaged site, or the PAC-D unit may simply be present prior to potential damage, so that this can be repaired very rapidly. Some of our current studies on these systems are described.     

Fitting bent lines to data, with applications to allometry

Change-point models, in which a linear or non-linear relation is generalized by allowing it to change at a point not fixed in advance, are of growing importance in allometric and other types of modeling. Frequently, the change-point is picked "by eye" and separate regressions are run for each resultant subdomain. This procedure is deficient, however, for the following reasons: first, a repeatable and objective procedure for estimating the change-point has not been used; second, the subsequent analysis usually does not take into account the fact that the change-point is estimated from the data; and last, the usually desirable requirement of continuity at the change-point is ignored. This paper describes various methods for jointly estimating linear relations and the intervening change-point from the data. In the simplest case, with normal errors and a linear relation of one variable upon another, this amounts to fitting a "bent line" via least squares techniques. In addition, tests and graphical diagnostics for the presence of change-points are presented. An example is given where a change-point and slopes are estimated for the relation of running speed with size among land mammals. In the past, these data have been fit with a straight line or a parabola. It is shown here that superior fit and interpretability are achieved using a change-point model.     

From disease to development to cell biology and back

Nowadays, the focus of developmental studies is shifting away from formal models of developmental pathways that are characterised by flow charts of controlling factors connected by arrows, to mechanistic models that explain developmental processes at the cellular level. Surprisingly, this shift towards a cellular view of developmental biology is occurring simultaneously across a range of model organisms. One consequence of taking such a cell biological view of development is that many model organisms are now becoming good models for studies of human disease and therapy.     

How to Design PET Experiments to Study Neurochemistry: Application to Alcoholism

Positron Emission Tomography (PET) (and the related Single Photon Emission Computed Tomography) is a powerful imaging tool with a molecular specificity and sensitivity that are unique among imaging modalities. PET excels in the study of neurochemistry in three ways: 1) It can detect and quantify neuroreceptor molecules; 2) it can detect and quantify changes in neurotransmitters; and 3) it can detect and quantify exogenous drugs delivered to the brain. To carry out any of these applications, the user must harness the power of kinetic modeling. Further, the quality of the information gained is only as good as the soundness of the experimental design. This article reviews the concepts behind the three main uses of PET, the rationale behind kinetic modeling of PET data, and some of the key considerations when planning a PET experiment. Finally, some examples of PET imaging related to the study of alcoholism are discussed and critiqued.     

From humanized mice to human disease: guiding extrapolation from model to target

Extrapolation from a well-understood base population to a less-understood target population can fail if the base and target populations are not sufficiently similar. Differences between laboratory mice and humans, for example, can hinder extrapolation in medical research. Mice that carry a partial or complete human physiological system, known as humanized mice, are supposed to make extrapolation more reliable by simulating a variety of human diseases. But what justifies our belief that these mice are similar enough to their human counterparts to simulate human disease? I argue that, unless three requirements are met in the process of humanizing mice, very little does. My requirements are not meant to provide necessary and sufficient conditions that guarantee a particular outcome. Instead, they serve as a heuristic for guiding scientific judgments involving extrapolation. In developing each requirement, I engage with philosophical issues concerning the nature of model-based science and the mechanistic approach (and its limits) to making generalizations in the life sciences.     

Numerical approaches to lake classification with special reference to macrophyte communities

Summary Various approaches to the classification of S. Swedish lakes on the basis of their macrophyte composition are discussed. A main division is made between species or life-form, and synusia or community approaches. Three steps in the classification are discussed: primary clustering of sites to arrive at a local classification, secondary classification of clusters of sites to arrive at general community types and classification of lakes according to their community composition.The estimation of species performance is discussed and a logarithmic transformation advocated. The analysis of belt transects is suggested as a satisfactory general approach.Some problems regarding synusial independence and possible synusial interactions are mentioned. A survey of boundary types in lakes is given.Examples are given of classifications based on single life-forms and life-form combinations. Helophyte community composition of lakes is effectively shown by a combination of classification and ordination. The idea of aquatic community complexes is discussed with reference to the recent sigma-syntaxon approach in phytosociology.Nomenclature follows Lid (1974).     

Targeting drugs to APJ receptor: From signaling to pathophysiological effects

G-protein-coupled receptors (GPCRs) are recognized as the largest protein receptor superfamily, which are widely distributed in various tissues and organs. In addition, GPCRs are involved in many physiological and pathological longitudinal responses. Studies have indicated that putative receptor protein related to AT1 (APJ receptor) is an orphan GPCRs until its endogenous ligand apelin is found. Recently, Elabela, a new APJ receptor endogenous ligand was also found. Some evidence showed that the APJ receptor is distributed in the central nervous and cardiovascular systems. Moreover, the APJ receptor and its ligand are involved in many physiological functions and pathophysiological effects, making it a promising drug target for future treatment of diseases such as ischemic heart disease, hypertension, heart failure, and others. Although APJ is closely associated with many diseases, there are no drugs that can activate or inhibit APJ directly. In the current review, we try our best to summarize all agonists and antagonists targeting APJ, including peptides and small molecules. Given the role of apelin/APJ and Elabela/APJ in cardiovascular and other diseases, we believe that the combination of these agonists and antagonists with apelin and Elabela will play a corresponding role in various pathophysiological effects with further development of research.     

Modelling the work to be done by Escherichia coli to adapt to sudden temperature upshifts

AIMS: This paper studies and models the effect of the amplitude of a sudden temperature upshift DeltaT on the adaptation period of Escherichia coli, in terms of the work to be done by the cells during the subsequent lag phase (i.e., the product of growth rate mumax and lag phase duration lambda). METHODS AND RESULTS: Experimental data are obtained from bioreactor experiments with E. coli K12 MG1655. At a predetermined time instant during the exponential growth phase, a sudden temperature upshift is applied (no other environmental changes take place). The length of the (possibly) induced lag phase and the specific growth rate after the shift are quantified with the growth model of Baranyi and Roberts (Int J Food Microbiol 23, 1994, 277). Different models to describe the evolution of the product lambda x mumax as a function of the amplitude of the temperature shift are statistically compared. CONCLUSIONS: The evolution of lambda x mumax is influenced by the amplitude of the temperature shift DeltaT and by the normal physiological temperature range. As some cut-off is observed, the linear model with translation is preferred to describe this evolution. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to the characterization of microbial lag phenomena, in this case for E. coli K12 MG1655, in view of accurate predictive model building.     

Coming to grips with integrin binding to ligands

Integrins are alphabeta heterodimeric cell-surface receptors that are vital to the survival and function of nucleated cells. They recognize aspartic-acid- or a glutamic-acid-based sequence motifs in structurally diverse ligands. Integrin recognition of most ligands is divalent cation dependent and conformationally sensitive. In addition to this common property, there is an underlying binding specificity between integrins and ligands for which there has been no structural basis. The recently reported crystal structures of the extracellular segment of an integrin in its unliganded state and in complex with a prototypical Arg-Gly-Asp (RGD) ligand have provided an atomic basis for cation-mediated binding of aspartic-acid-based ligands to integrins. They also serve as a basis for modelling other integrins in complex with larger physiologic ligands. These models provide new insights into the molecular basis for ligand binding specificity in integrins and its regulation by activation-driven tertiary and quaternary changes.     

Using Hoechst 33342 to target radioactivity to the cell nucleus

We have explored the use of Hoechst 33342 (H33342) to carry radioactivity to the cell nucleus. H33342 enters cells and targets DNA at adenine-thymine-rich regions of the minor groove. Considerable membrane blebbing and ruffling occur in CHO cells within minutes after its addition to the culture medium in micromolar quantities. Blue vesicles are apparent in the cell cytoplasm, and by 30 min the nuclei are stained dark blue. Upon its binding to DNA, a visible emission shift of the dye can be observed with fluorescence microscopy. We have radioiodinated (125I) H33342 and specifically irradiated nuclear DNA by incubating CHO cells with 125I-H33342 at 37 degrees C and accumulating 125I decays at -90 degrees C. At various times, the cells are thawed and assayed for survival (clonogenicity) and DSB (gamma-H2AX) formation. 125I-H33342 decay leads to a monoexponential decrease in cell survival with a D0 of 122 125I decays per cell and a linear increase in DNA DSB induction (equivalent to 15 gamma-H2AX foci/cell). Cell death is not modified by the radioprotective effects of H33342 because we use considerably lower concentrations than those that provide a slight protection against gamma radiation. We conclude that cell killing by 125I-H33342 and the induction of gamma-H2AX foci are highly correlated.     

Biological progression from adult bone marrow to mononucleate muscle stem cell to multinucleate muscle fiber in response to injury

Adult bone marrow-derived cells (BMDC) are shown to contribute to muscle tissue in a step-wise biological progression. Following irradiation-induced damage, transplanted GFP-labeled BMDC become satellite cells: membrane-ensheathed mononucleate muscle stem cells. Following a subsequent exercise-induced damage, GFP-labeled multinucleate myofibers are detected. Isolated GFP-labeled satellite cells are heritably myogenic. They express three characteristic muscle markers, are karyotypically diploid, and form clones that can fuse into multinucleate cells in culture or into myofibers after injection into mouse muscles. These results suggest that two temporally distinct injury-related signals first induce BMDC to occupy the muscle stem cell niche and then to help regenerate mature muscle fibers. The stress-induced progression of BMDC to muscle satellite cell to muscle fiber results in a contribution to as many as 3.5% of muscle fibers and is due to developmental plasticity in response to environmental cues.     

Elatinaceae are sister to Malpighiaceae; Peridiscaceae belong to Saxifragales

Phylogenetic data from plastid (ndhF and rbcL) and nuclear (PHYC) genes indicate that, within the order Malpighiales, Elatinaceae are strongly supported as sister to Malpighiaceae. There are several putative morphological synapomorphies for this clade; most notably, they both have a base chromosome number of X = 6 (or some multiple of three or six), opposite or whorled leaves with stipules, unicellular hairs (also uniseriate in some Elatinaceae), multicellular glands on the leaves, and resin (Elatinacae) or latex (Malpighiaceae). Further study is needed to determine if these features are synapomorphic within the order. Malpighiaceae have previously been inferred as sister to Peridiscaceae based on rbcL sequence data, but the rbcL sequence of Whittonia is a chimera of two sequences, neither of which appears to be Whittonia. Our data from plastid (atpB, rbcL) and nuclear (18S rDNA) genes instead place Peridiscaeace as a member of the Saxifragales.     

Improvement to the air-injection technique to estimate xylem vulnerability to cavitation

Several techniques have been developed to quantify the degree of embolism of the xylem using hydraulic conductance. Although there have been several improvements to these techniques, their reliability is still questionable and many technical pitfalls persist. We are proposing here a manometric approach to improve the accuracy of xylem cavitation measurement by the original air-injection technique which uses twigs exposed to pressurized air to cause cavitation. The measured parameter is air bubble production (P _b) caused by xylem cavitation in birch (Betula pendula Roth) twigs from which the percent increase in bubble production is calculated to quantify xylem cavitation. Data produced by three different methods (bench-drying, air-injection, and manometric approach) are compared. Xylem vulnerability curves (VCs) constructed by the reference and reliable bench-drying technique and the manometric approach show similar sigmoid “S” shape, but a small anomaly appeared in the VC constructed by the original air-injection technique. The xylem pressure inducing 50% of embolism (P ₅₀) was the same with the three techniques. Furthermore, there was a strong positive correlation between the estimators of xylem cavitation measured by the three different methods. For its reliability, precision and ease we recommend the manometric technique as an improved version of the original hydraulic air-injection method.     

Legal obligation of physicians to disclose information to patients.

Obtaining a patient''s consent is a routine daily process for physicians, although many are unaware of the scope of this legal obligation. In 1980 the Supreme Court of Canada changed the law relating to informed consent; promotion of patient autonomy shifted the focus from a standard of professional disclosure to one of a "reasonable patient." Physicians have a legal obligation to disclose to patients specific information, the scope of which is determined by a court on the basis of a reasonable patient''s expectation and the circumstances of the case. This gives rise to many controversies in the practice of clinical medicine. It is difficult for physicians to know which treatment risks require disclosure, since this is decided by a court in a retrospective analysis of the evidence. Will the court recognize exceptions to the duty of disclosing information? If several health care professionals are involved in a patient''s care who has the duty to disclose information? Can this duty be delegated? This paper provides physicians with guidelines that are consistent with the promotion of patient autonomy and comply with the doctrine of informed consent. In addition, it suggests ways of improving awareness of the doctrine and procedures to ease its application.     

Leishmania amastigotes: resistance to complement-mediated lysis is not due to a failure to fix C3

Amastigote forms of Leishmania major are sensitive to lysis by fresh serum, whereas those of L. donovani are resistant. To understand the basis for this resistance we have examined the interaction of complement with amastigotes of seven strains of leishmania. Complement activation was determined by measuring the ability of amastigotes to consume complement from normal serum and by identifying parasite surface-bound C3. All of the strains that were tested activated complement, including both those that are resistant and those that are susceptible to inactivation by fresh serum. Complement consumption by amastigotes was measured as a decrease in the ACH 50 titers of serum exposed to parasites. L. major, L. donovani, and L. mexicana mexicana (strain 1VLM) amastigotes decrease titers by 35.7, 33.5, and 40.3%, respectively. The binding of C3 to amastigotes was judged qualitatively by immunofluorescence and quantitatively by a C3 radiobinding assay. L. major amastigotes bind an average of 6.6 X 10(4) molecules of C3 per parasite. L. mexicana amazonensis, L. mexicana mexicana, and L. donovani bind an average of 3.9 X 10(4), 5.9 X 10(4), and 3.7 X 10(4) molecules, respectively. In all cases, C3 binding is the result of alternative pathway activation requiring Mg++ but not Ca++. Amastigotes of the disseminating strains of leishmania represent the first example of a group of protozoa that activate early complement components leading to fixation of C3, but that are resistant to inactivation by complement.     

INCENP binds directly to tubulin and requires dynamic microtubules to target to the cleavage furrow

Inner centromere protein (INCENP) is a chromosomal passenger protein with an essential role in mitosis. At the metaphase/anaphase transition, some INCENP transfers from the centromeres to the central spindle; the remainder then transfers to the equatorial cortex prior to cleavage furrow formation. The molecular associations dictating INCENP behavior during mitosis are currently unknown. Here we show that targeting INCENP to the cleavage plane requires dynamic microtubules, but not F-actin. When microtubules are eliminated, INCENP is dispersed across the entire cell cortex. Yeast two-hybrid and in vitro binding data demonstrate that INCENP binds directly to beta-tubulin via a conserved domain encompassing residues 48-85. Furthermore, INCENP binds to microtubules polymerized from purified tubulin in vitro and appears to bundle microtubules when expressed in the interphase cytoplasm. These data indicate that INCENP is a microtubule-binding protein that targets to the equatorial cortex through interactions requiring microtubules.     

Proteins containing the UBA domain are able to bind to multi-ubiquitin chains

The UBA domain is a motif found in a variety of proteins, some of which are associated with the ubiquitin-proteasome system. We describe the isolation of a fission-yeast gene, mud1+, which encodes a UBA domain containing protein that is able to bind multi-ubiquitin chains. We show that the UBA domain is responsible for this activity. Two other proteins containing this motif, the fission-yeast homologues of Rad23 and Dsk2, are also shown to bind multi-ubiquitin chains via their UBA domains. These two proteins are implicated, along with the fission-yeast Pus1(S5a/Rpn10) subunit of the 26 S proteasome, in the recognition and turnover of substrates by this proteolytic complex.