Social interactions between the rural and urban monkeys,Macaca mulatta

Social interactions between urban and rural monkeys were studied both in competitive and non-competitive situations. In a competitive foodgetting situation, the urban monkeys dominated the rural ones to a significant degree. In contrast, in all the test situations the urban animals were found to be highly aggressive in their interactions with those of the rural group. Dominance of the urban over the rural monkeys was interpreted as the result of a highly aggressive nature acquired from their prior exposure to the highly competitive environment prevailing in urban areas.This study was concluded at the Panjab University, Chandigarh, India, and was partly financed by NIH grant M-5263. I am thankful to Professor Harry F. Harlow for initiating me into this project.     

Directed Evolution of Insoluble Arabidopsis thaliana Zeta Class Glutathione S-Transferase Mutants for Higher Solubility in Escherichia coli

Expression of recombinant protein in Escherichia coli (E.coli) is generally considered as one of the ideal systems to produce proteins for industrial production.However,the majority of proteins usually fail to fold into their native state and accumulate as insoluble inclusion bodies with no biological activity in E.coli (Yang et al.,2003).Although numerous strategies and approaches are proposed to solve the problem (Nygaard and Harlow,2001;Mogk et al.,2002;Austin,2003),they still fail to improve the solubility of protein and are not ideal for high-throughput applications (Waldo,2003).Furthermore,with the expression condition becoming stricter,the procedures become more complex and the costs grow higher,thus making them inappropriate for application in industrial production.     

Cloning of a guanosine-inosine kinase gene of Escherichia coli and characterization of the purified gene product.

We attempted to clone an inosine kinase gene of Escherichia coli. A mutant strain which grows slowly with inosine as the sole purine source was used as a host for cloning. A cloned 2.8-kbp DNA fragment can accelerate the growth of the mutant with inosine. The fragment was sequenced, and one protein of 434 amino acids long was found. This protein was overexpressed. The overexpressed protein was purified and characterized. The enzyme had both inosine and guanosine kinase activity. The Vmaxs for guanosine and inosine were 2.9 and 4.9 mumol/min/mg of protein, respectively. The Kms for guanosine and inosine were 6.1 microM and 2.1 mM, respectively. This enzyme accepted ATP and dATP as a phosphate donor but not p-nitrophenyl phosphate. These results show clearly that this enzyme is not a phosphotransferase but a guanosine kinase having low (Vmax/Km) activity with inosine. The sequence of the gene we have cloned is almost identical to that of the gsk gene (K.W. Harlow, P. Nygaard, and B. Hove-Jensen, J. Bacteriol. 177:2236-2240, 1995).     

Modeling menstrual cycle length using a mixture distribution

In reproductive health studies, epidemiologists are often interested in examining the effects of covariates on menstrual cycle length which is a convenient, noninvasive measure of women's ovarian and reproductive function. Previous literature (Harlow and Zeger, 1991) suggests that the distribution of cycle length is a mixture of a major symmetric distribution and a component featuring a long right tail. Motivated by the shape of this marginal distribution, we propose a mixture distribution for cycle length, representing standard cycles from a Normal distribution and nonstandard cycles from a shifted Weibull distribution. The parameters are estimated using an estimating equation derived from the score function of an independence working model. The fitted mixture distribution agrees well with the distribution estimated using nonparametric approaches. We propose two measures to help determine whether a cycle is standard or nonstandard, developing tools necessary to identify characteristics of the menstrual cycles that are biologically indicative of ovarian dysfunction. We model the effect of a woman's age on the mean and variation of both standard and nonstandard cycle lengths using multiple measurements of women.     

Validation of a Static Franz Diffusion Cell System for <Emphasis Type="Italic">In Vitro</Emphasis> Permeation Studies

Over the years, in vitro Franz diffusion experiments have evolved into one of the most important methods for researching transdermal drug administration. Unfortunately, this type of testing often yields permeation data that suffer from poor reproducibility. Moreover, this feature frequently occurs when synthetic membranes are used as barriers, in which case biological tissue-associated variability has been removed as an artefact of total variation. The objective of the current study was to evaluate the influence of a full-validation protocol on the performance of a tailor-made array of Franz diffusion cells (GlaxoSmithKline, Harlow, UK) available in our laboratory. To this end, ibuprofen was used as a model hydrophobic drug while synthetic membranes were used as barriers. The parameters investigated included Franz cell dimensions, stirring conditions, membrane type, membrane treatment, temperature regulation and sampling frequency. It was determined that validation dramatically reduced derived data variability as the coefficient of variation for steady-state ibuprofen permeation from a gel formulation was reduced from 25.7% to 5.3% (n = 6). Thus, validation and refinement of the protocol combined with improved operator training can greatly enhance reproducibility in Franz cell experimentation.     

Mechanics and control of the cytoskeleton in Amoeba proteus.

Many models of the cytoskeletal motility of Amoeba proteus can be formulated in terms of the theory of reactive interpenetrating flow (Dembo and Harlow, 1986). We have devised numerical methodology for testing such models against the phenomenon of steady axisymmetric fountain flow. The simplest workable scheme revealed by such tests (the minimal model) is the main preoccupation of this study. All parameters of the minimal model are determined from available data. Using these parameters the model quantitatively accounts for the self assembly of the cytoskeleton of A. proteus: for the formation and detailed morphology of the endoplasmic channel, the ectoplasmic tube, the uropod, the plasma gel sheet, and the hyaline cap. The model accounts for the kinematics of the cytoskeleton: the detailed velocity field of the forward flow of the endoplasm, the contraction of the ectoplasmic tube, and the inversion of the flow in the fountain zone. The model also gives a satisfactory account of measurements of pressure gradients, measurements of heat dissipation, and measurements of the output of useful work by amoeba. Finally, the model suggests a very promising (but still hypothetical) continuum formulation of the free boundary problem of amoeboid motion. by balancing normal forces on the plasma membrane as closely as possible, the minimal model is able to predict the turgor pressure and surface tension of A. proteus. Several dynamical factors are crucial to the success of the minimal model and are likely to be general features of cytoskeletal mechanics and control in amoeboid cells. These are: a constitutive law for the viscosity of the contractile network that includes an automatic process of gelation as the network density gets large; a very vigorous cycle of network polymerization and depolymerization (in the case of A. proteus, the time constant for this reaction is approximately 12 s); control of network contractility by a diffusible factor (probably calcium ion); and control of the adhesive interaction between the cytoskeleton and the inner surface of the plasma membrane.     

The mechanics of motility in dissociated cytoplasm.

We stimulate the dynamical behavior of dissociated cytoplasm using the Reactive Flow Model (Dembo, M., and F. Harlow, 1986, Biophys. J., 50:109-121). We find that for the most part the predicted dynamical behavior of the cytoplasm is governed by three nondimensional numbers. Several other nondimensional parameters, the initial conditions, and boundary conditions are found to have lesser effects. Of the three major nondimensional parameters, one (D#) controls the percentage of ectoplasm, the second (C#) controls the sharpness of the endoplasm-ectoplasm boundary, and the third (R#) controls the topological complexity of the endoplasm-ectoplasm distribution. If R# is very small, then the cytoplasm contracts into a single uniform mass, and there is no bulk streaming. If R# is very large, then the cytoplasmic mass breaks up into a number of clumps scattered throughout the available volume. Between these clumps the solution undergoes turbulent or chaotic patterns of streaming. Intermediate values of R# can be found such that the mass of cytoplasm remains connected and yet undergoes coherent modes of motility similar to flares (Taylor, D.L., J.S. Condeelis, P.L. Moore, and R.D. Allen, 1973, J. Cell Biol., 59:378-394) and rosettes (Kuroda, K., 1979, Cell Motility: Molecules and Organization, 347-362).     

Requirement of Cyclin E-Cdk2 Inhibition in p16INK4a-Mediated Growth Suppression

Loss-of-function mutations of p16^INK4a have been identified in a large number of human tumors. An established biochemical function of p16 is its ability to specifically inhibit cyclin D-dependent kinases in vitro, and this inhibition is believed to be the cause of the p16-mediated G₁ cell cycle arrest after reintroduction of p16 into p16-deficient tumor cells. However, a mutant of Cdk4, Cdk4^N158, designed to specifically inhibit cyclin D-dependent kinases through dominant negative interference, was unable to arrest the cell cycle of the same cells (S. van den Heuvel and E. Harlow, Science 262:2050–2054, 1993). In this study, we determined functional differences between p16 and Cdk4^N158. We show that p16 and Cdk4^N158 inhibit the kinase activity of cellular cyclin D1 complexes through different mechanisms. p16 dissociated cyclin D1-Cdk4 complexes with the release of bound p27^KIP1, while Cdk4^N158 formed complexes with cyclin D1 and p27. In cells induced to overexpress p16, a higher portion of cellular p27 formed complexes with cyclin E-Cdk2, and Cdk2-associated kinase activities were correspondingly inhibited. Cells engineered to express moderately elevated levels of cyclin E became resistant to p16-mediated growth suppression. These results demonstrate that inhibition of cyclin D-dependent kinase activity may not be sufficient to cause G₁ arrest in actively proliferating tumor cells. Inhibition of cyclin E-dependent kinases is required in p16-mediated growth suppression.     

Chloroplast‐targeted bacterial RecA proteins confer tolerance to chloroplast DNA damage by methyl viologen or UV‐C radiation in tobacco (Nicotiana tabacum) plants

The nature and importance of the DNA repair system in the chloroplasts of higher plants under oxidative stress or UV radiation‐induced genotoxicity was investigated via gain‐of‐functional approaches exploiting bacterial RecAs. For this purpose, transgenic tobacco (Nicotiana tabacum) plants and cell suspensions overexpressing Escherichia coli or Pseudomonas aeruginosa RecA fused to a chloroplast‐targeting transit peptide were first produced. The transgenic tobacco plants maintained higher amounts of chloroplast DNA compared with wild‐type (WT) upon treatments with methyl viologen (MV), a herbicide that generates reactive oxygen species (ROS) in chloroplasts. Consistent with these results, the transgenic tobacco leaves showed less bleaching than WT following MV exposure. Similarly, the MV‐treated transgenic Arabidopsis plants overexpressing the chloroplast RecA homologue RECA1 showed weak bleaching, while the recA1 mutant showed opposite results upon MV treatment. In addition, when exposed to UV‐C radiation, the dark‐grown E. coli RecA‐overexpressing transgenic tobacco cell suspensions, but not their WT counterparts, resumed growth and greening after the recovery period under light conditions. Measurements of UV radiation‐induced chloroplast DNA damage using DraI assays (Harlow et al. 1994) with the chloroplast rbcL DNA probe and quantitative PCR analyses showed that the transgenic cell suspensions better repaired their UV‐C radiation‐induced chloroplast DNA lesions compared with WT. Taken all together, it was concluded that RecA‐overexpressing transgenic plants are endowed with an increased chloroplast DNA maintenance capacity and enhanced repair activities, and consequently have a higher survival tolerance to genotoxic stresses. These observations are made possible by the functional compatibility of the bacterial RecAs in chloroplasts.     

Cadmium transport, resistance, and toxicity in bacteria, algae, and fungi

Cadmium is an important environmental pollutant and a potent toxicant to bacteria, algae, and fungi. Mechanisms of Cd toxicity and resistance are variable, depending on the organism. It is very clear that the form of the metal and the environment it is studied in, play an important role in how Cd exerts its effect and how the organism(s) responds. A wide range of Cd concentrations have been used to designate resistance in organisms. To date, no concentration has been specified that is applicable to all species studied under standardized conditions. Cadmium exerts its toxic effect(s) over a wide range of concentrations. In most cases, algae and cyanobacteria are the most sensitive organisms, whereas bacteria and fungi appear to be more resistant. In some bacteria, plasmid-encoded resistance can lead to reduced Cd2+ uptake. However, some Gram-negative bacteria without plasmids are just as resistant to Cd as are bacteria containing plasmids encoding for Cd resistance. According to Silver and Misra (1984), there is no evidence for enzymatic or chemical transformations associated with Cd resistance. Insufficient information is available on the genetics of Cd uptake and resistance in cyanobacteria and algae. Mechanisms remain largely unknown at this point in time. Cadmium is toxic to these organisms, causing severe inhibition of such physiological processes as growth, photosynthesis, and nitrogen fixation at concentrations less than 2 ppm, and often in the ppb range (Tables 2 and 3). Cadmium also causes pronounced morphological aberrations in these organisms, which are probably related to deleterious effects on cell division. This may be direct or indirect, as a result of Cd effects on protein synthesis and cellular organelles such as mitochondria and chloroplasts. Cadmium is accumulated internally in algae (Table 4) as a result of a two-phase uptake process. The first phase involves a rapid physicochemical adsorption of Cd onto cell wall binding sites, which are probably proteins and (or) polysaccharides. This is followed by a lag period and then a slow, steady intracellular uptake. This latter phase is energy dependent and may involve transport systems used to accumulate other divalent cations, such as Mn2+ and Ca2+. Some data indicate that Cd resistance, and possibly uptake, in algae and cyanobacteria is controlled by a plasmid-encoded gene(s). Although considerable information is available on Cd toxicity to, and uptake in fungi, further work is clearly needed in several areas. There is little information about Cd uptake by filamentous fungi, and even in yeasts, information on the specificity, kinetics, and mechanisms of Cd uptake is limited.(ABSTRACT TRUNCATED AT 400 WORDS)     

Earth, sky, wind, and weather

This paper seeks to understand what it means to live 'in the open'. It begins with an account of experiments that test whether children have acquired a scientifically correct understanding of the shape of the earth, according to which people live all around on the outside of a solid sphere. This understanding cannot accommodate the phenomenon of the sky, in relation to which the earth can appear only as the ground of human habitation. James Gibson's ecological approach to perception offers a possible alternative, depicting earth and sky as complementary hemispheres. Yet for Gibson, this earth-sky can be inhabited only insofar as it is furnished with objects. To that extent, it ceases to be open. Drawing on elements of Merleau-Ponty's phenomenology, it is argued that in the open world persons and things relate not as closed forms but by virtue of their common immersion in the generative fluxes of the medium – in wind and weather. Fundamental to life is the process of respiration, by which organisms continually disrupt any boundary between earth and sky, binding substance and medium together in forging their own growth and movement. Thus to inhabit the open is not to be stranded on the outer surface of the earth but to be caught up in the transformations of the weather-world.     

Pain, paradox, and value

The author argues that "as soon as one begins to study the understanding and management of pain in science, human medicine, and veterinary medicine, one begins to encounter a variety of apparent paradoxes." He contends that these paradoxes, ten of which he identifies and discusses in this essay, are based on flawed philosophical and valuational assumptions underlying science and medicine. Rollins concludes that, as social morality increasingly has an impact on science, a new ideology will evolve that is more receptive to the moral universe and more capable of a "coherent vision of pain, one which acknowledges that the medical notion of adequacy of anaesthesia is as much a moral as a scientific one."     

LBL, a novel, developmentally regulated, laminin-binding lectin.

A 190/220-kDa complex found in integrin preparations was purified, and monoclonal antibodies were raised against it. The immunoaffinity-purified complex appears to be a trimer of very similar or identical 70-kDa subunits. It is a novel extracellular matrix molecule as determined by its subunit composition, N-terminal amino acid sequence, and in vivo localization. It is distributed widely in basement membranes including those from muscle, nerve, and kidney. It is also present in connective tissue regions such as perineurium and perimysium. It has the unusual property that it is initially expressed very late in avian development near the time of hatching. This protein is found to copurified with integrin because it binds to the carbohydrate support in Sepharose. Hemagglutination assays with mono- and disaccharides show that it functions as a lectin with galactoside-binding specificity. This protein is also found to bind strongly and specifically to laminin at a site distinct from its lectin activity, but does not bind to fibronectin or type IV collagen. The protein appears to be conserved and is a common contaminant of many laminin preparations. We call this novel protein "LBL" for laminin-binding lectin.     

Silverleaf nightshade,Solarium elaeagnifolium,origin, distribution,and relation to man

Silverleaf nightshade (Solanum elaeagnifolium) is a perennial weed that has become increasingly troublesome over the past several decades. Extensive use of soil-applied herbicides, accompanied by a reduction in annual weed competition and reduced tillage, have contributed to its spread and establishment as a serious pest. Crop plants are affected directly via competition and allelopathy or indirectly as the nightshade plants serve as hosts for destructive phytophagous insects or fungal pathogens. Probably native to the southwestern United States and northern Mexico,S. elaeagnifolium is now found in many semiarid regions of the world. Plants were used by the Pima, Kiowa and Navajo Indians in the preparation of food and in the tanning of leather. Containing the toxic glycoalkaloids solanine and solasonine, plants can cause livestock poisonings. The fruits, however, are a source of solasodine, which is used in the commercial manufacture of steroidal hormones.