Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli.

The construction and purification of recombinant baculovirus vectors for the expression of foreign genes in insect cells by standard transfection and plaque assay methods can take as long as 4 to 6 weeks. This period can be reduced to several days by using a novel baculovirus shuttle vector (bacmid) that can replicate in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. The bacmid is a recombinant virus that contains a mini-F replicon, a kanamycin resistance marker, and attTn7, the target site for the bacterial transposon Tn7. Expression cassettes comprising a baculovirus promoter driving expression of a foreign gene that is flanked by the left and right ends of Tn7 can transpose to the target bacmid in E. coli when Tn7 transposition functions are provided in trans by a helper plasmid. The foreign gene is expressed when the resulting composite bacmid is introduced into insect cells.     

Anthramycin inhibition of restriction endonuclease cleavage and its use as a reversible blocking agent in DNA constructions.

Anthramycin can form a stable complex with DNA which does not dissociate upon repeated ethanol precipitations. The complex forms in less than one hour at pH 5.5. Bound anthramycin seems to be located in the minor groove of the DNA helix in the anthramycin DNA complex, since methylation of adenosine residues at N-3 by dimethylsulfate is reduced. The anthramycin-DNA complex is resistant to digestion by an excess of a number of restriction enzymes. Anthramycin can be removed from DNA by incubation at acid pH. The released DNA can then be cleaved by restriction enzymes. Anthramycin-DNA complexes can be acted upon by T4 polynucleotide ligase to form longer DNA molecules. The ability of anthramycin to form a stable but reversible complex which is not cleaved by restriction enzymes but can engage in joining reactions may allow a wider variety of DNA fragments to be more readily constructed in vitro.     

What is this stuff called fitness?

This paper considers a variety of attempts to define fitness in such a way as to defend the theory of evolution by natural selection from the criticism that it is a circular argument. Each of the definitions is shown to be inconsistent with the others. The paper argues that the environment in which an animal evolves can be defined only with respect to the properties of the phenotype of the animal and that it is therefore not illuminating to try to explain the phenotypic properties of the animal in terms of adaptation to an environment that is defined by those very properties. Furthermore, since there is no way that the environment can be defined independently of the presence of the animal there is no way that the quality of an animal can be assessed; and there can be no objective criteria by whichany form of selection can be carried out, therefore there can be no criteria by whichnatural selection can be carried out. It is proposed that fitness is nothing more than the production of offspring, that this is a phenotypic property like all the others, and if it is heritable then the offspring of the parents that produce the most offspring will themselves produce the most offspring, and that in principle it is impossible to account for this in terms of the other phenotypic properties of the fittest animals except by circular argument. Differential rates of reproduction are the causes of evolution and the phenotypic causes are strictly inexplicable.     

Visual analysis of trabecular bone structure

Acquiring image data of bone biopsies by a micro-CT scanner is today a common technique. The amount of data to be assessed is huge. The task to assess quantitative measures requires a concise visualization. We present visualization techniques that can be used interactively on state-of-the-art PCs and demonstrate how the frontier can be pushed further. A skeletonization process is applied to the image of the bone to create the central surface. After triangulation this surface can be renderd at interactive frame rates. When the surface is additionally colored by local measures (mean grey value of image data, local thickness) the overall structure and details can be recognized at the same time. This can facilitate the exploration of the biopsy and can help finding special features.     

Use of bioluminescence in nucleic acid hybridization reactions

Luminescence reactions can be used to detect specific nucleic acid sequences hybridized with a nucleic probe. Different labels such as cytidine sulphone, fluorescein, and biotin can be incorporated into DNA or oligonucleotide molecules and detected by antibody or avidin conjugates coupled to glucose-6P dehydrogenase. On supports such as nitrocellulose filters, sensitivity is not greatly increased using luminescence, but detection is rapid and easy to perform using polaroid film. Moreover, hybridization can be performed with different labelled probes on the same sample. In solution, luminescence can be used to monitor sandwich reactions. The method is less sensitive than detection on filters but can easily be automated. The performance of these assays can be increased considerably by enzymatic amplification of the target catalysed by a thermostable polymerase.     

A new procedure for enzymatic semisynthesis of human insulin by hydrolysis of single-chain des-(b-30)-lnsulin precursor with lysyl endopeptidase

It has been shown that the single-chain des-(B-30)-insulin precursor (SCI) can be converted into human insulin ester by transpeptidation using trypsin in the presence of a threonine derivative. The present study demonstrates that Achromobacter lyticus protease 1 (lysyl endopeptidase) can catalyze the transpeptidation reaction more efficiently than can trypsin. It is also shown that des-(B-30)-insulin (DAI) can be produced by hydrolysis of SCI with the lysyl endopeptidase. Since it is well known that SCI can be produced by gene technology, the following method is recommended for industrial production of human insulin ester: hydrolysis of SCI with lysyl endopeptidase followed by coupling of the resulting DAI with a threonine derivative using trypsin or lysyl endopeptidase.     

Effects of a fecapentaene on protein kinase C

Protein kinase C (PKC) is a Ca2(+)- and phospholipid-dependent serine and threonine protein kinase which binds and is activated by tumor promoters such as the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA). PKC can be activated in vitro by phosphatidylserine (PS) plus Ca2+. We report here that the compound fecapentaene-12 can replace the requirement for PS in the activation of PKC by Ca2+. In addition, at low concentrations fecapentaene-12 can enhance the activation of PKC by Ca2+ and PS. It can also either enhance or inhibit activation of PKC by the tumor promoter teleocidin, depending on the assay conditions. These results are of interest since fecapentaene is known to be a potent mutagen that is produced by Bacteroides species present in the lumen of the human colon. The present studies raise the possibility that this compound might also play a role in colon cancer by altering the activity of PKC.     

Biomimetics of Campaniform Sensilla: Measuring Strain from the Deformation of Holes

We present a bio-inspired strategy for designing embedded strain sensors in space structures. In insects, the campaniform sensillum is a hole extending through the cuticle arranged such that its shape changes in response to loads. The shape change is rotated through 90 by the suspension of a bell-shaped cap whose deflection is detected by a cell beneath the cuticle. It can be sensitive to displacements of the order of 1 nm. The essential morphology, a hole formed in a plate of fibrous composite material, was modelled by Skordos et al. who showed that global deformation of the plate (which can be flat, curved or a tube) induces higher local deformation of the hole due to its locally higher compliance. Further developments reported here show that this approach can be applied to groups of holes relative to their orientation.The morphology of the sensillum in insects suggests that greater sensitivity can be achieved by arranging several holes in a regular pattern; that if the hole is oval it can be "aimed" to sense specific strain directions; and that either by controlling the shape of the hole or its relationship with other holes it can have a tuned response to dynamic strains.We investigate space applications in which novel bio-inspired strain sensors could successfully be used.     

Biomimetics of Campaniform Sensilla： Measuring Strain from the Deformation of Holes

We present a bio-inspired strategy for designing embedded strain sensors in space structures. In insects, the campaniform sensillum is a hole extending through the cuticle arranged such that its shape changes in response to loads. The shape change is rotated through 90° by the suspension of a bell-shaped cap whose deflection is detected by a cell beneath the cuticle. It can be sensitive to displacements of the order of 1 nm. The essential morphology, a hole formed in a plate of fibrous composite mate- rial, was modelled by Skordos et al. who showed that global deformation of the plate （which can be flat, curved or a tube） induces higher local deformation of the hole due to its locally higher compliance. Further developments reported here show that this approach can be applied to groups of holes relative to their orientation. , The morphology of the sensillum in insects suggests that greater sensitivity can be achieved by arranging several holes in a regular pattern; that if the hole is oval it can be ＂aimed＂ to sense specific strain directions; and that either by controlling the shape of the hole or its relationship with other holes it can have a tuned response to dynamic strains. We investigate space applications in which novel bio-inspired strain sensors could successfully be used.     

Neural Coding with Graded Membrane Potential Changes and Spikes

The neural encoding of sensory stimuli is usually investigated for spike responses, although many neurons are known to convey information by graded membrane potential changes. We compare by model simulations how well different dynamical stimuli can be discriminated on the basis of spiking or graded responses. Although a continuously varying membrane potential contains more information than binary spike trains, we find situations where different stimuli can be better discriminated on the basis of spike responses than on the basis of graded responses. Spikes can be superior to graded membrane potential fluctuations if spikes sharpen the temporal structure of neuronal responses by amplifying fast transients of the membrane potential. Such fast membrane potential changes can be induced deterministically by the stimulus or can be due to membrane potential noise that is influenced in its statistical properties by the stimulus. The graded response mode is superior for discrimination between stimuli on a fine time scale.     

Cooperative Regulation of Cellular Proliferation by Intercellular Diffusion

A theoretical model for the cooperative control of cellular kinetics is investigated. A critical substance A is produced by the cells whose concentration in a given cell determines whether that cell can divide. The substance A can leak out of the cells into the surrounding medium as well as be reabsorbed by the cells. This feature then implies communication between the cells since all concentrations will be functions of the population density. The substance A also has a lifetime, i.e. decays, for example, by denaturation. This system can be described by three coupled nonlinear differential equations which can be solved analytically in certain limiting cases and can, of course, be studied in detail by computer techniques. Our investigations have shown that (a) there is a critical initial cell population density below which cell proliferation will not occur, (b) cell proliferation can be stimulated by supplying substance A to the medium and there is a critical initial concentration in the medium for initiating proliferation when the cell population density is subcritical, and (c) a well-defined induction period prior to exponential growth may exist whose length depends on the system parameters and initial conditions.     

Serum amyloid A and protein AA: Molecular mechanisms of a transmissible amyloidosis

Systemic AA-amyloidosis is a complication of chronic inflammatory diseases and the fibril protein AA derives from the acute phase reactant serum AA. AA-amyloidosis can be induced in mice by an inflammatory challenge. The lag phase before amyloid develops can be dramatically shortened by administration of a small amount of amyloid fibrils. Systemic AA-amyloidosis is transmissible in mice and may be so in humans. Since transmission can cross species barriers it is possible that AA-amyloidosis can be induced by amyloid in food, e.g. foie gras. In mice, development of AA-amyloidosis can also be accelerated by other components with amyloid-like properties. A new possible risk factor may appear with synthetically made fibrils from short peptides, constructed for tissue repair.     

An efficient and site-specific gene trimming method

Trimming a DNA strand into a precisely determined fragment can be carried out efficiently by an improved method involving a site-specific trim-primer and a single-stranded DNA template which is generated from a multifunctional vector, pTZ18R, and linearized by using an Eco RI-pTZ18R splinter. A complementary DNA strand is synthesized by DNA polymerase I (Klenow), and the 3'-end of the template upstream from the annealed primer is trimmed by subsequent T4 DNA polymerase reaction. An ATG translation initiator codon or a termination codon can be incorporated into the trim-primer, providing versatility to this single-stranded DNA-initiated gene trimming method that can be applied to subcloning and expression of any DNA fragment with known terminal sequences.     

On the theory of noncovalent binding

It is widely accepted that the binding constant of a receptor and ligand can be written as a two-body integral involving the interaction energy of the receptor and the ligand. Interestingly, however, three different theories of binding in the literature dictate three distinct integrals. The present study uses theory, as well as simulations of binding experiments, to test the validity of the three integrals. When binding is measured by a signal that detects the ligand in the binding site, the most accurate results are obtained by an integral of the Boltzmann factor, where the bound complex is defined in terms of an exclusive binding region. A novel prediction of this approach, that expanding a ligand can increase its binding constant, is borne out by the simulations. The simulations also show that abnormal binding isotherms can be obtained when the region over which the signal is detected deviates markedly from the exclusion zone. Interestingly, the binding constant measured by equilibrium dialysis, rather than by monitoring a localized signal, can yield a binding constant that differs from that obtained from a signal measurement, and that is matched best by the integral of the Mayer factor.     

The stimulation of human synovial fibroblast plasminogen activator activity. Involvement of cyclic AMP and cyclooxygenase products

The plasminogen activator activity of human synovial fibroblasts is raised by a monocyte-derived polypeptide, synovial activator and also by all-trans retinoic acid. The elevation of the synovial cell plasminogen activator activity by the two stimuli is potentiated both by agents which can raise cellular cyclic AMP levels, namely prostaglandin E2, cholera toxin and 3-isobutyl-1-methylxanthine, and also by exogenous 8-bromocyclic AMP. These findings suggest that there might be a substrate, which is phosphorylated by a cyclic AMP-dependent protein kinase and which is important in the modulation of the synovial cell plasminogen activator activity by the two stimuli. Prostanoids can be important in the stimulation of the synovial fibroblast plasminogen activator activity by mononuclear cell supernatants, since indomethacin can inhibit the increase in proteinase activity.     

Fluorescence lifetime distributions in proteins.

The fluorescence lifetime value of tryptophan residues varies by more than a factor of 100 in different proteins and is determined by several factors, which include solvent exposure and interactions with other elements of the protein matrix. Because of the variety of different elements that can alter the lifetime value and the sensitivity to the particular environment of the tryptophan residue, it is likely that non-unique lifetime values result in protein systems. The emission decay of most proteins can be satisfactorily described only using several exponential components. Here it is proposed that continuous lifetime distributions can better represent the observed decay. An approach based on protein dynamics is presented, which provides fluorescence lifetime distribution functions for single tryptophan residue proteins. First, lifetime distributions for proteins interconverting between two conformations, each characterized by a different lifetime value, are derived. The evolution of the lifetime values as a function of the interconversion rate is studied. In this case lifetime distributions can be obtained from a distribution of rates of interconversion between the two conformations. Second, the existence of a continuum of energy substates within a given conformation was considered. The occupation of a particular energy substate at a given temperature is proportional to the Boltzmann factor. The density of energy states of the potential well depends upon the width of the well, which determines the degree of freedom the residue can move in the conformational space. Lifetime distributions can be obtained by association of each energy substate with a different lifetime value and assuming that the average conformation can change as the energy of the substate is increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dendritic hold and read: a gated mechanism for short term information storage and retrieval

Two contrasting theories have been proposed to explain the mechanistic basis of short term memory. One theory posits that short term memory is represented by persistent neural activity supported by reverberating feedback networks. An alternate, more recent theory posits that short term memory can be supported by feedforward networks. While feedback driven memory can be implemented by well described mechanisms of synaptic plasticity, little is known of possible molecular and cellular mechanisms that can implement feedforward driven memory. Here we report such a mechanism in which the memory trace exists in the form of glutamate-bound but Mg(2+)-blocked NMDA receptors on the thin terminal dendrites of CA1 pyramidal neurons. Because glutamate dissociates from subsets of NMDA receptors very slowly, excitatory synaptic transmission can leave a silent residual trace that outlasts the electrical activity by hundreds of milliseconds. Read-out of the memory trace is possible if a critical level of these bound-but-blocked receptors accumulates on a dendritic branch that will allow these quasi-stable receptors to sustain a regenerative depolarization when triggered by an independent gating signal. This process is referred to here as dendritic hold and read (DHR). Because the read-out of the input is not dependent on repetition of the input and information flows in a single-pass manner, DHR can potentially support a feedforward memory architecture.     

The function of melanin or six blind people examine an elephant.

The pigment melanin is found in all living kingdoms and in many different structures and forms. When its various functions are examined separately, its behaviors seem disparate and conflicting. It has a clear role in camouflage and sexual display. Other major roles are examined critically. It can act as a sun screen but is not a very effective one. It can also scavenge active chemical species, but this, too, is not done very effectively. It produces active radicals that can damage DNA. It binds to drugs in ways that are either beneficial or deleterious. Aside from camouflage, its other roles can be brought together by a unifying hypothesis as first proposed by Proctor and McGinness nearly 20 years ago. Melanin is envisaged as an energy transducer with the properties of an amorphous semiconductor. It can absorb many different types of energy and dissipate them in the form of heat. However, if the energy input is too great, the output can be expressed in the form of activated chemical species that can damage cellular macromolecules resulting in cell death, mutations and cancer. The protective aspect of melanin in dark skin is seen as resulting from its high concentration and its confinement to ellipsoidal and densely packed organelles that can effectively shield the nucleus. In light skin, its radical nature is seen as potentially participating in the carcinogenic process, particularly when overwhelmed by intense episodes of sunburn.     

Spontaneous action potentials due to channel fluctuations.

A theoretical and numerical analysis of the Hodgkin-Huxley equations with the inclusion of stochastic channel dynamics is presented. It is shown that the system can be approximated by a one-dimensional bistable Langevin equation. Spontaneous action potentials can arise from the channel fluctuations and are analogous to escape by a particle over a potential barrier. The mean firing rate can be calculated using Kramers' classic result for barrier escape. The probability density function of the interspike intervals can also be estimated. The analytical results compare favorably with numerical simulations of the complete stochastic system.