Advances in broad bandwidth light sources for ultrahigh resolution optical coherence tomography.

Novel ultra-broad bandwidth light sources enabling unprecedented sub-2 microm axial resolution over the 400 nm-1700 nm wavelength range have been developed and evaluated with respect to their feasibility for clinical ultrahigh resolution optical coherence tomography (UHR OCT) applications. The state-of-the-art light sources described here include a compact Kerr lens mode locked Ti:sapphire laser (lambdaC = 785 nm, delta lambda = 260 nm, P(out) = 50 mW) and different nonlinear fibre-based light sources with spectral bandwidths (at full width at half maximum) up to 350 nm at lambdaC = 1130 nm and 470 nm at lambdaC = 1375 nm. In vitro UHR OCT imaging is demonstrated at multiple wavelengths in human cancer cells, animal ganglion cells as well as in neuropathologic and ophthalmic biopsies in order to compare and optimize UHR OCT image contrast, resolution and penetration depth.

Cloning of a catalytic subunit of cAMP-dependent protein kinase from the honeybee (Apis mellifera) and its localization in the brain.

In the honeybee the cAMP-dependent signal transduction cascade has been implicated in processes underlying learning and memory. The cAMP-dependent protein kinase (PKA) is the major mediator of cAMP action. To characterize the PKA system in the honeybee brain we cloned a homologue of a PKA catalytic subunit from the honeybee. The deduced amino acid sequence shows 80-94% identity with catalytic subunits of PKA from Drosophila melanogaster, Aplysia californica and mammals. The corresponding gene is predominantly expressed in the mushroom bodies, a structure that is involved in learning and memory processes. However, expression can also be found in the antennal and optic lobes. The level of expression varies within all three neuropiles.

Reversible downregulation of protein kinase A during olfactory learning using antisense technique impairs long-term memory formation in the honeybee, Apis mellifera.

In this study, we examined the role of cAMP-dependent protein kinase (PKA) in associative olfactory learning of the honeybee, Apis mellifera. In the bee, specific interference with molecules to clarify their role in a certain behavior is difficult, because genetic approaches, such as mutants or transgenic animals, are not feasible at the moment. As a new approach in insects in vivo, we report the use of short antisense oligonucleotides. We show that phosphorothioate-modified oligodeoxynucleotides complementary to the mRNA of a catalytic subunit of PKA directly injected into the bee brain cause a reversible and specific downregulation of both the amount of the catalytic subunit and of PKA activity by 10-15%. The amounts of the regulatory subunit of PKA, as well as PKC, are not affected. The slight "knockdown" of PKA activity during the training procedure, a classical olfactory conditioning of the proboscis extension reflex, neither affects acquisition nor memory retention 3 or 6 hr after training. However, it causes an impairment of long-term memory retention 24 hr after training. Downregulation of PKA 3 hr after training has no detectable effect on memory formation. We conclude that PKA contributes to the induction of a long-term memory 24 hr after training when activated during learning. Second, we demonstrate that the antisense technique is feasible in honeybees in vivo and provides a new and powerful tool for the study of the molecular basis of learning and memory formation in insects.

Differential impact of substitution of amino acids 9-13 and 91-101 of human CD14 on soluble CD14-dependent activation of cells by lipopolysaccharide.

The soluble form of the endotoxin receptor CD14 is required for the LPS-induced activation of cells lacking membrane-bound CD14. It has been shown that a deletion mutant of human CD14 consisting of the N-terminal 152 amino acids has the capacity to mediate the stimulation of different cell types by LPS. To identify the structural domains of the molecule related to this functional property, we screened a set of alanine substitution mutants using CD14-negative U373 astrocytoma cells. We show that 3 of 18 soluble mutants of human CD14 failed to mediate the LPS-induced IL-6 production in U373 cells. These mutants were located in two regions of the molecule (aa 9-13 and 91-101) that are not essential for LPS binding. In addition, the mutants had a reduced capacity to mediate LPS-stimulated IL-6 production in human vascular endothelial and SMC. In contrast, the potential of sCD14(91-94,96)A, and sCD14(97-101)A to signal LPS-induced activation of human PBMC was not significantly reduced. These results show that the regions 9-13 and 91-101 are involved in the sCD14-dependent stimulation of cells by LPS but that the mechanisms by which different cell types are activated may not be identical.

IA in Kenyon cells of the mushroom body of honeybees resembles shaker currents: kinetics, modulation by K+, and simulation.

Cultured Kenyon cells from the mushroom body of the honeybee, Apis mellifera, show a voltage-gated, fast transient K+ current that is sensitive to 4-aminopyridine, an A current. The kinetic properties of this A current and its modulation by extracellular K+ ions were investigated in vitro with the whole cell patch-clamp technique. The A current was isolated from other voltage-gated currents either pharmacologically or with suitable voltage-clamp protocols. Hodgkin- and Huxley-style mathematical equations were used for the description of this current and for the simulation of action potentials in a Kenyon cell model. Activation and inactivation of the A current are fast and voltage dependent with time constants of 0.4 +/- 0.1 ms (means +/- SE) at +45 mV and 3.0 +/- 1.6 ms at +45 mV, respectively. The pronounced voltage dependence of the inactivation kinetics indicates that at least a part of this current of the honeybee Kenyon cells is a shaker-like current. Deactivation and recovery from inactivation also show voltage dependency. The time constant of deactivation has a value of 0.4 +/- 0.1 ms at -75 mV. Recovery from inactivation needs a double-exponential function to be fitted adequately; the resulting time constants are 18 +/- 3.1 ms for the fast and 745 +/- 107 ms for the slow process at -75 mV. Half-maximal activation of the A current occurs at -0.7 +/- 2.9 mV, and half-maximal inactivation occurs at -54.7 +/- 2.4 mV. An increase in the extracellular K+ concentration increases the conductance and accelerates the recovery from inactivation of the A current, affecting the slow but not the fast time constant. With respect to these modulations the current under investigation resembles some of the shaker-like currents. The data of the A current were incorporated into a reduced computational model of the voltage-gated currents of Kenyon cells. In addition, the model contained a delayed rectifier K+ current, a Na+ current, and a leakage current. The model is able to generate an action potential on current injection. The model predicts that the A current causes repolarization of the action potential but not a delay in the initiation of the action potential. It further predicts that the activation of the delayed rectifier K+ current is too slow to contribute markedly to repolarization during a single action potential. Because of its fast activation, the A current reduces the amplitude of the net depolarizing current and thus reduces the peak amplitude and the duration of the action potential.

Enhanced expression of Ki-67, topoisomerase IIalpha, PCNA, p53 and p21WAF1/Cip1 reflecting proliferation and repair activity in UV-irradiated melanocytic nevi.

To investigate the effect of ultraviolet (UV) irradiation on the expression of cell cycle-associated proteins, melanocytic nevi from healthy volunteers were partially covered, irradiated with a defined UV dose, and excised 1 week thereafter. The irradiated and the protected parts were examined separately by conventional microscopy and immunohistochemistry using the antibodies Ki-S11 (Ki-67), Ki-S7 (topoisomerase IIalpha), PC10 (proliferating cell nuclear antigen [PCNA]), DO-7 (p53), 6B6 (p21WAF1/Cip1), and the melanocytic marker HMB-45. DNA nick-end labeling was used as a marker of apoptosis. Irradiation resulted in morphological changes and increased HMB-45 reactivity. Proliferation, as assessed by Ki-67 and topoisomerase IIalpha expression, was also clearly enhanced in the UV-exposed areas. This was confirmed by the appearance of occasional mitotic figures. PCNA expression levels markedly exceeded those of the proliferation markers and did not correlate with the latter in most cases. p21 immunolabeling indices were also consistently augmented after UV exposure; hence it is likely that growth-inhibitory mechanisms partly compensate for the proliferative impulse, and the disproportional rise in PCNA expression probably reflects DNA repair activity. Enhanced p53 immunostaining in four cases suggests that the induction of p21 after irradiation may be p53 mediated, whereas no concomitant apoptotic events were observed. We conclude that UV light can stimulate the proliferative activity of melanocytes in melanocytic nevi, but that simultaneously cell cycle inhibitors are activated to permit DNA repair.

Gene activation by the AraC protein can be inhibited by DNA looping between AraC and a LexA repressor that interacts with AraC: possible applications as a two-hybrid system.

The Escherichia coli activator and repressor proteins AraC and LexA bind DNA as homodimers. Here we show that their heterodimerization through fused cognate dimerization domains results in repression of AraC-dependent gene activation by LexA. Repression also requires a LexA operator half-site located several helical turns downstream of the AraC operator. This requirement for a specific spatial organization of the operators suggests the formation of a DNA loop between operator-bound Ara/LexA heterodimers, and we propose that heterodimerization with the AraC hybrid provides co-operativity for operator binding and repression by the LexA hybrid. Consistent with a mechanism that involves DNA looping, repression increases when the E. coli DNA looping and transcriptional effector protein IHF binds between the AraC and LexA operators. Thus, we have combined the functions of three distinct transcriptional effector proteins to achieve a new mode of gene regulation by DNA looping, in which the activator protein is an essential part of the repressor complex. The flexibility of the DNA loop may facilitate this novel combinatorial arrangement of those proteins on the DNA. The requirement for protein interactions between the AraC and LexA hybrids for gene regulation suggests that this regulatory circuit may prove useful as an E. coli-based two-hybrid system.

Low endotoxic potential of Legionella pneumophila lipopolysaccharide due to failure of interaction with the monocyte lipopolysaccharide receptor CD14.

Legionella pneumophila, a gram-negative bacterium causing Legionnaires' disease and Pontiac fever, was shown to be highly reactive in in vitro gelation of Limulus lysate but not able to induce fever and the local Shwartzman reaction in rabbits and mice. We analyzed the capacity of purified L. pneumophila lipopolysaccharide (LPS-Lp) to induce activation of the human monocytic cell line Mono Mac 6, as revealed by secretion of proinflammatory cytokines and desensitization to subsequent LPS stimulation. We showed that despite normal reactivity of LPS-Lp in the Limulus amoebocyte lysate assay, induction of cytokine secretion in Mono Mac 6 cells and desensitization to an endotoxin challenge required LPS-Lp concentrations 1,000 times higher than for LPS of Salmonella enterica serovar Minnesota. Therefore, we examined the interaction of LPS-Lp with the LPS receptor CD14. We demonstrated that LPS-Lp did not bind to membrane-bound CD14 expressed on transfected CHO cells, nor did it react with soluble CD14. Our results suggest that the low endotoxic potential of LPS-Lp is due to a failure of interaction with the LPS receptor CD14.

The molecular basis for therapeutic concepts utilizing CD14.

The CD14 molecule is a key receptor on myeloid lineage cells involved in the recognition of lipopolysaccharide (LPS) and Gram-negative bacteria. The application of its soluble form, sCD14, has been shown to protect mice from lethality in LPS-induced shock. Therefore the protein or its derivatives may be considered as a possible therapeutic alternative for the treatment of patients suffering from Gram-negative septic shock. In this study we performed an alanine scan of amino acids 1 to 152 of human CD14. Twenty-three substitution mutants were generated and stably transfected into CHO-cells. In each mutant five amino acids were substituted by alanine. We analyzed (a) whether mutant proteins expressed on the surface of transfectants were recognized by a panel of anti-CD14 monoclonal antibodies (mAb's), (b) the ability of mCD14-mutants to bind LPS and E. coli in a serum- or LBP-dependent manner, and (c) the capacity of soluble mutants to mediate the LPS-induced IL 6 release of U 373 astrocytoma cells. Twenty-one CD14-mutants were expressed on the surface of transfectants and 18 were present as soluble forms in the culture supernatants. We demonstrated that only CD14(39-41,43-44)A completely lacked the ability to bind LPS and E. coli. In addition, a combined mutant CD14(9-13/57,59,61-63)A had very limited capacity to interact with LPS indicating that the LPS-binding site of human CD14 is a conformational epitope. Analysis of LPS-induced activation of CD14-negative U 373 cells revealed that the regions 9-13 and 91-101 are most important for sCD14-mediated signalling.

A fluorescence polarization competition immunoassay for tyrosine kinases.

We have recently reported a homogeneous, nonradioactive fluorescence polarization method to assay protein tyrosine kinase activity. Our original approach can only be used with a peptide substrate and requires large amounts of anti-phosphotyrosine antibody. To overcome these problems an alternate fluorescence polarization competition immunoassay was designed and evaluated. In this assay, phosphorylated peptide or protein produced by kinase reaction will compete with a fluorescent phosphopeptide used as a tracer for immunocomplex formation with phosphotyrosine antibody. In this format kinase activity will result in the loss of the polarization signal. To validate the fluorescence polarization competition immunoassay, Lck activity was compared with a more commonly used 32PO4-transfer assay using Lck peptide or enolase as the substrate. In both the assays, Lck activity showed a similar dependence on ATP, Lck enzyme, and the peptide/enolase substrate concentrations with the FP signal inversely proportional to the amount of 32PO4 transferred to the substrate. Inhibition by staurosporine and the Lck inhibitor 4-amino-5-(methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine was similar in these two assays. The advantages of this assay over other kinase assays include use of nonisotopic substrates and a more simple procedure in which the kinase assay is done in a single tube (well of a microtiter plate), without separation, precipitation, or washing. This method is easily automated for high-throughput drug discovery screening.

A homogeneous, fluorescence polarization assay for src-family tyrosine kinases.

A nonradioactive, simple, sensitive fluorescence polarization assay was developed to assay protein tyrosine kinase activity. This assay involves incubation of a fluorescenylated peptide substrate with the kinase, ATP, and anti-phosphotyrosine antibody. The phosphorylated peptide product is immunocomplexed with the anti-phosphotyrosine antibody resulting in an increase in the polarization signal as measured in a fluorescence polarization analyzer. Among several anti-phosphotyrosine antibodies examined, monoclonal antibody PY54 was found to give the best polarization signal with the test peptide. For validation of the fluorescence polarization assay, Lck activity was compared with a 32PO4 transfer assay. In both the fluorescence polarization and 32PO4 transfer assays, Lck activity showed a similar dependence on ATP, Lck enzyme, and peptide substrate concentrations. Both assays gave similar inhibition constants with a known tyrosine kinase inhibitor staurosporine and the Lck inhibitor, 4-amino-5-(methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. These results show that the fluorescence polarization assay can detect inhibitors and is comparable to the 32PO4 transfer assay. The fluorescence polarization method is advantageous compared to the 32PO4 transfer assay or ELISA or DELFIA because it is a one-step assay that does not involve several washings, liquid transfer, and sample preparation steps. It has the added advantage of using nonisotopic substrates. The fluorescence polarization assay thus is environmentally safe and minimizes handling problems. The homogeneous nature of the assay makes it readily adaptable to high-throughput screening for small-molecule drug discovery.

Learning and memory in honeybees: from behavior to neural substrates.

Learning and memory in honeybees is analyzed on five levels, using a top-down approach. (a) Observatory learning is applied during navigation and dance communication. (b) Local cues at the feeding site are learned associatively. (c) Classical conditioning of the proboscis extension response to olfactory stimuli provides insight into behavioral, neural, and neuropharmacological mechanisms of associative learning. (d) At the neural level, the pathways coding the conditioned and the unconditioned stimulus are identified. The reinforcing function of the unconditioned stimulus is traced to a particular neuron. (e) At the cellular level, the cAMP pathway is found to be critically involved. Nitric oxide is an essential mediator for the transfer from short- to long-term memory.