Induced coherence, vacuum fields, and complementarity in biphoton generation.

It is well established that spontaneous parametric down-conversion with induced coherence across two coupled interferometers results in high-visibility single-photon interference. We describe experiments in which additional photon channels are introduced such that "which-path" information is made possible and the fringe visibility in single-photon interference is reduced in accordance with basic notions of complementarity. However, these additional pathways result in nearly perfect visibility when photons are counted in coincidence. A simplified theoretical model accounts for these observations and attributes them directly to the vacuum fields at the different crystals.

Mechanisms, functions and ecology of colour vision in the honeybee.

Research in the honeybee has laid the foundations for our understanding of insect colour vision. The trichromatic colour vision of honeybees shares fundamental properties with primate and human colour perception, such as colour constancy, colour opponency, segregation of colour and brightness coding. Laborious efforts to reconstruct the colour vision pathway in the honeybee have provided detailed descriptions of neural connectivity and the properties of photoreceptors and interneurons in the optic lobes of the bee brain. The modelling of colour perception advanced with the establishment of colour discrimination models that were based on experimental data, the Colour-Opponent Coding and Receptor Noise-Limited models, which are important tools for the quantitative assessment of bee colour vision and colour-guided behaviours. Major insights into the visual ecology of bees have been gained combining behavioural experiments and quantitative modelling, and asking how bee vision has influenced the evolution of flower colours and patterns. Recently research has focussed on the discrimination and categorisation of coloured patterns, colourful scenes and various other groupings of coloured stimuli, highlighting the bees' behavioural flexibility. The identification of perceptual mechanisms remains of fundamental importance for the interpretation of their learning strategies and performance in diverse experimental tasks.

Visual generalization in honeybees: evidence of peak shift in color discrimination.

In the present study, we investigated color generalization in the honeybee Apis mellifera after differential conditioning. In particular, we evaluated the effect of varying the position of a novel color along a perceptual continuum relative to familiar colors on response biases. Honeybee foragers were differentially trained to discriminate between rewarded (S+) and unrewarded (S-) colors and tested on responses toward the former S+ when presented against a novel color. A color space based on the receptor noise-limited model was used to evaluate the relationship between colors and to characterize a perceptual continuum. When S+ was tested against a novel color occupying a locus in the color space located in the same direction from S- as S+, but further away, the bees shifted their stronger response away from S- toward the novel color. These results reveal the occurrence of peak shift in the color vision of honeybees and indicate that honeybees can learn color stimuli in relational terms based on chromatic perceptual differences.

Biosorption of process-equipment-related leachables (PERLs) in biomanufacturing: A quantitative approach to study partitioning of PERLs in a cell culture system.

The assessment and potential risk of process equipment-related leachables (PERLs) in the production of biopharmaceuticals and cell therapeutics using single-use (SU) equipment has been discussed previously. However, potential interactions of cells with PERLs have not yet been considered. Here, we present a quantitative adsorption study of neutral, organic small-molecule leachable compounds - known for extractables & leachables (E&L) analysis of SU equipment - in aqueous suspensions of CHO and T cells. The solid-water partition coefficient Kd was obtained for all compounds that showed adsorption. The findings implied that hydrophobic interactions are dominant; however, there was no unambiguous correlation between the derived adsorption coefficient Kd and the octanol-water partition coefficient Kow. Interestingly, a maximum affinity of both cell types to the leachable bis(2,4-di-tert-butylphenyl)phosphate, which is known to be detrimental to cell development, was observed. A comparison of both cell types revealed that they generally interact with the same compounds in most cases but to different extents. Using partition coefficients enables estimation of the concentrations of leachable compounds associated with the biomass phase and in the aqueous suspensions and could be used for risk assessment of SU systems in biopharmaceutical and cell therapy (CT) manufacturing processes.

Evidence of red sensitive photoreceptors in Pygopleurus israelitus (Glaphyridae: Coleoptera) and its implications for beetle pollination in the southeast Mediterranean.

A very well-documented case of flower-beetle interaction is the association in the Mediterranean region between red bowl-shaped flowers and beetles of the family Glaphyridae. The present study examines the visual mechanisms by which Pygopleurus israelitus (Glaphyridae: Scarabaeoidea: Coleoptera) would perceive the colors of flowers they visit by characterizing the spectral sensitivity of its photoreceptors. Our measurements revealed the presence of three types of photoreceptors, maximally sensitive in the UV, green and red areas of the spectrum. Using color vision space diagrams, we calculated the distribution of beetle-visited flower colors in the glaphyrid and honeybee color space and evaluated whether chromatic discrimination differs between the two types of pollinators. Respective color loci in the beetle color space are located on one side of the locus for green foliage background, whereas in the honeybee the flower color loci surround the locus occupied by green foliage. Our results represent the first evidence of a red sensitive photoreceptor in a flower-visiting coleopteran species, highlighting Glaphyridae as an interesting model group to study the role of pollinators in flower color evolution.

The flight paths of honeybees recruited by the waggle dance.

In the 'dance language' of honeybees, the dancer generates a specific, coded message that describes the direction and distance from the hive of a new food source, and this message is displaced in both space and time from the dancer's discovery of that source. Karl von Frisch concluded that bees 'recruited' by this dance used the information encoded in it to guide them directly to the remote food source, and this Nobel Prize-winning discovery revealed the most sophisticated example of non-primate communication that we know of. In spite of some initial scepticism, almost all biologists are now convinced that von Frisch was correct, but what has hitherto been lacking is a quantitative description of how effectively recruits translate the code in the dance into flight to their destinations. Using harmonic radar to record the actual flight paths of recruited bees, we now provide that description.

Homing in the ant Cataglyphis bicolor.

Cataglyphis bicolor, an ant widely distributed in North Africa and the Near East, orient to the sun as well as to visual patterns of the environment. These two mechanisms can be separated. Foraging ants (hunters) orient to terrestrial cues as long as possible, and only after these have become ineffective do they switch over to the menotactical sun orientation. In the digging individuals, however, the visual knowledge of locality is significantly inferior to that of the hunters. Diggers vary considerably in size, but hunters belong to the largest size group. In addition, the largest and smallest individuals orient differently toward black and white areas and stripe patterns.

Associative learning modifies neural representations of odors in the insect brain.

Recording brain activity in vivo during learning is fundamental to understanding how memories are formed. We used functional calcium imaging to track odor representations in the primary chemosensory center of the honeybee, the antennal lobe, while training animals to discriminate a rewarded odor from an unrewarded one. Our results show that associative learning transforms odor representations and decorrelates activity patterns for the rewarded versus the unrewarded odor, making them less similar. Additionally, activity for the rewarded but not for the unrewarded odor is increased. These results indicate that neural representations of the environment may be modified through associative learning.

The glomerular code for odor representation is species specific in the honeybee Apis mellifera.

Odors are coded by glomerular activity patterns in the insect antennal lobe (AL) and in the mammalian olfactory bulb. We measured glomerular responses to 30 different odors in the AL of honeybees using calcium-sensitive dyes. By subsequently staining glomeruli and identifying individual glomerular outlines, we were able to compare the patterns between animals. Regardless of whether the odors were mixtures or pure substances, environmental odors or pheromones, their representations were highly conserved among individuals. Therefore, it may be possible to create a functional atlas of the AL in which particular molecular receptive ranges are attributed to each glomerulus.

Natural organic matter (NOM) has the potential to modify the multixenobiotic resistance (MXR) activity in freshwater amphipods Eulimnogammarus cyaneus and E. verrucosus.

Based on the chemical features of natural organic matter (NOM) with its variety of functional groups, we hypothesized that NOM will modify the multixenobiotic-resistance (MXR) of an organism as xenobiotic chemicals do. The MXR system is a general first rather non-specific line of defense against environmental contaminants. The aim of this study was to compare the impacts on MXR activity in amphipod species (Eulimnogammarus cyaneus and E. verrucosus, from Lake Baikal) stressed by cadmium chloride or dissolved NOM for 24 h. NOM exposure concentrations were environmentally realistic. MXR activity was assessed based on rhodamine B efflux; its specificity was proven by a verapamil inhibition assay. It was shown that both NOM and CdCl(2) lead to substantial reduction of the rhodamine B efflux. This suggests that NOM may be regarded as a chemosensor which is able to reduce the efficiency of the MXR system. Possible mechanisms of direct NOM impact on MXR processes are discussed, such as peroxidation of the membranes (including P-glycoproteins) or internal blockage of the MXR pump by bioconcentrated NOM. In general, our results show that well-developed depuration pathways of freshwater organisms in contaminated environments may be impaired by strong chemical stressors and, more important, by natural biogeochemical matrices such as humic substances--humic substances are present in all freshwater systems.

Insect visual perception: complex abilities of simple nervous systems.

Despite their relatively simple nervous systems, insects display a rich behavioural repertoire, in which vision plays a major role. In the past two years, much knowledge has been gained about how insects are capable of a variety of flexible, visually guided tasks that involve a high level of complexity. From long-range navigation to median-range orientation and close-up recognition, insects apply different strategies that complement each other, that are used sequentially during their approach flight towards their goals, and that may replace each other, depending on the salience of, and the attention towards, particular visual cues.

Odour perception in honeybees: coding information in glomerular patterns.

Major advances have been made during the past two years in understanding how honeybees process olfactory input at the level of their first brain structure dealing with odours, the antennal lobe (the insect analogue of the mammalian olfactory bulb). It is now possible to map physiological responses to morphologically identified olfactory glomeruli, allowing for the creation of a functional atlas of the antennal lobe. Furthermore, the measurement of odour-evoked activity patterns has now been combined with studies of appetitive odour learning. The results show that both genetically determined components and learning-related plasticity shape olfactory processing in the antennal lobe.

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.

DNA supercoiling and bacterial adaptation: thermotolerance and thermoresistance.

When bacterial cells are shifted to higher temperatures their degree of DNA supercoiling changes. Topoisomerases are involved in bacterial adaptation to environmental changes requiring rapid shifts in gene expression. This role in heat shock has been elucidated by genetic studies on the Escherichia coli topA gene and its sigma 32-dependent promoter, P1. Other studies have shown that certain gyrA mutants have increased thermoresistance.

Proliferation of intracellular membrane structures upon homologous overproduction of cytochrome P-450 in Candida maltosa.

In an alkane-assimilating yeast, Candida maltosa, a cultivation on alkane causes both induction of endoplasmic reticulum (ER)-resident membrane proteins, such as cytochrome P-450, and proliferation of ER. In this study, individual genes for alkane-inducible forms of cytochrome P-450 (P-450alk) were homologously overexpressed in C. maltosa using a galactose-inducible expression system developed in this yeast. Immunoelectron microscopy revealed that, upon the overexpression, a dramatic proliferation of ER occurred, in which overproduced P-450alk protein accumulated. The proliferated membranes were mainly tubular forms and stacks of paired membranes were also observed after prolonged expression. The tubular forms were morphologically very similar to the proliferated ER in alkane-induced C. maltosa cells. The observed proliferation of ER membranes by homologous overproduction of P-450alk, here depicted, will provide a unique opportunity for investigating the mechanisms by which cells regulate ER biogenesis, in comparison with the intrinsic form of ER proliferation.

Localization of MODY3 to a 5-cM region of human chromosome 12.

Maturity-onset diabetes of the young (MODY) is a heterogeneous disorder that appears to be characterized by a primary defect in insulin secretion. Mutations in an unknown locus (MODY1) on chromosome 20 and the glucokinase gene (MODY2) on chromosome 7 can cause this form of non-insulin-dependent diabetes. Recent genetic studies have identified a third locus on chromosome 12 (MODY3) that is linked to MODY in a group of French families. We have identified three families from Denmark, Germany, and the U.S. (Michigan) showing evidence of linkage with MODY3 and a family from Japan showing suggestive evidence. Analysis of key recombinants in these families localized MODY3 to a 5-cM interval between the markers D12S86 and D12S807/D12S820.

Mutations in the hepatocyte nuclear factor-1alpha gene in MODY and early-onset NIDDM: evidence for a mutational hotspot in exon 4.

We have recently shown that mutations in the gene encoding the transcription factor hepatocyte nuclear factor (HNF)-1alpha are the cause of one form of maturity-onset diabetes of the young (MODY3). Here, we report the exon-intron organization and partial sequence of the human HNF-1alpha gene. In addition, we have screened the ten exons and flanking introns of this gene for mutations in a group of 25 unrelated white subjects from Germany who presented with NIDDM before 35 years of age and had a first-degree relative with NIDDM. Mutations were identified in nine of these individuals, suggesting that mutations in the HNF-1alpha gene are a common cause of diabetes in German subjects with early-onset NIDDM and a family history of diabetes. Thus, screening for mutations in this gene may be indicated in subjects with early-onset NIDDM. Interestingly, three of the nine mutations occurred at the same site in exon 4 with insertion of a C in a polyC tract, centered around codon 290 (designated Pro291fsinsC), thereby resulting in a frameshift during translation and premature termination. Analyses of linked DNA polymorphisms in the HNF-1alpha gene indicated that the Pro291fsinsC mutation was present on a different haplotype in each subject, implying that the polyC tract represents a mutational hot spot. We have also identified the mutation in the HNF-1alpha gene in the Jutland pedigree, one of the original MODY pedigrees reported in the literature, as being a T-->G substitution in codon 241, resulting in the replacement of a conserved Cys by Gly (C241G). The information on the sequence of the HNF-1alpha gene and its promoter region will facilitate the search for mutations in other subjects and studies of the role of the gene in determining normal beta-cell functions.

Inhibition of DNA gyrase activity in an in vitro transcription-translation system stimulates gyrA expression in a DNA concentration dependent manner. Evidence for the involvement of factors which may be titrated.

Stimulation of gyrA expression in an in vitro transcription-translation system by novobiocin, a DNA gyrase inhibitor, depends on the DNA concentration in the extract. At low DNA concentrations (less than 20 micrograms/ml) we note significant stimulation (2 to 25 x) upon the addition of novobiocin; at high DNA concentrations, stimulation is minimal. This observation is not due to the limited capacity of the system to transcribe or relax DNA. Using an extract prepared from a novobiocin-resistant strain of Escherichia coli, we were able to show that DNA gyrase mediates the novobiocin-enhanced expression. In an experiment with a fixed level of a gyrA-lac template, we found that the addition of a second non-Lac template increased expression from the gyrA-lac template, while concomitantly decreasing the extent of novobiocin stimulation. These observations are consistent with an inhibitory factor that can be titrated by increasing the DNA concentration and whose effects are minimized when the DNA template is in a relaxed conformation. The results of a mixed-extract experiment using two extracts that differ in their activities and degrees of novobiocin stimulation are also consistent with an inhibitory factor that mediates the relaxation-induced stimulation of transcription.

Regulation of Escherichia coli topA gene transcription: involvement of a sigmaS-dependent promoter.

To investigate the regulation of Escherichia coli topA gene transcription, primer extension was employed to determine the transcription initiation sites from the chromosomal topA gene. When cells were grown in LB medium to log phase, four transcription initiation sites could be identified. Three of these sites corresponded to promoters P1, P2 and P4 previously characterized using topA-galK fusion plasmids. The P3 promoter that is active on the plasmid was not utilized at the chromosomal topA gene under the conditions employed. There was a new transcription initiation site corresponding to a new promoter Px1. When cells started to enter stationary phase, promoter Px1 gradually became the major transcription initiation site for topA, while transcription from promoters P2 and P4 decreased. In an E. coli mutant lacking sigmaS (the rpoS gene product), the stationary phase specific sigma factor, the induction of transcription from promoter Px1 was abolished. In another mutant lacking H-NS activity, resulting in increased sigmaS level in log-phase, the transcription from promoter Px1 during log phase was increased. Thus Px1 appeared to be regulated by sigmaS. The activity of promoter P1 on the chromosome increased during heat shock, consistent with the previous result obtained using the topA-galK fusion plasmid showing that P1 is a sigma32-dependent heat shock promoter. Promoters P2 and P4 were most likely to be recognized by sigma70. The total level of topoisomerase I protein in the rpoS mutant was not reduced significantly in stationary phase due to increased transcription initiation from the other topA promoters. The utilization of multiple sigma factors for transcription initiation of topA could be important for adaptation of E. coli to change in growth conditions.

Hfr formation directed by tn10.

The transposable drug-resistance element, Tn10, can serve as a region of homology to direct the insertion of an F'ts114 lac plasmid into the chromosome of Salmonella typhimurium. Derivatives of F'ts114 lac were constructed that carry Tn10 insertions; these plasmids were transferred to strains having a Tn10 insertion in the chromosome. Under these circumstances, Hfr formation requires homologous recombination between plasmid-borne and chromosomal Tn10 elements. The process is dependent on recA function and on the presence of both Tn10 elements. All Hfr's isolated from a given merodiploid show the same direction of transfer. Depending on the orientation of Tn10 in the F' plasmid, Hfr's transferring in either direction can be obtained from any chromosomal Tn10 insertion. Since Tn10 insertions can be generated in any region of the chromosome, this method permits the isolation of Hfr's with either direction of transfer having their origin at almost any predetermined site. The Hfr's constructed by this method are sufficiently stable for standard genetic mapping crosses, and they have also been used to generate new F' plasmids. Implicit in the results above is the possibility of determining the orientation of any chromosomal Tn10 insertion by constructing an Hfr using a standard F' Tn10 plasmid and determining the direction of chromosome transfer. The general approaches described here are applicable to other transposable elements and other bacterial systems.