Combined AFM and confocal fluorescence microscope for applications in bio-nanotechnology.

We present a custom-designed atomic force fluorescence microscope (AFFM), which can perform simultaneous optical and topographic measurements with single molecule sensitivity throughout the whole visible to near-infrared spectral region. Integration of atomic force microscopy (AFM) and confocal fluorescence microscopy combines the high-resolution topographical imaging of AFM with the reliable (bio)-chemical identification capability of optical methods. The AFFM is equipped with a spectrograph enabling combined topographic and fluorescence spectral imaging, which significantly enhances discrimination of spectroscopically distinct objects. The modular design allows easy switching between different modes of operation such as tip-scanning, sample-scanning or mechanical manipulation, all of which are combined with synchronous optical detection. We demonstrate that coupling the AFM with the fluorescence microscope does not compromise its ability to image with a high spatial resolution. Examples of several modes of operation of the AFFM are shown using two-dimensional crystals and membranes containing light-harvesting complexes from the photosynthetic bacterium Rhodobacter sphaeroides.

Assembly of light-harvesting bacteriochlorophyll in a model transmembrane helix in its natural environment.

The transmembrane, bacteriochlorophyll-binding region of a bacterial light-harvesting complex, (LH2-alpha from the photosynthetic bacterium Rhodobacter sphaeroides) was redesigned and overexpressed in a mutant of Rb. sphaeroides lacking LH2. Bacteriochlorophyll served as internal probe for the fitness of this new region for the assembly and energy transfer function of the LH2 complex. The ability to absorb and transfer light energy is practically undisturbed by the exchange of the transmembrane segment, valine -7 to threonine +6, of LH2-alpha with a 14 residue Ala-Leu sequence. This stretch makes up the residues of the transmembrane helix that are in close contact (< or =4.5 A) with the bacteriochlorophyll molecules that are coordinated through His of both the alpha and beta-subunits. In this Ala-Leu stretch, neither alpha-His0, which binds the bacteriochlorophyll, nor the adjacent alpha-Ile-1, were replaced. Novel LH2 complexes composed of LH2-alpha with a model transmembrane sequence and a normal LH2-beta are assembled in vivo into a complex, the biochemical and spectroscopic properties of which closely resemble the native one. In contrast, the additional insertion of four residues just outside the C-terminal end of the model transmembrane helix leads to complete loss of functional antenna complex. The results suggest that light energy can be harvested and transferred efficiently by bacteriochlorophyll molecules attached to only few key residues distributed over the polypeptide, while residues at the bacteriochlorophyll-helix interface seem to be largely dispensable for the functional assembly of this membrane protein complex. This novel antenna with a simplified transmembrane domain and a built-in probe for assembly and function provides a powerful model system for investigation of the factors that contribute to the assembly of chromophores in membrane-embedded proteins.

The long-range supraorganization of the bacterial photosynthetic unit: A key role for PufX.

Bacterial photosynthesis relies on the interplay between light harvesting and electron transfer complexes, all of which are located within the intracytoplasmic membrane. These complexes capture and transfer solar energy, which is used to generate a proton gradient. In this study, we identify one of the factors that determines the organization of these complexes. We undertook a comparison of the organization of the light-harvesting complex 1 (LH1)/reaction center (RC) cores in the LH2(-) mutant of Rhodobacter sphaeroides in the presence or absence of the PufX protein. From polarized absorption spectra on oriented membranes, we conclude that PufX induces a specific orientation of the reaction center in the LH1 ring, as well as the formation of a long-range regular array of LH1-RC cores in the photosynthetic membrane. From our data, we have constructed a precise model of how the RC is positioned within the LH1 ring relative to the long (orientation) axis of the photosynthetic membrane.

Rational antibiotic therapy for respiratory disorders in dogs and cats.

Therapy of respiratory tract infections presents some unique challenges to the veterinary practitioner. These infections often involve underlying disease processes that have predisposed the patient to secondary bacterial infection and may complicate the response to therapy. Because of the diversity of microbial organisms that may colonize and invade the respiratory tract, treatment targeted at the infecting pathogens is best accomplished with bacterial culture and susceptibility testing. When these data are unavailable, rational antibiotic treatment should be based on familiarity with historical data and clinical experience. Optimal drug selection is based on predicted microbial susceptibility, drug distribution in the respiratory tract, and safety of the patient. Instituting the appropriate dosage regimen and duration of therapy maximizes the opportunity for a successful resolution of bacterial infections.

Site-directed modification of the ligands to the bacteriochlorophylls of the light-harvesting LH1 and LH2 complexes of Rhodobacter sphaeroides.

The core light-harvesting LH1 complex of Rhodobactersphaeroides consists of an assembly of membrane-spanning alpha and beta polypeptides, each of which binds one bacteriochlorophyll molecule. In this study we have used site-directed mutagenesis to demonstrate that the B880 bacteriochlorophyll binding site of LH1 shows a high degree of specificity for the residue that provides the ligand to the Bchl Mg2+ ion. alpha His0 (alphaH0) was changed to Asn, Leu, and Tyr, and beta His0 (betaH0) to Asn, Gln, Leu, and Tyr; the mutated genes were expressed to yield both LH1-only and LH1 + RC strains, in two different carotenoid backgrounds. None of the alphaH0 mutations formed an LH1 complex either in isolation or with RCs. Of the mutations of betaH0 those to Asn and Gln formed LH1 complexes but in the latter case the complex was very unstable and occurred at very low cellular levels. In a similar study, the alphaH0 and betaH0 residues of the LH2 complex were changed to Asn. However, no complex was formed in either case. FT Raman spectroscopy of the betaH0N mutant LH1 shows perturbation of the interaction state of the keto carbonyl of one Bchl which sheds light on the possible H-bond partners for these keto oxygens. These data directly address the B880 binding site of the core LH1 complex and show that it is subtly different from the B850 binding site of the peripheral LH2 complex. A model of this binding site may be proposed from these results.

Functions of conserved tryptophan residues of the core light-harvesting complex of Rhodobacter sphaeroides.

We have examined mutants in the core light-harvesting complex of Rhodobacter sphaeroides in which the tryptophan residues located at positions alpha+11, beta+6, and beta+9 have been mutated to each of the three other aromatic amino acids, namely tyrosine, phenylalanine, and histidine. We confirm that the alpha+11 residue and show that the beta+9 residue each form a hydrogen bond to a C2-acetyl group of a BChl molecule. Mutation of either of these residues to a phenylalanine results in a breakage of the normal hydrogen bond, whereas a histidine in either of these positions is able to form a hydrogen bond to the BChl. Comparison of the absorption spectra with the hydrogen bonding of the C2-acetyl groups for the various mutants demonstrates a role for this molecular interaction in the tuning of the absorption properties of the complex. We further demonstrate that there is a consistent linear relationship between the downshift in the C2-acetyl stretching mode and the red shift in the absorption maximum, in both core and peripheral antenna complexes. This linear relationship allows us to estimate the contribution of H bonding to the red shifts of these complexes. Though the residue beta+6 is found not to be directly involved in interactions with the pigment molecules, mutation of this residue is shown in some cases to result in both a destabilization of the complex and a decrease in the binding site homogeneity. Finally, a consideration of the amount of antenna complex present in the various mutants shows an important role for the reaction center and/or the pufX gene product in the assembly or stabilization of this membrane protein.

Evaluation of structure-function relationships in the core light-harvesting complex of photosynthetic bacteria by reconstitution with mutant polypeptides.

Seven mutant LH1 polypeptides of Rhodobactor sphaeroides have been isolated, and their behaviors in in vitro reconstitution of LH1 and its subunit complex have been characterized. Two mutants were selected to address the increased stability of the subunit complex of Rb. sphaeroides compared with that of Rhodobacter capsulatus. We found that this difference can be largely ascribed to the existence of Tyr at position +4 in the beta-polypeptide (the numbering system used assigns position 0 to the His which provides the coordinating ligand to bacteriochlorophyll) of the former bacterium compared to Met in that position in the latter. The amount of energy involved in the increased interaction was 1.6 kcal/mol, which would be consistent with a hydrogen bond involving Tyr. Mutation of the His at position 0 to Asn allows an estimate of the binding energy for subunit formation contributed by coordination of the imidazole group of His to the Mg atom of bacteriochlorophyll of >4.5 kcal/mol per BChl. Finally, an evaluation of the role of amino acids in the C-terminal region of the alpha-polypeptide was begun. Reconstitution of a mutant alpha-polypeptide in which Trp at position +11 was changed to Phe resulted in optimal formation of an LH1-type complex whose lambda(max) was blue-shifted to 853 nm, the same as observed in the intact bacterium harboring this mutation. These results provide further confirmation that the environment of BChl in reconstituted LH1 complexes is the same as in vivo and support the assignment of this residue to a role in hydrogen bonding with the C3(1) carbonyl group of BChl. Two other mutants of the alpha-polypeptide in which 5 and 14 amino acids in the C-terminus were deleted were also examined. These were of interest because the latter mutant, unlike the former, resulted in a low level of expression of LH1 in intact cells. However, with both of these mutant polypeptides, reconstitution appeared identical to that of the native system. In the case of the mutant shortened by 14 amino acids, a small blue-shift in lambda(max) to 861 nm was observed, again reproducing the blue-shift exhibited by the intact cells. Thus, these results suggest that the lowered levels of in vivo expression observed in these two mutants are due to reduced incorporation of the alpha-polypeptide into the membrane or its increased degradation, rather than to decreased stabilization of the LH1 complex.

Protein structure modelling of the bacterial light-harvesting complex.

Protein structure modelling offers a method of obtaining 3-dimensional information that can be tested and used to plan mutagenesis experiments when a crystallographically determined structure is not available. At its simplest a model may consist of little more than a secondary structure prediction coupled with a determination of the likely regions of transmembrane/membrane surface/globular configuration. These methods can yield an interesting topology map of the protein, which places the residues in their likely positions with respect to, for example, the membrane interface. If it is a member of a large family of related proteins then aligned protein sequences can be used to predict the residues that have an important function as these will be largely conserved in the alignments. Using all these methods a model can be constructed (using for example, the Nicholson Molecular Modelling Kit) to visualize the proposed structure in three dimensions following the premise of good design, that is, avoiding obvious steric clashes, packing of helices in a realistic manner, observing the correct H-bond lengths, etc. In this latter exercise the review of Chothia (Annu. Rev. Biochem. 53, 537-572, 1984) of the principles of protein structure is particularly helpful as it clearly sets out how proteins pack and their preferred configuration. There is a wealth of information about individual amino acid conformational preferences and observed frequencies of occurrence in known protein structures, which can help decide how the residues in the model can be oriented. In this article we have collated the various protein models of the bacterial light-harvesting complexes and present our own model, which is a synthesis of the available biophysical data and theoretical predictions, and show its performance in explaining recent results of site-directed mutants of the LH1 and LH2 light-harvesting complexes of Rhodobacter sphaeroides.

Modification of a hydrogen bond to a bacteriochlorophyll a molecule in the light-harvesting 1 antenna of Rhodobacter sphaeroides.

Site-directed mutagenesis has been used to examine the function of a highly conserved aromatic residue, alpha Trp43, in the light-harvesting 1 antenna of the photosynthetic bacterium Rhodobacter sphaeroides. In this antenna alpha Trp43 is thought to be located near the putative binding site for bacteriochlorophyll; in this work it was changed to both Tyr and Phe, and in each case the main near-infrared absorbance peak was shifted to the blue, from 876 nm to 865 nm and then to 853 nm, respectively. Resonance Raman spectroscopy of the resulting complexes shows a shift of one component of the 1640-cm-1 peak to 1632 cm-1 for the Tyr mutant and to 1660 cm-1 for the Phe mutant. This demonstrates a strengthening of an existing H bond for the Tyr change and a breakage of this bond for the change to Phe. The 1640-cm-1 peak has been previously assigned to H-bonded C2 acetyl carbonyl groups of both bacteriochlorophylls in the light-harvesting 1 antenna dimer [Robert, B. & Lutz, M. (1985) Biochim. Biophys. Acta 807, 10-21]. These results indicate that one of these H bonds is to alpha Trp43, placing this residue in close proximity to the bacteriochlorophyll a macrocycle with which it interacts. The existence of this bond places constraints on the conformation of the alpha polypeptide, and a model of an alpha beta heterodimer is presented incorporating these data.

Mutants of Rhodobacter sphaeroides lacking one or more pigment-protein complexes and complementation with reaction-centre, LH1, and LH2 genes.

The photosynthetic apparatus of Rhodobacter sphaeroides is comprised of three types of pigment-protein complex: the photochemical reaction centre and its attendant LH1 and LH2 light-harvesting complexes. To augment existing deletion/insertion mutants in the genes coding for these complexes we have constructed two further mutants, one of which is a novel double mutant which is devoid of all three types of complex. We have also constructed vectors for the expression of either LH1, LH2 or reaction-centre genes. The resulting system allows each pigment-protein complex to be studied either as part of an intact photosystem or as the sole complex in the cell. In this way we have demonstrated that reaction centres can assemble independently of either light-harvesting complex in R. sphaeroides. In addition, the isolation of derivatives of the deletion/insertion mutants exhibiting spontaneous mutations in carotenoid biosynthesis provides an avenue for examining the role of carotenoids in the assembly of the photosynthetic apparatus. We show that the LH1 complex is assembled regardless of the carotenoid background, and that the type of carotenoid present modifies the absorbance of the LH1 bacteriochlorophylls.

Comparison of larkspur alkaloid extract and lithium chloride in maintaining cattle aversion to larkspur in the field.

Lithium chloride (LiCl) and larkspur (Delphinium barbeyi) alkaloid extract were compared in their effect as an emetic to create taste aversions to fresh larkspur. They were further compared in the field to determine whether the indigenous larkspur alkaloids were more effective in maintaining the aversion when conditioned cattle were subjected to the social pressure (social facilitation) of control cattle eating larkspur. Taste aversions were produced in two groups of 1-yr-old cattle by offering fresh larkspur and then gavaging with either LiCl at 200 mg/kg of BW or larkspur alkaloid extract at 1.1 to 1.6 mL/kg of BW. The third group (control) was gavaged with water. The alkaloid group was slower to form an aversion than the lithium group, requiring four doses compared with two doses. All groups were then taken to larkspur-infested rangeland to test the aversion in the field. In the first field trial in which groups grazed separately, both aversion-induced groups generally abstained from eating larkspur. In the second trial in which all groups grazed together, both aversion-induced groups consumed less than half as much larkspur as the controls, but neither group abstained completely. Larkspur alkaloids did not maintain the aversion to larkspur to a greater degree than did LiCl when aversion-induced cattle were subjected to social facilitation.

DNA sequencing and complementation/deletion analysis of the bchA-puf operon region of Rhodobacter sphaeroides: in vivo mapping of the oxygen-regulated puf promoter.

Within the photosynthetic gene cluster of Rhodobacter sphaeroides the genes encoding light-harvesting LHI and reaction-centre complexes are transcriptionally linked in the order pufBALMX. The region stretching 1.6 kb upstream of pufB has been examined by DNA sequencing and by complementation/deletion analysis. These studies demonstrate that three open reading frames are located upstream of pufB. One open reading frame, designated bchA, terminates just inside pufQ, which is located proximal to pufB. BchA contains a 37 bp region that functions as the oxygen-regulated promoter for pufQ, and probably for the puf operon as a whole. We also demonstrate that the protein encoded by pufQ appears to play a role in bacteriochlorophyll biosynthesis.

Toxicity of extracts of tall larkspur (Delphinium barbeyi) in mice, hamsters, rats and sheep.

Larkspur consumption is a major source of cattle losses on mountain and high plains rangelands of western North America. Our objective was to find a suitable laboratory animal model for measuring larkspur toxicity for subsequent use as an estimator of toxicity to cattle. The LD50 for subcutaneous injection and for oral gavage of extract from Delphinium barbeyi, a highly toxic and troublesome rangeland tall larkspur, was compared for mice, hamsters, rats and sheep. Similarity of primary clinical signs of poisoning and lack of significant difference in slope of the dose-response curves implied that the overall effect of the larkspur alkaloids was the same for all rodent species tested. Sheep were the most susceptible to poisoning by subcutaneous injection of larkspur extract with decreasing susceptibility in hamsters, mice and rats, but sheep had least susceptibility when comparing response to oral (by gavage) doses. Also, death occurred rapidly after gavage as compared with subcutaneous doses in mice, hamsters and rats, but was nearly the same for sheep. We conclude that, of the animals tested, mice would be the best choice for a bioassay of the toxicity of larkspur because of their high susceptibility, rapid response time, and small dose requirement.

Description of a scale for rating the clinical response of cattle poisoned by larkspur.

Larkspur poisoning is a major cause of acute death of cattle on mountain and high plains rangelands of western United States. A nonlethal method to quantify dose response in cattle is needed to better estimate the toxicity of larkspur plants and the response of cattle to larkspur poisoning and to provide a basis for reference during studies. A numerical system of rating the clinical signs of larkspur poisoning was developed and used to describe the response of 10 Hereford cows given a repeated single daily dose of larkspur (Delphinium occidentale x barbeyi) by gavage. Larkspur poisoning resulted from a short-term cumulative effect, and a statistically significant increase in score was essentially maximal by 4 days. At the dose given, this effect did not persist for more than 4 days after cessation of dosing. Poisoning was most severe between 5 and 9 hours after dosing. Early signs of poisoning can be subtle and sometimes brief. The effect of larkspur poisoning can be exacerbated temporarily by exertion. Therefore, cattle could probably repeatedly consume an otherwise toxic daily dose, without manifesting marked signs of poisoning, if consumption decreased to a sufficient degree intermittently at 2- to 4-day intervals.

Toxicity of pyrrolizidine alkaloids from Riddell groundsel (Senecio riddellii) to cattle.

The toxicity of Riddell groundsel (Senecio riddellii) gavaged to calves at a known lethal rate was compared with the toxicity of riddelliine and riddelliine N-oxide, the pyrrolizidine alkaloids isolated from the plant, which were fed by intraruminal infusion. Doses of the alkaloids were adjusted to the amount determined to be in the plant and fed individually and in combination. The relative toxicosis in the calves was measured by clinical signs, serum enzyme changes, survival time to morbidity, and histologic changes. Calves fed Senecio riddellii by gavage for 20 consecutive days to provide 45 mg of total pyrrolizidine alkaloids/kg of body weight/d developed clinical signs and serum enzyme changes typical of seneciosis, with 100% morbidity. However, calves receiving riddelliine at 4.5 mg/kg/d for 20 days had neither serum enzyme changes nor clinical signs of pyrrolizidine alkaloidosis. Calves treated with riddelliine N-oxide (40.5 mg/kg/d), and with riddelliine (4.5 mg/kg/d) and riddelliine N-oxide (40.5 mg/kg/d) in combination, had 100% morbidity, although the latter group had fewer liver lesions. These results establish that the N-oxide form of the alkaloid alone is capable of inducing typical Senecio toxicosis in cattle and that the free base level of the plant cannot be considered to be the sole factor in assessing the toxicity of S riddellii.

Adverse influence of social facilitation and learning context in training cattle to avoid eating larkspur.

Through conditioned food aversion learning, livestock can be trained to avoid eating harmful plants. The objectives of this study were to determine whether social facilitation will extinguish an aversion to larkspur (a poisonous plant on mountain rangelands) and to determine whether the aversion can be reinforced to withstand social facilitation in a group-feeding and field-grazing situation. Two groups of heifers offered fresh larkspur were simultaneously infused intraruminally with lithium chloride (LiCl) to create an association between the taste of larkspur and LiCl-induced gastrointestinal distress. A third control group was infused with water. Heifers from one averted group (extinction) were paired with nonaverted controls and offered larkspur. When the extinction group sampled larkspur, and LiCl was not infused, the aversion was extinguished rapidly. Heifers in the other averted group (reinforcement), being infused with LiCl whenever they sampled larkspur, abstained from eating larkspur in the group-feeding situation. Heifers were then taken to larkspur-infested rangeland. After the control heifers began eating larkspur, the averted heifers started to sample it and the aversion was extinguished in three of four heifers. However, the aversion was renewed when the heifers were returned to the pen- and group-feeding situation where the aversion was created. Reinforced aversion was overcome by social facilitation in an unfamiliar field-grazing environment.

Giant benign mesothelioma.

Pleural mesothelioma is a rare neoplasm that is usually highly malignant, but benign mesotheliomas do occur; approximately 400 cases are described in the literature. We report the case of a young woman with a massive benign mesothelioma that filled the entire left hemithorax but was successfully resected with full reexpansion of the lung.

Photosynthetic antenna proteins: 100 ps before photochemistry starts.

All photosynthetic organisms require a light harvesting system to funnel excitation energy towards the photosynthetic reaction centre, a process which can take 100 ps. Laser spectroscopy allows us to measure rates of energy transfer between pigments of the light harvesting system for the first time. These rates are correlated with models of the light harvesting apparatus.

Plant-animal interactions in larkspur poisoning in cattle.

Larkspur (Delphinium spp.) toxicity in cattle seriously impedes the efficient use of productive mountain rangelands. Larkspurs contain complex diterpenoid alkaloids that cause acute intoxication and death from respiratory paralysis. Alkaloids and their concentrations vary among larkspur species, plant parts and phenological growth stages, thus causing great variability in toxicity. Ingestion rate of larkspur by the cow, alkaloid toxicity and concentration in the plant and the kinetics of absorption and excretion interact to determine whether a cow is poisoned. Plant and animal factors influencing consumption and subsequent intoxication must be further elucidated to devise management strategies to reduce liverstock losses.