TBX18 overexpression enhances pacemaker function in a rat subsidiary atrial pacemaker model of sick sinus syndrome.

KEY POINTS: The sinoatrial node (SAN) is the primary pacemaker of the heart. SAN dysfunction, or 'sick sinus syndrome', can cause excessively slow heart rates and pauses, leading to exercise limitation and syncope, currently treated by implantation of an electronic pacemaker. 'Biopacemaking' utilises gene therapy to restore pacemaker activity by manipulating gene expression. Overexpressing the HCN pacemaker ion channel has been widely used with limited success. We utilised bradycardic rat subsidiary atrial pacemaker tissue to evaluate alternative gene targets: the Na+ /Ca2+ exchanger NCX1, and the transcription factors TBX3 and TBX18 known to be involved in SAN embryonic development. TBX18 overexpression restored normal SAN function, as assessed by increased rate, improved heart rate stability and restoration of isoprenaline response. TBX3 and NCX1 were not effective in accelerating the rate of subsidiary atrial pacemaker tissue. Gene therapy targeting TBX18 could therefore have the potential to restore pacemaker function in human sick sinus syndrome obviating electronic pacemakers. ABSTRACT: The sinoatrial node (SAN) is the primary pacemaker of the heart. Disease of the SAN, sick sinus syndrome, causes heart rate instability in the form of bradycardia and pauses, leading to exercise limitation and syncope. Biopacemaking aims to restore pacemaker activity by manipulating gene expression, and approaches utilising HCN channel overexpression have been widely used. We evaluated alternative gene targets for biopacemaking to restore normal SAN pacemaker physiology within bradycardic subsidiary atrial pacemaker (SAP) tissue, using the Na+ /Ca2+ exchanger NCX1, and the transcription factors TBX3 and TBX18. TBX18 expression in SAP tissue restored normal SAN function, as assessed by increased rate (SAN 267.5 ± 13.6 bpm, SAP 144.1 ± 8.6 bpm, SAP-TBX18 214.4 ± 14.4 bpm; P < 0.001), improved heart rate stability (standard deviation of RR intervals fell from 39.3 ± 7.2 ms to 6.9 ± 0.8 ms, P < 0.01; root mean square of successive differences of RR intervals fell from 41.7 ± 8.2 ms to 6.1 ± 1.2 ms, P < 0.01; standard deviation of points perpendicular to the line of identity of Poincaré plots (SD1) fell from 29.5 ± 5.8 ms to 7.9 ± 2.0 ms, P < 0.05) and restoration of isoprenaline response (increases in rates of SAN 65.5 ± 1.3%, SAP 28.4 ± 3.4% and SAP-TBX18 103.3 ± 10.2%; P < 0.001). These changes were driven by a TBX18-induced switch in the dominant HCN isoform in SAP tissue, with a significant upregulation of HCN2 (from 1.01 × 10-5  ± 2.2 × 10-6 to 2.8 × 10-5  ± 4.3 × 10-6 arbitrary units, P < 0.001). Biophysically detailed computer modelling incorporating isoform-specific HCN channel electrophysiology confirmed that the measured changes in HCN abundance could account for the observed changes in beating rates. TBX3 and NCX1 were not effective in accelerating the rate of SAP tissue.

Dr Pugh: a poisoner?

On 16 February 1845 the Reverend W. H. Browne, rector of St John's Church in Launceston, Van Diemen's Land, wrote in his journal, "My dear Wife died very suddenly almost immediately after and in consequence of taking a preparation of Hyd. Cyan. Acid prepared & supplied by Dr Pugh". This journal entry raises a number of questions. Was Dr Pugh treating a condition which he thought merited that treatment or was it a ghastly mistake? Was Caroline Browne suffering from pulmonary tuberculosis? Was hydrocyanic acid an accepted treatment at that time? Did Mrs Browne take the wrong dose? Was an incorrect concentration of the drug prepared by Dr Pugh? Did he use the wrong pharmacopoeia in preparing the hydrocyanic acid? Why was there no inquest? Only some of these questions can be answered.

Enhancement of the radiation response of EMT-6 tumours by a copper octabromotetracarboranylphenylporphyrin.

OBJECTIVE: The carborane-containing porphyrin, copper (II) 2,3,7,8,12,13,17,18-octabromo-5,10,15,20-tetrakis(3-[1,2-dicarba-closo-dodecaboranyl]methoxyphenyl)-porphyrin (CuTCPBr), was investigated as a potential radiation enhancing agent for X-ray radiotherapy (XRT) in a subcutaneously implanted EMT-6 murine carcinoma. METHOD: The biodistribution and toxicological profile of this porphyrin has been shown to be favourable for another bimodal radiotherapy technique, boron neutron-capture therapy. For the XRT studies, CuTCPBr was formulated in either 9% Cremophor (BASF Corporation, Ludwigschafen, Germany) EL and 18% propylene glycol (9% CRM) or a revised formulation comprising 1% Cremophor ELP, 2% Tween 80 (JT Baker, Mansfield, MA), 5% ethanol and 2.2% PEG 400 (CTEP formulation), which would be more clinically acceptable than the original 9% CRM formulation. Using the 9% CRM formulation of CuTCPBr, doses of 100, 210 or 400 mg kg(-1) of body weight were used in combination with single doses of 25-35 Gy 100 kVp X-rays. RESULTS: While doses of 100 mg kg(-1) and 210 mg kg(-1) did not result in any significant enhancement of tumour response, the 400 mg kg(-1) dose did. A dose modification factor of 1.20±0.10 was obtained based on the comparison of doses that produced a 50% local tumour control probability. With the CTEP formulation of CuTCPBr, doses of 83 and 170 mg kg(-1) produced significant radiation enhancement, with dose modification factors based on the TCP(50) of 1.29±0.15 and 1.84±0.24, respectively. CONCLUSION: CuTCPBr significantly enhanced the efficacy of XRT in the treatment of EMT-6 carcinomas in mice. The CTEP formulation showed a marked improvement, with over 9% CRM being associated with higher dose modification factors. Moreover, the radiation response in the skin was not enhanced.

Nature of weak magnetism in SrTiO3/LaAlO3 multilayers.

We report the observation of weak magnetism in superlattices of LaAlO(3)/SrTiO(3) using ß-detected nuclear magnetic resonance. The spin lattice relaxation rate of ^{8}Li in superlattices with a spacer layers of 8 and 6 unit cells of LaAlO(3) exhibits a strong peak near ~35 K, whereas no such peak is observed in a superlattice with spacer layer thickness of 3 unit cells. We attribute the observed temperature dependence to slowing down of weakly coupled electronic moments at the LaAlO(3)/SrTiO(3) interface. These results show that the magnetism at the interface depends strongly on the thickness of the spacer layer, and that a minimal thickness of ~4-6 unit cells is required for the appearance of magnetism. A simple model is used to determine that the observed relaxation is due to small fluctuating moments (~0.002µ(B)) in the two samples with a larger LaAlO(3) spacer thickness.

The radiobiological principles of boron neutron capture therapy: a critical review.

The radiobiology of the dose components in a BNCT exposure is examined. The effect of exposure time in determining the biological effectiveness of γ-rays, due to the repair of sublethal damage, has been largely overlooked in the application of BNCT. Recoil protons from fast neutrons vary in their relative biological effectiveness (RBE) as a function of energy and tissue endpoint. Thus the energy spectrum of a beam will influence the RBE of this dose component. Protons from the neutron capture reaction in nitrogen have not been studied but in practice protons from nitrogen capture have been combined with the recoil proton contribution into a total proton dose. The relative biological effectiveness of the products of the neutron capture reaction in boron is derived from two factors, the RBE of the short range particles and the bio-distribution of boron, referred to collectively as the compound biological effectiveness factor. Caution is needed in the application of these factors for different normal tissues and tumors.

Real-time polymerase chain reaction (PCR) assays for the specific detection and quantification of seven Eimeria species that cause coccidiosis in chickens.

Coccidiosis of chickens is an economically important disease caused by infection with species of Eimeria. The oocysts of some of the seven recognized species are difficult to distinguish morphologically and for this reason diagnostic laboratories are increasingly utilizing DNA-based technologies for the specific identification of Eimeria. The real-time PCR provides both sensitivity and speed for the analysis of DNA samples, and the approach has the capability of quantifying DNA. Together with a protocol for the extraction of DNA directly from faecal samples, real-time PCR assays have been established for the detection and quantification of seven species of Eimeria that infect chickens in Australia. The assays target one genetic marker, the second internal transcribed spacer of nuclear ribosomal DNA (ITS-2), use TaqMan MGB technology with species-specific probes, and can be multiplexed in pairs such that the seven species of Eimeria can be screened in four reaction tubes. A test screen of commercial flocks identified more Eimeria-infected chickens than were detected by coproscopic examination for oocysts. These molecular assays can also be used for the quality control of mixed-species vaccines. The ability to multiplex the assays makes them particularly practical for screening samples from chickens with mixed-species infections where the relative abundance of each Eimeria species present is required.

Biotechnological advances in the diagnosis of avian coccidiosis and the analysis of genetic variation in Eimeria.

Coccidiosis is an intestinal disease of chickens caused by various species of protozoan parasites within the genus Eimeria. This disease has a major economic impact to growers and to the poultry industry world-wide. The diagnosis and genetic characterization of the different species of Eimeria are central to the prevention, surveillance and control of coccidiosis, particularly now given the major problems with wide-spread resistance of Eimeria species against anticoccidial drugs (coccidiostats) and the residue problems associated with these compounds. While traditional methods have had major limitations in the specific diagnosis of coccidiosis, there have been significant advances in the development of molecular-diagnostic tools. The present article provides a background on coccidiosis, reviews the main molecular methods which have been used and describes recent advances in the establishment of polymerase chain reaction (PCR)-coupled electrophoretic approaches for the specific diagnosis of coccidiosis as well as the genetic characterization of species of Eimeria. These biotechnological advances are considered to represent a significant step toward the improved prevention and control of this important disease of poultry.

Porphyrin-mediated boron neutron capture therapy: a preclinical evaluation of the response of the oral mucosa.

Preclinical studies are in progress to determine the potential of boron neutron capture therapy (BNCT) for the treatment of carcinomas of the head and neck. Recently, it has been demonstrated that various boronated porphyrins can target a variety of tumor types. Of the porphyrins evaluated so far, copper tetracarboranylphenyl porphyrin (CuTCPH) is potentially a strong candidate for clinical use. In the present investigation, the response of the oral mucosa to CuTCPH-mediated boron neutron capture (BNC) irradiation was assessed using the ventral surface of the tongue of adult male Fischer 344 rats, a standard rodent model. CuTCPH was administered by intravenous infusion, at a dose of 200 mg/kg body weight, over a 48-h period. Three days after the end of the administration of CuTCPH, biodistribution studies indicated very low levels of boron (<2 microg/g) in the blood. Levels of boron in tongue tissue were 39.0 +/- 3.8 microg/g at this time. This was the time selected for irradiation with single doses of thermal neutrons from the Brookhaven Medical Research Reactor. The estimated level of boron-10 in the oral mucosa was used in the calculation of the physical radiation doses from the 10B(n,alpha)7Li reaction. This differs from the approach using the present generation of clinical boron carriers, where boron levels in blood at the time of irradiation are used for this calculation. Dose-response curves for the incidence of mucosal ulceration were fitted using probit analysis, and the doses required to produce a 50% incidence of the effect (ED50 +/- SE) were calculated. Analysis of the dose-effect data for CuTCPH-mediated BNC irradiation, compared with those for X rays and thermal neutrons alone, gave a compound biological effectiveness (CBE) factor of approximately 0.04. This very low CBE factor would suggest that there was relatively low accumulation of boron in the key target epithelial stem cells of the oral mucosa. As a consequence, with low levels of boron (<2 microg/g) in the blood, the response of the oral mucosa to CuTCPH-mediated BNCT will be governed primarily by the radiation effects of the thermal neutron beam and not from the boron neutron capture reaction [10B(n,alpha)7Li].

Lack of evidence for co-speciation in a parasitic nematode of grey kangaroos.

Multilocus enzyme electrophoresis was used to compare specimens of the parasitic nematode Cloacina obtusa from the stomach of the eastern grey kangaroo, Macropus giganteus and the western grey kangaroo, M. fuliginosus. Allelic variation among nematodes was detected at 17 (85%) of 20 loci, but there was only a single fixed genetic difference (at the locus for isocitrate dehydrogenase, IDH) between C. obtusa from M. fuliginosus and those from M. giganteus in areas where each host occurred in allopatry. However, this fixed difference was not apparent within the zone of host sympatry. Although electrophoretic data indicate genetic divergence among allopatric populations of C. obtusa in the two host species, the magnitude of the electrophoretic difference (5%) between these populations does not refute the hypothesis that C. obtusa represents a single species. The 'usual' situation for parasitic helminths of grey kangaroos is that pairs of parasite species occur in the two host species. This situation differs for C. obtusa, where there has been a lack of speciation following a speciation event in its macropodid marsupial hosts. This finding suggests that a speciation event in the host does not necessarily lead to a speciation event for all its parasites and further highlights our lack of understanding of which processes drive speciation in parasites.

The effect of dexamethasone on the uptake of p-boronophenylalanine in the rat brain and intracranial 9L gliosarcoma.

The steroid dexamethasone sodium phosphate (DEX) is routinely used to treat edema in brain tumor patients. The objective of the present study was to evaluate the effects of DEX on the uptake of boronophenylalanine (BPA) using the rat 9L gliosarcoma tumor model and surrounding brain tissue. Two steroid dosage protocols were used. The high-dose DEX protocol involved five 3mg/kg intraperitoneal injections at 47, 35, 23, 11 and 1 h prior to the administration of the BPA for a total dose of 15 mg DEX/kg rat. The low-dose DEX administration protocol involved two doses of 1.5mg/kg at 17 h and 1h prior to BPA injection for a total dose of 3mg DEX/kg rat. The control animals received no pretreatment, prior to the administration of BPA. Seventeen days after tumor implantation, rats were injected i.p. with 0.014 ml/g body weight BPA solution (1200 mg BPA/kg; approximately 59 mg (10)B/kg). In all groups, rats were euthanized at 3h after BPA injection. Administration of the steroid had an effect on tumor weight, which decreased to approximately 78% (p > 0.05) of the control weight in the low-dose DEX group, and approximately 48% (p < 0.001) of the control weight in the high-dose DEX group. At 3 h after the administration of BPA, the concentration of boron in tumor was comparable (p > 0.1) in the control and high-dose DEX groups. The lowest mean value (73.8+/-1.6 microg/g) was obtained in the low-dose DEX group. This was significantly lower (p > 0.02) than the tumor boron contents in the high-dose DEX and control groups, which were 81.1+/-1.9 and 79.9+/-1.7 microg/g, respectively. Tumor:blood boron partition ratios for the control, low- and high-dose DEX groups were 2.3, 2.3 and 2.5, respectively. Boron concentrations were also measured in the normal brain and in the zone of brain adjacent to the tumor exhibiting edema. Although treatment with DEX had no appreciable effect on boron uptake in the normal brain of the rat, after the administration of BPA, it did impact on the boron levels in the zone of peritumoral edema. After the high-dose DEX administration protocol, boron levels in the zone of edema were reduced by approximately 14% (p < 0.02). This finding suggests that BPA targeting of tumor cells in the peritumoral zone could be compromised by DEX. These cells appear to play a critical role in tumor recurrence after BNCT or conventional radiotherapy.

Synthesis of copper octabromotetracarboranylphenylporphyrin for boron neutron capture therapy and its toxicity and biodistribution in tumour-bearing mice.

Copper tetracarboranyltetraphenylporphyrin (CuTCPH) is a minimally toxic carborane-containing porphyrin that has safely delivered high concentrations of boron for experimental boron neutron capture therapy (BNCT). Copper octabromotetracarboranylphenylporphyrin (CuTCPBr), synthesized by bromination of CuTCPH, is one of several new minimally toxic analogues of CuTCPH being studied in our laboratory, which could possess comparable or better tumour-targeting properties with enhanced tumour cytotoxicity. Its biodistribution, biokinetics and toxicity in mice with subcutaneous EMT-6 (mammary) or SCCVII (squamous cell) carcinomas were compared with those of CuTCPH. The administration of approximately 200 mg kg(-1) of either porphyrin in six intraperitoneal injections over 2 days had no apparent effect, but administration of approximately 400 mg kg(-1) slightly lowered body weights, elevated alanine and aspartate transaminase activities in blood plasma, and depressed blood platelet counts for several days. Enzymes and platelets returned to normal within 5 days after those injections and body weights returned to normal within 2 weeks. High average concentrations of boron from either porphyrin were achieved in the two tumour models from a total dose of approximately 200 mg kg(-1). The high tumour boron concentration decreased slowly while concentrations in blood decreased rapidly. Boron concentrations in brain and skin were consistently lower than in tumour by a factor of 10 or more. Although either CuTCPH or CuTCPBr can be labelled with (64)Cu for imaging by positron emission tomography (PET), CuTCPBr can also be labelled by (76)Br, another PET-imageable nuclide.

Porphyrin-mediated boron neutron capture therapy: evaluation of the reactions of skin and central nervous system.

PURPOSE: Recently, various boronated porphyrins have been shown to preferentially target a variety of tumour types. Of the different porphyrins evaluated, copper tetra-phenyl-carboranyl porphyrin (CuTCPH) is a strong candidate for future preclinical evaluation. In the present study, the responses of two critical normal tissues, skin and central nervous system (CNS), to boron neutron capture (BNC) irradiation in the presence of this porphyrin were evaluated. MATERIALS AND METHODS: Standard models for the skin and spinal cord of adult male Fischer 344 rats were used. CuTCPH was administered by intravenous infusion at a dose of 200 mg x kg(-1) body weight, over 48 h. The thermal beam at the Brookhaven Medical Research Reactor was used for the BNC irradiations. The 20-mm diameter irradiation field, for both the skin and the spinal cord, was located on the mid-dorsal line of the neck. Dose-response data were fitted using probit analysis and the doses required to produce a 50% incidence rate of early and late skin changes or myeloparesis (ED(50) +/- SE) were calculated from these curves. RESULTS: Biodistribution studies indicated very low levels of boron (<3 microg x g(-1)) in the blood 3 days after the administration of CuTCPH. This was the time point selected for radiation exposure in the radiobiological studies. Levels of boron in the CNS were also low (2.8 +/- 0.6 microg x g(-1)) after 3 days. However, the concentration of boron in the skin was considerably higher at 22.7 +/- 2.6 microg x g(-1). Single radiation exposures were carried out using a thermal neutron beam. The impact of CuTCPH-mediated BNC irradiation on the normal skin and CNS at therapeutically effective exposure times was minimal. This was primarily due to the very low blood boron levels (from CuTCPH) at the time of irradiation. Analysis of the relevant dose-effect data gave compound biological effectiveness factors of about 1.8 for skin (moist desquamation) and about 4.4 for spinal cord (myeloparesis) for CuTCPH. These values were based on the BNC radiation doses to tissues calculated using the blood boron levels at the time of irradiation. CONCLUSIONS: CuTCPH-mediated BNC irradiation will not cause significant damage to skin and CNS at clinically relevant radiation doses provided that blood boron levels are low at the time of radiation exposure.

Evaluation of the relative biological effectiveness of a clinical epithermal neutron beam using dog brain.

This investigation was designed to determine the relative biological effectiveness (RBE) of an epithermal neutron beam (FiR 1 beam) using the brains of dogs. The FiR 1 beam was developed for the treatment of patients with glioma using boron neutron capture therapy. Comparisons were made between the effects of whole-brain irradiation with epithermal neutrons and 6 MV photons. For irradiations with epithermal neutrons, three dose groups were used, 9.4 +/- 0.1, 10.2 +/- 0.1 and 11.5 +/- 0.2 Gy. These physical doses were given as a single exposure and are quoted at the 90% isodose. Four groups of five dogs were irradiated with single doses of 10, 12, 14 or 16 Gy of 6 MV photons to the 100% isodose. Different reference isodoses were used to obtain the most comparable dose distribution in the brain for the two different irradiation modalities. Sequential magnetic resonance images (MRI) were taken for 77-115 weeks after irradiation to detect changes in the brain. Dose-effect relationships were established for changes in the brain as detected either by MRI or by subsequent gross morphology and histology. The doses that caused a specified response in 50% of the animals (ED(50)) were calculated from these dose-effect curves for each end point, and these values were used to calculate the RBE values for the different end points. The RBE values for the FiR 1 beam, based on changes observed on MRI, were in the range 1.2-1.3. For microscopic and gross pathological lesions, the values were in the range 1.2-1.4. The corresponding RBE values for the MRI and pathological end points for the high-LET components (protons from nitrogen capture and recoil protons from fast neutrons) were in the ranges 3.5-4.0 and 3.4-4.4, respectively. This assumed a dose-rate reduction factor of 0.6 for the low-dose-rate gamma-ray component of this beam. Finally, a comparison was made between experimentally derived photon doses, for a specified end point, with calculated photon equivalent doses, which were obtained using the weighting factors for clinical studies on the epithermal neutron beam on the Brookhaven Medical Research Reactor (BNL) in New York. This indicated that the radiation-induced lesions seen in the present study were, on average, detected at a 12% lower photon dose than predicted by the use of the BNL clinical weighting factors. This indicates the need for caution in the extrapolation of results from one reactor-based epithermal neutron beam to another.

Long-term infusions of p-boronophenylalanine for boron neutron capture therapy: evaluation using rat brain tumor and spinal cord models.

Rat 9L gliosarcoma cells infiltrating the normal brain have been shown previously to accumulate only approximately 30% as much boron as the intact tumor after administration of the boronated amino acid p-boronophenylalanine (BPA). Long-term i.v. infusions of BPA were shown previously to increase the boron content of these infiltrating tumor cells significantly. Experiments to determine whether this improved BPA distribution into infiltrating tumor cells after a long-term i.v. infusion improves tumor control after BNCT in this brain tumor model and whether it has any deleterious effects in the response of the rat spinal cord to BNCT are the subjects of the present report. BPA was administered in a fructose solution at a dose of 650 mg BPA/kg by single i.p. injection or by i.v. infusion for 2 h or 6 h, at 330 mg BPA/kg h(-1). At 1 h after the end of either the 2-h or the 6-h infusion, the CNS:blood (10)B partition ratio was 0.9:1. At 3 h after the single i.p. injection, the ratio was 0.6:1. After spinal cord irradiations, the ED(50) for myeloparesis was 14.7 +/- 0.4 Gy after i.p. administration of BPA and 12.9 +/- 0.3 Gy in rats irradiated after a 6-h i.v. infusion of BPA; these values were significantly different (P < 0.001). After irradiation with 100 kVp X rays, the ED(50) was 18.6 +/- 0.1 Gy. The boron compound biological effectiveness (CBE) factors calculated for the boron neutron capture dose component were 1.2 +/- 0.1 for the i.p. BPA administration protocol and 1.5 +/- 0.1 after irradiation using the 6-h i.v. BPA infusion protocol (P < 0.05). In the rat 9L gliosarcoma brain tumor model, the blood boron concentrations at 1 h after the end of the 2-h infusion (330 mg BPA/kg h(-1); n = 15) or after the 6-h infusion (190 mg BPA/kg h(-1); n = 13) were 18.9 +/- 2.2 microg 10B/g and 20.7 +/- 1.8 microg 10B/g, respectively. The irradiation times were adjusted individually, based on the preirradiation blood sample, to deliver a predicted 50% tumor control dose of 8.2 Gy ( approximately 30 photon-equivalent Gy) to all tumors. In the present study, the long-term survival was approximately 50% and was not significantly different between the 2-h and the 6-h infusion groups. The mode of BPA administration and the time between administration and irradiation influence the 10B partition ratio between the CNS and the blood, which in turn influences the measured CBE factor. These findings underline the need for clinical biodistribution studies to be carried out to establish 10B partition ratios as a key component in the evaluation of modified administration protocols involving BPA.

Boron microquantification in oral muscosa and skin following administration of a neutron capture therapy agent.

Clinical trials of boron neutron capture therapy (BNCT) for intracranial tumours using boronophenylalanine-fructose undertaken at Harvard-MIT and Brookhaven National Laboratory have observed acute normal tissue reactions in the skin and oral mucosa. Because the range of the 10B(n, alpha)7Li reaction products is very short, 10-14 microns combined, knowledge of the 10B microdistribution in tissue is critical for understanding the microdosimetry and radiobiology of BNCT. This paper reports measurements of the microdistribution of 10B in an animal model, rat skin and tongue, using high resolution quantitative autoradiography (HRQAR), a neutron-induced etched track autoradiographic technique. The steep spatial gradient and high absolute value relative to blood of the 10B concentration observed in some strata of the rat tongue epithelium and skin are important for properly evaluating the radiobiology and the biological effectiveness factors for normal tissue reactions such as oral mucositis, which are generally assessed using the blood boron concentration rather than the tissue boron concentration.

Recognition templates for predicting adenylate-binding sites in proteins.

Recognition templates encapsulate the structural and energetic features for the specific recognition of a given ligand by a protein active site. These templates identify the major interactions used for specific recognition and may be used to find specific binding sites in proteins of unknown function. We present a grid-based method for deriving recognition templates for adenylate groups from a set of diverse nucleotide-binding proteins. The templates reveal the basis of specific binding of adenylate, including tight shape complementarity, specific hydrogen bonds, and underscoring the importance of a key steric contact for excluding guanylate from adenylate-specific sites. We demonstrate the utility of recognition templates in identifying specific adenylate-binding sites in a diverse set of dinucleotide-binding proteins.

Boron neutron capture therapy: effects of split dose and overall treatment time.

New clinical protocols are being developed that will entail the administration of considerably higher doses of the boron delivery agent boronophenylalanine (BPA) than those in current clinical use. Fractionation (2 or 4 fractions) of BPA mediated boron neutron capture therapy (BNCT) is also under consideration at some clinical centres. Given the considerably higher infusion volumes that will be entailed in the delivery of BPA in the new high dosage protocols, there will be a requirement to extend the gap between fractions to 2 or more days. In order to assess the effects of a 2 fraction protocol on the therapeutic efficacy of BPA mediated BNCT, a series of split dose irradiations (two equal fractions) were undertaken using the rat intracranially implanted 9L gliosarcoma model. A single dose exposure to BPA mediated BNCT of 3.0 Gy resulted in long term survival levels of 50%. Survival levels increased to 71% and 77% with a 3 and 5 day gap between dose fractions (two equal fractions), respectively, using the same total dose. A further increase in the time interval between dose fractions to 7 days resulted in a reduction in survival to 36%. However, there was no significant difference between the single dose and the 3, 5 and 7 day survival data (P > 0.1) The difference between the 5 and 7 day split dose survival data was of border-line significance (P = 0.05). It is anticipated that mucositis, could become a potential problem in future BNCT clinical protocols involving higher doses, larger field sizes or multiple fields. The potential sparing of the oral mucosa, due to repopulation during the interval between the two fractions, was investigated using a series of split dose BPA mediated BNC irradiations. The ventral surface of the rat tongue was utilised as a model for oral mucosa. The ED50 (50% incidence) values for the ulceration end point were 3.0+/-0.1, 3.2+/-0.1, 3.0+/-0.1 and 3.6+/-0.1 Gy, for 3, 5, 7 and 9 day splits between doses, respectively. It is evident from this data that there were no significant changes in the ED50 (p < 0.001) until the 9 day dose split, when the ED50 value was 20% higher than the ED50 value after a 7 day split. It was concluded that the two fraction BNCT protocol, with dose splits of up to 5 days, did not diminish the therapeutic response of the rat 9L gliosarcoma, when compared with a single dose BNCT protocol. Tolerance of the oral mucosa to BNC irradiation was not increased until there was a 9 day gap between fractions. However, the beneficial effects of dose sparing at this time interval between doses, would probably be counteracted by a reduction in the therapeutic effectiveness of the BNCT modality, due to repopulation of tumour clonogens between doses.

Crystal structure of a neutralizing human IGG against HIV-1: a template for vaccine design.

We present the crystal structure at 2.7 angstrom resolution of the human antibody IgG1 b12. Antibody b12 recognizes the CD4-binding site of human immunodeficiency virus-1 (HIV-1) gp120 and is one of only two known antibodies against gp120 capable of broad and potent neutralization of primary HIV-1 isolates. A key feature of the antibody-combining site is the protruding, finger-like long CDR H3 that can penetrate the recessed CD4-binding site of gp120. A docking model of b12 and gp120 reveals severe structural constraints that explain the extraordinary challenge in eliciting effective neutralizing antibodies similar to b12. The structure, together with mutagenesis studies, provides a rationale for the extensive cross-reactivity of b12 and a valuable framework for the design of HIV-1 vaccines capable of eliciting b12-like activity.

Response of avian embryonic brain to spatially segmented x-ray microbeams.

Duck embryo was studied as a model for assessing the effects of microbeam radiation therapy (MRT) on the human infant brain. Because of the high risk of radiation-induced disruption of the developmental process in the immature brain, conventional wide-beam radiotherapy of brain tumors is seldom carried out in infants under the age of three. Other types of treatment for pediatric brain tumors are frequently ineffective. Recent findings from studies in Grenoble on the brain of suckling rats indicate that MRT could be of benefit for the treatment of early childhood tumors. In our studies, duck embryos were irradiated at 3-4 days prior to hatching. Irradiation was carried out using a single exposure of synchrotron-generated X-rays, either in the form of parallel microplanar beams (microbeams), or as non-segmented broad beam. The individual microplanar beams had a width of 27 microm and height of 11 mm, and a center-to-center spacing of 100 microm. Doses to the exposed areas of embryo brain were 40, 80, 160 and 450 Gy (in-slice dose) for the microbeam, and 6, 12 and 18 Gy for the broad beam. The biological end point employed in the study was ataxia. This neurological symptom of radiation damage to the brain developed within 75 days of hatching. Histopathological analysis of brain tissue did not reveal any radiation induced lesions for microbeam doses of 40-160 Gy (in-slice), although some incidences of ataxia were observed in that dose group. However, severe brain lesions did occur in animals in the 450 Gy microbeam dose groups, and mild lesions in the 18 Gy broad beam dose group. These results indicate that embryonic duck brain has an appreciably higher tolerance to the microbeam modality, as compared to the broad beam modality. When the microbeam dose was normalized to the full volume of the irradiated tissue. i.e., the dose averaged over microbeams and the space between the microbeams, brain tolerance was estimated to be about three times higher to microbeam irradiation as compared with broad beam irradiation.

Changes in epidermal radiosensitivity with time associated with increased colony numbers.

Epidermal clonogenic cell survival and colony formation following irradiation were investigated and related to radiosensitivity. A rapid in vivo/in vitro assay was developed for the quantification of colonies arising from surviving clonogenic cells in pig epidermis after irradiation. Bromodeoxyuridine (BrdU)-labelled cells in full thickness epidermal sheets were visualized using standard immunohistochemistry. In unirradiated skin, approximately 900 BrdU-positive cells mm(-2) were counted. In a time sequence experiment, BrdU-positive cell numbers increased from an average of 900 cells mm(-2) to approximately 1400 cells mm(-2) after BrdU-labelling for 2-24 h. In irradiated skin, colonies containing >/=16 BrdU-positive cells were seen for the first time at days 14/15 after irradiation. The number of these colonies per cm(2) as a function of skin surface dose yielded a cell survival curve with a D(0)-value (+/-SE) of 3.9+/-0.6 Gy. This relatively high D(0)-value is possibly due to a rapid fall off in depth dose distribution for the iridium-192 source and consequently a substantial contribution of hair follicular epithelium to colony formation. At 14/15 days after irradiation, the ED(50) level of 33.6 Gy for the in vivo response of moist desquamation corresponded with 2.7 colonies cm(-2). Surprisingly, the number of colonies increased with time after irradiation with an estimated doubling time of approximately 4 days, while the D(0)-value remained virtually unchanged. This increase in colony numbers could be due to migration of clonogenic cells, to the recruitment of dormant clonogenic cell survivors by elevated levels of cytokines, or to both. Although frequent biopsying caused increased cytokine levels, which had a systemic effect on unirradiated skin, it had no influence on colony formation in irradiated skin. Smaller colonies, containing 4-8 cells or 9-15 cells, were abundant, particularly after higher doses, which resulted in higher D(0)-values. The majority of these small colonies were abortive and did not progress to larger colonies. There was no statistical evidence for significant variations in the interanimal responses.