Monte Carlo simulation of single-plane magnetically focused narrow proton beams.

We present Monte Carlo simulations of magnetically focused proton beams shaped by a single quadrapole magnet. Such beams are narrowly focused in one longitudinal plane but fan out in the perpendicular plane producing elongated elliptical beam spots (a 'screwdriver' shape). The focused beams were compared to passively collimated beams (the current standard of delivery for small radiosurgery beams). Beam energies considered were relevant to functional radiosurgery and standard radiosurgery clinical applications. Three monoenergetic beams (100, 125, and 150 MeV) and a modulated beam were simulated. Monoenergetic magnetically focused beams demonstrated 28 to 32% lower entrance doses, 31 to 47% larger central peak to entrance depth dose ratios, 26 to 35% smaller integral dose, 25 to 32% smaller estimated therapeutic ratios, 19 to 37% smaller penumbra volumes, and 38 to 65% smaller vertical profile lateral penumbras at Bragg depth, compared to the collimated beams. Focused modulated beams showed 31% larger central peak to entrance dose ratio, and 62 to 65% smaller vertical lateral penumbras over the plateau of the spread out Bragg peak. These advantages can be attributed to the directional acceleration of protons in the transverse plane due to the magnetic field. Such beams can be produced using commercially available assemblies of permanent rare earth magnets that do not require electric power or cryrogenic cooling. Our simulations suggest that these magnets can be inexpensively incorporated into the beam line to deliver reduced dose to normal tissue, and enhanced dose to elongated elliptical targets with major and minor axes on the order of a few centimeters and millimeters, respectively. Such beams may find application in novel proton functional and standard radiosurgery treatments in and around critical structures.

Brief stimulation of the peroneal nerve attenuates the exercise pressor reflex in anaesthetised cats.

We recently demonstrated that applying capsaicin to the common peroneal nerve, thereby activating small diameter afferent neurons, caused a substantial rise in mean arterial pressure (MAP) and heart rate (HR) that lasted approximately 20 min. In addition, this application of capsaicin transiently attenuated the exercise pressor reflex (EPR). The purpose of the current study was to test the hypothesis that stimulating the peroneal nerve at an intensity that activated both myelinated and unmyelinated axons for a short duration (1 min) causes a similar attenuation of the EPR. Cats were anaesthetised with alpha-chloralose and urethane, the popliteal fossa was exposed, and static contraction was induced by stimulating the tibial nerve. The ipsilateral peroneal nerve was cut and placed on a stimulating electrode. Prior to peroneal nerve stimulation, static contraction of the triceps surae muscle for 1 min increased MAP 48+/-8 mmHg and HR 16+/-3 bpm. Electrical stimulation of the central end of the cut peroneal nerve for 1 min (100 x motor threshold; 40 Hz; 0.1 ms) increased MAP and HR by 62+/-11 mmHg and 28+/-4 bpm, respectively. These increases returned to prestimulation levels within 1 min. Two minutes after the peroneal stimulation was stopped, the EPR was markedly reduced as muscle contraction increased MAP and HR by 20+/-4 mmHg and 7+/-2 bpm, respectively. Repeating the muscle contraction approximately 25 min after peroneal stimulation increased MAP and HR by 38+/-8 mmHg and 12+/-2 bpm, indicating some recovery of the EPR. These results show that brief (1 min) electrical stimulation of afferent neurons in the peroneal nerve attenuates the EPR. This supports the hypothesis that strong activation of small diameter afferent neurons stimulates a nervous system mechanism that diminishes the sensory input from skeletal muscle involved in cardiovascular regulation.

QTL analysis of an intervarietal set of substitution lines in Brassica napus: (i) Seed oil content and fatty acid composition.

Backcross breeding with marker-assisted selection was used to construct an intervarietal set of part chromosome substitution lines in Brassica napus, formed from a cross between two winter varieties of oilseed rape: Tapidor and Victor. A total of 22 lines from this substitution library were examined over a 3-year period, in a total of nine field trials, for seed oil fatty acid composition and seed oil content. Trialing of the substitution lines gave evidence for the existence of 13 quantitative trait loci (QTL). All 13 QTL affected fatty acid composition of the seed, and were distributed among linkage groups 1, 3, 6, 7, 8, 11, 13, 14, 18, and 19. Seven of these QTL, on linkage groups 3, 6, 8, 13, 14, 18, and 19, also affected total seed oil content. The positions of these QTL are compared to those in the published literature and with respect to erucic acid QTL previously identified in a backcross population of the same cross. The substitution line approach gives increased precision and sensitivity for QTL mapping compared to other methods.

Molecular analysis of the structure of the maize B-chromosome.

The overall composition of the maize B is similar to that of the standard chromosome complement (A-chromosomes). This has been demonstrated by the use of several methods including: (a) genomic in situ hybridization (GISH), (b) analysis of highly repetitive sequences by the comparison of restriction digests of 0B and +B DNA and (c) the characterization of middle-repetitive cloned sequences. By the use of the technique of random amplification of polymorphic DNA-polymerase chain reaction, we have identified a B-specific repetitive sequence family. Sequence analysis of the B-specific clone reveals a relationship to the PREM-1 family of maize retroelements, which are preferentially transcribed in pollen. These results suggest an internal origin of the B-chromosome from within the maize genome, but demonstrate also that specific sequences can evolve by rapid processes of genomic turnover. Models for the origin of the maize B are discussed in the context of the present data.

Molecular marker analysis of Helianthus annuus L. 2. Construction of an RFLP linkage map for cultivated sunflower.

A detailed linkage map of Helianthus annuus was constructed based on segregation at 234 RFLP loci, detected by 213 probes, in an F2 population of 289 individuals (derived from a cross between the inbred lines HA89 and ZENB8). The genetic markers covered 1380 centiMorgans (cM) of the sunflower genome and were aranged in 17 linkage groups, corresponding to the haploid number of chromosomes in this species. One locus was found to be unlinked. Although the average interval size was 5.9 cM, there were a number of regions larger than 20 cM that were devoid of markers. Genotypic classes at 23 loci deviated significantly from the expected ratios (1∶2∶1 or 3∶1), all showing a reduction in the ZENB8 homozygous class. The majority of these loci were found to map to four regions on linkage groups G, L and P.

Molecular marker analysis of Helianthus annuus L. 1. Restriction fragment length polymorphism between inbred lines of cultivated sunflower.

cDNA and PstI genomic clones have been used to assess levels of restriction fragment length polymorphism (RFLP) in Helianthus annuus and to determine the inter-relationships between a diverse set of 24 inbred lines. Of the cDNA clones screened 45% were useful as RFLP probes, compared to less than 20% from the PstI library, which showed high levels of redundancy for high copy sequences. Fifty-seven low-copy DNA probes (23 PstI and 34 cDNA clones) were used to fingerprint 12 maintainer (B) lines and 12 restorer (R) lines. The average number of RFLP variants per probe was found to be 3.2, with a mean polymorphic index of 0.49, indicating that high levels of nuclear DNA polymorphism are to be found in cultivated sunflower. Cluster and principal coordinate analysis of the fingerprinting data clearly separated the maintainer and restorer lines, but there was a degree of association between 2 unbranched R-lines and the B-line germ plasm pool.

RFLP analysis of complex traits in crop plants.

Detailed genetic maps, based upon molecular markers (in particular, on restriction fragment length polymorphisms-RFLPs) have now been constructed for a number of crop plant species, and permit a range of genetic analyses hitherto considered impossible. The availability of such maps has made it possible to approach the dissection and manipulation of both simply-inherited and complex characteristics. Even those characters that show apparently 'quantitative' inheritance (displaying essentially continuous variation within a segregating family) can frequently be resolved into a handful of major gene effects. Once tagged with molecular markers, the genes can be assembled in any desired combination, permitting the testing of hypotheses on gene action/interaction, or the construction of varieties of plant species with improved agronomic performance. The next technological challenge is to 'walk' from RFLP markers to isolate the actual genes responsible for the complex trait, by a combination of genetical and physical mapping techniques. Such analyses will begin to clarify our picture of the relationship between genetic and physical maps, of recombination, and of the arrangement of diverse families of DNA sequences in plant genomes.

The isolation and characterisation of plant sequences homologous to human hypervariable minisatellites.

We have isolated DNA probes, homologous to the human hypervariable minisatellite sequence 33.15, from the genome of rice (Oryza sativa). These probes are capable of producing a multilocus rice DNA fingerprint. The rice sequence has a tandem repeating structure based on a 12 bp GC-rich repeat which shows homology to its human counterpart. This probe detects up to 30 loci which are at a number of unlinked chromosomal sites. The GC-rich sequence is invariably associated with an open reading frame (ORF) of unknown function. The ORF is probably a member of a small multigene family.

Cage design and configuration for an arboreal species of primate.

The squirrel monkey (genus Saimiri) is an arboreal primate from equatorial South America. This species forms large social groups that consist of multiple females and males of varying ages, from infant to adult. As the use of squirrel monkeys in research continues to grow, an understanding of optimal cage design and environment is essential. The University of South Alabama Primate Research Laboratory houses a breeding colony of 350 squirrel monkeys. Each group cage, measuring 4.5 X 2.5 X 1.5 meters, can contain up to 20 animals. A breeding group consists of one adult male, eight to ten adult females, and varying numbers of infant and juvenile animals. In order to determine the most suitable cage environment for the squirrel monkey, a series of studies were carried out to compare various perch materials and cage configurations. Squirrel monkeys preferred a poly-vinyl-chloride pipe perch (rigid) over rope perches (non-rigid). When provided with multiple levels of perches, all levels were used. Males tended to distribute their activities randomly at different levels. In a two tiered perch arrangement, females concentrated 67% of their social activity on the top tier. In a triple tier configuration, females concentrated 66% of their travel on the top tier. These results indicate that by creating a cage environment with multiple tiers of horizontal perches the effective cage space can be doubled or tripled. This provides an effective means of reducing population density without enlarging the dimensions of the cage or reducing social group size.

An enzymic method of preparing plant chromosomes for in situ hybridization.

A method of preparing chromosomes from plant root tips for in situ hybridization with tritiated DNA is described. The technique relies on the enzymic hydrolysis of plant cell walls with a pectinase-cellulase mixture. It is shown that, despite the enzymic mixture possessing nuclease activity, there is no detectable degradation of DNA within fixed root tips. To demonstrate the suitability of this method of preparing plant chromosomes for in situ hybridization, a cloned repetitive DNA sequence has been hybridized to Allium sativum chromosomes. Chromosomes prepared using this technique also can be readily C-banded.

Interrelationship of cultivated rices Oryza sativa and O. glaberrima with wild O. perennis complex : Analysis of fraction 1 protein and some repeated DNA sequences.

Phylogenetic relationship of the cultivated rices Oryza sativa and O. glaberrima with the O. perennis complex, distributed on the three continents of Asia, Africa and America, and O. australiensis has been studied using Fraction 1 protein and two repeated DNA sequences as markers. Fraction 1 protein isolated from the leaf tissue of accessions of different species was subjected to isoelectric focusing. All the species studied have similar nuclear-encoded small subunit polypeptides and chloroplast-encoded large subunit polypeptides, except two of the O. perennis accessions from South America and O. australiensis, which have a different pattern for the chloroplast subunit. Two DNA sequences were isolated from Eco R1 restriction endonuclease digests of total DNA from O. sativa. One of the sequences has been characterized as highly repeated satellite DNA, and the other one as a moderately repeated DNA sequence. These sequences were used as probes in DNA/DNA hybridization with restriction endonuclease digested DNA from some accessions of the different species. Those accessions that are divergent for large subunit polypeptides of Fraction 1 protein (O. australiensis and two of the four South American O. perennis accessions) also lack the satellite DNA and have a different hybridization pattern with the moderately repeated sequence. All other accessions, irrespective of their geographical origin, are similar. We propose that various accessions of O. perennis from Africa and Asia are closely related to O. sativa and O. glaberrima, and that the dispersal of cultivated and O. perennis rices to different continents may be quite recent. The American O. perennis is a heterogeneous group. Some of the accessions ascribed to this group are closely related to the Asian and African O. perennis, while others have diverged.

Organization and evolution of repeated DNA sequences in closely related plant genomes.

In common with many other eukaryotic species, the genomes of species in the genus Allium contain a high proportion of repeated DNA sequences, which may be implicated in the considerable differences in genome size that are seen between even very closely related species. The gross organization of repetitive sequences within the genome of Allium sativum and of some other related species has been investigated using DNA/DNA hybridization studies. Such studies show that there has been much modulation in the amounts of different repeated DNA families during the evolution of the genus Allium; these repetitive elements are interspersed in all species with sequences of low repetition. The organization and distribution of one particular repetitive family within the genus has been examined using a cloned hybridization probe. Hybridization of this probe to DNA from related genomes reveals that this element is present in all Allium species examined, but with large-scale modulation of its abundance, and some considerable changes in its sequence environment. The evolution of such genome-specific arrangements of common repetitive elements and the possible mechanisms by which they might be maintained are discussed.

Noninvasive Doppler determination of cardiac output in man. Clinical validation.

A noninvasive technique for assessing cardiac output (CO) was evaluated by comparing it with thermodilution determinations in patients in the intensive care unit. The new method uses pulsed ultrasound to measure aortic diameter and continuous-wave Doppler ultrasound to obtain aortic blood velocity. An initial study evaluating just the velocity measurement showed that changes of the Doppler index of output (DI) correlated well with those of thermodilution cardiac output (TDCO). Linear regression analysis yielded delta DI = 0.87 delta TDCO + 0.14 (r = 0.83, n = 95). Using a university research instrument these measurements were possible in 54 of 60 patients (90%). A second study using a prototype commercial device incorporated the diameter measurement. Ultrasonic cardiac output (UCO), calculated as the time integral of velocity multiplied by the aortic area, was compared to TDCO. The data, obtained from 45 of 53 patients (85%), are described by the linear regression UCO = 0.95TDCO + 0.38 (r = 0.94, n = 110) over a range of 2-11 l/min. Patients with aortic stenosis, aortic insufficiency or a prosthetic valve have been excluded from the second study due to conditions likely to violate the assumptions upon which the calculation of absolute cardiac output is based. These results indicate that accurate CO can be measured by noninvasive ultrasound in most patients. The technique may be useful for extended CO monitoring in acute care patients and for CO assessment in many other types of patients undergoing diagnostic studies and therapeutic interventions.

The distribution of satellite and main-band DNA components in the melanogaster species subgroup of Drosophila. I. Fractionation of DNA in actinomycin D and distamycin A density gradients.

Fractionation of total adult DNA of five of the seven species of the melanogaster species sub-group of Drosophila in actinomycin D and distamycin A caesium density gradients has revealed the presence of three main-band DNA components, common to all species, and ten satellite DNAs that are distributed between the species. Satellite DNAs are either unique to a species or common to two or more species. The abundance of a common satellite DNA varies between species. There is no simple relationship between the presence of a satellite DNA and a branch point of phylogenetic divergence; nevertheless the arrangement of the species in a phylogeny that is based on the numbers of satellites held in common accurately reflects the pattern of relationships between the same species based on differences in inversions of polytene chromosomes. The species can be similarly arranged according to the compositions of their mitochondrial DNAs. It is possible that the same basic set of sequences, each of low frequency, is common to all species with arbitrary or selected amplification of particular sequences to differing extents in individual species. The conservation of satellites in the group and the close parallel between the distributions of satellites and inversions between the species suggests that either the processes that operate to change both chromosomal phenomena are similarly time-dependent and occurring at relatively low rates or that their rates of change are restricted according to some undetermined functions of these aspects of the genome.