Hepatobiliary circulation and dominant urinary excretion of homogentisic acid in a mouse model of alkaptonuria.

Altered activity of specific enzymes in phenylalanine-tyrosine (phe-tyr) metabolism results in incomplete breakdown of various metabolite substrates in this pathway. Increased biofluid concentration and tissue accumulation of the phe-tyr pathway metabolite homogentisic acid (HGA) is central to pathophysiology in the inherited disorder alkaptonuria (AKU). Accumulation of metabolites upstream of HGA, including tyrosine, occurs in patients on nitisinone, a licenced drug for AKU and hereditary tyrosinaemia type 1, which inhibits the enzyme responsible for HGA production. The aim of this study was to investigate the phe-tyr metabolite content of key biofluids and tissues in AKU mice on and off nitisinone to gain new insights into the biodistribution of metabolites in these altered metabolic states. The data show for the first time that HGA is present in bile in AKU (mean [±SD] = 1003[±410] µmol/L; nitisinone-treated AKU mean [±SD] = 45[±23] µmol/L). Biliary tyrosine, 3(4-hydroxyphenyl)pyruvic acid (HPPA) and 3(4-hydroxyphenyl)lactic acid (HPLA) are also increased on nitisinone. Urine was confirmed as the dominant elimination route of HGA in untreated AKU, but with indication of biliary excretion. These data provide new insights into pathways of phe-tyr metabolite biodistribution and metabolism, showing for the first time that hepatobiliary excretion contributes to the total pool of metabolites in this pathway. Our data suggest that biliary elimination of organic acids and other metabolites may play an underappreciated role in disorders of metabolism. We propose that our finding of approximately 3.8 times greater urinary HGA excretion in AKU mice compared with patients is one reason for the lack of extensive tissue ochronosis in the AKU mouse model.

Developing Decision-Making Expertise in Professional Sports Staff: What We Can Learn from the Good Judgement Project.

Success within performance sports is heavily dependent upon the quality of the decisions taken by educated and experienced staff. Multi-disciplinary teams (MDTs) typically collate voluminous data, and staff typically undergo extensive and rigorous technical and domain-specific training. Although sports professionals operate in sometimes volatile, uncertain, complex and ambiguous decision-making environments, a common assumption seems to be that education and experience will automatically lead to enhanced and effective decision-making capabilities. Accordingly, there are few formal curriculums, in coaching or sports science contexts, focussed on translating the extensive research on judgement and decision-making expertise to professional sports staff. This article aims to draw on key research findings to offer insights and practical recommendations to support staff working within professional performance contexts. Through this distillation, we hope to enhance understanding of the factors underpinning effective decision-making in dynamic, high-stakes professional sporting environments. Broadly, the conclusions of this research demonstrate that decision-making efficacy is enhanced through application of three specific strategies: (i) Design of more engaging professional cultures harnessing the power of collectives encouraging diverse opinions and perspectives, and fostering and promoting collaborative teamwork, (ii) education specifically targeting debiasing training, designed to counter the most common cognitive pitfalls and biases and, (iii) the implementation of evaluation strategies integrating rigorous testing and real-time feedback.

On fragmenting, densely mineralised acellular protrusions into articular cartilage and their possible role in osteoarthritis.

High density mineralised protrusions (HDMP) from the tidemark mineralising front into hyaline articular cartilage (HAC) were first described in Thoroughbred racehorse fetlock joints and later in Icelandic horse hock joints. We now report them in human material. Whole femoral heads removed at operation for joint replacement or from dissection room cadavers were imaged using magnetic resonance imaging (MRI) dual echo steady state at 0.23 mm resolution, then 26-µm resolution high contrast X-ray microtomography, sectioned and embedded in polymethylmethacrylate, blocks cut and polished and re-imaged with 6-µm resolution X-ray microtomography. Tissue mineralisation density was imaged using backscattered electron SEM (BSE SEM) at 20 kV with uncoated samples. HAC histology was studied by BSE SEM after staining block faces with ammonium triiodide solution. HDMP arise via the extrusion of an unknown mineralisable matrix into clefts in HAC, a process of acellular dystrophic calcification. Their formation may be an extension of a crack self-healing mechanism found in bone and articular calcified cartilage. Mineral concentration exceeds that of articular calcified cartilage and is not uniform. It is probable that they have not been reported previously because they are removed by decalcification with standard protocols. Mineral phase morphology frequently shows the agglomeration of many fine particles into larger concretions. HDMP are surrounded by HAC, are brittle, and show fault lines within them. Dense fragments found within damaged HAC could make a significant contribution to joint destruction. At least larger HDMP can be detected with the best MRI imaging ex vivo.

Cushing's disease: a single centre's experience using the linear accelerator (LINAC) for stereotactic radiosurgery and fractionated stereotactic radiotherapy.

Cushing's disease is hypercortisolaemia secondary to an adrenocorticotrophic hormone secreting pituitary adenoma. Primary management is almost always surgical, with limited effective medical interventions available. Adjuvant therapy in the form of radiation is gaining popularity, with the bulk of the literature related to the Gamma Knife. We present the results from our own institution using the linear accelerator (LINAC) since 1990. Thirty-six patients who underwent stereotactic radiosurgery (SRS), one patient who underwent fractionated stereotactic radiotherapy (FSRT) and for the purposes of comparison, 13 patients who had undergone conventional radiotherapy prior to 1990, were included in the analysis. Serum cortisol levels improved in nine of 36 (25%) SRS patients and 24 hour urinary free cortisol levels improved in 13 of 36 patients (36.1%). Tumour volume control was excellent in the SRS group with deterioration in only one patient (3%). The patient who underwent FSRT had a highly aggressive tumour refractory to radiation.

Acromegaly: a single centre's experience of stereotactic radiosurgery and radiotherapy for growth hormone secreting pituitary tumours with the linear accelerator.

Primary treatment for growth-hormone secreting pituitary adenomas usually involves surgery, with treatment options for recurrent and persistent disease including repeat surgery, medication and radiation therapy. The majority of previously published series for radiation therapy in acromegaly in the past 20 years have been based on Gamma-Knife (Elekta, Stockholm, Sweden) surgery. To our knowledge, we present the largest series of linear accelerator-based treatment for this disease, with a review of 121 patients treated at our institution; since 1990, 86 patients underwent stereotactic radiosurgery (SRS), 10 patients underwent fractionated stereotactic radiotherapy (FSRT), and for the purposes of comparison we also reviewed 25 patients who underwent conventional radiotherapy prior to 1990. Tumour volume control in all three groups was excellent and consistent with previously reported literature - only three of 86 (4%) patients undergoing SRS had a documented increase in tumour size, and none of the patients undergoing FSRT had a documented increase in size following a median follow-up of 5.5 and 5.1 years for SRS and FSRT, respectively. Target growth hormone levels of <2.5 ng/mL were met by 12 of 86 (14%) of the SRS group, and by two of 10 (20%) in the FSRT group. Target insulin-like growth factor-1 levels of age and sex matched controls were achieved in 16 of 86 patients (18.6%) post-SRS and five of 10 patients (50%) post-FSRT. New hormonal deficits requiring replacement therapy were identified in 17 of 86 (19.8%) patients post-SRS which is consistent with previously published radiosurgical series. Identified non-hormonal morbidity was low (<5%).

The use of radial symmetry to localize retinal landmarks.

Locating the optic disc center and the fovea in digital fundus images is surprisingly difficult due to the variation range in color and contrast and the possible presence of pathologies creating bright spots or changing the appearance of retinal landmarks. These reasons make it difficult to find good templates of optic disc and fovea shape and color for pattern matching. In this paper we propose radial symmetry as the principal cue to locate both optic disc and macula centers. Centers of bright and dark circularly symmetrical regions with arbitrary radii, can be found robustly against changes in brightness and contrast by using the Fast Radial Symmetry transform. Detectors based on this transform coupled with a weak hypothesis on vessel density (optic disc intersects large vessels while the fovea lies in an avascular region), can provide a fast location of both OD and macula with accuracy similar or better than state-of-the-art methods. The approach has been chosen as the default technique for fast localization of the two landmarks in the VAMPIRE software suite.

Ochronosis in a murine model of alkaptonuria is synonymous to that in the human condition.

OBJECTIVE: Alkaptonuria (AKU) is a rare genetic disease which results in severe early onset osteoarthropathy. It has recently been shown that the subchondral interface is of key significance in disease pathogenesis. Human surgical tissues are often beyond this initial stage and there is no published murine model of pathogenesis, to study the natural history of the disease. The murine genotype exists but it has been reported not to demonstrate ochronotic osteoarthropathy consistent with the human disease. Recent anecdotal evidence of macroscopic renal ochronosis in a mouse model of tyrosinaemia led us to perform histological analysis of tissues of these mice that are known to be affected in human AKU. DESIGN: The homogentisate 1,2-dioxygenase Hgd(+/)(-)Fah(-)(/)(-) mouse can model either hereditary tyrosinaemia type I (HT1) or AKU depending on selection conditions. Mice having undergone Hgd reversion were sacrificed at various time points, and their tissues taken for histological analysis. Sections were stained with haematoxylin eosin (H&E) and Schmorl's reagent. RESULTS: Early time point observations at 8 months showed no sign of macroscopic ochronosis of tissues. Macroscopic examination at 13 months revealed ochronosis of the kidneys. Microscopic analysis of the kidneys revealed large pigmented nodules displaying distinct ochre colouration. Close microscopic examination of the distal femur and proximal fibula at the subchondral junctions revealed the presence of numerous pigmented chondrocytes. CONCLUSIONS: Here we present the first data showing ochronosis of tissues in a murine model of AKU. These preliminary histological observations provide a stimulus for further studies into the natural history of the disease to provide a greater understanding of this class of arthropathy.

A single centre's experience of stereotactic radiosurgery and radiotherapy for non-functioning pituitary adenomas with the Linear Accelerator (Linac).

Non-functioning pituitary adenomas are primarily a surgically managed pathology, but recurrence or regrowth is not uncommon. Previous large series have retrospectively validated the use of the Gamma Knife (GK) as an adjuvant treatment. To our knowledge, we present the largest case series to date with the Linear Accelerator (Linac) for the management of this pathology. In this study we review the clinical course of 118 patients, 51 of whom had stereotactic radiosurgery (SRS) and 67 who had fractionated stereotactic radiotherapy (FSRT); the discriminatory feature being proximity to the optic chiasm. For comparison purposes a population of 53 patients who had conventional radiotherapy (CRT) is included. The local control rates at 5 years for SRS, FRST and CRT were 100%, 93% and 87% respectively. Treatment-related morbidity was low. These data confirm that Linac SRS and FSRT are safe and effective for the treatment of non-functioning pituitary adenomas.

The role of calcified cartilage and subchondral bone in the initiation and progression of ochronotic arthropathy in alkaptonuria.

OBJECTIVE: Alkaptonuria is a genetic disorder of tyrosine metabolism, resulting in elevated circulating concentrations of homogentisic acid. Homogentisic acid is deposited as a polymer, termed ochronotic pigment, in collagenous tissues, especially cartilages of weight-bearing joints, leading to a severe osteoarthropathy. We undertook this study to investigate the initiation and progression of ochronosis from the earliest detection of pigment through complete joint failure. METHODS: Nine joint samples with varying severities of ochronosis were obtained from alkaptonuria patients undergoing surgery and compared to joint samples obtained from osteoarthritis (OA) patients. Samples were analyzed by light and fluorescence microscopy, 3-dimensional scanning electron microscopy (SEM), and the quantitative backscattered electron mode of SEM. Cartilage samples were mechanically tested by compression to determine Young's modulus of pigmented, nonpigmented, and OA cartilage samples. RESULTS: In alkaptonuria samples with the least advanced ochronosis, pigment was observed intracellularly and in the territorial matrix of individual chondrocytes at the boundary of the subchondral bone and calcified cartilage. In more advanced ochronosis, pigmentation was widespread throughout the hyaline cartilage in either granular composition or as blanket pigmentation in which there is complete and homogenous pigmentation of cartilage matrix. Once hyaline cartilage was extensively pigmented, there was aggressive osteoclastic resorption of the subchondral plate. Pigmented cartilage became impacted on less highly mineralized trabeculae and embedded in the marrow space. Pigmented cartilage samples were much stiffer than nonpigmented or OA cartilage as revealed by a significant difference in Young's modulus. CONCLUSION: Using alkaptonuria cartilage specimens with a wide spectrum of pigmentation, we have characterized the progression of ochronosis. Intact cartilage appears to be resistant to pigmentation but becomes susceptible following focal changes in calcified cartilage. Ochronosis spreads throughout the cartilage, altering the mechanical properties. In advanced ochronosis, there is aggressive resorption of the underlying calcified cartilage leading to an extraordinary phenotype in which there is complete loss of the subchondral plate. These findings should contribute to better understanding of cartilage-subchondral interactions in arthropathies.

Wildlife forensics: "supervised" assignment testing can complicate the association of suspect cases to source populations.

Forensic science techniques are an important component of investigations for wildlife-related offences. In particular, DNA analyses can be used to characterize several attributes of biological evidence including sex, individual and species identification. Additionally, genetic assignment testing has enabled forensic biologists to identify the local population from which an individual may have originated. This technique has proved useful in situations where animals have been illegally harvested from areas/populations where hunting is prohibited. For this report, we used individual-based clustering (IBC), in the program Structure 2.2, under both "supervised" and "unsupervised" approaches to assess whether three suspected, illegally harvested moose originated from an endangered population. Atypical circumstances, with Nova Scotia having two moose sub-species in its jurisdiction, enabled strong IBC assignment testing results to determine the source population of the suspected samples. We found differences between the "unsupervised" and "supervised" modeling approaches to define genetic structure among the a priori characterized populations in our data set. Our findings illustrate the fact that individual clustering assignment tests can assist wildlife forensic cases to identify the source population of illegally harvested animals. However, the accuracy of results are highly dependant on the model choice used to define genetic clusters, as well as on the availability of a thorough database of samples throughout the managed area to accurately identify all genetic populations. Further, it is clear from our analyses that political jurisdictions do not accurately reflect isolated populations and we recommend using unsupervised IBC modeling for biological accuracy.

VAMPIRE: Vessel assessment and measurement platform for images of the REtina.

We present VAMPIRE, a software application for efficient, semi-automatic quantification of retinal vessel properties with large collections of fundus camera images. VAMPIRE is also an international collaborative project of four image processing groups and five clinical centres. The system provides automatic detection of retinal landmarks (optic disc, vasculature), and quantifies key parameters used frequently in investigative studies: vessel width, vessel branching coefficients, and tortuosity. The ultimate vision is to make VAMPIRE available as a public tool, to support quantification and analysis of large collections of fundus camera images.

Screening for diabetic retinopathy: a comparative trial of photography and scanning laser ophthalmoscopy.

AIMS: To evaluate the sensitivity and specificity of wide-field scanning laser ophthalmoscopy (WSLO) in the detection of referable diabetic eye disease, and to compare its performance with digital retinal photography. METHODS: Patients enrolled into the study underwent non-mydriatic WSLO imaging, then single- and dual-field mydriatic digital retinal photography, and examination with slit lamp biomicroscopy, the reference standard. Grading of retinopathy was performed in a masked fashion. RESULTS: A total of 380 patients (759 eyes) were recruited to the study. Technical failure rates for dilated single-field retinal photography, dual-field retinal photography and undilated WSLO were 6.3, 5.8 and 10.8%, respectively (0.005 < p < 0.02 for photography vs. WSLO). The respective indices for screening sensitivity were 82.9, 82.9 and 83.6% (p > 0.2). Specificity was 92.1, 91.1 and 89.5%, respectively (p > 0.2). CONCLUSIONS: Sensitivity and specificity for WSLO were similar to retinal photography. The technical failure rate was greater for the WSLO used in this study.

Calculi and intracellular ochronosis in the submandibular tissues from a patient with alkaptonuria.

Alkaptonuria (AKU) is a rare autosomal recessive condition caused by deficiency of the enzyme homogentisate 1,2 dioxygenase, resulting in widespread deposition of oxidised homogentisic acid (HGA) polymer, primarily in joint tissues but also in other connective tissues. Macroscopic pigmentation of connective tissues in AKU is well documented and is the end point of a process that is not understood. Deposition in less common regions may provide clues to the pigment formation process. This is the first report of detection of ochronotic pigment in acinar cells and lumina in the submandibular gland of a patient with AKU. Deposition was noted in the apical region of the cells. A lobar duct presented a large calculus with unusual deposits possibly associated with calcium salts. This report highlights the effect that local and intracellular factors may have on converting HGA into polymeric derivatives in the absence of an extracellular matrix.

Is there safety in numbers? The effect of cattle herding on biting risk from tsetse flies.

In sub-Saharan Africa, tsetse (Glossina spp.) transmit species of Trypanosoma which threaten 45-50 million cattle with trypanosomiasis. These livestock are subject to various herding practices which may affect biting rates on individual cattle and hence the probability of infection. In Zimbabwe, studies were made of the effect of herd size and composition on individual biting rates by capturing tsetse as they approached and departed from groups of one to 12 cattle. Flies were captured using a ring of electrocuting nets and bloodmeals were analysed using DNA markers to identify which individual cattle were bitten. Increasing the size of a herd from one to 12 adults increased the mean number of tsetse visiting the herd four-fold and the mean feeding probability from 54% to 71%; the increased probability with larger herds was probably a result of fewer flies per host, which, in turn, reduced the hosts' defensive behaviour. For adults and juveniles in groups of four to eight cattle, > 89% of bloodmeals were from the adults, even when these comprised just 13% of the herd. For groups comprising two oxen, four cows/heifers and two calves, a grouping that reflects the typical composition of communal herds in Zimbabwe, approximately 80% of bloodmeals were from the oxen. Simple models of entomological inoculation rates suggest that cattle herding practices may reduce individual trypanosomiasis risk by up to 90%. These results have several epidemiological and practical implications. First, the gregarious nature of hosts needs to be considered in estimating entomological inoculation rates. Secondly, heterogeneities in biting rates on different cattle may help to explain why disease prevalence is frequently lower in younger/smaller cattle. Thirdly, the cost and effectiveness of tsetse control using insecticide-treated cattle may be improved by treating older/larger hosts within a herd. In general, the patterns observed with tsetse appear to apply to other genera of cattle-feeding Diptera (Stomoxys, Anopheles, Tabanidae) and thus may be important for the development of strategies for controlling other diseases affecting livestock.

PPAR agonists modulate human osteoclast formation and activity in vitro.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear steroid hormone superfamily and exist in three isoforms: PPARalpha, beta and gamma, each with specific functions. In this study, we have investigated the expression of PPARs by human osteoclast precursors and osteoclasts generated in vitro. In addition, the effects of fibrates and isoform-specific PPAR agonists on osteoclast formation and resorption in vitro were determined. Human peripheral blood mononuclear cells (PBMCs) were stimulated with human recombinant RANKL and M-CSF to generate osteoclasts. RNA was extracted at days 0, 7, 14 and 21 and RT-PCR for all three PPAR isoforms demonstrated their expression throughout this culture period. To determine the effect on osteoclast formation, PPAR agonists (10(-8) M to 10(-5) M) were added from the beginning of the culture until day 14 and the number of multinucleated osteoclasts counted. The effect of PPAR agonists on osteoclast function was similarly determined by treating mature, multinucleated osteoclasts cultured on dentine wafers with PPAR agonists (10(-8) M to 10(-5) M) for 7 days and quantifying resorption. Bezafibrate and fenofibrate, which non-discriminately activate all PPAR isoforms, significantly inhibited the formation of multinucleated osteoclasts from PBMC in vitro. Bezafibrate treatment of mature osteoclast resulted in 50% inhibition (at 10(-8) M and 10(-7) M) of resorption, yet fenofibrate had no significant effect. Activation of individual PPARs with isoform-specific agonist (GW9578, L165041 and ciglitizone which preferentially activate PPARalpha, beta and gamma respectively) resulted in significant dose-dependent inhibition of multinucleated osteoclast formation. Divergent effects on osteoclast resorption were observed; GW9578 had no significant effect on resorption, whereas ciglitizone and L165041 dose-dependently inhibited and stimulated resorption, respectively. These data show for the first time expression of all three PPAR isoforms throughout the development and maturation period of osteoclasts generated from human PBMCs. In addition, we demonstrate that isoform-specific PPAR agonists have strong effects on multinucleation and highly variable effects on bone resorption. In conclusion, this study highlights the potential of PPARs as therapeutic targets in diseases with accelerated osteoclast formation and resorption.

Plant chromosome homology: hypotheses relating rendezvous, recognition and reciprocal exchange.

Many higher eukaryotes have dispersed repetitive DNA and multiple instances of segmental duplications. As well, many plants and lower animals are polyploids. Thus restricting reciprocal genetic exchange to truly homologous chromosomes is likely a multi-step process. We propose the following sequence of events. First the ability to form a synaptonemal complex (SC) prematurely (i.e. before homology checking/recognition) is precluded by the organization of chromosomes during premeiotic S phase. Next rough alignment is accomplished regionally by having key allelic transcription units brought to the same transcription center. Once rough alignment is accomplished, close alignment can occur in conjunction with homology checking/recognition. Successful homology checking produces changes that now permit SC formation within the region of the check. Some organisms (with challenges to true homology such as dispersed repetitive DNA and segmental duplications) may require that, for a region to be competent to form an SC, successful homology checks must occur at both ends of the region. Successful early SC formation may provide an environment in which recombination intermediates can be earmarked for resolution into crossovers. Later in prophase I SC formation can occur nonhomologously, if two unsynapsed chromosomal axes meet.

Application of DNA markers to identify the individual-specific hosts of tsetse feeding on cattle.

Primer sets for five different ungulate loci were used to obtain individual microsatellite DNA profiles for 29 Mashona cattle from a herd in Zimbabwe. There were 3-13 alleles for each locus and, using the entire suite of five loci, each animal within the herd, including closely related individuals, could be unequivocally distinguished. Wild-caught Glossina pallidipes Austen (Diptera: Glossinidae) were fed on specific cattle and the bloodmeal was profiled 0.5-72 h after feeding. The individual specific sources of the bloodmeals, including mixe meals produced by allowing tsetse to feed on two different cattle, were reliabl identified up to 24 h after feeding. The technique was used in field studies of hos selection by G. pallidipes and G. morsitans morsitans Westwood (Diptera Glossinidae) attracted to pairs of cattle. When the pair comprised an adult and a calf, 100% of meals were from the adult. For some pairs of adult cattle, tsetse were biased significantly towards feeding on one animal, whereas for other pairs there was no such bias. In general, feeding was greater on the animal known to have lower rate of host defensive behaviour. Results suggest that relatively slight differences in the inherent defensive behaviour of cattle produce large difference in host-specific feeding rates when the hosts are adjacent. For flies attracted to pair of cattle, < 2% contained blood from both hosts. The DNA profiling technique will be useful in studying the epidemiology of vector-borne diseases of livestock.

Postexposure rabies prophylaxis protocol for domestic animals and epidemiologic characteristics of rabies vaccination failures in Texas: 1995-1999.

OBJECTIVE: To determine whether postexposure rabies prophylaxis (PEP) in domestic animals, as mandated by the state of Texas, has continued to be effective and to evaluate PEP and preexposure rabies vaccination failures from 1995 through 1999. DESIGN: Retrospective study. ANIMALS: 830 unvaccinated domestic animals (621 dogs, 78 horses, 71 cats, and 60 cattle) that received PEP and 4 animals (3 dogs and 1 horse) that had preexposure rabies vaccination failure. PROCEDURE: Zoonotic incident case reports from 1995 through 1999 were reviewed for information regarding unvaccinated domestic animals that received PEP according to state protocol after exposure to a rabid animal; reports were also reviewed for information regarding preexposure rabies vaccination failures. The PEP recommendations were to immediately vaccinate the animal against rabies, isolate the animal for 90 days, and administer booster vaccinations during the third and eighth weeks of the isolation period. Rabies vaccines used in the PEP protocol were administered via the route prescribed by the USDA. RESULTS: From 1995 through 1999, 830 animals received PEP; 4 failures were recorded. Additionally, 4 preexposure rabies vaccination failures were recorded. CONCLUSIONS AND CLINICAL RELEVANCE: Results of this study indicate that an effective PEP protocol for unvaccinated domestic animals exposed to rabies includes immediate vaccination against rabies, a strict isolation period of 90 days, and administration of booster vaccinations during the third and eighth weeks of the isolation period. This PEP schedule has proven to be effective for control of rabies in domestic animals.

M2 delta-to-oxalate pi* conjugation in oxalate-bridged complexes containing M-M quadruple bonds.

The electronic structures of oxalate-bridged, quadruply-bonded dimolybdenum and ditungsten compounds have been investigated by a variety of computational methods employing density function theory (gradient corrected and time-dependent) which reveal the consequences of strong mixing of M2 delta and oxalate pi orbitals within extended chains and cyclic structures.

Evidence that kinetochore fibre microtubules shorten predominantly at the pole in anaphase flea-beetle spermatocytes.

Kinetochore spindle fibres in flea-beetle metaphase primary spermatocytes have two regions with distinct morphologies. As seen after staining with antibodies against tubulin, the kinetochore microtubules are tightly bundled in the 5 microm closest to the kinetochore but they splay out in the region closest to the pole. This morphology persists throughout anaphase. This distinct morphology allows one to deduce the site where kinetochore microtubules depolymerise during anaphase. During poleward movement of the autosomes in anaphase, the bundled region shortens by about 0.25 microm for each 1 microm the chromosome moves poleward; this suggests that, during anaphase, 75% of the kinetochore microtubule shortening occurs at the pole end. Sex chromosomes in metaphase cells are separated from the autosomes and do not move poleward at the same time as the autosomes: they are reported to move poleward when the autosomes are in metaphase, to stop part way to the poles, and to move poleward again as the autosomes do (Virkki 1970). Kinetochore microtubules of the sex chromosomes also have bundled and splayed regions; measurements of those regions suggest that these chromosomes may move poleward before the autosomes enter anaphase, but not afterwards.