Hyperpolarization moves S4 sensors inward to open MVP,a methanococcal voltage-gated potassium channel

MVP, a Methanococcus jannaschii voltage-gated potassium channel, was cloned and shown to operate in eukaryotic and prokaryotic cells. Like pacemaker channels, MVP opens on hyperpolarization using S4 voltage sensors like those in classical channels activated by depolarization. The MVP S4 span resembles classical sensors in sequence, charge, topology and movement, traveling inward on hyperpolarization and outward on depolarization (via canaliculi in the protein that bring the extracellular and internal solutions into proximity across a short barrier). Thus, MVP opens with sensors inward indicating a reversal of S4 position and pore state compared to classical channels. Homologous channels in mammals and plants are expected to function similarly.     

A technique for respiratory-gated radiotherapy treatment verification with an EPID in cine mode

Respiratory gating based on external surrogates is performed in many clinics. We have developed a new technique for treatment verification using an electronic portal imaging device (EPID) in cine mode for gated 3D conformal therapy. Implanted radiopaque fiducial markers inside or near the target are required for this technique. The markers are contoured on the planning CT set, enabling us to create digitally reconstructed radiographs (DRRs) for each treatment beam. During the treatment, a sequence of EPID images can be acquired without disrupting the treatment. Implanted markers are visualized in the images and their positions in the beam's eye view are calculated off-line and compared to the reference position by matching the field apertures in corresponding EPID and DRR images. The precision of the patient set-up, the placement of the beam-gating window, as well as the residual tumour motion can be assessed for each treatment fraction. This technique has been demonstrated with a case study patient, who had three markers implanted in his liver. For this patient, the intra-fractional variation of all marker positions in the gating window had a 95% range of 4.8 mm in the SI direction (the primary axis of motion). This was about the same (5 mm) as the residual motion considered in the planning process. The inter-fractional variation of the daily mean positions of the markers, which indicates the uncertainty in the set-up procedure, was within +8.3 mm/-4.5 mm (95% range) in the SI direction for this case.     

Cerebellar Golgi cells in the rat receive multimodal convergent peripheral inputs via the lateral funiculus of the spinal cord

We recently showed that the activity of cerebellar Golgi cells can be powerfully modulated by stimulation of peripheral afferents, in a pattern different to local Purkinje cells. Here we have examined the pathways underlying these responses. Graded electrical stimulation of muscle and cutaneous nerves revealed that long-lasting depressions and short-lasting excitations of Golgi cells were evoked by stimulation of cutaneous nerves at stimulus intensities that activated large mechanoreceptive afferents, and grew as additional afferents were recruited. In contrast, none of the neurones responded to stimulation of muscle nerves at intensities that activated group I afferents, although about half responded with long-lasting depressions, but not excitations, to stimuli that recruited group II and III afferents. Selective lesions of the spinal dorsal columns did not affect either of these types of response. After lesions of one lateral funiculus in the lumbar cord the responses evoked by stimulation of the hindlimb contralateral to the lesion were reduced or abolished, leaving responses evoked by ipsilateral hindlimb afferents unaltered. Since both ipsi- and contralateral afferents generate responses in Golgi cells, the convergence from the two sides must occur supraspinally. It is difficult to reconcile these properties with any of the direct spinocerebellar pathways or spinoreticulocerebellar pathways that have been described. Instead, it is likely that the responses are evoked via the multimodal 'wide dynamic range' neurones of the anterolateral system. Golgi cell activity may thus be powerfully enhanced or depressed during arousal via the anterolateral system.     

Progression of self-reported symptoms in laboratory animal allergy

BACKGROUND: Laboratory animal allergy is a common illness among workers exposed to laboratory animals and can progress to symptoms of asthma. OBJECTIVES: This study evaluates the continuum of disease from allergy symptoms to asthma symptoms in a dynamic cohort of workers exposed to animals in a pharmaceutical company. METHODS: Data arose from annual questionnaires administered to workers in a surveillance program established to monitor exposure to animals and the development of allergy. The life-table method was used to compare asthma-free survival between workers with and without symptoms of allergy. A Cox proportional hazards model was used to examine the effects of covariates on the development of asthma. RESULTS: A total of 603 workers contributed 2527.4 person-years to the study over the 12.3-year period. The probabilities of experiencing asthma symptoms by the 11th year of follow-up were 0.367 for workers with allergy symptoms and 0.052 for those without allergy symptoms. The hazard ratio for asthma symptoms when comparing workers with and without allergy symptoms was 7.39 (95% CI, 3.29-16.60) after adjustment for sex and family history of allergy. Female subjects developed asthma at a rate 3.4 times that of male subjects. CONCLUSIONS: This study supports the hypothesis that laboratory animal allergy symptoms are a major risk factor for the development of asthma. It also suggests a heightened risk of asthma for women who work with laboratory animals, a finding that has not been previously reported.     

Interaction of Dr adhesin with collagen type IV is a critical step in Escherichia coli renal persistence

The pathogenic mechanism of recurrent or chronic urinary tract infection is poorly understood. Escherichia coli cells bearing Dr fimbriae display unique tropism to the basement membrane (BM)-renal interstitium that enables the bacteria to cause chronic pyelonephritis in experimental mice. The renal receptors for Dr-fimbriated E. coli are type IV collagen and decay-accelerating factor (DAF). We hypothesized that type IV collagen receptor-mediated BM-interstitial tropism is essential for E. coli to cause chronic pyelonephritis. To test the role of the type IV collagen tropism of Dr-fimbriated E. coli in renal persistence, we constructed an isogenic mutant in the DraE adhesin subunit that was unable to bind type IV collagen but retained binding to DAF and examined its virulence in the mouse model. The collagen-binding mutant DrI113T was eliminated from the mouse renal tissues in 6 to 8 weeks, while the parent strain caused persistent renal infection that lasted at least 14 weeks (P < or = 0.02). Transcomplementation with the intact Dr operon restored collagen-binding activity, BM-interstitial tropism, and the ability to cause persistent renal infection. We conclude that type IV collagen binding mediated by DraE adhesin is a critical step for the development of persistent renal infection in a murine model of E. coli pyelonephritis.     

Effect of glass composition on the degradation properties and ion release characteristics of phosphate glass--polycaprolactone composites

A series of polycaprolactone and ternary-based (Na(2)O)(0.55-x)(CaO)(x)(P(2)O(5))(0.45) glass composites were created, each containing 20% volume percentage of glass with various calcium compositions. A short-term degradation study was carried out to investigate the physical and ion release behaviour of these composites, utilising analytical techniques such as dynamical mechanical analysis, and ion chromatography. All the composites experienced significant loss of weight and stiffness throughout the study, with the 24 mol% calcium composites losing the greatest amount of weight and stiffness. The pH profile of the aqueous solutions in which the composites were placed were initially acidic, but began to neutralise mid-way through the study, with the 36 mol% solution achieving the most acidic conditions. The ion release behaviour mirrored the mass loss behaviour of the glass component of the composites. The cations (sodium and calcium ions) release was comparable with the initial stages of composite mass degradation, both of which exhibited almost immediate release when placed into solution. The 24 mol% composites underwent rapid rates of cation release, while the 36 mol% experienced the slowest rates of release. By contrast, anion (phosphates and polyphosphates) release showed a dissimilar trend, with rapid release of the P(2)O(7) and P(3)O(10) occurring during the first few hours in solution, whilst the P(3)O(9) structure released steadily during the first 48 h in solution. Finally, PO(4) release was at a constant rate over the duration of the study, releasing up to 300 ppm from the 32 and 36 mol% samples by the end of 200 h. To summarise, these results show that by combining phosphate glasses with biodegradable polymer, it is possible to create composites whose rate of degradation can be controlled to meet the needs of their end application.     

Characterization of Hprt mutations in cDNA and genomic DNA of T-cell mutants from control and 1,3-butadiene-exposed male B6C3F1 mice and F344 rats

A multiplex PCR procedure for analysis of genomic DNA mutations in the mouse hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene was developed and then used with other established methods for the coincident identification of large- and small-scale genetic alterations in the Hprt gene of mutant T-cell isolates propagated from sham- and 1,3-butadiene (BD)-exposed mice and rats. The spectra data for RT-PCR/cDNA analysis and multiplex PCR of genomic DNA from Hprt mutants were combined, and statistical analyses of the mutant fractions for the classes of mutations identified in control versus exposed animals were conducted. Under the assumption that the mutant fractions are distributed as Poisson variates, BD exposure of mice significantly increased the frequencies of (1) nearly all types of base substitutions; (2) single-base deletions and insertions; and (3) all subcategories of deletions. Significantly elevated fractions of G:C-->C:G and A:T-->T:A transversions in the Hprt gene of BD-exposed mice were consistent with the occurrence of these substitutions as the predominant ras gene mutations in multiple tumor types increased in incidence in carcinogenicity studies of BD in mice. BD exposure of rats produced significant increases in (1) base substitutions only at A:T base pairs; (2) single-base insertions; (3) complex mutations; and (4) deletions (mainly 5' partial and complete gene deletions). Future coincident analyses of large- and small-scale mutations in rodents exposed to specific BD metabolites should help identify species differences in the sources of deletion mutations and other types of mutations induced by BD exposures in mice versus rats.     

Fatal fat embolism following amphotericin B lipid complex injection

A case of amphotericin B lipid complex induced fatal fat embolism is described. A 41-year-old Caucasian man with AIDS was undergoing treatment for cryptococcal meningitis with amphotericin B. His course was complicated by renal failure necessitating a change in therapy to amphotericin B lipid complex (Abelcet). At approximately 48 h, the patient developed tachycardia, tachypnea, respiratory failure, decline in hematocrit, thrombocytopenia, and alteration in mental status. Autopsy findings included fat emboli involving heart, lungs, kidney, and brain. To our knowledge, this is the first case report of a fatal fat embolism caused by intravenous liposome drug delivery.