Mitochondrial tRNA gene clusters in Aspergillus nidulans: Organization and nucleotide sequence

The organization of tRNA genes on the circular 32 kb mitochondrial genome of the ascomycete Aspergillus nidulans has been studied by gel transfer hybridization and by DNA sequencing. Most of the tRNA genes are tightly clustered within two regions (1 kb each) flanking the split gene for the large ribosomal subunit RNA. The upstream cluster contains nine genes, the downstream cluster eleven genes. The twenty tRNA genes are on the same strand as the two rRNA genes and are separated from each other by AT-rich spacer sequences, usually consisting of only a few nucleotides. Two tRNA genes (leul and ala) are joined end to end. The occurrence of two tRNA^Gty genes is the first exception to the observation that in mitochondria all four-codon families are read by a single tRNA. Both genes are adjacent and show extensive sequence homology, suggesting relatively recent origin by gene duplication. The product of glyl has a U in the wobble position as do all other tRNA gene products specific for four-codon families, whereas the gly2 product, which has a rare A in the same position, should read only the codon GGU. The products of metl and thr have an A and G in positions 18 and 55, respectively, like the mitochondrial tRNA^fMet and tRNA^Thr of Neurospora crassa. Other unusual features are the replacement of the invariant G-C pair at positions 53 and 61 by A-T in met2, glyl and gly2, the replacement of the invariant T at position 8 by A in phe and G in pro and the deletion of a nucleotide at position 9 in ser2.     

A Basic Positron Emission Tomography System Constructed to Locate a Radioactive Source in a Bi-dimensional Space

A simple Positron Emission Tomography (PET) prototype has been constructed to fully characterize its basic working principles. The PET prototype was created by coupling plastic scintillator crystals to photomultipliers or PMT''s which are placed at opposing positions to detect two gamma rays emitted from a radioactive source, of which is placed in the geometric center of the PET set-up. The prototype consists of four detectors placed geometrically in a 20 cm diameter circle, and a radioactive source in the center. By moving the radioactive source centimeters from the center the system one is able to detect the displacement by measuring the time of flight difference between any two PMT''s and, with this information, the system can calculate the virtual position in a graphical interface. In this way, the prototype reproduces the main principles of a PET system. It is capable to determine the real position of the source with intervals of 4 cm in 2 lines of detection taking less than 2 min.     

The function and role of two types of mechanoreceptive ‘free’ nerve endings in the head skin of amphibian embryos

Summary The properties of trigeminal ganglion sensory neurones innervating the head skin of lateXenopus laevis embryos have been studied using extracellular recordings. Two types of mechanosensory neurones were found:Rapid-transient detectors which responded with few impulses to rapid, local, indentation of the skin with a fine probe (10–25 m diameter), andMovement detectors which responded with a slowly adapting discharge to even very slow distortion of the skin (5 m· s^–1) with small or large probes. Receptive fields over the whole head surface as far back as the gill rudiments were plotted for both types of neurone. The areas for the two types were similar (means of 0.015 mm² for rapid-transient and 0.017 mm² for movement).Comparative observations on embryos ofRana temporaria andTriturus helveticus showed a very similar division of trigeminal sensory neurones into two types. InXenopus embryos stimuli which only excite the movement detectors were found to have inhibitory effects on behaviour. They would stop swimming and responses to other excitatory stimuli. Stimuli which excited the rapid-transient detectors normally evoked swimming. The division of the somatosensory system inXenopus embryos into two subsystems with different sensitivities and inhibitory, or excitatory effects on behaviour is discussed and related to findings in other groups of animals.I thank the MRC for support.     

Mutational analysis of the S21 pinholin

Lambdoid phage 21 has the prototype pinholin‐SAR endolysin lysis system, which is widely distributed among phages. Its prototype pinholin, S²¹68, triggers at an allele‐specific time to form small, heptameric lesions, or pinholes, in the cytoplasmic membrane, thus initiating lysis. S²¹68 has two transmembrane domains, TMD1 and TMD2. Only TMD2 is required for the formation of pinholes, whereas TMD1 acts as an inhibitor of TMD2 and must be externalized to the periplasm in the lytic pathway. Previously we provided evidence that S²¹68 first accumulates as inactive dimers with both transmembrane domains embedded in the bilayer. Here we analyse an extensive collection of S²¹ mutants to identify residues and domains critical to the function and regulation of the pinholin. Evidence is presented indicating that, within the inactive dimer, TMD1 acts in trans as an inhibitor of the lethal function of TMD2. A wide range of phenotypes, from absolute lysis defectives to accelerated lysis triggering, are observed for mutations mapping to each topological domain. The pattern of phenotypes allows the generation of a model for the structure of the inactive dimer. The model identifies the faces of the two transmembrane domains involved in intramolecular and intermolecular interactions, as well as interaction with the lipid.     

Effect of concentration on the aggregation of poly(γ-benzyl-L-glutamate) in dioxane: Critical concentration and maximum length for linear head to tail aggregates

Dielectric relaxation studies in the frequency range 100 Hz to 2 MHz of poly(γ-benzyl-L -glutamate) in dioxane have been carried out over a range of concentration 10^?4–10^?2g/g. The structure of aggregates is analyzed in terms of dipole moment and relaxation time. A critical concentration (? 10^?3 g/g for the studied molecular weights) has been determined below which the aggregates are found to have linear head to tail type structure. Above the critical concentration a different structure of aggregates is apparent which could not be fully analyzed by these measurements alone. Possible forms of aggregation above the critical concentration are discussed. Formation of long range order which would lead to nematic liquid crystalline phase at higher concentrations has been discussed as one of the possible explanations for the observed behavior above the critical concentration. Maximum length of linear head to tail type aggregates for poly(γ-benzyl-L -glutamate) in dioxane as determined from these results correspond to an α-helix of molecular weight 210,000. A slight difference in the purity of dioxane has been shown to have an influence on the reproducibility of the state of aggregation as well as on the rate of disaggregation on dilution.     

Exploring TOF capabilities of PET detector blocks based on large monolithic crystals and analog SiPMs

Monolithic scintillators are more frequently used in PET instrumentation due to their advantages in terms of accurate position estimation of the impinging gamma rays both planar and depth of interaction, their increased efficiency, and expected timing capabilities. Such timing performance has been studied when those blocks are coupled to digital photosensors showing an excellent timing resolution.In this work we study the timing behaviour of detectors composed by monolithic crystals and analog SiPMs read out by an ASIC. The scintillation light spreads across the crystal towards the photosensors, resulting in a high number of SiPMs and ASIC channels fired. This has been studied in relation with the Coincidence Timing Resolution (CTR). We have used LYSO monolithic blocks with dimensions of 50 × 50 × 15 mm³ coupled to SiPM arrays (8 × 8 elements with 6 × 6 mm² area) which compose detectors suitable for clinical applications.While a CTR as good as 186 ps FWHM was achieved for a pair of 3 × 3 × 5 mm³ LYSO crystals, when using the monolithic block and the SiPM arrays, a raw CTR over 1 ns was observed. An optimal timestamp assignment was studied as well as compensation methods for the time-skew and time-walk errors. This work describes all steps followed to improve the CTR. Eventually, an average detector time resolution of 497 ps FWHM was measured for the whole thick monolithic block. This improves to 380 ps FWHM for a central volume of interest near the photosensors. The timing dependency with the photon depth of interaction and planar position are also included.     

The neuronal MAP kinase cascade: a biochemical signal integration system subserving synaptic plasticity and memory

The mitogen-activated protein kinase (MAP kinase, MAPK) cascade, as the name implies, was originally discovered as a critical regulator of cell division and differentiation. As further details of this signaling cascade were worked out, it became clear that the MAPK cascade is in fact a prototype for a family of signaling cascades that share the motif of three serially linked kinases regulating each other by sequential phosphorylation. Thus, a revised nomenclature arose that uses the term MAPK to refer to the entire superfamily of signaling cascades (comprising the erks, the JNKs and the p38 stress activated protein kinases), and specifies the prototype MAPK as the extracellular signal-regulated kinase (erk). The two erk MAPK isoforms, p44 MAPK and p42 MAPK, are referred to as erk1 and erk2, respectively.The erks are abundantly expressed in neurons in the mature central nervous system, raising the question of why the prototype molecular regulators of cell division and differentiation are present in these non-dividing, terminally differentiated neurons. This review will describe the beginnings of an answer to this question. Interestingly, the general model has begun to emerge that the erk signaling system has been co-opted in mature neurons to function in synaptic plasticity and memory. Moreover, recent insights have led to the intriguing prospect that these molecules serve as biochemical signal integrators and molecular coincidence detectors for coordinating responses to extracellular signals in neurons. In this review I will first outline the essential components of this signal transduction cascade, and briefly describe recent results implicating the erks in mammalian synaptic plasticity and learning. I will then proceed to outline recent results implicating the erks as molecular signal integrators and, potentially, coincidence detectors. Finally, I will speculate on what the critical downstream effectors of the erks are in neurons, and how they might provide a readout of the integrated signal.     

Assessment of skeletal age in short and tall children

Skeletal age assessment has been carried out on 843 children between the ages of 31 and 122 months, selected as the tallest 2.5%, shortest 2.5% and median 5% of the Child Health and Development Studies population. Skeletal age was read from a roentgenogram of the left hand and wrist individually for each of the 30 ossification centers with reference to the Greulich and Pyle Atlas, second edition. The readings were performed blind, by one observer and a 20% sample was read a second time blind to determine the replicability of the assessments. Duplicate readings were within two months of each other in mean bone age and almost always within nine months of each other for individual centers. Estimates of weighted mean bone age vary considerably with stature. Mean bone ages of Negro children tend to be higher than those of White children; those of Chinese and Japanese boys tend to be lower than those of White boys. Generally, similar differences were found with respect to the estimated mean ages of onset of ossification for the ulna, lunate, scaphoid, trapezium and trapezoid.     

Visual associative memory and the orientation-contingent color after-effect

The traditional explanation of the McCollough effect (ME) by selective adaptation of single detectors selective to color and orientation suffers from a number of inconsistencies: 1) the ME lasts much longer (from several days up to 3 months) than the ordinary adaptation, the decay of the effect being completely arrested by night sleep or occluding the eye for a long time; 2) the strength of the ME practically does not depend on the intensity of adapting light; and 3) a set of related pattern-contingent after-effects discovered later required for such an explanation new detectors, specific for other patterns. These properties can be explained, however, in the framework of associative memory and novelty filters. A computational model has been developed, which consists of 1) an input layer of two (left and right eyes) square matrices with two analog receptors (red and green) in each pixel, 2) an isomorphic associative neural layer, each analog neuron being synaptically connected with all receptors of both eyes, and 3) an output layer (novelty filter). The modification of synaptic efficacies conforms to the Hebb learning rule. The function of the model was examined by simulation. After a few presentations of colored gratings, the model displays the ME that is slowly destroyed by subsequent presentations of random pictures. With a sufficiently large receptor matrix, the effect lasts a thousand times longer than the period of adaptation. Continuous darkness does not change the strength of the effect. Like in real ME, the model does not display interocular transfer. The model can account for different pattern-contingent color after-effects without assuming any predetermined specific detectors. Such detectors are constructed in the course of adaptation to specific stimuli (gratings).     

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca^2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish⁵. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria ‘dipstick’, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function^1,6-9.     

Sensitivity of the IQM and MatriXX detectors in megavolt photon beams

AimThis study focused on evaluating the sensitivity of integral quality monitoring (IQM^®) system and MatriXX detectors. These two detectors are recommended for radiotherapy pre-treatment quality assurance (QA).BackgroundIQM is a large wedged-shaped ionisation chamber mounted to the linear accelerator (linac) head in practice. MatriXX consists of an array of ionisation chambers also attached to the linac head.Materials and methodsIn this study, the dosimetric performance and sensitivity of MatriXX and IQM detectors were evaluated using the following characteristics: reproducibility, linearity, error detection capability and three-dimensional conformal radiotherapy (3D-CRT) plans of the head and neck, thorax and pelvic regions.ResultsThis study indicates that the signal responses of the large ionisation chamber device (IQM) and the small pixel array of ionisation chambers device (MatriXX) are reproducible, linear and sensitive to MLC positional errors, backup jaw positional errors and dose errors. The local percentage differences for dose errors of 1%, 2%, and 3% were, respectively, within 0.35–8.23%, 0.78–16.21%, and 1.10–24.41% for the IQM device. While for the MatriXX detector, the ranges were between 0.24–3.19, 0.57–6.43 and 0.81–12.95, respectively. Since IQM is essentially a double wedge-shaped large ionisation chamber, its reproducibility and detection capability are competitive to that of MatriXX. In addition, the sensitivity of the two QA systems increases with an increase in escalation percentage, and the signal responses are patient plan specific.ConclusionsThe two detectors response signals have good correlations and they are accurate for pre-treatment QA. Statistically, (P < 0.05) there is a significant difference between the IQM and MatriXX response to dose errors.     

Identification of sperm morphometric subpopulations in the canine ejaculate: do they reflect different subpopulations in sperm chromatin integrity?

A statistical approach using sequentially principal component analysis (PCA) clustering and discriminant analysis was developed to disclose morphometric sperm subpopulations. In addition, we used a similar approach to disclose subpopulations of spermatozoa with different degrees of DNA fragmentation. It is widely accepted that sperm morphology is a strong indicator of semen quality and since the sperm head mainly comprises the sperm DNA, it has been proposed that subtle changes in sperm head morphology may be related to abnormal DNA content. Semen from four mongrel dogs (five replicates per dog) were used to investigate DNA quality by means of the sperm chromatin structure assay (SCSA), and for computerized sperm morphometry (ASMA). Each sperm head was measured for nine primary parameters: head area (A), head perimeter (P), head length (L), head width (W), acrosome area (%), midpiece width (w), midpiece area (a), distance (d) between the major axes of the head and midpiece, angle (theta) of divergence of the midpiece from the head axis; and four parameters of head shape: FUN1 (L/W), FUN2 (4pi A/P2), FUN3 ((L - W)/(L + W)) and FUN 4 (pi LW/4A). The data matrix consisted of 2361 observations, (morphometric analysis on individual spermatozoa) and 63,815 observations for the DNA integrity. The PCA analysis revealed five variables with Eigen values over 1, representing more than 79% of the cumulative variance. The morphometric data revealed five sperm subpopulations, while the DNA data gave six subpopulations of spermatozoa with different DNA integrity. Significant differences were found in the percentage of spermatozoa falling in each cluster among dogs (p < 0.05). Linear regression models including sperm head shape factors 2, 3 and 4 predicted the amount of denatured DNA within each individual spermatozoon (p < 0.001). We conclude that the ASMA analysis can be considered a powerful tool to improve the spermiogram.     

Measurement of <Superscript>131</Superscript>I activity in thyroid of nuclear medical staff and internal dose assessment in a Polish nuclear medical hospital

This paper presents results of ¹³¹I thyroid activity measurements in 30 members of the nuclear medicine personnel of the Department of Endocrinology and Nuclear Medicine Holy Cross Cancer Centre in Kielce, Poland. A whole-body spectrometer equipped with two semiconductor gamma radiation detectors served as the basic research instrument. In ten out of 30 examined staff members, the determined ¹³¹I activity was found to be above the detection limit (DL = 5 Bq of ¹³¹I in the thyroid). The measured activities ranged from (5 ± 2) Bq to (217 ± 56) Bq. The highest activities in thyroids were detected for technical and cleaning personnel, whereas the lowest values were recorded for medical doctors. Having measured the activities, an attempt has been made to estimate the corresponding annual effective doses, which were found to range from 0.02 to 0.8 mSv. The highest annual equivalent doses have been found for thyroid, ranging from 0.4 to 15.4 mSv, detected for a cleaner and a technician, respectively. The maximum estimated effective dose corresponds to 32% of the annual background dose in Poland, and to circa 4% of the annual limit for the effective dose due to occupational exposure of 20 mSv per year, which is in compliance with the value recommended by the International Commission on Radiological Protection.     

Myosin structure. Proximity measurements by fluorescence energy transfer

The results of energy transfer experiments on the proximity of six sites on the globular head region of myosin are discussed. A large hydrophobic crevice has been detected on each myosin head which is sufficiently large to accommodate six aromatic rings simultaneously. In the crevice is located a thiol residue not involved in activation of myosin Ca²⁺ ATPase and a lysine residue which is specifically trinitrophenylated with 2, 4, 6-trinitrobenzenesulfonic acid. A second sulfhydryl whose modification activates the Ca²⁺ ATPase is located near the hydrophobic thiol site. The tryptophan whose fluorescence is enhanced by ATP binding is sufficiently close to the thiols and lysine residue to quantitatively transfer its energy to probes at these sites. The site of myosin ATPase has been tentatively located as being near the other five sites by energy transfer to or from synthetic chromophoric substrates. Implications of these results on the possibility of determining the location of the myosin light chain and actin binding sites are discussed.     

A Model of CatSper Channel Mediated Calcium Dynamics in Mammalian Spermatozoa

CatSpers are calcium (Ca²⁺) channels that are located along the principal piece of mammalian sperm flagella and are directly linked to sperm motility and hyperactivation. It has been observed that Ca²⁺ entry through CatSper channels triggers a tail to head Ca²⁺ propagation in mouse sperm, as well as a sustained increase of Ca²⁺ in the head. Here, we develop a mathematical model to investigate this propagation and sustained increase in the head. A 1-d reaction-diffusion model tracking intracellular Ca²⁺ with flux terms for the CatSper channels, a leak flux, and plasma membrane Ca²⁺ clearance mechanism is studied. Results of this simple model exhibit tail to head Ca²⁺ propagation, but no sustained increase in the head. Therefore, in this model, a simple plasma membrane pump-leak system with diffusion in the cytosol cannot account for these experimentally observed results. It has been proposed that Ca²⁺ influx from the CatSper channels induce additional Ca²⁺ release from an internal store. We test this hypothesis by examining the possible role of Ca²⁺ release from the redundant nuclear envelope (RNE), an inositol 1,4,5-trisphosphate (IP₃) gated Ca²⁺ store in the neck. The simple model is extended to include an equation for IP₃ synthesis, degradation, and diffusion, as well as flux terms for Ca²⁺ in the RNE. When IP₃ and the RNE are accounted for, the results of the model exhibit a tail to head Ca²⁺ propagation as well as a sustained increase of Ca²⁺ in the head.     

Artificial vision with wirelessly powered subretinal electronic implant alpha-IMS

This study aims at substituting the essential functions of photoreceptors in patients who are blind owing to untreatable forms of hereditary retinal degenerations. A microelectronic neuroprosthetic device, powered via transdermal inductive transmission, carrying 1500 independent microphotodiode-amplifier-electrode elements on a 9 mm² chip, was subretinally implanted in nine blind patients. Light perception (8/9), light localization (7/9), motion detection (5/9, angular speed up to 35 deg s⁻¹), grating acuity measurement (6/9, up to 3.3 cycles per degree) and visual acuity measurement with Landolt C-rings (2/9) up to Snellen visual acuity of 20/546 (corresponding to decimal 0.037 or corresponding to 1.43 logMAR (minimum angle of resolution)) were restored via the subretinal implant. Additionally, the identification, localization and discrimination of objects improved significantly (n = 8; p < 0.05 for each subtest) in repeated tests over a nine-month period. Three subjects were able to read letters spontaneously and one subject was able to read letters after training in an alternative-force choice test. Five subjects reported implant-mediated visual perceptions in daily life within a field of 15° of visual angle. Control tests were performed each time with the implant''s power source switched off. These data show that subretinal implants can restore visual functions that are useful for daily life.     

A conjugate counting method to determine [75Se]SeHCAT retention in the human body

To evaluate the functional integrity of the distal part of the ileum the retention of a γ-labelled bile acid (SeHCAT) in the human body can be measured with a detector. Due to the lack of a whole body counter at our institution a two detector system was designed to measure SeHCAT retention and an evaluation of such a system has been made. The detectors are positioned on either side of a patient lying supine on a hospital trolley. The trolley is stepped forward in 100 mm steps, to determine the SeHCAT activity in the patient. With these counts the location of the SeHCAT activity and total activity present in the body can be determined. A water rilled phantom and a phantom consisting of nine 1-L saline bags with ⁷⁵Se activity placed in them was used to determine system performance. Four patients with no history of bowel disease were compared with published data for normals. Results showed that the system performed satisfactorily, and accurate quantitative measurements could be made, showing that this inexpensive system could be used where a whole body counter is not available.     

Methods for the continuous measurement of O2 consumption and H2 production by nodulated legume root systems

The details of two systems for measuring O2 uptake and H2 production in flow-through gas systems used to study nodule physiology and biochemistry are presented here. Both are constructed from commercially available fuel cells. Oxygen uptake measurements are based upon the differential signal from paired detectors exposed to sample and blank gas streams. Because of the very small signal:background ratio needed to detect O2 uptake against atmospheric O2 concentration, these detectors were mounted in thermally controlled aluminium blocks designed to avoid changes in back pressure. Also to avoid apparent concentration differences arising because of pressure differences at the detectors it is important that sample, control and calibration gas flow rates are the same. Hydrogen detectors were mounted in an aluminium block similar to that used for O2, although the requirements for temperature and back pressure control are much less stringent for this application.The limit for differential detection of O2 uptake in air was about 25 ppm (1 mol 1^-l) and for H2 production 1 ppm (0l04 mol l^-1). Linearity checks for the two detectors over the range 4-90% O2 and 15-750 ppm H2 gave regression coefficients of >0.999 and neither detector was significantly affected by changing the background mixing gas from N2 to Ar or He provided that flow rates and back pressures remained constant. Water vapour had no effect on the H2 detectors, but caused small baseline shifts during O2 uptake measurements which were obviated with silica gel drying filters. Changes in gas stream pO2 produced small baseline shifts with the H2 detectors, but did not effect the magnitude of the H2 signal.Two examples are provided of the use of these detectors together with the soyabean/USDA 16 symbiosis in a flow-through system furnished with an infrared gas analyser (IRGA) to measure CO2 production.     

Na(I) binding by tartaric acid derivatives

A study of the binding of Na⁺ by two readily available tartaric acid derivatives, dimethyl tartrate and dipyrrolidine tartramide, has been carried out. These binding studies reveal binding stoichiometries of 1:1 for the dimethyl tartrate and 2:1 for the dipyrrolidine tartramide, and binding affinities (association constants) of 6.61 M⁻¹ for the dimethyl tartrate and 70.4 M⁻² for the dipyrrolidine tartramide indicating weak binding of Na⁺ in both cases. An X-ray crystal structure of a NaI complex of dipyrrolidine tartramide has also been determined. The binding stoichiometry is 1:1 in the solid state as opposed to the 2:1 binding stoichiometry that is observed in solution. The 1:1 binding in the solid state results in a coordination polymer in which half of the carbonyl oxygens and half of the hydroxy oxygens of the dipyrrolidine tartramide ligand bridge between adjacent Na⁺ cations. This allows each Na⁺ cation to achieve an octahedral coordination geometry. The iodides are ordered in a linear fashion, and each column of iodides is separated from the other columns by the coordination polymer and by linear columns of water molecules.