Inflation of 430-parsec bipolar radio bubbles in the Galactic Centre by an energetic event.

The Galactic Centre contains a supermassive black hole with a mass of four million Suns1 within an environment that differs markedly from that of the Galactic disk. Although the black hole is essentially quiescent in the broader context of active galactic nuclei, X-ray observations have provided evidence for energetic outbursts from its surroundings2. Also, although the levels of star formation in the Galactic Centre have been approximately constant over the past few hundred million years, there is evidence of increased short-duration bursts3, strongly influenced by the interaction of the black hole with the enhanced gas density present within the ring-like central molecular zone4 at Galactic longitude |l| < 0.7 degrees and latitude |b| < 0.2 degrees. The inner 200-parsec region is characterized by large amounts of warm molecular gas5, a high cosmic-ray ionization rate6, unusual gas chemistry, enhanced synchrotron emission7,8, and a multitude of radio-emitting magnetized filaments9, the origin of which has not been established. Here we report radio imaging that reveals a bipolar bubble structure, with an overall span of 1 degree by 3 degrees (140 parsecs × 430 parsecs), extending above and below the Galactic plane and apparently associated with the Galactic Centre. The structure is edge-brightened and bounded, with symmetry implying creation by an energetic event in the Galactic Centre. We estimate the age of the bubbles to be a few million years, with a total energy of 7 × 1052 ergs. We postulate that the progenitor event was a major contributor to the increased cosmic-ray density in the Galactic Centre, and is in turn the principal source of the relativistic particles required to power the synchrotron emission of the radio filaments within and in the vicinity of the bubble cavities.

A review of COVID vaccines: success against a moving target.

BACKGROUND: Multiple vaccine platforms against COVID-19 have been developed and found safe and efficacious at a record speed. Although most are effective, they vary in their ease of production and distribution, their potential speed of modification against new variants, and their durability of protection and safety in certain target groups. SOURCES OF DATA: Our discussion is based on published reports of clinical trials and analyses from national and global health agencies. AREAS OF AGREEMENT: The production of neutralizing antibodies against the viral spike protein is protective, and all vaccines for which published data exist have been found to be effective against severe disease caused by the viral strain they target. AREAS OF CONTROVERSY: The degree to which vaccines protect against emerging variants, moderate disease and asymptomatic infection remains somewhat unclear. GROWING POINTS: Knowledge of the duration of protection and its decay is increasing, and discussions of booster frequency and target strains are ongoing. AREAS TIMELY FOR DEVELOPING RESEARCH: The global effort to combat transmission and disease continues to rely upon intense epidemiological surveillance, whilst real-world data and clinical trials shape vaccination schedules and formulae.

Campylobacter outbreak associated with raw drinking milk, North West England, 2016.

In December 2016, Public Health England investigated an outbreak of campylobacteriosis in North West England, with 69 cases in total. Epidemiological, microbiological and environmental investigations associated the illness with the consumption of unpasteurised cows' milk from Farm X, where milk was predominantly sold from a vending machine. Campylobacter was detected in milk samples which, when sequenced, were identical in sequence type as pathogens isolated from cases (Clonal Complex ST-403, Sequence Type 7432). The farm was served with a Hygiene Emergency Prohibition Order to prevent further cases. To our knowledge, this is the first outbreak of campylobacter associated with unpasteurised milk in England since 1996. Our findings highlighted several important lessons, including that the current testing regime in England for unpasteurised milk is not fit for purpose and that the required warning label should include additional wording, underscoring the risk to vulnerable groups. There has been a substantial increase in both the volume of unpasteurised milk consumed in England and the use of vending machines to sell unpasteurised milk over the last 10 years, making unpasteurised milk more readily accessible to a wider population. The evidence generated from outbreaks like this is therefore critical and should be used to influence policy development.

Elucidation of the K32 Capsular Polysaccharide Structure and Characterization of the KL32 Gene Cluster of Acinetobacter baumannii LUH5549.

Capsular polysaccharide (CPS), isolated from Acinetobacter baumannii LUH5549 carrying the KL32 capsule biosynthesis gene cluster, was studied by sugar analysis, Smith degradation, and one- and two-dimensional 1H and 13C NMR spectroscopy. The K32 CPS was found to be composed of branched pentasaccharide repeats (K units) containing two residues of ß-D-GalpNAc and one residue of ß-D-GlcpA (ß-D-glucuronic acid) in the main chain and one residue each of ß-D-Glcp and α-D-GlcpNAc in the disaccharide side chain. Consistent with the established CPS structure, the KL32 gene cluster includes genes for a UDP-glucose 6-dehydrogenase (Ugd3) responsible for D-GlcA synthesis and four glycosyltransferases that were assigned to specific linkages. Genes encoding an acetyltransferase and an unknown protein product were not involved in CPS biosynthesis. Whilst the KL32 gene cluster has previously been found in the global clone 2 (GC2) lineage, LUH5549 belongs to the sequence type ST354, thus demonstrating horizontal gene transfer between these lineages.

Structure of the K82 Capsular Polysaccharide from Acinetobacter baumannii LUH5534 Containing a d-Galactose 4,6-Pyruvic Acid Acetal.

Type K82 capsular polysaccharide (CPS) was isolated from Acinetobacter baumannii LUH5534. The structure of a linear tetrasaccharide repeating unit of the CPS was established by sugar analysis along with one- and two-dimensional 1H and 13C NMR spectroscopy. Proteins encoded by the KL82 capsule gene cluster in the genome of LUH5534 were assigned to roles in the synthesis of the K82 CPS. In particular, functions were assigned to two new glycosyltransferases (Gtr152 and Gtr153) and a novel pyruvyltransferase, Ptr5, responsible for the synthesis of d-galactose 4,6-(R)-pyruvic acid acetal.

Evaluation of a surveillance programme for women with a family history of breast cancer.

AIM: To establish health related costs and benefits of clinical services for women at increased familial risk of breast cancer. METHODS: Analysis of costs and outcomes for one UK regional service, supplemented with data from a multinational collaborative study. Main outcome measures were aggregate costs for regular clinical examination, mammographic screening and further investigations; breast cancer incidence; proportion of cancers detected at "early" or "late" stage, compared with corresponding data for unscreened women of comparable age; survival in relation to stage at diagnosis; itemised and aggregate costs of management for "early" and "late" stage breast cancer; hence direct health care costs per quality adjusted life-year (QALY) gained. RESULTS: The surveillance programme costs pound1500 (euro1600, US$2100) per woman (over 15 years). Breast cancer incidence is close to 6 per thousand examinations; 75% of tumours are detected through screening and 77% are "early" (path stage 1 or 2). Corresponding figures for unscreened women (including relatives of those attending the breast cancer family clinic) indicate that surveillance achieves a beneficial "stage shift", with reduction in treatment costs and improvement in survival, in about 22% of cases. CONCLUSIONS: The current clinical service for women at familial risk of breast cancer costs about pound4800 (euro5200, US$6800) per QALY gained. That figure is sensitive to the rate of detection of breast cancer and the degree of beneficial stage shift achieved. Within the realistic range of estimates for these two parameters, the cost per QALY may be as high as pound14,000 (euro15,300, US$20,000) or as low as pound1000 (euro1100, US$1400).

Roles of mitochondria and temperature in the control of intracellular calcium in adult rat sensory neurons.

We recorded Ca2+ current and intracellular Ca2+ ([Ca2+](i)) in isolated adult rat dorsal root ganglion (DRG) neurons at 20 and 30 degrees C. In neurons bathed in tetraethylammonium and dialyzed with cesium, warming reduced resting [Ca2+](i) from 87 to 49 nM and the time constant of the decay of [Ca2+](i) transients (tau(r)) from 1.3 to 0.99s (Q(10)=1.4). The Buffer Index, the ratio between Ca2+ influx and Delta[Ca2+](i) (f I(ca)d(t)/Delta[Ca2+]i) , increased two- to threefold with warming. Neither inhibition of the plasma membrane Ca2+ -ATPase by intracellular sodium orthovanadate nor inhibition of Ca2+ uptake by the endoplasmic reticulum by thapsigargin plus ryanodine were necessary for the effects of warming on these parameters. In contrast, inhibition of the mitochondrial Ca2+ uniporter by intracellular ruthenium red largely reversed the effects of warming. Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 500 nM) increased resting [Ca2+](i) at 30 degrees C. Ten millimolar intracellular sodium prolonged the recovery of [Ca2+](i) transients to 10-40s. This effect was reversed by an inhibitor of mitochondrial Na(+)/Ca2+ -exchange (CGP 37157, 10 microM). Thus, mitochondrial Ca2+ uptake is necessary for the temperature-dependent increase in Ca2+ buffering and mitochondrial Ca2+ fluxes contribute to the control of [Ca2+](i) between 50 and 150 nM at 30 degrees C.

Selenium (IV,VI) reduction and tolerance by fungi in an oxic environment.

Microbial processes are known to mediate selenium (Se) oxidation-reduction reactions, strongly influencing Se speciation, bioavailability, and transport throughout the environment. While these processes have commonly been studied in anaerobic bacteria, the role that aerobic fungi play in Se redox reactions could be important for Se-rich soil systems, dominated by microbial activity. We quantified fungal growth, aerobic Se(IV, VI) reduction, and Se immobilization and volatilization in the presence of six, metal-tolerant Ascomycete fungi. We found that the removal of dissolved Se was dependent on the fungal species, Se form (i.e., selenite or selenate), and Se concentration. All six species grew and removed dissolved Se(IV) or Se(VI) from solution, with five species reducing both oxyanions to Se(0) biominerals, and all six species removing at least 15%-20% of the supplied Se via volatilization. Growth rates of all fungi, however, decreased with increasing Se(IV,VI) concentrations. All fungi removed 85%-93% of the dissolved Se(IV) within 10 d in the presence of 0.01 mm Se(IV), although only about 20%-30% Se(VI) was removed when grown with 0.01 mm Se(VI). Fungi-produced biominerals were typically 50- to 300-nm-diameter amorphous or paracrystalline spherical Se(0) nanoparticles. Our results demonstrate that activity of common soil fungi can influence Se form and distribution, and these organisms may therefore play a role in detoxifying Se-polluted environments.

New method for detection of heart allograft rejection: validation of sensitivity and reliability in a rat heterotopic allograft model.

BACKGROUND: Patients with inflammatory heart muscle diseases would benefit from a safe, convenient, rapidly performed diagnostic technique with real-time results not involving tissue removal. We have performed a detailed evaluation of detection of heart allograft rejection by autofluorescence in a heterotopic abdominal rat heart allograft model ex vivo. METHODS AND RESULTS: Recipient rats with allograft (Lewis to Fisher 344; n=71) and isograft (Lewis to Lewis; n=33) hearts, treated with cyclosporine or untreated, were killed at days 2, 4, 7, 14, 21, 28, and 56 after transplant. Nontransplant controls with (n=24) or without (n=24) immunosuppressive therapy were also studied. When the rats were killed, autofluorescence spectra were acquired under blue-light excitation from midtransverse ventricular sections of native and transplanted hearts. Corresponding sections were then evaluated pathologically by a modified International Society for Heart and Lung Transplantation (ISHLT) grading schema. The spectral differences between rejecting and nonrejecting hearts were quantified by linear discriminant functions, producing scores that decreased progressively with increasing severity of tissue rejection. Mean+/-SD discriminant function scores were 2.9+/-1.6, 1.8+/-2.2, -0.1+/-2.8, -1.2+/-2.3, and -2.3+/-3.0 for isografts and allograft ISHLT grades 0, I, II, and III, respectively (Spearman rank-order correlation -0.6; P<0.001, test for trend). Cyclosporine had no detectable effect on the spectra. CONCLUSIONS: The correlation between changes in autofluorescence spectra and ISHLT rejection grade strongly supports the possibility of catheter-based, fluorescence-guided surveillance of rejection.

Influence of chloride, potassium, and tetraethylammonium on the early outward current of sheep cardiac Purkinje fibers.

In voltage clamp studies of cardiac Purkinje fibers, a large early outward current is consistently observed during depolarizations to voltages more positive than -20 mV. After the outward peak of the current, the total membrane current declines slowly. Dudel et al. (1967. Pfluegers Arch. Eur. J. Physiol. 294:197--212) reduced the extracellular chloride concentration and found that the outward peak and the decline of the current were abolished. They concluded that the total membrane current at these voltages was largely determined by a time- and voltage-dependent change in the membrane chloride conductance. We reinvestigated the chloride sensitivity of this current, taking care to minimize possible sources of error. When the extracellular chloride concentration was reduced to 8.6% of control, the principal effect was a 20% decrease in the peak amplitude of the outward current. This implies that the membrane chloride conductance is not the major determinant of the total current at these voltages. The reversal potential of current tails obtained after a short conditioning depolarization was not changed by alterations in the extracellular chloride or potassium concentrations. We suspect that the tail currents contain both inward and outward components, and that the apparent reversal potential of the net tail current largely reflects the kinetics of the outward component, so that this experiment does not rule out potassium as a possible charge carrier. The possibility that potassium carries much of the early outward current was further investigated using tetraethylammonium, which blocks potassium currents in nerve and skeletal muscle. This drug substantially reduced the early outward current, which suggests that much of the early outward current is carried by potassium ions.

4-Aminopyridine and the early outward current of sheep cardiac Purkinje fibers.

We have studied the effects of the potassium-blocking agent 4-aminopyridine (4-AP) on the action potential and membrane currents of the sheep cardiac Purkinje fiber. 4-AP slowed the rate of phase 1 repolarization and shifted the plateau of the action potential to less negative potentials. In the presence of 4-AP, the substitution of sodium methylsulfate or methanesulfonate for the NaCl of Tyrode's solution further slowed the rate of phase 1 repolarization, even though chloride replacement has no effect on the untreated preparation. In voltage clamp experiments, 4-AP rapidly and reversibly reduced the early peak of outward current that is seen when the Purkinje fiber membrane is voltage-clamped to potentials positive to -20 mV. In addition, 4-AP reduced the steady outward current seen at the end of clamp steps positive to -40 mV. 4-AP did not appear to change the slow inward current observed over the range of -60 to -40 mV, nor did it greatly change the current tails that have been used as a measure of the slow inward conductance at more positive potentials. 4-AP did not block the inward rectifying potassium currents, IK1 and IK2. A phasic outward current component that was insensitive to 4-AP was reduced by chloride replacement. We conclude that the early outward current has two components: a chloride-sensitive component plus a 4-AP-sensitive component. Since a portion of the steady-state current was sensitive to 4-AP, the early outward current either does not fully inactivate or 4-AP blocks a component of time-independent background current.

Calcium- and voltage-activated plateau currents of cardiac Purkinje fibers.

We have used the two-microelectrode voltage-clamp technique to investigate the components of membrane current that contribute to the formation of the early part of the plateau phase of the action potential of calf cardiac Purkinje fibers. 3,4-Diaminopyridine (50 microM) reduced the net transient outward current elicited by depolarizations to potentials positive to -30 mV but had no consistent effect on contraction. We attribute this effect to the blockade of a voltage-activated transient potassium current component. Ryanodine (1 microM), an inhibitor of sarcoplasmic reticulum calcium release and intracellular calcium oscillations in Purkinje fibers (Sutko, J.L., and J.L. Kenyon. 1983. Journal of General Physiology. 82:385-404), had complex effects on membrane currents as it abolished phasic contractions. At early times during a depolarization (5-30 ms), ryanodine reduced the net outward current. We attribute this effect to the loss of a component of calcium-activated potassium current caused by the inhibition of sarcoplasmic reticulum calcium release and the intracellular calcium transient. At later times during a depolarization (50-200 ms), ryanodine increased the net outward current. This effect was not seen in low-sodium solutions and we could not observe a reversal potential over a voltage range of -100 to +75 mV. These data suggest that the effect of ryanodine on the late membrane current is attributable to the loss of sodium-calcium exchange current caused by the inhibition of sarcoplasmic reticulum calcium release and the intracellular calcium transient. Neither effect of ryanodine was dependent on chloride ions, which suggests that chloride ions do not carry the ryanodine-sensitive current components. Strontium (2.7 mM replacing calcium) and caffeine (10 mM), two other treatments that interfere with sarcoplasmic reticulum function, had effects in common with ryanodine. This supports the hypothesis that the effects of ryanodine may be attributed to the inhibition of sarcoplasmic reticulum calcium release.

Effects of low-chloride solutions on action potentials of sheep cardiac Purkinje fibers.

The rapid repolarization during phase 1 of the action potential of sheep cardiac purkinje fibers has been attributed to a time- and voltage-dependent chloride current. In part, this conclusion was based on experiments that showed a substantial slowing of phase 1 when larger, presumably impermeant, anions were substituted for chloride in tyrode's solution. We have re- examined the electrical effects of low-chloride solutions. We recorded action potentials of sheep cardiac purkinje fibers in normal tyrode's solution and in low-chloride solutions made by substituting sodium propionate, acetylglycinate, methylsulfate, or methanesulfonate for the NaCl of Tyrode's solution. Total calcium was adjusted to keep calcium ion activity of test solutions equal to that of control solutions. Propionate gave qualitatively variable results in preliminary experiments; it was not tested further. Low-chloride solutions made with the other anions gave much more consistent results: phase 1 and the notch that often occurs between phases 1 and 2 were usually unaffected, and the action potential duration usually increased. The only apparent change in the resting potential was a transient 3-6 mV depolarization when low-chloride solution was first admitted to the chamber, and a symmetrical transient hyperpolarization when chloride was returned to normal. If a time- and voltage-dependent chloride current exists in sheep cardiac purkinje fibers, our results suggest that it plays little role in generating phase 1 of the action potential.

Ryanodine modification of cardiac muscle responses to potassium-free solutions. Evidence for inhibition of sarcoplasmic reticulum calcium release.

To test whether ryanodine blocks the release of calcium from the sarcoplasmic reticulum in cardiac muscle, we examined its effects on the aftercontractions and transient depolarizations or transient inward currents developed by guinea pig papillary muscles and voltage-clamped calf cardiac Purkinje fibers in potassium-free solutions. Ryanodine (0.1-1.0 microM) abolished or prevented aftercontractions and transient depolarizations by the papillary muscles without affecting any of the other sequelae of potassium removal. In the presence of 4.7 mM potassium and at a stimulation rate of 1 Hz, ryanodine had only a small variable effect on papillary muscle force development and action potential characteristics. In calf Purkinje fibers, ryanodine (1 nM-1 microM) completely blocked the aftercontractions and transient inward currents without altering the steady state current-voltage relationship. Ryanodine also abolished the twitch in potassium-free solutions, but it enhanced the tonic force during depolarizing voltage-clamp steps. This latter effect was dependent on the combination of ryanodine and potassium-free solutions. The slow inward current was not blocked by 1 microM ryanodine, but ryanodine did appear to abolish an outward current that remained in the presence of 0.5 mM 4-aminopyridine. Our observations are consistent with the hypothesis that ryanodine, by inhibiting the release of calcium from the sarcoplasmic reticulum, prevents the oscillations in intracellular calcium that activate the transient inward currents and aftercontractions associated with calcium overload states.

Delays in inactivation development and activation kinetics in myxicola giant axons.

Na inactivation was studied in Myxicola (two-pulse procedure, 6-ms gap between conditioning and test pulses). Inactivation developed with an initial delay (range 130-817 microseconds) followed by a simple exponential decline (time constant tau c). Delays (deviations from a simple exponential) are seen only for brief conditioning pulses were gNa is slightly activated. Hodgkin-Huxley kinetics with series resistance, Rs, predict deviations from a simple exponential only for conditioning pulses that substantially activate gNa. Reducing INa fivefold (Tris substitution) had no effect on either tau c or delay. Delay in not generated by Rs or by contamination from activation development. The slowest time constant in Na tails is approximately 1 ms (Goldman and Hahin, 1978) and the gap was 6 ms. Shortening the gap to 2 ms had no effect on either tau c or delay. Delay is a true property of the channel. Delay decreased with more positive conditioning potentials, and also decreased approximately proportionally with time to peak gNa during the conditioning pulse, as expected for sequentially coupled activation and inactivation. In a few cases the difference between Na current values for brief conditioning pulses and the tau c exponential could be measured. Difference values decayed exponentially with time constant tau m. The inactivation time course is described by a model that assumes a process with the kinetics of gNa activation as a precursor to inactivation.

Tietz syndrome (hypopigmentation/deafness) caused by mutation of MITF.

Patients with Tietz syndrome have congenital profound deafness and generalised hypopigmentation, inherited in a fully penetrant autosomal dominant fashion. The pigmentary features and complete penetrance make this syndrome distinct among syndromes with pigmentary anomalies and deafness, which characteristically have patchy depigmentation and variable penetrance. Only one family has been reported with the exact features described in the original report of this syndrome. This family was reascertained and a missense mutation was found in the basic region of the MITF gene in family members with Tietz syndrome. Mutations in other regions of this gene have been found to produce Waardenburg syndrome type 2 (WS2), which also includes pigmentary changes and hearing loss, but in contrast to Tietz syndrome, depigmentation is patchy and hearing loss is variable in WS2.

The spatial relationship between Ca2+ channels and Ca2+-activated channels and the function of Ca2+-buffering in avian sensory neurons.

In order to learn about the endogenous Ca2+-buffering in the cytoplasm of chick dorsal root ganglion (DRG) neurons and the distance separating the ryanodine receptor Ca2+ release channels (RyRs) from the plasma membrane, we monitored the amplitude and time course of Ca2+-activated Cl- currents (I(ClCa)) in protocols that manipulated Ca2+-buffering. I(ClCa)was activated by Ca2+ influx via voltage-gated Ca2+ channels or by Ca2+ release via RyRs activated by 10 mM caffeine. I(ClCa)was measured in neurons at 20 degrees C and 35 degrees C using the amphotericin perforated patch technique that preserves endogenous Ca2+-buffering, or at 20 degrees C in neurons dialyzed with pipette solutions designed to replace the endogenous Ca2+ buffers. The amplitude of I(ClCa)activated by Ca2+ influx or Ca2+ at 20 degrees C was similar in the amphotericin neurons and neurons dialyzed with an 'unbuffered' pipette solution containing 10 mM citrate and 3 mM ATP as the only Ca2+ binding molecules. Thus, endogenous mobile Ca2+ buffers are relatively unimportant in chick DRG neurons. Warming the neurons from 20 degrees C to 35 degrees C increased the amplitude and the rate of deactivation of I(ClCa)consistent with an increased rate of Ca2+ buffering by fixed endogenous Ca2+-buffers. Dialysis with 2 mM EGTA/0.1 microM free Ca2+ reduced the amplitude and increased the rate of deactivation of I(ClCa)activated by Ca2+ influx and abolished I(ClCa)activated by Ca2+ release. Dialysis with 2 mM BAPTA/0.1 microM free Ca2+ abolished I(ClCa)activated by Ca2+ influx or release. Dialysis with 42 mM HEEDTA/0.5 microM free Ca2+ caused the persistent activation of I(ClCa). Calculations using a Ca2+-diffusion model suggest that the voltage-gated Ca2+ channels and the Ca2+-activated Cl- channels are separated by 50-400 nm and that the RyRs are more than 600 nm from the plasma membrane.

Temperature dependencies of Ca2+ current, Ca(2+)-activated Cl- current and Ca2+ transients in sensory neurones.

We recorded Ca2+ current (ICa) and Ca(2+)-activated Cl- current (ICl(Ca)) in isolated chick dorsal root ganglion neurons. At room temperature, ICl(Ca) is activated by Ca2+ influx (e.g. ICa) or by caffeine-stimulated release of Ca2+ via ryanodine receptors. Warming from room temperature to 37 degrees C increased the amplitude of ICa as well as the amplitude and rate of deactivation of ICl(Ca) activated by Ca2+ influx. In contrast, the activation of ICl(Ca) by caffeine-stimulated release of Ca2+ from intracellular stores abruptly failed between 19 and 28 degrees C. Warning from 22 to 37 degrees C reduced the amplitude of [Ca2+]i transients (measured with Indo-1) in chick neurons by more than 50% and reduced [Ca2+]i transients in mouse neurons by more than 40%. We investigated the role of mitochondria in these phenomena using carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) to inhibit mitochondrial Ca2+ uptake. 1-4 microM FCCP slowed the deactivation of ICa-activated ICl(Ca) at 20 degrees C and at 36 degrees C, having a greater effect at the higher temperature. In the presence of FCCP, the rate of deactivation of ICl(Ca) was relatively insensitive to temperature in this protocol. In contrast, FCCP had little effect on ICl(Ca) activated by caffeine at warmer temperatures (> 22 degrees C) but prolonged ICl(Ca) at cooler temperatures (< 22 degrees C). Thus, we find that warming reduces the ability of Ca2+ release to raise [Ca2+]i increases the effect of mitochondria on the deactivation of ICl(Ca) if ICl(Ca) is activated by Ca2+ influx, and reduces the effect of mitochondria if ICl(Ca) is activated by caffeine-stimulated Ca2+ release.

Phylogenetic analysis of mitochondrial DNA in Japanese pedigrees of sensorineural hearing loss associated with the A1555G mutation.

Thirteen Japanese families (ten of which were from the northern part of Japan), with sensorineural hearing loss associated with the 1555 A to G (A1555G) mitochondrial mutation, a known cause of non-syndromic hearing loss, were phylogenetically analysed using data obtained by restriction fragment length polymorphism (RFLP) and D-loop sequencing of mitochondrial DNA (mtDNA). Various types of mtDNA polymorphism were detected by restriction enzymes and D-loop sequence. No common polymorphic pattern throughout the 13 families was found, though three families exhibited the same restriction patterns and the same sequence substitution in the D-loop. To find where each of the 13 families are situated in the phylogenetic tree, the 482-bp of D-loop sequence were compared with those of 62 normal Japanese subjects. Despite the three families mentioned above appearing to be clustered, the remaining 10 families were scattered along the phylogenetic tree. This indicates that there was no common ancestor for the 13 Japanese families bearing the A1555G mutation except three families, and that the A1555G mutation occurred sporadically and multiplied through evolution of the mtDNA in Japan. The present results showed that the common pathogenicity (hearing loss associated with the A1555G mutation) can occur sporadically in families which have different genetic backgrounds, even in the Japanese population.

Analysis of the 5' regulatory region of the human Norrie's disease gene: evidence that a non-translated CT dinucleotide repeat in exon one has a role in controlling expression.

Analysis of the 5', upstream regions of the Norrie's disease gene (NDP) from 1380bp to +428 (relative to the putative transcription start site) has been undertaken by transfection analysis. Constructs in which a deletion series of sequences from the region were linked to a luciferase reporter gene have been introduced by electroporation into the retinoblastoma cell line, WeriB. Both positive and negative regulatory elements have been identified, and WeriB proved to be a useful cell-line in which to study some aspects of the regulation of this retinal expressed locus. The analysis, therefore, also provides an indication of additional sequences for mutation detection in those patients in which no alterations in the three exons of the gene have been detected.