Does the addition of virtual reality training to a standard program of inpatient rehabilitation improve sitting balance ability and function after stroke? Protocol for a single-blind randomized controlled trial.

BACKGROUND: Sitting ability and function are commonly impaired after stroke. Balance training has been shown to be helpful, but abundant repetitions are required for optimal recovery and patients must be motivated to perform rehabilitation exercises repeatedly to maximize treatment intensity. Virtual reality training (VRT), which allows patients to interact with a virtual environment using computer software and hardware, is enjoyable and may encourage greater repetition of therapeutic exercises. However, the potential for VRT to promote sitting balance has not yet been explored. The objective of this study is to determine if supplemental VRT-based sitting balance exercises improve sitting balance ability and function in stroke rehabilitation inpatients. METHODS/DESIGN: This is a single-site, single-blind, parallel-group randomized control trial. Seventy six stroke rehabilitation inpatients who cannot stand independently for greater than one minute but can sit for at least 20 minutes (including at least one minute without support) are being recruited from a tertiary-care dedicated stroke rehabilitation unit. Participants are randomly allocated to experimental or control groups. Both participate in 10-12 sessions of 30-45 minutes of VRT performed in sitting administered by a single physiotherapist, in addition to their traditional therapy. The experimental group plays five games which challenge sitting balance while the control group plays five games which minimize trunk lean. Outcome measures of sitting balance ability (Function in Sitting Test, Ottawa Sitting Scale, quantitative measures of postural sway) and function (Reaching Performance Scale, Wolf Motor Function Test, quantitative measures of the limits of stability) are administered prior to, immediately following, and one month following the intervention by a second physiotherapist blind to the participant's group allocation. DISCUSSION: The treatment of sitting balance post-stroke with VRT has not yet been explored. Results from the current study will provide important evidence for the use of low-cost, accessible VRT as an adjunct intervention to increase sitting balance in lower-functioning patients receiving inpatient rehabilitation. The motivating and enjoyable attributes of VRT may increase exercise dosage, leading to improved function and optimal results from rehabilitation. TRIAL REGISTRATION: https://clinicaltrials.gov/; Identifier: NCT02285933. Registered 06 November 2014. Funded by the Heart & Stroke Foundation of Canada and a generous donation from Tony & Elizabeth Graham.

Normal esophageal high-resolution manometry and impedance values in the supine and sitting positions in the population of Northern China.

The aim of this study was to investigate the normal high-resolution manometry and impedance (HRiM) values in the supine and sitting positions in the population of Northern China, and to investigate the influence of different body positions and bolus consistency on esophageal HRiM findings. In this study, healthy volunteers in the supine position underwent esophageal HRiM examination of 10 swallows of 5 mL normal saline solution and 10 swallows of 5 mL synthetic gel of known viscosity, and in the sitting position of an additional five swallows of a synthetic gel of known viscosity. Total bolus transit time (TBTT), complete bolus transit rate (CBTR), distal contractile integral (DCI), distal esophageal amplitude (DEA), and integrated relaxation pressure (IRP) were measured. Sixty-two healthy volunteers were examined in the supine position and 45 of these performed additional swallows of the viscous gel in the sitting position. In the supine position, normal values for swallowing the liquid and viscous boli were as follows: TBTT 6.9 ± 0.9 and 8.0 ± 1.2 s (P < 0.001), CBTR 90.3 ± 14.0 and 77.9 ± 20.3% (P < 0.001), DCI 1891.5 ± 1131.9 and 1967.8 ± 1140.1 mmHg.s.cm (P = 0.227), DEA 95.3 ± 35.4 and 98.7 ± 37.5 mmHg (P = 0.148), and IRP 10.4 ± 4.9 and 9.0 ± 4.2 mmHg (P < 0.001), respectively. For swallows of the viscous boli in the sitting position, TBTT, DCI, DEA, and IRP were significantly decreased, while CBTR was unchanged (P = 0.075). Normal HRiM values of the population of Northern China were established. Esophageal transit times of viscous boli were significantly slower, more often incomplete and produced less normal peristalsis in the supine position than swallows of liquid boli. Independent reference values for different manometric systems, body positions, and population need to be established before clinical application.

Audit and review for evidence-based red cell wastage reduction measures.

Stocks of red blood cells (RBC) are held to ideally match supply and demand; hold too great a stock and unnecessary wastage occurs; too low a stock results in delay or lack of blood for the patient. Blood is a precious resource and its supply needs to be managed effectively. The aim was to identify how RBC units are wasted and propose laboratory-based reduction measures that would not compromise the clinical requirements of the patient. Wastage of RBC was investigated using a 'dashboard' query of a laboratory information management system. By employing service improvement tools, proposals were made to reduce unnecessary RBC waste while ensuring an adequate supply to the patient. The efficacy of those proposals was examined using the same dashboard to compare similar periods before and after their introduction. The reduction in RBC wastage for all groups during an eight month period (December to July) was from 6.4% (5.3% non-AB or B RhD-positive) pre-implementation to 4.4% (2.5% non-AB/B RhD-positive) post-implementation. Group O RhD-negative wastage reduced from 10.4% to 4.4% after introduction of waste-saving proposals. However, there was an increase in staff time required to introduce the changes and in associated Group and Screen testing (3.4 to 3.8 per unit issued). RBC wastage was significantly reduced (P<0.0001) by 32.8% (52%, non-AB/B RhD-positive), saving approximately 225 RBC units per annum. Financially, increased associated costs did not negate the savings made by the measures introduced.

Impact of early screening echocardiography and targeted PDA treatment on neonatal outcomes in "22-23" week and "24-26" infants.

The hemodynamically significant patent ductus arteriosus (hsPDA) is a controversial topic in neonatology, particularly among neonates at the earliest gestational ages of 22+0-23+6 weeks. There is little, to no data on the natural history or impact of the PDA in extremely preterm babies. In addition, these high-risk patients have typically been excluded from randomized clinical trials of PDA treatment. In this work, we present the impact of early hemodynamic screening (HS) of a cohort of patients born 22+0-23+6 weeks gestation who either were diagnosed with hsPDA or died in the first postnatal week as compared to a historical control (HC) cohort. We also report a comparator population of 24+0-26+6 weeks gestation. All patients in the HS epoch were evaluated between 12-18h postnatal age and treated based on disease physiology whereas the HC patients underwent echocardiography at the discretion of the clinical team. We demonstrate a two-fold reduction in the composite primary outcome of death prior to 36 weeks or severe BPD and report a lower incidence of severe intraventricular hemorrhage (n=5, 7% vs n=27, 27%), necrotizing enterocolitis (n=1, 1% vs n=11, 11%) and first-week vasopressor use (n=7, 11% vs n=40, 39%) in the HS cohort. HS was also associated with an increase in survival free of severe morbidity from the already high rate of 50% to 73% among neonates <24 weeks gestation. We present a biophysiological rationale behind the potential modulator role of hsPDA on these outcomes and review the physiology relevant to neonates born at these extremely preterm gestations. These data highlight the need for further interrogation of the biological impact of hsPDA and impact of early echocardiography directed therapy in infants born less than 24 weeks gestation.

Does F4TCNQ Adsorption on Cu(111) Form a 2D-MOF?

The results of a quantitative experimental structural investigation of the adsorption phases formed by 2,3,5,6-tetrafluoro-7,7',8,8'-tetracyanoquinodimethane (F4TCNQ) on Cu(111) are reported. A particular objective was to establish whether Cu adatoms are incorporated into the molecular overlayer. A combination of normal incidence X-ray standing waves, low-energy electron diffraction, scanning tunneling microscopy, and X-ray photoelectron spectroscopy measurements, complemented by dispersion-inclusive density functional theory calculations, demonstrates that F4TCNQ on Cu(111) does cause Cu adatoms to be incorporated into the overlayer to form a two-dimensional metal-organic framework (2D-MOF). This conclusion is shown to be consistent with the behavior of F4TCNQ adsorption on other coinage metal surfaces, despite an earlier report concluding that the adsorption structure on Cu(111) is consistent with the absence of any substrate reconstruction.

Effective electromagnetic noise cancellation with beamformers and synthetic gradiometry in shielded and partly shielded environments.

The major challenge of MEG, the inverse problem, is to estimate the very weak primary neuronal currents from the measurements of extracranial magnetic fields. The non-uniqueness of this inverse solution is compounded by the fact that MEG signals contain large environmental and physiological noise that further complicates the problem. In this paper, we evaluate the effectiveness of magnetic noise cancellation by synthetic gradiometers and the beamformer analysis method of synthetic aperture magnetometry (SAM) for source localisation in the presence of large stimulus-generated noise. We demonstrate that activation of primary somatosensory cortex can be accurately identified using SAM despite the presence of significant stimulus-related magnetic interference. This interference was generated by a contact heat evoked potential stimulator (CHEPS), recently developed for thermal pain research, but which to date has not been used in a MEG environment. We also show that in a reduced shielding environment the use of higher order synthetic gradiometry is sufficient to obtain signal-to-noise ratios (SNRs) that allow for accurate localisation of cortical sensory function.

Coronary spasm induced by capecitabine mimicks ST elevation myocardial infarction.

Capecitabine is a chemotherapeutic prodrug that is metabolised to 5-fluorouracil. Supported by the National Institute for Health and Clinical Excellence guidance it is now first-line adjuvant treatment for metastatic colorectal cancer in the UK. Although cardiac chest pain and myocardial ischaemia are well recognised side effects of 5-fluorouracil, their association with capecitabine is not widely appreciated. Two cases are described of coronary spasm secondary to capecitabine in patients referred for emergency invasive treatment of presumed ST elevation myocardial infarction (STEMI). The contemporary treatment of acute coronary syndromes involves aggressive antiplatelet therapy, anticoagulation and cardiac catheterisation. This treatment, although beneficial in most patients, is associated with a small but significant risk of bleeding complications. A wider appreciation of the potential for capecitabine to induce spasm mimicking STEMI is important in order to reduce the risk of the administration of thrombolytics and other potentially dangerous drugs and have a higher threshold for referral for emergency angiography.

Regional gastrointestinal pH profile is altered in patients with type 1 diabetes and peripheral neuropathy.

BACKGROUND: Gastrointestinal (GI) symptoms, such as nausea and bloating, are common in people with type 1 diabetes (T1DM). Autonomic dysfunction can lead to changes in the GI secreto-motor function which can be associated with GI symptom development. We hypothesized that regional pH profiles in T1DM differs from health and would be associated with objective physiological/clinical markers. METHODS: Forty-seven T1DM with confirmed diabetic sensory peripheral neuropathy and 41 healthy age-matched subjects underwent standardized wireless motility capsule testing. T1DM completed the gastroparesis cardinal symptom index (GCSI) and the gastrointestinal symptom rating scale. Disease duration, glycemic control, insulin usage, and 24-hour heart rate variability testing were evaluated. KEY RESULTS: In comparison to healthy subjects, gastric, and large bowel median pH were lower in T1DM (1.8 ± 1.6 vs 2.9 ± 1.5, P = 0.001 and 6.7 ± 0.6 vs 7.0 ± 0.5, P = 0.003, respectively). Additionally, change in pH across the pylorus was lower while change in pH across the ileocecal junction was higher in T1DM (5.2 ± 1.5 vs 5.8 ± 0.5, P = 0.003 and 1.8 ± 0.4 vs 1.3 ± 0.4, P < 0.0001, respectively). No difference was found in small bowel median pH. Gastric median pH was associated with small bowel transit time (r = 0.30, P = 0.049). Change in pH across the pylorus was negatively associated with fasting glycose (r = -0.35, P = 0.027). Small bowel median pH was associated with nausea (r = 0.42, P = 0.005) and small bowel transit time (r = 0.48, P = 0.0007). Large bowel median pH was associated with nausea (r = 0.35, P = 0.018) and the total GCSI score (r = 0.34, P = 0.023). CONCLUSIONS AND INFERENCES: The GI pH profile in T1DM with DSPN is different from healthy subjects. Changes in pH profile may have important therapeutic implications and influence pharmacotherapeutic bioavailability.

Regional gastrointestinal contractility parameters using the wireless motility capsule: inter-observer reproducibility and influence of age, gender and study country.

BACKGROUND: The wireless motility capsule concurrently measures temperature, pH and pressure as it traverses the gastrointestinal tract. AIMS: To describe normative values for motility/contractility parameters across age, gender and testing centres. METHODS: Healthy participants underwent a standardised wireless motility capsule assessment following an overnight fast and consumption of a meal of known nutritional content. Traces were divided into regions of interest and analysed using 2 software packages (MotiliGI and GIMS Data Viewer). Inter-observer agreement was independently assessed by 2 investigators. RESULTS: Normative data for motility/contractility parameters (maximum amplitude, mean peak amplitude, contraction frequency and motility index) are presented for 107 individuals (62 male, median age 40 years, range 18-78). MotiliGI-Gastric, small bowel and colonic maximal contraction amplitude correlated with age (r = .24, P = .01; r = .22, P = .02; and r = .2, P = .04 respectively). Small bowel motility index was higher in females than males (150.4 ± 12 vs 122 ± 7.6, P = .04). Inter-observer agreement was excellent for transit times, pH and contractility/motility parameters. GIMS Data viewer-Gastric, small bowel and colonic loge motility index correlated with the respective area under the contraction curve, total contractions, sum of amplitudes and contraction frequency (all r>.35, P < .0003) but not with transit times. CONCLUSIONS: Our analysis provides normative data for motility/contractility parameters. Log motility index summarises a number of measures. In future, the measurement of contractile activity with the wireless motility capsule may potentially aid in the diagnosis of disease states such as visceral myopathic disorders.

Frequent genital herpes simplex virus 2 shedding in immunocompetent women. Effect of acyclovir treatment.

Reactivation of herpes simplex virus type 2 (HSV-2) occurs intermittently as perceived clinically and by viral culture. We performed a series of studies to evaluate the frequency and pattern of HSV-2 reactivation using both viral isolation and HSV PCR assay. Daily samples of genital secretions were obtained from 27 HSV-2 seropositive women; a subset of subjects obtained samples while receiving oral acyclovir 400 mg PO twice a day. HSV DNA was detected in genital swab specimens on 28% of 1,410 d compared with 8.1% of days by viral isolation. 11 of 20 women had HSV DNA detected on > 20% of days, 4 on > 50%, and 2 on > 75% of days; in contrast, none of the women shed on > 21% of days by viral isolation. The daily administration of oral acyclovir promptly reduced the frequency of HSV DNA detection by a median of 80%. Within 3-4 d of discontinuing daily acyclovir, HSV DNA again appeared in the genital area. HSV-2 shedding in the genital mucosa occurs much more frequently than previously appreciated. This frequent reactivation likely plays a role in the epidemic spread of genital herpes worldwide.

Neurokinin-1 receptor antagonism in a human model of visceral hypersensitivity.

BACKGROUND: Substance P acting via the neurokinin-1 receptor is involved in the development of hyperalgesia, although studies using neurokinin-1 receptor antagonists (NK-1RA) in human somatic pain have been disappointing. AIM: To evaluate whether Substance P is involved in the development of human visceral pain/hyperalgesia using a selective NK-1RA. METHODS: Using a validated human model of acid-induced oesophageal allodynia, pain thresholds to electrical stimulation (mA) were measured in the proximal oesophagus and the foot (somatic control), pre- and for 4 h postdistal oesophageal acid in 14 healthy subjects, using a double-blind, randomized, two-period, crossover study. Measurements were taken on the third day of dosing with either an oral NK-1RA or matching placebo, with 2 weeks washout between periods. RESULTS: Baseline pain threshold did not differ between treatments (proximal oesophagus 37 +/- 7.4 mA NK-1RA vs. 38 +/- 10.1 placebo P = 0.81, foot 40 +/- 15 mA NK-1RA vs. 38 +/- 14 placebo P = 0.68). NK-1RA did not attenuate the reduction in pain threshold in the proximal oesophagus postacid infusion (AUC-394 +/- 279 NK-1RA vs. -262 +/- 397 placebo P = 0.54). CONCLUSIONS: The lack of effect of NK-1RA on oesophageal pain threshold in our model does not support a role for Substance P in the development of acid-induced oesophageal allodynia.

Exploring the neurophysiological basis of chest wall allodynia induced by experimental oesophageal acidification - evidence of central sensitization.

In somatic models of central sensitisation (CS) allodynia develops following changes to somatic A-beta fibres, allowing these afferents which normally only process innocuous sensations to encode pain. The aim of this study was to determine whether somatic allodynia induced by visceral sensitisation occurs via N-Methyl-D-Aspartate (NMDA) receptor mediated changes to the neurophysiological characteristics of somatic A-beta fibres. Twelve healthy subjects had oesophageal, chest wall and foot pain thresholds (PT) to electrical stimulation measured, and chest wall evoked potentials (CEP) recorded before and 30 minutes after distal oesophageal acidification on 2 separate visits. Intravenous ketamine (an NMDA receptor antagonist) or saline was given 30 minutes post acid with repeated oesophageal and chest wall PT measurements and CEP recordings. Distal oesophageal acidification reduced PT to electrical stimulation on the anterior chest wall (37 +/- 10 mA v 29 +/- 7 mA p = 0.01) and proximal oesophagus (46 +/- 10 mA v 33 +/- 11 mA p = 0.001) but not the foot (37 +/- 25 mA v 39 +/- 23 mA p = 0.12). The induction of chest wall somatic allodynia was accompanied by a reduction in the latency of the P1 (36 +/- 3 ms to 30 +/- 4 ms p = 0.016) and P2 (87 +/- 7 ms to v 76 +/- 7 ms p = 0.049) components of the CEP. NMDA receptor antagonism reversed both visceral and somatic pain hypersensitivity but did not affect CEP latencies. These data provide objective neurophysiological evidence that CS contributes to the development of somatic allodynia following visceral sensitisation.

Assessing the temporal reproducibility of human esophageal motor-evoked potentials to transcranial magnetic stimulation.

BACKGROUND: Although the electrophysiological properties and reproducibility of somatic limb motor evoked potentials (MEPs) to transcranial magnetic stimulation (TMS) are well characterized, little is known about the reproducibility of MEPs for viscerosomatic structures such as the esophagus. AIM: To determine the temporal reproducibility of esophageal MEPs to TMS. METHODS: MEPs to TMS were recorded from the proximal esophagus, using a swallowed catheter housing a pair of electrodes, in eight healthy subjects at five stimulus intensities (SI) (motor threshold [MT] to 20% above MT). For each SI, 20 consecutive TMS stimuli at 5-second intervals were delivered over a single scalp site (dominant hemisphere at site exhibiting MT at lowest SI) and repeated 40 and 80 minutes thereafter. MEP amplitudes and latencies were measured, and means were sequentially calculated for each SI and then log-transformed. The repeatability coefficients (RC) for the three time points were calculated across each set of 20 stimuli and presented as an exponential ratio. RESULTS: Best RC (amplitude/latency) were achieved at 120% SI relative to MT, being 1.8/1.2 (optimal = 1.0). For lower intensities of 115%, 110%, 105%, and 100% SI, the RC were 2.1/1.2, 2.1/1.1, 2.4/1.2, and 2.6/1.4, respectively. For all SI, the greatest reductions in RC occurred over the first 10 stimuli, with little additional gain beyond this number. CONCLUSIONS: Latencies of esophageal MEP to TMS across intensities are highly reproducible, whereas amplitudes are more stimulus intensity-dependent, being most reliable and reproducible at the highest stimulus strengths. SIGNIFICANCE: Using careful parameters, TMS can be used reliably in future studies of viscerosomatic structures, although the size of the response variability needs to be taken into account when assessing changes in cortico-fugal activity.

Cortical processing of human somatic and visceral sensation.

Somatic sensation can be localized precisely, whereas localization of visceral sensation is vague, possibly reflecting differences in the pattern of somatic and visceral input to the cerebral cortex. We used functional magnetic resonance imaging to study the cortical processing of sensation arising from the proximal (somatic) and distal (visceral) esophagus in six healthy male subjects. Esophageal stimulation was performed by phasic distension of a 2 cm balloon at 0.5 Hz. For each esophageal region, five separate 30 sec periods of nonpainful distension were alternated with five periods of similar duration without distension. Gradient echoplanar images depicting bold contrast were acquired using a 1.5 T GE scanner. Distension of the proximal esophagus was localized precisely to the upper chest and was represented in the trunk region of the left primary somatosensory cortex. In contrast, distension of the distal esophagus was perceived diffusely over the lower chest and was represented bilaterally at the junction of the primary and secondary somatosensory cortices. Different activation patterns were also observed in the anterior cingulate gyrus with the proximal esophagus being represented in the right midanterior cingulate cortex (BA 24) and the distal esophagus in the perigenual area (BA32). Differences in the activation of the dorsolateral prefrontal cortex and cerebellum were also observed for the two esophageal regions. These findings suggest that cortical specialization in the sensory-discriminative, affective, and cognitive areas of the cortex accounts for the perceptual differences observed between the two sensory modalities.

CH4 and N2O from mechanically turned windrow and vermicomposting systems following in-vessel pre-treatment.

Methane (CH4) and nitrous oxide (N2O) are included in the six greenhouse gases listed in the Kyoto protocol that require emission reduction. To meet reduced emission targets, governments need to first quantify their contribution to global warming. Composting has been identified as an important source of CH4 and N2O. With increasing divergence of biodegradable waste from landfill into the composting sector, it is important to quantify emissions of CH4 and N2O from all forms of composting and from all stages. This study focuses on the final phase of a two stage composting process and compares the generation and emission of CH4 and N2O associated with two differing composting methods: mechanically turned windrow and vermicomposting. The first stage was in-vessel pre-treatment. Source-segregated household waste was first pre-composted for seven days using an in-vessel system. The second stage of composting involved forming half of the pre-composted material into a windrow and applying half to vermicomposting beds. The duration of this stage was 85 days and CH4 and N2O emissions were monitored throughout for both systems. Waste samples were regularly subjected to respirometry analysis and both processes were found to be equally effective at stabilising the organic matter content. The mechanically turned windrow system was characterised by emissions of CH4 and to a much lesser extent N2O. However, the vermicomposting system emitted significant fluxes of N2O and only trace amounts of CH4. In-vessel pre-treatment removed considerable amounts of available C and N prior to the second stage of composting. This had the effect of reducing emissions of CH4 and N2O from the second stage compared to emissions from fresh waste found in other studies. The characteristics of each of the two composting processes are discussed in detail. Very different mechanisms for emission of CH4 and N2O are proposed for each system. For the windrow system, development of anaerobic zones were thought to be responsible for CH4 release. High N2O emission rates from vermicomposting were ascribed to strongly nitrifying conditions in the processing beds combined with the presence of de-nitrifying bacteria within the worm gut.

Regional gastrointestinal transit and pH studied in 215 healthy volunteers using the wireless motility capsule: influence of age, gender, study country and testing protocol.

BACKGROUND: The wireless motility capsule (WMC) offers the ability to investigate luminal gastrointestinal (GI) physiology in a minimally invasive manner. AIM: To investigate the effect of testing protocol, gender, age and study country on regional GI transit times and associated pH values using the WMC. METHODS: Regional GI transit times and pH values were determined in 215 healthy volunteers from USA and Sweden studied using the WMC over a 6.5-year period. The effects of test protocol, gender, age and study country were examined. RESULTS: For GI transit times, testing protocol was associated with differences in gastric emptying time (GET; shorter with protocol 2 (motility capsule ingested immediately after meal) vs. protocol 1 (motility capsule immediately before): median difference: 52 min, P = 0.0063) and colonic transit time (CTT; longer with protocol 2: median 140 min, P = 0.0189), but had no overall effect on whole gut transit time. Females had longer GET (by median 17 min, P = 0.0307), and also longer CTT by (104 min, P = 0.0285) and whole gut transit time by (263 min, P = 0.0077). Increasing age was associated with shorter small bowel transit time (P = 0.002), and study country also influenced small bowel and CTTs. Whole gut and CTTs showed clustering of data at values separated by 24 h, suggesting that describing these measures as continuous variables is invalid. Testing protocol, gender and study country also significantly influenced pH values. CONCLUSIONS: Regional GI transit times and pH values, delineated using the wireless motility capsule (WMC), vary based on testing protocol, gender, age and country. Standardisation of testing is crucial for cross-referencing in clinical practice and future research.

Distribution patterns of HP1, a heterochromatin-associated nonhistone chromosomal protein of Drosophila.

We have previously reported the identification of a nonhistone chromosomal protein (nhcp-19; now called HP1) preferentially associated with the heterochromatin of Drosophila melanogaster. A detailed study of the HP1 distribution pattern on polytene chromosomes by immunofluorescent staining, using monoclonal antibody C1A9, has been carried out. The results indicate that this protein is found within the centric beta-heterochromatin, in cytological regions 31, 41 and 80, and throughout polytene chromosome 4. Staining of telomeres is frequently observed, those of chromosome arms 2R and 3R and the X chromosome being the most conspicuous. Analysis of a fourth chromosome insertional translocation T(3;4)f/In(3L)P confirms an autonomous interaction with chromosome 4 material. Similarly, the beta-heterochromatin distal to light on chromosome arm 2L, moved to position 97D2 on chromosome arm 3R in the rearrangement ltx13, is prominently stained using the C1A9 antibody. Staining of intact salivary glands indicates that this rearranged segment of beta-heterochromatin is not associated with the polytene chromocenter, but provides an independent structural reference point. HP1 is not observed in the nuclei of the early syncytial embryo, but becomes concentrated in the nuclei at the syncytial blastoderm stage (ca. nuclear division cycle 10). This suggests that heterochromatin formation occurs at approximately the same stage at which nuclei first become transcriptionally competent. Thus, the C1A9 antibody may serve as a useful marker for both structural and functional studies of the Drosophila nucleus.

Overproduction of the membrane-bound components of the histidine permease from Salmonella typhimurium: identification of the M protein.

The periplasmic histidine permease of Salmonella typhimurium is composed of a soluble histidine-binding protein and three membrane-bound components. These latter are produced in very small amounts and only two, the Q and the P protein, have been previously identified. This paper describes the construction of a plasmid carrying the hisQ, hisM, and hisP genes under the control of the lambda PL promoter, thus allowing great overproduction of those gene products. The M protein has been identified in such overproducing strains and its nature confirmed by constructing in vitro hisM deletions within the plasmid. With these results the identification of all components of the histidine permease has been completed.

Generation of soluble recombinant human acute phase serum amyloid A2 (A-SAA2) protein and its use in development of an A-SAA specific ELISA.

Human acute phase serum amyloid A (the A-SAA2 isoform) was expressed at high levels using the pGEX bacterial expression system. A-SAA2 protein was expressed in E. coli NM544 as part of a fusion protein facilitating rapid purification. A-SAA2 was cleaved from the fusion moiety in the presence of a non-ionic detergent (Triton X-100) to release a soluble A-SAA2. Further purification using ion exchange chromatography yielded a pure A-SAA2 (3 mg per litre of culture). Antibodies generated against recombinant A-SAA2 were specific for the acute phase SAAs, A-SAA1 and A-SAA2 and showed no cross-reactivity with the constitutively expressed SAA (C-SAA). These antibodies were used to develop a rapid enzyme-linked immunosorbent assay (ELISA) specific for the measurement of A-SAA in serum.

Downregulation of cGMP phosphodiesterase induced by expression of GTPase-deficient cone transducin in mouse rod photoreceptors.

PURPOSE: Photoexcitation of vertebrate retinal rod photoreceptors stimulates GTP binding to the transducin alpha subunit. Like other GTP-binding proteins, transducin restores itself to an inactive form by hydrolyzing its bound GTP. The role of GTP hydrolysis in phototransduction was investigated. METHODS: A mutant form of cone transducin alpha deficient in its ability to hydrolyze bound GTP was expressed in mouse rod photoreceptors. RESULTS: Expression of the mutant cone transducin alpha at levels threefold to sixfold higher than endogenous rod transducin alpha led to a specific depletion of the transducin target, cGMP phosphodiesterase, and a decrease in the cGMP level. Suction electrode recordings revealed abnormally prolonged flash responses, decreased maximal response amplitudes, and a shift in the stimulus-response relation to higher flash strengths. CONCLUSIONS: Rods expressing high levels of GTPase-deficient cone transduction alpha have reduced levels of phosphodiesterase catalytic subunits and cGMP. These changes are associated with prolonged flash responses, reduced dark current, and decreased sensitivity to light.