Dissociation of the stimulatory activities of staphylococcal enterotoxins for T cells and monocytes.

The staphylococcal enterotoxins (SEs) are homologous proteins related in their capacity for stimulating both T cells and monocytes. To assess the importance of conserved structure and sequence to functional activity, the role of the disulfide loop and adjacent sequence in these toxins was evaluated. Contrary to previous reports, we demonstrate here that the disulfide loop was required for the mitogenic activity of SEA and SEB. While T cell-stimulatory activity was compromised, reduced and alkylated SEs retained major histocompatibility complex class II-binding and monocyte-stimulatory activities, suggesting that their inability to induce T cell proliferation was due to failure to interact with T cell receptor (TCR) rather than with class II molecules. Reduction and alkylation did not affect the far-ultraviolet circular dichroic spectrum of SEA, suggesting that the loss of mitogenic activity was not associated with significant changes in secondary structure. The disulfide linkage imparts considerable stability to these toxins as peptide cleavages within the loop of SEB were not associated with detectable loss of function, although cleavage in the conserved sequence outside the loop of SEA resulted in loss of mitogenic activity. This report thus establishes a functional role for a conserved element in SEs, the disulfide loop, and further indicates that their class II- and TCR-binding activities can be dissociated.

In vitro motility of native thin filaments from Drosophila indirect flight muscles reveals that the held-up 2 TnI mutation affects calcium activation.

A procedure for the isolation of regulated native thin filaments from the indirect flight muscles (IFM) of Drosophila melanogaster is described. These are the first striated invertebrate thin filaments to show Ca-regulated in vitro motility. Regulated native thin filaments from wild type and a troponin I mutant, held-up-2, were compared by in vitro motility assays that showed that the mutant troponin I caused activation of motility at pCa values higher than wild type. The held-up2 mutation, in the sole troponin I gene (wupA) in the Drosophila genome, is known to cause hypercontraction of the IFM and other muscles in vivo leading to their eventual destruction. The mutation causes substitution of alanine by valine at a homologous and completely conserved troponin I residue (A25) in the vertebrate skeletal muscle TnI isoform. The effects of the held-up 2 mutation on calcium activation of thin filament in vitro motility are discussed with respect to its effects on hypercontraction and dysfunction. Previous electron microscopy and 3-dimensional reconstruction studies showed that the tropomyosin of held-up 2 thin filaments occupies positions associated with the so-called 'closed' state, but independently of calcium concentration. This is discussed with respect to calcium dependent regulation of held-up-2 thin filaments in in vitro motility.

Temporal constancy of zodiacal light.

Measurements over a 4-year period from the satellite OSO-5 have failed to show any temporal variations in the surface brightness and polarization of zodiacal light.

Discrete Light Sources Observed by Satellite OSO-B.

A comparison is made between the intensities, as measured by satellite OSO-B, of various discrete light sources on the earth (lightning, city lights, flare gas from oil wells).

Satellite observations of lightning.

Analysis of the positions of nighttime thunderstorms as determined from the detection of optical radiation by satellite OSO-B reveals that ten times as many lightning storms occur over land areas as over the sea.

Going paperless: improved cataract surgery outcome data quality in a new fully electronic unit.

OBJECTIVES: To report outcome data on the first 5000 consecutive cataract cases at a new paperless eye unit and benchmark against the Royal College of Ophthalmologists' National Ophthalmology Database (RCOphth NOD). METHODS: Using the in-built audit tool of the electronic medical records system, data from all cataract operations performed between 1 April 2014 and 13 January 2017 were compiled. RESULTS: Five thousand and eight cases were recorded of which the overall intra-operative complication rate was 2.4%, the most common being posterior capsular rupture-1.14%. Follow-up data on post-operative complications were recorded in 98.6% of cases. Pre- and post-operative visual acuities was measured in 98.0% of cases. In all, 40.8% of eyes achieved a visual acuity of 6/6 or better and 90.7% achieved 6/12 or better. CONCLUSIONS: A data set of >5000 consecutive cataract operations was obtained in this eye department. The recording of pre- and post-operative visual acuity in 98% of cases compare very favourably to the RCOphth NOD Audit Report 2017 where pre- and post-operative visual acuities were recorded in only 57.1% of operations. Despite this difference, the outcome measures from this unit and RCOphth NOD were very similar, validating the results of the RCOphth NOD audit reports. Significantly, when applying the RCOphth NOD audit criteria for measuring post-operative visual acuity, approximately 15% of cases were excluded from the data set, reducing the completeness of the data set. Paperless ophthalmology units are feasible in today's NHS and can produce near complete cataract data sets; this can ultimately lead to more comprehensive and reliable aggregate cataract outcome data.

The Royal College of Ophthalmologists' National Ophthalmology Database Study of cataract surgery. Report 5: Clinical outcome and risk factors for posterior capsule rupture and visual acuity loss following cataract surgery in patients aged 90 years and older.

BACKGROUND: Older age is commonly associated with an increased risk of surgical complications and comparatively poor outcomes. PURPOSE: To report cataract surgery outcomes and risk indicators for patients aged 90 years and older. METHODS: Data collected as part of routine cataract care in 34 centres contributing to the United Kingdom Royal College of Ophthalmologists' National Ophthalmology Database (NOD) were analysed. Very elderly people undergoing cataract surgery were profiled in terms of demographics, pre- and postoperative best-measured visual acuity (VA), ocular co-morbidities, intraoperative posterior capsule rupture (PCR) or vitreous loss or both, and risk indicators for operative PCR and adverse VA outcome. RESULTS: 25,856 cataract operations in 19,166 people of 90 years or older between 2000 and 2014 are reported. Preoperative VA was available for 82.4% eyes, being 0.30 LogMAR or better in 21.5%. Postoperative VA was available for 61.8% eyes, being 0.30 LogMAR or better in 74.4%. For those without ocular co-morbidity, postoperative VA was 0.30 LogMAR or better in 84.7%. Various co-morbidities were present in 49% and contributed to an adverse VA outcome. PCR data were available for all operations and occurred in 2.7%. Significant risk indicators for PCR included pseudoexfoliation/phakodonesis, mature cataract, smaller pupil and worse preoperative VA. CONCLUSIONS: Slightly poorer cataract surgery outcome results were noted in patients of 90 years or older, more so in patients with ocular co-morbidity which was highly prevalent. However, surgeons should not be deterred from offering cataract surgery to the very elderly as successful visual rehabilitation remains achievable.

Correction of the disease phenotype in the mouse model of Stargardt disease by lentiviral gene therapy.

Autosomal recessive Stargardt disease (STGD1) is a macular dystrophy caused by mutations in the ABCA4 (ABCR) gene. The disease phenotype that is most recognized in STGD1 patients, and also in the Abca4-/- mouse (a disease model), is lipofuscin accumulation in retinal pigment epithelium. Here, we tested whether delivery of the normal (wt) human ABCA4 gene to the subretinal space of the Abca4 -/- mice via lentiviral vectors would correct the disease phenotype; that is, reduce accumulation of the lipofuscin pigment A2E. Equine infectious anemia virus (EIAV)-derived lentiviral vectors were constructed expressing either the human ABCA4 gene or the LacZ reporter gene under the control of the constitutive (CMV) or photoreceptor-specific (Rho) promoters. Abca4-/- mice were injected subretinally with 1 microl ( approximately 5.0 x 10(5) TU) of each EIAV vector in one eye at postnatal days 4 and 5. An injection of saline, an EIAV-null vector, or an uninjected contralateral eye served as a control. Mice were killed at various times after injection to determine photoreceptor (PR) transduction efficiency and A2E concentrations. EIAV-LacZ vectors transduced from 5 to 20% of the PRs in the injected area in mice. Most importantly, a single subretinal injection of EIAV-CMV-ABCA4 to Abca4-/- mouse eyes substantially reduced disease-associated A2E accumulation compared to untreated and mock-treated control eyes. Treated eyes of Abca4-/- mice accumulated 8-12 pmol per eye (s.d.=2.7) of A2E 1 year after treatment, amounts comparable to wt controls, whereas mock-treated or untreated eyes had 3-5 times more A2E (27-39 pmol per eye, s.d.=1.5; P=0.001-0.005). Although extrapolation to humans requires caution, the high transduction efficiency of both rod and cone photoreceptors and the statistically significant reduction of A2E accumulation in the mouse model of STGD1 suggest that lentiviral gene therapy is a potentially efficient tool for treating ABCA4-associated diseases.

Cat-PROM5: a brief psychometrically robust self-report questionnaire instrument for cataract surgery.

PurposeTo develop a short, psychometrically robust and responsive cataract patient reported outcome measure suitable for use in high-volume surgical environments.MethodsA prospective study in which participants completed development versions of questionnaires exploring the quality of their eyesight using items harvested from two existing United Kingdom developed parent questionnaires. Participants were 822 patients awaiting cataract surgery recruited from 4 cataract surgical centres based in the UK. Exclusion criteria were other visually significant comorbidities and age <50 years. An iterative multi-stage process of evaluation using Rasch and factor analyses with sequential item reduction was undertaken.ResultsA definitive item set of just five items delivered performance in accordance with the requirements of the Rasch model: no threshold disordering, no misfitting items, Rasch-based reliability 0.90, person separation 2.98, Cronbach's α 0.89, good targeting of questions to patients with cataract with pre-operative item mean -0.41 logits and absence of significant floor or ceiling effects, minor deviations of item invariance, and confirmed unidimensionality. The test-re-test repeatability intra-class correlation coefficient was 0.89 with excellent responsiveness to surgery, Cohen's d -1.45 SD. Rasch calibration values are provided for Cat-PROM5 users.ConclusionsA psychometrically robust and highly responsive five-item cataract surgery patient reported outcome measure has been developed, which is suitable for use in high-volume cataract surgical services.

Cataract surgery patient-reported outcome measures: a head-to-head comparison of the psychometric performance and patient acceptability of the Cat-PROM5 and Catquest-9SF self-report questionnaires.

Background Cataract surgery is the most frequently undertaken NHS surgical procedure. Visual acuity (VA) provides a poor indication of visual difficulty in a complex visual world. In the absence of a suitable outcome metric, recent efforts have been directed towards the development of a cataract patient-reported outcome measure (PROM) of sufficient brevity, precision, and responsiveness to be implementable in routine high volume clinical services.Aim To compare and contrast the two most promising candidate PROMs for routine cataract surgery.Method The psychometric performance and patient acceptability of the recently UK developed five-item Cat-PROM5 questionnaire was compared with the English translation of the Swedish nine-item Catquest-9SF using Rasch-based performance metrics and qualitative semistructured interviews.Results Rasch-based performance was assessed in 822 typical NHS cataract surgery patients across four centres in England. Both questionnaires demonstrated good to excellent performance for all metrics assessed, including Person Reliability Indices of 0.90 (Cat-PROM5) and 0.88 (Catquest-9SF), responsiveness to surgery (Cohen's standardized effect size) of 1.45 SD (Cat-PROM5) and 1.47 SD (Catquest-9SF) and they were highly correlated with each other (R=0.85). Qualitative assessments confirmed that both questionnaires were acceptable to patients, including in the presence of ocular comorbidities. Preferences were expressed for the shorter Cat-PROM5, which allowed patients to map their own issues to the questions as opposed to the more restrictive specific scenarios of Catquest-9SF.Conclusion The recently UK developed Cat-PROM5 cataract surgery questionnaire is shorter, with performance and patient acceptability at least as good or better than the previous 'best of class' Catquest-9SF instrument.

In vivo interaction of synthetic acylated apopeptides with high density lipoproteins in rat.

The metabolism of synthetic peptide analogues of high density lipoprotein (HDL) apoproteins has been studied in the rat. These compounds are 15-amino acid lipid associating peptides (LAPs) bearing acyl chains of various lengths (0-16 carbon units). After injection of each 125I-LAP, the serum decay curves suggested a two-compartment process with a clearance rate decreasing when the acyl chain lengths increased. The similarity between the apparent half-life of C16-LAP and that of apoprotein A-I as well as the chromatographic analysis of rat serum were consistent with a partitioning of the LAPs between HDL and the aqueous phase. This was strongly dependent upon the acyl chain length of the LAPs. The distribution volumes of the 125I-LAPs in organs were measured 10 min after injection. The results were analyzed using a model explicitly predicting the organ distribution volumes of HDL and the equilibrium constant (Keq) of the binding of each LAP to HDL. HDL distributed significantly in the adrenals (250 microliters/g), liver (80 microliters/g), and ovaries (55 microliters/g), but not in the kidneys. This suggests that the binding of HDL apoproteins to kidneys, reported by others, was due to the uptake of free apoproteins. The Keqs exhibited a log-linear relationship with respect to the acyl chain length of the LAPs. Each carbon unit added to the acyl chain decreased the free energy of association by a constant value (0.3 kcal mol-1). This clearly showed a strict hydrophobic effect similar to that previously observed in vitro.

Performance of the 24-2-5 frequency doubling technology screening test: a prospective case study.

AIM: To evaluate the performance of the frequency doubling technology (FDT) 24-2-5 screening test by comparison with the established N-30-5 FDT screening test for detection of glaucoma. METHOD: A prospective random sample of individuals referred for possible glaucoma were tested with FDT screening tests 24-2-5 and N-30-5 using the Humphrey Matrix perimeter in addition to standard clinical examination relevant to glaucoma detection. Discriminatory power, reliability and test time of these tests were assessed and compared. The case definition for glaucoma was made by patient according to the established clinical diagnosis. RESULTS: Of 63 referred eligible individuals, 53 (84%) were recruited. Sensitivity and specificity for the N-30-5 screening test was 78 and 85% respectively, compared with 83% and 75% for the 24-2-5 with areas under a receiver operator characteristic curve being 0.87 and 0.92. Differences between these indices were not statistically significant. For a specificity of 95%, sensitivity values were 76% and 56% for the 24-2-5 and N-30-5 respectively. Mean (standard deviation) test duration for the FDT 24-2-5 and N-30-5 screening tests were 111 (13) and 39 (10) seconds respectively (p<0.001). A total of 19 subjects (36%) produced unreliable test results in one or both eyes when tested with the 24-2-5 screening test compared with 5 subjects (9%) with the N-30-5 (p<0.0005). CONCLUSION: Minimal discriminatory power differences existed between the two screening tests evaluated, with both screening tests exhibiting high discriminatory power for detection of individuals with glaucoma. More individuals produced unreliable results on the 24-2-5 screening, which also took longer to perform.

A tropomyosin-2 mutation suppresses a troponin I myopathy in Drosophila.

A suppressor mutation, D53, of the held-up(2) allele of the Drosophila melanogaster Troponin I (wupA) gene is described. D53, a missense mutation, S185F, of the tropomyosin-2, Tm2, gene fully suppresses all the phenotypic effects of held-up(2), including the destructive hypercontraction of the indirect flight muscles (IFMs), a lack of jumping, the progressive myopathy of the walking muscles, and reductions in larval crawling and feeding behavior. The suppressor restores normal function of the IFMs, but flight ability decreases with age and correlates with an unusual, progressive structural collapse of the myofibrillar lattice starting at the center. The S185F substitution in Tm2 is close to a troponin T binding site on tropomyosin. Models to explain suppression by D53, derived from current knowledge of the vertebrate troponin-tropomyosin complex structure and functions, are discussed. The effects of S185F are compared with those of two mutations in residues 175 and 180 of human alpha-tropomyosin 1 which cause familial hypertrophic cardiomyopathy (HCM).

Trk C signaling is required for retinal progenitor cell proliferation.

Although neurotrophin actions in the survival of specific retinal cell types have been identified, the biological functions for neurotrophin-3 (NT-3) in early retinal development remain unclear. Having localized NT-3 and trk C expression at early developmental stages when retinal neuroepithelial progenitor cells predominate, we sought to modulate NT-3 signaling in these cells by overexpressing a truncated isoform of the NT-3 receptor, trk C. We have demonstrated that this non-catalytic receptor can inhibit NT-3 signaling when coexpressed with the full-length kinase-active trk C receptor. Using a replication-deficient retrovirus to ectopically express the truncated trk C receptor to limited numbers of progenitor cells in ovo, we examined the effects of disrupted trk C signaling on the proliferation or differentiation of retinal cells. Clones expressing truncated trk C exhibited a 70% reduction in clone size, compared with clones infected with a control virus, indicating that inhibition of trk C signaling decreased the clonal expansion of cells derived from a single retinal progenitor cell. Additionally, impaired NT-3 signaling resulted in a reduction of all retinal cell types, suggesting that NT-3 targets retinal precursor cells rather than differentiated cell types. BrdU labeling studies performed at E6 indicate that this reduction in cell number occurs through a decrease in cell proliferation. These studies suggest that NT-3 is an important mitogen early in retinal development and serves to establish the size of the progenitor pool from which all future differentiated cells arise.

Structure of a biologically active fragment of human serum apolipoprotein C-II in the presence of sodium dodecyl sulfate and dodecylphosphocholine.

We have studied the three-dimensional structure of a biologically active peptide of apolipoprotein C-II (apoC-II) in the presence of lipid mimetics by CD and NMR spectroscopy. This peptide, corresponding to residues 44-79 of apoC-II, has been shown to reverse the symptoms of genetic apoC-II deficiency in a human subject. A comparison of alpha-proton secondary shifts and CD spectroscopic data indicates that the structure of apoC-II(44-79) is similar in the presence of dodecylphosphocholine and sodium dodecyl sulfate. The three-dimensional structure of apoC-II(44-79) in the presence of sodium dodecyl sulfate, determined by relaxation matrix calculations, contains two amphipathic helical domains formed by residues 50-58 and 67-75, separated by a non-helical linker centered at Tyr63. The C-terminal helix is terminated by a loop formed by residues 76-79. The C-terminal helix is better defined and has a larger hydrophobic face than the N-terminal helix, which leads us to propose that the C-terminal helix together with the non-helical Ile66 constitute the primary lipid binding domain of apoC-II(44-79). Based on our structure we suggest a new mechanism of lipoprotein lipase activation in which both helices of apoC-II(44-79) remain lipid bound, while the seven-residue interhelical linker extends away from the lipid surface in order to project Tyr63 into the apoC-II binding site of lipoprotein lipase.

Plasma factors affecting the in vitro conversion of high-density lipoproteins labeled with a non-transferable marker.

We studied the in vitro conversion of HDL3 labeled with a radioiodinated diacyl lipid associating peptide (diLAP). DiLAP was previously shown to be nontransferable, which permitted its' use as a reliable marker of HDL particles. DiLAP-labeled HDL3 was incubated for 23 h at 37 degrees C in human or rat plasma or in reconstituted media containing delipidated plasma and/or lipoproteins and/or partially purified CETP. At the end of the incubations, the samples were adjusted to a density of 1.125 g/ml and ultracentrifuged. The two resulting fractions containing HDL2 and HDL3, respectively, were analyzed by gradient gel electrophoresis. Depending upon experimental conditions, diLAP-labeled HDL3 was converted into HDL2b- and/or small HDL3c-like particles. LCAT inhibition and to a lesser extent CETP promoted the formation of small HDL3c. Reactivation of LCAT led to the disappearance of small HDL3c. No HDL3c formed from HDL2 even in the absence of LCAT activity. When the incubations were performed in the presence of 100 mM thimerosal, which inhibited PLTP but not CETP activity, the conversion of diLAP-labeled HDL3 into HDL2 was almost completely blocked. Collective consideration of these data indicates that the formation of small HDL is moderately facilitated by CETP; that small HDL are converted to larger HDL species by LCAT and that the transformation of HDL3 into HDL2 is a process which largely depends upon PLTP activity.

Plasma lipid transport in the preruminant calf, Bos spp: primary structure of bovine apolipoprotein A-I.

The preruminant calf (Bos spp.) is a model of considerable interest with regard to hepatic and intestinal lipoprotein metabolism (Bauchart et al., J. Lipid Res. (1989) 30, 1499-1514 and Laplaud et al., J. Lipid Res. (1990) 31, 1781-1792). As a preliminary step towards future experiments dealing with HDL metabolism in the calf, we have purified apoA-I from this animal and determined its complete amino acid sequence. Thus, approx. 10% of calf apoA-I was shown to contain a propeptide, with the sequence Arg-His-Phe-Trp-Gln-Gln. Enzymatic cleavage of apoA-I resulted in 10 proteolytic peptides. The complete apoA-I sequence was obtained after alignment of peptides on the basis of their homologies with those from rabbit apoA-I. Thus calf apoA-I consists of 241 amino acid residues, and exhibits high sequence homology with all mammalian apoA-I's studied to date. The bovine protein contained 10 hydrophobic amphipathic helical regions, occurring between residues 43-64, 65-86, 87-97, 98-119, 120-141, 142-163, 164-184, 185-206, 207-217 and 218-241. A computer-constructed phylogenetic tree showed that bovine apoA-I was more closely related to its dog counterpart, including the presence of a single methionine, than to the corresponding macaque and human proteins. Comparative predictions of the respective antigenic structures of human and bovine apoA-I's using the Hopp-Woods algorithm indicated similar positions for all 13 detectable antigenic sites, among which 7 were of identical, or closely related, amino acid composition. This finding was confirmed by demonstration of partial immunological identity between the two proteins upon immunodiffusion analysis, a result obtained using a monospecific rabbit antiserum against bovine apoA-I. Finally, comparison of sequence homology between bovine apoA-I and the lecithin:cholesterol acyl transferase (LCAT) activating region of human apoC-I suggests that several LCAT activating domains may be present in calf apoA-I.

Measurement and prediction of the rates of spontaneous transfer of phospholipids between plasma lipoproteins.

The purpose of this report is to develop a correlation between the hydrophobicity of a phospholipid as measured by reversed-phase high-performance liquid chromatography and its rate of spontaneous transfer and to use this correlation to predict the rate of transfer of any homologous lipid from any lipoprotein. We have studied the mechanism of transfer of a series of fluorescent or radiolabeled phospholipids among natural and reassembled serum lipoproteins. Fluorescent phosphatidylcholines included those with 9-(1-pyrenyl)nonanoic acid in the sn-2 position and lauric, myristic, palmitic, stearic, oleic or linoleic acid at sn-1. The radioactive phosphatidylcholines contained [3H]oleic acid in the sn-2 position and lauric, myristic, or palmitic acid at sn-1. The kinetics of transfer of the pyrene-labeled lipid were followed by changes in the excimer fluorescence, and that of the radioactive lipids by separation of the donor (lipid-apolipoprotein recombinant) from the acceptor (single bilayer vesicles) on a column of Sephacryl S-200. The retention time of each lipid was measured by high-performance hydrophobic chromatography through a Waters radially compressed C18 column eluted with 75% isopropanol and 25% triethylammonium phosphate (0.15 M). A linear relationship was observed between the rate-constant of transfer and the retention time which suggest that the rate of desorption of phosphatidylcholines from lipoproteins and vesicles is controlled predominately by the hydrophobic effect. For a homologous series of lipids, the rate of transfer can be predicted from retention times obtained from hydrophobic chromatography. The kinetics of transfer of 1-lauroyl-2-[9-(1-pyrenyl)nonanoyl] phosphatidylcholine between isolated human serum lipoproteins exhibits a linear correlation between the transfer half-time and the size of the donor lipoproteins. As a consequence, transfer from very-low-density lipoprotein is 10-times slower than that observed from high-density lipoproteins. The observed correlations between phospholipid transfer rates and both the Stokes radius of the donor and the retention time of the phospholipid on a hydrophobic column permit one to calculate the rate of transfer of homologous molecules between lipid-protein complexes. The results predict that the spontaneous transfer of phospholipids between plasma lipoproteins would be too slow to be a physiologically important phenomena.

Ischemia in the ambulatory setting--the total ischemic burden: relation to exercise testing and investigative and therapeutic implications.

To establish the relation between treadmill exercise testing and ambulatory St segment monitoring in the detection of ischemia in patients with coronary artery disease, and to assess whether standard medical therapy affects any such relation, 277 patients with stable angina and angiographically documented coronary artery disease were studied with treadmill exercise testing and 48 h ambulatory ST segment monitoring. One hundred forty-six patients (52%) were studied while receiving no routine antianginal therapy, and 131 (48%) while receiving standard medical therapy. In 187 patients (67%) the exercise test was positive for ischemia. During 11,964 h of ambulatory monitoring, 881 episodes of ischemia (645 [73%] silent) were recorded, of which 809 (92%) occurred in patients with a positive exercise test. The mean heart rate at the onset of ischemic episodes during ambulatory monitoring was significantly less than that at the onset of 1 mm ST segment depression during exercise testing (94.5 versus 105.9 beats/min, p less than 0.0001). However, the frequency of ambulatory ischemic episodes was strongly related to a positive exercise test (p less than 0.001), and this relation was similar for both silent and painful ischemia (p less than 0.0001 for both) and in patients who were and were not receiving therapy (p less than 0.0001 for both). The total duration of ischemia was similarly related to a positive exercise test (p less than 0.0001). Only one patient with a negative exercise test had frequent (greater than 5/day) episodes of ischemia on ambulatory monitoring and had documented coronary artery spasm. Thus, exercise testing identifies the majority of patients likely to have significant ischemia during their daily activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional and ultrastructural effects of a missense mutation in the indirect flight muscle-specific actin gene of Drosophila melanogaster.

A single-site mutation of the flight-muscle-specific actin gene of Drosophila melanogaster causes a substitution of glutamic acid 93 by lysine in all the actin encoded in the indirect flight muscle (IFM). In these Act88FE93K mutants, myofibrillar bundles of thick and thin filaments are present but lack Z-discs and all sarcomeric repeats. Dense filament bundles, which are probably aberrant Z-discs, are seen in myofibrils of pupal flies, but early in adult life these move to the periphery of the fibrils and are not seen in skinned adult fibres. Consistent with this observation, alpha-actinin and other high molecular weight proteins, possibly associated with Z-discs, are not detected on SDS/polyacrylamide gels or Western blots of skinned adult IFM. The mutation lies at the beginning of a loop in the small domain of actin, near the myosin binding region. However, that the mutant actin binds myosin heads is shown by (1) rigor crossbridges in electron micrographs, (2) the appropriate rise in stiffness when ATP is withdrawn in mechanical experiments, and (3) equal protection against tryptic digestion provided by rigor binding between actin and myosin in both wild-type and mutant fibres. Reversal of rigor chevron angle along some thin filaments reflects reversal of thin-filament polarity due to lattice disorder. The absence of Z-discs, alpha-actinin and two high molecular weight proteins, and binding studies by others, suggest that the substitution at residue 93 affects the binding of the mutant actin to a protein, possibly alpha-actinin, which is necessary for Z-disc assembly or maintenance.