Experimental demonstration of a soft x-ray self-seeded free-electron laser.

The Linac Coherent Light Source has added a self-seeding capability to the soft x-ray range using a grating monochromator system. We report the demonstration of soft x-ray self-seeding with a measured resolving power of 2000-5000, wavelength stability of 10(-4), and an increase in peak brightness by a factor of 2-5 across the photon energy range of 500-1000 eV. By avoiding the need for a monochromator at the experimental station, the self-seeded beam can deliver as much as 50-fold higher brightness to users.

Alterations in the PITSLRE protein kinase gene complex on chromosome 1p36 in childhood neuroblastoma.

p58cdc2L1, a protein kinase implicated in apoptotic signaling, is one of eight separate kinases encoded by three tandemly duplicated and linked genes, which we have termed PITSLRE A, B and C. One allele of this complex on chromosome 1 was either deleted or translocated in each of 18 neuroblastoma cell lines with cytogenetically apparent 1p alterations. A protein encoded by this locus, PITSLRE gamma 1, was absent in three of the lines and a smaller, apparently truncated, PITSLRE polypeptide was found in another line. These findings identify a novel gene complex on chromosome 1 that encodes a protein kinase subfamily. We suggest that the PITSLRE locus may harbour one or more tumour suppressor genes affected by chromosome 1p36 modifications in neuroblastoma.

Prevalence of autoantibodies to the 65- and 67-kD isoforms of glutamate decarboxylase in insulin-dependent diabetes mellitus.

We investigated the presence of autoantibodies to baculovirus-expressed human recombinant 65- and 67-kD isoforms of glutamate decarboxylase (GAD65 and GAD67) in insulin-dependent diabetes mellitus (IDDM). In the immunoprecipitation test using [35S]methionine-labeled GADs antibodies to GAD65 were detected in 13/15 (87%) islet cell antibody (ICA)-positive and in 1/35 (2.9%) ICA-negative first-degree relatives of patients with IDDM, in 6/11 (54.5%) ICA-positive nondiabetic schoolchildren, and in 35/50 (70%) patients with newly diagnosed IDDM. GAD67 antibodies were positive only in five (33%) of the ICA-positive relatives (P < 0.05) and in nine (18%) IDDM patients at onset (P < 0.00001). After onset of IDDM antibodies to GAD65 and GAD67 declined but were still positive in 25 and 9.4% of subjects with long-standing IDDM (> 10 yr). In all study groups antibodies to GAD67 were only detected in GAD65 antibody-positive sera. An immunotrapping enzyme activity assay for GAD65 antibodies was positive in 64/75 (85.3%) of sera that were GAD antibody positive in the immunoprecipitation test (r = 0.870, P < 0.0001). In two (2.7%) sera GAD65 antibodies that block GAD enzyme activity were found. Our data suggest that antibodies to GAD65 but not to GAD67 represent sensitive markers for preclinical and overt IDDM. The immunotrapping assay here described represents a valuable technique for specific and sensitive screening for GAD antibodies.

Both TEL and AML-1 contribute repression domains to the t(12;21) fusion protein.

t(12;21) is the most frequent translocation found in pediatric B-cell acute lymphoblastic leukemias. This translocation fuses a putative repressor domain from the TEL DNA-binding protein to nearly all of the AML-1B transcription factor. Here, we demonstrate that fusion of the TEL pointed domain to the GAL4 DNA-binding domain resulted in sequence-specific transcriptional repression, indicating that the pointed domain is a portable repression motif. The TEL pointed domain functioned equally well when the GAL4 DNA-binding sites were moved 600 bp from the promoter, suggesting an active mechanism of repression. This lead us to demonstrate that wild-type TEL and the t(12;21) fusion protein bind the mSin3A corepressor. In the fusion protein, both TEL and AML-1B contribute mSin3 interaction domains. Deletion mutagenesis indicated that both the TEL and AML-1B mSin3-binding domains contribute to repression by the fusion protein. While both TEL and AML-1B associate with mSin3A, TEL/AML-1B appears to bind this corepressor much more stably than either wild-type protein, suggesting a mode of action for the t(12;21) fusion protein.

ETO, a target of t(8;21) in acute leukemia, makes distinct contacts with multiple histone deacetylases and binds mSin3A through its oligomerization domain.

t(8;21) and t(16;21) create two fusion proteins, AML-1-ETO and AML-1-MTG16, respectively, which fuse the AML-1 DNA binding domain to putative transcriptional corepressors, ETO and MTG16. Here, we show that distinct domains of ETO contact the mSin3A and N-CoR corepressors and define two binding sites within ETO for each of these corepressors. In addition, of eight histone deacetylases (HDACs) tested, only the class I HDACs HDAC-1, HDAC-2, and HDAC-3 bind ETO. However, these HDACs bind ETO through different domains. We also show that the murine homologue of MTG16, ETO-2, is also a transcriptional corepressor that works through a similar but distinct mechanism. Like ETO, ETO-2 interacts with N-CoR, but ETO-2 fails to bind mSin3A. Furthermore, ETO-2 binds HDAC-1, HDAC-2, and HDAC-3 but also interacts with HDAC-6 and HDAC-8. In addition, we show that expression of AML-1-ETO causes disruption of the cell cycle in the G(1) phase. Disruption of the cell cycle required the ability of AML-1-ETO to repress transcription because a mutant of AML-1-ETO, Delta469, which removes the majority of the corepressor binding sites, had no phenotype. Moreover, treatment of AML-1-ETO-expressing cells with trichostatin A, an HDAC inhibitor, restored cell cycle control. Thus, AML-1-ETO makes distinct contacts with multiple HDACs and an HDAC inhibitor biologically inactivates this fusion protein.

TEL, a putative tumor suppressor, modulates cell growth and cell morphology of ras-transformed cells while repressing the transcription of stromelysin-1.

TEL is a member of the ETS family of transcription factors that interacts with the mSin3 and SMRT corepressors to regulate transcription. TEL is biallelically disrupted in acute leukemia, and loss of heterozygosity at the TEL locus has been observed in various cancers. Here we show that expression of TEL in Ras-transformed NIH 3T3 cells inhibits cell growth in soft agar and in normal cultures. Unexpectedly, cells expressing both Ras and TEL grew as aggregates. To begin to explain the morphology of Ras-plus TEL-expressing cells, we demonstrated that the endogenous matrix metalloproteinase stromelysin-1 was repressed by TEL. TEL bound sequences in the stromelysin-1 promoter and repressed the promoter in transient-expression assays, suggesting that it is a direct target for TEL-mediated regulation. Mutants of TEL that removed a binding site for the mSin3A corepressor but retained the ETS domain failed to repress stromelysin-1. When BB-94, a matrix metalloproteinase inhibitor, was added to the culture medium of Ras-expressing cells, it caused a cell aggregation phenotype similar to that caused by TEL expression. In addition, TEL inhibited the invasiveness of Ras-transformed cells in vitro and in vivo. Our results suggest that TEL acts as a tumor suppressor, in part, by transcriptional repression of stromelysin-1.

Regional changes in capillary supply in skeletal muscle of high-intensity endurance-trained rats.

The objective of this study was to test the hypothesis that an endurance training program designed to produce recruitment of all extensor muscle fiber types during each exercise bout would stimulate capillary angiogenesis throughout rat gastrocnemius and soleus muscles. Male Sprague-Dawley rats were exercise trained 5 days/wk for 12-14 wk with exercise bouts consisting of a combination of high intensity (32 m/min on a 15% incline) and long duration (90 min/day). On completion of high-intensity endurance training (HIET) or cage activity [sedentary (Sed)], rat hindquarters were vascularly isolated and perfusion fixed with a modified Karnovsky's fixative. Capillary supply was measured in soleus and gastrocnemius muscles by using Olympus Cue 2 image-analyzer software. Capillary supply was reflected in measurements of capillary-to-fiber ratio, capillary numerical density, capillary surface area density, and capillary volume density on transversely cut tissue sections. HIET increased citrate synthase activity by 20 and 42% in the medial and long heads of the triceps brachii, respectively. Sarcomere lengths were similar in gastrocnemius and soleus muscles of Sed and HIET rats after fixation. All four indexes of capillary supply were significantly greater throughout the gastrocnemius muscle of HIET rats compared with Sed values. The relative increase in capillarity was greater in white than in red gastrocnemius muscle of HIET rats. HIET also increased capillary supply of soleus muscle. However, only capillary numerical density was statistically greater (19%) in HIET soleus compared with Sed. These results support the hypothesis that this training program would produce an increase in capillary supply in all extensor muscles.

Skeletal muscle biochemical adaptations to exercise training in miniature swine.

The primary purpose of this study was to test the hypothesis that endurance exercise training induces increased oxidative capacity in porcine skeletal muscle. To test this hypothesis, female miniature swine were either trained by treadmill running 5 days/wk over 16-20 wk (Trn; n = 35) or pen confined (Sed; n = 33). Myocardial hypertrophy, lower heart rates during submaximal stages of a maximal treadmill running test, and increased running time to exhaustion during that test were indicative of training efficacy. A variety of skeletal muscles were sampled and subsequently assayed for the enzymes citrate synthase (CS), 3-hydroxyacyl-CoA dehydrogenase, and lactate dehydrogenase and for antioxidant enzymes. Fiber type composition of a representative muscle was also determined histochemically. The largest increase in CS activity (62%) was found in the gluteus maximus muscle (Sed, 14.7 +/- 1.1 mumol.min-1.g-1; Trn, 23.9 +/- 1.0; P < 0.0005). Muscles exhibiting increased CS activity, however, were located primarily in the forelimb; ankle and knee extensor and respiratory muscles were unchanged with training. Only two muscles exhibited higher 3-hydroxyacyl-CoA dehydrogenase activity in Trn compared with Sed. Lactate dehydrogenase activity was unchanged with training, as were activities of antioxidant enzymes. Histochemical analysis of the triceps brachii muscle (long head) revealed lower type IIB fiber numbers in Trn (Sed, 42 +/- 6%; Trn, 10 +/- 4; P < 0.01) and greater type IID/X fiber numbers (Sed, 11 +/- 2; Trn, 22 +/- 3; P < 0.025). These findings indicate that porcine skeletal muscle adapts to endurance exercise training in a manner similar to muscle of humans and other animal models, with increased oxidative capacity. Specific muscles exhibiting these adaptations, however, differ between the miniature swine and other species.

Training induces nonuniform increases in eNOS content along the coronary arterial tree.

Exercise training produces enhanced nitric oxide (NO)-dependent, endothelium-mediated vasodilator responses of porcine coronary arterioles but not conduit coronary arteries. The purpose of this study was to test the hypothesis that exercise training increases the amount of endothelial NO synthase (eNOS) in the coronary arterial microcirculation but not in the conduit coronary arteries. Miniature swine were either exercise trained or remained sedentary for 16--20 wk. Exercise-trained pigs exhibited increased skeletal muscle oxidative capacity, exercise tolerance, and heart weight-to-body weight ratios. Content of eNOS protein was determined with immunoblot analysis in conduit coronary arteries (2- to 3-mm ID), small arteries (301- to 1,000-microm ID), resistance arteries (151- to 300-microm ID), and three sizes of coronary arterioles [large (101- to 150-microm ID), intermediate (51- to 100-microm ID), and small (<50-microm ID)]. Immunoblots revealed increased eNOS protein in some sizes of coronary arteries and arterioles but not in others. Content of eNOS was increased by 60--80% in small and large arterioles, resistance arteries, and small arteries; was increased by 10--20% in intermediate-sized arterioles; and was not changed or decreased in conduit arteries. Immunohistochemistry revealed that eNOS was located in the endothelial cells in all sizes of coronary artery. We conclude that exercise training increases eNOS protein expression in a nonuniform manner throughout the coronary arterial tree. Regional differences in shear stress and intraluminal pressures during exercise training bouts may be responsible for the distribution of increased eNOS protein content in the coronary arterial tree.

Mechanisms of transcriptional repression by the t(8;21)-, t(12;21)-, and inv(16)-encoded fusion proteins.

AML-1 is one of the most frequently translocated genes in human leukemia. AML-1 binds DNA and activates or represses transcription, while the chromosomal translocation fusion proteins in acute myeloid leukemia subvert these functions. The t(8;21) is the second most frequent translocation in acute myeloid leukemia and creates a fusion between the DNA binding domain of AML-1 and the ETO (also known as MTG8) corepressor. The t(12;21) is found in up to 25% of pediatric B cell acute lymphoblastic leukemias and fuses the ETS family transcription factor TEL to the amino terminus of AML-1. In addition, the inv(16), the most frequent translocation in acute myeloid leukemia, fuses the AML-1 cofactor CBFbeta to the smooth muscle myosin heavy chain MYH11. Both the t(8;21) and t(12;21) create transcriptional repressors that impair AML-1 target gene expression. We demonstrated that the fusion proteins encoded by these translocations contact the nuclear hormone corepressors (N-CoR/SMRT), mSin3A, and histone deacetylases. We have also found that both TEL and AML-1 interact with mSin3A. TEL also binds N-CoR and histone deacetylase-3, indicating that wild-type TEL is a transcriptional repressor. The t(12;21) fuses the mSin3A interaction domain of TEL to AML-1 to transform AML-1 from a regulated to an unregulated transcriptional repressor. The recognition that AML-1 interacts with mSin3A to repress transcription suggested that the inv(16) fusion protein might also repress the transcription of AML-1-target genes. In fact, the inv(16) encodes a protein that cooperates with AML-1 to repress transcription. The inv(16) fusion protein was found in a ternary complex with AML-1 and mSin3A, suggesting that the inv(16) also acts by recruiting transcriptional corepressors and histone deacetylases.

Trials in children.

Over 20% of epilepsies in children are intractable, and are frequently associated with deterioration of motor and cognitive functions. Some are similar to those seen in adults, but many age-related epilepsies occur only in children. Therefore, a sizeable proportion of intractable epilepsies of childhood are different from those encountered in adults, and there is an urgent need for antiepileptic drug trials to be undertaken in children at an early stage of development. An ethical framework within which these trials can be conducted has been defined.

Effect of cataract extraction with intraocular lens implantation on ocular hemodynamics.

PURPOSE: To investigate the effect of cataract extraction on ocular hemodynamics. SETTING: University Eye Clinic of Ulm, Germany. METHODS: In 51 consecutive patients assigned for cataract surgery, pulse amplitude, pulse volume, and pulsatile ocular blood flow were measured 1 day before and 3 days and 12 months after cataract extraction using an ocular blood flow tonograph. Statistical analysis was performed with Student's t-test and Wilcoxon signed-rank test. RESULTS: In the study eyes, 3 days after cataract surgery pulse amplitude, pulse volume, and pulsatile ocular blood flow had decreased from 2.5 to 2.1 mm Hg (P = .0014), 5.0 to 4.4 microliters (P = .0059), and 836.2 to 728.0 microliters/min (P = .0017), respectively. No statistically significant change between preoperative and 3 day postoperative measurements occurred in the fellow eyes. There was no significant difference in systemic blood pressure, heart rate, or IOP in study and fellow eyes before and 3 days after cataract surgery. The early reduction of pulse amplitude, pulse volume, and pulsatile ocular blood flow in the study eyes was not present 1 year postoperatively. CONCLUSION: Uncomplicated cataract extraction is associated with a temporary ipsilateral impairment of ocular hemodynamics. A neural mechanism triggered by cataract extraction may be involved in these temporary changes.

Restenosis is associated with decreased coronary artery nitric oxide synthase.

The purpose of the present study was to test the hypothesis that restenosis is associated with decreased constitutive nitric oxide synthase activity. Male miniswine with moderately elevated serum cholesterol levels underwent cardiac catheterization and oversized balloon injury to the right and left circumflex coronary arteries, followed 2 weeks later by repeat injury on the same coronary segments. After 4 weeks, the coronary arteries were either immediately frozen in liquid nitrogen or pressure-perfusion fixed and prepared for histologic examination. Constitutive nitric oxide synthase activity was quantified using a fibroblast reporter cell method, while constitutive nitric oxide synthase protein was compared between balloon-injured and non-balloon-injured arteries using Western blot analysis. Immunohistochemical studies were performed using a specific antibody against constitutive nitric oxide synthase protein. Following balloon injury, there was decreased constitutive nitric oxide synthase activity in balloon-injured coronary arteries, compared to distal non-balloon-injured segments from the same artery. Histological examination demonstrated an intact endothelium. Specific antibody staining revealed that there was less constitutive nitric oxide synthase protein reactivity by immunohistochemical analysis. Western analysis confirmed less constitutive nitric oxide synthase protein. The data are consistent with the hypothesis that restenosis is associated with decreased endothelial cell nitric oxide production. The data suggest this is secondary to a decreased amount of constitutive nitric oxide synthase enzyme in the endothelium. A deficiency in constitutive nitric oxide synthase enzyme may contribute to the impaired second messenger and paracrine functions of the endothelium observed during restenosis following balloon injury, including abnormal vasomotion, extracellular matrix formation, and platelet aggregation.

Restenosis following placement of an intracoronary heparin treated tantulum stent in the hyperlipidemic miniature swine model.

Restenosis persists as an important factor limiting a favorable long term outcome following mechanical revascularization. The objective of the present study was to compare the effects of an intracoronary heparin treated tantalum prototype stent and balloon angioplasty on intimal hyperplasia, luminal diameter, and thrombosis in a porcine restenosis model. Male miniswine maintained on a high cholesterol diet and 325 mg aspirin per day underwent cardiac catheterization and oversized balloon injury to the right and left circumflex coronary arteries. Two weeks later one artery was either balloon injured again or implanted with a stent. No additional anticoagulation following stent placement was given, however aspirin was continued throughout the study. At four weeks, the coronary arteries were harvested and prepared for histologic examination and blinded quantitative morphometric analysis. The prototype stent was successfully deployed in 10 coronary arteries. Histological examination at explant revealed no evidence for thrombus or platelet aggregation. The angiographic luminal diameter of stented vessels was not significantly different from the diameter measured prior to implantation. In contrast, the angiographic diameter of balloon injured vessels was significantly decreased (4.4 +/- 0.4 mm2, balloon injured, vs. 5.8 +/- 3.3 mm2, control; p < 0.05). Stented arteries showed significantly more intimal hyperplasia, compared to balloon injured vessels (2.99 +/- 0.58 mm2 intimal area, stented arteries vs. 0.38 +/- 0.15 mm2 intimal area, control arteries; p < 0.05). In conclusion, heparin treated tantalum wire prototype intracoronary stents were successfully deployed in swine coronary arteries with no evidence for thrombus formation. Despite a significant intimal response, luminal diameter was preserved in stented vessels. The data suggest that a heparin treated tantalum wire prototype intracoronary stent may be an effective method of coronary revascularization that results in the preservation of luminal diameter without thrombotic occlusion.

Muscle hemodynamics in hereditary myopathy of Labrador retrievers.

Morphologic lesions seen in six 8-month-old Labrador Retrievers with hereditary myopathy were predominantly small- and large-group atrophy of muscle cells of all fiber types. The dogs were intolerant of exercise and fatigued rapidly. An isolated gracilis muscle preparation was used to study the hemodynamic features of the microvasculature. Isogravimetric capillary pressure as well as arterial and venous pressures in the isolated gracilis muscle preparation obtained during maximal vasodilatation were within the range reported for healthy, mixed-breed dogs, as were precapillary, postcapillary, and total vascular resistances. Capillary filtration and osmotic reflection coefficients were not different from those reported in other studies on healthy dogs. All measurements and calculations were repeated during reperfusion, subsequent to a 4-hour period of global ischemia. Postischemic vascular responses were similar to the pattern previously reported in healthy dogs. These studies did not support the hypothesis of a vascular defect as a cause of hereditary myopathy in Labrador Retrievers.

Distribution and implications of beta-endorphin and ACTH-immunoreactive cells in the intermediate lobe of the hypophysis in healthy equids.

The distribution of cells that stain positive for beta-endorphin and ACTH immunoreactivity was studied in the pars intermedia (PI) of the hypophysis in 3 healthy horses and 2 healthy ponies. Serial sections treated with commercial antibodies generated against beta-endorphin or ACTH were processed for immunocytochemical studies, using the avidin biotin immunoperoxidase-complex method. Distribution patterns of cells reacting with antibodies were similar in cells from all equids. Cells immunostained for ACTH were numerous and widely distributed in the PI. Cells immunopositive for ACTH probably contain corticotrophin-like intermediate lobe peptide that cross-reacts with antisera to ACTH. Cells immunopositive for beta-endorphin were fewer in number and had a more limited distribution in the PI. Most beta-endorphin-positive cells were located along the border of the PI adjacent to the lobus nervosus and had abundant eosinophilic cytoplasm when stained with hematoxylin and eosin. Cells with prominent eosinophilic cytoplasm were not common in other areas of the PI. When serial sections were examined, cells that stained positive for beta-endorphin immunoreactivity also appeared positive for ACTH.

Myotonia in a chow chow.

Congenital myotonia was diagnosed in an inbred Chow Chow pup with severe muscle stiffness that regressed with exercise. Voluntary movement, percussion, or needle insertion caused sustained contraction of the muscles involved. Electromyographic recordings from several muscles contained myotonic discharges. Creatine kinase activity was mildly increased. Slight myofiber hypertrophy and a few atrophic fibers were seen in muscle biopsy specimens. Treatment of the pup with procainamide caused a marked decrease in clinical signs. Myotonia congenita in the Chow Chow appears to be inherited as an autosomal recessive trait. This condition can be diagnosed on the basis of clinical signs. Satisfactory clinical management of myotonia congenita can be achieved with procainamide.

Radiation treatment for thymoma in a dog.

A 10-year-old Toy Poodle was admitted for evaluation of a chronic cough caused by a cranial mediastinal mass. The mass was diagnosed, by needle biopsy, as a thymoma. Detection of serum acetylcholine receptor antibodies suggested that the dog had subclinical myasthenia gravis. The dog underwent orthovoltage radiation treatment, which resulted in an approximate 60% reduction in tumor mass and freedom from clinical signs for 6 months. Concurrent use of prednisolone may have been associated with disappearance of the acetylcholine receptor antibodies.

Anury in two Cairn terriers.

Congenital anury (taillessness) was observed in 2 Cairn Terriers. Fecal staining of the hind limbs was evident in both dogs. A mating of the affected dogs produced a litter of 2 apparently normal pups. The small litter size may have been attributable to lethal gene combinations similar to those described in mice. The radiographic appearance of the caudal and sacral vertebrae was atypical in both tailless dogs. Results of myelography and electrodiagnostic testing of anal sphincter function were normal in one of the dogs. One of the tailless dogs was euthanatized and was found to lack specific muscles and muscle parts responsible for normal defecation, which resulted in fecal staining of the hind limbs. Pedigree analysis revealed the tailless dogs to be closely related and slightly inbred, suggesting a hereditary etiologic factor for anury.

Congenital and acquired neuromuscular disease of young dogs and cats.

Neuromuscular disorders in small animals include a diverse group of congenital and acquired diseases. The prognosis will vary according to the disorder and the portion of the motor unit affected. A number of diseases might be satisfactorily treated (for example, myasthenia gravis, congenital myotonia), whereas others may be self-limiting (for example, hereditary myopathy of Labrador Retrievers). Accurate diagnosis is necessary for establishing a prognosis and treatment plan suitable to the patient and client. Specific diagnosis in the absence of specialized tests is difficult, although not always impossible (for example, congenital myotonia in the Chow Chow). A knowledge of the neuromuscular diseases that might affect small animals, a detailed history, and a thorough physical examination will help in the presumptive diagnosis. Specialized laboratory examinations may need to be applied (for example, antiacetylcholine receptor antibody titer for acquired myasthenia gravis). Referral may be necessary for more detailed diagnostic workup (for example, electromyographic examination, nerve or muscle biopsy examination). In the case of inherited neuromuscular disorders, a knowledge of inheritance patterns will allow genetic counseling to avoid future problem breedings.