Unstable expansion of CAG repeat in hereditary dentatorubral-pallidoluysian atrophy (DRPLA).

Hereditary dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurologic disorder characterized by variable combinations of myoclonus, epilepsy, cerebellar ataxia, choreoathetosis and dementia. By specifically searching published brain cDNA sequences for the presence of CAG repeats we identified unstable expansion of a CAG in a gene on chromosome 12 in all the 22 DRPLA patients examined. A good correlation between the size of the CAG repeat expansion and the ages of disease onset is found in this group. Patients with earlier onset tended to have a phenotype of progressive myoclonus epilepsy and larger expansions. We propose that the wide variety of clinical manifestations of DRPLA can now be explained by the variable unstable expansion of the CAG repeat.

Suppression of aggregate formation and apoptosis by transglutaminase inhibitors in cells expressing truncated DRPLA protein with an expanded polyglutamine stretch.

To elucidate the molecular mechanisms whereby expanded polyglutamine stretches elicit a gain of toxic function, we expressed full-length and truncated DRPLA (dentatorubral-pallidoluysian atrophy) cDNAs with or without expanded CAG repeats in COS-7 cells. We found that truncated DRPLA proteins containing an expanded polyglutamine stretch form filamentous peri- and intranuclear aggregates and undergo apoptosis. The apoptotic cell death was partially suppressed by the transglutaminase inhibitors cystamine and monodansyl cadaverine (but not putrescine), suggesting involvement of a transglutaminase reaction and providing a potential basis for the development of therapeutic measures for CAG-repeat expansion diseases.

Identification of the spinocerebellar ataxia type 2 gene using a direct identification of repeat expansion and cloning technique, DIRECT.

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant, neurodegenerative disorder that affects the cerebellum and other areas of the central nervous system. We have devised a novel strategy, the direct identification of repeat expansion and cloning technique (DIRECT), which allows selective detection of expanded CAG repeats and cloning of the genes involved. By applying DIRECT, we identified an expanded CAG repeat of the gene for SCA2. CAG repeats of normal alleles range in size from 15 to 24 repeat units, while those of SCA2 chromosomes are expanded to 35 to 59 repeat units. The SCA2 cDNA is predicted to code for 1,313 amino acids-with the CAG repeats coding for a polyglutamine tract. DIRECT is a robust strategy for identification of pathologically expanded trinucleotide repeats and will dramatically accelerate the search for causative genes of neuropsychiatric diseases caused by trinucleotide repeat expansions.

Expanded polyglutamine stretches interact with TAFII130, interfering with CREB-dependent transcription.

At least eight inherited neurodegenerative diseases are caused by expanded CAG repeats encoding polyglutamine (polyQ) stretches. Although cytotoxicities of expanded polyQ stretches are implicated, the molecular mechanisms of neurodegeneration remain unclear. We found that expanded polyQ stretches preferentially bind to TAFII130, a coactivator involved in cAMP-responsive element binding protein (CREB)-dependent transcriptional activation, and strongly suppress CREB-dependent transcriptional activation. The suppression of CREB-dependent transcription and the cell death induced by polyQ stretches were restored by the co-expression of TAFII130. Our results indicate that interference of transcription by the binding of TAFII130 with expanded polyQ stretches is involved in the pathogenetic mechanisms underlying neurodegeneration.

Can hypodermal passage cell distribution limit root penetration by mycorrhizal fungi?

* The basis for significant interspecific variability in colonization by arbuscular mycorrhizal fungi is poorly understood. Limited evidence suggests that, for species with a dimorphic hypodermis, colonization of the root cortex occurs only through hypodermal passage cells. Therefore, the hypothesis that interspecific variability in mycorrhizal colonization is accounted for by interspecific variation in passage cell distribution was tested. * The arbuscular mycorrhizal colonization and distribution of fungal penetration points and hypodermal passage cells in the root systems of eight species (seven plant families) possessing a dimorphic hypodermis were characterized. * Mycorrhizal fungal penetration of the hypodermis occurred exclusively through passage cells. Moreover, the proportion of root length with passage cells explained nearly 99% of the variability among the eight plant species in the proportion of root length with penetration points. * In dimorphic hypodermal species, passage cells appear to be key determinants of mycorrhizal colonization because they are the cells through which fungal penetration of the hypodermis occurs. Variation among such species in mycorrhizal colonization may be at least partly determined by variation in the proportion of root length with passage cells.

Trinucleotide repeats in 202 families with ataxia: a small expanded (CAG)n allele at the SCA17 locus.

BACKGROUND: Ten neurodegenerative disorders characterized by spinocerebellar ataxia (SCA) are known to be caused by trinucleotide repeat (TNR) expansions. However, in some instances the molecular diagnosis is considered indeterminate because of the overlap between normal and affected allele ranges. In addition, the mechanism that generates expanded alleles is not completely understood. OBJECTIVE: To examine the clinical and molecular characteristics of a large group of Portuguese and Brazilian families with ataxia to improve knowledge of the molecular diagnosis of SCA. PATIENTS AND METHODS: We have (1) assessed repeat sizes at all known TNR loci implicated in SCA; (2) determined frequency distributions of normal alleles and expansions; and (3) looked at genotype-phenotype correlations in 202 unrelated Portuguese and Brazilian patients with SCA. Molecular analysis of TNR expansions was performed using polymerase chain reaction amplification. RESULTS: Patients from 110 unrelated families with SCA showed TNR expansions at 1 of the loci studied. Dominantly transmitted cases had (CAG)(n) expansions at the Machado-Joseph disease gene (MJD1) (63%), at SCA2 (3%), the gene for dentatorubropallidoluysian atrophy (DRPLA) (2%), SCA6 (1%), or SCA7 (1%) loci, or (CTG)(n) expansions at the SCA8 (2%) gene, whereas (GAA)(n) expansions in the Freidreich ataxia gene (FRDA) were found in 64% of families with recessive ataxia. Isolated patients also had TNR expansions at the MJD1 (6%), SCA8 (6%), or FRDA (8%) genes; in addition, an expanded allele at the TATA-binding protein gene (TBP), with 43 CAGs, was present in a patient with ataxia and mental deterioration. Associations between frequencies of SCA2 and SCA6 and a frequency of large normal alleles were found in Portuguese and Brazilian individuals, respectively. Interestingly, no association between the frequencies of DRPLA and large normal alleles was found in the Portuguese group. CONCLUSIONS: Our results show that (1) a significant number of isolated cases of ataxia are due to TNR expansions; (2) expanded DRPLA alleles in Portuguese families may have evolved from an ancestral haplotype; and (3) small (CAG)(n) expansions at the TBP gene may cause SCA17.

Atrophy of the cerebellum and brainstem in dentatorubral pallidoluysian atrophy. Influence of CAG repeat size on MRI findings.

To elucidate how the size of the expanded CAG repeat of the gene for dentatorubral pallidoluysian atrophy (DRPLA) and other factors affect the atrophy of the brainstem and cerebellum, and the appearance of high-intensity signals on T2-weighted MRI of the cerebral white matter of patients with DRPLA, we quantitatively analyzed the MRI findings of 26 patients with DRPLA, the diagnosis of which was confirmed by molecular analysis of the DRPLA gene. When we classified the patients into two groups based on the size of the expanded CAG repeat of the DRPLA gene (group 1, number of CAG repeat units > or = 66; group 2, number of CAG repeat units < or = 65), we found strong inverse correlations between the age at MRI and the areas of midsagittal structures of the cerebellum and brainstem in group 1 but not in group 2. Multiple regression analysis, however, revealed that both the patient's age at MRI and the size of the expanded CAG repeat correlated with the areas of midsagittal structures. Involvement of the cerebral white matter as detected on T2-weighted images was observed more frequently in patients belonging to group 2 than in group 1 patients. Furthermore it was demonstrated that high-intensity signals can be detected on T2-weighted images of the cerebral white matter of patients with a largely expanded CAG repeat (group 1) in their thirties. These results suggest that patient age as well as the size of the expanded CAG repeat are related to the degree of atrophy of the brainstem and cerebellum, and the white matter changes in patients with DRPLA.

Extraradical hyphae of the mycorrhizal fungus Glomus intraradices can hydrolyse organic phosphate.

Organic phosphorus sources make up a large fraction of the total P in some soils. Vesicular-arbuscular mycorrhizal fungi provide a large surface area for the absorption of inorganic P. The question of whether or not they have direct access to organic P by producing extracellular phosphatases has hitherto been controversial because experiments had not been performed in the absence of other soil microorganisms. We used a split-dish in vitro carrot mycorrhiza system free from contaminating microorganisms. The extraradical hyphae of Glomus intraradices hydrolysed both 5-bromo-4-chloro-3-indolyl phosphate and phenolphthalein diphosphate. Moreover, they transferred significantly more P to roots when they had access to inositol hexaphosphoric acid (phytate) than when they did not. Thus we show unequivocally that extraradical hyphae of G. intraradices can hydrolyse organic P, and, further, that the resultant inorganic P can be taken up and transported to host roots.

Component growth efficiencies of mycorrhizal and nonmycorrhizal plants.

Two mycotrophic species (Lactuca sativa and Abutilon theophrasti) and one nonmycotrophic species (Beta vulgaris) were grown in a P-deficient soil, and the effects of mycorrhizal inoculation on three variables that determine growth rate were assessed for each. The phosphorus-use efficiency (PUE, dW/dP) is the ratio of d. wt increase to P content increase. Plant P is the amount of P (the limiting resource) controlled by the plant, which can be allocated to various purposes. The phosphorus efficiency index (PEI, dP/Pdt) is the efficiency with which plant P is used to acquire P from the soil. Inoculated and control plants of a given species initially contained the same amount of P because all plants were grown from seed. Mycorrhizal colonization significantly increased the PEI of Lactuca and Abutilon (by 23 and 32%, respectively). As expected, mycorrhizal inoculation did not significantly increase the PEI of Beta. As a result, mycorrhizal inoculation significantly increased the P content of Lactuca and Abutilon, but not Beta. Mycorrhizal colonization decreased the PUE of lettuce, but did not significantly affect that of Abutilon or Beta. Mycorrhizal inoculation therefore slightly increased the growth rate of Lactuca, greatly increased the growth rate of Abutilon, and ultimately had no significant effect on the growth rate of Beta.

Interactions between needles of Pinus resinosa and ectomycorrhizal fungi.

Relatively little is known about the factors controlling ectomycorrhizal fungal communities. One possible factor is forest litter chemistry. In a series of experiments we demonstrated that the growth of ectomycorrhizal fungi able to colonize red pine (Pinus resinosa Ait.) are differentially affected by red pine needles and needle chemical components. For example, water extracts of pine needles stimulated the growth of Suillus intermedius (Smith & Thiers) Smith & Thiers and inhibited the growth of Amanita rubescens Pers. Catechin and epicatechin gallate, components of the water extract, acted similarly to the extract. The volatile compounds α- and ß-pinene also had differential effects on the growth of the various species of ectomycorrhizal fungi. Our results suggest that forest litter chemistry has the potential differentially to affect the growth of ectomycorrhizal fungal species and so could affect the structure of ectomycorrhizal fungal communities.

Electron microscopic observation of pituitary adenylate cyclase-activating polypeptide (PACAP)-containing neurons in the rat retina.

The distribution and localization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the rat retina were studied by immunocytochemistry with both light and electron microscopy. PACAP-like immunoreactivity (PACAP-LI) was detected in the amacrine and horizontal cells as well as in the inner plexiform layer, the ganglion cell layer and the nerve fiber layer. PACAP-LI seemed to be concentrated predominantly in the neuronal perikarya and their processes, but not in other cells in the retina. At the ultrastructural level, PACAP-LI was visible in the plasma membranes, rough endoplasmic reticulum, and cytoplasmic matrix in the PACAP-positive neurons in the inner nuclear layer. In the inner plexiform layer, PACAP-positive amacrine cell processes made synaptic contact with immunonegative amacrine cell processes, bipolar cell processes, and ganglion cell terminals. These findings suggest that PACAP may function as a neurotransmitter and/or neuromodulator.

Glutamate-stimulated proliferation of rat retinal pigment epithelial cells.

We investigated the effects of glutamate on cell proliferation and the expression of basic fibroblast growth factor (bFGF) and its receptor (FGF-R1) mRNA in cultured rat retinal pigment epithelial (RPE) cells. The number of primary RPE cells was significantly higher after treatment with 0.2 to 1.0 mM glutamate (maximum at 1.0 mM) for 7 days than in controls. Glutamate-stimulated cell proliferation was abolished by (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), but not by 6,7-dinitroquinoxaline-2,3-dione or L(+)-2-amino-3-phosphonopropionic acid. Proliferation was increased to a similar extent by N-methyl-D-aspartate (NMDA), but not by kainate, alpha-amino-3-hydroxy-3-methyl-4-isoxazolepropionic acid or trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid. NMDA-receptor-like immunoreactivity was detected in most cells cultured. Treatment of cells with glutamate increased the level of bFGF mRNA and, to a lesser extent, that of FGF-R1 mRNA, which peaked 2 and 4 days, respectively, after glutamate was added. The increase in bFGF mRNA induced by glutamate was inhibited by MK-801. These findings suggest that glutamate might stimulate proliferation of RPE cells through activation of NMDA receptors and expression of bFGF and further suggest that glutamate may be involved in the proliferative changes of RPE cells in retinal wound healing.

Distribution and ultrastructural localization of a receptor for pituitary adenylate cyclase activating polypeptide and its mRNA in the rat retina.

Localization and gene expression of pituitary adenylate cyclase activating polypeptide receptor (PACAPR) in the rat retina were studied by immunocytochemistry and in situ hybridization, respectively. Antisera were raised against a synthetic peptide that corresponds to the carboxy-terminal cytoplasmic domain which is found in all subtypes of PACAPR. Strong PACAPR mRNA expression and PACAPR-like immunoreactivity (PACAPR-LI) were detected in ganglion cells, amacrine cells, and in the inner plexiform layer. PACAPR-LI appeared to be concentrated predominantly in the neuronal perikarya and processes. At the ultrastructural level, strong immunostaining for PACAPR was visible in the plasma membranes, rough endoplasmic reticulum and cytoplasmic matrix in neurons. This study provides the basis for a better understanding of the functions of PACAP in the rat retina.

No mutation in the entire coding region of the alpha-synuclein gene in pathologically confirmed cases of multiple system atrophy.

To determine whether mutations in the coding region of the alpha-synuclein gene are relevant in cases of multiple system atrophy (MSA), detailed nucleotide sequence analysis of the alpha-synuclein gene was performed using total RNA obtained from autopsied brain specimens of 11 pathologically confirmed cases of MSA. The brain specimens used in this study contained both gray and white matter, which were dissected from the frontal, temporal or occipital lobe. No nucleotide alterations were found in the entire coding region of the alpha-synuclein gene in any of the cases. While mutations in the regulatory or intronic regions of the gene were not ruled out, our results suggest that mutations in the coding region of the alpha-synuclein gene are unlikely to contribute to the pathogenesis of MSA.

Diplopia after cataract surgery.

PURPOSE: To evaluate the cause of diplopia after cataract surgery. SETTING: Cataract surgery at 7 hospitals and examination of diplopia at a central eye hospital. METHODS: This study comprised 18 eyes of 17 patients with diplopia that developed after cataract surgery in which retrobulbar anesthesia was used. The Hess screen test was done to diagnose oculomotor dysfunction. RESULTS: Several cases showed superior or inferior deviation of the globe, but most patients had nonuniform disturbances of eye movement. Examination of 3 patients by the Hess chart within 1 week after surgery showed paralysis of eye muscles but an overaction at a later stage, evident by reversal of eye position 1 month later. Surgery for strabismus was performed in 6 cases. One case with diplopia improved spontaneously 3 months after cataract surgery and achieved good alignment. CONCLUSIONS: The Hess screen test was useful for comparing changes in oculomotor function before and after surgery. Oculomotor dysfunction after cataract surgery may be caused directly by traumatic injury during administration of anesthesia or surgery using bridle sutures or indirectly from sensitivity to anesthetic agents.

Intraocular lens power calculation for microphthalmos.

PURPOSE: To evaluate the refractive results and accuracy of intraocular lens (IOL) power calculation formulas in eyes with microphthalmos. SETTING: Department of Ophthalmology, Showa University Hospital, Tokyo, Japan. METHODS: The accuracy of IOL power calculated using the SRK, SRK II, S-SRK, SRK/T, Holladay, and Hoffer Q formulas was evaluated in six eyes with axial lengths less than 19.0 mm. RESULTS: Postoperative measurement of refraction showed a tendency toward hypermetropia compared with the refraction predicted by each formula. The best predicted refraction was calculated using the SRK/T formula. The tendency for hyperopic estimation was related to the axial length, particularly in eyes with a shorter axial length. However, there was no relationship between the refractive power of the cornea and the error in the predicted refraction by the SRK/T formula. Two eyes with an IOL power of 30.0 diopters (D) had severe hypermetropia. CONCLUSION: Theoretical formulas were more accurate than empirical ones in eyes with microphthalmos. The severe hypermetropia in the two eyes with a 30.0 D IOL indicates that such patients require a higher IOL power.

Analysis of preoperative factors predictive of visual acuity in axial myopia.

PURPOSE: To identify the factors predicting visual acuity after cataract surgery in patients with high myopia. SETTING: Departments of Ophthalmology, Showa University School of Medicine and Showa University Fujigaoka Hospital, Kanagawa, Japan. METHODS: Stepwise regression analysis was used to identify the factors determining the visual acuity in 940 eyes with an axial length of 27.0 mm or longer having cataract surgery. Using a formula derived from the stepwise regression analysis, the predicted postoperative visual acuity was compared with the actual value measured in another group of 104 eyes. RESULTS: Five factors were identified to significantly determine postoperative visual acuity: axial length, age, corneal opacity, refractive power of the cornea, and history of retinal detachment surgery. There was a significant relationship between predicted and actual postoperative visual acuities (r = .51, P < .001). Postoperative visual acuity was similar in 63% of cases. CONCLUSION: The results showed that at least five factors determine visual acuity after cataract surgery in patients with high myopia.

Gopher mound soil reduces growth and affects ion uptake of two annual grassland species.

Portions of an annual serpentine grassland community in California are subject to frequent gopher mound formation. Consequently, studies were undertaken to characterize the effects of mound soils on plant growth and ion uptake. For two of the dominant annual species (Bromus mollis L. and Plantago erecta Morris), growth was reduced in gopher mound soil relative to that in inter-mound soil. A similar reduction in growth was found for plants grown in soils collected at a depth corresponding to the depth of gopher burrowing. This reduction in growth was associated with lower total P and N contents of the soil which were reflected in lower shoot contents of N and P. Additional experiments, however, showed that reduced N and P availabilities in mound soil were not entirely responsible for the growth reduction. Similarly, shoot Ca/Mg ratios were reduced in mound soil but additions of Ca improved the Ca/Mg ratio without improving growth. Growth reductions were associated with altered shoot concentrations of microelements, particularly elevated levels of Mn. A competition experiment between Plantago and Bromus showed that Bromus was more competitive than Plantago in mound and inter-mound soils and that soil type had only small affects on the nature of the interaction between the two species.

Serial determination of serum neuron-specific enolase in patients with neuroblastoma and other pediatric tumors.

The importance of determination of serum neuron-specific enolase (NSE) in patients with neuroblastoma has been emphasized by several authors. However, the specificity and sensitivity of NSE have not yet been well studied in tumors of infancy and childhood, nor is the role of serial determination of NSE in monitoring these patients fully understood. Concentrations of serum NSE were determined by a newly developed radioimmunoassay technique in 241 samples from 111 patients. NSE was also assayed in sera of nude mice bearing human pediatric tumors (16 samples), as well as in 30 tumor specimens. Eighty-two serum samples from 19 patients with neuroblastoma all showed NSE values (mean 120.2 ng/mL, range 16.2 to 722.0 ng/mL) elevated beyond the upper border of the normal range (14.6 ng/mL), even though four of the 19 patients had normal urinary excretion of 3-methoxy-4-hydroxymandelic acid (VMA) and 3-methoxy-4-hydroxy-phenylacetic acid (HVA). Twelve of these patients were monitored with serial NSE determinations, and their serum NSE were found to correlate well with the tumor burden, but were transiently modified by chemotherapeutically induced cell death. All 68 samples from nine patients, free of neuroblastoma at assessment, showed NSE values within the normal range. Thirteen of 25 patients with tumors other than neuroblastoma, however, showed serum NSE values mildly elevated beyond the upper border of the normal range (mean of the 25 patients 36.7 ng/mL, range 5.0 to 234.0 ng/mL). Results from our nude mouse study and from NSE analysis of the tumor extracts paralleled the clinical results.(ABSTRACT TRUNCATED AT 250 WORDS)

Ocular hypotensive effects of monoamine oxidase-A inhibitors in rabbit.

The effects of monoamine oxidase-A (MAO-A) inhibitors with epinephrine on intraocular pressure in the pigmented rabbit were studied. MAO-A inhibitors were used topically with or without various concentrations of epinephrine. For the measurement of intraocular pressure, applanation pneumatonography was used and tissue MAO activities were determined by radiometric assay. After topical administration with clorgyline, MAO-A activities in the bulbar conjunctiva and the iris-ciliary body were remarkably inhibited, whereas MAO-B inhibition was minimal. Maximal reduction of intraocular pressure with 0.05% epinephrine was 3.2 mmHg. Single administration of clorgyline, amiflamine, moclobemide or CGP 11305-A caused decreases in the intraocular pressure of 2.0, 2.5, 1.8 and 2.4 mmHg, respectively. In the coadministration experiments with epinephrine, the ocular hypotensive effects of epinephrine were potentiated with clorgyline, amiflamine, moclobemide and CGP 11305-A (6.6, 4.8, 5.6 and 5.8 mmHg). On the contrary, they were not influenced by the MAO-B inhibitor deprenyl. These results indicated that MAO-A inhibitors potentiated the ocular hypotensive effects of epinephrine, and that the coadministration of a reversible MAO-A inhibitor with epinephrine might be useful for patients with glaucoma.