RESUMO
alpha-Syntrophin is a scaffolding adapter protein expressed primarily on the sarcolemma of skeletal muscle. The COOH-terminal half of alpha-syntrophin binds to dystrophin and related proteins, leaving the PSD-95, discs-large, ZO-1 (PDZ) domain free to recruit other proteins to the dystrophin complex. We investigated the function of the PDZ domain of alpha-syntrophin in vivo by generating transgenic mouse lines expressing full-length alpha-syntrophin or a mutated alpha-syntrophin lacking the PDZ domain (Delta PDZ). The Delta PDZ alpha-syntrophin displaced endogenous alpha- and beta 1-syntrophin from the sarcolemma and resulted in sarcolemma containing little or no syntrophin PDZ domain. As a consequence, neuronal nitric oxide synthase (nNOS) and aquaporin-4 were absent from the sarcolemma. However, the sarcolemmal expression and distribution of muscle sodium channels, which bind the alpha-syntrophin PDZ domain in vitro, were not altered. Both transgenic mouse lines were bred with an alpha-syntrophin-null mouse which lacks sarcolemmal nNOS and aquaporin-4. The full-length alpha-syntrophin, not the Delta PDZ form, reestablished nNOS and aquaporin-4 at the sarcolemma of these mice. Genetic crosses with the mdx mouse showed that neither transgenic syntrophin could associate with the sarcolemma in the absence of dystrophin. Together, these data show that the sarcolemmal localization of nNOS and aquaporin-4 in vivo depends on the presence of a dystrophin-bound alpha-syntrophin PDZ domain.
Assuntos
Aquaporinas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Óxido Nítrico Sintase/metabolismo , Sarcolema/metabolismo , Animais , Aquaporina 4 , Proteínas de Ligação ao Cálcio/metabolismo , Distrofina/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Proteínas Musculares/química , Proteínas Musculares/genética , Músculo Esquelético/química , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Óxido Nítrico Sintase Tipo I , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Sarcolema/enzimologia , Canais de Sódio/metabolismoRESUMO
Seven rhesus monkeys (14 eyes) were exposed to 1064-nanometer radiation in single pulses of 25 to 35 picoseconds fromn a mode-locked Nd: YA G laser. Threshold injury resulted from single pulses with a mnean energy of 13 +/- 3 mnicrojoules. Electron microscopy of the retina revealed that damnage was highly localized in the photoreceptor and pigmented epithelial cells at the oluter retina. Membrane disruption, distorted outer segmtzents, and abnormnal melanin granules resembling fetal premelanosomnies were observed.
Assuntos
Lasers , Retina/lesões , Animais , Retículo Endoplasmático , Traumatismos Oculares/patologia , Lisossomos , Macaca , Melaninas , Microscopia Eletrônica , Mitocôndrias , Retina/patologiaRESUMO
BACKGROUND: Fumarylacetoacetate hydrolase (FAH) catalyzes the final step of tyrosine and phenylalanine catabolism, the hydrolytic cleavage of a carbon-carbon bond in fumarylacetoacetate, to yield fumarate and acetoacetate. FAH has no known sequence homologs and functions by an unknown mechanism. Carbon-carbon hydrolysis reactions are essential for the human metabolism of aromatic amino acids. FAH deficiency causes the fatal metabolic disease hereditary tyrosinemia type I. Carbon-carbon bond hydrolysis is also important in the microbial metabolism of aromatic compounds as part of the global carbon cycle. RESULTS: The FAH crystal structure has been determined by rapid, automated analysis of multiwavelength anomalous diffraction data. The FAH polypeptide folds into a 120-residue N-terminal domain and a 300-residue C-terminal domain. The C-terminal domain defines an unusual beta-strand topology and a novel 'mixed beta-sandwich roll' structure. The structure of FAH complexed with its physiological products was also determined. This structure reveals fumarate binding near the entrance to the active site and acetoacetate binding to an octahedrally coordinated calcium ion located in close proximity to a Glu-His dyad. CONCLUSIONS: FAH represents the first structure of a hydrolase that acts specifically on carbon-carbon bonds. FAH also defines a new class of metalloenzymes characterized by a unique alpha/beta fold. A mechanism involving a Glu-His-water catalytic triad is suggested based on structural observations, sequence conservation and mutational analysis. The histidine imidazole group is proposed to function as a general base. The Ca(2+) is proposed to function in binding substrate, activating the nucleophile and stabilizing a carbanion leaving group. An oxyanion hole formed from sidechains is proposed to stabilize a tetrahedral alkoxide transition state. The proton transferred to the carbanion leaving group is proposed to originate from a lysine sidechain. The results also reveal the molecular basis for mutations causing the hereditary tyrosinemia type 1.
Assuntos
Carbono/química , Hidrolases/química , Aminoácidos/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Dimerização , Histidina , Humanos , Hidrolases/metabolismo , Hidrólise , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , ÁguaRESUMO
The photopathology of retinal lesions produced by extended exposure (1000 sec) to low corneal power levels (62 microW) of blue light (441 nm) was investigated by light microscopy in 20 rhesus eyes over an interval ranging from 1 hr to 90 days after exposure. Results indicate a nonthermal type of photochemical lesion originating in the retinal pigment epithelium and leading to a histological response with hypopigmentation which requires 48 hr to appear. This type of lesion helps to explain solar retinitis and eclipse blindness and has significance for aging and degenerative changes in the retina.
Assuntos
Oftalmopatias/patologia , Luz/efeitos adversos , Doenças Retinianas/patologia , Animais , Cegueira/etiologia , Oftalmopatias/etiologia , Haplorrinos , Macaca mulatta , Fotoquímica , Epitélio Pigmentado Ocular/patologia , Doenças Retinianas/etiologia , Retinite/etiologia , Luz SolarRESUMO
Under conditions of nonthermal radiant exposure to blue light (440 nm) the primate retina can suffer photic injury by a mechanism that must be photochemical in nature. We have examined the effects of elevated blood oxygen (pO2 of 270 mmHg) on the retinal photosensitivity to blue light in two macaque monkeys by histologic analysis of 12 lesions at 1 to 57 days after irradiation. The retinal image diameter from a xenon arc lamp source was 1 mm, the duration of exposure was 100 sec, and the radiant exposures ranged from 11 to 36 J/cm2. When blood oxygenation is not elevated experimentally, the threshold radiant exposure for a blue light lesion to be visible funduscopically at 2 days postexposure is about 30 J/cm2. At a high blood pO2 level, a radiant exposure of only 11 J/cm2 gave a funduscopically visible lesion at 1-day postexposure. This large increase in retinal sensitivity to blue light damage appears to be due to photodynamic action. The only direct effect of elevated blood pO2 on the retina observed histologically was the presence of numerous granules in the cells of the retinal pigment epithelium (RPE). However, there was no apparent histopathology associated with the elevation of blood pO2 alone. Analysis of the various photic lesions showed only moderate damage to the neural retina, but a strong response was seen in the RPE. This is the histopathologic pattern of a typical blue light lesion shown in previous studies but more severe. So the effect of elevated blood O2 is to increase retinal sensitivity to photic damage, to lower the damage threshold, and to increase the severity of damage at a given radiant exposure. The status of lesions at 23 and 57 days postexposure suggests that such injuries are repairable.
Assuntos
Oxigênio/sangue , Retina/fisiopatologia , Animais , Luz , Macaca , Epitélio Pigmentado Ocular/fisiopatologia , Doenças Retinianas/fisiopatologiaRESUMO
We found that the action spectrum for retinal damage (determined by the fundus photographic appearance of a minimal lesion immediately after exposure) extends into the near-ultraviolet by exposing three aphakic eyes from rhesus monkeys to 405-, 380-, 350-, and 320-nm wavelengths produced by a 2,500-W xenon lamp equipped with quartz optics and 10-nm interference filters. Exposure times were 100 and 1,000 seconds and the spot diameter on the retina was 500 micrometers. The retina was six times more sensitive to 350- and 325-nm wavelengths than to blue light (441 nm). Both ophthalmoscopic and histologic data showed that near-ultraviolet lesions differed in important respects from blue-light lesions. Near-ultraviolet produced irreparable damage to rod and cone photoreceptors.
Assuntos
Afacia Pós-Catarata/complicações , Doses de Radiação , Retina/efeitos da radiação , Animais , Macaca mulatta , Lesões Experimentais por Radiação/complicações , Lesões Experimentais por Radiação/patologia , Retina/patologiaRESUMO
Extended exposure (100s) of the macaque retina to blue light (400-500nm) produces a photochemical type or types of lesion. The basic mechanisms responsible for such photic damage are unknown but the toxic combination of light and oxygen leading to the free radicals O-.2, H2O2, OH., and O2(1 delta) have been suggested as a possible source of the phototoxicity. To test this hypothesis, the radiant exposure (J. cm-2) to short wavelength light (435-445nm) required for minimal damage in the macaque retina is under investigation as a function of oxygenation and after administration of substances known to either inhibit/scavenge radicals or act as anti-inflammatory/anti-oxidant agents. Substances under study include beta-carotene, steroids, catalase and SOD. Here we report radiant exposure in J.cm-2 needed to produce a minimal lesion vs oxygenation as measured by partial pressure of O2 in arterial blood (Po2). There is a sharp drop in the radiant exposure threshold with increase in the partial pressure of O2 in arterial blood, e.g. 30 J.cm-2 at 75 torr to 10 J.cm-2 at 271 torr, a factor of 3. Methylprednisolone injected intravenously one hour before exposure (125 mg) has been shown to raise the threshold for retinal damage in two macaques by a factor of approximately 2. Another animal fed beta-carotene (7.5 mg daily) over a period of 3 months has been exposed to blue light at several levels of oxygenation. The results suggest a protective effect.
Assuntos
Retina/efeitos da radiação , Animais , Carotenoides/farmacologia , Radicais Livres , Luz/efeitos adversos , Macaca , Metilprednisolona/farmacologia , Oxigênio/farmacologia , Oxigênio/efeitos da radiação , Fotoquímica , Retina/efeitos dos fármacos , Retina/metabolismo , beta CarotenoRESUMO
Threshold damage in the macaque retina is shown to be equivalent for the argon-krypton (Ar-Kr) 647 nm and the helium-neon (He-Ne) 632.8-nm lines for exposures to continuous wave (CW) radiation from 1 to 1,000 s. This equivalence allows interpolation from experiments with 647-nm, exposures at power levels that are unavailable with the He-Ne laser. To simulate He-Ne laser scanner exposures, 40-microseconds pulses of 647-nm light transmitted through a revolving disk with holes in the periphery were used to expose the retinas of monkeys under deep anesthesia at pulse repetition frequencies (PRFs) of 100, 200, 400, and 1,600 Hz for exposure durations of 1, 10, 100, and 1,000 s. The thresholds between laser exposure at 488 nm (Ar-Kr) and between laser exposure at 647 nm (Kr) are compared to assess thermal versus photochemical effects on the retina. The threshold for 488-nm pulses was consistently lower than that for 647-nm pulses at all PRFs and exposure times. The difference in thresholds increased with exposure time and PRF. The sharp decreases in 488-nm thresholds at 100-s exposure times for each PRF can be interpreted as a basically photochemical effect. The radiant exposure required for damage at 647 nm was several orders of magnitude above the radiant exposure from typical He-Ne scanner applications. From the similarity of the macaque retina to the human retina, it is concluded that no realistic ocular hazard exists from exposure to scanning laser systems of 1 mW or less, operating at higher than 100 Hz.
Assuntos
Lasers/efeitos adversos , Retina/lesões , Animais , Humanos , MacacaAssuntos
Queimaduras Oculares/etiologia , Lasers/efeitos adversos , Retina , Animais , Córnea , Haplorrinos , Hélio , NeônioAssuntos
Luz , Retina/efeitos da radiação , Animais , Haplorrinos , Lasers , Análise Espectral , TemperaturaAssuntos
Carcinoma Broncogênico/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Fotorradiação com Hematoporfirina , Hematoporfirinas/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Fotoquimioterapia , Idoso , Carcinoma Broncogênico/mortalidade , Carcinoma Broncogênico/radioterapia , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Terapia Combinada , Éter de Diematoporfirina , Feminino , Fotorradiação com Hematoporfirina/efeitos adversos , Humanos , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/radioterapia , Masculino , Pessoa de Meia-Idade , Cuidados Paliativos , Fotoquimioterapia/efeitos adversos , Distribuição AleatóriaRESUMO
An apparatus has been designed and built which simultaneously measures the energy incident on a biological system which is being exposed to a laser beam. The advantages of the direct reading system are: (1) no attention is required of the laser operator, (2) a permanent record is produced, (3) true integration of the pulse train is accomplished, (4) high inherent accuracy, (5) in conjunction with a fast oscilloscope, it reproduces pulse waveforms with minimum distortion, and (6) calibration is simple and direct. Basically the system employs a fast high-current capability photodiode, a high-quality integrating capacitor, an emission-limited pump diode with a servo-motor amplifier combination for dark current balance. The photodiode cathode surface is S-1 so that both ruby and neodymium wavelengths 694.3 mmicro and 1060 mmicro, respectively, may be measured. Reproducibility is better than 3% when compared with a blackbody receiver (cone radiometer). A Tektronix 585A oscilloscope and P-80 cathode follower probe fitted with a 50-Omega load resistor can be plugged into the photodiode housing in place of the integrating capacitor for waveform observation and power-level measurements. Calibration on an absolute basis is easily accomplished by simultaneous comparison with a calibrated blackbody receiver (cone radiometer).
RESUMO
This is a case report of a 13-year-old boy with an osteoid-osteoma located in the pedicle of the third lumbar vertebra. The lesion could not be visualized on routine roentgenograms but with the use of technetium polyphosphate bone scan as a guide a suggestive bone lesion was found with tomograms. Histologic examination confirmed the diagnosis of osteoid-osteoma. This case illustrates the value of skeletal scanning in occult bone lesions.