Avery H. Weiss

Shotgun identification of protein modifications from protein complexes and lens tissue

Large-scale genomics has enabled proteomics by creating sequence infrastructures that can be used with mass spectrometry data to identify proteins. Although protein sequences can be deduced from nucleotide sequences, posttranslational modifications to proteins, in general, cannot. We describe a process for the analysis of posttranslational modifications that is simple, robust, general, and can be applied to complicated protein mixtures. A protein or protein mixture is digested by using three different enzymes: one that cleaves in a site-specific manner and two others that cleave nonspecifically. The mixture of peptides is separated by multidimensional liquid chromatography and analyzed by a tandem mass spectrometer. This approach has been applied to modification analyses of proteins in a simple protein mixture, Cdc2p protein complexes isolated through the use of an affinity tag, and lens tissue from a patient with congenital cataracts. Phosphorylation sites have been detected with known stoichiometry of as low as 10%. Eighteen sites of four different types of modification have been detected on three of the five proteins in a simple mixture, three of which were previously unreported. Three proteins from Cdc2p isolated complexes yielded eight sites containing three different types of modifications. In the lens tissue, 270 proteins were identified, and 11 different crystallins were found to contain a total of 73 sites of modification. Modifications identified in the crystallin proteins included Ser, Thr, and Tyr phosphorylation, Arg and Lys methylation, Lys acetylation, and Met, Tyr, and Trp oxidations. The method presented will be useful in discovering co- and posttranslational modifications of proteins.

Traumatic Retinoschisis in Battered Babies

Five infants who were victims of physical abuse had extensive bilateral retinal hemorrhages on initial evaluation and subsequently developed signs of permanent retinal damage. None showed external evidence of trauma to the eyes. Vitreous hemorrhage developed after a delay of several days or more in three cases that were followed closely from the time of the traumatic incident. In several eyes, apparent intraretinal blood-filled cavities were seen acutely in the macular region and elsewhere. Late scarring of the macula typically had a cystic or crater-like configuration. Electroretinography showed loss or reduction of the positive B-wave with preservation of the negative A-wave in every case. We propose that splitting of the retina resulting from the direct mechanical effects of violent shaking was responsible for all of these findings.

Acute conjunctivitis in childhood

We obtained specimens for culture from the lids and conjunctivae of 95 patients with acute conjunctivitis and 91 control children of similar age and, in addition, stained the conjunctival scrapings with Giemsa and Gram stains. The conjunctivitis was attributed to bacterial infection in 76 patients, viral infection in 12 children, and allergy in 2 patients; no cause was identified in the remaining 5 patients. In most cases the etiologic diagnosis was based on the results of laboratory studies. By separately culturing microorganisms in specimens from the lids and conjunctivae of patients and control subjects, we could distinguish normal flora from pathogens, and blepharitis from conjunctivitis. Staphylococci, corynebacteria, and alpha-hemolytic streptococci were the predominant organisms recovered from the lids of control subjects. In contrast, Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis were the major pathogens cultured from the conjunctival specimens from patients with bacterial conjunctivitis. Gram stains of conjunctival scrapings provided a rapid means of predicting the pathogen in 51 of 55 cases of bacterial conjunctivitis. Giemsa stains of conjunctival scrapings provided etiologic information in 81 of 84 cases, showing neutrophilia in bacterial infections, lymphocytosis in viral infections, and eosinophilia in allergic disease. These results indicate that most cases of acute conjunctivitis in children can be diagnosed on the basis of differential cultures of microorganisms from the lid and conjunctiva, together with Giemsa stains of conjunctival scrapings.

Methotrexate for resistant chronic uveitis in children with juvenile rheumatoid arthritis

We used low-dose methotrexate to treat seven children with juvenile rheumatoid arthritis-associated uveitis complicated by cataract and glaucoma or resistant to topical corticosteroid. The use of methotrexate decreased the severity of uveitis in six of seven patients and allowed for the discontinuation or reduction of corticosteroid drops.

Bacterial periorbital and orbital cellulitis in childhood.

The clinical features, microbiologic data, complications, and treatment in 137 children with periorbital cellulitis and 21 children with orbital cellulitis is presented. Periorbital cellulitis was more frequent (87%) than orbital cellulitis (13%). Periorbital cellulitis is a heterogeneous disease that may complicate trauma of the eyelids, external ocular infection, and upper respiratory infection. Children with periorbital cellulitis related to trauma or external infection tended to be less than 5 years old with negative blood cultures (99%) and positive cultures of percutaneous aspirates (42%); while children with periorbital cellulitis related to upper respiratory infection also tended to be less than 5 years of age, but blood cultures were frequently positive (42%) and cultures of percutaneous aspirates were usually negative (92%). Three children in the latter group developed meningitis. Intravenous antibiotic alone was effective treatment in most patients (90%). Orbital cellulitis was more frequent in children older than 5 years and frequently associated with sinusitis (90%). Blood and skin cultures were usually negative. Intravenous antibiotics alone were effective management in many patients (62%), but a significant proportion required paranasal sinus or orbital surgery (38%).

Visual Sensory Disorders in Congenital Nystagmus

Congenital nystagmus (CN) is a common disorder indicative of a primary disturbance of the ocular motor or visual sensory systems. The authors prospectively evaluated 81 patients with CN, structurally normal eyes, and minimal or no abnormalities of the optic nerve, macula, and retinal pigment epithelium (RPE). Seventy-four (91%) patients were found to have a disorder of the visual sensory system. Thirty-four patients had albinism, 37 had a congenital or early onset disorder of the retinal photoreceptors, and 3 had abnormalities of the optic nerve. The remaining seven (9%) patients had motor CN. Most patients presenting with CN have visual loss and should be evaluated for an underlying disorder of the visual sensory system.

Adrenal Suppression After Corticosteroid Injection of Periocular Hemangiomas

Two infants with symptomatic periocular hemangiomas received 1-ml intralesional injections of a 50:50 mixture of triamcinolone acetonide (40 mg/ml) and betamethasone (8 mg/ml). Both developed adrenal suppression as evidenced by the immediate depression of their serum cortisol and adrenocorticotrophic hormone levels. One patient remained adrenal suppressed for five months and, concurrently, her growth rate and weight dropped from normal to below the fifth percentile. Adrenal suppression should be added to the growing list of complications caused by the corticosteroid injection of periocular hemangiomas.

Development of the spatio-chromatic visual evoked potential (VEP): a longitudinal study

Most prior visual evoked potential (VEP) research on the development of color vision has employed pattern-reversing stimuli that are not optimal for producing chromatic responses. We measured infant VEPs using low spatial frequency, onset-offset stimuli, modulated along the three axes of a cone-based color space (Derrington et al. [J. Physiol 1984;357, 241-265.]). Three color-normal infants were tested in a longitudinal design over the first postnatal year. One red/green color-deficient infant was also tested at 197 days. We found that VEP responses to S-axis (tritan) stimuli have their initial onset later than responses to red/green (L-M) or achromatic stimuli, and that developmental changes in VEP waveforms are more complex and longer lasting for chromatic than for achromatic stimuli. Possible mechanisms underlying these changes are discussed.

Development of pattern visual evoked potentials: longitudinal measurements in human infants.

Purpose. This experiment used longitudinal testing to trace the emergence of the major components of pattern visual evoked potentials (VEPs) in infants, using two paradigms: large-checkerboard pattern reversal and low spatial frequency pattern onset. Methods. Testing with both pattern-reversal and pattern-onset stimuli was performed on the same infants. Testing was conducted at weekly intervals during the first three postnatal months, and at intervals of 2 weeks to 1 month thereafter. Results. The pattern-reversal and early pattern-onset responses recorded within individual subjects showed remarkably systematic developmental sequences. The broad, positive component seen at 200 to 250 ms in infants could be traced readily through the developmental sequence, to become the more sharply tuned positive component seen at about 100 ms in adults. Responses to low spatial frequency pattern onsets in infants were larger and more reliable than those in adults. The late components of the pattern-onset response, generally attributed to pattern offset, emerged later and with more complex changes. In all cases, response amplitude was much more variable than response latency, both within and between subjects. Conclusions. Frequent VEP recording in a longitudinal design can reveal systematic and detailed transitions of wave-form during development.

Longitudinal measures of visual function, tumor volume, and prediction of visual outcomes after treatment of optic pathway gliomas.

PURPOSE To examine longitudinal changes in visual acuity, tumor volume, and visual evoked potentials (VEP) before and after treatment in children with optic pathway gliomas. DESIGN Retrospective cohort study. PARTICIPANTS Twenty-one patients (0.7-9 years of age). METHODS Patients initially were treated either by chemotherapy (n = 18) or radiotherapy (n = 3). Patients were followed up with serial magnetic resonance imaging, age-corrected visual acuity measurements in logarithm of the minimum angle of resolution (logMAR) units, and pattern VEP. Longitudinal visual outcome data were obtained on average for 9 years (range, 4-16 years). Tumor volumes before and after treatment were estimated in 15 patients. Multivariate regression was used to predict visual outcomes. MAIN OUTCOME MEASURES Visual acuity, relative tumor volumes, and VEP. RESULTS Before treatment, 81% of patients had reduced visual acuity and 81% had optic nerve pallor, whereas all had a reduced VEP in 1 or both eyes. After initial treatment, tumor volume decreased in 53%, stabilized in 27%, and progressively increased in 20%. Treatment arrested the rapid decline in visual acuity loss and stabilized visual acuity for 4 to 5 years. The rate of visual acuity decline was not correlated with tumor shrinkage. Sixty-two percent of patients required additional treatment with either chemotherapy or radiation because of tumor growth or progressive loss of visual function. Visual acuity at last examination was stable or improved in 33% of patients, but on average declined 0.4 logMAR units. Visual acuity was 20/200 or better in 1 eye of 62% of patients. The rate of visual acuity decline was predicted weakly by tumor volume at presentation (R(2) = 0.19; P<0.009). Visual acuity at last examination was predicted best by visual acuity and tumor volume at presentation (R(2) = 0.66; P<0.001). CONCLUSIONS Systemic chemotherapy arrested the decline in visual acuity and stabilized vision on average for 5 years. At presentation, VEPs were a more sensitive indicator of optic pathway damage than visual acuity or optic nerve appearance. Although tumor reduction or stabilization was achieved in 80% of patients, pre-existing visual damage, indexed by objective measures of tumor volume and visual function, limited visual outcomes. FINANCIAL DISCLOSURE(S) The author(s) have no proprietary or commercial interest in any materials discussed in this article.