Sequence analysis of a cDNA for lysyl hydroxylase isolated from human skin fibroblasts from a normal donor: differences from human placental lysyl hydroxylase cDNA.

Using polymerase chain reaction, we have isolated and sequenced a 3-kb cDNA for lysyl hydroxylase (LH) from human skin fibroblasts from an normal donor. Apart from two polymorphic sites, no differences were observed between the 2184 nt coding regions of LH cDNA from fibroblasts and placenta. However, four differences were observed in the 3' non-coding regions of the two cDNAs; three were single base changes and the fourth a deletion of a single base. The absence of the single nucleotide in the LH cDNA from fibroblasts resulted in the loss of an HpaII site that is present in the placental LH cDNA; this was confirmed in HpaII digests of fibroblast and placental LH cDNAs from the same donor. Northern blots showed that the LH gene was strongly expressed in fibroblasts and placenta and, to a lesser extent, in aorta, lung, vein, cartilage, and artery.

The expression of a functional, secreted human lysyl hydroxylase in a baculovirus system.

This study reports the expression of functional human lysyl hydroxylase (LH), a post-translational modifying enzyme that catalyzes the hydroxylation of the lysine residues essential for cross-linking in collagen biosynthesis. We have developed a novel baculovirus system for the expression of LH, a protein that exists normally within the lumen of the endoplasmic reticulum, using a powerful baculovirus signal sequence for secretion. The supernatant from Sf9 cells infected with the viral recombinant showed significant LH activity that increased linearly with supernatant concentration, whereas there was no detectable LH activity in the cell pellet. Silver staining of the fractions purified from the active supernatant by concanavalin A Sepharose chromatography and separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis demonstrated an 85-kDa protein (the expected size of the LH subunit) that was most prominent in those fractions with the highest LH activity. N-terminal amino acid sequencing verified that the N-terminal primary structure of this 85-kDa protein was identical to human LH. Moreover, the activity of the expressed protein was shown to be dependent on the presence of Fe++, ascorbate, and alpha-ketoglutarate, three essential cofactors for LH activity. We have therefore successfully developed a novel expression system that produces functional human LH and enables this normally nonsecretory enzyme to be secreted, facilitating its separation from the intracellular proteins of insect cells. Future applications should allow characterization of the LH active site by crystallographic studies and site-directed mutagenesis for structure-function comparison.

Primary intraocular posttransplantation lymphoproliferative disorder.

We report a case of posttransplantation lymphoproliferative disorder manifesting as an isolated, unilateral iris tumor. A 2-year-old boy who had undergone liver transplantation for biliary atresia at age 4 months was seen with a 2-month history of an enlarging iris nodule. Histopathologic examination of the iris lesion demonstrated a mixed population of lymphoid cells. To our knowledge, this is the youngest patient with posttransplantation lymphoproliferative disorder isolated to the eye.

Back impairment and disability determination. Another attempt at objective, reliable rating.

Present disability evaluation schedules for the low back are not scientifically based and produce very great interexaminer differences. The authors have developed a new impairment schedule based on a comprehensive review of the medical literature and the collected opinions of a large number of back specialists. Tests of the new schedule show a marked decrease in interexaminer differences compared to the prior California disability rating schedule. The impairment schedule can be readily adapted to any legal system of disability rating and can be modified easily to reflect new medical knowledge. The result should be a disability rating which is more objective, more scientifically valid, and more consistent, reducing litigation, with fairness to both the low-back impaired worker and the employer.

A time-lapse, image digitization videomicroscope system based on a mini computer with large peripheral memory.

We describe a time-lapse image digitization videomicroscope system that uses a mini computer as the main processing unit in conjunction with a large peripheral memory for storing multiple digitized images. The advantage of the system lies in its ability to facilitate acquisition and analysis of data relative to size, location and optical density of cells and cellular structure using elementary image processing techniques. This system can also potentially control microscope stage movement, enabling acquisition of larger amounts of data per time-lapse experiment.

Nerve tension and blood flow in a rat model of immediate and delayed repairs.

In vivo studies of rat sciatic nerves in models of immediate and delayed repairs demonstrated the viscoelastic properties of the nerve and the inverse correlation between nerve blood flow and tension. In both the proximal and distal segments of the divided nerve in models of immediate and delayed repairs, the nerve blood flow decreased approximately 50% with substantial recovery in 30 minutes after 8% elongation, whereas 15% elongation produced approximately an 80% reduction in blood flow with minimal recovery. However, the baseline blood flow of the nerves in the delayed-repair model was nearly two times higher than that of the acutely injured nerves. Maximal decrease in nerve tension and corresponding increase in blood flow occurred within the first 20 minutes after elongation. The suture pull-out with failure of the repairs occurred at more than 15% elongation for all nerves. The previously divided nerves had a sixfold greater decrease in length than the acutely divided nerves (p less than 0.02). For repairs of large nerves where vascular ingrowth is likely to be incomplete, elongation of more than 8% may cause ischemia that is detrimental to nerve regeneration. Mechanical failure of the repairs occurs after elongation of 16% to 17%. The combination of nerve ischemia and mechanical failure of suture repairs suggests that surgeons should be careful to limit the use of elongation in acute and delayed repairs.

Treatment strategies and visual acuity outcomes in chronic postoperative Propionibacterium acnes endophthalmitis.

PURPOSE: To report the treatment strategies and visual acuity outcomes of chronic postoperative endophthalmitis caused by Propionibacterium acnes. DESIGN: Retrospective noncomparative case series. PARTICIPANTS: All patients presenting 8 or more weeks after cataract surgery with intraocular inflammation caused by culture-proven P. acnes infection and treated at two institutions from 1974 through 1996 were included. METHODS: Patients underwent three different initial treatment strategies. The study did not have a defined treatment protocol, but all patients received intraocular antibiotics. Patients were not randomly assigned to the various treatment strategies. MAIN OUTCOME MEASURES: Final visual acuity and effectiveness of various treatment procedures either as initial or follow-up therapy were assessed. RESULTS: Using the 3 initial strategies, 36 patients were treated: (1) intraocular antibiotic injection alone (IOAB; n = 12); (2) pars plana vitrectomy and IOAB injection (PPV; n = 10); and (3) PPV with subtotal capsulectomy and IOAB injection (PPV-PC; n = 14). The number of patients with recurrent or persistent inflammation after one of the three initial treatment strategies were as follows: (1) IOAB alone, 12 (100%); (2) PPV, 5 (50%); and (3) PPV-PC, 2 (14%). None of the patients that underwent subsequent PPV, total capsular bag removal, IOAB injection, and either intraocular lens (IOL) exchange or removal had persistent or recurrent intraocular inflammation. Overall, final visual acuity was 20/40 or better in 18 patients (50%), and a total of 28 patients (78%) retained 20/400 or better vision. The mean follow-up after the last treatment was 2.9 years. CONCLUSIONS: In this series of chronic P. acnes endophthalmitis, initial treatment with IOAB injection alone or vitrectomy without capsulectomy was associated with high rates of recurrent or persistent intraocular inflammation. Pars plana vitrectomy, partial capsulectomy, and IOAB injection without IOL exchange was usually successful on long-term follow-up. In patients with recurrent intraocular inflammation, pars plana vitrectomy, total capsular bag removal, IOAB injection, and IOL exchange or removal was a uniformly successful strategy. In contrast to other types of postoperative endophthalmitis, IOL exchange can be considered in these patients after total capsular bag removal.

Suggested guidelines for rating cardiac disability in workers' compensation. Medical and Chiropractic Advisory Committee to the Administrative Director of the California Division of Industrial Accidents.

Cardiac disability ratings in workers' compensation cases currently lack any consistent scientific basis, with varying medical evidence used by different examiners in the same case. Opinions about the extent of disability may differ with the same patient, delaying resolution and the delivery of benefits. We describe guidelines for determining cardiac impairment and suggest a schedule for rating disability based on evidence. Our experience is in California, but arriving at equitable ratings for disability purposes is a nationwide challenge. Exercise stress testing provides the best reproducible data to test the heart's ability to do work. When exercise stress testing is not possible or adequate, alternative or supplemental testing is necessary. Certain conditions, such as hypertension, arrhythmias, coronary artery spasm, and a history of coronary artery operations or myocardial infarction, may affect "cardiac disability" but may not necessarily be reflected in exercise testing.