Protein oxidative damage in a transgenic mouse model of familial amyotrophic lateral sclerosis

The Gly93-->Ala mutation in the Cu,Zn superoxide dismutase (Cu,Zn-SOD) gene (SOD1) found in some familial amyotrophic lateral sclerosis (FALS) patients has been shown to result in an aberrant increase in hydroxyl radical production by the mutant enzyme that may cause oxidative injury to spinal motor neurons. In the present study, we analyzed the extent of oxidative injury to lumbar and cervical spinal cord proteins in transgenic FALS mice that overexpress the SOD1 mutation [TgN(SOD1-G93A)G1H] in comparison with nontransgenic mice. Total protein oxidation was examined by spectrophotometric measurement of tissue protein carbonyl content by the dinitrophenylhydrazine (DNPH) assay. Four ages were investigated: 30 (pre-motor neuron pathology and clinical disease), 60 (after initiation of pathology, but pre-disease), 100 (approximately 50% loss of motor neurons and function), and 120 (near complete hindlimb paralysis) days. Protein carbonyl content in 30-day-old TgN(SOD1-G93A)G1H mice was twice as high as the level found in age-matched nontransgenic mice. However, at 60 and 100 days of age, the levels were the same. Then, between 100 and 120 days of age, the levels in the TgN(SOD1-G93A)G1H mice increased dramatically (557%) compared with either the nontransgenic mice or transgenic animals that overexpress the wild-type human Cu,Zn-SOD [TgN(SOD1)N29]. The 100-120-day increase in spinal cord protein carbonyl levels was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoretic separation and western blot immunoassay, which enabled the identification of heavily oxidized individual proteins using a monoclonal antibody against DNPH-derivatized proteins. One of the more heavily oxidized protein bands (14 kDa) was identified by immunoprecipitation as largely Cu,Zn-SOD. Western blot comparison of the extent of Cu,Zn-SOD protein carbonylation revealed that the level in spinal cord samples from 120-day-old TgN(SOD1-G93A)G1H mice was significantly higher than that found in age-matched nontransgenic or TgN(SOD1)N29 mice. These results suggest that the increased hydroxyl radical production associated with the G93A SOD1 mutation and/or lipid peroxidation-derived radical species (peroxyl or alkoxyl) causes extensive protein oxidative injury and that the Cu,Zn-SOD itself is a key target, which may compromise its antioxidant function.     

Cataracts in cichlid fish

Bilateral cataracts were detected in a group of aquarium-raised tilapia (Sarotherodon mossambicus). Vision impairment was manifested as random swimming into objects, but the fish appeared to function normally otherwise. Most of the lens was opaque, with the outer extent of the cortex and subcapsular region remaining transparent. Skeletal abnormalities, ie, deformed fins, abnormal jaws, and curvature of the spine, were observed in affected individuals. Histologically, there was progressive degeneration of the cataractous portion as the affected fish matured. Preliminary breeding experiments indicated that the condition may be inherited.     

Ambulatory urodynamics

Ambulatory monitoring of lower and upper urinary tract continues to develop. The addition of electronic urine-loss measurements to indicate the exact time-related loss is an important new feature. In the near future, quantitative urine-loss measurements will become available. The additional possibility of plugging in a flowmeter completes the technique with respect to pressure-flow analysis. At present, however, ambulatory urodynamics is still confined to specialized urodynamic centers. Ambulatory urodynamic monitoring has shown that de-novo detrusor instability after a suspension operation is frequently missed as a preoperative diagnosis of detrusor overactivity. The first steps are being taken toward giving a quantitative analysis of detrusor activity during the filling phase. This justifies a more widespread use of this ambulatory monitoring.     

Patient compliance in mobile screening mammography

RATIONALE AND OBJECTIVES: We assessed the follow-up behavior of women who had abnormal results of screening mammograms taken on a mobile van. METHODS: A retrospective cohort study was conducted between 1988 and 1991 of all women served by a mobile mammography van in rural North Carolina. Compliance with radiologist recommendations for follow-up was assessed through review of patient records and mail surveys of patients with incomplete records. RESULTS: Compliance was 44% for negative or benign mammograms, 57% for indeterminate mammograms, and 62% for probably malignant or malignant mammograms. Women who had a previous mammogram or had a malignant finding were more likely to comply with follow-up recommendations (p < .0001) than women with normal or benign results and no history of mammography. Compliers and noncompliers did not differ with respect to family history of breast cancer or personal history of breast discomfort. CONCLUSION: Compliance with recommendations in this setting was lower than expected. This may be because rural women using mobile van mammography have limited access to the resources needed for appropriate follow-up. Further research is needed to examine explanations for poor compliance in this setting.     

Isolation of yeast artificial chromosomes free of endogenous yeast chromosomes: construction of alternate hosts with defined karyotypic alterations

An intrinsic feature of yeast artificial chromosomes (YACs) is that the cloned DNA is generally in the same size range (i.e., approximately 200-2000 kb) as the endogenous yeast chromosomes. As a result, the isolation of YAC DNA, which typically involves separation by pulsed-field gel electrophoresis, is frequently confounded by the presence of a comigrating or closely migrating endogenous yeast chromosome(s). We have developed a strategy that reliably allows the isolation of any YAC free of endogenous yeast chromosomes. Using recombination-mediated chromosome fragmentation, a set of Saccharomyces cerevisiae host strains was systematically constructed. Each strain contains defined alterations in its electrophoretic karyotype, which provide a large-size interval devoid of endogenous chromosomes (i.e., a karyotypic "window"). All of the constructed strains contain the kar1-delta 15 mutation, thereby allowing the efficient transfer of a YAC from its original host into an appropriately selected window strain using the kar1-transfer procedure. This approach provides a robust and efficient means to obtain relatively pure YAC DNA regardless of YAC size.     

Prostate specific antigen in the expressed prostatic fluid of men with benign prostatic hyperplasia and prostate carcinoma

PURPOSE: We tested the hypothesis that the histochemically demonstrated prostate specific antigen (PSA) content of prostate carcinoma cells does not necessarily reflect PSA production and secretion by evaluating expressed prostatic fluid. MATERIALS AND METHODS: Expressed prostatic fluid and serum from 152 men with clinical benign prostatic hypertrophy (BPH), 132 with histologically proved BPH and 46 with prostate carcinoma were analyzed with the Hybritech PSA assay. RESULTS: Expressed prostatic fluid PSA levels from carcinoma patients (median 1.70 mg./ml., mean 2.25) were significantly higher than in the histologically proved BPH group (median 1.28 mg./ml., mean 1.42, p < 0.05). CONCLUSIONS: PSA concentration is increased in the expressed prostatic fluid of prostates of men with carcinoma compared to those with histological BPH. This finding may be a functional manifestation of a field change or paracrine effects within the prostate.     

Epithelial antibiotics induced at sites of inflammation

The role of antimicrobial peptides in epithelial defense is not fully understood. An epithelial beta-defensin, lingual antimicrobial peptide (LAP), was isolated from bovine tongue and the corresponding complementary DNA cloned. LAP showed a broad spectrum of antibacterial and antifungal activities. LAP messenger RNA abundance was markedly increased in the epithelium surrounding naturally occurring tongue lesions. This increase coincided with the cellular hallmarks of acute and chronic inflammation in the underlying lamina propria, supporting a role for epithelial antimicrobial peptides as integral components of the inflammatory response.     

Potentiation of lysis of leukaemia cells by a bispecific antibody to CD33 and CD16 (Fc gamma RIII) expressed by human natural killer (NK) cells

Bispecific antibodies recognizing tumour-associated antigens and trigger molecules expressed on immune effector cells have been shown to redirect cytotoxicity of several types of peripheral blood cells against relevant tumour targets. Among various effector cells, natural killer (NK) cells appear to play a role in defence against leukaemia. Here we report the successful chemical conjugation of monoclonal antibodies to CD33 and CD16 to create a bispecific antibody (BsAb 251 x 3G8). This bispecific antibody is capable of augmenting the killing of otherwise resistant leukaemia cells by peripheral blood lymphocytes (PBL), purified resting NK (R-NK) cells, and activated NK (A-NK) cells. BsAb 251 x 3G8 may play a role in the therapy of acute myeloid leukaemia (AML) through redirecting the cytotoxic activity of endogenous or adoptively transferred NK cells.