Structure of an affinity-matured inhibitory recombinant fab against urokinase plasminogen activator reveals basis of potency and specificity.

Affinity maturation of U33, a recombinant Fab inhibitor of uPA, was used to improve the affinity and the inhibitory effect compared to the parental Fab. Arginine scanning of the six CDR loops of U33 was done to identify initial binding determinants since uPA prefers arginine in its primary substrate binding pocket. Two CDR loops were selected to create an engineered affinity maturation library of U33 that was diversified around ArgL91 (CDR L3) and ArgH52 (CDR H2). Biopanning of the randomized U33 library under stringent conditions resulted in eight Fabs with improved binding properties. One of the most potent inhibitors, AB2, exhibited a 13-fold decrease in IC50 when compared to U33 largely due to a decrease in its off rate. To identify contributions of interfacial residues that might undergo structural rearrangement upon interface formation we used X-ray footprinting and mass spectrometry (XFMS). Four residues showed a pronounced decrease in solvent accessibility, and their clustering suggests that AB2 targets the active site and also engages residues in an adjacent pocket unique to human uPA. The 2.9 Å resolution crystal structure of AB2-bound to uPA shows a binding mode in which the CDR L1 loop inserts into the active site cleft and acts as a determinant of inhibition. The selectivity determinant of this binding mode is unlike previously identified inhibitory Fabs against uPA related serine proteases, MTSP-1, HGFA and FXIa. CDRs H2 and L3 loops aid in interface formation and provide critical salt-bridges to remodel loops surrounding the active site of uPA providing specificity and further evidence that antibodies can be potent and selective inhibitors of proteolytic enzymes.

Increasing axial resolution of 3D data sets using deconvolution algorithms.

Deconvolution algorithms are tools for the restoration of data degraded by blur and noise. An incorporation of regularization functions into the iterative form of reconstruction algorithms can improve the restoration performance and characteristics (e.g. noise and artefact handling). In this study, algorithms based on Richardson-Lucy deconvolution algorithm are tested. The ability of these algorithms to improve axial resolution of three-dimensional data sets is evaluated on model synthetic data. Finally, unregularized Richardson-Lucy algorithm is selected for the evaluation and reconstruction of three-dimensional chromosomal data sets of Drosophila melanogaster. Problems concerning the reconstruction process are discussed and further improvements are proposed.

Distribution of volumes of individual glomeruli in kidneys at autopsy: association with age, nephron number, birth weight and body mass index.

BACKGROUND: Glomerular hypertrophy occurs in a number of normal and pathological states. Glomerular volume in kidneys at autopsy is usually indirectly derived from estimates of total glomerular mass and nephron number, and provides only a single value per kidney, with no indication of the range of volumes of glomeruli within the kidney of any given subject. We review findings of the distribution of volumes of different glomeruli within subjects without kidney disease, and their correlations with age, nephron number, birth weight and body mass index (BMI). METHODS: The study describes findings from autopsy kidneys of selected adult white males from the Southeast USA who had unexpected deaths, and who did not have renal scarring or renal disease. Total glomerular (nephron) number and total glomerular volume were estimated using the disector/fractionator combination, and mean glomerular volume (Vglom) was derived. The volumes of 30 individual glomeruli (IGV) in each subject were determined using the disector/Cavalieri method. IGV values were compared by categories of age, nephron number, birth weight and BMI. RESULTS: There was substantial variation in IGV within subjects. Older age, lower nephron number, lower birth weight and gross obesity were associated with higher mean IGV and with greater IGV heterogeneity. High Vglom and high IGVs were associated with more glomerulosclerosis. However, amongst the generally modest numbers of sclerosed glomeruli, the pattern was uniformly of ischemic collapse of the glomerular tuft. There was no detectable focal segmental glomerular tuft injury. DISCUSSION: In this series of people without overt renal disease, greater age, nephron deficit, lower birth weight and obesity were marked by glomerular enlargement and greater glomerular volume heterogeneity within individuals.

A developmental nephron deficit in rats is associated with increased susceptibility to a secondary renal injury due to advanced glycation end-products.

AIMS/HYPOTHESIS: The aim of this study was to investigate the effects of a secondary renal insult, due to chronic infusion of AGEs on renal function, and on early pathological markers in rats with a developmental nephron deficit. METHODS: Female Wistar-Kyoto rats were fed a low-protein diet (LPD; 8.7% casein) or a normal-protein diet (NPD; 20% casein) during pregnancy and lactation. Nephron number was estimated in 4-week-old female offspring. Male offspring were allowed to grow to 20 weeks of age, when AGEs derived from BSA (AGE-BSA) or BSA was infused subcutaneously (20 mg kg(-1) day(-1)) for 4 weeks. At 24 weeks, blood pressure, renal function and circulating and renal AGEs were assessed. Real-time PCR was used to investigate early molecular markers of renal pathology. RESULTS: As expected, maternal protein restriction led to reduced nephron endowment in LPD offspring. This alone did not affect blood pressure or lead to hyperfiltration in adulthood. However, when coupled with the secondary renal insult, the expression of the genes encoding transforming growth factor-beta(1) and procollagen III was significantly upregulated in the kidneys. In addition, there was renal accumulation of AGEs in LPD offspring, and this was exacerbated by AGE infusion. CONCLUSIONS/INTERPRETATION: Our results demonstrate that the adult kidney with a reduced nephron endowment is more vulnerable to secondary renal insult from AGE-BSA. Since AGE formation is markedly elevated with hyperglycaemia, our findings suggest that a developmental or acquired deficit may render the kidney susceptible to diabetic renal disease.

Milkborne campylobacter infection in Hungary.

In April 1998, an annual 2-day animal farm sale was held in Hódmezóvásárhely, where 500 to 600 visitors consumed unpasteurized milk. The first signs of disease began 2 days after the end of the sale. Fifty-two people from a wide age range fell ill, primarily with inflammatory enteritis. These cases included 34 with Campylobacter positivity: 30 with Campylobacter jejuni and 4 with Campylobacter coli. Environmental samples (raw milk, udder swabs, and rectal swabs from 12 cows in the suspected herd) were tested 2 weeks after the first signs of the disease, and two rectal swabs were found to be positive for C. jejuni. Initially, the epidemic seemed to be sporadic and, accordingly, only 26 human and 2 animal Campylobacter isolates were reserved for randomly amplified polymorphic DNA analysis. This comparative analysis verified that fecally contaminated milk was the source of the outbreak. The DNA-banding patterns of 20 C. jejuni isolates (19 human and 1 animal) were identical. The antibiotic susceptibilities of the Campylobacter isolates were determined, and only six C. jejuni (human) isolates, one C. coli (human) isolate, and one C. jejuni (animal) isolate were resistant to tetracycline, both by disk diffusion and by E test (antimicrobial gradient strip for the quantitative determination of susceptibility or resistance of microorganisms). No plasmid was detected in these tetracycline-resistant isolates. The endotoxin production of Campylobacter isolates was examined via the cytopathogenic effect on the Vero cell line. This effect exhibited various degrees of positivity in 19 cases. Only two human C. jejuni isolates displayed + + + + positivity. Both isolates were from patients who had required antibiotic therapy and hospital care.

Cytogenetic examination of people working with agrochemicals in the southern region of Hungary.

The team performed medical examinations, including cytogenetic examinations, on 55 people working professionally with agrochemicals in eight farmers' cooperatives of County Csongrád in the southern region of Hungary. The people exposed to spraying in a closed space showed no increase in chromosome aberrations. There was an increase in chromosome aberrations in workers exposed to these agrochemicals in open fields. No conclusions regarding workers' health can be drawn from these data. Regulations designed to prevent accidents with agrochemicals are more effective for closed spaces than for the open fields.

Biological monitoring and health surveillance of a group of greenhouse pesticide sprayers.

Repeated extensive medical examinations were carried out in 11 workers spraying organophosphate (OP), carbamate and pyrethroid pesticides in greenhouses during the whole year and in 10 control persons. On a questionnaire their social, health and family status and their working conditions were recorded. Before and after a regular spraying period with pyrethroids for 3 months the following tests were carried out: urinalysis, haematology, immunoglobulin (Ig) G, IgA and IgM levels, whole blood cholinesterase (ChE) activity, serum gamma-glutamyltransferase (GGT) activity, chromosome analysis and electrocardiography (ECG). Atmospheric concentrations of pyrethroids as well as their concentration in the blood and urine of pesticide workers were determined by means of gas-liquid chromatography (GLC). No abnormalities related to the work were found in the tests.

[The Listeria monocytogenes carrier state of the staff of maternity centres in non epidemic periods (author's transl)]. / Listeria monocytogenes-Trägertum von an Geburtshilflichen Abteilungen Tätigen in epidemiefreien Perioden

9 of 177 examined healthy adults working in maternity centers were found to be Listeria monocytogenes carriers, every examined person worked by profession in maternity departments in areas free of human epidemic. After one year (4 degrees c), the bacterium was cultivated from their faeces in culture media containing either rivanol or trypaflavin.