Agarose drop method for loading thin polyacrylamide gels

The gels (<1 mm) can be loaded conveniently, rapidly, and quantitatively by suspending the sample to be analyzed in a drop of agarose gel and simply placing the solidified drop on top of the stacking gel. With this method there is no lower limit to the size of the sample to be loaded or to the thinness of the gel to be employed. Using the silver stain quantitation of between 1 and 1000 ng/sample is easily achieved.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23819/1/0000058.pd

Uromucoid (Tamm-Horsfall glycoprotein) forms different polymeric arrangements on a filter surface under different physicochemical conditions

Normal human urine cannot be forced through a 0.2 [mu]m filter. To investigate the reason for this phenomenon, uromucoid (Tamm-Horsfall protein) was purified from human urine and its capacity to block a 0.2 [mu]m Millipore filter was measured under different conditions. In the presence of cations (H+, Na+, Ca2+) uromucoid blocked the filter. The blocking varied with cation concentration. Scanning electron microscopy of the filter surface revealed different arrangements of polymerized uromucoid coating the filter surface depending on ionic conditions. In the presence of 100 mmol/l NaCl or 1 mmol/l CaCl2 uromucoid polymers were present in a fibrous arrangement. In the presence of both NaCl and CaCl2 a dence mat of uromucoid polymers was present together with clumps of aggregated polymer. In the absence of ions uromucoid formed a homogeneous coat on the filter surface (as demonstrated by scanning electron microscopy, Western blotting and 125I-uromucoid binding studies) but did not block the filter. Similar fibrous and highly aggregated arrangements of uromucoid polymer were seen in hyaline casts from urine. These data are consistent with the concept that the uromucoid glycoprotein can exist in several different polymeric forms under different ionic conditions.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26802/1/0000358.pd

Contrast‐Enhanced Diagnostic Ultrasound Causes Renal Tissue Damage in a Porcine Model

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135402/1/jum201029101391.pd

A map of urine proteins based on one-dimensional SDS-polyacrylamide gel electrophoresis and Western blotting using one microliter of unconcentrated urine

A sensitive one-dimensional SDS-polyacrylamide gel electrophoretic system was devised whereby the proteins in 1 [mu]l of unconcentrated urine could be visualized by silver staining over the range 9 000-900 000 molecular weight. Identification of urine proteins was confirmed by Western blotting using peroxidase labelled antibodies. A map of the major proteins visualized in urine from individuals with renal disease was constructed. We conclude that the information available from the simple analysis of proteins according to their size is limited to general conclusions regarding whether proteinuria is likely to be of tubular or glomerular or mixed origin. More specific identification of individual proteins is not feasible because simple protein staining is not sufficiently reliable to identify individual proteins. The reasons for this conclusion are as follows: (a) many proteins in urine migrate with similar apparent molecular weights, (b) some proteins are not visualized by silver staining, and (c) albumin polymeric complexes and fragments can be present at almost any molecular weight. However, one-dimensional SDS-polyacrylamide gel electrophoresis together with Western blotting does provide reliable information which might be clinically and experimentally useful.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26100/1/0000176.pd

Polymeric complexes and fragments of albumin in normal human plasma

Nitrocellulose blots of normal human plasma proteins separated by sodium dodecyi sulfate-polyacrylamide gel electrophoresis were examined for polymeric complexes and fragments of albumin using an immunoperoxidase-labelled mouse monoclonal anti-human albumin antibody. Under reducing conditions, no polymeric complexes were seen. Under non-reducing conditions, polymeric complexes were detected at the following molecular weights: 210000, 168000, 147000, 132000, and 110000. These probably represent both homo- and heteropolymers of albumin. Fresh plasma samples were also analyzed by S-200 chromatography with the same results indicating that detection of polymeric complexes was not an artifact of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique. In quantitative terms, polymeric complexes constituted 0.3-2.8% of the total albumin present. Fragments of albumin were also seen in normal human plasma with molecular weights of 45000, 28000 and 19000. These fragments probably represent breakdown products of albumin in normal blood, and they constituted less than 2% of the total albumin present.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24635/1/0000046.pd

Fragmentation and polymeric complexes of albumin in human urine

Analysis of urine proteins of some individuals with proteinuria by SDS-PAGE and silver staining revealed protein bands in urine which did not appear to be present in plasma. The bands migrated with apparent molecular weights of 260 000, 180 000, 110 000, 45 000, 40 000, 30 000, 24 000, 18 000 and 11 000. These bands were shown to be albumin polymer and fragments by using a polyclonal antibody to (a) immunoprecipitate radiolabelled urine proteins, and (b) identify bands blotted from SDS-PAGE gels onto nitrocellulose paper. The specificity of the polyclonal antialbumin antibody was confirmed by using two mouse monoclonal antibodies raised against human albumin which, between them, recognized the same protein bands on nitrocellulose paper as did the polyclonal antibody. The results of these studies of albumin in human urine confirm that albumin exists as polymer and also show that albumin fragmentation occurs in urine. Fragmentation occurs by proteolysis of the albumin molecule both at sites within and outside disulfide loops. The predominant cleavage site appears to be approximately two-fifths of the distance from one end of the albumin molecule to produce disulfide-linked fragments of about 45 000 and 30 000 molecular weight.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25619/1/0000167.pd

Feasibility of applying ultrasound strain imaging to detect renal transplant chronic allograft nephropathy

Feasibility of applying ultrasound strain imaging to detect renal transplant chronic allograft nephropathy.Chronic renal transplant fibrosis, often termed Chronic Allograft Nephropathy, may progress undetected. Since renal fibrosis may be accompanied by a change in measurable elastic tissue properties, ultrasound strain measurements may be useful in its detection. Ultrasound strain imaging was performed for two subjects with renal transplants; one with normal renal function and one with mild renal insufficiency and biopsy demonstrated fibrosis. Subjects underwent ultrasound examination with application of a controlled deformation using phase-sensitive, two-dimensional speckle tracking to evaluate internal tissue motion to measure tissue displacement and strain. Measurements over multiple beams for an equivalent deformational stress showed there was a threefold differences in renal cortical strain between the two subjects. These data suggest that ultrasound elasticity imaging may prove useful in measuring mechanical changes related to fibrosis within the transplant kidney

Regulation of macrophage tumor necrosis factor production by prostaglandin E2

We have studied the role of prostaglandin E2 on the modulation of tumor necrosis factor by immunologically elicited and lipopolysaccharide treated murine macrophages. Indomethacin, a potent inhibitor of prostaglandin E2 production, caused a dose dependent augmentation of lipopolysaccharide induced tumor necrosis factor production (2-3 fold at 10-7 molar). Tumor necrosis factor was released into the extracellular environment and no activity was found to be associated with membrane or cytosolic fractions. Prostaglandin E2 added to the lipopolysaccharide treated cultures suppressed tumor necrosis factor in a dose dependent manner. In these studies, 10-7 molar PGE2 reduced tumor necrosis factor production to basal levels. These data suggest that PGE2 may be a potent autoregulatory factor that dramatically influences tumor necrosis factor production.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26166/1/0000243.pd

ARHGDIA mutations cause nephrotic syndrome via defective RHO GTPase signaling

Nephrotic syndrome (NS) is divided into steroid-sensitive (SSNS) and -resistant (SRNS) variants. SRNS causes end-stage kidney disease, which cannot be cured. While the disease mechanisms of NS are not well understood, genetic mapping studies suggest a multitude of unknown single-gene causes. We combined homozygosity mapping with whole-exome resequencing and identified an ARHGDIA mutation that causes SRNS. We demonstrated that ARHGDIA is in a complex with RHO GTPases and is prominently expressed in podocytes of rat glomeruli. ARHGDIA mutations (R120X and G173V) from individuals with SRNS abrogated interaction with RHO GTPases and increased active GTP-bound RAC1 and CDC42, but not RHOA, indicating that RAC1 and CDC42 are more relevant to the pathogenesis of this SRNS variant than RHOA. Moreover, the mutations enhanced migration of cultured human podocytes; however, enhanced migration was reversed by treatment with RAC1 inhibitors. The nephrotic phenotype was recapitulated in arhgdia-deficient zebrafish. RAC1 inhibitors were partially effective in ameliorating arhgdia-associated defects. These findings identify a single-gene cause of NS and reveal that RHO GTPase signaling is a pathogenic mediator of SRNS.ope

Who will make the 'best' use of Africa's land? Lessons from Zimbabwe

Conflict over African land – between small holders and large industrial farmers and between domestic farmers and global agribusinesses – raises key questions about who will make the best use of African land and which farmers do most to decrease poverty and produce more food, industrial inputs, and exports. Zimbabwe has already gone through two major changes in land occupation, and thus provides an important test of what is the 'best' use of the land. Three measures of 'best' use have been cited in Zimbabwe: reward for military victory, poverty reduction, and agricultural production. Initial evidence indicates that commercial small holder production is a better use of the land than larger, more mechanised farming