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K J Assmann

Publications and source records attributed to K J Assmann.

At least 19 recordsLinked to original sources
PubMed1996

Acute passive anti-glomerular basement membrane nephritis in P-selectin-deficient mice.

P-selectin present on surfaces of activated endothelium and platelets mediates neutrophil-endothelial and neutrophilplatelet interactions. The role of P-selectin in vivo was examined in a model of acute passive anti-GBM nephritis in P-selectin-deficient and wild-type mice which was induced by intravenous injection of anti-GBM serum. There were two major differences between P-selectin-deficient and wild-type mice. Firstly, mutant mice had approximately two fold more glomerular PMNs and albuminuria than wild-type animals at the peak of neutrophil influx and proteinuria. Secondly, Lipoxin A4 (LXA4), an eicosanoid which inhibits leukocyte-endothelial adhesion in vitro, and is generated primarily by transcellular biosynthetic routes during P-selectin-mediated platelet-PMN interaction [1], was approximately 60% of wild type levels in nephritic kidneys of P-selectin-deficient mice. Injection of wild-type platelets into P-selectin-null mice restored LXA4 to wild-type levels. The corresponding PMN influx approximated PMN levels in wild-type mice receiving platelets but urine albuminuria remained higher. Although these two P-selectin-dependent events cannot be directly linked, our results point to the importance of considering both platelet and endothelial P-selectin in determining the cellular events in inflammation.

Acute Disease↗
PubMed1996

Different mediator systems in biphasic heterologous phase of anti-GBM nephritis in mice.

BACKGROUND: After the injection of rabbit anti-mouse glomerular basement membrane (GBM) antibody into normal C57BL/6J mice severe albuminuria develops, which reaches a peak at 24 h. This early albuminuria is dependent on polymorphonuclear granulocytes (PMN) and is completely absent in the congenic beige mutant strain (C57BL/6J, bg/bg), which is genetically deficient in leukocytic neutral proteinase activity. We now studied the development of anti-GBM nephritis in beige mice during the later heterologous phase. METHODS: In untreated beige mice we assessed the albuminuria and glomerular lesions on days 1-5 after i.v. injection of anti-GBM antibody. Secondly, effector mechanisms involved in the later days of the heterologous phase were studied by substitution of whole anti-GBM antibodies by F(ab')2 fragments, by leukocyte depletion (total body irradiation), scavenging of reactive oxygen metabolites (dimethylsulfoxide treatment), and complement depletion (cobra venom factor treatment). RESULTS: In the later part of the heterologous phase (days 2-5), when there is still no sign of autologous antibody formation, i.v. injection of anti-GBM antibodies in beige mice induces nephritis with gradually increasing albuminuria, that reaches levels similar to those in non-deficient, congenic controls by day 3. This late albuminuria did not occur after injection of F(ab')2 fragments of the antibody, could be prevented by leukocyte depletion, and was greatly reduced by treatment with dimethylsulfoxide, a scavenger of hydroxyl radicals. The late albuminuria was not influenced by complement depletion with cobra venom factor. Histologic and immunohistologic studies gave no indication for a role of glomerular macrophages or lymphocytes. CONCLUSIONS: The heterologous phase in murine anti-GBM nephritis is a biphasic process, with sequential involvement of different and independent mediating systems: both phases are PMN-dependent, but only the early albuminuria depends on leukocytic neutral proteinase activity, whereas the albuminuria and the glomerular damage at later days are effected by reactive oxygen metabolites, most probably originating from PMN accumulating in the glomerulus.

Albuminuria↗
PubMed1996

Organ distribution of aminopeptidase A and dipeptidyl peptidase IV in normal mice.

The hydrolases aminopeptidase A and dipeptidyl peptidase IV, both present in the kidney on the brush borders of the proximal tubule epithelial cells and podocytes, are involved in the induction of experimental membranous glomerulonephritis in the mouse. However, little is known about their (co)distribution in other tissues and their function in health and disease. A detailed insight into the localization of these two enzymes is a prerequisite to elucidation of their function. Therefore, we investigated the presence and co-distribution of aminopeptidase A and dipeptidyl peptidase IV by immunohistology with two different rat monoclonal antibodies, the specificity of which was determined by an immunodepletion technique. In addition, the molecular weight of the hydrolases; was analyzed by SDS-PAGE after isolation by solid-phase immunoprecipitation from glomeruli, renal brush borders, and thymus. Both hydrolases showed different molecular weights in renal corpuscle, renal brush borders, and thymic cells. A widespread organ distribution of the two hydrolases was observed, with co-localization in kidney, liver, small intestine, thymus, brain, spleen, and lymph nodes, either on the same cells or on different cells in the same organ. This distribution and partial co-localization suggests that the two hydrolases, acting either alone or in concert, have a role in many diverse biological processes.

Aminopeptidases↗
PubMed1995

Selective proteinuria in diabetic nephropathy in the rat is associated with a relative decrease in glomerular basement membrane heparan sulphate.

In the present study we investigated whether glomerular hyperfiltration and albuminuria in streptozotocin-induced diabetic nephropathy in male Wistar-Münich rats are associated with changes in the heparan sulphate content of the glomerular basement membrane. Rats with a diabetes mellitus duration of 8 months, treated with low doses of insulin, showed a significant increase in glomerular filtration rate (p < 0.01) and effective renal plasma flow (p < 0.05), without alterations in filtration fraction or mean arterial blood pressure. Diabetic rats developed progressive albuminuria (at 7 months, diabetic rats (D): 42 +/- 13 vs control rats (C): 0.5 +/- 0.2 mg/24 h, p < 0.002) and a decrease of the selectivity index (clearance IgG/clearance albumin) of the proteinuria (at 7 months, D: 0.20 +/- 0.04 vs C: 0.39 +/- 0.17, p < 0.05), suggesting loss of glomerular basement membrane charge. Light- and electron microscopy demonstrated a moderate increase of mesangial matrix and thickening of the glomerular basement membrane in the diabetic rats. Immunohistochemically an increase of laminin, collagen III and IV staining was observed in the mesangium and in the glomerular basement membrane, without alterations in glomerular basement membrane staining of heparan sulphate proteoglycan core protein or heparan sulphate. Glomerular basement membrane heparan sulphate content, quantitated in individual glomerular extracts by a new inhibition ELISA using a specific anti-glomerular basement membrane heparan sulphate monoclonal antibody (JM403), was not altered (median (range) D: 314 (152-941) vs C: 262 (244-467) ng heparan sulphate/mg glomerulus). However, the amount of glomerular 4-hydroxyproline, as a measure for collagen content, was significantly increased (D: 1665 (712-2014) vs C: 672 (515-1208) ng/mg glomerulus, p < 0.01). Consequently, a significant decrease of the heparan sulphate/4-hydroxyproline ratio (D: 0.21 (0.14-1.16) vs C: 0.39 (0.30-0.47), p < 0.05) was found. In summary, we demonstrate that in streptozotocin-diabetic rats glomerular hyperfiltration and a progressive, selective proteinuria are associated with a relative decrease of glomerular basement membrane heparan sulphate. Functionally, a diminished heparan sulphate-associated charge density within the glomerular basement membrane might explain the selective proteinuria in the diabetic rats.

Albumins↗
PubMed1995

Reduction of heparan sulphate-associated anionic sites in the glomerular basement membrane of rats with streptozotocin-induced diabetic nephropathy.

Heparan sulphate-associated anionic sites in the glomerular basement membrane were studied in rats 8 months after induction of diabetes by streptozotocin and in age- adn sex-matched control rats, employing the cationic dye cuprolinic blue. Morphometric analysis at the ultrastructural level was performed using a computerized image processor. The heparan sulphate specificity of the cuprolinic blue staining was demonstrated by glycosaminoglycan-degrading enzymes, showing that pretreatment of the sections with heparitinase abolished all staining, whereas chondroitinase ABC had no effect. The majority of anionic sites (74% in diabetic and 81% in control rats) were found within the lamina rara externa of the glomerular basement membrane. A minority of anionic sites were scattered throughout the lamina densa and lamina rara interna, and were significantly smaller than those in the lamina rara externa of the glomerular basement membrane (p<0.001 and p<0.01 for diabetic and control rats, respectively). Diabetic rats progressively developed albuminuria reaching 40.3 (32.2-62.0) mg/24 h after 8 months in contrast to the control animals (0.8 (0.2-0.9) mg/24 h, p<0.002). At the same time, the number of heparan sulphate anionic sites and the total anionic site surface (number of anionic sites x mean anionic site surface) in the lamina rara externa of the glomerular basement membrane was reduced by 19% (p<0.021) and by 26% (p<0.02), respectively. Number and total anionic site surface in the remaining part of the glomerular basement membrane (lamina densa and lamina rara interna) were not significantly changed. We conclude that in streptozotocin-diabetic rats with an increased urinary albumin excretion, a reduced heparan sulphate charge barrier/density is found at the lamina rara externa of the glomerular basement membrane.

Albuminuria↗
PubMed1995

Familial glomerulonephritis characterized by massive deposits of fibronectin.

In recent years more than 150 cases of glomerulonephritis characterized by deposits of irregularly arranged fibrils have been documented. In the majority of these cases immunoglobulins and complement are the prime constituents of these deposits. We recently made a diagnosis of fibrillary glomerulonephritis without immunoglobulin deposition in two members of a family, a father and a son. In the father, proteinuria was first discovered 18 years ago. In 1985 he was referred to our outpatient clinic because of hypertension and increasing proteinuria. From that time onward he was regularly seen for blood pressure control. Nephrotic-range proteinuria persisted, without hardly any evidence of deterioration of renal function. Renal biopsies were performed in 1985 and 1993. His son underwent a renal biopsy in 1993 because of moderate proteinuria. The biopsies of both patients disclosed a distinct form of fibrillary glomerulonephritis that was characterized by massive deposits of a homogeneous, eosinophilic material in the mesangial and subendothelial areas. Staining for amyloid was negative. Immunofluorescence revealed that the biopsy specimens only stained faintly for immunoglobulins, complement factors C1q and C3, the extracellular matrix proteins, collagen IV, and laminin. However, they strongly stained for fibronectin. Using monoclonal antibodies specific for cell-derived fibronectin (IST-9) and plasma- and cell-derived fibronectin (IST-4), in the biopsy of the son we demonstrated that the fibronectin deposited in the glomeruli was mainly derived from the plasma, and to a lesser extent from resident glomerular cells. In addition, a moderate staining for amyloid P and vitronectin also was present. No or minor enhanced staining for collagen I, III, or V, heparan sulfate proteoglycan or its glycosaminoglycan side chains, tenascin, or thrombospondin could be observed. By electron microscopy the deposits in the mesangium and the subendothelial spaces appeared focally to be composed of irregularly arranged fibrils or microtubules 10 to 12 nm in diameter. Fibrillary glomerulonephritis with massive deposits of fibronectin represents a rare form of familial glomerulonephritis. In our patients the glomerulonephritis has an indolent course with hardly any deterioration of renal function.

Adult↗
PubMed1995

Antineutrophil cytoplasmic autoantibodies with myeloperoxidase specificity in a patient with anti-glomerular basement membrane disease.

An unusual case is described of a patient with both anti-glomerular basement membrane (GBM) disease and anti-neutrophil cytoplasmic autoantibodies with myeloperoxidase specificity (MPO-ANCA) presenting with acute renal failure. Four years before a seronegative arthritis of the left wrist was diagnosed. Tests for ANCA by indirect immunofluorescence were repeatedly negative. The diagnosis was made by renal biopsy and by testing the serum with specific enzyme-linked immunosorbent assays (ELISA) for MPO-ANCA and anti-GBM antibodies. To our knowledge, this is the first patient presenting with such findings in the Dutch literature.

Acute Kidney Injury↗
PubMed1995

Role of macrophages and colony-stimulating factor-1 in murine antiglomerular basement membrane glomerulonephritis.

Macrophages have been shown to mediate glomerular injury in antiglomerular basement membrane (anti-GBM) glomerulonephritis in rats and rabbits. To evaluate the role of macrophages and the macrophage-related cytokines, colony stimulating factor-1 (CSF-1), monocyte chemoattractant protein-1 (MCP-1) and RANTES, accelerated anti-GBM nephritis was studied in op/op mutant mice, which lack CSF-1 and are severely macrophage deficient, and in heterozygous op/+ control mice. Observations were made 24 h and 3 days after the injection of rabbit anti-mouse GBM antibody in mice preimmunized with rabbit immunoglobulin G. Proteinuria rose progressively in both groups but did not differ between them (urine protein/creatinine ratio at 3 days: 1.01 +/- 0.38 in op/op versus 1.45 +/- 0.43 in op/+; P, not significant). In both op/op and op/+ mice, anti-GBM nephritis was associated with renal expression of mRNA for RANTES and MCP-1 and barely detectable levels of mRNA for CSF-1. In contrast, these cytokines were not expressed in sham-injected mice. Morphologic lesions appeared earlier in op/op mice but were comparable by Day 3. Glomerular injury consisted of capillary thrombosis and endothelial cell damage associated with mild to moderate leukocyte infiltration. Despite enhanced expression of mRNA for RANTES and MCP-1, glomerular macrophage infiltration was not increased in op/+ mice. It was concluded that, in mice, in contrast to rats and rabbits, accelerated anti-GBM nephritis may develop in the absence of both CSF-1 and macrophage infiltration.

Animals↗
PubMed1995

Cyclosporin A reduces albuminuria in experimental anti-GBM nephritis independently from changes in GFR.

Cyclosporin A (CsA) can reduce proteinuria in various forms of human and experimental glomerulonephritis. This antiproteinuric effect of CsA may be the consequence of diminution of immunological damage, a drug-induced decrease of GFR, or changes in permselectivity of the glomerular basement membrane (GBM). We studied the antiproteinuric effect of CsA in the heterologous phase of a passively induced anti-GBM nephritis in the mouse. This passive model is characterized by acute exudative glomerular lesions and a dose-dependent albuminuria. Rabbit anti-mouse GBM antibodies were administered intravenously in C57B110 mice at day 4, after 3 days of pretreatment with either CsA (75 mg/kg body weight) (n = 15) or olive oil (OO, controls, n = 15) orally. CsA did not influence the severity of the histological lesions. Albuminuria was substantially reduced by CsA (CsA 1.6 +/- 1.8; OO 5.6 +/- 3.2 mg/18 h; P < 0.002). There was a considerable concomitant reduction of the GFR by CsA, as measured with a 51Cr-EDTA single-shot plasma clearance technique before (day-1) and during treatment (day 4): GFR ratio day 4/day-1 for CsA, 0.4 +/- 0.1; for OO controls, 1.1 +/- 0.6; P < 0.01. This drug-induced decrease of GFR was prevented by simultaneous treatment with phenoxybenzamine (PB) twice daily 45 micrograms orally for 4 days (GFR ratio day 4/day-1 for PB and CsA, 0.9 +/- 0.4; controls (PB and OO), 1.0 +/- 0.4; P = NS). Although the CsA-induced GFR reduction was prevented, CsA still reduced albuminuria significantly (PB and CsA, 2.2 +/- 1.8; controls (PB and OO), 5.6 +/- 1.8 mg/18 h; P = 0.003).(ABSTRACT TRUNCATED AT 250 WORDS)

Albuminuria↗
PubMed1995

Decrease of heparan sulfate staining in the glomerular basement membrane in murine lupus nephritis.

Recently we found in biopsies of human lupus nephritis a nearly complete loss of heparan sulfate (HS) staining in the glomerular basement membrane (GMB). To clarify the relationship between HS staining and albuminuria in lupus nephritis, we studied MRL/lpr mice with short (< 7 days) or prolonged duration of albuminuria (14-21 days) and compared these with mice of different ages without albuminuria. Kidney sections were stained for mouse immunoglobulin (Ig), HS, heparan sulfate proteoglycan (HSPG)-core protein and laminin in immunofluorescence. In mice with prolonged albuminuria HS staining in the glomerular capillary loops had almost completely disappeared, whereas staining was unaltered in non-albuminuric mice (P = 0.001). In mice with short duration of albuminuria, there was a tendency toward a decrease of HS staining (P = 0.06). The expression of HSPG-core protein and other extra cellular matrix (ECM) components was unaltered in all groups. HS staining correlated inversely with albuminuria (rs = -0.55; P < 0.001) and with staining of Ig deposits in the capillary loops (rs = -0.74; P < 0.001). Despite the nearly complete loss of HS staining in the GBM in mice with prolonged albuminuria, there was no change in glomerular HS content as assessed by agarose electrophoresis and HS inhibition ELISA. We conclude that the development of albuminuria in MRL/lpr mice is accompanied by a loss of HS staining in the GBM, probably due to the masking of HS by deposits of Ig. In vitro studies revealed that autoantibodies complexed to nucleosomal antigens can inhibit the binding of the anti-HS monoclonal antibody to HS. Whether this also occurs in vivo remains to be determined.

Albuminuria↗
PubMed1994

[Tubulointerstitial nephritis caused by mesalazine (5-ASA) agents].

In five patients (4 men aged 34, 20, 22 and 29 years, and a woman of 34 years) tubulo-interstitial nephritis developed during treatment with 5-aminosalicylic acid (5-ASA) containing drugs for colitis ulcerosa, proctocolitis or Crohn's disease. Two patients had an impaired renal function before treatment with 5-ASA medication. In all patients there was only an incomplete recovery of renal function after cessation of the therapy. It is therefore necessary to monitor renal function regularly in patients receiving 5-ASA containing preparations.

Adult↗
PubMed1994

The extent of peritubular CD14 staining in renal allografts as an independent immunohistological marker for acute rejection.

Previously, we demonstrated that in acute interstitial rejection, immunohistological staining of renal allograft biopsies with the CD14 mAb WT14, reacting with human monocytes/macrophages, shows a characteristic peritubular increase of positive cells. To test the diagnostic value of this CD14 positivity, we compared, in 154 unselected renal allograft biopsies, the extent of peritubular WT14 staining with (a) the original histological diagnosis, made with knowledge of clinical data, (b) the retrospectively and blindly scored histological diagnosis according to the criteria of the Banff classification, and (c) the eventual clinical diagnosis, which included evaluation of the response to therapy. The extent of peritubular WT14 positivity, blindly scored on cryostat sections of the frozen part of the biopsies, correlated positively with the probability of acute rejection (AR). When using a cutoff of 70% WT14 positivity for the diagnosis of AR, as extracted from a receiver operating characteristic curve, the WT14 diagnosis had a positive predictive value of 91% and a negative predictive value of 56%, compared with the original histological diagnosis. Compared with the Banff diagnosis of AR (grade I-III), these values were 95% and 47%, and compared with the clinical diagnosis, 84% and 63%, respectively. The WT14 diagnosis essentially corrected the original histological diagnosis in 7 cases, and was consistent with the eventual diagnosis in 5 equivocal cases. We conclude that the extent of peritubular CD14 positivity can be used as a marker for AR and can serve as a valuable additional criterion for AR in the histological examination of renal allograft biopsies.

Acute Disease↗
PubMed1994

Expression of glomerular extracellular matrix components in human diabetic nephropathy: decrease of heparan sulphate in the glomerular basement membrane.

Diabetic nephropathy is characterized by albuminuria which proceeds to overt proteinuria. The highly negatively stained HS side chain of heparan sulphate proteoglycan (HSPG) is a major determinant of the charge-dependent permeability of the GBM. We set out to study the presence of HS and HSPG in the GBM of patients with diabetic nephropathy using newly developed monoclonal antibodies, and to compare HSPG expression to the expression of other previously investigated glomerular extracellular matrix compounds. Immunohistochemically, glomerular extracellular matrix components were analysed in 14 renal biopsies of patients with diabetic nephropathy and compared with those of normal control subjects. Monoclonal antibodies used were: JM403 against the HS side chain of GBM HSPG and JM72 against the HSPG-core protein. Also, a polyclonal antiserum (B31) against human GBM-HSPG-core protein was used. Additionally, antibodies were used against collagen types I, III, IV and against alpha 1 (IV)NC, alpha 3(IV)NC and fibronectin. Staining was scored for intensity and for staining pattern by four independent observers who had no previous knowledge of the sample origin. No glomerular staining was seen for collagen type I. Collagen type III was present in some diabetic nodules. Anti-collagen type IV showed a decreased GBM staining in patients with diabetic nephropathy (p = 0.04). With anti-alpha 1 (IV)NC no changes in GBM staining intensity were observed; with anti-alpha 3 (IV)NC brilliant GBM staining was seen in both groups. Increased mesangial staining (p = 0.003) was seen with anti-collagen type IV in biopsies with nodular lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1994

A 4-year-old boy with neurofibromatosis and severe renovascular hypertension due to renal arterial dysplasia.

A 4-year-old boy had severe hypertension, cardiac failure, and signs of neurofibromatosis. Arteriography disclosed renal artery stenosis in both kidneys with signs of ischemia, particularly in the right kidney. Because of insufficient response to antihypertensive therapy, a right-sided nephrectomy was performed. Histological examination of this kidney showed segmental stenosis in all branches of the renal artery. The vascular lesions were characterized by an intimal proliferation of spindle cells in a mucoid matrix with destruction of the internal elastic membrane frequently accompanied by loss or attenuation of the media and fibrosis of the adventitia. Occasionally, a nodular arrangement of the spindle cells at the interface between intima and media was observed. Immunohistochemical studies demonstrate a smooth-muscle cell origin for these cells.

Angiography↗
PubMed1994

Anti-nucleosome antibodies complexed to nucleosomal antigens show anti-DNA reactivity and bind to rat glomerular basement membrane in vivo.

Histones can mediate the binding of DNA and anti-DNA to the glomerular basement membrane (GBM). In ELISA histone/DNA/anti-DNA complexes are able to bind to heparan sulfate (HS), an intrinsic constituent of the GBM. We questioned whether histone containing immune complexes are able to bind to the GBM, and if so, whether the ligand in the GBM is HS. Monoclonal antibodies (mAbs) complexed to nucleosomal antigens and noncomplexed mAbs were isolated from culture supernatants of four IgG anti-nuclear mAbs. All noncomplexed mAbs showed strong anti-nucleosome reactivity in ELISA. One of them showed in addition anti-DNA reactivity in noncomplexed form. The other three mAbs only showed anti-DNA reactivity when they were complexed to nucleosomal antigens. After renal perfusion a fine granular binding of complexed mAbs to the glomerular capillary wall and activation of complement was observed in immunofluorescence, whereas noncomplexed mAbs did not bind. Immuno-electron microscopy showed binding of complexes to the whole width of the GBM. When HS in the GBM was removed by renal heparinase perfusion the binding of complexed mAb decreased, but did not disappear completely. We conclude that anti-nucleosome mAbs, which do not bind DNA, become DNA reactive once complexed to nucleosomal antigens. These complexed mAbs can bind to the GBM. The binding ligand in the GBM is partly, but not solely, HS. Binding to the GBM of immune complexes containing nucleosomal material might be an important event in the pathogenesis of lupus nephritis.

Animals↗
PubMed1994

Monoclonal antibodies against the protein core and glycosaminoglycan side chain of glomerular basement membrane heparan sulfate proteoglycan: characterization and immunohistological application in human tissues.

We raised monoclonal antibodies (MAb) against the core protein and the heparan sulfate (HS) side chain of heparan sulfate proteoglycan (HSPG) from glomerular basement membranes (GBM). Anti-HSPG-core MAb were obtained after immunization of mice with HSPG purified from human GBM and the anti-HS MAb after immunization of mice with HSPG from rat glomeruli, which crossreacted with human HS and GBM HSPG. The specificity of the MAb was demonstrated by ELISA studies, Western blotting, inhibition experiments, and indirect immunofluorescence (IF) on kidney cryostat sections pre-treated with glycosaminoglycan (GAG)-degrading enzymes. Indirect IF on normal human kidney tissue showed prominent GBM staining for both MAb, with variable staining of the other renal basement membranes (BMs). By indirect immunoelectron microscopy (IEM), most intense staining was observed at the endothelial side of the GBM for both MAb, although the staining patterns were not identical. Both MAb were used to localize HSPG in human tissues by indirect IF. They bound to antigens present in the BMs of most tissues examined, including those of epithelia and endothelia. Differences between both MAb were observed for BMs of muscle cells, since the anti-HSPG core protein MAb (JM-72) staining was negative, whereas the anti-HS MAb (JM-403) clearly stained these structures. Comparison of our staining patterns in human tissues with the distribution of other anti-BM HSPG antibodies suggests that there are at least two types of BM HSPG, which have common epitopes on the HS side chains recognized by JM-403.

Animals↗
PubMed1994

Characterization of a 43 kD protein associated to aminopeptidase A from murine kidney.

SDS-PAGE of affinity-purified APA under reducing conditions showed in addition to the specific APA band of M(r) 130 kD, a second band of M(r) 43 kD. Internal amino acid sequencing of three tryptic peptides from this second band, that was cut out of the polyacrylamide gel, matched the actin sequence. The identity of the 43 kD band was also confirmed by Western blotting.

Actins↗