Angiotensin converting enzyme gene polymorphism in primary vesicoureteral reflux.

We studied the insertion/deletion (I/D) polymorphism of the angiotensin converting enzyme (ACE) gene in 78 patients with primary vesicoureteral reflux (VUR), and examined renal function by dimercaptosuccinate (DMSA) renoscintigraphy and diethylenetriamine-penta-acetic acid (DTPA) renogram in each genotype. Patients were classified into three genotypes according to the ACE gene I/D polymorphisms: 32 in II genotype, 36 in ID, and 10 in DD. The incidence of presumably congenital unilateral small kidneys was high in DD patients (70%). Glomerular filtration rate obtained from DTPA renogram was 120.7+/-35.7 ml/min (expressed as mean+/-SD) in II genotype, 111.7+/-33.3 in ID, and 88.0+/-18.0 in DD. The total quantitative DMSA tracer uptake of both kidneys was also low in patients with the D allele. This study shows that the D allele of ACE gene is closely related to small congenital kidneys with refluxing ureters in patients with primary VUR, and in accordance with previous reports, this allele is also related to the progression of reflux nephropathy.

Apoptosis of renal tubular cells in Shiga-toxin-mediated hemolytic uremic syndrome.

In order to clarify the mechanism of unusual renal tubular dysfunction seen in a child with Shiga toxin (Stx)-mediated hemolytic uremic syndrome (HUS), we studied the renal biopsy specimens for Stx binding and apoptosis of renal tubular cells. A 7-year-old boy with Stx-2-mediated HUS demonstrated extensive renal tubular damage characterized by nonoliguric acute renal failure, increased urinary tubular enzymes and defective urine-concentrating capacity. His renal biopsy specimens were analyzed for Stx binding and apoptotic cell death. Seven kidney tissue specimens obtained from patients without HUS served as controls. Detection of Stx binding to renal sections and apoptotic cells were performed using mouse monoclonal anti-Stx 2 antibody and the TUNEL method, respectively. Positive staining was observed predominantly in renal tubular cells, while the 7 kidney tissue specimens from control patients did not show positive staining. To the best of our knowledge, this is the first case to show Stx binding and apoptotic cell death in renal tubules on biopsy specimens obtained from a child with Stx-mediated HUS. In conclusion, this case suggests that vascular endothelial cells are not the sole nor the consistent target for Stx-mediated cell injury and that significant renal tubular damage other than glomerular damage might occur in some children with Stx-mediated HUS.

Non-lupus nephropathy associated with antiphospholipid antibodies.

Renal biopsy was performed in a 12-year-old girl with hematuria and proteinuria which was first detected at the age of 7, and the findings were the mesangial proliferative glomerulonephritis with IgG and C3 deposits. The routine blood examination for the biopsy disclosed the presence of the prolonged activated partial thromboplastin time and the biological false positive reaction in the syphilis test. These results led us to the further investigation, which revealed the presence of high titers of anticardiolipin antibodies. Since this girl presented no extra-renal symptoms of systemic lupus erythematosus (SLE) and had negative serologic tests for SLE, we hypothesize that her nephritis is closely related to antiphospholipid antibodies.

Suppression of morphological transformation by radicicol is accompanied by enhanced gelsolin expression.

Radicicol, an inhibitor of Src-family protein-tyrosine kinases, causes morphological reversion of v-src- and v-Ha-ras-transformed fibroblasts and arrest of the cell cycle at both the G1 and the G2 phases. Radicicol was found to inhibit the growth of several other oncogene-transformed cell lines and human carcinoma cell lines and to revert their cell morphology to be flat. In the radicicol-treated flat cells, actin stress fiber bundles were reorganized. Since this effect of radicicol on these cell lines was inhibited by cycloheximide, de novo protein synthesis is required for the morphological reversion. Screening of cellular proteins enhanced in response to radicicol by two-dimensional gel electrophoresis suggested that the amount of gelsolin, an actin regulatory protein, was distinctly increased upon radicicol treatment. Western blot and Northern blot analyses showed that radicicol enhanced transcription of the gelsolin gene in human carcinoma cell lines, as a result of which the amount of gelsolin was increased several folds. Injection with an anti-gelsolin antibody into cells and successive treatment with radicicol resulted in approximately 80% reduction of the number of flat cells with stress fibers in comparison with controls treated with an irrelevant antibody. These results show that elevated expression of gelsolin is associated, at least in part, with the suppression of transformation and the restoration of actin stress fibers in human carcinoma cells by radicicol.

Quantitative analysis of low molecular weight G-actin-binding proteins, cofilin, ADF and profilin, expressed in developing and degenerating chicken skeletal muscles.

A large amount of G-actin is pooled in the cytoplasm of young embryonic skeletal muscle and, although its concentration is reduced as muscle develops, the total amount of actin in muscle cells increases remarkably. Three G-actin-binding proteins, cofilin, ADF and profilin, are known to be involved in creating the G-actin pool in the embryonic muscle. To better understand how they are responsible for the regulation of assembly and disassembly of actin in developing and degenerating muscles, we measured the amounts of the three G-actin-binding proteins by means of quantitative immunoblotting and compared them with that of G-actin. The sum of the amounts of the three actin-binding proteins was insufficient at early developmental stages but sufficient at later stages to account for the pool of G-actin in young muscle cells. It decreased in parallel with the decrease in the G-actin pool as muscle developed. Expression of thymosin beta 4, which is known to be extremely important for G-actin-sequestering in a variety of non-muscle cells, was detected at a considerable level in young embryonic but not in adult skeletal muscles according to Northern and Western blotting. In degenerating denervated and dystrophic muscles, cofilin and profilin, but not ADF, were significantly increased in amount. From these results, we conclude that the G-actin pool in young embryonic skeletal muscle is mainly due to cofilin, ADF, profilin and thymosin beta 4, but thymosin beta 4 as well as ADF becomes less important as muscle develops. Cofilin and profilin may also be involved in the redistribution of actin during myofibrillogenesis and in the process of actin disassembly in degenerating muscles.

Differential assembly of cytoskeletal and sarcomeric actins in developing skeletal muscle cells in vitro.

Monoclonal antibodies (McAb) to actin were prepared to analyze the assembly of actin isoforms in developing muscle cells in vitro. One of the antibodies (SkA-06) was specific for alpha-sarcomeric actin isoforms in skeletal and cardiac muscles, while the others recognized cytoskeletal (beta, gamma) actin isoforms in smooth muscle and non-muscle tissues as well as the sarcomeric (alpha) actins. Using SkA-06 and a polyclonal antibody (PcAb) specific for cytoskeletal actins, the subcellular localization of the actin isoforms was examined by immunocytochemical methods. While in developing young myotubes, cytoskeletal and sarcomeric actins were co-localized in nascent myofibrils or stress-fiber-like structures, sarcomeric actins predominated in striated myofibrils in more developed myotubes. When FITC-labeled cytoskeletal and sarcomeric actins were introduced into young myotubes by a microinjection method, the latter became detectable in striated structures sooner than the former but they were finally incorporated into striated myofibrils. These results suggest that alpha-actin(s) as well as beta- and gamma-actins can be incorporated into myofibrils, but alpha-actin(s) is assembled preferentially into myofibrils in developing muscle cells.

Site-directed mutagenesis of the phosphorylation site of cofilin: its role in cofilin-actin interaction and cytoplasmic localization.

It has been demonstrated that the activity of ADF and cofilin, which constitute a functionally related protein family, is markedly altered by phosphorylation, and that the phosphorylation site is Ser 3 in their amino acid sequences [Agnew et al., 1995: J. Biol. Chem. 270:17582-17587; Moriyama et al., 1996: Genes Cells 1:73-86]. In order to clarify the function of the phosphorylated and unphosphorylated forms of cofilin in living cells especially in the process of cytokinesis, we generated analogs of the unphosphorylated form (A3-cofilin) and phosphorylated form (D3-cofilin) by converting the phosphorylation site (Ser 3) of cofilin to Ala and Asp, respectively. The mutated proteins were produced in an Escherichia coli expression system, and conjugated with fluorescent dyes. In in vitro functional assay, labeled A3-cofilin retained the authentic ability to bind to and sever F-actin, while labeled D3-cofilin failed to interact with actin. They were then injected into living cells to examine their cellular distribution. They exhibited distinct localization patterns in the cytoplasm; A3-cofilin was highly concentrated at the membrane ruffles and cleavage furrow, where endogenous cofilin is also known to be enriched. In contrast, D3-cofilin showed only diffuse distribution both in the cytoplasm and nucleus. These results suggest that the subcellular distribution of cofilin as well as its interacting with actin in vivo is regulated by its phosphorylation and dephosphorylation.

Changes of intracellular electrolyte contents in rat skeletal muscle during body suspension.

The CNS-mediated inhibition of active Na(+)-K+ transport in both "type S" muscle, soleus (SOL), and "type FF" muscle, extensor digitorum longus (EDL) was investigated in rats suspended horizontally. Plasma Na+ and K+ contents did not change during the suspension period. The relative wet weight of SOL decreased more than that of EDL by suspension. There was significant intracellular Na+ accumulation and K+ loss in both SOL and diaphragm of suspended rats. However, cerebrum, cerebellum, medulla oblongate, ventricle, liver, pancreas, kidney, intestine, aorta and EDL were spared from the intracellular Na+ accumulation and K+ loss. Sciatic nerve sectioning or cervical spinal cord transection recovered the Na+ and K+ contents in the SOL of suspended rats. The results indicate the existence of neural inhibition of the active Na(+)-K+ transport in skeletal muscle of the suspended rats.

Effects of cofilin on actin filamentous structures in cultured muscle cells. Intracellular regulation of cofilin action.

The previous investigation (Abe et al. (1989) J. Biochem. 106, 696-702) suggested that cofilin is deeply involved in the regulation of actin assembly in developing skeletal muscle. In this study, to examine further the function of cofilin in living myogenic cells in culture, recombinant cofilin having extra Cys residues at the N terminus was produced in Escherichia coli and was labeled with tetramethylrhodamine-iodoacetamide (IATMR). When the cofilin labeled with IATMR (IATMR-cofilin) was introduced into myogenic cells, actin filaments in the cytoplasm or nascent myofibrils were promptly disrupted, and many cytoplasmic rods which contained both IATMR-cofilin and actin were generated. Sarcomeric myofibrillar structures were not disrupted but tropomyosin was dissociated from the structures by the exogenous cofilin, and the IATMR-cofilin became localized in I-band regions. 24 hours after the injection, however, the actin-cofilin rods disappeared completely and the IATMR-cofilin became diffused in the cytoplasm as endogenous cofilin. Concomitantly, actin filaments were recovered and tropomyosin was re-associated with sarcomeric I-bands. At this point, the IATMR-cofilin in the cells still retained the functional activity to form intranuclear actin-cofilin rods in response to stimulation by DMSO just as endogenous cofilin. FITC-labeled actin introduced into myogenic cells at first failed to assemble into filamentous structures in the presence of the exogenous cofilin, but was gradually incorporated into myofibrils with time. The drastic effects of the exogenous cofilin on actin assembly were suppressed by phosphatidylinositol 4,5-bisphosphate (PIP2). These results indicate that the exogenous cofilin is active and alters actin dynamics remarkably in muscle cells, but its activity in the cytoplasm gradually becomes regulated by the action of some factors including PIP2-binding.

Concentration of cofilin, a small actin-binding protein, at the cleavage furrow during cytokinesis.

Cofilin is a small actin-binding protein which regulates actin polymerization in a pH-dependent manner. Immunofluorescence microscopy with a monoclonal antibody for cofilin revealed that this protein is temporarily concentrated at the contractile ring during cytokinesis. Cofilin appeared to accumulate rapidly at the contractile ring during late stages of furrowing, and was finally enriched at the midbody. The concentration of cofilin at the contractile ring was observed in several kinds of cultured cells. Furthermore, cofilin introduced into living cells by a microinjection method was also concentrated at the contractile ring. These results suggest that cofilin is involved in actin reorganization during cytokinesis.

Intracellular Na+ and K+ shifts induced by contractile activities of rat skeletal muscles.

The effects of direct and indirect electrical stimulation on intracellular potassium and sodium contents ([K]i and [Na]i, respectively) in rat soleus muscle (SOL) and extensor digitorum longus muscle (EDL) were investigated under in vivo conditions. The changes of [K]i and [Na]i contents in both muscles which were stimulated indirectly reached respective values at 30 min or 1 hr after the beginning of stimulation, whereas those of EDL stimulated with 60 Hz changed gradually through 2 hr stimulation. The shifts of [K]i and [Na]i in EDL occurred during the twitch contraction at considerably lower frequency stimulation (0.5-10 Hz), whereas those in SOL were observed during the tetanus contraction at high frequency stimulation (10-40 Hz). The difference of change in cationic shifts between EDL and SOL under low frequency stimulation was reduced by ouabain treatment, though the difference was still significant. When the muscles were indirectly stimulated 6000 times at 1, 5, 10 and 20 Hz, the cationic shifts in EDL were greater than those in SOL, extending over all frequencies. It was concluded that such a difference in ionic shift between contracting EDL and SOL may be primarily due to the difference in unidirectional ionic fluxes per stimulation and, secondly, to the difference in Na(+)-K+ pump activity.

Cytoplasmic localization and nuclear transport of cofilin in cultured myotubes.

We used immunofluorescence methods to examine the cellular distribution of cofilin in chicken myotubes in primary culture. Cofilin showed mainly diffuse distribution in the cytoplasm except for rather strong staining around the nuclei and faint striated patterns along myofibrils, but did not stain inside the nuclei. Neither stress fiber-like structures nor myofibrils were clearly stained. In the presence of 10% dimethyl sulfoxide (DMSO), intranuclear actin-cofilin rods, which were composed of alpha-actin isoform and cofilin, were formed in all the nuclei of individual myotubes. In the cofilin sequence, a putative nuclear localization signal (NLS) was observed. We examined the NLS activity of this portion by using a synthetic peptide corresponding to the putative NLS. When the NLS peptide conjugated with bovine serum albumin was microinjected into the cytoplasm of myotubes, it was rapidly accumulated into the nuclei. The same result was obtained with in vitro a nuclear protein import assay system with digitonin-permeabilized myotubes. Therefore, we suggest that this portion is responsible for the nuclear transport of cofilin. In myotubes, the majority of cofilin was present in an unphosphorylated form and this form remained unchanged after the DMSO treatment. Thus, we suggest that the phosphorylation of cofilin itself is not directly involved in its nuclear transport at least in myotubes.

Colocalization of ADF and cofilin in intranuclear actin rods of cultured muscle cells.

Immunofluorescence microscopy revealed that two actin-binding proteins of low molecular weight with different functional activity, ADF and cofilin, are transported into nuclei of cultured myogenic cells to form rod structures there together with actin, when the cells were incubated in medium containing dimethylsulfoxide. In most cases, ADF and cofilin colocalized in the same nuclear actin rods, but ADF appeared to predominate in mononucleated cells, while cofilin was present in multinucleated myotubes. In some mononucleated cells, the nuclear actin rods were composed of ADF and actin but devoid of cofilin. An ADF homologue in mammals, destrin, was also translocated into nuclear actin rods under similar conditions. As a nuclear transport signal sequence exists in cofilin and ADF but not in actin, ADF and/or cofilin may be responsible for the nuclear import of actin in myogenic cells under certain conditions.

Change of intracellular K+ activity in rat soleus muscle during hypokalemia.

The relationship between intracellular total K+ concentration [( K]i) as determined by a flame spectrophotometer and intracellular K+ activity (aKi) as determined by an ion-selective microelectrode was studied in soleus muscle of rats on a diet deficient in K+ for 40 days. [K]i began to fall immediately from the initial stage of hypokalemia, while aKi was well-maintained for 15 days. Then, aKi decreased gradually. The measured resting potential (Em) hyperpolarized beyond the EK was calculated from aKi in hypokalemic rat muscle from day 20 to 40. A rapid increase in aKi occurred over 3 hours in soleus muscle of hypokalemic rats for 5 to 6 weeks. It was concluded that the bound intracellular K+ acts as a buffer for aKi in hypokalemic rat muscle, that Em exceeds EK because the Na+-K+ pump is stimulated by increased [Na]i and that the increase in aKi after denervation is due to the removal of a Na+-K+ pump inhibitor normally released from nerve ending.

Active sodium-potassium transports in skeletal muscles of deoxycorticosterone hypertensive rats.

CNS-induced suppressions of active Na+, K+ transport was investigated in both 'tonic' muscles, soleus (SOL), and 'twitch' muscle, extensor digitorum longus (EDL) of deoxycorticosterone acetate (DOCA) hypertensive rats. There was a marked K+ loss and Na+ accumulation in the skeletal and smooth muscles of DOCA hypertensive rats. The cellular K+ loss was in the order of SOL greater than EDL greater than diaphragm greater than intestine greater than aorta. However, liver, kidney and CNS organs such as cerebrum, cerebellum and medulla oblongata were spared from this K+ fall. Sciatic nerve sectioning or cervical transection activated the active Na+, K+ transport in SOL during DOCA hypertension but inhibited further the pump activity in EDL. The application of tetrodotoxin on the sciatic nerve also activated the Na+, K+ transport in SOL but inhibited the transport in EDL. The facilitatory effect of denervation on the pump activity in SOL was abolished by pretreatment with ouabain. Injection of curare had no effect on Na+ and K+ contents in both SOL and EDL. These results indicate that the CNS is involved differently on the neural regulations of the active Na+, K+ transport systems in SOL and EDL of DOCA hypertensive rats.