Human 76p: A new member of the gamma-tubulin-associated protein family.

The role of the centrosomes in microtubule nucleation remains largely unknown at the molecular level. gamma-Tubulin and the two associated proteins h103p (hGCP2) and h104p (hGCP3) are essential. These proteins are also present in soluble complexes containing additional polypeptides. Partial sequencing of a 76- kD polypeptide band from these complexes allowed the isolation of a cDNA encoding for a new protein (h76p = hGCP4) expressed ubiquitously in mammalian tissues. Orthologues of h76p have been characterized in Drosophila and in the higher plant Medicago. Several pieces of evidence indicate that h76p is involved in microtubule nucleation. (1) h76p is localized at the centrosome as demonstrated by immunofluorescence. (2) h76p and gamma-tubulin are associated in the gamma-tubulin complexes. (3) gamma-tubulin complexes containing h76p bind to microtubules. (4) h76p is recruited to the spindle poles and to Xenopus sperm basal bodies. (5) h76p is necessary for aster nucleation by sperm basal bodies and recombinant h76p partially replaces endogenous 76p in oocyte extracts. Surprisingly, h76p shares partial sequence identity with human centrosomal proteins h103p and h104p, suggesting a common protein core. Hence, human gamma-tubulin appears associated with at least three evolutionary related centrosomal proteins, raising new questions about their functions at the molecular level.

Decrease of bradykinin-induced glomerular contraction in diabetic rat: a new cellular interpretation.

The contractile response to bradykinin (BK), measured by the reduction of the planar surface area, was studied in glomeruli and mesangial cells (MC) isolated from diabetic rats (D) one week after diabetes induction with injection of streptozotocin (STZ; 60 mg kg-1, i.p.). Results were compared with age and weight-matched untreated rats (N) and were expressed by two parameters of cell activity, the mean maximum contraction (MMC) and the proportion of contractile cells (PCC). Glomerular and mesangial contraction were found to be clearly reduced in diabetic rats in response to 100 nM BK. The lower contractile response was associated with a decrease of both glomerular calcium uptake and mesangial cell intracellular calcium mobilization. The fact that cell pretreatment with two protein kinase C (PKC) inhibitors, phorbol 12-13 myristate acetate and calphostin, lowered normal cell contraction at the level of that found in diabetic MC without any significant effect in the latter, suggests the involvement of a PKC pathway, perhaps by a decrease of activatable PKC in diabetes. In addition, our results led to the first description of a possible role of the kallikrein-kinin system in the early glomerular hemodynamic changes occurring in diabetes. Insulin (1-200 nM) increased the contractile response of cultured diabetic cells (MMC), and in this case, it also increased the PCC. It must be stressed that the effect of 1 nM insulin on the former (88% increase) was very much smaller than its effect on the latter (103% increase). The combination of the two parameters (contraction index, CI) provided a realistic evaluation of the contractile capacities of the cell population of the cultures as a whole. The differences in this index between normal and diabetic cell populations, in the absence or presence of insulin, were strictly parallel to those found in intact glomeruli. Finally, our results further confirm (Ouardani et al., Biol. Cell 86, 127, (1996)) the limit of the first five cell passages within which cultured MC can be reasonably used for the study of contractile abnormalities occurring in the early steps of diabetic state.

Loss of differences in mesangial cell phenotype between diabetic and normal rats: role of culture passages.

In mesangial cells (MC) isolated from streptozotocin (STZ)-induced diabetic rat kidneys, sensitivity to bradykinin (BK) for the induction of cell division and collagen synthesis, was found to be lower than in normal MC. Nevertheless, decreased activities could be reverted in vitro by insulin, at non-proliferative concentration (Girolami et al (1995), Can J Physiol Pharmacol 73, 848-853). The aim of the present study was to determine whether differences in the properties of diabetic MC could be ascribed to the diabetic state per se, and/or to experimental conditions, ie culture replating. Through successive cell replating, normal and diabetic types of MC were compared in terms of proliferation, contraction, free calcium concentration in response to KCl depolarization, and in relation to the expression of two cytoskeleton proteins specific to muscle cells, myosin and dystrophin. Studies of proliferation, contraction and free calcium concentration consistently showed that passage 5 was a limit beyond which differences between the two MC types were very small and sometimes non-significant. We found that the mean maximum contraction (MMC) and especially the proportion of contractile cells (PCC) among diabetic cells was lower than in normal MC. In addition, loss in proliferation activity and in [Ca2+]i concentrations were also found to occur during these five early passages. Dystrophin, a new marker of contractile phenotype recently described in smooth muscle cell (Leis et al (1994) Cell Biol Toxicol 10, 305), was first localized in MC and was compared with myosin also expressed in MC. However, during the course of cell replating and/or with the diabetic state, no visible quantitative changes were detected in the expression of the two contractile proteins. We conclude that cultured mesangial cells undergo phenotype modulations, as observed in other cells, in particular smooth muscle cells and consequently, comparative studies between normal and diabetic MC should not be carried out after the 5th passage.

Comparison of B1 and B2 receptor activation on intracellular calcium, cell proliferation, and extracellular collagen secretion in mesangial cells from normal and diabetic rats.

The mesangial cell is a contractile secreting cell found in a key position in the renal glomerulus. Several kidney and systemic diseases are associated with dysfunctions of the mesangial cells. We compared the effect of bradykinin (BK; B2 agonist) and des-Arg9-bradykinin (DBK; B1 agonist) on intracellular calcium mobilization, cell proliferation, and collagen secretion of mesangial cells from normal and streptozotocin-induced diabetic rats. Stimulation of mesangial cells with BK and DBK caused an increase in intracellular calcium (Ca2+). However, the patterns of the Ca2+ increases induced by BK and DBK were different, indicating that DBK induced a major Ca2+ influx, whereas BK preferentially released Ca2+ from intracellular pools. Stimulation with BK and DBK did not show any heterologous desensitization, thus indicating the presence of two distinct binding sites. In normal cells, DBK stimulated cell proliferation more than BK, and this action was potentiated by insulin. No effect of BK or DBK was found in cells harvested from diabetic rats. The proliferation effect of BK and DBK was restored by insulin. DBK stimulated more collagen synthesis than BK in normal cells. In cells harvested from diabetic rats the collagen secretion was increased, but BK and DBK no longer had any effect. Insulin reduced basal collagen secretion in normal cells and cells harvested from diabetic rats. Insulin also blunted stimulation by BK and DBK in normal cells but did not restore the response to BK and DBK in cells harvested from diabetic rats. Our results show that the sensitivity to DBK and BK decreases during the course of insulin-dependent diabetes, indicating modulation by insulin.

Cystatin C secretion by rat glomerular mesangial cells: autocrine loop for in vitro growth-promoting activity.

Cystatin C, the major inhibitor of the cysteine proteinases found in human and rat body fluids, is particularly abundant in seminal plasma and cerebrospinal fluid. In a precedent report, we have evidenced noteworthy levels of cystatin C in rat kidney cortex. In the present study, we show that rat mesangial glomerular cells produce cystatin C. Immunoprecipitation of extracts of metabolically labeled cells and culture media showed that the synthesized cystatin C is a 15.5 +/- 0.5 kDa protein. The protein was released into the culture supernatant (1.6 +/- 0.26 micrograms/10(6) cells/24 h). Urinary rat cystatin C and PPPR synthetic peptide (5-8 N-terminal sequence of rat cystatin C) increased mesangial cell proliferation. Affinity chromatography on Ultrogel-avidin-biotin-PPPR of extracts of metabolically labeled cells indicate the existence of a PPPR binding protein of 46 kDa. The results described in this work suggest, for glomerular rat mesangial cells in vitro, an autocrine regulation of proliferation by cystatin C.

Cystatin C levels in sera of patients with human immunodeficiency virus infection. A new avidin-biotin ELISA assay for its measurement.

A solid-phase enzyme-linked immunosorbent assay (ELISA) for determining human serum cystatin C is described. In 50 normal samples, cystatin C concentration was 1247 +/- 224 micrograms/L (mean +/- SD) which is in agreement with previously reported levels. Serum levels of cystatin C and beta 2-microglobulin (beta 2-M) were investigated in a time-course study during the development of human immunodeficiency virus (HIV) infection. We found a persistent and uniform increase in the beta 2-M concentration (2762 +/- 1239 micrograms/L). In contrast to beta 2-M, on the basis of cystatin C levels, we found two distinct populations, one of which demonstrated an increased concentration (1620 +/- 618 micrograms/L). Interestingly a second group (21% of patients) exhibited an initial significant decrease in cystatin C concentration with a mean value of 377 (range 55-850) micrograms/L, followed by an increase. The biphasic pattern of cystatin C serum, a major cysteine proteinase inhibitor, during the course of HIV infection suggests a possible role for these proteinases (or proteinase inhibitors) in the development of this syndrome.

Neutrophil chemotactic activity is modulated by human cystatin C, an inhibitor of cysteine proteases.

Cystatin C, a cysteine proteinase inhibitor has recently been suggested to be a potent regulator of inflammatory processes and may act in defense against viral and bacterial infections. Two common forms of the protein were purified from the urine of a patient having received a renal transplant. The slow form of cystatin C possessed the N-terminal tetrapeptide Lys Pro Pro Arg, which was cleaved in the fast form. This peptide sequence, called postin, was synthesized. The three molecules, slow and fast forms of cystatin and the synthetic peptide, were tested for their effects on the migration activity of human polymorphonuclear neutrophils (PMNs). The slow form was found to display both chemotactic and chemokinetic activities, while the fast form and postin were only chemokinetic. Nevertheless, all the substances could induce a "motile" morphology. In addition, the two forms of cystatin C were powerful inhibitors of PMN chemotaxis induced by complement-derived chemotactic factors. This suggests that cystatin C in its two different cleaved forms and the N-terminal tetrapeptide can modulate PMN locomotion. Cysteine proteases may therefore play a role in neutrophil migration activity.

Modulation of phagocytosis-associated respiratory burst by human cystatin C: role of the N-terminal tetrapeptide Lys-Pro-Pro-Arg.

Cystatin C, a cysteine protease inhibitor, has recently been suggested to be a potent regulator in inflammatory processes. Human cystatin C was isolated from the urine of one patient suffering from tubular disorders and was tested for its effects on two functions of human polymorphonuclear neutrophils (PMN): O2- release and phagocytosis. Slow-form or (des 1-4) cystatin C and fast-form or (des 1-8) cystatin C differed by the presence in (des 1-4) cystatin C only of the N-terminal tetrapeptide Lys-Pro-Pro-Arg. Whereas (des 1-8) cystatin C did not seem to interfere with PMN functions at physiological concentrations, (des 1-4) cystatin C induced an inhibition of PMN phagocytosis-associated respiratory burst in response to opsonized zymosan particles. The inhibition may be attributed to the tetrapeptide Lys-Pro-Pro-Arg which has been synthesized and shown to have the same inhibitor effects, at concentrations similar to those required for (des 1-4) cystatin C. These results support a potential role for cystatin C as a modulator during inflammation.

Direct radioimmunoassay of rat cystatin C: increased urinary excretion of this cysteine proteases inhibitor during chromate nephropathy.

Rat cystatin C was purified to apparent homogeneity from rat urine after induction of a tubular dysfunction with sodium chromate. The two-steps purification procedure included a Carboxymethyl-papain affinity chromatography and anion exchange chromatography. The purified protein was identified as rat cystatin C by the following criteria: firstly retained on a Cm-papain affinity column, secondly an apparent molecular weight of 15 kDa and pI of 10.2. Antisera raised in rabbits against our purified rat cystatin C did not cross-react with other urinary proteins such as rat albumin and rat kallikrein, but partially cross-reacted with human cystatin C. A direct radioimmunoassay was developed and it enabled 8.32 fmol/ml of rat cystatin C to be detected. The detection range was between 0.125 and 62.5 ng/ml, with 10% intra-assay variation and 14% inter-assay variation. Physiological rat cystatin C excretion (40 +/- 18 micrograms/24 h) was found by the direct assay. In the chromate-intoxicated rat, urinary excretion increased twenty-fivefold (1017 +/- 391 micrograms/24 h) and returned to normal level one week after intoxication. This RIA will allow the study of rat cystatin C metabolism particularly during renal dysfunction.

A rapid two-step purification of rat cystatin C, one major inhibitor of cysteine proteinases.

Rat cystatin C was purified to apparent homogeneity from rat urine after induction of a tubular dysfunction with sodium chromate. Twentyfold concentrated urine was chromatographed by a rapid purification procedure. A two-step purification including affinity chromatography on carboxymethyl papain- Sepharose and high-resolution anion exchange chromatography was developed. The purified protein has an apparent molecular mass of 15 kDa and pI of 10.2; its aminoacid composition was similar to human cystatin C. As opposed to previous data, purified urinary rat cystatin C did not contain significant amounts of carbohydrate. Antisera against rat cystatin C, raised in rabbits, partially cross-reacted with human and mouse cystatin C, indicating their antigenic similarities. Like human cystatin C, native rat cystatin C, named slow form, is degraded into a more acidic form, called fast form, by a loss of N-terminal amino acids; fast form displayed a pI of 9.4.

Relationship between cellular adhesiveness and metastatic activity in polyomavirus-transformed FR3T3 rat cell lines.

A series of polyomavirus-transformed rat cells with varying tumorigenic potential were tested for biophysical parameters possibly related to metastatic properties: adhesive capacity and strength of adhesion to different substrates (laminin, fibronectin and albumin), cell deformability and spreading. Two groups of cell lines were defined according to their higher or lower adhesive capacity. Adhesivity did not appear to be related to cell deformability and spreading. A weak correlation was suggested between low adhesivity and high metastatic potential. A selection method was devised to separate cell samples into 3 subpopulations with different adhesive strength. Two cell lines, originally different, were chosen for this study: Py-tsa A25 cells were less adherent and highly metastatic, and Py-WTA2 cells were more adherent and less metastatic. After s.c. inoculation into syngeneic Fisher rats, the 3 selected subpopulations of the 2 cell lines induced pulmonary nodules to varying degrees, but only the less adherent ones were able to induce visceral metastasis located in stomach and intestine. In this case, animal survival time was 30% lower than for the highly adherent selected cells. After 10 culture passages, the same subpopulations were able to metastasize only in the lungs. However, when the selection procedure was repeated, the less adherent cells were again able to yield visceral nodules. Tumorigenicity remained unchanged in all cases. Study of cell dissemination and arrest in vivo showed a rapid targeting of labelled tumor cells toward lungs and stomach 5 hr after intradermal injection, where they remained up to 72 hr. More adherent cells displayed delayed localization after injection (24 hr) and radioactivity decreased more rapidly.

Alloantibody bipolar bridging; a new mechanism of cell surface activation.

Bipolar bridging of cellular membrane receptors and epitopes by alloantibodies (Fab bridging the MHC antigens and Fc the Fc receptors) has been shown on a murine mast cell model to be a way of cell signaling and activation. In order to test a possible general significance of this phenomenon, another model was studied, namely guinea pig neutrophils. It was found l(1) that neutrophils from S2, S13 and BIO-AD strains both express class I (B) and class II (Ia) antigens on their surface, as detected by a Prot.A-SRBC rosetting method, after cell incubation with related alloantibodies; (2) that Fc receptors for IgG (Fc gamma R) were specific for IgG2 subclass, as determined by the same rosetting method after binding of preformed immune complexes (IgG1, IgG2 and F(ab')2 anti-DNP-DNP25 BSA); and (3) that specific alloantibodies of IgG2 subclass were able to specifically activate the neutrophil oxidative metabolism as shown by superoxide anion (O2-) release, detected by the luminol-dependent chemiluminescence method. Neither the IgG1 nor F(ab')2 portion were able to trigger O2- release. This demonstrates a second situation of a cell membrane activation through alloantibody bipolar bridging.

Chemotaxis inhibition induced in polymorphonuclear neutrophils by soluble immune complexes.

Complement-independent chemotactic factor(s) may be generated in fresh guinea pig serum by contact with soluble Ab1Ag1 immune complexes. This activated serum is equally efficient in inducing an unresponsive state in polymorphonuclear neutrophils (PMN) to subsequent chemotaxis challenge with sera containing C-dependent or C-independent chemotactic factors. The unresponsiveness persists long after the removal of serum. Reagents which are inactive on complement but which prevent the generation of C-independent chemotactic factors in fresh serum (diisopropyl fluorophosphate, synthetic esters, kaolin) inhibit both the induction of PMN chemotaxis and the PMN deactivation. Conversely, serum from a guinea pig decomplemented in vivo retains its ability to generate C-independent factors active in PMN attraction and desensitization. The opposition of two pathways for the production of chemotactic factors in serum, one depending on complement, the other on the contact system of coagulation, is again emphasized. A different procedure for inducing unresponsiveness in PMN with soluble complexes in the absence of serum is also presented here.

Defective leukocytotaxia and recurrent staphylococcal infecion: deficiency of leukocytotaxia and abnormal granulocytes associated with increase serum IgE levels in an adult with recurrent staphylococcal infection.

A man who was suffering from recurrent staphylococcal infection had antecedent symptoms of severe pruritus. Laboratory investigations showed leukocytosis with eosinophilia, hyperimmunoglobulinemia of all fractions, but particularly of IgE, and a deficiency of cell-mediated immunity on in vivo testing. Phagocytosis and bactericidal activity of polymorphonuclear leukocytes were normal, but a cellular and serum-associated defect in leukocytotaxia was present. Ultrastructural changes were observed in polymorphonuclear leukocytes. Association of impaired leukocytotaxia and elevated levels of IgE is not uncommon. Recurrent bacterial infections in the patient described are probably related to defective chemotaxis.

Chemotaxis for polymorphonuclear leucocytes induced by soluble antigen- antibody complexes.

Guinea-pig soluble immune complexes formed either by simply mixing antibody and antigen in excess or by redissolving a washed immune precipitate with antigen, after incubation with fresh serum, could induce a migration of polymorphonuclear leucocytes in vitro. This chemotactic effect of soluble complexes, although less than that of insoluble complexes, persisted despite experimental changes in the specificity, the dose and the class of antibodies. Soluble complexes of various molecular compositions induced chemotaxis but the most efficient complex was of Ab 1 Ag 1 formula. Unlike larger complexes, the Ab 1 Ag 1 complexes induced little or no complement fixation. Another source of chemotactic mediators was needed, apparently related to the esterases of the contact system of coagulation.