Development of a novel, nearly insoluble antiadhesive membrane.

BACKGROUND: Sodium hyaluronate/carboxymethylcellulose (HA/CMC) is difficult to use in a moist environment because of its susceptibility to moisture. METHODS: We developed the three-layered nDM-14R membrane. The surface layers are composed of 1-lactide, glycolide and e-caprolactone copolymers. HA/CMC and nDM-14R were used in all these studies. (1) The central region of 1 × 10 cm specimens (n = 5) was moistened for 0, 5, 10, 20, 30 or 60 s, after which the tensile strength was determined; (2) one side of specimens of 1 × 10 cm (n = 5) was moistened with agar gel for 5, 10, 15 or 30 s, after which the adhesion strength was determined, and (3) Rat cecum (n = 10) was scratched, 3 × 3 cm specimens were placed on the scratched area, and adhesions were evaluated on postoperative day 14. RESULTS AND CONCLUSION: (1) The tensile strength of nDM-14R after contact for 10-30 s was greater than that of HA/CMC. (2) The adhesive strength of HA/CMC after contact for 5-10 s was greater than that of nDM-14R. (3) Adhesion scores in treatment groups were significantly lower than in the control group. The results suggest that nDM-14R has the same antiadhesive effect and allows easier placement under moist conditions than HA/CMC.

Physical and functional interactions between STAT3 and KAP1.

Signal transducers and activators of transcription (STATs) mediate cell proliferation, differentiation and survival in immune responses, hematopoiesis, neurogenesis and other biological processes. For example, STAT3 has been reported to be constitutively activated in numerous cancer cells. To clarify the molecular mechanisms underlying the STAT activation, we performed yeast two-hybrid screening and identified KAP1/TIF1beta as a novel STAT-binding partner. KAP1 is a universal corepressor protein for the Kruppel-associated box zinc-finger protein superfamily of transcriptional repressors. We found endogenous KAP1 associated with endogenous STAT3 in vivo. Importantly, small-interfering RNA-mediated reduction of KAP1 expression enhanced interleukin (IL)-6-induced STAT3-dependent transcription and gene expression. Furthermore, reduction of KAP1 expression resulted in the marked accumulation of STAT3 phosphorylated on Ser727 in the nucleus, a modification that regulates its transcriptional activation. These results indicate that KAP1 may serve as a transcriptional regulator of the IL-6/STAT3 signaling pathway.

Glycosylation of human CRLR at Asn123 is required for ligand binding and signaling.

Calcitonin receptor-like receptor (CRLR) constitutes either a CGRP receptor when complexed with receptor activity-modifying protein 1 (RAMP1) or an adrenomedullin receptor when complexed with RAMP2 or RAMP3. RAMP proteins modify the glycosylation status of CRLR and determine their receptor specificity; when treated with tunicamycin, a glycosylation inhibitor, CHO-K1 cells constitutively expressing both RAMP2 and CRLR lost the capacity to bind adrenomedullin. Similarly, in HEK293 EBNA cells constitutively expressing RAMP1/CRLR receptor complex CGRP binding was remarkably inhibited. Whichever RAMP protein was co-expressing with CRLR, the ligand binding was sensitive to tunicamycin. There are three putative Asn-linked glycosylation sites in the extracellular, amino terminal domain of CRLR at positions 66, 118 and 123. Analysis of CRLR mutants in which Gln was substituted for selected Asn residues showed that glycosylation of Asn123 is required for both the binding of adrenomedullin and the transduction of its signal. Substituting Asn66 or Asn118 had no effect. FACS analysis of cells expressing FLAG-tagged CRLRs showed that disrupting Asn-linked glycosylation severely affected the transport of the CRLR protein to the cell surface on N66/118/123Q mutant, and slightly reduced the level of the cell surface expression of N123Q mutant compared with wild-type CRLR. But other single mutants (N66Q, N118Q) had no effect for other single mutants. Our data shows that glycosylation of Asn66 and Asn118 is not essential for ligand binding, signal transduction and cell surface expression, and Asn123 is important for ligand binding and signal transduction rather than cell surface expression. It thus appears that glycosylation of Asn123 is required for CRLR to assume the appropriate conformation on the cell surface through its interaction with RAMPs.

Replication protein A1 reduces transcription of the endothelial nitric oxide synthase gene containing a -786T-->C mutation associated with coronary spastic angina.

We recently reported that a mutation (-786T-->C) in the promoter region of the endothelial nitric oxide synthase (eNOS) gene reduced transcription of the gene and was strongly associated with coronary spastic angina and myocardial infarction. To elucidate the molecular mechanism for the reduced eNOS gene transcription, we have now purified a protein that specifically binds to the mutant allele in nuclear extracts from HeLa cells. The purified protein was identical to replication protein A1 (RPA1), known as a single-stranded DNA binding protein essential for DNA repair, replication and recombination. In human umbilical vein endothelial cells, inhibition of RPA1 expression using antisense oligonucleotide restored transcription driven by the mutated promoter sequence, whereas, conversely, overexpression of RPA1 further reduced it. RPA1 was similarly detected in placenta and eNOS mRNA levels in placentas carrying the -786T-->C mutation were significantly lower than in placentas without it. The functional importance of the diminished eNOS expression was revealed by the finding that serum nitrite/nitrate levels among individuals carrying the -786T-->C mutation were significantly lower than among those without the mutation. RPA1 thus apparently functions as a repressor protein in the -786T-->C mutation-related reduction of eNOS gene transcription associated with the development of coronary artery disease.

Genetic risk factors for coronary artery spasm: significance of endothelial nitric oxide synthase gene T-786-->C and missense Glu298Asp variants.

BACKGROUND: We recently identified two endothelial nitric oxide synthase (eNOS) gene polymorphisms, Glu298Asp and T-786-->C, which are independently associated with coronary spasm. eNOS gene intron 4b/a polymorphism is also reported to be involved in smoking-dependent coronary artery disease. The genetic linkage among these polymorphisms remains unknown. Also, it is unclear which variant is most responsible for coronary spasm. In the present study, we first examined the genetic linkage among these three variants. Next, we studied the risk factors of coronary spasm by using all significant genetic and conventional risk factors in a large-scale study. METHODS: The genotype and allele frequencies for the T-786-->C, intron 4b/a, and Glu298Asp variants were assessed in 423 randomly selected DNA samples to examine their genetic linkages. The relative capacities of all risk factors to predict coronary spasm were then analyzed using multiple logistic regression in 201 patients with coronary spasm and 345 volunteers. RESULTS: Comparison of allele frequencies revealed that the eNOS intron 4a allele was significantly linked to the T-786-->C mutation (P < 0.00001), whereas there was not a linkage between the intron 4a allele and the Glu298Asp variant (P = 0.1437) or between the Glu298Asp variant and the T-786-->C mutation (P = 0.1996). Multiple logistic regression revealed that the most predictive independent risk factor for coronary spasm was the T-786-->C mutation (P < 0.001), followed by cigarette smoking (P < 0.001), hypertension (P = 0.004), and the Glu298Asp variant (P = 0.028). CONCLUSIONS: We found that the T-786-->C mutation and the intron 4a allele are in linkage disequilibrium. We previously showed that the T-786-->C mutation reduced eNOS gene promoter activity. In that context, our results strongly suggest that the T-786-->C mutation underlies the functional characteristics of the intron 4a allele. Further, multiple logistic regression analysis revealed that the T-786-->C mutation is the most predictive risk factor for coronary spasm, followed by cigarette smoking. Given that those effects are potentially additive, patients carrying the eNOS gene variants should be strongly cautioned against smoking.

Effects of cardiotrophin-1 on hemodynamics and endocrine function of the heart.

Cardiotrophin-1 (CT-1), a member of the interleukin-6 superfamily of cytokines, possesses hypertrophic actions and atrial natriuretic peptide (ANP)-producing activity in vitro. The goal of our study is to elucidate whether CT-1 affects the cardiovascular system in vivo. Intravenous injection of CT-1 (4-100 microg/kg) in conscious rats evoked significant declines in blood pressure and reflex increases in heart rate (HR) in a dose-dependent manner. CT-1 induced no significant change in cardiac output (from 260.7 +/- 11.0 to 264.7 +/- 26.6 ml. min(-1). kg(-1), P = not significant), which was compatible with the results from isolated perfused rat hearts; HR, change in pressure over time, left ventricular developed pressure, and perfusion pressure were unaffected. Northern blot and RT-PCR analyses revealed that CT-1 increased expression of inducible nitric oxide synthase (iNOS) in lung and aorta but not in heart or liver. Pretreatment with aminoguanidine, a specific iNOS inhibitor, inhibited both iNOS mRNA production and the depressor effect of CT-1. Interestingly, CT-1 increased ventricular expression of ANP and brain natriuretic peptide (BNP). The data demonstrate that CT-1 elicits its hypotensive effect via a nitric oxide-dependent mechanism and that CT-1 induces ANP and BNP mRNA expression in vivo.

Tol1, a fission yeast phosphomonoesterase, is an in vivo target of lithium, and its deletion leads to sulfite auxotrophy.

Lithium is the drug of choice for the treatment of bipolar affective disorder. The identification of an in vivo target of lithium in fission yeast as a model organism may help in the understanding of lithium therapy. For this purpose, we have isolated genes whose overexpression improved cell growth under high LiCl concentrations. Overexpression of tol1(+), one of the isolated genes, increased the tolerance of wild-type yeast cells for LiCl but not for NaCl. tol1(+) encodes a member of the lithium-sensitive phosphomonoesterase protein family, and it exerts dual enzymatic activities, 3'(2'),5'-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase. tol1(+) gene-disrupted cells required high concentrations of sulfite in the medium for growth. Consistently, sulfite repressed the sulfate assimilation pathway in fission yeast. However, tol1(+) gene-disrupted cells could not fully recover from their growth defect and abnormal morphology even when the medium was supplemented with sulfite, suggesting the possible implication of inositol polyphosphate 1-phosphatase activity for cell growth and morphology. Given the remarkable functional conservation of the lithium-sensitive dual-specificity phosphomonoesterase between fission yeast and higher-eukaryotic cells during evolution, it may represent a likely in vivo target of lithium action across many species.

Outside-in signalling of fibronectin stimulates cardiomyocyte hypertrophy in cultured neonatal rat ventricular myocytes.

Cardiac hypertrophy involves the accumulation of extracellular matrix proteins, such as fibronectin, leading to increasing myocardial stiffness, ventricular dysfunction and heart failure. To better understand the possible role of extracellular matrix-evoked intracellular signalling in ventricular myocytes, we investigated the effect of fibronectin on myocyte hypertrophic responses using cell culture models. Cell size in myocytes cultured on fibronectin-coated dishes was three times larger than that grown on non-coated dishes. However, the number of cells on fibronectin-coated dishes was not changed throughout the experiment. Protein synthesis was significantly increased by fibronectin, as were synthesis of atrial and brain natriuretic peptides. Fibronectin also elicited actin reorganization, co-localization of beta 1 integrin and vinculin, formation of focal adhesions and tyrosine phosphorylation of focal adhesion kinase in myocytes. These fibronectin-mediated effects were inhibited in a dose-dependent manner by GRGDSP, a competitive antagonist of the fibronectin receptors; GRGDSP had no effect on cell number or viability. Blocking antibody for beta 1 and beta 3 integrin significantly suppressed fibronectin-induced secretion of natriuretic peptides. Myocyte hypertrophy was observed in myocyte-nonmyocyte co-culture that reflects more closely the myocyte environment in vivo. GRGDSP may also suppress the myocyte hypertrophic response in the co-culture. These findings demonstrate that the interaction of fibronectin and RGD-dependent integrins is involved in the hypertrophic responses of myocyte in vitro, and suggest that extracellular matrix proteins such as fibronectin are not merely passive adhesive molecules but are active participants in processes leading to myocyte hypertrophy.

Involvement of cardiotrophin-1 in cardiac myocyte-nonmyocyte interactions during hypertrophy of rat cardiac myocytes in vitro.

BACKGROUND: The mechanism responsible for cardiac hypertrophy is currently conceptualized as having 2 components, mediated by cardiac myocytes and nonmyocytes, respectively. The interaction between myocytes and nonmyocytes via growth factors and/or cytokines plays an important role in the development of cardiac hypertrophy. We found that cardiac myocytes showed hypertrophic changes when cocultured with cardiac nonmyocytes. Cardiotrophin-1 (CT-1), a new member of the interleukin-6 family of cytokines, was identified by its ability to induce hypertrophic response in cardiac myocytes. In this study, we used the in vitro coculture system to examine how CT-1 is involved in the interaction between cardiac myocytes and nonmyocytes during the hypertrophy process. METHODS AND RESULTS: RNase protection assay revealed that CT-1 mRNA levels were 3. 5 times higher in cultured cardiac nonmyocytes than in cultured cardiac myocytes. We developed anti-CT-1 antibodies and found that they significantly inhibited the increased atrial and brain natriuretic peptide secretion and protein synthesis characteristic of hypertrophic changes of myocytes in the coculture. In addition, non-myocyte-conditioned medium rapidly elicited tyrosine phosphorylation of STAT3 and induced an increase in natriuretic peptide secretion and protein synthesis in cultured cardiac myocytes; these effects were partially suppressed by anti-CT-1 antibodies. Finally, the hypertrophic effects of CT-1 and endothelin-1, which we had previously implicated in the hypertrophic activity in the coculture, were additive in cardiac myocytes. CONCLUSIONS: These results show that CT-1 secreted from cardiac nonmyocytes is significantly involved in the hypertrophic changes of cardiac myocytes in the coculture and suggest that CT-1 is an important local regulator in the process of cardiac hypertrophy.

The RAMP2/CRLR complex is a functional adrenomedullin receptor in human endothelial and vascular smooth muscle cells.

Adrenomedullin, a potently hypotensive peptide isolated from human pheochromocytoma, is known to elicit a rise in cAMP levels within mammalian endothelial and smooth muscle cells. Until now, however, little has been known about the adrenomedullin receptor. Recently, a group called receptor activity-modifying proteins that complex with the calcitonin receptor-like receptor, and thereby regulate its transport and ligand specificity, were identified. Here we show that mRNA for both the calcitonin receptor-like receptor and the receptor activity-modifying protein 2, but not the receptor activity-modifying protein 1 or receptor activity-modifying protein 3, are expressed in human endothelial and vascular smooth muscle cells. We also found that adrenomedullin increased cAMP levels in HeLa EBNA and 293 EBNA cells, expressing both the receptor activity-modifying protein 2 and calcitonin receptor-like receptor proteins. Thus, the receptor activity-modifying protein 2/calcitonin receptor-like receptor complex apparently serves as a functional adrenomedullin receptor in human endothelial and vascular smooth muscle cells.

Endothelial nitric oxide synthase gene is positively associated with essential hypertension.

Essential hypertension has a genetic basis. Accumulating evidence, including findings of elevation of arterial blood pressure in mice lacking the endothelial nitric oxide synthase (eNOS) gene, strongly suggests that alteration in NO metabolism is implicated in hypertension. There are, however, no reports indicating that polymorphism in the eNOS gene is associated with essential hypertension. We have identified a missense variant, Glu298Asp, in exon 7 of the eNOS gene and demonstrated that it is associated with both coronary spastic angina and myocardial infarction. To explore the genetic involvement of the eNOS gene in essential hypertension, we examined the possible association between essential hypertension and several polymorphisms including the Glu298Asp variant, variable number tandem repeats in intron 4 (eNOS4b/4a), and two polymorphisms in introns 18 and 23. We performed a large-scale study of genetic association using two independent populations from Kyoto (n=458; 240 normotensive versus 218 hypertensive subjects) and Kumamoto (n=421; 223 normotensive versus 187 hypertensive subjects), Japan. In both groups, a new coding variant, Glu298Asp, showed a strong association with essential hypertension (Kyoto: odds ratio, 2.3 [95% confidence interval, 1.4 to 3.9]; Kumamoto: odds ratio, 2.4 [95% confidence interval, 1.4 to 4.0]). The allele frequencies of 298Asp in hypertensive subjects were significantly higher than those in normotensive subjects in both groups (Kyoto: 0.103 versus 0.050, P<0.0017; Kumamoto: 0.120 versus 0.058, P<0.0013, respectively). No such disequilibrium between genotypes was significantly associated with any other polymorphisms we examined; the Glu298Asp variant was also not linked to any other polymorphisms. In conclusion, the Glu298Asp missense variant was significantly associated with essential hypertension, which suggests that it is a genetic susceptibility factor for essential hypertension.

Endothelin-1 and cardiotrophin-1 induce brain natriuretic peptide gene expression by distinct transcriptional mechanisms.

Cardiotrophin-1 (CT-1) a novel IL-6-related cytokine, induces distinct hypertrophic responses to endothelin-1 (ET-1) on cultured neonatal rat cardiac myocytes. We found that ET-1 and CT-1 show a distinct pattern of gene induction of natriuretic peptides. Elucidation of the transcriptional mechanisms of brain natriuretic peptide (BNP) gene induction by. ET-1 or CT-1 will provide better information for our understanding of the molecular mechanisms of cardiac hypertrophy. In this study, reporter constructs containing the human BNP 5' flanking sequence were transfected into neonatal rat cardiac myocytes and the cells were stimulated with ET-1 or CT-1. A total of 1813 bp of the human BNP 5' flanking sequence conferred an ET-1 inducibility on the reporter gene. However, it did not confer CT-1 inducibility. These results show that distinct mechanisms are involved in BNP gene induction by ET-1 or CT-1, and (in this study) that the CT-1 responsive element is not located in the region examined.

Interaction of myocytes and nonmyocytes is necessary for mechanical stretch to induce ANP/BNP production in cardiocyte culture.

In cardiac hypertrophy or ventricular remodeling, enlargement of myocytes and interstitial or perivascular fibrosis are observed simultaneously, which suggests an interaction between cardiac myocytes and fibroblasts. In this study we examined the mechanism of cyclic mechanical stretch-induced myocytic hypertrophy, focusing on the interaction between myocytes and cardiac nonmyocytes, mostly fibroblasts. Ventricular myocytes (MCs) and cardiac nonmyocytes (NMCs) were separately extracted from neonatal rat ventricles by the discontinuous Percoll gradient method and primary cultures of cardiac cells were prepared. When MCs were co-cultured with NMCs, the size of MCs and the ANP/BNP secretion were significantly increased. This hypertrophic change of MCs in the co-culture was significantly suppressed by BQ-123, an endothelin-A (ETA) receptor antagonist. Cyclic stretch did not induce hypertrophic responses in MC culture. However, it further increased ANP/BNP production in MC-NMC co-culture (2.2-fold and 2.1-fold increases vs. non-stretch group after 48-h incubation). This increase in ANP/BNP production in the co-culture was significantly suppressed by CV-11974, an angiotensin II (Ang II) type 1 receptor antagonist. This study raises the possibility that NMCs regulate cardiocyte hypertrophy via secretion of endothelin-1 and that Ang II is involved in the interaction between MCs and NMCs during the course of hypertrophic response of cardiocytes to mechanical stretch.

Vertical stab wound to the lumbo-sacral spinal canal: report of a case.

A 63-year-old man was stabbed by an iron bar in the perineum. On admission, the external aspect of this wound appeared to be only a tiny hole. However, the bar had penetrated the rectum, sacrum, and lumbar spinal canal up to the third lumbar vertebral body. This patient was eventually treated with sacral wound drainage, a closure of the rectal wound, and pelvic drainage. Lumbar surgery was not performed because computed tomography (CT) and magnetic resonance imaging (MRI) did not show a progression of inflammatory changes in either the lumbar canal or perispinal hollow caused by the iron bar during the hospital stay. The clinical, CT, and MRI findings thus provided important information in the diagnosis and treatment of stab wounds.

Role of cardiac nonmyocytes in cyclic mechanical stretch-induced myocyte hypertrophy.

In cardiac hypertrophy or ventricular remodeling, not only the enlargement of myocytes but also interstitial or perivascular fibrosis are observed simultaneously, which suggests an interaction between cardiac myocytes and fibroblasts. In this study, we examined the mechanism of cyclic mechanical stretch-induced myocyte hypertrophy, highlighting the interaction between myocytes and cardiac nonmyocytes, mostly fibroblasts. Ventricular myocytes (MC) and cardiac nonmyocytes (NMC) were separately extracted from neonatal rat ventricles by the discontinuous Percoll gradient method and primary cultures of cardiac cells were prepared. Cyclic mechanical stretch was applied to the cultures with a Flexercell Stress Unit. In addition to cell size, we examined atrial natriuretic peptide/brain natriuretic peptide (ANP/BNP) production as the most sensitive biological markers for MC hypertrophy. Cyclic stretch did not induce hypertrophic responses in MC when they were cultured without NMC. In contrast, when MC were co-cultured with NMC, cyclic stretch induced further increase in ANP/BNP production (2.2-fold and 2.1-fold increases versus non-stretch group, after 48-h incubation). This increase in ANP/BNP production in the co-culture was significantly suppressed by CV-11974, an angiotensin II type 1 receptor antagonist. Moreover, ANP/BNP production in the co-culture was significantly suppressed by BQ-123, an endothelin A receptor antagonist, whether cyclic stretch was applied or not. This study raised the possibility that NMC mediate the hypertrophic effect of mechanical stress on MC by increasing endothelin production. It was also suggested that, in this process, angiotensin II is involved in the crosstalk between MC and NMC.

The gene encoding a new mitogenic factor in a Streptococcus pyogenes strain is distributed only in group A streptococci.

We recently cloned a gene encoding a new mitogenic factor (MF) from Streptococcus pyogenes NY-5. In the present study, we determined the distribution of this MF gene (mf) by PCR based upon its sequence. Of 371 streptococcal group A strains isolated from clinical specimens, 370 (99.7%) were positive for mf. The strain that was negative for the MF gene was also negative for the streptolysin O gene (slo). Some streptococcal strains belonging to groups C and G were negative for mf but positive for slo. Group B strains were negative for both. Furthermore, we examined the presence of mf in 54 strains belonging to 28 families and found mf only in group A streptococci. These results indicate that mf is distributed specifically in group A streptococci and the presence of mf in clinical samples strongly suggests infection with group A streptococci.

Therapeutic effect of neuraminidase-treated LAK cells on liver metastasis of Colon 26.

To improve the lymphokine-activated killer (LAK) cell therapy for liver metastasis, two methods which enhance accumulation of LAK cells in the liver were examined for their effects on the liver metastasis of Colon 26 cancer cells in BALB/c mice. Distribution of LAK cells in the mice was examined by the 51Cr labeling method. Portal vein infusion of LAK cells or tail vein infusion of neuraminidase treated-LAK (N-LAK) cells showed an augmented accumulation of infused cells in the liver. In the first experiment, LAK cells (5 x 10(7) cells) were infused in the portal vein or tail vein at days 3 and 7 after the inoculation of 5 x 10(4) tumor cells and 1 x 10(4) units of IL-2 were given three times a day from day 3 to day 7. The portal infusion of LAK cells produced a greater reduction of liver metastases compared with the peripheral infusion. In the second experiment, 5 x 10(7) LAK cells or N-LAK cells were infused via the tail vein on days 1 and 3, and 1 x 10(4) units of IL-2 were given once a day from day 1 to day 5 after the inoculation of 1 x 10(4) tumor cells. The therapeutic effect of N-LAK cells was greater than non-treated LAK cells on the number of metastatic lesions and the survival time of mice. Since access to the human portal vein is difficult and risky in clinical situation, peripheral infusion of N-LAK cells is preferable.

In vitro catalysis of oxidative folding of disulfide-bonded proteins by the Escherichia coli dsbA (ppfA) gene product.

It was shown previously that the Escherichia coli gene ppfA (dsbA) encodes a periplasmic protein, and its inactivation leads to a deficiency in disulfide bond formation of envelope proteins (Kamitani, S., Akiyama, Y., and Ito, K. (1992) EMBO J. 11, 57-62; Bardwell, J. C. A., McGovern, K., and Beckwith, J. (1991) Cell 67, 581-589). The DsbA/PpfA protein was overproduced, purified, and examined for its activities in vitro. Its abundance in a wild-type cell was estimated to be about 850 molecules which probably exist as homodimers as suggested by size exclusion chromatography. Purified DsbA markedly stimulated disulfide bond formation of E. coli alkaline phosphatase, either in vitro synthesized or purified and denatured, as well as of reduced bovine ribonuclease A. The DsbA-catalyzed rapid disulfide bond formation occurred after a lag period which appeared to be determined by the redox state of the reaction mixture and concentration of DsbA. Inclusion of higher concentrations of oxidized glutathione or DsbA shortened the lag period. We propose that DsbA, which proved to directly catalyze disulfide bond formation, may also have a role in maintaining the bacterial periplasm oxidative.

Identification and characterization of an Escherichia coli gene required for the formation of correctly folded alkaline phosphatase, a periplasmic enzyme.

Tn5 insertion mutations of Escherichia coli were isolated that impaired the formation of correctly folded alkaline phosphatase (PhoA) in the periplasm. The PhoA polypeptide synthesized in the mutants was translocated across the cytoplasmic membrane but not released into the periplasmic space. It was susceptible to degradation by proteases in vivo and in vitro. The wild-type counterpart of this gene (named ppfA) has been sequenced and shown to encode a periplasmic protein with a pair of potentially redox-active cysteine residues. PhoA synthesized in the mutants indeed lacked disulfide bridges. These results indicate that the folding of PhoA in vivo is not spontaneous but catalyzed at least at the disulfide bond formation step.