Search

Background: We determined the diagnostic value of the trypsin inhibitor, uristatin, that is commonly found in urine and plasma in patients with infections or inflammations of any kind., Methods: We collected urine specimens from patients with infections of the urinary or upper respiratory tract and from healthy controls. We also collected blood from patients with a likely upper respiratory tract infection and healthy controls. A bacterial count of >10(5) organisms/ml in urine was considered to represent infection rather than contamination., Results: The uristatin dipstick test in urine showed acceptable negative predictive values (NPV of up to 93%) for patients without infection or inflammation. Here, the dipsticks could eliminate some urine cultures. For those with infection or inflammation, the positive predictive values (PPV) of the dipsticks were lower (up to 57%). Including the leukocyte esterase and nitrite values increased the PPV of the dipsticks for those with disease., Conclusions: The uristatin strip was more accurate than the leukocyte and nitrite dipsticks for predicting upper respiratory infections (URI) and C-reactive protein for those with infection or inflammation. The uristatin dipstick was able to detect both the bikunin and uristatin inhibitors.

We previously reported a novel small gene, designated hepatocyte growth factor activator inhibitor type 2 (HAI-2) related small peptide (H2RSP), in the process of the search for splicing variant forms of HAI-2 by 3(')-rapid amplification of cDNA ends method [Biochem. Biophys. Res. Commun. 288 (2001) 390]. Human H2RSP gene consisted of four exons spanning approximately 1kbp and was located in 11kbp downstream of HAI-2 gene. In this study, we cloned and characterized the mouse counterpart of H2RSP gene, which was located in 6.6kbp downstream of mouse HAI-2 gene, and analyzed the transcripts generated from both genes. Similar to human, mouse H2RSP mRNA (0.5kb) was detected abundantly in various tissues including the gastrointestinal tract, and has nuclear localization signal (NLS) in the lysine-rich region (exon 4), which was well-conserved between human and mouse genes. However, chimeric mRNA transcribed from both HAI-2 (exons 1-7) and H2RSP (exons 2-4) genes, which was found in the kidney, prostate, and placenta of human by Northern blot analysis, was not detected in mouse tissue even by a reverse transcription-polymerase chain reaction (RT-PCR). Instead of the chimeric mRNA, a novel splicing variant lacking putative transmembrane domain of HAI-2 was found in mouse but not in human as a putative secrete form of HAI-2. These results suggest that the organization of H2RSP and HAI-2 gene complex is well-conserved, but the usage of these genes was quite different between human and mouse.

Background: Renal cells are not fully differentiated at birth, representing a major risk in preterm infants. We evaluated glomerular and tubular functional integrity as well as structural integrity of renal tubules among healthy full-term and preterm infants as well as diseased preterm infants., Methods: A total of 50 newborns (10 healthy full-term, 10 healthy preterm, and 30 diseased preterm, at 38.9 +/- 1.10, 34.2 +/- 0.92, and 32 +/- 2.47 weeks gestational age, respectively) were included in the present study. Glomerular function was assessed by measuring urinary levels of both microalbumin and immunoglobulin G as well as serum creatinine levels, whereas the proximal tubular function was investigated by measuring the urinary levels of both alpha1-microglobulin and beta2-microglobulin as well as retinol-binding protein. Also, distal tubular reabsorption capacity was investigated by assessing fractional excretion of sodium. Moreover, the structural integrity of renal proximal tubules was studied by measuring the urinary activities of both the brush-border membrane enzyme leucine-aminopeptidase (LAP) and the lysosomal enzyme N-acetyl-beta-D-glucosaminidase. The preceding investigations were done on both the first and third days of life of all 50 newborns., Results: Glomerular and tubular function and structure was relatively impaired at birth among both healthy and diseased preterm as well as healthy full-term neonates and improved rapidly thereafter. The diseased preterm neonates showed worse renal function and structure with minimal improvement regardless of the underlying sickness., Conclusion: Renal insufficiency and renal immaturity could be evaluated using enzymuria and low- and high-molecular-weight proteinuria as noninvasive methods.

The expression pattern of the alpha(1)-microglobulin/bikunin precursor (AMBP) gene, and its two protein products were studied in mouse embryos of 8.5-15.5 days of embryonic development by in situ hybridization and immunohistochemistry. AMBP mRNA is strongly transcribed in liver parenchyma, pancreas, and intestine epithelium. Sites of weaker expression are the vessels of the umbilical cord, the developing vertebral bodies, and kidney. The alpha(1)-microglobulin and bikunin proteins are accordingly present in developing hepatocytes, pancreas, kidney, and gut. However, additional sites of protein distribution were found that do not correlate to mRNA localization: alpha(1)-microglobulin was found in myocytes and bikunin in cardiac muscle, nervous system microvasculature, and connective tissue. Both proteins were found in brain mesenchyme and meninges. Thus, a restricted expression of the AMBP mRNA in a few organs contrasts to a widespread and unique distribution of each of the two proteins., (Copyright 2002 Elsevier Science Ireland Ltd.)

alpha-1-Microglobulin (A1M) and bikunin are two plasma glycoproteins encoded by an alpha-1-microglobulin/bikunin precursor (AMBP) gene. Despite their lack of any structural or functional relationship, both A1M and bikunin originate from AMBP cleavage by a furin-like protease that releases the two mature molecules. The AMBP gene maintains a tight control over its expression by a unique enhancer, which is controlled by several hepatocyte-enriched nuclear factors; however, the mechanisms of regulation of the intracellular levels of the AMBP protein are currently unknown. We report the ability of the AMBP protein to self-associate and form a dimer in a yeast environment using the yeast two-hybrid system and an in vitro dimerization assay. We also show that the A1M protein binds to its precursor protein, AMBP, whereas bikunin does not. This observation warrants further investigations for a dimerization-dependent intracellular control that AMBP may be involved in. The relevance of AMBP dimerization and its possible biological significance are postulated.

Hepatocyte growth factor activator inhibitor type 2 (HAI-2) is a serine proteinase inhibitor containing two Kunitz-type inhibitor domains, initially identified as a potent inhibitor of hepatocyte growth factor activator (HGFA). In a previous study (Biochem. Biophys. Res. Commun. 255, 740-748, 1999), we reported that a predominant transcript of mouse HAI-2 is a splicing variant lacking the first Kunitz domain (KD-1). Since KD-1 was reported to be responsible for the inhibition of HGFA in human HAI-2 and the second Kunitz domain (KD-2) of human HAI-2 was much less inhibitory against HGFA, it has been suggested that most of mouse HAI-2 may be ineffective in inhibiting HGFA. In this study, we have performed functional characterization of Kunitz domains in mouse HAI-2 by using recombinant proteins synthesized by Chinese hamster ovary cells without or with point mutation in the putative reactive site of each Kunitz domain. The results revealed that, unlike human HAI-2, KD-2 of mouse HAI-2 efficiently inhibits HGFA. Therefore, the major mouse HAI-2 protein that consists only of KD-2 can be a potent inhibitor of HGF activation in vivo.

A novel small gene, designated hepatocyte growth factor activator inhibitor type 2 (HAI-2)-related small peptide (H2RSP) was cloned and characterized in the process of the search for splicing variant forms of HAI-2 by 3'-rapid amplification of cDNA ends (RACE). The gene consisted of 4 exons spanning approximately 1 kb and was located in 11 kb downstream of HAI-2 gene (19q.13.11). The novel transcript identified by 3'-RACE was thought to be chimerically transcribed from both HAI-2 (exons 1-7) and H2RSP (exons 2-4) genes. Wild-type H2RSP mRNA (0.5 kb) was detected abundantly in various tissues including the gastrointestinal tract, whereas chimeric mRNA (1.5 kb) was found mainly in the kidney, prostate, and placenta by Northern blot analysis. The predicted amino acid sequence of H2RSP contained two unique domains, namely the serine-rich region (exon 3) and the lysine-rich region (exon 4). Transfection of deleted series of H2RSP cDNAs fused to enhanced green fluorescent protein (EGFP) into HeLa cells revealed that H2RSP has nuclear localization signal in the lysine-rich region. Immunohistochemical study using anti-H2RSP polyclonal antibody indeed revealed the nuclear localization of this peptide in vivo. These results suggest that H2RSP and H2RSP/HAI-2 chimeric peptides might function as a transcriptional regulatory peptide at the nucleus., (Copyright 2001 Academic Press.)

Background: Our previous studies have demonstrated that renal proximal tubular epithelial cells (PTCs) may contribute to renal interstitial fibrosis by the generation of transforming growth factor-beta1 (TGF-beta1). In these in vitro experiments, TGF-beta1 was, however, secreted in its latent form. Plasmin has been implicated as a potential physiological activator of TGF-beta1. The inter-alpha-trypsin inhibitor (IalphaI) family of serum protease inhibitors together with tumor necrosis factor-stimulated gene 6 (TSG-6) recently have been implicated in the regulation of this protease pathway. The aim of the current study was to examine PTC synthesis of these proteins and to relate it to alterations of plasmin-protease activity., Methods: PTCs were grown to confluence and stimulated under serum-free conditions with either interleukin-1beta (IL-1beta) or 25 mmol/L D-glucose. Alterations in IalphaI and TSG-6 generation were detected by Western analysis of both membrane extracts and supernatant samples. Alterations in gene expression were examined by reverse transcription-polymerase chain reaction. The effect of alteration in synthesis of TSG-6 on plasmin activity was determined by quantitating plasmin inhibitory activity of supernatant samples by in vitro calorimetric assay prior to and following TSG-6 immunoprecipitation., Results: The data demonstrate that human PTCs constitutively express mRNA for bikunin and heavy chain 3 (H3) of IalphaI. Neither IL-1beta (1 ng/mL) nor 25 mmol/L D-glucose influenced their mRNA expression nor protein synthesis. In contrast, the addition of either IL-1beta or 25 mmol/L D-glucose increased TSG-6 mRNA expression. This was accompanied by an early up-regulation of TSG-6 protein expression following IL-1beta stimulation (24 h) and a late up-regulation after the addition of 25 mmol/L D-glucose (96 h) in the cell culture supernatant and associated with the cell membranes. Early induction of TSG-6 mRNA by IL-1beta was unaffected by the addition of the protein synthesis inhibitor cycloheximide. In contrast, the later glucose-stimulated induction of TSG-6 mRNA was abrogated by the addition of cycloheximide. Stimulation of TSG-6 by either IL-1beta or 25 mmol/L D-glucose was associated with an inhibition of total percentage plasmin activity. Immunoprecipitation of TSG-6 in these samples returned plasmin activity to control levels., Conclusions: : The data demonstrate that human PTCs constitutively express the bikunin and H3 components of the IalphaI family of serum protease inhibitors. Moreover, the addition of IL-1beta or 25 mmol/L D-glucose up-regulates the expression of TSG-6 in these cells, resulting in an inhibition of plasmin activity.

In recent years, significant progress has been made in uncovering the molecular basis of endoderm specification in Xenopus. Much less is understood, however, about endodermal patterning and how endoderm-derived organs such as the liver are formed. Progress has been hampered by the lack of good molecular markers of presumptive liver tissue. Here, we have examined the embryonic expression of a number of marker genes during liver organogenesis, including the transcription factors hex, sox17alpha, and hnf3beta, as well as a number of proteins specific to the adult liver. Interestingly, sox17alpha appears to specifically mark the gall bladder precursors. At 7 days of development expression of the liver differentiation markers albumin, alpha1-microglobulin/bikunin precursor, fibrinogen, transferrin and transthyretin is restricted to the differentiating liver bud. Surprisingly, however, at 3 days of development most of these genes have a more widespread endodermal expression pattern. In addition to expression in the undifferentiated liver bud they were expressed extensively throughout the presumptive intestinal tissue, which may reflect some general feature of how the hepatic gene program is developmentally regulated.

Bikunin is a Kunitz-type proteinase inhibitor, which is cross-linked to heavy chains via a chondroitin sulfate chain, forming inter-alpha-inhibitor and related molecules. Rat bikunin was produced by baculovirus-infected insect cells. The protein could be purified with a total yield of 20 mg/liter medium. Unlike naturally occuring bikunin the recombinant protein had no galactosaminoglycan chain. Endoglycosidase digestion also suggested that the recombinant form lacked N-linked oligosaccharides. Bikunin is translated as a part of a precursor, alpha1-microglobulin/bikunin, but the functional significance of the cotranslation is unknown. Our results indicate that the proteinase inhibitory function of bikunin is not regulated by the alpha1-microglobulin-part of the alpha1-microglobulin/bikunin precursor since recombinant bikunin had the same trypsin inhibitory activity as the recombinant precursor. Both free bikunin and the precursor were also functional as a substrate in an in vitro xylosylation system. This demonstrates that the alpha1-microglobulin-part is not necessary for the first step of galactosaminoglycan assembly.

The 28-kd plasma protein alpha(1)-microglobulin is found in the blood of mammals and fish in a free, monomeric form and as high-molecular-weight complexes with molecular masses above 200 kd. In this study, iodine 125-labeled free and high-molecular weight rat alpha(1)-microglobulin (a mixture of alpha(1)-microglobulin/alpha(1)-inhibitor-3 and alpha(1)-microglobulin/fibronectin complexes) were injected intravenously into rats. The distribution of the proteins was measured by using scintillation camera imaging. Both forms of (125)I-labeled alpha(1)-microglobulin were rapidly cleared from the blood, with a half-life of 2 and 16 minutes for the initial and late phase, respectively, for free alpha(1)-microglobulin; and a half-life of 3 and 130 minutes for the initial and late phase, respectively, for the complexes. After 45 minutes, 6%, 16%, 27%, 13%, and 34% of the free (125)I-labeled alpha(1)-microglobulin and 18%, 21%, 6%, 10%, and 42% of the (125)I-labeled alpha(1)-microglobulin complexes were found in the blood, gastrointestinal tract, kidneys, liver, and the remainder of the body, respectively. The local distribution of injected (125)I-labeled alpha(1)-microglobulin in intestines and kidneys was investigated by microscopy and autoradiography. In the intestine, both forms were distributed in the basal layers, villi, and luminal contents. The results also suggested intracellular labeling of epithelial cells. Well-defined local regions containing higher concentrations of injected protein could be seen in the intestine. In the kidneys, both forms were found mostly in the cortex. Free (125)I-labeled alpha(1)-microglobulin was found predominantly in epithelial cells of a subset of the tubules, whereas the (125)I-labeled complexes were more evenly distributed. Intracellular labeling was indicated for both alpha(1)-microglobulin forms. The results thus indicate a rapid transport of (125)I-labeled alpha(1)-microglobulin from the blood to most tissues.

Background: The selectivity of proteinuria, introduced in clinical nephrology in 1960 and useful in predicting steroid responsiveness in nephrotic syndrome, found little place in clinical practice in subsequent decades, since its assessment did not appear to help predict histologic diagnosis or determine prognosis. The amount of proteinuria and the degree of tubulointerstitial damage appeared to be better predictors of functional outcome. A correlation between them has been found, referred to some toxicity of proteinuria on tubular cells, but so far no single feature or component of proteinuria has been identified as being responsible for this toxicity., Methods: We evaluated 89 patients with nephrotic syndrome [9 with minimal change disease (MCD), 29 with primary focal segmental glomerulosclerosis (FSGS), and 51 with idiopathic membranous glomerulonephritis (MGN)] to determine if the selectivity of proteinuria was associated with tubulointerstitial damage. A semiquantitative grading of histologic lesions and qualitative evaluation of the "tubular" component of proteinuria expressed as a pattern of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and as fractional excretion of the low molecular weight (LMW) protein alpha1-microglobulin (FE alpha1m) were used. A second aim of the study was to assess the predictive value on functional outcome [remission or progression to chronic renal failure (CRF)] and response to therapy of the selectivity of proteinuria, considered alone and in combination with FE alpha1m., Results: Proteinuria was classified as highly selective [selectivity index (SI) < or = 0.10, N = 15], moderately selective (SI > or = 0.11 < or = 0.20, N = 34), or nonselective (SI > or = 0.21, N = 40). A significant relationship was found between the SI and the histologic degree of tubulointerstitial damage (score 0 to 1 vs. score > or =2, P = 0.000), severity of the tubular component of proteinuria (mixed SDS-PAGE pattern with LMW proteins not lower than 23 kD vs. mixed pattern with LMW proteins up to 20 to 10 kD, P = 0.000), and FE alpha1m (values below vs. above a defined cut-off, P = 0.000). The functional outcome was evaluated in 60 patients with baseline normal renal function (serum creatinine 0.97 +/- 0.19 mg/dL). The patients with high, moderate, or nonselective proteinuria had 100, 50, and 29% of complete or partial remission (P = 0.0001) and 0, 25, and 35% of progression to CRF, respectively (P = 0.050). In 45 patients with moderately selective (N = 28) and nonselective (N = 17) proteinuria, according to some arbitrary cutoffs for FE alpha1m (MGN, < or = vs. > 0. 240% of creatinine clearance; FSGS and MCD, < or = vs. > 0.350%), the remission rate was 62 versus 6% in patients with FE alpha1m below or above the cutoffs (P = 0.0001), and progression to CRF was 7 and 69%, respectively (P = 0.0001). The response to therapy (complete or partial remission at the last observation), evaluated retrospectively in 40 patients, was 100, 67, and 33% in high, moderate, and nonselective proteinuria (P = 0.0002); in 30 patients with moderate and nonselective proteinuria, according to an FE alpha1m value that was < or = or > the cutoffs, the response rate was 75 versus 10% (P = 0.001)., Conclusions: There is a significant relationship between selectivity of proteinuria and tubulointerstitial damage. Moreover, the selectivity of proteinuria has a predictive value on functional outcome. When proteinuria is highly selective, the tubulointerstitial damage is rather infrequent, and 100% of patients develop clinical remission. When proteinuria is moderately selective or nonselective, increasing numbers of patients develop tubulointerstitial damage; in these patients, the functional outcome and response to therapy is partly dependent on tubulointerstitial involvement, and the best predictor of functional outcome is the combination of SI and FE alpha1m.

Background: Nephropathia epidemica (NE) is a mild form of hemorrhagic fever with renal syndrome caused by Puumala hantavirus. Its long-term prognosis is considered favorable. Some reports suggest, however, that a previous hantavirus infection increases the risk of hypertension., Methods: We studied 46 previously healthy subjects (26 males and 20 females, mean age of 44 years) who had serologically confirmed NE three to seven years previously, and 38 healthy, seronegative controls (22 males and 16 females, mean age of 44 years). Ambulatory blood pressure (ABP) was monitored. Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were determined by 51CrEDTA and 131I-hippurate clearances, respectively. The filtration fraction (FF) was calculated. Quantitative 24-hour urinary protein excretion (UprotE) and timed overnight urinary excretion of alpha1-microglobulin were measured., Results: The NE patients had a higher mean ambulatory systolic BP than the controls (123 +/- 13 vs. 117 +/- 9 mm Hg, P = 0. 008). GFR and FF were increased in patients compared with controls (GFR, 120 +/- 20 vs. 109 +/- 14 mL/min/1.73 m2, P = 0.006; FF, 19 +/- 3 vs. 18 +/- 3%, P = 0.030), but ERPF did not differ between the groups. The patients also had higher UPE than the controls (median 0. 18 g/day, range 0.12 to 0.38 vs. median 0.14 g/day, range 0.09 to 0. 24, P < 0.001, respectively). The overnight urinary excretion rate of alpha1-microglobulin exceeded 7 microg/min in nine patients., Conclusion: Three to seven years after NE, the patients had higher GFR and FF, more proteinuria, and higher ambulatory systolic BP compared with the healthy controls. NE may thus cause mild renal lesions and alterations in BP in some patients.

Inter-alpha-inhibitor (IalphaI) is a human plasma serine proteinase inhibitor. It contains one light peptide chain called bikunin that exerts antiproteinase activity and other antiinflammatory functions. Bikunin is covalently linked to two heavy chains that, after tissular diffusion, stabilize the extracellular matrix. Owing to its negative acute-phase reactant character and its susceptibility to proteolysis, IalphaI has been implicated in the pathophysiology of sepsis. Moreover, IalphaI has been shown to exert a protective effect on a pig model of endotoxic shock. Twenty patients admitted to the intensive care unit (ICU) for a septic syndrome were included in the present study. IalphaI and antithrombin III (ATIII) levels were measured on admission. Sequential measurements of IalphaI could be done in 4 patients. We demonstrate that IalphaI levels are significantly decreased in plasma samples collected on admission from patients with sepsis (59 +/- 32 mg/L vs 241 +/- 70 mg/L; P < .0001). This decrease was greater in severe sepsis and septic shock than in sepsis. Death was not predictable from initiol IalphaI levels. In 2 patients with a favorable course, IalphaI values regularly increased during the ICU stay. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot analysis and microsequencing, we characterized IalphaI-related components in plasma from several patients; they obviously arise from IalphaI through proteolytic cleavage. Thus, systemic proteolysis and decreased biosynthesis both contribute to the fall in the plasma level of IalphaI. Because IalphaI is very sensitive to proteolysis by polymorphonuclear granulocytes (PMNs) that are stimulated during sepsis, we suggest that IalphaI plasma level would be a useful marker for neutrophil proteinase activity. ATIII, as well as IalphaI, is considered a negative acute phase protein. Because in vitro ATIII is less susceptible than IalphaI to proteolysis by PMNs and because their relative levels weakly correlated, we suggest that an unspecific systemic proteolysis is not significantly involved in the ATIII deficiency occurring in sepsis.

Bikunin is a plasma proteinase inhibitor that has received little attention in the past, probably because its activity towards various proteinases was found to be relatively weak in early work. It was recently discovered, however, that bikunin effectively inhibits a proteinase that seems to be involved in the metastasis of tumour cells--cell surface plasmin--and that a fragment of bikunin inhibits two proteinases of the coagulation pathway--factor Xa and kallikrein. Furthermore, it has been found that bikunin has other properties, such as the ability to modulate cell growth and to block cellular calcium uptake. Most of the bikunin in the blood occurs as a covalently linked subunit of the proteins pre- and inter-alpha-inhibitor. In this form bikunin lacks some of its known activities, and there is evidence that its release by partial proteolytic degradation may function as a regulatory mechanism. Although the physiological function of bikunin still remains to be established, current data suggest that this protein plays a role in inflammation. Further studies could therefore lead to results of therapeutical value.

Bikunin, which is an inhibitor of serine proteases, is widely distributed in human tissues, including liver, kidney, and mucous membranes of the stomach and colon. The aim of this study was to clarify whether bikunin is expressed in human epidermis and its appendages. Immunoblot analysis using a specific polyclonal antibody to bikunin revealed that a single 43 kDa protein is present in the cell lysate from the human keratinocyte cell line HaCaT. Immunohistochemically, dotted reaction products stained with anti-bikunin antibody were localized on the cell boundary in both basal and spinous cell layers, except on the cell boundary of the basal cells facing the basal membrane. There were no reaction products in the granular-horny cell layers. Reaction products stained with anti-bikunin antibody were also observed on the hair bulb cells and eccrine sweat gland cells, but not on apocrine sweat glands. Also, reaction products were observed on the luminal surface of the renal proximal tubules and in the cytoplasm of these cells. In immunoelectron microscopy, gold particles were observed on the cell membranes close to the desmosomal structures. Reverse transcription-polymerase chain reaction and northern blot analyses showed that mRNA specific for bikunin was expressed in HaCaT cells and human epidermal keratinocytes obtained from suction blisters, and was contained in a commercially available human keratinocyte cDNA preparation. These findings indicate that bikunin is expressed in keratinocytes and may play an important part in regulating keratinocytes in either mitosis or inflammation.

A homozygous cross-reacting material negative factor XII-deficient patient with 3% antigen and activity levels of factor XII was screened for the identification of a mutation at the genomic level. Low-ionic strength single-stranded conformation polymorphism (SSCP) analysis and sequence analysis showed that the proband's gene for factor XII had an A-->G substitution at nucleotide position 7832 in exon 3, resulting in a Tyr34 to Cys substitution in the NH2-terminal type II domain of factor XII. We designated this mutation as factor XII Tenri. Mutagenic polymerase chain reaction (PCR), followed by KpnI digestion, showed a homozygous mutation in the proband's gene and heterozygous mutations in his parents and sister. Immunoprecipitation and Western blot analyses of plasma samples from the factor XII Tenri family indicated that the proband had a trace amount of variant factor XII with an apparent molecular mass of 115 kD, which was converted to the normal 80-kD form after reduction, suggesting that factor XII Tenri was secreted as a disulfide-linked heterodimer with a approximately 35-kD protein, which we identified as alpha1-microglobulin by immunoblotting. Pulse-chase experiments using baby hamster kidney (BHK) cells showed that Tenri-type factor XII was extensively degraded intracellularly, but the addition of cystine resulted in increased secretion of the mutant. Using membrane-permeable inhibitors, we observed that the degradation occurred in the pre-Golgi, nonlysosomal compartment and a proteasome appeared to play a major role in this process. On the basis of these in vitro results, we speculate that the majority of the factor XII Tenri is degraded intracellularly through a quality control mechanism in the endoplasmic reticulum (ER), and a small amount of factor XII Tenri that formed a disulfide-linked heterodimer with alpha1-microglobulin is secreted into the blood stream.

Hepatocyte growth factor activator inhibitor type 2 (HAI-2) is a new Kunitz-type serine protease inhibitor, which is purified and cloned from human stomach cancer cell line MKN45. The mature HAI-2 protein contains two Kunitz domains and the first domain is mainly responsible for the inhibitory activity against hepatocyte growth factor activator (HGFA). In this study, we identified the mouse homolog of HAI-2 (mHAI-2) by screening the data base of public expressed sequence tag (dbEST). In addition to a full-length cDNA corresponding to human HAI-2, a shorter size of mHAI-2 cDNA was obtained from mouse kidney by reverse-transcription polymerase chain reaction (RT-PCR). Sequence analysis of this shorter cDNA revealed that the region encoding the first Kunitz domain was completely deleted. Analysis of mouse genomic DNA showed that the deleted cDNA was generated by an alternative splicing mechanism. Surprisingly, the spliced form lacking the first Kunitz domain was a predominant transcript in all tissues of mice tested but not in those of human as assessed by RT-PCR analysis. This phenomenon is also confirmed by Western blot analysis using the specific antiserum against human HAI-2 protein. These results suggest that most of HAI-2 expressed in various tissues of mice may be unable to inhibit HGFA efficiently., (Copyright 1999 Academic Press.)

Tissue distribution of bikunin mRNA, which encodes a Kunitz-type serine protease inhibitor of the inter-alpha-inhibitor family (IalphaI), was studied in rats and mice by the reverse-transcripsion polymerase chain reaction (RT-PCR). We found that the liver as well as other tissues, such as the kidney, testis and adrenal gland, expressed bikunin mRNA. Although signals of bikunin mRNA were faint in the whole brain of rats and mice, distinct signals were found in limited portions of rat brain, such as the hippocampus, cerebral cortex and pituitary, but undetectable in cerebellum, medulla oblongata, hypothalamus, striatum, midbrain and choroid plexus. In three distinct types of cells, such as neurons, astrocytes and meningeal cells, in primary cultures isolated from the cerebral cortex and meninges of 1-day-old newborn rats, only neurons positively expressed bikunin mRNA. These results suggest that, in addition to peripheral tissues, neurons in the hippocampus and cerebral cortex produce bikunin, suggesting a potential role of bikunin/IalphaI family in these brain regions.

We recently reported that peritoneal fluid mainly contains two proteoglycans; one is the interstitial proteoglycan referred to as decorin, and the other an uncharacterized small chondroitin sulfate proteoglycan. In the present study, we have used a two-step process to isolate the small chondroitin sulfate proteoglycan free of decorin. The purified molecule ran as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent molecular mass 50 kd made up of a chondroitin-4-sulfate glycosaminoglycan chain and a 30-kd core protein. NH2-terminal analysis of the core protein showed significant sequence homology with bikunin, a component of the human inter-alpha-trypsin inhibitor (IalphaI) family. A Western blot analysis using anti-human inter-alpha-trypsin inhibitor confirmed the identity of the small chondroitin sulfate proteoglycan as bikunin, and a trypsin inhibitor counterstain assay confirmed its anti-trypsin activity. Examination of serum from patients receiving continuous peritoneal dialysis suggests that free bikunin in peritoneal fluid may be the result of leakage of serum proteins into the peritoneum. Our findings further show that the interaction of serum with peritoneal mesothelial cells offers a new and novel explanation for the presence of bikunin in peritoneal fluid.

The urinary glycoprotein that inhibits calcium oxalate (CaOx) crystallization in vitro shows a structural similarity to urinary trypsin inhibitor (UTI; recently termed bikunin), the light chain of inter-alpha-trypsin inhibitor (I alpha I). The functional domains of I alpha I involved in its inhibitory activity of CaOx crystallization have been investigated using isolated intact domains of I alpha I produced from controlled proteolytic digests of the protein. The fragments investigated include the heavy chains of I alpha I, UTI, chondroitinase AC-treated UTI, and the carboxyl-terminal domain of UTI (termed HI-8). The effects of I alpha I and its fragments on the inhibitory activity of CaOx crystallization were evaluated in vitro using CaOx crystal aggregation and growth assays, and seeded crystal generation assay as well as using crystal matrix protein generation assay. UTI, but not the heavy chains of I alpha I, had a discernible effect on CaOx crystallization inhibitory activity. Less requirement of the carbohydrate moiety of UTI is implicated by the observation that chondroitinase AC-treated UTI fragment was also found to inhibit CaOx crystallization with almost the same activity as UTI. HI-8 also efficiently inhibited CaOx crystallization, while I alpha I showed a weak inhibitory activity. The results are almost consistent with a seed crystal generation assay and a crystal adsorption inhibition assay, in which I alpha I or its derivatives inhibits prothrombin fragment 1 (F1) adsorption to CaOx crystals. In conclusion, these results suggest that the part of the I alpha I protein responsible for inhibition of CaOx crystallization is the carboxyl-terminal domain of UTI.

Bikunin is a serine protease inhibitor found in the blood serum and urine of humans and other animals. Its sequence shows internal repetition, suggesting that it contains two domains that resemble bovine pancreatic trypsin inhibitor (BPTI). A fragment of bikunin has been crystallised, its structure solved and subsequently refined against 2.5 A data. The two BPTI-like domains pack closely together and are related by an approximate 60 degrees rotation combined with a translation. These domains are very similar to each other and other proteins with this fold. The largest variations occur in the loops responsible for protease recognition. The loops of the first domain are unobstructed by the remaining protein. However, the loops of the second domain are close to the first domain and it is possible that protease binding may be affected or, in some cases, abolished by the presence of the first domain. Thus, cleavage of the two domains could alter the substrate specificity of domain II. Bikunin has a hydrophobic patch close to the N terminus of domain I, which is the most likely site for cell-surface receptor binding. In addition, there is a basic patch at one end of domain II that may be responsible for the inhibition of calcium oxalate crystallization in urine.

Bikunin is a chondroitin-sulphate containing serum protein with a Stokes-Einstein radius and a negative net charge close to those of serum albumin. The plasma half life of bikunin is about 10 minutes, and approximately half of its clearance occurs in the kidneys. The quantitative role of glomerular filtration in the renal clearance of this protein has not been determined. To assess the glomerular permeability of bikunin we used isolated rat kidneys that were perfused with an albumin solution. The metabolic activities of the tubuli were inhibited by low temperature (8 degrees C). The clearances of radiolabeled bikunin and albumin were repeatedly determined under identical conditions. The fractional clearance of bikunin was found to be 80 times higher than that of albumin: 15% +/- 1% versus 0.18% +/- 0.02%. This value for bikunin can fully account for its renal clearance in vivo. It has previously been shown that uncharged flexible solutes, such as dextrans, have higher renal clearances than globular molecules with similar radii. The high glomerular permeability of bikunin is therefore probably due to its elongated and flexible configuration. Moreover, the observed clearance value of the anionic molecule bikunin is close to that of a neutral flexible dextran of similar size, indicating that the charge of bikunin is of little importance for its glomerular permeability.

Polymorphonuclear leukocytes contain well-defined proteolytic enzymes in their azurophilic granules that can be released into tissues during inflammation, producing a localized excess of proteases that causes a protease-antiprotease imbalance with subsequent tissue destruction. The antiproteolytic compounds of the epidermis, such as the protease inhibitors elafin and antileukoprotease, are thought to counteract the proteolytic tissue damage. We investigated the urine of patients suffering from inflammatory skin conditions (e.g., erysipelas, psoriasis) for the presence of urinary antiprotease activities. Purification of elastase-inhibitory activities from pooled urine samples by cation exchange high-performance liquid chromatography and preparative and analytical reverse-phase high-performance liquid chromatography yielded two different types of inhibitors. One was a cationic, acid-stable, and elastase-specific inhibitor of M(r) 6,000 by size-exclusion high-performance liquid chromatography. N-terminal amino acid sequence analysis of the first 28 residues showed identity with elafin, an elastase-specific inhibitor recently isolated from psoriatic scales. The second anti-protease activity was due to two forms of urinary bikunin, the inhibitory subunit of inter-alpha-inhibitor. Both bikunin fragments, with M(r) 4,000 and 16,000, were identified by N-terminal amino acid sequence analysis of the first 10 residues and were characterized by an antiproteolytic profile against human leukocyte elastase, cathepsin G, and trypsin. Urinary protease inhibitors may serve as diagnostic markers of inflammatory diseases.

One of the major sulfated proteins secreted by rat hepatocytes contains a low-sulfated chondroitin sulfate chain and its apparent molecular mass upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis shifts from 40 to 28 kDa upon chondroitinase ABC treatment (E. M. Sjöberg and E. Fries, 1990, Biochem. J. 272, 113-118). These properties suggest that this protein is the rat homologue of the major trypsin inhibitor of human urine which was recently named bikunin. In serum, bikunin occurs mainly as a subunit of the pre-alpha-inhibitor and the inter-alpha-inhibitor; in these proteins it is covalently linked to the other polypeptides through its chondroitin sulfate chain. Bikunin has been shown to be synthesized by liver cells as a 42-kDa precursor, in which it is linked to alpha 1-microglobulin by two basic amino acids. We have isolated bikunin from rat urine and prepared antibodies against it. In rat hepatocytes pulse-labeled with [35S]methionine, these antibodies precipitated a labeled protein of 42 kDa. Upon chase, three different labeled proteins were recognized by the antibodies in the medium: one protein of 40 kDa (free bikunin), one of 125 kDa (presumably pre-alpha-inhibitor), and one greater than 240 kDa (possibly a protein related to the inter-alpha-inhibitor). Pulse-chase experiments with [35S]sulfate showed that these proteins occurred intracellularly as precursors containing alpha 1-microglobulin. These results demonstrate that the completion of the chondroitin sulfate chain and its coupling to other polypeptide chains occur before the cleavage of the alpha 1-microglobulin/bikunin precursor.

The amyloid beta peptide (A beta P) is the major constituent of the amyloid deposits that accumulate extracellularly in the brain of patients with Alzheimer's disease. This peptide is obtained from transmembrane amyloid protein precursors (APP) which sometimes contain a Kunitz protease inhibitor (KPI) insert in their extracellular domain and therefore are able to inhibit serine proteases. Expression of the transmembrane and the secreted APP containing the KPI domain was obtained by transient transfection of COS-1 cells. The overexpressed proteins were detected in immunoblotting experiments and inhibition of trypsin was analyzed using reverse enzymography. Our results indicate that post-translational modifications including glycosylation improve the inhibition of trypsin by the APP containing the KPI domain.

A 3000--6000-fold purified kallikrein was obtained from human serum in 10--25% yield by chromatography on QAE-Sephadex A-50, Molselect CM-50 and on soybean trypsin inhibitor (SBTI)-AH-Sepharose 4-B. The enzyme had a specific activity of 14--23 U, as measured by BAEE hydrolysis. Some properties of highly purified kallikrein are described.