Edwin Fink

Amino acid sequence elucidation of human acrosin‐trypsin inhibitor (HUSI‐II) reveals that Kazal‐type proteinase inhibitors are structurally related to β‐subunits of glycoprotein hormones

The amino acid sequence of the acrosin‐trypsin inhibitor HUSI‐II from human seminal plasma is presented which unequivocally identifies HUSI‐II as being of Kazal‐type. In addition, the HUSI‐II sequence shows a striking similarity to the middle part of glycoprotein hormone β‐subunits thus revealing a hitherto unknown structural and evolutionary relationship between Kazal‐type inhibitors and glycoprotein hormones.

Human Keratinocyte Cell Lines Differ in the Expression of the Collagenolytic Matrix Metalloproteinases-1, -8, and -13 and of TIMP-1

Abstract We investigated cells and conditioned media of the three human keratinocyte cell lines HaCaT (nontumorigenic), A5 (benign, tumorigenic) and II-4RT (malignant, tumorigenic) with regard to production and secretion of the collagenases-1 to -3 (MMP-1, MMP-8 and MMP-13) and TIMP-1 using seminested RTPCR, Western blots, ELISA, immunocytochemistry and casein zymography. Transcripts of MMP-1, -8, -13 and TIMP-1 were detected in all cell lines by RTPCR and the corresponding proteins were found in the cytoplasm of all three cell lines by Western blot analysis and/or immunocytochemistry. The conditioned media of the malignant II-4RT cells contain significantly more MMP-1 and MMP-8 than those of HaCaT or A5 as evidenced by immunoblotting and ELISA. In addition to the presence of latent MMP-1, zymography also detected the active form of this enzyme. TIMP-1 was found only in extracts of all three cell lines, predominantly in A5. This study clearly indicates that the epithelial tumor cells synthesize different collagenases and TIMP-1. The malignant clone secretes increased amounts of distinct collagenases compared to the nontumorigenic cell line, thereby verifying a correlation between biological behaviour and the amount of collagenases. In addition, we provide clear evidence that MMP-8 is not exclusively found in polymorphonuclear granulocytes, but also in keratinocyte cell lines.

Matrix metalloproteinases-2,-3,-7,-9 and-10, but not MMP-11, are differentially expressed in normal, benign tumorigenic and malignant human keratinocyte cell lines.

Abstract In order to investigate the correlations between constitutive proteinase expression and the degree of tumorigenicity of cancer cells we have studied a model system of three keratinocyte cell lines. RT-PCR studies showed that the cell lines express the genes of matrix metalloproteinase-2, -3, -7, -9, -10 and -11, indicating that they are able to synthesize the corresponding enzymes. Actual MMP synthesis was proven by zymography and Western blotting. In conditioned media gelatinolytic activities or immunoreactive forms of MMP-2, -3, -7, -9, -10 and -11 were detected. The signal intensities showed that MMP secretion increases in the order HaCaT < A5 ≤ II-4RT, whereas only MMP-11 is secreted by all cell lines in equal amounts. Intracellularly, enhanced levels of one or both of the tumorigenic variants were only found for MMP-3, -9 and -10, suggesting special functions of these intracellular MMP pools for the tumorigenic cell lines. For MMP-11 exclusive expression in stromal fibroblasts of tumor tissues is widely accepted; however, our results and three other recent reports demonstrate that this concept is not generally valid. In conclusion, the three keratinocyte cell lines investigated here represent an excellent model for studying constitutive expression and secretion of MMPs in correlation to the degree of in vivo tumorigenicity.

PURIFICATION AND PRIMARY STRUCTURE DETERMINATION OF A BOWMAN-BIRK TRYPSIN INHIBITOR FROM TORRESEA CEARENSIS SEEDS

A Bowman-Birk-type trypsin inhibitor (TcTI) was purified from seeds of Torresea cearensis, a Brazilian native tree of the Papilionoideae sub-family of Leguminosae. Three forms of the inhibitor were separated by anion exchange chromatography. The major form with 63 amino acids was entirely sequenced; it shows a high structural similarity to the Bowman-Birk inhibitors from other Leguminosae. The putative reactive sites of the inhibitor are a lysine residue at position 15 and a histidine at position 42 as identified by alignment to related inhibitors, direct chemical modification and specific enzymatic degradation. Immunoprecipitation with antibodies raised in rats is reduced significantly if TcTI is complexed with chymotrypsin and, to a lesser degree, if complexed with trypsin. TcTI forms a ternary complex with trypsin and chymotrypsin. The binary complexes with trypsin or chymotrypsin were isolated by gel filtration. Dissociation constants of the complexes with trypsin, plasmin, chymotrypsin, and factor XIIa are 1, 36, 50, 1450 nM, respectively; human plasma kallikrein, human factor Xa, porcine pancreatic kallikrein and bovine thrombin are not inhibited. TcTI prolongs blood clotting time of the contact phase activation pathway by inhibition of FXIIa.

Inhibition of Plasma Kallikrein with Aprotinin in porcine endotoxin Shock

Activation of the contact phase of coagulation has been implicated in the pathogenesis of septic shock. We wanted to determine if inhibition of plasma kallikrein can prevent arterial hypotension and liberation of kinins from kininogen, induced by an infusion of bacterial lipopolysaccharide (LPS) in anesthetized, ventilated 20-kg pigs. The LPS was given IV in a dose of 5 micrograms/kg/h for 8 hours. The plasma kallikrein inhibitor aprotinin, 537 mumol, was given IV during 8 hours, resulting in plasma levels above 10 mumol/L. Ten animals (SA) received LPS and aprotinin and ten randomized controls (SC) received LPS and saline. Kinin-containing kininogen was determined on the basis of the amount of kinin releasable in plasma samples by incubation with trypsin. Kininogen decreased to 58% +/- 4% of the baseline value without any difference between groups. This may indicate participation of other processes than degradation by plasma kallikrein in the decrease of kininogen. Arterial blood pressure was higher at 7 hours in the SA animals than in the SC group (101% +/- 11% vs. 68% +/- 8%; mean +/- SEM; p = 0.026). Fibrin monomer and C3adesArg plasma levels were attenuated by aprotinin treatment. These findings underscore the important role of the contact system in LPS shock.

Role of Bradykinin in Anaphylactoid Reactions during Hemodialysis with AN69 Dialyzers

In vitro experiments have related anaphylactoid reactions in patients treated with angiotensin-converting enzyme (ACE) inhibitors during dialysis with AN69 membranes to excessive bradykinin generation using this negatively charged dialysis membrane. In the present clinical trial plasma bradykinin levels were followed during the early phase of dialysis in 10 patients, not being treated with ACE inhibitors, using AN69, cuprophane, and polysulfone membranes. Bradykinin was measured after extraction by radioimmunoassay. During this study one episode of anaphylaxis occurred during dialysis with the AN69 membrane. Blood samples were collected during the first 5 min of the adverse reaction and showed a more than 100-fold increase in the venous effluent of the AN69 dialyzer (baseline 40 +/- 3 vs. 4,900 +/- 130 fmol/ml after 5 min). Even though none of the patients received ACE inhibitors, there were 4 more asymptomatic individuals who displayed a more than two-fold increase in their plasma bradykinin concentrations in the venous effluent of the AN69 dialyzer. When these patients were treated either with cuprophane or with polysulfone dialyzers, no significant bradykinin formation was detected, nor were there any adverse events. Taken together, these findings show that anaphylactoid reactions with the AN69 membrane are due to excessive bradykinin generation which even may occur in the absence of ACE inhibitors.

The Hyaluronan-Binding Serine Protease from Human Plasma Cleaves HMW and LMW Kininogen and Releases Bradykinin

Abstract The influence of the hyaluronanbinding protease (PHBSP), a plasma enzyme with FVII and prourokinase activating potency, on components of the contact phase (kallikrein/kinin) system was investigated. No activation or cleavage of the proenzymes involved in the contact phase system was observed. The procofactor high molecular weight kininogen (HK), however, was cleaved in vitro by PHBSP in the absence of any charged surface, releasing the activated cofactor and the vasoactive nonapeptide bradykinin. Glycosoaminoglycans strongly enhanced the reaction. The cleavage was comparable to that of plasma kallikrein, but clearly different from that of coagulation factor FXIa. Upon extended incubation with PHBSP, the light chain was further processed, partially removing about 60 amino acid residues from the Nterminus of domain D5 of the light chain. These cleavage site(s) were distinct from plasma kallikrein or FXIa cleavage sites. PHBSP and, more interestingly, also plasma kallikrein could cleave low molecular weight kininogen in vitro, indicating that domains D5H and D6H are no prerequisite for kininogen cleavage. PHBSP was also able to release bradykinin from HK in plasma where the procofactor circulates predominantly in complex with plasma kallikrein or FXI. In conclusion, PHBSP represents a novel kininogencleaving and bradykininreleasing enzyme in plasma that shares significant catalytic similarities with plasma kallikrein. Since they are structurally unrelated in their heavy chains (propeptide), their similar in vivo catalytic activities might be directed at distinct sites where PHBSP could induce processes that are related to the kallikrein/kinin system.

Molecular cloning and sequence analysis of the cDNA encoding the human acrosin-trypsin inhibitor (HUSI-II)

A complete cDNA clone encoding the human acrosin‐trypsin inhibitor HUSI‐II has been isolated from a cDNA library of human testis and completely sequenced. The cDNA of 594 bp contained an open reading frame of 252 base pairs, The deduced amino acid sequence comprised the complete amino acid sequence of HUSI‐II[1] and a putative signal peptide. Northern blotting analysis revealed that HUSI‐II is synthesized in testis, epididymis and seminal vesicle, but not in the prostate gland.

Identification and occurrence of mRNAs for components of the kallikrein-kinin system in human skin and in skin diseases.

Bradykinin and kallidin are released during dermal injury and inflammation as a result of activation of kallikreins which cleave high- and low-molecular weight kininogen (HMW and LMW kininogen, respectively). In the skin, kinins are involved, e.g., as co-mitogens in cellular proliferation or in processes propagating pain and inflammation. The aim of our study was to investigate the specific occurrence of mRNAs for components of the kallikrein-kinin system in normal human skin and in skin biopsies of patients with selected skin diseases (psoriasis, lichenificated atopic eczema, basalioma). In normal skin, reverse transcription polymerase chain reaction (RT-PCR) with specific primer pairs followed by separation of products by polyacrylamide gel electrophoresis (PAGE) revealed the presence of mRNAs for tissue kallikrein, for the B2 and the B1 bradykinin receptors, but not for kininogen. In biopsies of lichenificated atopic eczema and basalioma, additionally, the mRNAs for HMW and LMW kininogen were detected, whereas in psoriatic skin mRNA for HMW kininogen was not expressed. These differences in mRNA expression may reflect the different contribution of kallikrein-kinin system components to the maintenance of chronic skin diseases like psoriasis. In acute dermal reactions occurring in lichenificated atopic eczema or in basalioma, tissue mRNA for HMW kininogen appears to be arisen from sources not pre-existing in normal skin.

Acid-Stable Proteinase Inhibitors from Human Seminal Plasma

Publisher Summary Antitryptic activity in human seminal plasma was first detected by Rasmussen and Albrechtson. Haendle et al. described the occurrence of acid-stable trypsin inhibitors in testes, epididymis, and seminal vesicles as well as in the seminal plasma of many mammals, including man. Fink et al. and Suominen and Niemi showed that the antitryptic activity in human seminal plasma is due to two different trypsin inhibitots, the human seminal plasma inhibitors (HUSI) I and II. HUSI-I, trypsin-chymotrypsin inhibitor, and HUSI-II, a trypsin-acrosin inhibitot, were further characterized in laboratory. α-Antitrypsin, an acid-unstable glycoprotein, is also present in human seminal plasma. Trypsin inhibitors isolated from human spermatozoa show very similar characteristics to those of HUSI-I and HUSI-II. This chapter discusses the assay methods like: Trypsin Inhibition, Chymotrypsin Inhibition and Acrosin Inhibition. It also discusses the purification procedure and properties.