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Objectives: To identify a 50.8-kDa biomarker to perform a preliminary clinical evaluation of its utility as an aid in the early detection of prostate cancer., Methods: The 50.8-kDa protein, previously called NMP48, was partially purified from the serum of an individual with prostate cancer and identified by peptide mass fingerprinting of tryptic peptides from an in-gel digest. Serum samples were obtained from men with biopsy-confirmed prostate cancer, high-grade prostatic intraepithelial neoplasia, and benign histologic features, from men with clinically defined benign prostatic hyperplasia, and from controls without prostatic disease. These samples were analyzed for the presence of the biomarker by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry., Results: The 50.8-kDa protein was identified by peptide mass fingerprinting as being related to vitamin D-binding protein. It was found in 96% of the sera from individuals with prostate cancer (n = 52) including 11 of 12 specimens that exhibited prostate-specific antigen values of less than 4 ng/mL. The 50.8-kDa protein was found in 10 of 19 samples from men with prostatic intraepithelial neoplasia; however, it was not detected in the sera of 5 (75%) of 20 individuals with benign prostatic histologic features, 7 (70%) of 10 with clinical benign prostatic hyperplasia, 8 (80%) of 10 patients who had previously undergone radical prostatectomy, or 48 (96%) of 50 specimens from healthy controls., Conclusions: Although the study cohort was relatively small, the data suggest that an assay for the 50.8-kDa protein may be useful for the early detection of prostate cancer. Additional elucidation of its structure may yield insight into the development of this disease.

Prostate cancer (CaP) patients with disseminated disease often suffer from severe cachexia, which contributes to mortality in advanced cancer. Human cachexia-associated protein (HCAP) was recently identified from a breast cancer library based on the available 20-amino acid sequence of proteolysis-inducing factor (PIF), which is a highly active cachectic factor isolated from mouse colon adenocarcinoma MAC16. Herein, we investigated the expression of HCAP in CaP and its potential involvement in CaP-associated cachexia. HCAP mRNA was detected in CaP cell lines, in primary CaP tissues and in its osseous metastases. In situ hybridization showed HCAP mRNA to be localized only in the epithelial cells in CaP tissues, in the metastatic foci in bone, liver and lymph node, but not in the stromal cells or in normal prostate tissues. HCAP protein was detected in 9 of 14 CaP metastases but not in normal prostate tissues from cadaveric donors or patients with organ-confined tumors. Our Western blot analysis revealed that HCAP was present in 9 of 19 urine specimens from cachectic CaP patients but not in 19 urine samples of noncachectic patients. HCAP mRNA and protein were also detected in LuCaP 35 and PC-3M xenografts from our cachectic animal models. Our results demonstrated that human CaP cells express HCAP and the expression of HCAP is associated with the progression of CaP and the development of CaP cachexia., (Copyright 2003 Wiley-Liss, Inc.)

The monoclonal antibody (MAb) A6H, originally developed to fetal renal tissues, was found to be highly reactive to renal cell carcinoma and was subsequently demonstrated to co-stimulate a subpopulation of T cells. The A6H antigen had not been identified heretofore. Antigen from detergent extracts of renal cell carcinoma cells (7860) was immunoabsorbed with A6H-agarose, and the resin-bound proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The antigen had a molecular weight of approximately 120 kDa as determined by Western blots. The 120-kDa protein band was excised and subjected to in-gel tryptic digestion, and the resulting peptides were separated and analyzed by liquid chromatography tandem mass spectrometry (LC MS\MS). The tandem mass spectra of the eluting peptides were used in combination with the SEQUEST computer program to search a human National Cancer Institute (NCI) protein database for the identity of the protein. The target antigen was shown to be dipeptidyl peptidase IV (DPP IV), which is also known as the cluster differentiation antigen CD26. Flow analysis of the expression of the A6H antigen and of CD26 on 7860 cells and on peripheral blood lymphocytes supported the identification of the A6H antigen as DPP IV. Recognition that the A6H antigen is DPP IV/CD26 afforded the opportunity to compare previous studies on A6H with those on other anti-CD26 antibodies in terms of expression in cancer cell lines and various tissues and as co-stimulators of T-cell activation.

Recently we described the generation of the prostate tissue-specific monoclonal antibody (MAb) 107-1A4, its expression pattern and preliminary targeting of human prostate cancer xenografts. In this report we demonstrate that the target antigen for MAb 107-1A4 is prostate-specific membrane antigen (PSMA) using immunoaffinity absorption followed by SDS-PAGE and mass spectrometric analysis of peptides produced by in-gel tryptic digestion. The identity of the antigen has been confirmed by Western blots using MAbs of known specificity. MAb 107-1A4 is not reactive on Western blots. The conformational epitope for 107-1A4 is on the extracellular domain of PSMA. In competition studies, the binding of MAb 107-1A4 to LNCaP cells is inhibited by itself but not by any other of several other anti-PSMA MAbs, suggesting that the epitope may be unique. These results suggest that 107-1A4 is reactive to a conformational epitope in the external domain of PSMA that is unique among the panel of anti-PSMA MAbs in this study. Furthermore this work demonstrates the ability of mass spectroscopy to elucidate antibody-ligand interaction., (Copyright 2001 Wiley-Liss, Inc.)

We have recently reported that complement factor H, a negative regulator of complement-mediated cytotoxicity, is produced and secreted by most bladder cancers. This observation was exploited in the development of the BTA stat and BTA TRAK diagnostic assays, both of which make use of two factor H-specific monoclonal antibodies in sandwich format. Here we show that both antibodies exert interesting effects on the biochemistry of complement activation in in vitro systems. Antibody X13.2 competes with C3b for association with factor H and strongly inhibits factor H/factor I-mediated cleavage of C3b, thereby evidently inactivating a negative regulator of complement; yet, the antibody strongly inhibits complement-mediated lysis as well. Conversely, antibody X52. 1, which does not compete with C3b and has no effect on solution-phase cleavage of C3b, is capable of enhancing complement-mediated lysis of various cell types, including cancer cells, by over 10-fold. Our observations indicate that it is possible to deconvolute the biochemical roles of factor H in complement by means of appropriate inhibitors, a finding with potentially valuable implications for both basic research and cancer therapy.

The BTAstat and BTA TRAK tests are new immunoassays that detect and measure an antigen in the urine of individuals diagnosed with bladder cancer. As described in this report, the monoclonal antibodies used in these kits were developed by immunizing mice with partially purified protein preparations derived from the urine of patients with bladder cancer. The antigen that is recognized by the monoclonal antibodies was purified from the urine of bladder cancer patients by immunoaffinity chromatography and identified as being either complement factor H (FH) or a closely related protein (CFHrp) by partial amino acid sequence analysis. Like serum FH, the urine antigen was demonstrated to have a complement factor C3b binding site and to accelerate the degradation of C3b in the presence of complement factor I. The culture supernatants from several human bladder, cervical, and renal cancer cell lines contained antigen as determined by immunoassay, and antigen affinity-purified from HeLaS3 culture media was shown to have FH activity. Moreover, the cell lines were shown to make products of the expected sizes by reverse transcription-PCR using FH-specific primers. In contrast, normal human epithelial keratinocytes, a myeloid leukemia cell line, and the colon cancer line LS174T were negative for production of a FH-like protein (CFHrp). We propose that the expression of proteins with FH-like activities may confer a selective growth advantage to cancer cells in vivo by decreasing complement activity, thus aiding their escape from lysis by immune surveillance. Identification of these proteins as cancer products also suggests avenues of chemotherapy or immunotherapy of some cancers.

In this preliminary study, we report that an enzyme-linked immunofluorescence assay (EFLA) was developed for the determination of PR92 antigen in prostatic fluid, utilizing anti-PR92 monoclonal antibody. Fluid samples from 64 patients were assayed. PR92 antigen was expressed as unit per microgram (U/microgram) of prostatic fluid proteins. One hundred percent of men (7 out of 7) less than 50 years of age demonstrated concentrations less than 25 U/micrograms; 91% of men (10 out of 11) with documented carcinoma, and only 9.5% of men (2 out of 21) with benign prostatic hyperplasia, demonstrated concentrations above 230 U/micrograms. The mean concentration of PR92 antigen in prostatic fluid of a group of patients suspected of having prostate cancer (high-risk group; 227 +/- 42 U/micrograms) was significantly greater than that of those with benign prostatic hyperplasia (87 +/- 23 U/micrograms; P = 0.05). Further evaluation of this potential marker and of other antigens within the prostatic fluid is warranted.

Background: Endometrial carcinoma is generally diagnosed only after the onset of postmenopausal bleeding. Although most patients with Stage I disease can be cured, the prognosis worsens significantly when the tumor is no longer confined to the uterine corpus. Serum CA 125 is elevated in only 10-20% cases of Stage I and II endometrial carcinoma. A serum tumor marker that can detect early stage endometrial cancer might aid in management of the disease., Methods: An OVX1 double-determinant radioimmunoassay was used to detect an epitope on a high-molecular-weight mucinlike glycoprotein found in the sera of 45 patients with endometrial cancer., Results: Apparently healthy persons had serum OVX1 antigen levels of 2.23 plus or minus 2.48 U/ml (mean +/- standard deviation). Elevated levels of OVX1 antigen (> 7.2 U/ml) were found in 5% of 184 healthy persons and in 64% of 45 patients with endometrial cancer. OVX1 antigen was elevated in 64% of 36 patients with Stage I, 50% of 2 patients with Stage II, 60% of 5 patients with Stage III, and each of 2 patients with Stage IV endometrial cancer, but only 8.6% of 58 patients with endometriosis. Elevation of serum OVX1 was found more frequently in patients with deep myometrial invasion and with poorly differentiated tumors (P < 0.01)., Conclusions: The OVX1 antigen deserves further evaluation as a marker for early detection of endometrial cancers and as a prognostic factor for women with apparent early stage disease.

Purpose: At second-look surgical surveillance procedures, normal CA-125 levels can be associated with persistent disease in 50% to 60% of patients. A novel radioimmunoassay (RIA) has been evaluated for the ability to identify patients with persistent disease who have normal levels of CA-125., Materials and Methods: The OVX1 double-determinant assay used a murine monoclonal antibody to detect an epitope on a high-molecular weight mucin-like glycoprotein., Results: Apparently healthy individuals had serum OVX1 levels of 2.23 +/- 2.48 U/mL (mean +/- SD). Elevated serum OVX1 levels (> 7.2 U/mL) were found in 5% of 184 normal individuals and in 70% of 93 epithelial ovarian cancer patients with clinically evident disease. Among sera from these ovarian cancer patients, OVX1 was elevated in 68% of 76 samples with CA-125 levels more than 35 U/mL and in 76% of 17 samples with CA-125 levels less than 35 U/mL. In serum samples obtained at the time of positive second-look laparotomy, 59% of 41 patients with CA-125 levels less than 35 U/mL had elevated OVX1 antigen levels, whereas 41% of 22 patients with CA-125 levels more than 35 U/mL had elevated serum OVX1 levels. In patients with negative second-look laparotomies, false-positive results were eliminated by increasing the threshold of OVX1 to 10.5 U/mL. At this level, 32% of 41 patients with positive second-look operations had an elevated OVX1 level, despite a normal CA-125 level. When used in combination, CA-125 (> 35 U/mL) and OVX1 (> 10.5 U/mL) detected persistent disease in 56% of 63 patients with positive surveillance procedures, compared with 35% when CA-125 was used alone (P < .05)., Conclusion: An elevated OVX1 level can alert oncologists to the possibility that ovarian cancer has persisted, despite the return of CA-125 to a normal range.

Genes encoding the four principal polypeptide domains (N, A1-B1, A2-B2, and A3-B3) of carcinoembryonic antigen (CEA) were synthesized and expressed in Escherichia coli as fusion products with bacterial CMP-KDO synthetase (CKS). The four synthetic fusion proteins were purified in high yield and used as targets in Western blots for 11 anti-CEA MAbs and to compete with immobilized CEA for binding to four of these MAbs. Each of the MAbs showed strong binding to one or more of the fusion proteins. In Western blots, MAbs H19C91 and 4230 bound only to CKS-N. MAbs H8C2 and H11C35 bound only CKS-A1-B1, and MAbs T84.66, H46C136, and H21C83 appeared to be specific for CKS-A3-B3. None of the MAbs tested bound only to CKS-A2-B2. However, two MAbs bound both CKS-A1-B1 and CKS-A3-B3 and one MAb (3519) bound to all three of the repeated domains. Since these three domains exhibit over 90% amino acid sequence homology, the latter results were not surprising. The competition studies largely confirmed the results of Western blots but did show some MAb-fusion protein interactions not observed in Western blots. These competition studies also allowed estimation of the relative affinities of the MAbs for the synthetic domains and for native CEA. These studies demonstrated that epitopes in CEA recognized by the MAbs in this study are peptide in nature and that the fusion proteins are of utility in the localization of the epitopes on the polypeptide chain of CEA.

The carboxypeptidase inhibitor from Russet Burbank potatoes (C. A. Ryan et al. (1974b), J. Biol. Chem 249, 5495) is a mixture of approximately equal amounts of two polypeptide chains containing 38 and 39 amino acid residues, respectively. The chains differ in their amino terminal sequence only, one beginning with smaller than Glu-His-Ala ... and the other with smaller than Glu-Gln-His-Ala ..... Specific cleavage procedures utilized in determining the complete amino acid sequence of the inhibitor included acid cleavage of the aspartyl-proline bond and tryptic and chymotryptic digestion. Mass spectrometry, automatic Edman degradation, and subtractive Edman degradation were employed in sequencing the resulting peptide fragments.

High concentrations of ethanol are effective in reducing the intermolecular cross-linking which predominates when the reactions of tetranitromethane with the carboxypeptidase inhibitor from potatoes, a chymotrypsin inhibitor also from potatoes, and insulin are carried out under standard conditions. Including reagents such as ethanol may be of general utility in the preparation of monomeric nitro-derivatives of hydrophobic, low-molecular-weight proteins.

The total amount of carboxypeptidase inhibitor was estimated in extracts of the leaves, stems, and sprouts of Solanum tuberosum L. cv. Russet Burbank. Although the tuber contained the highest levels on a dry weight basis, inhibitor was also detected in the leaves, sprouts, and upper stems. The relative amounts of each of three carboxypeptidase isoinhibitor families were estimated in several plant tissues by purifying the mixture of isoinhibitors using immobilized carboxypeptidase and then resolving the individual families by polyacrylamide gel electrophoresis. These data demonstrated both that differences in distribution are found in the potato plant and that the three isoinhibitor families described previously (Hass GM, JE Derr, DJ Makus, CA Ryan 1979 Plant Physiol 64: 1022-1028) account for essentially all of the carboxypeptidase inhibitor activity in the tissues studied.

The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A subunit I has been determined by automated Edman degradation of the cyanogen bromide fractions derived from the precursor protein. The activation peptide contains 94 amino acid residues in a unique sequence which precedes directly the amino-terminal alanine residue of carboxypeptidase A alpha. A notable feature of the activation peptide is the presence of acidic amino acid residues immediately preceding the site of activation. The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A shows extensive similarity to those of the corresponding porcine and rat enzymes.

The carboxypeptidase inhibitor from Russet Burbank potatoes was subjected to a variety of chemical modifications and their effects on inhibitory activity toward carboxypeptidases A and B were determined. The importance of the alpha carboxylate of glycine-39 to the enzyme-inhibitor interaction was demonstrated by the observation that a derivative in which all four carboxyls were modified was inactive whereas a derivative in which only the beta carboxylates of aspartic acid residues 5, 16, and 17 were masked retained full inhibitory activity. In addition to these three aspartic acid residues, lysine residues 10 and 13, histidine residues 3 and 15, and arginine-32 were modified and residues 1-5 removed with little effect on inhibitory activity. Tryptophan residues 22 and 28 did not react with 2-hydroxy-5-nitrobenzyl bromide or o-nitrophenylsulfenyl chloride, and thus are presumed to be buried in the interior of the inhibitor molecule. Although tyrosine-37 was acetylated without affecting binding characteristics, both carboxypeptidases A and B protected against deacetylation by hydroxylamine. These studies indicate that the carboxyl terminal region of the inhibitor is in contact with enzyme in the complex. The parallel effects of modifications on inhibitory activity toward carboxypeptidases A and B support previous evidence that both enzymes utilize the same binding site on the inhibitor [C. A. Ryan (1971), Biochem. Biophys. Res. Commun. 44, 1265].

Lead encephalopathy was induced in suckling rats by administering lead to the mother. The brains were studied by light and electron microscopy, and the results were compared with observations in the human disease as well as in cases of cerebral ischemia in children. In their severe forms, both human and experimental lead encephalopathies are characterized by exudative extracellular edema and perivascular PAS-positive globules. The latter consist of osmiophilic non-membrane-limited cytoplasmic inclusions located, in the rat exclusively and in the human predominantly, in perivascular astrocytes. Intervascular strands are also found in both forms of the disease. In the rat these consist of basement membrane surrounding endothelial cytoplasm. Chemically, experimental lead encephalopathy with morphologically demonstrable edema is associated with an increase in brain water, sodium and serum albumin. Relative to the serum concentration, the increase in water is disproportionately greater than the sodium or albumin. There were no demonstrable changes in chloride or potassium.

Two pregnancy-specific proteins were detected by immunoelectrophoresis using antisera developed to homogenates of bovine extraembryonic membranes. Antisera also reacted to extracts of endometrium from pregnant cows and extraembryonic fluids. However, antisera did not react with a preparation presumed to be bovine placental lactogen, fetuin, extracts of various somatic tissues from pregnant cows or extracts of endometrium from nonpregnant cows. One of the proteins had an estimated molecular weight of 65,000-70,000, an isoelectric point of 4.6-4.8 and yielded a reaction of identity with bovine alpha 1-fetoprotein by immunodiffusion. The second protein yielded a reaction of identity with bovine alpha 1-fetoprotein by immunodiffusion. The second protein had no immunological cross-reactivity with the known proteins or organ extracts which were tested. The molecular weight and isoelectric point was 47,000-53,000 and 4.0-4.4, respectively. These data demonstrate the presence of at least 2 pregnancy-specific proteins in cattle.

To test whether proteolytic events are involved in natural killer (NK) cell mediated lysis of tumour cells, twenty-three different protease inhibitors were added to in vitro assays of natural killer cell reactivity. Of all of the materials tested, only tosyl-L-lysine chloromethyl ketone (TLCK), tosyl-L-phenylalanine chloromethyl ketone (TPCK) and benzamidine unequivocally inhibited killing at concentrations approaching those needed to affect appropriate purified proteases. All of the effective inhibitors, and none of the others tested, inhibited binding of effector to target cells. The action of TLCK was focused on both effector and target cells, in that cytolysis was completely inhibited by a 1 hr pretreatment of effectors with 10(-4) M TLCK, and 60% inhibited by a 1 hr treatment of targets only.

Three zones of carboxypeptidase inhibitory activity were observed when heat-stable extracts of potato tubers (cv. Russet Burbank) were chromatographed on carboxymethyl cellulose. The isoinhibitors found in these zones were denoted I, II, and III based upon their order of elution from this column. The predominant form (II) had previously been suggested to be a mixture of two polypeptides (IIa and IIb) differing in that IIa possessed an additional residue of glutamine (Hass et al. 1975 Biochemistry 14: 1334). These closely related isoinhibitors (IIa and IIb) were separated by equilibrium ion exchange chromatography and characterized. Isoinhibitor I was shown to be identical to II except for two replacements, Ser-30 --> Ala and Arg-32 --> Gly. These replacements had no significant effect on apparent K(i) values toward either carboxypeptidase A or B. Isoinhibitor III, which was identical to II except that it lacked the amino terminal pyrrolidone carboxylic acid and following glutamine residue, was also functionally indistinguishable from II in inhibition studies. It was concluded that at least two and possibly as many as five genes code for the various isoinhibitor species which are present in potato tubers.

The amino acid sequence of a 37 residue carboxypeptidase inhibitor from tomato fruit has been determined. The amino terminus was shown to be 2-oxopyrrolidine-5-carboxylic acid by digestion of reduced and S-carboxymethylated inhibitor with pyroglutamate aminopeptidase. The remainder of the sequence was assigned by analysis of peptides which had been generated by specific cleavage at the Asp4-Pro5 bond under acid conditions and by treatment with trypsin. The amino acid sequence of this inhibitor is identical with that of an analogous inhibitor from potatoes in 26 positions, and two of the replacements are highly conservative. The identification of the nonconservative replacements has been used to better define regions of the inhibitor which are not believed to contribute significantly to the free energy of association of the enzyme-inhibitor complex.

Local vesical absorption of organic compounds may play a role in the pathogenesis of bladder cancer. An associated increased absorption of tryptophan in patients with bladder cancer has been demonstrated.

The mechanism of action of a carboxypeptidase inhibitor from potatoes has been probed by studying its interaction with derivatives of carboxypeptidase A containing modified residues at the active site. Arsanilazocarboxypeptidase A, a derivative containing a chromophore attached to tyrosine 248, exhibits a circular dichroism spectrum which is sensitive to the presence of ligands at the active site (Kagan, H.M., and Vallee, B.L. (1969), Biochemistry 8, 4223). Since the spectral change attending binding of the carboxypeptidase inhibitor to arsanilazocarboxypeptidase A is similar to that produced by small substrates and inhibitors, the enzyme-inhibitor interaction also involves the enzyme active site. Catalytic activity is not required for inhibitor binding. Complexes of the inhibitor with apocarboxypeptidase A anc carboxypeptidase A which was inactivated by treatment with the affinity label, N-bromoacetyl-N-methyl-L-phenylalanine, are demonstrated by gel filtration experiments. Morever, competitive binding studies reveal that the latter derivative, in which the binding pocket is presumably blocked by reagent, binds inhibitor nearly as strongly as does the native enzyme, and differences in free energy of association being only 0.4 kcal/mol of a total binding energy of - 11 kcal/mol. A model is proposed to account for both the tight binding of inhibitor to the N-bromoacetyl-N-methyl-L-phenylalanine derivative and the involvement of the active site of arsanilazocarboxypeptidase A. It is suggested that the inhibitor fits into a shallow depression at the active site of the enzyme but does not penetrate into the binding pocket.

Carboxypeptidases from animal, plant, fungal, and bacterial sources were tested for their ability to bind to the carboxypeptidase inhibitor from Russet Burbank potatoes. Enzymes which participate in the degradation of dietary protein were partially purified from animal species as diverse as the cow and the limpet, and all were potently affected by the inhibitor. However, several zymogens of the enzymes in this group were tested and shown not to bind immobilized inhibitor. With the exception of an enzyme from mast cells and a novel carboxypeptidase A-like enzyme from bovine placenta, all animal carboxypeptidases which were not of digestive tract origin were not affected by the inhibitor. The inhibitor had no effect on the enzymic activities of all plant and most microbial carboxypeptidases. However, a strong association between the inhibitor and Streptomyces griseus carboxypeptidase has been noted previously and a low affinity (K(i) about 10 micromolar) for a carboxypeptidase G(1) from an acinetobacterium was found in this study.

If Sprague-Dawley rats, 25-30 days old, are fed a diet containing 4-5 mg% of Mg, about 25% of survivors develop a large tumor of the thymus within 6-12 weeks. The tumor is composed of lymphoblasts, which seem to arise from the thymic reticuloendothelial system and, at times, disseminate as an acute T cell lymphoma-leukemia of unknown etiology. If the tumor cells are transmitted intraperitoneally to rats, 14-16 days pregnant, a local invasive and generalized disease is established in the mother but not in the fetuses or their domain. However, if the neoplastic cells are injected into the fetal domain, they colonize the fetal tissues. The colonization by tumor cells is most impressive in the extravascular structure of the placental labyrinth but not in the placental syncytiotrophoblastic zone at the maternal-placental junction. This raises the question as to whether this zone may functionally mediate not only the well-known absolute intrauterine fetal defense against maternal metastatic neoplasia, but also the defense of the fetus against maternal immunologic rejection.

The determination of the covalent structure of a carboxypeptidase inhibitor from potatoes containing 39 amino acid residues has been completed by analysis of the pairing of the six half-cystine residues. Since the native inhibitor is resistant to fragmentation by proteases, the protein was first subjected to cleavage at aspartic acid residues by exposure to 0.03 N HCl at 110 degrees C for 10h to yield a fragment containing two chains (residues 6-15 and residues 18-39)held together by three disulfide bonds. Digestion with subtilisin and Pronase, respectively, yielded sets of peptides from which, by diagonal electrophoresis and amino acid analysis, the paired cystinyl residues were identified as Cys-8 to Cys-24, Cys-12 to Cys-27, and Cys-18 to Cys-34. Charge-transfer titration of the native inhibitor with N-methylnicotinamide chloride suggests that one of the two tryptophan residues and the single tyrosine residue are exposed to the solvent.

Rabbits maintained for several weeks on a regimen of modest amounts of vitamin D and dietary cholesterol were placed in three groups in accordance with their response to repeated subcutaneous injections of nicotine in mineral oil. The group that had the greatest increase in plasma FFA following nicotine injections gradually developed, over a period of about 12 weeks, severe calcific atheroarteriosclerosis with peripheral thromboarteritis. Those that had a moderate increase in plasma FFA following nicotine injections developed calcific atheroarteriosclerosis but no thromboarteritis. Those that had the least increase in plasma FFA following nicotine injections developed no arterial lesions. Comparable or much greater increases in plasma FFA occurred in rabbits on the vitamin D-cholesterol regimen when adrenalin, ACTH or heparin was injected rather than nicotine. These animals did not develop calcific atheroarteriosclerotic thromboarteritis or any other lesions which could be correlated with the increased levels of plasma FFA. Inasmuch as nicotine, vitamin D or dietary cholesterol in the amounts used were innocuous when used alone, the interactions between the effects of at least these three factors need to be known in individual animals before the pathogenesis of the calcific atheroarteriosclerotic lesions with thrombosis can eventually be understood.