Management of osteoradionecrosis of the jaws with pentoxifylline-tocopherol: a systematic review of the literature and meta-analysis.

Osteoradionecrosis (ORN) of the jaws remains among the most commonly encountered and challenging complications of radiotherapy to the head and neck. The purpose of this study was to provide a review of the medical management for ORN and evaluate the reported outcomes with the use of pentoxifylline and tocopherol (PENTO), by means of a systematic review and meta-analysis. The predictor variable was the use of PENTO in the treatment of ORN. The outcome variable was the proportion of full recovery or significant improvement not requiring further intervention. The likelihood function was used to combine the studies and estimate the proportion and standard deviation of each outcome by the maximum likelihood estimation. Seven studies met the inclusion criteria. A total 211 patients were treated. One hundred twenty-six patients recovered fully or improved significantly not requiring further intervention. Sixty patients remained the same, 10 were lost to follow-up, and the disease progressed in 15. The current literature supports the use of PENTO in the treatment of ORN of the jaws. Additional well-designed prospective studies are needed in order to further validate the regimen that can then be employed in the treatment of ORN.

In vitro digestion of purified ß-casein variants A(1), A(2), B, and I: effects on antioxidant and angiotensin-converting enzyme inhibitory capacity.

Genetic polymorphisms of bovine milk proteins affect the protein profile of the milk and, hence, certain technological properties, such as casein (CN) number and cheese yield. However, reports show that such polymorphisms may also affect the health-related properties of milk. Therefore, to gain insight into their digestion pattern and bioactive potential, ß-CN was purified from bovine milk originating from cows homozygous for the variants A(1), A(2), B, and I by a combination of cold storage, ultracentrifugation, and acid precipitation. The purity of the isolated ß-CN was determined by HPLC, variants were verified by mass spectrometry, and molar extinction coefficients at λ=280nm were determined. ß-Casein from each of the variants was subjected to in vitro digestion using pepsin and pancreatic enzymes. Antioxidant and angiotensin-converting enzyme (ACE) inhibitory capacities of the hydrolysates were assessed at 3 stages of digestion and related to that of the undigested samples. Neither molar extinction coefficients nor overall digestibility varied significantly between these 4 variants; however, clear differences in digestion pattern were indicated by gel electrophoresis. In particular, after 60min of pepsin followed by 5min of pancreatic enzyme digestion, one ≈4kDa peptide with the N-terminal sequence (106)H-K-E-M-P-F-P-K- was absent from ß-CN variant B. This is likely a result of the (122)Ser to (122)Arg substitution in variant B introducing a novel trypsin cleavage site, leading to the changed digestion pattern. All investigated ß-CN variants exhibited a significant increase in antioxidant capacity upon digestion, as measured by the Trolox-equivalent antioxidant capacity assay. After 60min of pepsin + 120min of pancreatic enzyme digestion, the accumulated increase in antioxidant capacity was ≈1.7-fold for the 4 ß-CN variants. The ACE inhibitory capacity was also significantly increased by digestion, with the B variant reaching the highest inhibitory capacity at the end of digestion (60min of pepsin + 120min of pancreatic enzymes), possibly because of the observed alternative digestion pattern. These results demonstrate that genetic polymorphisms affect the digestion pattern and bioactivity of milk proteins. Moreover, their capacity for radical scavenging and ACE inhibition is affected by digestion.

Platelet apoptosis by cold-induced glycoprotein Ibα clustering.

BACKGROUND: Cold-storage of platelets followed by rewarming induces changes in Glycoprotein (GP) Ibα-distribution indicative of receptor clustering and initiates thromboxane A(2) -formation. GPIbα is associated with 14-3-3 proteins, which contribute to GPIbα-signaling and in nucleated cells take part in apoptosis regulation. OBJECTIVES AND METHODS: We investigated whether GPIbα-clustering induces platelet apoptosis through 14-3-3 proteins during cold (4 h 0 °C)-rewarming (1 h 37 °C). RESULTS: During cold-rewarming, 14-3-3 proteins associate with GPIbα and dissociate from Bad inducing Bad-dephosphorylation and activation. This initiates pro-apoptosis changes in Bax/Bcl-x(L) and Bax-translocation to the mitochondria, inducing cytochrome c release. The result is activation of caspase-9, which triggers phosphatidylserine exposure and platelet phagocytosis by macrophages. Responses are prevented by N-acetyl-D-glucosamine (GN), which blocks GPIbα-clustering, and by O-sialoglycoprotein endopeptidase, which removes extracellular GPIbα. CONCLUSIONS: Cold-rewarming triggers apoptosis through a GN-sensitive GPIbα-change indicative of receptor clustering. Attempts to improve platelet transfusion by cold-storage should focus on prevention of the GPIbα-change.

Phosphatidylserine exposure and procoagulant activity in acute promyelocytic leukemia.

BACKGROUND: Acute promyelocytic leukemia (APL) frequently causes disseminated intravascular coagulation that can worsen with cytotoxic chemotherapy but improve with the therapeutic differentiating agents, all trans retinoic acid (ATRA) and arsenic trioxide (As(2)O(3)). APL cells display tissue factor but the relationship of tissue factor and other procoagulant activity to phosphatidylserine (PS) exposure is largely unknown. METHODS: Lactadherin, a milk protein with stereospecific binding to phosphatidyl-L-serine, was used as a probe for PS exposure on an immortalized APL cell line (NB4) and on the cells of eight patients with APL. PS exposure was evaluated with flow cytometry, confocal microscopy, coagulation assays, and purified prothrombinase and factor (F) Xase assays. RESULTS: Plasma procoagulant activity of NB4 and APL cells increased approximately 15-fold after exposure to etoposide or daunorubicin and decreased 80% after treatment with ATRA or As(2)O(3). Procoagulant activity corresponded to exposed PS on viable APL cells. PS exposure decreased after treatment with ATRA or As(2)O(3) and increased after treatment with daunorubicin or etoposide. Excess lactadherin inhibited 80-85% of intrinsic FXase, FVIIa-tissue factor and prothrombinase activities on both NB4 cells and APL cells. Confocal microscopy identified membrane patches that stained with lactadherin, but not annexin V, demonstrating focal, low-level PS exposure. CONCLUSIONS: PS is exposed on viable APL cells and is necessary for approximately 80% of procoagulant activity.

Characterization of human mucin (MUC15) and identification of ovine and caprine orthologs.

The glycoprotein MUC15 (mucin 15) was initially isolated from the bovine milk fat globule membrane. The present work demonstrates the existence of immunologically similar proteins ( approximately 130 kDa) in ovine, caprine, porcine, and buffalo milk samples. Purification and N-terminal amino acid sequencing confirmed the presence of ovine and caprine MUC15 orthologs in milk fat globule membranes. Expression of MUC15 in human milk was demonstrated by immunostaining ( approximately 150 kDa) as well as by mass spectrometry. Screening of a human multiple tissue expression array showed abundant MUC15 gene expression in placenta, salivary gland, thyroid gland, trachea, esophagus, kidney, testis, and the leukemia K-562 cell line. Furthermore, moderate expression was seen in the pancreas, adult and fetal lung, fetal kidney, lymph node, adult and fetal thymus, and parietal lobe. Structural motifs for interactions (epidermal growth factor receptor and Src homology 2 domains) are identified in the intracellular region. Implication of the mucin in signal transduction and the potential physiological function of MUC15 are discussed.

Lactadherin blocks thrombosis and hemostasis in vivo: correlation with platelet phosphatidylserine exposure.

BACKGROUND: Platelet membrane phosphatidylserine (PS) is considered to be essential for hemostasis and thrombosis, but the in vivo topography of platelet PS has not been characterized. We hypothesized that platelet PS exposure would be identified on adherent platelets at the site of vascular injury and that blockade of PS would impede hemostasis and thrombosis. OBJECTIVE: To localize and estimate the extent of platelet PS exposure and evaluate the impact of PS blockade in vivo. METHODS: Lactadherin, a PS-binding milk protein, was utilized together with annexin V to detect both partial and complete membrane PS exposure on platelets in a mouse model of thrombosis and to evaluate the functional need for PS. Preliminary experiments were performed with synthetic membranes and with purified platelets. RESULTS: The number of lactadherin-binding sites on synthetic membranes was proportional to PS content, whereas annexin V required a threshold of 2.5-8% PS. Approximately 95% of thrombin-stimulated platelets exposed PS, but the quantity was below the threshold for annexin V binding at physiologic Ca(2+) concentrations. In mice, most adherent and aggregated platelets on the walls of ferric chloride-treated mesenteric veins exposed low levels of PS, rather than having complete exposure. In mice, blockade of PS with lactadherin inhibited platelet prothrombinase and factor Xase activity, and prolonged tail bleeding time and the time to carotid artery thrombosis. CONCLUSIONS: In vivo PS exposure contributes to both hemostasis and thrombosis. In this model of vascular injury, most platelets exhibit partial rather than complete PS exposure.

Characterization of carbohydrate structures of bovine MUC15 and distribution of the mucin in bovine milk.

The present work reports the characterization of carbohydrate structures and the distribution of the newly identified mucin MUC15, a highly glycosylated protein associated with the bovine milk fat globule membrane (MFGM). Distribution of MUC15 was investigated in various fractions of bovine milk by densitometric scanning of Western blots. In raw milk, MUC15 was shown to constitute 0.08% (wt) of the protein and approximately 1.5% (wt) of the MFGM-associated proteins. Surprisingly, this study showed that in addition to the fat-containing fractions, such as MFGM and buttermilk, MUC15 was present in nonfat-containing fractions as well, such as skim milk and whey. Compositional and structural studies of the carbohydrates of bovine milk MUC15 showed that the glycans are composed of fucose, galactose, mannose, N-acetylgalactosamine, N-acetylglycosamine, and sialic acid. The carbohydrate was shown to constitute 65% of the total molecular weight, and the molar ratios of the individual sugars to protein of the O-linked glycans were determined. The glycan structures of MUC15 were further studied by enzymatic deglycosylation experiments using different endo- and exoglycosidases as well as a panel of lectins. The N-linked glycans were shown to contain mainly hybrid-type N-glycans. In addition, the N-glycans were shown to be sialylated and contain terminal poly-lactosamine structures. The O-linked glycans were found to constitute some unsubstituted Core-1 structures and a substantial number of sialylated Core-1 O-linked glycans. By comparing the results of peanut agglutinin lectin binding, enzymatic deglycosylation, and monosaccharide composition analysis, we concluded that bovine MUC15 also contains more complex O-glycans containing high amounts N-acetylglucosamine residues. Furthermore, a small subset of the O-linked glycans is decorated with lactosamine on their terminal ends.

Inhibitory activities of bovine macromolecular whey proteins on rotavirus infections in vitro and in vivo.

Rotavirus is a major cause of infantile viral gastroenteritis and can lead to severe and sometimes lethal dehydration. Previous studies have shown that breast-fed children are better protected against symptomatic infections, and that the milk fat globule protein lactadherin might be at least partly responsible for this effect. In vitro studies have shown that human lactadherin, in contrast to the bovine ortholog, could inhibit rotavirus infectivity, and that bovine MUC1 and a commercially available bovine macromolecular whey protein (MMWP) fraction proved to be effective. The present work describes the versatility of MMWP against the infection of 2 human intestinal cell lines (Caco-2 and FHs 74 Int) by 4 different rotavirus strains (Wa, RRV, YM, RF). Isolation of a protein fraction (CM3Q3) from MMWP that effectively inhibits rotavirus infectivity in vitro is documented. Purification was achieved by monitoring the rotaviral inhibitory activity in fractions obtained from 2 consecutive steps of ion-exchange chromatography. The major component of CM3Q3 was shown to be bovine IgG, and the attenuating capacity of this fraction is most properly linked to this component. The capacity of MMWP, MUC1, lactadherin, and the CM3Q3 fraction to inhibit the infectivity of the murine EMcN rotavirus strain was analyzed in adult BALB/c mice by using 2 different amounts of virus (10 and 100 times more than 50% the viral shedding doses). Only CM3Q3 was able to significantly affect the shedding of rotavirus in the stools of experimentally infected mice when the high viral dose was given. Detection of rotavirus-specific serum antibodies showed that the high dose infected all groups of mice. Experiments with the low dose of virus implied that all the tested milk proteins could affect the viral shedding in stools; in addition, use of MUC1, MMWP, and CM3Q3 prevented the appearance of serum viral antibodies. The advantages of using bovine immunoglobulins to induce passive immunity against rotavirus have been substantially investigated, although studies have mainly focused on the use of derivatives from immunized cows, especially colostrum. This report associates considerable activity against rotavirus infectivity with an ordinary whey product, suggesting that there might be alternatives to colostral-derived products.

Activated scramblase and inhibited aminophospholipid translocase cause phosphatidylserine exposure in a distinct platelet fraction.

Platelet procoagulant activity is mainly determined by the extent of surface-exposed phosphatidylserine (PS), controlled by the activity of aminophospholipid translocase and phospholipid scramblase. Here, we studied both transport activities in single platelets upon stimulation with various agonists. Besides the formation of procoagulant microparticles, the results show that a distinct fraction of the platelets exposes PS when stimulated. The extent of PS exposure in these platelet fractions was similar to that in platelets challenged with Ca2+-ionophore, where all cells exhibit maximal attainable PS exposure. The size of the PS-exposing fraction depends on the agonist and is proportional to the platelet procoagulant activity. Scramblase activity was observed only in the PS-exposing platelet fraction, whereas translocase activity was exclusively detectable in the fraction that did not expose PS. We conclude that, irrespective of the agonist, procoagulant platelets exhibit maximal surface exposure of PS by switching on scramblase and inhibiting translocase activity.

Inhibitory effects of human and bovine milk constituents on rotavirus infections.

Among etiologic agents, rotavirus is the major cause of severe dehydration diarrhea in infant mammals. In vitro and in vivo studies have indicated that the human milk-fat globule protein lactadherin inhibits rotavirus binding and protects breast-fed children against symptomatic rotavirus infection. The present work was conducted to evaluate the effect of lactadherin, along with some other milk proteins and fractions, on rotavirus infections in MA104 and Caco-2 cell lines. It is shown that human, and not bovine, lactadherin inhibits Wa rotavirus infection in vitro. Human lactadherin seems to act through a mechanism involving protein-virus interactions. The reason for the activity of human lactadherin is not clear, but it might lie within differences in the protein structure or the attached oligosaccharides. Likewise, in our hands, bovine lactoferrin did not show any suppressive activity against rotavirus. In contrast, MUC1 from bovine milk inhibits the neuraminidase-sensitive rotavirus RRV strain efficiently, whereas it has no effect on the neuraminidase-resistant Wa strain. Finally, a bovine macromolecular whey protein fraction turned out to have an efficient and versatile inhibitory activity against rotavirus.

frizzled 9 is expressed in neural precursor cells in the developing neural tube.

The wnt signaling pathway has important functions in nervous system development. To better understand this process we have cloned and analyzed the expression of the wnt receptor, frizzled 9, in the developing nervous system in mouse, chick and zebrafish. The earliest expression of mouse frizzled 9 mRNA expression begins at E8.5 with expression throughout the entire rostral-caudal neuraxis. This early expression pattern within the neural tube appears to be conserved between chick and zebrafish. Expression becomes restricted to a ventral domain in the mouse ventricular zone at E11.5, a region specified to give rise to neurons and glia. Using a polyclonal antibody to MFZ9 further shows expression limited to neural restricted precursors cells.

Regulation of eye development by frizzled signaling in Xenopus.

Eye development in both invertebrates and vertebrates is regulated by a network of highly conserved transcription factors. However, it is not known what controls the expression of these factors to regulate early eye formation and whether transmembrane signaling events are involved. Here we establish a role for signaling via a member of the frizzled family of receptors in regulating early eye development. We show that overexpression of Xenopus frizzled 3 (Xfz3), a receptor expressed during normal eye development, functions cell autonomously to promote ectopic eye formation and can perturb endogenous eye development. Ectopic eyes obtained with Xfz3 overexpression have a laminar organization similar to that of endogenous eyes and contain differentiated retinal cell types. Ectopic eye formation is preceded by ectopic expression of transcription factors involved in early eye development, including Pax6, Rx, and Otx2. Conversely, targeted overexpression of a dominant-negative form of Xfz3 (Nxfz3), consisting of the soluble extracellular domain of the receptor, results in suppression of endogenous Pax6, Rx, and Otx2 expression and suppression of endogenous eye development. This effect can be rescued by coexpression of Xfz3. Finally, overexpression of Kermit, a protein that interacts with the C-terminal intracellular domain of Xfz3, also blocks endogenous eye development, suggesting that signaling through Xfz3 or a related receptor is required for normal eye development. In summary, we show that frizzled signaling is both necessary and sufficient to regulate eye development in Xenopus.

Purification of MUC1 from bovine milk-fat globules and characterization of a corresponding full-length cDNA clone.

The highly glycosylated protein MUC1 was purified from bovine milk-fat globule membranes by a procedure involving detergent extraction, ion-exchange chromatography and reverse-phase chromatography. The identity of the purified mucin protein was confirmed by N-terminal sequencing and partial amino acid sequences obtained by peptide mapping. The complete amino acid sequence of MUC1 was determined by cloning and sequencing the corresponding bovine mammary gland cDNA, which was shown to encode a protein of 580 amino acid residues comprising a cleavable signal peptide of 22 residues. The deduced amino acid sequence demonstrated structural features characteristic for mucins, including an extracellular tandem repeat region with 11 partially conserved repeats (20 amino acids each), a membrane-proximal SEA module, a transmembrane domain, and a cytoplasmic C-terminal region. Monosaccharide composition determinations suggested significant structural differences between O-linked glycans of MUC1 originating from either bovine or human milk. Interspecies differences of the consensus repeat sequence in MUC1 and the physiological functions are discussed.

SIMPSON: a general simulation program for solid-state NMR spectroscopy.

A computer program for fast and accurate numerical simulation of solid-state NMR experiments is described. The program is designed to emulate a NMR spectrometer by letting the user specify high-level NMR concepts such as spin systems, nuclear spin interactions, RF irradiation, free precession, phase cycling, coherence-order filtering, and implicit/explicit acquisition. These elements are implemented using the Tcl scripting language to ensure a minimum of programming overhead and direct interpretation without the need for compilation, while maintaining the flexibility of a full-featured programming language. Basically, there are no intrinsic limitations to the number of spins, types of interactions, sample conditions (static or spinning, powders, uniaxially oriented molecules, single crystals, or solutions), and the complexity or number of spectral dimensions for the pulse sequence. The applicability ranges from simple 1D experiments to advanced multiple-pulse and multiple-dimensional experiments, series of simulations, parameter scans, complex data manipulation/visualization, and iterative fitting of simulated to experimental spectra. A major effort has been devoted to optimizing the computation speed using state-of-the-art algorithms for the time-consuming parts of the calculations implemented in the core of the program using the C programming language. Modification and maintenance of the program are facilitated by releasing the program as open source software (General Public License) currently at http://nmr.imsb.au.dk. The general features of the program are demonstrated by numerical simulations of various aspects for REDOR, rotational resonance, DRAMA, DRAWS, HORROR, C7, TEDOR, POST-C7, CW decoupling, TPPM, F-SLG, SLF, SEMA-CP, PISEMA, RFDR, QCPMG-MAS, and MQ-MAS experiments.

Structural characterization of the fibroblast growth factor-binding protein purified from bovine prepartum mammary gland secretion.

A novel heparin-binding protein was purified to homogeneity from bovine prepartum mammary gland secretion using heparin-Sepharose chromatography and reverse-phase high performance liquid chromatography successively. Structural information obtained by N-terminal amino acid sequencing of a series of proteolytically generated peptides permitted the cloning of the corresponding cDNA. The isolated cDNA was 1170 base pairs long and consisted of an 83-base pair 5'-untranslated region followed by a 702-base pair coding region and a 385-base pair 3'-untranslated region. The open reading frame resulted in a protein comprising 234- amino acid residues, including a signal sequence. Instead of Lys(24) as the predicted N terminus, Edman degradation of the native protein revealed N-terminal processing at two sites as follows: a primary site between Arg(31)-Gly(32) and a secondary site between Arg(51)-Ser(52). The amino acid sequence showed a significant similarity with that of human (60%) and mouse (53%) fibroblast growth factor-binding protein (FGF-BP). Accordingly, ligand blotting experiments revealed that bovine FGF-BP bound FGF-2. The theoretical mass of the protein predicted from the cDNA sequence is 22.5 kDa. However, the molecular mass of the purified protein was estimated to 28.6 kDa by mass spectrometry and 36 kDa by electrophoresis. The apparent molecular weight differences are most likely due to post-transcriptional modifications, shown to involve N- and O-glycosylation of Asn(155) and Ser(172), respectively. All 10 cysteine residues in the protein participated in disulfide bonds, and the pattern was identified as Cys(71)-Cys(88), Cys(97)-Cys(130), Cys(106)-Cys(142), Cys(198)-Cys(234), and Cys(214)-Cys(222). As the 10 cysteines of the three known FGF-BPs are positionally conserved, the disulfide bond pattern of bovine FGF-BP may be regarded as representative for the FGF-BP family.

Functional analyses of two cellular binding domains of bovine lactadherin.

The glycoprotein bovine lactadherin (formerly known as PAS-6/7) comprises two EGF-like domains and two C-like domains found in blood clotting factors V and VIII. Bovine lactadherin binds to alpha(v)beta(5) integrin in an RGD-dependent manner and also to phospholipids, especially phosphatidyl serine. To define and characterize these bindings the interactions between lactadherin and different mammalian cell types were investigated. Using recombinant forms of bovine lactadherin, the human breast carcinomas MCF-7 cells expressing the alpha(v)beta(5) integrin receptor were shown to bind specifically to RGD containing lactadherin but not to a mutated RGE lactadherin. A monoclonal antibody against the alpha(v)beta(5) integrin receptor and a synthetic RGD-containing peptide inhibited the adhesion of MCF-7 cells to lactadherin. Green monkey kidney MA-104 cells, also expressing the alpha(v)beta(3) together with the alpha(v)beta(5) integrin, showed binding to bovine lactadherin via both integrins. To investigate the interaction of lipid with lactadherin two fragments were expressed corresponding to the C1C2 domains and the C2 domain. Both fragments bound to phosphatidyl serine in a concentration-dependent manner with an affinity similar to native lactadherin (K(d) = 1.8 nM). A peptide corresponding to the C-terminal part of the C2 domain inhibited the binding of lactadherin to phospholipid in a concentration-dependent manner, and finally it was shown that lactadherin mediates binding between artificial phosphatidyl serine membranes and MCF-7 cells. Taken together these results show that lactadherin can act as link between two surfaces by binding to integrin receptors through its N-terminus and to phospholipids through its C-terminus.

Cysteines involved in the interconversion between dehydrogenase and oxidase forms of bovine xanthine oxidoreductase.

Mammalian xanthine oxidoreductase exists intracellularly in its dehydrogenase form. However, outside of this reducing milieu the enzyme quickly transforms into an oxidase form. Interconversion can be controlled by sulfhydryl reactive reagents, suggesting that disulfide bridging is linked to this phenomenon. The present work identified cysteines involved in the interconversion process. Purified enzyme was subjected to mild reduction with 1,4-dithioerythriol to regain dehydrogenase activity, and the accessible cysteines were labeled with specific radioactive alkylation reagents, iodoacetic acid. This partial alkylation stabilizes the dehydrogenase form, presumable by hindering formation of disulfide bond(s). Six of 38 cysteines were found to be labeled (residues 169, 170, 535, 992, 1317, and 1325). The significance of this labeling of bovine xanthine oxidoreductase is discussed in relation to structural knowledge about the enzyme, and especially by comparison with the AA sequences of avian and invertebrate enzymes, which do not undergo conversion. Cysteines 535 and 992 are the most likely marked residues to be involved in the interconversion, whereas the other cysteines are located too far from the cofactorbinding areas in xanthine oxidoreductase.

Isolation of adipophilin and butyrophilin from bovine milk and characterization of a cDNA encoding adipophilin.

The milk fat globule membrane-associated proteins adipophilin (alias adipocyte differentiation-related protein) and butyrophilin were purified from bovine milk by reverse-phase chromatography. The nucleotide sequence of bovine adipophilin was obtained via peptide mapping and sequencing of a mammary gland cDNA clone, which comprises 1841 nucleotides and has an open reading frame of 450 amino acids. By peptide mapping, 19% of the amino acid sequence was confirmed. The obtained amino acid sequence has 87 and 80% identical residues with human and mouse adipophilin, respectively. Alignment with the proteins perilipin and TIP47 revealed two highly conserved segments, which may assemble into amphipathic alpha-helices.

Assignment of disulfide bridges in bovine CD36.

The multifunctional membrane protein CD36 is expressed on platelets, mature monocytes and macrophages, microvascular endothelial cells and mammary epithelial cells. The exact physiological function of this glycoprotein is unclear. In order to determine the number and pattern of disulfide bridges, CD36 was purified from bovine milk fat globule membranes. The purification procedure involved Triton X-114 extraction, DEAE-Sepharose ion-exchange chromatography and reverse-phase chromatography on a Resource RPC column. The CD36 preparation was used for characterization of the disulfide bridge pattern, which was determined by peptide mapping, amino acid sequence analysis, and matrix-assisted laser-desorption ionization/time of flight mass spectrometry. We have found that there are no free cysteines in CD36 and that the six centrally clustered cysteines are linked by disulfide bonds, Cys242-Cys310, Cys271-Cys332 and Cys312-Cys321, resulting in a 1-3, 2-6 and 4-5 arrangement of the disulfide bridges. These data are in agreement with a model where the protein is oriented so that it has two short intracellular segments (residues 1-6 and 461-471) and two transmembrane domains (residues 7-28 and 439-460), and with four cysteines expected to be acylated placed near the intracellular side of the membrane. The remaining part of CD36 is extracellular, comprising eight glycosylations and three disulfide bridges. In the CD36 family of membrane proteins, it is likely that a similar pattern of disulfide bridges can be found in the sensory neuron membrane protein-1 from the silk moth Antheraea polyphemus and the mammalian scavenger receptor class B type I, whereas lysosome membrane protein II, and epithelial membrane protein from Drosophila melanogaster are both lacking one cysteine in the area of interest.

A refined kinetic analysis of plasminogen activation by recombinant bovine tissue-type plasminogen activator indicates two interconvertible activator forms.

Bovine tissue-type plasminogen activator (tPA) was heterologously expressed in the methylotrophic yeast Pichia pastoris and characterized structurally and kinetically. The bovine single-chain tPA-mediated activation of bovine plasminogen was studied in the presence and absence of fibrinogen fragments. We have proposed a refined new method of kinetic analysis which allows examination of both stationary and prestationary phases of this process. The investigation revealed the presence of two interconvertible forms of the recombinant bovine tPA being in equilibrium at a 1 to 50 ratio. Only the minor form was able to bind and activate plasminogen. Saturation of the whole pool of tPA required high plasminogen concentration (Km >/= 5 microM) in order to reverse the equilibrium between the two forms. Fibrinogen fragments activated the single-chain tPA due to preferential binding and stabilization of the minor "active" form of the enzyme until all the molecules of tPA were converted. The same mechanism could be applied to human tPA as well. The Km values, obtained for recombinant bovine and human tPA in the presence of fibrinogen fragments, were found to be similar (Km = 0.1 microM) while kcat of human tPA was 5-10 times higher.