Rhizopus oryzae fungus cells producing L(+)-lactic acid: kinetic and metabolic parameters of free and PVA-cryogel-entrapped mycelium.

Spores of the filamentous fungus Rhizopus oryzae were entrapped in macroporous poly(vinyl alcohol) cryogel (PVA-cryogel). To prepare immobilised biocatalyst capable of producing L(+)-lactic acid (LA), the fungus cells were cultivated inside the carrier beads. The growth parameters and metabolic activity of the suspended (free) and immobilised cells producing LA in a batch process were comparatively investigated. The immobilised cells possessed increased resistance to high concentrations of accumulated product and gave much higher yields of LA in the iterative working cycle than the free cells did. Detailed kinetic analysis of the changes in the intracellular adenosine triphosphate concentration, specific rate of growth, substrate consumption and LA production showed that the fungus cells entrapped in PVA-cryogel are more attractive for biotechnological applications compared to the free cells.

Biostability and biocompatibility of a surface-grafted phospholipid monolayer on a solid substrate.

We have previously demonstrated phosphorylcholine monolayer chemically grafted onto a methacryloyl-terminated solid substrate by in situ polymerization. The in situ polymerization was carried out at the interface between a pre-assembled acrylated phospholipid monolayer produced by vesicle fusion and a methacryloyl-terminated substrate using a water-soluble initiator, 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPD). Herein, we examined the biostability and biocompatibility of a surface-grafted phospholipid monolayer (poly-PC) on a methacryloyl-terminated substrate using a "wash off' test, in vitro protein adsorption and in vivo cage implantation for time intervals of 4, 7, 14 and 21 days, respectively. In order to compare the biostability and biocompatibility of phospholipid surfaces on solid substrates, we used two types of phospholipid surfaces: a physically adsorbed phospholipid monolayer (PC) and a poly-PC. Atomic force microscopy and water contact angle measurements indicated that the poly-PC surface was more stable in PBS, Triton X-100 and to EO gas sterilization than the PC surface. The adsorption of proteins such as albumin, fibrinogen, IgG and human plasma proteins on the poly-PC surfaces were significantly reduced, in vitro. Moreover, the poly-PC surface greatly reduced macrophage adhesion and the formation of foreign body giant cells, in vivo.

Molecular basis of non-self recognition by the horseshoe crab tachylectins.

The self/non-self discrimination by innate immunity through simple ligands universally expressed both on pathogens and hosts, such as monosaccharides and acetyl group, depends on the density or clustering patterns of the ligands. The specific recognition by the horseshoe crab tachylectins with a propeller-like fold or a propeller-like oligomeric arrangement is reinforced by the short distance between the individual binding sites that interact with pathogen-associated molecular patterns (PAMPs). There is virtually no conformational change in the main or side chains of tachylectins upon binding with the ligands. This low structural flexibility of the propeller structures must be very important for specific interaction with PAMPs. Mammalian lectins, such as mannose-binding lectin and ficolins, trigger complement activation through the lectin pathway in the form of opsonins. However, tachylectins have no effector collagenous domains and no lectin-associated serine proteases found in the mammalian lectins. Furthermore, no complement-like proteins have been found in horseshoe crabs, except for alpha(2)-macroglobulin. The mystery of the molecular mechanism of the scavenging pathway of pathogens in horseshoe crabs remains to be solved.

cDNA cloning, tissue distribution, and subcellular localization of horseshoe crab big defensin.

A full-length cDNA for horseshoe crab big defensin with a strong antimicrobial activity was obtained from a hemocyte cDNA library. The open reading frame of the cDNA coded for an NH2-terminal signal sequence followed by a propeptide and the mature big defensin. The propeptide is linked to the mature protein through an -Arg-X-Lys/Arg-Arg- motif, the processing site for Kex2-like proteases. Northern blot analysis revealed that big defensin is expressed in all the tissues tested, suggesting that big defensin plays an important role not only in hemocytes but also in other tissues for host defense. The subcellular localization, determined by immunocytochemistry at ultrastructural level, confirmed the previous findings obtained by biochemical analysis that big defensin locates in both small and large granules in hemocytes. Big defensin is the first example to demonstrate the existence of broad tissue distribution in horseshoe crab.

Stratifin, a keratinocyte specific 14-3-3 protein, harbors a pleckstrin homology (PH) domain and enhances protein kinase C activity.

The intrinsic signal(s) responsible for the onset of human keratinocyte terminal differentiation is not yet fully understood. Evidence has been recently accumulated linking the phospholipase-mediated activation of protein kinase C to the coordinate changes in gene expression occurring during keratinocyte terminal differentiation. Here we report the purification of a keratinocyte-derived protein enhancing protein kinase C enzymatic activity. The stimulator eluted as a peak with estimated molecular mass of approximately 70 kDa, while analysis by SDS-PAGE showed a 30 kDa protein migrating as a distinct doublet, suggesting the formation of a 30 kDa homodimer. The amino acid sequence analysis allowed the unambigous identification of the protein kinase C stimulator as a mixture of the highly homologous sigma (stratifin) and zeta isoforms of 14-3-3 proteins, which are homodimers of identical 30 kDa subunits. Mono Q anion exchange chromatography and immunoblot analysis further confirmed that stratifin enhances protein kinase C activity. Stratifin was originally sequenced from a human keratinocyte protein database, but its function was unknown. The pleckstrin homology domain has been recently related to protein translocation to the cell membrane as well as to functional interactions of intracellular proteins involved in signal transduction. We show here that stratifin (and 14-3-3 zeta) harbors a pleckstrin homology domain, and the consequent functional implications will be discussed.

Scanning electron microscopic investigations of surface treated large-bore catheters used for extracorporeal detoxification methods.

Typical complications caused by surface properties of synthetic catheter implants are infection, thrombosis, and stenosis. New methods for surface modification with the aim of reducing such complications are ion beam-based technologies. In our study 109 large-bore catheters without (n = 42) and with treated surfaces with silver (n = 39) or silicone (n = 28) were inserted into the interna jugular and the subclavian veins and were used for extracorporeal detoxification methods. After removal, the catheters were investigated with scanning electron microscopy (SEM) and for bacterial colonization. In 42 large-bore catheters without surface treatment deposits of fibrin, protein and blood cells were seen on the inner and outer surface. Bacterial colonization was observed in 38.1%. In contrast, the catheters with treated outer surfaces showed a very low thrombogenicity and a low contamination rate of 8.9%. The ion beam-based technologies reduce the thrombogenicity and infection rates of the catheter surfaces. In comparison to catheters without treated surfaces, catheters with surface treatment are good alternatives in blood contacting applications ranging from hemodialysis to oncology.

Multiple isoforms of the human pentraxin serum amyloid P component.

Human serum amyloid P component (SAP) isolated from 20 healthy individuals was analyzed by anion exchange chromatography and isoelectric focusing (IEF) in order to investigate the existence of multiple forms of SAP and interindividual structural differences. Anion exchange chromatography showed one major and several minor subpopulations of SAP. IEF of all SAP isolates showed a previously unreported degree of heterogeneity with six isoelectric forms (pKi range 5.5-6.1) and with minor interindividual differences in respect of isoelectric points. Total enzymatic deglycosylation of SAP reduced the number of bands in IEF to two indicating the existence of two types of polypeptide chains.

Vascular changes and blood-brain barrier damage in the pathogenesis of polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (membranous lipodystrophy).

The histopathological, immunohistochemical and electron microscopic findings in eight patients with polycystic lipomembranous osteodysplasia and sclerosing leukoencephalopathy (PLO-SL) are described. This autosomally recessively inherited disease is first manifested by multiple bone cysts, which are later followed around the age of 30 by severe neuropsychiatric syndrome. The pathogenesis of PLO-SL has not been established, and the search for the most suspected error in lipid metabolism has been unsuccessful. The typical macroscopic features were marked hydrocephalus ex vacuo due to severe destruction of the white matter (WM) with extensive secondary astrocytic gliosis, and with relatively better preserved gray matter (GM). The basement membranes of blood vessels with plump endothelium were thickened and often multiplied, most prominently in the WM. Extravasation of plasma constituents was demonstrated immunohistochemically. On the basis of the vascular changes, also present in bone lesions, it is proposed that severe chronic vasogenic brain edema is the main pathogenetic mechanism of the severe leukoencephalopathy in this disease entity.

Hapten-tagged plasma proteins as immunocytochemical probes for the study of vascular permeability.

Bovine serum albumin and transferrin were covalently coupled with fluorescein isothiocyanate and digoxigenin, respectively, and intravenously co-injected in equal amounts in mouse. The derivation of the two proteins induces minor alterations of their physicochemical properties as well as of their physiological functions. The two tracers were revealed within vascular and extravascular compartments of diaphragm by quantitative postembedding immunocytochemistry, using antibodies against each of the haptens in conjunction with the protein AG-gold complexes. The influence of different fixatives and embedding protocols on the immunodetectability of the hapten-tagged proteins was assessed. Both resist reasonably well to osmication and embedding in Epon. None of the haptens reacted with the heterologous antibody. At 30 minutes after injection, the tracers were detected in blood plasma, interstitium, and endothelial plasmalemmal vesicles. The presence of both proteins within the interendothelial clefts was inconspicuous. The ratios between the labeling densities found over endothelium, interstitial space, and vascular lumen were similar for both tracers. This suggests that the endothelium of mouse diaphragm capillaries might exhibit comparable permeabilities towards serum albumin and transferrin which are similar in size and charge. The study shows that hapten-tagged polypeptides are close to the corresponding native macromolecules, and represent interesting tools for the morphological study of dynamic processes such as transcytosis.

Solvent/detergent-treated plasma: a virus-inactivated substitute for fresh frozen plasma.

Fresh frozen plasma (FFP) is prepared in blood banks world-wide as a by-product of red blood cell concentrate preparation. Appropriate clinical use is for coagulation factor disorders where appropriate concentrates are unavailable and when multiple coagulation factor deficits occur such as in surgery. Viral safety depends on donor selection and screening; thus, there continues to be a small but defined risk of viral transmission comparable with that exhibited by whole blood. We have prepared a virus sterilized FFP (S/D-FFP) by treatment of FFP with 1% tri(n-butyl)phosphate (TNBP) and 1% Triton X-100 at 30 degrees C for 4 hours. Added reagents are removed by extraction with soybean oil and chromatography on insolubilized C18 resin. Treatment results in the rapid and complete inactivation of greater than or equal to 10(7.5) infectious doses (ID50) of vesicular stomatitis virus (VSV) and greater than or equal to 10(6.9) ID50 of sindbis virus (used as marker viruses), greater than or equal to 10(6.2) ID50 of human immunodeficiency virus (HIV), greater than or equal to 10(6) chimp infectious doses (CID50) of hepatitis B virus (HBV), and greater than or equal to 10(5) CID50 of hepatitis C virus (HCV). Immunization of rabbits with S/D-FFP and subsequent adsorption of elicited antibodies with untreated FFP confirmed the absence of neoimmungen formation. Coagulation factor content was comparable with that found in FFP. Based on these laboratory and animal studies, together with the extensive history of the successful use of S/D-treated coagulation factor concentrates, we conclude that replacement of FFP with S/D-FFP, prepared in a manufacturing facility, will result in improved virus safety and product uniformity with no loss of efficacy.

[Serum factors regulating macrophage migration in chronic cicatricial stenosis of larynx and trachea in children]. / Sootnoshenie syvorotochnykh factorov, reguliruiushchikh migratsiiu makrofagov pri khronisheskom rubtsovom stenoze gortani i trakhei u detei.

Many children with chronic cicatricial stenosis of the larynx and trachea showed a high activity of the factor stimulating macrophage migration in vitro (MSF). The high MSF level is typical of patients with large scars in tissues rich in cellular elements. The MSF predominance was to a certain extent correlated with abnormal responses to surgical intervention. This was indicated by poor results of surgical treatment if the MSF was low before and immediately after it. With normal MSF the treatment was effective in at least 50% of cases. The therapeutic effectiveness was correlated with MSF variations at an early postoperative stage.

Morphologic characteristics of adsorbed human plasma proteins on vascular grafts and biomaterials.

Protein adsorption on the surfaces of clinically significant prosthetic vascular graft materials from human whole blood was independent of plasma concentration as determined morphologically by use of immunogold labels. Some proteins, such as fibrinogen, adsorbed in a multilayer pattern on expanded polytetrafluoroethylene and had a preference for particular surface features of the polymer. Other proteins, such as Hageman factor (factor XII), showed diffuse adsorption patterns. Physiologically significant proteins that have not been well studied, such as immunoglobulin G and factor VIII, adsorbed readily to the surface of expanded polytetrafluoroethylene. This finding may be significant since adsorbed proteins may activate coagulation mechanisms and immunologic responses, including platelet and monocyte adhesion and activation. Any human blood protein for which an antibody has been developed can be studied by use of this technique.

In vivo protein adsorption onto polymers: a transmission electron microscopic study.

This paper reports in vivo protein adsorption onto polymers, including Biomer, PEO grafted Biomer (B-PEO-4K), heparin immobilized Biomer with PEO spacers (B-PEO-4K-HEP), and HEMA-Styrene block copolymer (H-S). Vascular grafts (6 mm ID, 7 cm in length) were fabricated with Biomer, coated on their luminal surfaces with test polymers, and implanted into the abdominal aorta of dogs. After 3 weeks-1 month, the grafts were retrieved and processed for TEM and SEM. TEM measured the thickness of adsorbed protein layers stained with a OsO4 solution, and the distribution pattern of adsorbed proteins (albumin, IgG and fibrinogen) using the immunoperoxidase technique. Retrieved grafts of Biomer and B-PEO-4K showed mural thrombi along the graft length, while thrombus formation on B-PEO-4K-HEP and H-S grafts was limited to the anastomotic sites. SEM pictures of B-PEO-4-HEP and H-S surfaces demonstrated clear morphology, with minimal platelet adhesion and activation, and microthrombi. Biomer and B-PEO-4K demonstrated a thick proteinaceous layer (1000-2000 A), whereas B-PEO-4K-HEP and H-S showed what can be described as a monolayer protein thickness (200-300 A). B-PEO-4K-HEP and H-S showed a monolayer-like adsorbed protein pattern, with high concentrations of albumin and IgG, and less fibrinogen, while Biomer and B-PEO-4K showed multilayered patterns with relatively high concentrations of fibrinogen, and less albumin. These results suggest that the surface properties of polymer may control protein adsorption pattern, and the composition of adsorbed protein is essential to in vivo long-term blood compatibility.

Determination of the disulfide array in the human defensin HNP-2. A covalently cyclized peptide.

HNP-2 is a 29-residue peptide present in human neutrophils and is a member of the defensin family of antimicrobial peptides. All defensins contain an invariant disulfide infrastructure comprised of 6 half-cystine residues. The disulfide structure of HNP-2 was determined using a novel method to identify the cross-links involving the amino- and carboxyl-terminal cysteine residues. A derivative of HNP-2 was synthesized by covalent modification of the terminal cysteine residues. This derivative was purified, characterized, and subjected to exhaustive proteolytic digestion. Characterization of purified proteolytic fragments by amino acid analysis and/or sequence analysis identified an oligopeptide containing all 6 cystine residues. This oligopeptide was subjected to a single cycle of Edman degradation to cleave the peptide bond linking 2 adjacent cysteines. Purification and characterization of the Edman reaction products allowed for assignment of the disulfide array in HNP-2, revealing a cystine motif unique to the defensin peptide family. Further, the covalent structure of HNP-2 was found to be cyclic as one disulfide links the amino- and carboxyl-terminal cysteine residues. HNP-2 is the only polypeptide known to possess such a configuration.