Tubulization with chitosan guides for the repair of long gap peripheral nerve injury in the rat.

Biosynthetic guides can be an alternative to nerve grafts for reconstructing severely injured peripheral nerves. The aim of this study was to evaluate the regenerative capability of chitosan tubes to bridge critical nerve gaps (15 mm long) in the rat sciatic nerve compared with silicone (SIL) tubes and nerve autografts (AGs). A total of 28 Wistar Hannover rats were randomly distributed into four groups (n = 7 each), in which the nerve was repaired by SIL tube, chitosan guides of low (∼2%, DAI) and medium (∼5%, DAII) degree of acetylation, and AG. Electrophysiological and algesimetry tests were performed serially along 4 months follow-up, and histomorphometric analysis was performed at the end of the study. Both groups with chitosan tubes showed similar degree of functional recovery, and similar number of myelinated nerve fibers at mid tube after 4 months of implantation. The results with chitosan tubes were significantly better compared to SIL tubes (P < 0.01), but lower than with AG (P < 0.01). In contrast to AG, in which all the rats had effective regeneration and target reinnervation, chitosan tubes from DAI and DAII achieved 43 and 57% success, respectively, whereas regeneration failed in all the animals repaired with SIL tubes. This study suggests that chitosan guides are promising conduits to construct artificial nerve grafts.

Laser cutting: influence on morphological and physicochemical properties of polyhydroxybutyrate.

Polyhydroxybutyrate (PHB) is a biocompatible and resorbable implant material. For these reasons, it has been used for the fabrication of temporary stents, bone plates, nails and screws (Peng et al. Biomaterials 1996;17:685). In some cases, the brittle mechanical properties of PHB homopolymer limit its application. A typical plasticizer, triethylcitrate (TEC), was used to overcome such limitations by making the material more pliable. In the past few years, CO2-laser cutting of PHB was used in the manufacturing of small medical devices such as stents. Embrittlement of plasticized PHB tubes has been observed, after laser machining. Consequently, the physicochemical and morphological properties of laser-processed surfaces and cut edges of plasticized polymer samples were examined to determine the extent of changes in polymer properties as a result of laser machining. These studies included determination of the depth of the laser-induced heat affected zone by polariscopy of thin polymer sections. Molecular weight changes and changes in the TEC content as a function of distance from the laser-cut edge were determined. In a preliminary test, the cellular response to the processed material was investigated by cell culture study of L929 mouse fibroblasts on laser-machined surfaces. The heat-affected zone was readily classified into four different regions with a total depth of about 60 to 100 microm (Klamp, Master Thesis, University of Rostock, 1998). These results correspond well with the chemical analysis and molecular weight measurements. Furthermore, it was found that cells grew preferentially on the laser-machined area. These findings have significant implications for the manufacture of medical implants from PHB by laser machining.

Biocompatibility and surface structure of chemically modified immunoisolating alginate-PLL capsules.

Grafting of encapsulated living cells has the potential to cure a wide variety of diseases. Large-scale application of the technique, however, is hampered by insufficient biocompatibility of the capsules. A major factor in the biocompatibility of capsules is inadequate covering of the inflammatory poly-L-lysine (PLL) on the capsules' surface. In the present study, we investigate whether tissue responses against alginate-PLL capsules can be reduced by crosslinking the surface of the capsules with heparin or polyacrylic acid. Our transplant study in rats shows a tissue response composed of fibroblasts and macrophages on alginate-PLL-alginate and alginate-PLL-heparin capsules that was completely absent on alginate-PLL-polyacrylic acid capsules. Atomic force microscopy analyses of the capsules demonstrates that the improved biocompatibility of alginate-PLL-capsules by polyacrylic acid coating should not only be explained by a more adequate binding of PLL but also by the induction of a smoother surface. This study shows for the first time that biologic responses against capsules can be successfully deleted by chemically crosslinking biocompatible molecules on the surface of alginate-PLL capsules.

In vivo binding partners of the Lens culinaris lectin.

The immobilized lectin from the lentil (Lens culinaris) specifically binds two fractions out of the L. culinaris seed globulins. Both fractions are displaced from the lectin at low pH values. In addition, fraction I fails to interact at high ionic strengths, and fraction II in the presence of glucose or other lectin-specific sugars. The behaviour in zonal isoelectric precipitation and electrophoretical patterns indicate that both fractions represent subpopulations of the storage proteins. The interaction as demonstrated by affinity chromatography is corroborated by nephelometry: If the dissolved proteins (lectin plus fraction I or fraction II) are mixed under proper conditions the solutions become turbid. An even more pronounced interaction is observed if the lectin is reacted with both fractions at the same time. Seed albumins able to interact with the immobilized lectin include the dissolved lectin and two glycosidases (alpha-mannosidase, alpha-galactosidase) all of which are located in the protein bodies. A third glycosidase (beta-galactosidase) from outside of the protein bodies does not bind to the lectin. The results are discussed in view of the possibility that lectins may serve as packaging aids for other proteins in the protein bodies.

Improved membrane filtration media for enumeration of total coliforms and Escherichia coli from sewage and surface waters.

Two media were developed that allowed both a total coliform count and an Escherichia coli count to be determined on the same medium after 24 h of incubation at 35 degrees C. The new media were tested along with two standard media on 10 surface water and 7 sewage samples. The experimental media yielded equivalent or higher counts relative to the standard media and recovered more specifically the desired indicator groups as determined by colony identification.

Affinity chromatography on immobilized hog gastric mucin and ovomucoid. A general method for isolation of lectins.

Two affinity adsorbents of general applicability for isolating of plant lectins are presented. The isolation of 30 lectins from 27 plants is described. The method works quickly and is inexpensive. In particular, mixtures of different lectins occurring in some plants may be resolved in one run.

Characterization of Eubacterium coprostanoligenes sp. nov., a cholesterol-reducing anaerobe.

A small, anaerobic, gram-positive coccobacillus that reduces cholesterol to coprostanol was isolated from a hog sewage lagoon. This isolate, strain HLT (T = type strain) does not require cholesterol for growth, but it requires lecithin and has phospholipase activity. Much acid is produced by the fermentation of amygdalin, lactose, and salicin. Arabinose, cellobiose, fructose, glucose, mannose, and melibiose are fermented weakly. Acetic, formic, and succinic acids are produced, as is hydrogen. The isolate does not reduce nitrate, produce indole, or hydrolyze starch and gelatin. Esculin is hydrolyzed. The properties of strain HLT are most similar to those of members of the genus Eubacterium. Because strain HL (= ATCC 51222) has unique morphological and physiological properties, we propose that it should be the type strain of a new species in the genus Eubacterium, Eubacterium coprostanoligenes.

Interactions between lectins and other components of leguminous protein bodies.

Previous work from this laboratory had shown that Leguminosa seed extracts contain lectin-bound proteins. In the present paper, the isolation of protein bodies from the seeds of 7 Leguminosa species (Canavalia ensiformis, Lens culinaris, Pisum sativum, Glycine max, Sophora japonica, Wisteria floribunda and Phaseolus vulgaris) is described. Protein bodies were characterized microscopically and by their constituents, storage proteins, lectins and some glycosidases. From the protein bodies, lectin-bound proteins were isolated and were shown to be identical with those from whole seed extracts. This indicates a common localization of lectin-bound proteins and of lectins. Lectin-bound proteins belong to the storage proteins and to the proteins with glycosidase activity. The common localization of these proteins and interactions between them suggest a biological role of seed lectins: during maturation they may act as a packaging aid for storage proteins and enzymes into developing protein bodies. Lectins thus may contribute to an ordered construction and degradation of protein bodies.

A point mutation in the active site of Legionella pneumophila O-acetyltransferase results in modified lipopolysaccharide but does not influence virulence.

The majority of clinical isolates of Legionella pneumophila serogroup 1 produce lipopolysaccharide (LPS) that reacts with monoclonal antibody (MAb) 3/1. By using a negative cell sorting method, we isolated a spontaneous LPS mutant from L. pneumophila serogroup 1 strain Corby that lost reactivity with this MAb. The mutant contained a single nucleotide exchange in position 169 of the lag-1 gene that encodes an O-acetyltransferase that is responsible for O-acetylation of the L. pneumophila O-repeat unit (legionaminic acid). This mutation resulted in a single amino acid exchange in a highly conserved motif present in many O-acetyltransferase-like proteins. RT-PCR analysis revealed that the mutant lag-1 gene was transcribed, but the resulting protein lacked O-acetyltransferase activity. Chemical analysis of the mutant LPS revealed that it lacked 8-O-acetyl groups in legionaminic acid. In addition, the mutant failed to produce high-molecular-weight long-chain O-polysaccharide. Complementation of the mutant with the wild-type lag-1 gene restored reactivity with MAb 3/1 and the chemical structure of the wild-type LPS. Strain Corby and its MAb 3/1-negative mutant were indistinguishable in their serum resistance characteristics, and in uptake and intracellular multiplication in Acanthamoeba castellanii and macrophages.

Salt conservation in familial dysautonomia (Riley-Day syndrome).

In some patients with familial dysautonimia, plasma renin activity shows a paradoxical response to postural stimuli, i.e., levels of plasma renin activity are high when the patient is in the supine position and fall significantly during subsequent ambulation. Furthermore, there is no coordinated release of plasma renin activity and aldosterone. The aim of the present study was to determine whether these findings are accompanied by a disturbance of salt conservation. Six patients were studied in a summer camp while on normal and low-salt diets. Plasma and urinary aldosterone levels rose sharply and appropriately when four of the patients were placed on a low-sodium diet. In these subjects, urinary sodium output fell sharply although three of them failed to attain sodium equilibrium by the third day of the low-sodium regimen. Elevation of early morning plasma renin activity appeared to correlate with an inversion in the normal day-night rhythm in urinary volume.