Crosslinking of tissue-derived biomaterials in 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)

In contrast to bifunctional reagents such as glutaraldehyde or polyfunctional reagents such as polyepoxides, carbodiimides belong to the class of zero-length crosslinkers which modify amino acid side-groups to permit crosslink formation, but do not remain as part of that linkage. The authors have compared the effects of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and glutaraldehyde (the de facto industrial standard crosslinker) on the hydrothermal, biochemical, and uniaxial mechanical properties of bovine pericardium. EDC crosslinking was optimized for maximum increase in collagen denaturation temperature using variables of pH, concentration, and ratio of EDC to N-hydroxysuccinimide (NHS): a reagent for formation of activated esters. EDC and glutaraldehyde crosslinked materials were subjected to hydrothermal denaturation tests, biochemical degradation by enzymes (collagenase, trypsin) and CNBr, amino acid analysis for unreacted lysine, and to high strain rate mechanical tests including: large deformation stress-strain studies (0.1 to 10 Hz), stress relaxation experiments (loading time 0.1 s) and small deformation forced vibration (1 and 10 Hz). A protocol for EDC crosslinking was developed which used 1.15% EDC (2:1 EDC:NHS) at pH 5.5 for 24 h. The increase in denaturation temperature for EDC (from 69.7±1.2°C to 86.0±0.3°C) was equivalent to that produced by glutaraldehyde (85.3±0.4°C). Both treatments equivalently increased resistance to collagenase and CNBr degradation; however, after denaturation, the EDC-treated tissue was slightly more resistant to collagenase, and markedly more resistant to trypsin. EDC-treated materials were more extensible and more elastic than glutaraldehyde-treated materials. Despite the differences in crosslinking mechanism, EDC and glutaraldehyde-treated materials are very similar. Subtle but intriguing differences in biochemical structure remain to be investigated.     

Effect of caffeine and reduced temperature (20 degrees C) on the organization of the pre-Golgi and the Golgi stack membranes

In the present study we have dissected the transport pathways between the ER and the Golgi complex using a recently introduced (Kuismanen, E., J. J?ntti, V. M?kiranta, and M. Sariola. 1992. J. Cell Sci. 102:505-513) inhibition of transport by caffeine at 20 degrees C. Recovery of the Golgi complex from brefeldin A (BFA) treatment was inhibited by caffeine at reduced temperature (20 degrees C) suggesting that caffeine inhibits the membrane traffic between the ER and the Golgi complex. Caffeine at 20 degrees C did not inhibit the BFA-induced retrograde movement of the Golgi membranes. Further, incubation of the cells in 10 mM caffeine at 20 degrees C had profound effects on the distribution and the organization of the pre-Golgi and the Golgi stack membranes. Caffeine treatment at 20 degrees C resulted in a selective and reversible translocation of the pre- and cis-Golgi marker protein (p58) to the periphery of the cell. This caffeine-induced effect on the Golgi complex was different from that induced by BFA, since mannosidase II, a Golgi stack marker, remained perinuclearly located and the Golgi stack coat protein, beta-COP, was not detached from Golgi membranes in the presence of 10 mM caffeine at 20 degrees C. Electron microscopic analysis showed that, in the presence of caffeine at 20 degrees C, the morphology of the Golgi stack was altered and accumulation of numerous small vesicles in the Golgi region was observed. The results in the present study suggest that caffeine at reduced temperature (20 degrees C) reveals a functional interface between the pre-Golgi and the Golgi stack.     

Characterization of the chicken and quail homologues of the human gene responsible for the X-linked Kallmann syndrome

The human KAL gene, responsible for the X-linked Kallmann syndrome, was isolated previously. Southern blot analysis using human cDNA probes detected cross-hybridization with DNA from several organisms, including chicken and quail. The entire coding sequences of chicken and quail KAL cDNAs were determined. A comparison of these cDNAs with the human KAL cDNA reveals an overall identity of 73 and 72%, respectively. This results in 76 and 75% identity at the protein level. The highest conservation was found in the WAP four-disulfide core motif and in two of the four fibronectin type III repeats reported in the human protein. These results further support the hypothesis that the KAL protein is an extracellular matrix component with anti-protease and adhesion functions.     

Distinction between artefactually shrunken and truly degenerated 'dark' neurons by in situ fixation with microwave irradiation

Dark, shrunken neurons frequently occur as artefacts in immersion fixed tissue. Perfusion fixation will prevent artefacts of this type. However, morphologically identical neurons have been described as truly degenerated cells in perfusion fixed brains in various pathological conditions. Since adequate perfusion is difficult to obtain in some pathological conditions, the question still remains whether the dark neurons found in some of these situations are true in vivo phenomena or artefacts caused by inadequate fixation. In the present study rat brains with cryogenic lesions were fixed in situ by microwave irradiation. With this method no artefactually changed dark neurons were observed in the normal parts of the brains. In the cryogenic lesions, however, a narrow rim of dark, shrunken neurons occurred adjacent to the normal cortex. This zone was identical to that observed in perfusion fixed tissue. Since inadequate fixation due to uneven perfusion of the damaged tissue is prevented with this method, we suggest that the neuronal changes represent true in vivo phenomena. Fixation with microwave irradiation can thus be used to differentiate between artefactually changed and truly degenerated dark neurons in various pathological conditions.