Methods for the separation of peptides on a new type of thin-layer chromatography (TLC) sheet and blotting onto polyvinylidene difluoride (PVDF) membranes with subsequent gas phase sequencing are described. For validation, the A and B chain of insulin were chromatographed on Empore TLC sheets and either extracted or blotted onto PVDF membranes. The advantages and disadvantages of thin-layer chromatography on Empore sheets versus high performance liquid chromatography (HPLC) are discussed, along with the possibility of combining the two methods. In addition, TLC was combined with electrophoresis (fingerprinting) for the separation of complex peptide mixtures. Blotting from TLC sheets onto PVDF membranes was performed in two ways: contact diffusion and electrophoretic transfer. In our experiments electroblotting was more effective. Amino acid sequence determination of the B chain of insulin was possible both after extraction from the TLC sheet and after blotting onto PVDF membranes. In the former case, liquid phase sequencing and, in the latter case, gas phase sequencing was performed. The possibility to blot from TLC sheets onto membranes, e.g. PVDF, may prove useful in many fields, for example in biochemistry, and in molecular and cell biology.

Amino Acid Sequence↗

PubMedDate not supplied

Ultrastructural characteristics of cellular reaction after experimentally induced lesions in the arterial vessel.

UNLABELLED: Restenosis after angioplasty occurs with an incidence of 20-50% and remains a major drawback. Certain randomized studies suggest that a bigger post-angioplasty lumen predicts a better long-term outcome. Conversely other studies showed a better outcome with limited injury. The present study aimed to investigate the depth of the lesion and relate this to cellular alterations after graded vascular injury. METHOD: Vessel segments of 30 pigs underwent injury using a directional atherectomy catheter. Vessels were assigned according to the extent of injury to Group 1 (intima lesion) or Group 2 (media injury). 2 hours to 7 days after injury, 68 arteries showing 41 intimal and 27 media lacerations were excised and processed for histology and transmission electron microscopy. RESULTS: Immediately after injury, thrombus formation was found at the site of the altered segment. A marked, transient infiltration of polymorphonuclear leukocytes (PMN) occurred only if the media was lacerated, starting within the first hours and increasing up until the 12 hours time point. The cellular infiltration was followed by a transformation of contractile myocytes to a synthetic subtype. The ratio of myofilaments to organelles decreased. A pronounced myoproliferative response was found in Group 2 after 7 days (p < 0.01), whereas only moderate tissue hyperplasia was seen in Group 1. CONCLUSION: The data presented provide evidence that the cellular alteration of injured vessels begins immediately. Subsequent to an initial temporary PMN infiltration, an activation of local myocytes occurs at a very early stage. In particular, a myoproliferative response was found only after deep injury with rupture of the internal elastic lamina.

Animals↗

PubMedDate not supplied

Tooth wear in living Pongids.

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Animals↗

PubMedDate not supplied

Lectin binding and uptake in human (myelo)monocytic cell lines: HL60 and U937.

The terminal carbohydrate residues of the human (myelo) monocytic cell lines HL60 and two subclones of U937 were investigated by the use of lectins. Several terminal carbohydrate residues, including N-acetylglucosamine and carbohydrates of the complex type, were detected on all three cell lines. Except for galactose residues, the two subclones of U937 had almost identical terminal carbohydrate residues. The differences between the two U937 subclones and HL60 were more pronounced, the latter expressing fucose residues, which might be part of the CD15 cell adhesion molecules. Some of the differences of the carbohydrate residues between the cell lines could be attributed to their differentiation within the myelomonocytic cell lineage. Lectins that bound to the cell surface were internalized via either clathrin-coated pits and vesicles or nonspecific endocytosis, indicating that functionally different classes of cell surface glycoproteins are involved in lectin binding and internalization.

Cell Differentiation↗

PubMedDate not supplied

Release of membrane from milk fat globules by conjugated bile salts.

We investigated the capacity of conjugated bile salts to remove the membrane, a barrier to lipolysis, from milk fat globules. Cow, goat, and human globules were subjected to varying concentrations of the bile salt taurodeoxycholate at 37 degrees C for 2 min, and the released material was obtained by centrifugation at 2 degrees C and 50,000 g for 1 h. Sedimented pellets were analyzed for phospholipid and protein and were characterized further by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Pellets were examined in the electron microscope. Measurable amounts of membrane were sedimented from globules incubated at 37 degrees C for 2 min in 0.5 mM taurodeoxycholate. Taurocholate also exhibited this membrane-releasing ability. However, disintegration of milk fat globule membrane appears to be the principal effect of these salts at 37 degrees C. Our results show that conjugated bile salts within their normal concentration range (2-6 mM) in digesta of term and preterm infants are capable of removing membrane from milk fat globules.

Animals↗