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Abstract We have previously demonstrated that the nonspecific binding of unfractionated heparin (UFH) to plasma proteins has a marked modulating effect on its anticoagulant activity. Since some heparin-binding proteins are also acute-phase-reactant proteins, we explored the possibility that the induction of the acute-phase response can increase the plasma concentrations of heparin-binding proteins. The recovery of a fixed amount of UFH or low-molecular-weight heparin (LMWH) added in vitro to rat plasma samples obtained at various time intervals after the administration of intravenous endotoxin or subcutaneous turpentine was compared with that of saline-treated control animals. The anti–factor Xa activity was measured in the plasma samples before and after the addition of a chemically modified low-affinity heparin (LAH) to displace the proportion of the added heparin that is reversibly bound to plasma proteins. Our results show that at 6 hours post–endotoxin and at 24 hours post–turpentine treatment, virtually no anti–factor Xa activity could be measured in the plasma samples, while the expected levels were obtained for control plasma. After the addition of LAH to displace protein-bound UFH, essentially the same anti–factor Xa levels were measured in the plasma from all three treatment groups. These results indicate that induction of the acute-phase reaction can dramatically increase the levels of heparin-binding proteins in rat plasma. In addition, we compared the anti–factor Xa recovery of UFH with that of an LMWH from the plasma of endotoxin- and saline-treated rats and demonstrated that LMWH binds less to plasma proteins than UFH, even in plasma in which the levels of heparin-binding proteins are markedly elevated. The recovery of a fixed amount of UFH added in vitro to human plasma from septic patients was also reduced, but not to the same extent as seen in rat plasma. Removal of candidate heparin-binding and acute-phase proteins by immunodepletion indicated that vitronectin plays an important role in the nonspecific binding of UFH in patient plasma.

Cyclosporine A (CsA) has been shown to be an effective therapeutic agent for a wide variety of cutaneous diseases yet its exact mechanism of action is still unclear, although one well-defined effect of CsA is the inhibition of T-cell-derived cytokine expression. We recently demonstrated in vitro that CsA inhibits cell proliferation and suppresses cytokine gene expression in keratinocytes. In this study, we report the in vivo effects of CsA on skin cytokine gene expression as determined by reverse-transcriptase polymerase chain reaction. C57BL6 mice (female, 8-10 weeks old) were subcutaneously injected with CsA in olive oil (0, 5 and 10 mg/kg) every other day for 3 weeks. Treatment with 5 mg/kg CsA inhibited both interleu-kin (IL)-1Α and tumor necrosis factor Α gene expression by about 70 and 90%, respectively, relative to vehicle control levels. However, IL-6 gene expression did not significantly change. Injection of 10 mg/kg CsA inhibited expression of all three genes by 80-90% relative to control levels. These data show that CsA can inhibit constitutive cytokine gene expression in mouse skin.

trans-Urocanic acid (UCA) acts as a chromophore for UV radiation in the epidermis and isomerizes to cis-UCA which then initiates some of the changes leading to UV-induced immunosuppression. The mechanism of the immunomodulation by cis-UCA is unknown at present, but one possibility is that the interaction between cis-UCA and keratinocytes causes the release of immunosuppressive cytokines locally. To test this hypothesis, PAM-212 cells, a murine keratinocyte cell line, were incubated with 0.10-100 micrograms/mL trans- and cis-UCA for 6 or 24 h, respectively. The expression of interleukin (IL)-10, transforming growth factor (TGF)-beta and tumor necrosis factor (TNF)-alpha messenger RNA (mRNA) was then measured by reverse transcription-polymerase chain reaction in comparison with the mRNA for the house-keeping gene, beta-actin. No change or significant induction of any of the cytokine messages occurred. However, the expression of IL-10 messenger RNA (mRNA) was induced 24 h after UVB irradiation (300 J/m2) and that of TNF-alpha mRNA occurred 6 h after treatment with phorbol myristate acetate. The expression of IL-10 protein was also examined by immunostaining in both PAM-212 cells and B16-F10 murine melanoma cells between 3 and 48 h after incubation with 10 and 100 micrograms/mL cis- and trans-UCA. No alteration was seen with either isomer at either concentration. In contrast, UVB irradiation of both cell lines resulted in a marked increase in intracellular IL-10 protein at 24 and 48 h. Therefore the upregulation of the immunosuppressive cytokines, IL-10, TNF-alpha and TGF-beta, in keratinocytes is unlikely to be the mechanism by which cis-UCA induces immunosuppression in mice.

Previously, we demonstrated in a rat model of heparin-induced osteoporosis that low molecular weight heparin (LMWH) produces less bone loss than unfractionated heparin, and that only heparin increases osteoclast number and activity. In contrast, both heparin and LMWH were found to decrease osteoblast function to a similar extent, possibly because at the doses tested both agents produced maximal inhibition. To examine the relative effects of heparin and LMWH on osteoblast function more closely we used an in vitro bone nodule assay, together with measurements of alkaline phosphatase (ALP) activity. Both agents inhibited bone nodule formation and ALP activity in a concentration-dependent manner, but 6 to 8-fold higher concentrations of LMWH were required to achieve equivalent effects. The effect of heparin on osteoblast function was both chain-length and negative charge-dependent because the ability of defined heparin fragments to inhibit nodule formation correlated with their molecular weight (r = 0.98), and N-desulfated heparin was less inhibitory than heparin. In contrast, the effect of heparin on osteoblast function was pentasaccharide-independent because heparin with low affinity for antithrombin had similar activity to heparin with high antithrombin activity. These findings help to explain mounting clinical evidence that the risk of osteoporosis is lower with LMWH than with heparin.

The cDNA sequence of the 60 kDa heat-shock protein from mouse 3T3 cells has been determined. The deduced amino acid sequence of mouse hsp60 protein differs from the corresponding proteins from Chinese hamster and human cells in 7 and 13 residues, respectively, most of which are conservative replacements.

Using antibodies to a mitochondrial molecular chaperone class of protein, which is specifically altered in mutants resistant to microtubule (MT) inhibitors, the effect of a number of MT and mitochondrial inhibitors on the cellular distribution of mitochondria and various cytoskeletal filaments was examined. Treatment of Chinese hamster ovary (CHO) or chicken embryo fibroblast (CEF) cells with the MT inhibitors podophyllotoxin, colchicine, nocodazole and vinblastine caused depolymerization of cellular MTs, but had no significant effect on the distribution patterns of mitochondria. This is attributed to the association of mitochondria with intermediate filaments (IFs) which are not destroyed under these conditions. In contrast to MT inhibitors, treatment of CEFs with the potassium ionophores nonactin and valinomycin caused aggregation of mitochondria towards the perinuclear region of the cells, without having any apparent effect on cellular MTs. This observation suggests that mitochondrial membrane potential, which is abolished by these drugs, play a role in the cellular distribution of mitochondria. In cells recovering from the effects of MT inhibitors, mitochondria have been found to surround the MT organizing complexes and upon complete recovery a realignment of MTs with mitochondria takes place. These observations suggest that MT growth in cells does not occur in a completely random manner but that mitochondria may play some role in their directional growth.

In Chinese hamster ovary cells, stable mutants exhibiting specific resistance or collateral sensitivity towards the various microtubule inhibitors podophyllotoxin, colchicine, griseofulvin, taxol, nocodazole, vinblastine, and maytansine have been isolated. A number of independent mutants selected for resistance to podophyllotoxin and colchicine contain electrophoretically altered forms of two proteins, P1 and P2, of relative molecular masses of approximately 63 000 and 69 000, respectively. The proteins P1 and P2 have been shown to be microtubule related by a number of different genetic and biochemical criteria and are among the major proteins of Chinese hamster ovary cells, being present in approximately equimolar amounts with tubulin. In addition, a griseofulvin-resistant mutant contains a novel mutation (presumably nonsense) which reduces the relative molecular mass of a protein, P5 [Formula: see text], by about 20 000. Specific antibodies to protein P1 have been raised and cross-reactivity studies show that a similar protein is also present in cells from all vertebrate species examined, viz. man, monkey, mouse, Chinese hamster, Syrian hamster, and chicken. Immunofluorescence studies with anti-P1 and anti-tubulin antibodies show that, in interphase cells from various species, the P1 antibody reacts specifically with mitochondria whose overall cellular distribution is strikingly similar to the distribution of microtubules. The mitochondrial localization of the microtubule-related protein P1 provides strong suggestive evidence regarding the existence of a chemical and functional linkage between these two structures, with protein P1 playing an important role in this linkage. Some implication of these results are discussed and it is suggested that mitochondria play an important role in the dynamics of microtubules.