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Characteristic features of the persistent chlamydial developmental cycle, associated with chronic infections in both humans and animals, include the generation of non-replicative, morphologically aberrant bodies which are distinct from normal propagating reticulate bodies. Previous studies have correlated these morphological and metabolic changes with differential expression of diverse functional subsets of chlamydial genes. To further investigate these correlations, we compared mRNA expression of predicted chlamydial signal transduction genes between normal Chlamydophila pneumoniae A-03 infections in HEp-2 cells and those treated with gamma interferon (IFN-gamma) by using real-time RT-PCR. Inspection of the Cp. pneumoniae genome revealed at least 39 candidate signal transduction genes, of which 30 were differentially expressed in Cp. pneumoniae mediated persistence. Functional sub-groups of differentially expressed signal transduction genes include chlamydial GTPases (hflX, ychF, yhbZ and yphC), linked to bacterial cellular processes such as cell cycle control and ribosome assembly and stability. Other up-regulated signal transduction genes sharing similarity to bacterial stress response genes (htrA, surE, lytB and hrcA) were also detected. The transcriptional changes observed for the majority of signal transduction genes appear to be unique for Cp. pneumoniae, as similar changes were not observed in recent whole genomic analysis of C. trachomatis IFN-gamma mediated persistence. These results suggest that chlamydial signal transduction genes play potentially important roles in the establishment and maintenance of Cp. pneumoniae persistence, likely as part of the IFN-gamma response stimulon as described for C. trachomatis, but with considerable differences in the transcriptional profile.

A particularly intriguing role in communication within the nervous system is played by glycosyl phosphatidylinositol (GPI)-anchored glycoproteins. Although our understanding of how neuronal GPI-linked molecules transduce signals is still in its infancy, it is now possible to assemble a tentative model to describe their functioning. Shared features of these otherwise diverse receptors will be outlined, with particular emphasis given to the exclusively GPI-anchored contactin/F11 subgroup of the immunoglobulin superfamily. Contactin/F11 itself, with five extracellular ligands and an intracellular pathway involving Fyn, serves to illustrate the wide range of signalling possible through this group of receptors.

Two sites on tenascin mediate interactions with glycosaminoglycan chains of proteoglycans. One is situated on the fibrinogen-like domain, whereas the other lies within the fibronectin type III homology region (Aukhil, I., Joshi, P., Yan, Y. Z., and Erickson, H. P.(1993) J. Biol. Chem. 268, 2542-2553.). We now characterize the latter binding site more closely by means of recombinant protein fragments derived from the type III homology region of tenascin. Using a heparin-Sepharose column, we localize the second heparin binding site to the fifth fibronectin type III domain. This is confirmed in solid phase assays by incubation of fusion proteins with biotin-labeled heparin. In addition, we demonstrate the binding of heparan sulfate and dermatan sulfate to domain five. Molecular modelling of this domain reveals a conserved heparin-binding motif that we propose as the putative binding site. The fact, that different glycosaminoglycans may bind to this domain, implies that different classes of proteoglycans may in vivo compete for the same site.

Human α-1-antitrypsin (AT) was reacted with hydroxylamine at pH 9.0 giving cleavage at an Asn-Gly bond. A fragment of molecular weight 8,500 was released and this was isolated and sequenced. The fragment had the same carboxy terminal amino acid sequence as intact AT. The 80 residue polypeptide contained the Z variant mutation site and a portion of sequence identical to that found by others for the reactive site, inferring the presence in AT of two active sites. This sequence combined with prviously published work gives a continuous sequence of 152 amino acid residues from the carboxy terminal end of the AT molecule, including the mutation site of the S variant. The sequence shows strong homology with human antithrombin III.

α1-Antitrypsin has a microheterogeneity evident on isoelectric focusing as three major and several minor bands. We have identified the carbohydrate structures of the major bands; band 6 (isoform I) has three bi-antennary sidechains, band 4 (isoform II) has two bi- and one tri-antennary and band 2 (isoform III) has one bi- and two tri-antennary sidechains. The identity of the isoforms with the bands permitted their measurement in plasma by photometric scanning of the electrofocused gels. In healthy controls the levels of isoforms I, II and III were relatively constant and in the proportions of 5, 4 and 1, respectively. A marked change occured during inflammation and oestrogen stress with isoforms II and III accounting for most of the increase in α1-antitrypsin. One possible consequence of the changed proportions was shown to be the increased catabolism of the partia!!y desialylated tri-antennary isoforms compared to that of the predominant bi-antennary form of the healthy individual.

Type IX collagen has recently been shown to contain glycosaminoglycan chain(s) and furthermore to be immunologically identical with proteoglycan Lt (Vaughan, L., Winterhalter, K. H., and Bruckner, P. (1985) J. Biol. Chem. 260, 4758-4763). Here we demonstrate that the chondroitin sulfate carrying 115-kDa polypeptide of type IX collagen corresponds to the alpha 2(IX) chain. In addition the 84- and 68-kDa polypeptides were identified as the alpha 1(IX) and the alpha 3(IX) chains, respectively. This conclusion is based on a comparison of the tryptic fingerprints of the 84-, 115-, and 68-kDa chains of type IX collagen on high performance liquid chromatography with the similarly treated C2, C3, and C5 chains of the peptic fragment HMW. In addition, we provide evidence that both the C3 and C4 components of HMW are derived from the alpha 2(IX) chain.

Proteoglycan Lt (PG-Lt), isolated from 17-day-old chicken embryo sterna, appeared to differ from its counterpart from tibia and femur (Noro, A., Kimata, K., Oike, Y., Shinomura, T., Maeda, N., Yano, S., Takahashi, N., and Suzuki, S. (1983) J. Biol. Chem. 258, 9323-9331). The intact disulfide-bonded molecule of approximately 300 kDa was separable into three chains of 115, 84, and 68 kDa on reduction, the molecular masses being relative to those of collagen standards on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This is in contrast to tibial cartilage PG-Lt, from which there was no observed release of a 68-kDa chain (100 kDa relative to globular protein standards) after reduction. The 115-kDa chain of sternum PG-Lt consists of a core 68-kDa polypeptide to which the chondroitin sulfate chains are attached. The ratio of 4-sulfated to 6-sulfated disaccharides released after either chondroitinase ABC or AC digestion is 3:1. Identity of PG-Lt with type IX collagen was indicated by their similar elution profiles on DEAE-Trisacryl and by the presence in both proteins of co-migrating 84- and 68-kDa bands on SDS-PAGE. This identity was confirmed by immunoblotting PG-Lt after SDS-PAGE, with affinity-purified polyclonal antibodies specific for a triple helical domain (HMW) of type IX collagen. The nonreduced high molecular mass material and all three bands of the reduced PG-Lt were immunoreactive, giving immunostaining patterns similar to autoradiographs from the [14C]glycine-labeled protein.