An O-antigen study of Pseudomonas aeruginosa from serological group O11]

In the study of P. aeruginosa cultures, serogroup O11, strains agglutinated simultaneously by factor sera 11b and 11c have been detected. In experiments on cross agglutination and agglutinin adsorption the antigenic structure of these strains, viz. 11a, 11b and 11c, has been determined.     

The localization of heparin-binding fragments on human C4b-binding protein

C4b-binding protein (C4BP) is a multimeric plasma protein, which regulates the classical pathway of the C system. C4BP interacts with C C4b on a domain located in a 48-kDa chymotryptic fragment. We now demonstrate that C4BP contains heparin-binding fragments, which are located within the C4b binding domain. We have used an assay using heparin coupled to Sepharose CL-6B to show that 125I-C4BP binds to heparin in a time-dependent, saturable, and reversible manner. Binding could be inhibited by purified 48-kDa fragments and direct binding on the 48-kDa fragments to heparin-Sepharose was demonstrated by SDS-PAGE. mAb against native C4BP and the isolated 160-kDa central core fragment were evaluated for their ability to block the binding of 125I-C4BP to heparin and C4b. The relative efficacy of mAb against intact C4BP in blocking C4BP binding to heparin-Sepharose was similar to that for blocking 125I-C4BP binding to C4b. In addition, heparin blocked the binding of 125I-C4BP to C4b and vice versa. It is therefore likely that the heparin-binding fragments are localized on or close to the C4b-binding site of C4BP.     

Somatic growth processes: how are they altered in captivity?

The cellular growth mechanisms of captive cephalopods were examined to determine whether the growth processes in aquaria are the same as those of wild individuals. Mantle muscle tissue growth in cephalopods is a function of both the production of muscle fibres and the growth of existing fibres. After seven days, captive animals had thicker mantles, a greater proportion of mitochondria-rich tissue, muscle fibres with smaller mitochondrial cores and fewer small muscle fibres. This suggests a reduced rate of new fibre generation, indicating an alteration to the cellular growth mechanisms and not simply a change in the physiological rate of growth. Smaller individuals were affected to a greater extent. Such modifications to the actual mechanisms of growth may have the potential to alter the shape of an individual''s growth curve and can also affect final body size. Alterations to the proportion and structure of mantle components may impact upon many critical aspects of an individual''s biology, as the muscular mantle is central to locomotion, ventilation of gills, energy storage and possibly subcutaneous oxygen extraction.     

Kinetic mechanisms of inhibitor binding: relevance to the fast-acting slow-binding paradigm.

Although phlorizin inhibition of Na+-glucose cotransport occurs within a few seconds, 3H-phlorizin binding to the sodium-coupled glucose transport protein(s) requires several minutes to reach equilibrium (the fast-acting slow-binding paradigm). Using kinetic models of arbitrary dimension that can be reduced to a two-state diagram according to Cha's formalism, we show that three basic mechanisms of inhibitor binding can be identified whereby the inhibitor binding step either (A) represents, (B) precedes, or (C) follows the rate-limiting step in a binding reaction. We demonstrate that each of mechanisms A-C is associated with a set of unique kinetic properties, and that the time scale over which one may expect to observe mechanism C is conditioned by the turnover number of the catalytic cycle. In contrast, mechanisms A and B may be relevant to either fast-acting or slow-binding inhibitors. However, slow-binding inhibition according to mechanism A may not be compatible with a fast-acting behavior on the steady-state time scale of a few seconds. We conclude that the recruitment hypothesis (mechanism C) cannot account for slow phlorizin binding to the sodium-coupled glucose transport protein(s), and that mechanism B is the only alternative that may explain the fast-acting slow-binding paradigm.     

Alterations in C3 activation and binding caused by phosphorylation by a casein kinase released from activated human platelets.

A casein kinase released from activated human platelets phosphorylates a number of plasma proteins extracellularly, and that activation of platelets in systemic lupus erythematosus patients parallels an increase in the phosphate content of plasma proteins, including C3. The present study was undertaken to characterize this platelet protein kinase and to further elucidate the effect(s) on C3 function of phosphorylation by platelet casein kinase. The phosphate content of human plasma C3 was increased from 0.15 to 0.60 mol phosphate/mol of C3 after platelet activation in whole blood or platelet-rich plasma. The platelet casein kinase was distinct from other casein kinases in terms of its dependence on cations, inhibition by specific protein kinase inhibitors, and immunological reactivity. C3 that had been phosphorylated with platelet casein kinase was tested for its susceptibility to cleavage by trypsin or the classical and alternative pathway convertases and its binding to EAC and IgG. Phosphorylation did not affect the cleavage of C3 into C3a and C3b, but the binding of fragments from phosphorylated C3 to EAC14oxy2 cells and to IgG in purified systems and in serum was increased by 1.6-4.5 times over that of unphosphorylated C3. A covariation was seen between the enhanced binding of C3 fragments to IgG after phosphorylation and an increased ratio of glycerol/glycine binding, from 2.0 for unphosphorylated C3 to 4.9 for phosphorylated C3. The present study suggests that an overall effect of phosphorylation of C3 by platelet casein kinase is to enhance the opsonization of immune complexes.     

The J-related segment of tim44 is essential for cell viability: a mutant Tim44 remains in the mitochondrial import site, but inefficiently recruits mtHsp70 and impairs protein translocation.

Tim44 is a protein of the mitochondrial inner membrane and serves as an adaptor protein for mtHsp70 that drives the import of preproteins in an ATP-dependent manner. In this study we have modified the interaction of Tim44 with mtHsp70 and characterized the consequences for protein translocation. By deletion of an 18-residue segment of Tim44 with limited similarity to J-proteins, the binding of Tim44 to mtHsp70 was weakened. We found that in the yeast Saccharomyces cerevisiae the deletion of this segment is lethal. To investigate the role of the 18-residue segment, we expressed Tim44Delta18 in addition to the endogenous wild-type Tim44. Tim44Delta18 is correctly targeted to mitochondria and assembles in the inner membrane import site. The coexpression of Tim44Delta18 together with wild-type Tim44, however, does not stimulate protein import, but reduces its efficiency. In particular, the promotion of unfolding of preproteins during translocation is inhibited. mtHsp70 is still able to bind to Tim44Delta18 in an ATP-regulated manner, but the efficiency of interaction is reduced. These results suggest that the J-related segment of Tim44 is needed for productive interaction with mtHsp70. The efficient cooperation of mtHsp70 with Tim44 facilitates the translocation of loosely folded preproteins and plays a crucial role in the import of preproteins which contain a tightly folded domain.     

Proliferative and Differentiation Potential of Individual Clones Derived from Bone Marrow Stromal Precursor Cells

We present the results of a study on the proliferative and differentiation potential of individual clones of stromal fibroblasts growing in monolayer cultures of bone marrow cells. Each precursor cell yielding a large colony in primary culture is capable of up to 34 doublings in vitro. The transplantation of clones or monoclonal strains of stromal fibroblasts into the open system results in the formation of microenvironment consisting of the bone and reticular tissue and is suitable for the differentiation of all three lines of hemopoiesis. Evidence has been obtained that, in a closed system, individual clones are capable of differentiation into the bone, cartilaginous, and reticular tissues. In other words, the adult organism has a common cell precursor for these tissues.