Steady state kinetic mechanism of the Escherichia coli coenzyme A transferase.

Developing chloroplasts isolated from greening cotyledons and isolated etioplasts were capable of synthesizing and accumulating Mg-protoporphyrin IX monoester and longer wavelength metalloporphyrins when incubated in the dark in the presence of protoporphyrin and cofactors. These results constituted the first unambiguous demonstration of the insertion of magnesium into exogenous protoporphyrin in a cell-free system from higher plants. The metalloporphyrin synthetic activity did not occur in the absence of the plastids or when the plastids were heated in a 100 °C water bath for 2 min. It is thus suggested that, in higher plants, the in vitro insertion of magnesium into protoporphyrin is an enzymatic reaction.     

Use of the Airfuge for analysis and preparation of receptors incorporated into liposomes: Studies with the receptor for immunoglobulin E

A Beckman Airfuge has been employed for studying the interaction between lipids and the receptor for immunoglobulin E (IgE). For analytic experiments, samples were applied underneath a discontinuous sucrose gradient. After a 30-min centrifugation in a fixed-angle rotor, liposomes floated toward the top of the gradient whereas unincorporated receptor-IgE complexes remained at the bottom of the tube. Liposomes with incorporated receptors were also efficiently separated in the ACR-90 preparative rotor. These methods of "Airfuge flotation" can provide useful adjuncts to more traditional methods for density-gradient centrifugation especially when rapid analysis of small samples is desired.     

Solubilization and assay of an hepatic receptor for the haptoglobin-hemoglobin complex

Hemoglobin released into the bloodstream is tightly bound by haptoglobin. The resulting complex (HpHb) is promptly cleared from the circulation and accumulates in the liver. A binding protein with a high affinity for HpHb has been solubilized from an acetone powder of rat liver and freed from an endogenous inhibitor by passage over a column of immobilized hemoglobin. An assay procedure has been developed whereby the bound HpHb is selectively precipitated by polyethylene glycol 6000. Employing this assay, the binding reaction was shown to be linear and saturable with respect to the ligand. In contrast to several previously described receptors for glycoproteins, the carbohydrate moiety of haptoglobin did not appear to participate in the binding of HpHb by the soluble receptor.     

Regional Maritime Regime Building: Prospects in Northeast and Southeast Asia

Maritime issues are rising to the forefront of Asian security concerns. But maritime management regimes can constrain conflict and create confidence in co-operation. Such regimes can define the range of permissible state behavior and resolve dilemmas stemming from the sharing of common resources. Regimes originate through imposition, spontaneous processes, or negotiation and are supplied when there is sufficient demand for the functions they perform. Integrative forces that support regime formation are the existence of other international arrangements in the region; ethnic, cultural, or historical interstate relationships; and clear indications of benefits to be gained. Often a shock or crisis enhances regime formation or its robustness. Disintegrative forces include political or territorial differences, competition for leadership, and opposition to regionalism. In regional co-operation on maritime issues, Southeast Asia is clearly more advanced than Northeast Asia. However, the absence of robust multilateral maritime regimes in Asia reflects state perceptions that the costs outweigh the benefits. The primacy of dis-integrative factors argues strongly for an ad hoc, issue-specific, evolutionary process for multilateral maritime regime building in Asia.     

Ring opening of acylated β-d-arabinofuranose 1,2,5-orthobenzoates with nucleophiles allows access to novel selectively-protected arabinofuranose building blocks

β-d-Arabinofuranose 1,2,5-orthobenzoates with 3-O-acetyl, 3-O-benzoyl, and 3-O-chloroacetyl groups were prepared in an efficient manner starting from readily available crystalline methyl 2,3,5-tri-O-benzoyl-α-d-arabinofuranoside, and ring-opening reactions of these compounds with O- and S-nucleophiles were studied. Optimized conditions leading to the formation of the respective monosaccharide adducts (up to 96% isolated yields) and to α-(1→5)-linked disaccharide thioglycosides with 5′-OH unprotected (up to 30% isolated yields) were found. Basing on these results, a novel approach for effective differentiation of 3,5-diol system and 2-hydroxy group in arabinofuranose thioglycosides was proposed. The selectively protected derivatives prepared are valuable building blocks for the assembly of linear and branched oligoarabinofuranosides.     

Return to partial function of rabbit ovaries after cryopreservation in liquid nitrogen

Half of each ovary of seven rabbits was removed and deep-frozen in liquid nitrogen for two to three months before being thawed and reimplanted in the original animal. Three of the animals with cryopreserved demi-ovaries bred but did not conceive and these three plus one other had surviving ovarian tissue and normal uterine morphology at autopsy. In one of these cases the ovaries were smooth surfaced but, in two animals, follicles were present, and, in one, corpora haemoragica were observed. The three remaining rabbits would not breed after their frozen-thawed demi-ovaries were reimplanted and their uteri were atrophied at autopsy. No surviving ovarian tissue was detected. In the four cases where the organs survived cryopreservation, three sets of demi-ovaries were fast-frozen by a series of three plunges, respectively, into alcohol baths at ?7° C and ?38° C and then into liquid nitrogen at ?196° C. The fourth set was frozen at slower rates of 1° C per minute to ?7° C and then at 0.3° C per minute to ?38° C before being plunged into liquid nitrogen. One set of demi-ovaries was intact and the other three had the medulla cored out before freezing.We conclude that several protocols suffice to deep-freeze rabbit ovaries and permit the organs to return to partial function when reimplanted.     

Identification of side arm-modified DOTA scaffolds as multi-site binding ligands for cancer cells over normal cells

The metal-chelated 1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraacetic acid-tetraamide (DOTA) scaffold has been widely used as a contrast agent for diagnostic purposes in positron emission tomography (PET) and magnetic resonance imaging (MRI), but not as a biomarker targetable ligand. While the oxygen atoms at the stem of the four arms of the DOTA scaffold are needed for metal chelation, we previously introduced various physiochemical properties to extend these arms in a chemical library fashion to enhance the imaging contrast mechanism. We developed two such on-bead libraries, with 80 and 76 DOTA derivatives, where one arm was used to attach the DOTA scaffold onto resin beads and the other three arms were chemically modified. We now hypothesized that the chemical moieties used to modify these three arms can also recognize biomarkers on a cell surface. Therefore in this current study, we used such 76 derivatives of DOTA library to screen against HeLa cervical cancer cells. We found that two of the four ‘hits’ identified displayed higher binding towards HeLa cells than the unmodified parent DOTA. Furthermore, one of those ‘hits’ displayed better binding towards cervical and prostate cancer cells than lung and breast cancer cells and normal HBEC-3KT and RWPE1 cells. This indicates that this derivative can recognize a biomarker specific for certain types of cancer cells. If the compound has intrinsic activity, this can be used as a theranostic agent for real time therapy monitoring applications in the future. We believe that our DOTA derivative-based library approach can be applied to other types of cell and protein screens on various disease types in the future.     

An analysis of B cell memory. I. Interaction of helper and suppressor effects in the in vitro expression of IgM memory.

The pfc response of Srbc primed IgM memory cells has been characterized by limiting dilution analysis in vitro, in which LPS was used to maximize the response of spleen cells to Srbc. The analysis suggested that, even under these conditions, expression of B cell memory was not directly assayed and cell collaboration effects were still basic to the system.Two types of cells, as defined by function, appeared necessary to elicit optimal clonal proliferation of IgM B memory cells: firstly, helper T cells were essential for B cell proliferation even with LPS present in culture. Under appropriate conditions, helper activity could be provided by normal thymus cells. Secondly, activated T cells were required for the maximal conversion of normal thymus cells to helper cells. A third activity, T cell-dependent suppression, was observed at high cell doses. The implications of these results and the need for a comprehensive analysis of in vitro conditions for each individual type of experiment is discussed.     

A three dimensional model of the digestion of peptidoglycan by lysozyme

The digestion of single peptidoglycan chains of the recently proposed conformation (Formanek et al., 1974) can be described with the same enzymatic mechanism as proposed by Phillips for a hexasaccharide consisting of alternating N-acetylglucosamine, N-acetylmuramic acid residues (Phillips, 1966). It is shown by model building, that in a peptidoglycan lysozyme complex the peptide chains do not exhibit any sterical hindrance.The digestion of the peptidoglycan sacculus by lysozyme may occur at lattice defects of its paracrystalline structure. A slit of about 30 å lenght and 10–15 å width between peptidoglycan micells may be sufficient for the attachment of lysozyme.     

Primary structure of the hip gene of Escherichia coli and of its product,the β subunit of integration host factor

We describe the isolation and sequencing of the hip gene of Escherichia coli and show that it encodes the β subunit of integration host factor (IHFβ). In order to locate the coding region, we constructed a set of deletion mutants by exonucleolytic digestion of a fragment containing hip, determined which mutants were hip⁺ and which hip^? by complementation, and then sequenced the ends of the critical deletions. The 5′ end of the coding region was located precisely by comparing the deduced amino acid sequence to the actual N-terminal amino acid sequence of IHF. Our assignment of the coding region was further substantiated by the nucleotide sequences of a hip point mutant and of internal replacement mutations. We found a probable promoter for hip located about 85 base-pairs upstream from the initial AUG codon and about 75 base-pairs downstream from the 3′ end of the neighboring gene. rpsA, and we constructed an IHFβ overproducer by fusing the coding sequences to the λp_L promoter. A survey of known protein sequences revealed a close relationship between IHFβ and the type II prokaryotic DNA binding proteins (the “histone-like” proteins). This relationship is shared to a considerable extent by the other subunit of IHF, IHFα. A hip missense mutation that replaces a completely conserved glycine with aspartate has a null phenotype, suggesting that the conserved regions are functionally important.