Second-order correction to the Bigeleisen-Mayer equation due to the nuclear field shift

The nuclear field shift affects the electronic, rotational, and vibrational energies of polyatomic molecules. The theory of the shifts in molecular spectra has been studied by Schlembach and Tiemann [Schlembach, J. & Tiemann, E. (1982) Chem. Phys. 68, 21]; measurements of the electronic and rotational shifts of the diatomic halides of Pb and Tl have been made by Tiemann et al. [Tiemann, E., Kn?ckel, H. & Schlembach, J. (1982) Ber. Bunsenges. Phys. Chem. 86, 821]. These authors have estimated the relative shifts in the harmonic frequencies of these compounds due to the nuclear field shift to be of the order of 10(-6). I have used this estimate of the relative shift in vibrational frequency to calculate the correction to the harmonic oscillator approximation to the isotopic reduced partition-function ratio (208)Pb(32)S/(207)Pb(32)S. The correction is 0.3% of the harmonic oscillator value at 300 K. In the absence of compelling evidence to the contrary, it suffices to calculate the nuclear field effect on the total isotopic partition-function ratio from its shift of the electronic zero point energy and the unperturbed molecular vibration.     

Differential stimulation of c-fos expression in hypothalamic nuclei of the rat brain during short-term heat acclimation and mild dehydration

Evidence that multiple, probably non-endocytic mechanisms are involved in the uptake into mammalian cells of the alpha-helical amphipathic model peptide FLUOS-KLALKLALKALKAALKLA-NH2 (I) is presented. Extensive cellular uptake of N-terminally GC-elongated derivatives of I, conjugated by disufide bridges to differently charged peptides, indicated that I-like model peptides might serve as vectors for intracellular delivery of polar bioactive compounds. The mode of the cellular internalization of I comprising energy-, temperature-, pH- and ion-dependent as well as -independent processes suggests analogy to that displayed by small unstructured peptides reported previously (Oehlke et al., Biochim. Biophys. Acta 1330 (1997) 50-60). The uptake behavior of I also showed analogy to that of several protein-derived helical peptide sequences, recently found to be capable of efficiently carrying tagged oligonucleotides and peptides directly into the cytosol of mammalian cells (Derossi et al., J. Biol. Chem. 269 (1994) 10444-10450; Lin et al., J. Biol. Chem. 270 (1995) 14255-14258; Fawell et al., Proc. Natl. Acad. Sci. USA 91 (1994) 664-668; Chaloin et al., Biochemistry 36 (1997) 11179-11187; Vives et al., J. Biol. Chem., 272 (1997) 16010-16017).     

Probabilistic modeling of shear-induced formation and breakage of doublets cross-linked by receptor-ligand bonds

A model was constructed to describe previously published experiments of shear-induced formation and breakage of doublets of red cells and of latexes cross-linked by receptor-ligand bonds (. Biophys. J. 65:1318-1334; Tees and Goldsmith. 1996. Biophys. J. 71:1102-1114;. Biophys. J. 71:1115-1122). The model, based on McQuarrie's master equations (1963. J. Phys. Chem. 38:433-436), provides unifying treatments for three distinctive time periods in the experiments of particles in a Couette flow in which a doublet undergoes 1) formation upon two-body collision between singlets; 2) evolution of bonds at low shear rate; and 3) break-up at high shear rate. Neglecting the applied force at low shear rate, the probability of forming a doublet per collision as well as the evolution of probability distribution of bonds in a preformed doublet were solved analytically and found to be in quite good agreement with measurements. At high shear rate with significant force acting to accelerate bond dissociation, the predictions for break-up of doublets were obtained numerically and compared well with data in both individual and population studies. These comparisons enabled bond kinetic parameters for three types of particles cross-linked by two receptor-ligand systems to be calculated, which agreed well with those computed from Monte Carlo simulations. This work can be extended to analyze kinetics of receptor-ligand binding in cell aggregates, such as those of neutrophils and platelets in the circulation.     

Heat stress induces a glycosylation of membrane sterol in myxoamoebae of a true slime mold, Physarum polycephalum

To know the very early events occurring after heat shock, the changes of membrane lipids were examined. Heat stress induced the production of a certain glycolipid in the myxoamoebae of Physarum polycephalum in a few minutes. The purified glycolipid was determined to be a poriferasterol monoglucoside by structural studies that was previously reported to be expressed during the differentiation of Physarum cells from haploid myxoamoebae to diploid plasmodia (Murakami-Murofushi, K., Nakamura, K., Ohta, J., Suzuki, M., Suzuki, A., Murofushi, H., and Yokota, T. (1987) J. Biol. Chem. 262, 16719-16723). The activity of UDP-glucose:poriferasterol glucosyltransferase (Murakami-Murofushi, K., and Ohta, J. (1989) Biochim. Biophys. Acta 992, 412-415) was also expressed rapidly after heat shock. Thus, the activation of sterol glucosyltransferase and the production of sterol-glucoside were considered to be important events that were involved in the signal transduction system to induce some succeeding heat-shock responses, such as the synthesis of heat-shock proteins.     

Molecular cloning and structural characterization of the Arg-gingipain proteinase of Porphyromonas gingivalis. Biosynthesis as a proteinase-adhesin polyprotein

The identification of proteinases of Porphyromonas gingivalis that act as virulence factors in periodontal disease has important implications in the study of host-pathogen interactions as well as in the discovery of potential therapeutic and immunoprophylactic agents. We have cloned and characterized a gene that encodes the 50-kDa cysteine proteinase gingipain or Arg-gingipain-1 (RGP-1) described previously (Chen, Z., Potempa, J., Polanowski, A., Wikstrom, M., and Travis, J. (1992) J. Biol. Chem. 267, 18896-18901). Analysis of the amino acid sequence of RGP-1 deduced from the cloned DNA sequence showed that the biosynthesis of this proteinase involves processing of a polyprotein that contains multiple adhesin molecules located at its carboxyl terminus. This finding corroborates previous evidence (Pike R., McGraw, W., Potempa, J., and Travis, J. (1994) J. Biol. Chem. 269, 406-411) that RGP-1 is closely associated with adhesin molecules, and that high molecular weight forms of the proteinase are involved in the binding of erythrocytes.     

Biosynthesis of the escherichia coli K4 capsule polysaccharide. A parallel system for studies of glycosyltransferases in chondroitin formation

Escherichia coli K4 bacteria synthesize a capsule polysaccharide (GalNAc-GlcA(fructose))n with the carbohydrate backbone identical to chondroitin. GlcA- and GalNAc-transferase activities from the bacterial membrane were assayed with acceptors derived from the capsule polysaccharide and radiolabeled UDP-[14C]GlcA and UDP-[3H]GalNAc, respectively. It was shown that defructosylated oligosaccharides (chondroitin) could serve as substrates for both the GlcA- and the GalNAc-transferases. The radiolabeled products were completely degraded with chondroitinase AC; the [14C]GlcA unit could be removed by beta-D-glucuronidase, and the [3H]GalNAc could be removed by beta-N-acetylhexosaminidase. A fructosylated oligosaccharide acceptor tested for GlcA-transferase activity was found to be inactive. These results indicate that the chain elongation reaction of the K4 polysaccharide proceeds in the same way as the polymerization of the chondroitin chain, by the addition of the monosaccharide units one by one to the nonreducing end of the polymer. This makes the biosynthesis of the K4 polysaccharide an interesting parallel system for studies of chondroitin sulfate biosynthesis. In the biosynthesis of capsule polysaccharides from E. coli, a similar mechanism has earlier been demonstrated for polysialic acid (NeuNAc)n (Rohr, T. E., and Troy, F. A. (1980) J. Biol. Chem. 255, 2332-2342) and for the K5 polysaccharide (GlcAbeta1-4GlcNAcalpha1-4)n (Lidholt, K., Fjelstad, M., Jann, K., and Lindahl, U. (1994) Carbohydr. Res. 255, 87-101). In contrast, chain elongation of hyaluronan (GlcAbeta1-3GlcNAcbeta1-4)n is claimed to occur at the reducing end (Prehm, P. (1983) Biochem. J. 211, 181-189).     

The chemical structures of the capsular polysaccharides from Streptococcus pneumoniae types 32F and 32A

The structures of the capsular polysaccharides from Streptococcus pneumoniae types 32F and 32A have been determined by means of NMR spectroscopy as the principal method. It is concluded that both polysaccharides are composed of tetrasaccharide repeating units with a phosphorylcholine (PCho) group linked to the 3-position of the 4-substituted beta-L-rhamnose (Rha) residue. Both polysaccharides are substituted with one O-acetyl group at the 2-position of the same beta-L-rhamnose residue. In addition, the type-32A polysaccharide is substituted with another O-acetyl group at the 4-position of the 2,3-disubstituted alpha-D-glucose residue, i.e. the branch-point residue. An unusual detail in the structure is that the side chain is composed of a rhamnosyl phosphate. [chemical structure: see text] In the type-32F polysaccharide R=H, and in the type-32A polysaccharide R=Ac. The structure of C-polysaccharide found in our preparations of type-32F and type-32A capsular polysaccharides is in agreement with that published previously for the pneumococcal common antigen C-polysaccharide [Fischer, W., Behr, T., Hartmann, R., Peter-Katalinic, J. & Egge, H. (1993) Eur. J. Biochem. 215, 851-857; Kulakowska, M., Brisson, J.-R., Griffith, D. W., Young, N. M. & Jennings, H. J. (1993) Can. J. Chem. 71, 644-648].     

Structural studies of the cell envelope lipopolysaccharides from Haemophilus ducreyi strains ITM 2665 and ITM 4747

The structures of the lipopolysaccharides from Haemophilus ducreyi strains ITM 2665 and ITM 4747 have been investigated. Oligosaccharides were obtained from phenol/water-extracted lipopolysaccharide by mild acid hydrolysis and were studied with methylation analysis, fast atom bombardment-mass spectrometry, and NMR spectroscopy. The major oligosaccharide obtained from strain 2665 is a nonasaccharide with the following structure: beta-D-Galp-1-->4-beta-D-GlcNAcp-1-->3-beta-D-Galp-1-->4-D-alpha-D -Hepp- 1-->6-beta-D-Glcp-1-->(L-alpha-D-Hepp-1-->2-L-alpha-D-Hepp-1 -->3)-4-L-alpha- D-Hepp-Kdo, where the reducing terminal 3-deoxy-D-manno-octulosonic acid (or Kdo) exists in reduced anhydro forms. The proposed structure complements the preliminary structure described for Haemophilus ducreyi strain 35000 (Melaugh, W., Phillips, N. J., Campagnari, A. A., Karalus, R., and Gibson, B. W. (1992) J. Biol. Chem. 267, 13434-13439) with the missing anomeric configurations. The saccharide isolated from strain 4747 is a markedly simpler hexasaccharide with the following structure: beta-D-Galp-1-->4-beta-D-Glcp-1-->(L-alpha-D-Hepp-1-->2-L-alpha-D- Hepp- 1-->3)4-L-alpha-D-Hepp-Kdo. Apart from a different phosphorylation of the inner core region the proposed structure is identical to the structure of lipopolysaccharide from an only distantly related bacterium, viz. Haemophilus influenzae nontypable strain 2019 (Phillips, N. J., Apicella, M. A., Griffiss, J. M., and Gibson, B.W. (1992) Biochemistry 31, 4515-4526). The implications of these findings as regards the role of lipopolysaccharide as a virulence factor are discussed.     

Tissue-specific mRNA expression of a calcitonin receptor-like receptor during fetal and postnatal development

The distribution of calcitonin receptor-like receptor (CRLR) mRNA in developing rats was investigated by in situ hybridization. Signals were found in the piriform cortex, the central and basolateral amygdala and the amygdalostriatal transition area. Among peripheral organs, the CRLR was predominantly expressed in the lung. mRNA expression in blood vessels, liver, midgut, rectum and urethra was restricted to gestational days 16 and/or 20. The CRLR was thought to be a calcitonin gene-related peptide (CGRP) type 1 receptor (Aiyar et al., J. Biol. Chem., 271 (1996) 11325-11329). This contrasts with previously reported evidence that the CRLR is an orphan receptor with no identifiable interactions with CGRP and other related ligands (Flühmann et al., Biochem. Biophys. Res. Commun., 206 (1995) 341-347). In situ hybridization signals have not been detected in the cerebellum and the spleen known to present a high density of CGRP binding sites. The different regional distribution of CGRP receptor binding sites and CRLR mRNA implies the latter encoding a different CGRP receptor subtype.     

Cloning and characterization of a cDNA encoding a novel subtype of rat thyrotropin-releasing hormone receptor

A cDNA encoding a thyrotropin-releasing hormone (TRH) receptor expressed in the pituitary was previously cloned (De La Pena, P., Delgado, L. M., Del Camino, D., and Barros, F. (1992) Biochem. J. 284, 891-899; De La Pena, P., Delgado, L. M., Del Camino, D., and Barros, F. (1992) J. Biol. Chem. 267, 25703-25708; Duthie, S. M., Taylor, P. L., Anderson, J., Cook, J., and Eidne, K. A. (1993) Mol. Cell Endocrinol. 95, R11-R15). We now describe the isolation of a rat cDNA encoding a novel subtype of TRH receptor (termed TRHR2) displaying an overall homology of 50% to the pituitary TRH receptor. Introduction of TRHR2 cDNA in HEK-293 cells resulted in expression of high affinity TRH binding with a different pharmacological profile than the pituitary TRH receptor. De novo expressed receptors were functional and resulted in stimulation of calcium transient as assessed by fluorometric imaging plate reader analysis. The message for TRHR2 was exclusive to central nervous system tissues as judged by Northern blot analysis. Studies of the expression of TRHR-2 message by in situ hybridization revealed a pattern of expression remarkably distinct (present in spinothalamic tract, spinal cord dorsal horn) from that of the pituitary TRH receptor (present in hypothalamus, and ventral horn of the spinal cord, anterior pituitary). Therefore, we have identified a novel, pharmacologically distinct receptor for thyrotropin-releasing hormone that appears to be more restricted to the central nervous system particularly to the sensory neurons of spinothalamic tract and spinal cord dorsal horn, which may account for the sensory antinociceptive actions of TRH.     

Sulfhydryl reagents and cAMP-dependent kinase increase the sensitivity of the inositol 1,4,5-trisphosphate receptor in hepatocytes

Sulfhydryl reagents such as tert-butyl hydroperoxide (TBHP) have been shown to increase cytosolic Ca2+ concentration ([Ca2+]i) in rat hepatocytes in a way that resembles responses to Ca(2+)-mobilizing hormones (Saikada, I., Thomas, A. P., and Farber, J. L. (1991) J. Biol. Chem. 266, 717-722; Rooney, T. A., Renard, D. C., Sass, E. J., and Thomas, A. P. (1991) J. Biol. Chem. 266, 12272-12282) and to increase the amount of Ca2+ released by inositol 1,4,5-trisphosphate ((1,4,5)IP3) from permeable rat liver cells (Rooney et al., 1991, op. cit.; Missiaen, L., Taylor, C. W., and Berridge, M. J. (1991) Nature 352, 241-244; Renard, D. C., Seitz, M. B., and Thomas, A. P. (1992) Biochem. J. 284, 507-512). The effects of sulfhydryl reagents were studied in fura-2-injected rat and guinea pig hepatocytes and compared with the actions of cAMP (Burgess, G. M., Bird, G. St. J., Obie, J. F., and Putney, J. W., Jr. (1991) J. Biol. Chem. 261, 4772-4781). In rat liver cells, the increases in [Ca2+]i induced by TBHP and thimerosal were prevented by microinjection of the cells with the (1,4,5)IP3 receptor antagonist heparin. In guinea pig hepatocytes, TBHP was not able to increase [Ca2+]i unless the cells were pretreated with angiotensin II to raise endogenous levels of (1,4,5)IP3 or were first injected with a sub-threshold concentration of inositol 2,4,5-trisphosphate ((2,4,5)IP3). The responses to TBHP in (2,4,5)IP3-injected guinea pig cells were also blocked by heparin. In many respects, the actions of TBHP appeared to be similar to those of cAMP, which has previously been shown to increase sensitivity to (1,4,5)IP3 in intact guinea pig hepatocytes (Burgess et al., 1991, op. cit.). TBHP also mimicked the effect of cAMP-dependent kinase (PKA) in permeabilized guinea pig hepatocytes by increasing the amount of Ca2+ released by (1,4,5)IP3. The responses to TBHP and cAMP in (2,4,5)IP3-injected guinea pig hepatocytes differed, however, in that the increase in [Ca2+]i evoked by elevating intracellular cAMP was greatly reduced by Wiptide, an inhibitor of PKA, while Wiptide had no effect on the Ca2+ transients induced by TBHP. This provides evidence that the sensitizing effect of TBHP is not mediated by PKA and is more likely to be a direct effect on the inositol trisphosphate receptor. It is possible, however, that the sulfhydryl reagents and PKA act on a common regulatory site on the receptor protein.     

Expression of prolactin gene in mouse placenta during late pregnancy: detection of mRNA and its translation product

Threshold photoelectron spectra of N2+ were measured between 23.4 and 27.6 eV with high resolution and high intensity by using the penetrating field technique and synchrotron radiation. Five vibrational progressions were observed. The first of these progressions was the C2Sigmau+ state. The second progression was identified as the transition to the second state of 2Pig symmetry found by P. Baltzer, M. Larsson, L. Karlsson, B. Wannberg, and M. Carlsson (1992. Phys. Rev. A 46, 5545). The third progression, which was discovered by F. Merkt and P. M. Guyon (1993. J. Chem. Phys. 99, 3400), can be designated as the 2Sigmau- state by comparison with previous theoretical study (E. W. Thulstrup and A. Andersen, 1975. J. Phys. B 8, 965). The fourth and fifth progressions were designated as the 2Deltau and 2(2) Piu states by similar comparison with previous theories. Copyright 1997 Academic Press. Copyright 1997Academic Press     

Glycoprotein 330, a member of the low density lipoprotein receptor family, binds lipoprotein lipase in vitro

Glycoprotein 330 (gp330), a cell-surface protein that is localized in clathrin-coated pits, is structurally related to both the low density lipoprotein receptor (LDLR) and the LDLR-related protein/alpha 2-macroglobulin receptor (LRP). We recently demonstrated that gp330 and LRP may be functionally related as well; both bind the 39-kDa polypeptide referred to as receptor-associated protein (Kounnas, M. Z., Argraves, W. S., and Strickland, D. K. (1992) J. Biol. Chem. 267, 21162-21166). In this report, we tested several other LRP ligands for their ability to interact with human and rat gp330 in vitro. Gp330 did not exhibit detectable binding to the LRP ligands, alpha 2-macroglobulin protease complex or Pseudomonas aeruginosa exotoxin A. However, we found that gp330 (purified from human or rat) bound the lipolytic enzyme lipoprotein lipase (LPL) with high affinity (Kd = 6.1 and 2.7 nM, respectively). The binding was saturable, divalent cation dependent, and inhibited by heparin or receptor-associated protein. Because LRP has also been shown to bind LPL, the present findings further extend the functional similarities between gp330 and LRP. By analogy to the postulated role of the LRP-LPL interaction in facilitating hepatic clearance of LPL-associated lipoproteins from the blood (Beisiegel, U., Weber, W., and Bengtsson-Olivercrona, G. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 8342-8346; Chappell, D. A., Fry, G. L., Waknitz, M. A., Iverius, P. H., Williams, S. E., and Strickland, D. K. (1992) J. Biol. Chem. 267, 25764-25767), we speculate that the gp330-LPL interaction described herein may contribute to the uptake of LPL-associated lipoproteins in tissues expressing gp330. Consistent with this possibility, we found that LPL promoted in vitro binding of 125I-lipoproteins to gp330.     

Entropy-driven intermediate steps of oxygenation may regulate the allosteric behavior of hemoglobin

When the oxygen binding isotherms of human, bovine and fallow deer (Dama-Dama) hemoglobins are measured at different temperatures either by optical or calorimetric techniques, analyses according to the Adair's formalism show that at least one of the intermediate steps of ligation has a positive enthalpy change, i.e., absorbs rather than emitting heat, indicating that it is entropy rather than enthalpy driven (Bucci, E., et al. 1991. Biochemistry. 30:3195-3199; Bucci, E., et al. 1993. Biochemistry. 32:3519-3526; Johnson, C., et al. 1992. Biochemistry. 31:10074-10082; Johnson, C., et al. 1995. Biophys. Chem. 59:107-117). This phenomenon is confirmed in systems in which the beta82 lysines of human hemoglobin are covalently cross-linked by acylation with dicarboxylic acids of increasing length, namely the fumaryl (four-carbon-long), adipoyl (six-carbon-long), and sebacoyl (10-carbon-long) residues. Consistently in all of the systems here reported, the enthalpy excursions are masked by compensatory entropy changes, which keep the free energy of ligand binding constant for the first three steps of oxygenation. Furthermore, the cooperativity index and the overall oxygen affinity seem to be correlated to the positive enthalpy excursions of the intermediate steps of ligation. Fumaryl-Hb (hemoglobin cross-linked with a fumaryl residue, four carbons) with the lowest absorption of heat has the highest affinity and lowest cooperativity index. Adipoyl-Hb (hemoglobin cross-linked with an adipoyl residue, six carbons) has the highest absorption of heat and the highest cooperativity index. It appears that nonuniform heat release by the intermediates of oxygenation is part of the allosteric phenomena in hemoglobin systems. There is not enough information that would allow assigning these phenomena to the interplay of the various conformations described for hemoglobin besides the classic T (Fermi et al. 1984. J. Mol. Biol. 175:159-174) and R (Shanaan. 1983. J. Mol. Biol. 171:31-59), as listed at the end of the Discussion. The possibility cannot be excluded that entropy-driven steps characterize new conformational transitions still to be described.     

Genomic instability and catalase gene amplification induced by chronic exposure to oxidative stress

Chronic exposure (>200 days) of HA1 fibroblasts to increasing concentrations of H2O2 or O2 results in the development of a stable oxidative stress-resistant phenotype characterized by increased cellular antioxidant levels, particularly catalase (D. R. Spitz et al, Arch. Biochem. Biophys., 279: 249-260, 1990; D. R. Spitz et al., Arch. Biochem. Biophys., 292: 221-227, 1992; S. J. Sullivan et al., Am. J. Physiol. (Lung Cell. Mol. Physiol.), 262: L748-L756, 1992). Acutely stressed cells failed to develop a stably resistant phenotype or increased catalase activity, suggesting that chronic exposure is required for the development of this phenotype. This study investigates the mechanism underlying increased catalase activity in the H2O2- and O2-resistant cell lines. In H2O2- and O2-resistant cells, catalase activity was found to be 20-30-fold higher than that in the parental HA1 cells and correlated with increased immunoreactive catalase protein and steady-state catalase mRNA levels. Resistant cell lines also demonstrated a 4-6-fold increase in catalase gene copy number by Southern blot analysis, which is indicative of gene amplification. Chromosome banding and in situ hybridization studies identified a single amplified catalase gene site located on a rearranged chromosome with banding similarities to Z-4 in the hamster fibroblast karyotype. Simultaneous in situ hybridization with a Z-4-specific adenine phosphoribosyltransferase (APRT) gene revealed that the amplified catalase genes were located proximate to APRT on the same chromosome in all resistant cells. In contrast, HA1 cells contained only single copies of the catalase gene that were not located on APRT-containing chromosomes, indicating that amplification is associated with a chromosomal rearrangement possibly involving Z-4. The fact that chronic exposure of HA1 cells to either HO2 or 95% O2 resulted in gene amplification suggests that gene amplification represents a generalized response to oxidative stress, contributing to the development of resistant phenotypes. These results support the hypothesis that chronic exposure to endogenous metabolic or exogenous environmental oxidative stress represents an important factor contributing to gene amplification and genomic instability.     

Large kinetic isotope effects in enzymatic proton transfer and the role of substrate oscillations

We propose an interpretation of the experimental findings of Klinman and coworkers [Cha, Y., Murray, C. J. & Klinman, J. P. (1989) Science 243, 1325-1330; Grant, K. L. & Klinman, J. P. (1989) Biochemistry 28, 6597-6605; and Bahnson, B. J. & Klinman, J. P. (1995) Methods Enzymol. 249, 373-397], who showed that proton transfer reactions that are catalyzed by bovine serum amine oxidase proceed through tunneling. We show that two different tunneling models are consistent with the experiments. In the first model, the proton tunnels from the ground state. The temperature dependence of the kinetic isotope effect is caused by a thermally excited substrate mode that modulates the barrier, as has been suggested by Borgis and Hynes [Borgis, D. & Hynes, J. T. (1991) J. Chem. Phys. 94, 3619-3628]. In the second model, there is both over-the-barrier transfer and tunneling from excited states. Finally, we propose two experiments that can distinguish between the possible mechanisms.     

Proteoglycans from bovine proximal humeral articular cartilage. Structural basis for the polydispersity of proteoglycan subunit

Polydisperse proteoglycan subunit from bovine proximal humeral articular cartilage has been separated into a series of relatively monodisperse fractions which have been chemically and physically characterized. The proteoglycan subunit species of the lowest molecular weight contains the least chondroitin sulfate and had an amino acid composition relatively low in serine and glycine and relatively high in cysteine, methionine, and aspartic acid, almost identical to that of the hyaluronic acid-binding region of proteoglycan subunit isolated by Heinegard and Hascall (Heinegard, D., and Hascall, V.C. (1974) J. Biol. Chem. 249, 4250-4256). The molecular weight of proteoglycan subunit increases in proportion to its chondroitin sulfate content. As the molecular weight and chondroitin sulfate content of proteoglycan subunit increase, there is a parallel increase in the serine and glycine contents, and a decrease in the cysteine, methionine, and aspartic acid contents of proteoglycan subunit core protein. The pattern of polydispersity observed strongly supports the concept that proteoglycan subunit core protein contains a hyaluronic acid-binding region of constant size and composition and a polysaccharide attachment region of variable length and composition, composed of repeating peptide sequences containing serine and glycine in equimolar amounts.     

Magnitude of the equilibrium isotope effect on carbon-tritium bond synthesis

The exchange of tritium from 3HOH into the methyl group of pyruvate catalyzed by 6-phospho-2-keto-3-deoxygluconate aldolase (6-phospho-2-keto-3-deoxy-D-gluconate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.14) of Pseudomonas putida shows an equilibrium isotope effect of 0.78. From this value and the deuterium effect on the fumarase equilibrium (Thomson, J.F. (1960) Arch. Biochem. Biophys. 90, 1), one can calculate by use of the relative fractionation factors of Hartshorn and Shiner (Hartshorn, S.R. and Shiner, Jr., V.J. (1972) J. Am. Chem. Soc. 94, 9002), fractionation factors for transfer of deuterium or tritium from water to a number of organic molecules of interest.     

MacMARCKS is not essential for phagocytosis in macrophages

MacMARCKS (also known as myristoylated alanine-rich protein kinase C substrate (MARCKS)-related protein) is a member of the MARCKS family of protein kinase C substrates. MacMARCKS contains within it a basic effector domain that contains the serine residues that are phosphorylated by protein kinase C, as well as a calcium/calmodulin and actin-binding site. Two previous reports demonstrated that a macrophage cell line expressing a mutant form of MacMARCKS that lacks the effector domain is defective in phagocytosis and cell adhesion (Zhu, Z., Bao, Z., and Li, J. (1995) J. Biol. Chem. 270, 17652-17655; Li, J., Zhu, Z., and Bao, Z. (1996) J. Biol. Chem. 271, 12985-12990). We report here that macrophages from MacMARCKS null mice phagocytose and spread normally. Thus, although MacMARCKS is recruited to phagosomes, it is not absolutely required for phagocytosis.     

Chain elongation and joining of DNA synthesized during hydroxyurea treatment of Chinese hamster cells

We have previously presented evidence that hydroxyurea treatment of synchronized G1 Chinese hamster cells did not prevent the entry of cells into the DNA synthetic period but that the DNA synthesized during this period (in which total DNA synthesis was severely depressed) was quite small (Walters, R.A., Tobey, R.A. and Hildebrand, C.E. (1976) Biochem. Biophys. Res. Com. 69, 212-217). In view of the reported effects of hydroxyurea on deoxyribonucleoside metabolism and possible relationship to control of DNA replication (Bjursell, G. and Reichard, P. (1973) J. Biol. Chem. 248,3904-3909 and Walters, R.A., Tobey, R.A. and Ratliff, R.L. (1973) Biochim. Biophys. Acta 319, 336-347), we examined the fate of DNA synthesized during and shortly after hydroxyurea treatment to determine if this DNA exhibited any kinetic behavior which might be an indicator of aberrant synthesis. We found that, upon hydroxyurea removal, DNA grew at a linear rate of 0.98 +/- 0.12 - 10(6) dalton/min (0.98 +/- 0.12 mum/min) for about 2.3h. Beginning at 2.3 h, DNA with a molecular weight approx. 1.4 - 10(8) was very rapidly integrated into bulk DNA of greater than or equal to 3.5 - 10(8) daltons. The apparent growth rate of the 1.4 - 10(8) dalton DNA was approx. 10.6 mum/min. The data suggest that, at least for this DNA, joining into bulk DNA required one-third to one-half of the S period to begin and once begun, occurred very rapidly. The possibility of inegration of replicon clusters is considered.