Thermodynamic analysis of the nonspecific interaction of sodium dodecyl sulfate with swollen Bio-Gel beads

In frontal gel chromatography on Bio-Gel P-2, sodium dodecyl sulfate (SDS) at the concentrations below its critical micelle concentration (cmc) showed anomalously high partition coefficients (Kav obs) indicative of strong interactions with the swollen gel phase; further, Kav obs was found to increase with concentration and temperature. This preferential partition of SDS in the Bio-Gel phase was analyzed in terms of the transfer free energy of SDS from the mobile phase (0.1 M NaCl) to the swollen Bio-Gel phase. The results showed that the overall transfer process is primarily governed by hydrophobic free energy arising from the anomalous nature of hydrated water in the gel matrix; that is, in highly hydrated water "iceberg" formation is evidently limited and the hydrophobic free energy is accordingly lowered, resulting in the preferential partition of SDS in the swollen Bio-Gel phase. The increase in the negativity of transfer free energy with concentration, though relatively small, indicated a definite tendency for the formation of SDS clusters in the gel phase. Finally, a model illustrating the states of SDS molecules in the gel matrix is presented, which may also be pertinent to SDS-protein and SDS-amylose complexes.     

Purification and properties of reductases for aromatic aldehydes and ketones from guinea pig liver.

NADPH-dependent enzymatic reduction of aromatic aldehydes and ketones observed in the cytosol of guinea pig liver was mediated by at least three distinct reductases (AR 1, AR 2, and AR 3), which were separated by DEAE-cellulose chromatography. By several procedures AR 2 and AR 3 were purified to homogeneity, but AR 1 could be purified only 30-fold because of the small amount. These enzymes were found to have similar molecular weights of 34,000 to 36,000 and similar Stokes radii of about 2.5 nm. AR 3 was identical to aldehyde reductase [EC 1.1.1.2] in substrate specificity for aromatic aldehydes and D-glucuronate and specific inhibition by barbiturates. AR 1 and AR 2 acted on aromatic ketones and cyclohexanone as well as aromatic aldehydes at optimal pHs of 5.4 and 6.0, respectively, and were immunochemically distinguished from AR 3. AR 1 was the most sensitive to sulfhydryl reagents, and AR 2 was more stable at 50 degrees C than the other enzymes. Similar heterogeneity was observed in the kidney enzymes, but other tissues had little aldehyde reductase activity and contained only AR 3. In addition, lung contained a high molecular weight aromatic ketone reductase different from the above reductases.     

The peripheral inhibition of the tarsal sugar receptor by sodium chloride in the proboscis extension response of the blowfly,Phormia regina M.

Summary The proboscis extension reponse of the blowfly during stimulation of the tarsal sugar receptors was inhibited by the presence of NaCl. Acceptance thresholds for sucrose in various concentrations of NaCl were measured. The median acceptance thresholds for sucrose in mixtures of 0.01, 0.25, 0.5 and 1.0 M NaCl were 1.8 × 10^–3, 6.0 × 10^–3, 1.2 × 10^–2, and 2.0 × 10^–2 M, respectively. Concentration-response curves for sucrose in the tarsal D-type sugar receptor shifted to the right under the existence of high concentration of NaCl. Number of impulses per D-type sugar receptor at the median acceptance thresholds described above were 7.5, 8.4, 6.8 and 10.4 for the first 0.1 s of stimulation, respectively. The average number was 8.2 impulses per 0.1 s. Comparisons were made between the behavioral acceptance thresholds (1) on one leg exposed to sucrose mixed with 0.01 M NaCl and (2) on two contralateral legs, one of which was exposed to sucrose in 0.01 M NaCl and the other to 0.5 M NaCl alone. The acceptance thresholds from two experiments agreed with each other. The median threshold value was 1.7 × 10^–2 M sucrose. Behavioral inhibition by NaCl in mixtures with sucrose can be explained by its peripheral inhibition of sugar receptors.This research was supported in part by ITO foundation and Scientific Research Fund from the Ministry of Education of Japan.     

Interaction of antimannan with glycopeptides

Taka amylase A glycopeptide (TA-GP) strongly inhibited the interaction of antimannan (antibodies directed towards mannan from $\text{Saccharomyces cerevisiae}$) with yeast mannan, whereas ovalbumin glycopeptide (OA-GP) did so only poorly. We inferred that this is due to the strong reactivity of antimannan with terminal trimannosides composed of Manα1→2Man or Manα1→3Man linkages which occur in mannan and TA-GP. In contrast, TA-GP and OA-GP were nearly equally reactive with concanavalin A having the ability to interact with terminal mannose and 2-0-mannose residues which occur abundantly in these glycopeptides. Thus, antimannan should be useful as a probe for characterizing glycoproteins from extracellular fluids or cellular membranes.     

Enantiomeric cuparene-type sesquiterpenoids from Bazzania pompeana

Two enantiomeric cuparene-type sesquiterpenoids, (R)-(−)-cuparene (1) and (R)-(−)-δ-cuparenol (2), have been isolated from the liverwort, Bazzania pompeana. The structures and absolute configurations of the two compounds have been determined.     

KIT (c-kit oncogene product) pathway is constitutively activated in human testicular germ cell tumors

We investigated the expression of KIT (product of c-kit oncogene), gain-of-function mutations, and activation of its downstream signal transduction in human testicular cancers. KIT was expressed in 88% (22/25) of seminomas and in 44.4% (4/9) of non-seminomas compared to adjacent normal testicular tissue. Nine of the KIT-expressing seminomas had mutations (40.9%; 9/22) in the c-kit gene; two cases in exon 11 and 7 cases in exon 17. Two of these mutations in exon 17 were novel, and the other seven mutations were identical to the already known gain-of-function mutations which cause activation of KIT without ligand stem cell factor. All of the mutant KIT and 53.8% (7/13) of wild-type KIT were phosphorylated (activated) and associated with phosphorylated phosphatidylinositol 3-kinase (PI3K). Akt was also phosphorylated in these seminomas, suggesting that the KIT-PI3K-Akt pathway is activated in seminoma. These findings suggest that the KIT-PI3K-Akt pathway is constitutively activated in testicular germ cell tumors, due to overexpression of KIT protein and/or gain-of-function mutations in the c-kit gene.     

Fbxo45 Forms a Novel Ubiquitin Ligase Complex and Is Required for Neuronal Development

Fbxo45 is an F-box protein that is restricted to the nervous system. Unlike other F-box proteins, Fbxo45 was found not to form an SCF complex as a result of an amino acid substitution in the consensus sequence for Cul1 binding. Proteomics analysis revealed that Fbxo45 specifically associates with PAM (protein associated with Myc), a RING finger-type ubiquitin ligase. Mice deficient in Fbxo45 were generated and found to die soon after birth as a result of respiratory distress. Fbxo45^−/− embryos show abnormal innervation of the diaphragm, impaired synapse formation at neuromuscular junctions, and aberrant development of axon fiber tracts in the brain. Similar defects are also observed in mice lacking Phr1 (mouse ortholog of PAM), suggesting that Fbxo45 and Phr1 function in the same pathway. In addition, neuronal migration was impaired in Fbxo45^−/− mice. These results suggest that Fbxo45 forms a novel Fbxo45-PAM ubiquitin ligase complex that plays an important role in neural development.Ubiquitin-dependent proteolysis is indispensable for various biological processes (3, 40). Protein ubiquitylation is mediated by several enzymes that act in concert, with a ubiquitin ligase (E3) playing a key role in substrate recognition (14). E3 enzymes contain specific structural motifs that mediate recruitment of a ubiquitin-conjugating enzyme (E2), with these motifs including HECT, RING finger, U-box, and PHD finger domains (30). The SCF complex consists of Skp1 (adaptor subunit), Cul1 (scaffold subunit), an F-box protein (substrate recognition subunit), and Rbx1 (also known as Roc1 or Hrt1; RING finger-containing subunit). Whereas Skp1, Cul1, and Rbx1 are common to all SCF complexes, the F-box protein is variable (with ∼70 such proteins having been identified in humans) and confers substrate specificity.Fbxo45 is an F-box protein that was originally isolated as an estrogen-induced protein (47). Human and mouse Fbxo45 genes comprise three exons and possess several consensus binding sequences for the estrogen receptor in the promoter region. Fbxo45 mRNA is rapidly induced on exposure of MCF-7 cells to 17β-estradiol (47). FSN-1, the Caenorhabditis elegans ortholog of Fbxo45, binds to RPM-1 (regulator of presynaptic morphology 1) together with CUL-1 and SKR-1, the C. elegans orthologs of mammalian Cul1 and Skp1, respectively (21, 46). RPM-1 belongs to an evolutionarily conserved family of proteins (the PHR family) that include Highwire (HIW) (Drosophila melanogaster), Esrom (Danio rerio), Phr1 (Mus musculus), and protein associated with Myc (PAM) (Homo sapiens), each of which contains a RING-finger domain that is required for its E3 activity (7, 20, 21, 27, 44). Complete loss of function of fsn-1 in C. elegans results in defects that are characterized by the simultaneous presence of overdeveloped and underdeveloped neuromuscular junctions (NMJs) and which are similar to, but not as pronounced as, those observed in rpm-1^−/− mutants. These genetic findings support the notion that the functions of FSN-1 and RPM-1 are partially overlapping (21).Although PHR family members interact with many potential targets (11, 24, 26, 31), genetic data have shown that one key substrate of RPM-1 and HIW is the mitogen-activated protein kinase kinase kinase known as DLK (dual leucine zipper kinase) in C. elegans and known as Wallenda in D. melanogaster, respectively. The abundance of this kinase is increased in rpm-1 or hiw mutants, and synaptic defects in the mutant worms and flies are suppressed by a loss of DLK or Wallenda. Furthermore, an increase in the level of DLK or Wallenda is sufficient to phenocopy the synaptic defects of the rpm-1 or hiw mutants (5, 27). PAM has also been shown to catalyze the ubiquitylation of tuberin (TSC2) and to regulate signaling by mTOR (mammalian target of rapamycin) in human cells (12).To elucidate the physiological functions of Fbxo45 in mammals, we have now generated mice deficient in this protein. Analysis of the mutant mice revealed that Fbxo45 is required for normal neuromuscular synaptogenesis, axon pathfinding, and neuronal migration. Moreover, we found that Fbxo45 does not form an authentic SCF complex as a result of an amino acid substitution in the F-box domain, and we identified PAM as a binding partner of Fbxo45. The phenotype of Fbxo45^−/− mice was found to be similar to that of Phr1^−/− mice, especially with regard to the defects of neuromuscular synapse formation and of axon navigation. Our results indicate that three fundamental processes of neural development— axonal projection, synapse formation, and neuronal migration—may be linked by a common machinery consisting of the Fbxo45-Phr1 complex.