Protein Linewidth and Solvent Dynamics in Frozen Solution NMR

Solid-state NMR of proteins in frozen aqueous solution is a potentially powerful technique in structural biology, especially if it is combined with dynamic nuclear polarization signal enhancement strategies. One concern regarding NMR studies of frozen solution protein samples at low temperatures is that they may have poor linewidths, thus preventing high-resolution studies. To learn more about how the solvent shell composition and temperature affects the protein linewidth, we recorded ¹H, ²H, and ¹³C spectra of ubiquitin in frozen water and frozen glycerol-water solutions at different temperatures. We found that the ¹³C protein linewidths generally increase with decreasing temperature. This line broadening was found to be inhomogeneous and independent of proton decoupling. In pure water, we observe an abrupt line broadening with the freezing of the bulk solvent, followed by continuous line broadening at lower temperatures. In frozen glycerol-water, we did not observe an abrupt line broadening and the NMR lines were generally narrower than for pure water at the same temperature. ¹H and ²H measurements characterizing the dynamics of water that is in exchange with the protein showed that the ¹³C line broadening is relatively independent from the arrest of isotropic water motions.     

Nemertea inhabiting the Haploops (Amphipoda) community of the northern Øresund with special reference to the biology of Nipponnemertes pulcher (Hoplonemertea)

The densities of nemerteans and associated fauna on a soft-bottom sampling station (27–30 m deep) in the Øresund were determined from 47 cores (each 135 cm² in cross-section; 20 + cm deep) collected from September to December 1989; these data were compared with 14 cores taken from the same location in December 1982. Nine species of nemerteans were identified from cores and dredge samples: Palaeonemertea — Callinera-like sp.; Heteronemertea — Cerebratulus fuscus, C. marginatus, Lineus bilineatus, Micrura fasciolata, M. purpurea; Hoplonemertea — Amphiporus bioculatus, A. dissimulans, Nipponnemertes pulcher. Mean numbers of heteronemerteans were 32 and 10 m^?2 in 1982 and 1989, respectively, and hoplonemerteans were 90 and 71 m^?2 , respectively. Only one palaeonemertean was collected during both years. Mean densities of the dominant species, N. pulcher, were similar for the two years, 74 and 68 m^?2. The dominant groups of macrofauna (n m^?2) in 1989 were ostracods (1028), amphipods (618), polychaetes (514), and ophiuroids (449). Amphipods (>90% Haploops spp.) and polychaetes (at least 30 spp.) are the major potential prey for hoplonemerteans and heteronemerteans, respectively. Laboratory feeding experiments with N. pulcher revealed that it consumed amphipods (Haploops tenuis and H. tubicola) at a rate of 2.6 worm^?1 d^?1 during the first 12 hours, but after 36 hours and beyond the rate was maintained at approximately 0.2 worm^?1 d^?1. Beyond 12 hours this nemertean showed a tendancy to only partially evacuate its prey. It was demonstrated experimentally that N. pulcher has a supply of toxin capable of killing six amphipods in approximately one hour. Limited tests showed that N. pulcher fed on the cumacean Diastylis tumida, but not on the amphipod Maera loveni or the ostracod Philomedes globosus, and that Amphiporus dissimulans readily attacked Haploops spp., but not Maera or Philomedes. Although the results of laboratory experiments are tentative, they do suggest that suctorial hoplonemerteans can exert a potentially significant effect on benthic communities. Employing seven species of polychaetes as prey for Cerebratulus fuscus and Micrura fasciolata, only the latter responded positively to one of them, Glycera alba. The hermit crab Pagurus bernhardus violently rejected N. pulcher in all feeding trials.     

High levels of cathepsin B predict poor outcome in patients with breast cancer

Cathepsin B (CB) is a thiol-stimulated protease implicated in cancer invasion and metastasis. Other proteases involved in cancer spread such as urokinase-type plasminogen activator (uPA) and cathepsin D have previously been shown to be prognostic markers in breast cancer. CB was assayed by ELISA in 193 patients with primary breast cancer. CB levels were significantly higher in both primary and metastatic breast tumors than in fibroadenomas (p = 0.0001). In the primary carcinomas, CB levels showed no significant correlation with either nodal status, tumor size or estrogen receptor (ER) status. Patients with primary breast cancers containing high levels of CB had a significantly shorter disease-free interval (p = 0.01, chi-square = 6.61) and overall survival (p = 0.014, chi-square = 6.08) than patients with low levels of the protease. However, in multivariate analysis, using nodal status, tumor size, ER status and urokinase plasminogen activator (uPA), CB was not an independent prognostic marker. In contrast, nodal status, ER status and uPA were prognostic in multivariate analysis. In conclusion, CB, like certain other proteases implicated in cancer metastasis, correlates with poor outcome in patients with breast cancer. These results thus support the evidence from model systems linking CB to cancer spread. Inhibition of CB expression or activity might therefore be exploited for anti-metastatic therapies.     

Carotenoid biosynthesis in a Flavobacterium sp.: stereochemistry of hydrogen elimination in the desaturation of phytoene to lycopene, rubixanthin and zeaxanthin (Short Communication)

[2-(14)C,(2R)-2-(3)H(1)]- and [2-(14)C,(2S)-2-(3)H(1)]-Mevalonates were rapidly incorporated into phytoene, lycopene, rubixanthin and zeaxanthin in a Flavobacterium system obtained by disruption of the bacterial cells by shaking with glass beads. Four hydrogen atoms arising from the 2-pro-S-hydrogen atoms of mevalonate were lost in the desaturation of phytoene to lycopene, rubixanthin and zeaxanthin. The desaturation of phytoene involves trans-elimination of hydrogen in the introduction of the double bonds at C-7, C-11, C-7' and C-11'.     

Biochemical Studies of Bacterial Sporulation and Germination XIV. Phospholipids in Bacillus megaterium

The principal phospholipids of Bacillus megaterium throughout the cycle of growth and sporulation were found to be phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, and a hitherto unidentified isomer of glycosaminyl-phosphatidylglycerol. Phosphatidylglycerol predominated during vegetative cell growth and then declined as spores developed, whereas diphosphatidylglycerol became more prominent during spore maturation. The latter phosphatide was relatively inaccessible in the vegetative cell and was more accessible in the spore, as judged by solvent extraction under various conditions.     

Phosphorylation of the kinase suppressor of ras by associated kinases

The kinase suppressor of Ras (KSR) is a loss-of-function allele that suppresses the rough eye phenotype of activated Ras in Drosophila and the multivulval phenotype of activated Ras in Caenorhabditis elegans. The physiological role of mammalian KSR is not known. We examined the mechanisms regulating the phosphorylation of this putative kinase in mammalian cells. Wild-type mouse KSR and a mutated KSR protein predicted to create a kinase-dead protein are phosphorylated identically in intact cells and in the immune complex. Phosphopeptide sequencing identified 10 in vivo phosphorylation sites in KSR, all of which reside in the 539 noncatalytic amino terminal amino acids. Expression of the amino terminal portion of KSR alone demonstrated that it was phosphorylated in the intact cell and in an immune complex in a manner indistinguishable from that of intact KSR. These data demonstrate that the kinase domain of KSR is irrelevant to its phosphorylation state and suggest that the phosphorylation of KSR and its association with a distinct set of kinases may affect intracellular signaling.