An Improved Diluent for Rubella Hemagglutination and Hemagglutination-Inhibition Tests

Rubella hemagglutinating (HA) antigen was prepared in BHK-21 tissue as 5% cell suspensions and from unconcentrated and 20× concentrated infected supernatant fluids. In some instances, unconcentrated fluids were treated with Tween 80 and ether; cell suspensions were treated with ether alone. Preparations were tested for HA activity in dextrose-gelatin-Veronal (DGV) buffer solutions; 0.85% NaCl; Sorenson's phosphate-buffered saline, pH 7.2; and a diluent of 0.9% NaCl, 0.1% CaCl₂ (anhydrous), and 0.1% MgSO₄·7H₂O. HA titers were consistently two- to fourfold higher in the saline with added Ca⁺⁺ and Mg⁺⁺ than in DGV. Hemagglutination-inhibition titers of paired human sera were the same in either diluent. It is suggested that the interaction between rubella HA antigen and the red cells of young (less than 1-day-old) chicks may be at least partially ion dependent and that titers are enhanced by increased quantities of divalent cations.     

ANTIVIRAL ACTIVITY OF CULTURED BLUE-GREEN ALGAE (CYANOPHYTA)1

Lipophilic and hydrophilic extracts from approximately 600 strains of cultured cyanophytes, representing some 300 species, were examined for antiviral activity against three pathogenic viruses. Approximately 10% of the cultures produced substances that caused significant reduction in cytopathic effect normally associated with viral infection. The screening program identified the order Chroococcales as commonly producing antiviral agents.     

The Second Messenger, Cyclic AMP, Is Not Sufficient for Myelin Gene Induction in the Peripheral Nervous System

Abstract: The adenylyl cyclase-cyclic AMP (cAMP) second messenger pathway has been proposed to regulate myelin gene expression; however, a clear correlation between endogenous cAMP levels and myelin-specific mRNA levels has never been demonstrated during the induction or maintenance of differentiation by the myelinating Schwann cell. Endogenous cAMP levels decreased to 8–10% of normal nerve by 3 days after crush or permanent transection injury of adult rat sciatic nerve. Whereas levels remained low after transection injury, cAMP levels reached only 27% of the normal values by 35 days after crush injury. Because P₀ mRNA levels were 60% of normal levels by 14 days and 100% by 21 days after crush injury, cAMP increased only well after P₀ gene induction. cAMP, therefore, does not appear to trigger myelin gene induction but may be involved in myelin assembly or maintenance. Forskolin, an activator of adenylyl cyclase, increased endoneurial cAMP levels only in the normal nerve, and in the crushed nerve beginning at 16 days after injury, but at no time in the transected nerve. Only by treating transected nerve with 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cAMP phosphodiesterases, in combination with forskolin was it possible to increase cAMP levels. No induction of myelin genes, however, was observed with short- or long-term treatment with IBMX and forskolin in the transected nerve. A three-fold increase in phosphodiesterase activity was observed at 35 days after both injuries, and a nonmyelinated nerve was shown to have even higher activity. These experiments, therefore, suggest an important role for phosphodiesterase in the inactivation of this second messenger-dependent stimuli when Schwann cells are non-myelinating, such as after sciatic nerve injury or in the nonmyelinated nerve, which again implies that cAMP may be required for the maintenance of the myelin sheath.     

Facile removal of the N-indole-mesitylenesulfonyl protecting group using HF cleavage conditions

Summary Nitrogen indole protection of the -methyltryptophan side-chain residue is important for avoiding undesired side reactions during peptide synthesis. Of great importance is the choice of a side-chain protecting group for orthogonal peptide synthesis and its stability under a variety of chemical conditions required for synthesis of the four isomers of this unusual amino acid. We report here the successful use of the mesitylenesulfonyl (Mts) protecting group for -methyltryptophan in the synthesis of melanotropin and CCK peptide analogues and the ready cleavage of this protecting group under HF conditions.     

Suppression of tumorigenicity by the IncW R plasmid pSa in Agrobacterium tumefaciens

Summary The effect of the IncW R plasmid, pSa, on tumorigenicity and on the expression and maintenance of the Ti plasmid in tumorigenic strains of A. tumefaciens was determined. Plasmid pSa could be transferred into and stably maintained by both octopine-and nopaline-utilizing A. tumefaciens strains. The R plasmid had no effect on Ti plasmid maintenance or on Ti plasmid functions, such as octopine utilization or conjugal bacterial transfer. However, A. tumefaciens strains harboring both the R plasmid and the Ti plasmid in most instances failed to induce tumors on a number of plant species. This effect on tumorigenicity is specific to pSa. When pSa is cured from the A. tumefaciens transconjugants or when their Ti plasmids are genetically transferred to an appropriate recipient, the resultant strains lacking the R plasmid regain tumorigenicity. Restriction endonuclease analysis of plasmid DNA isolated from transconjugants harboring pSa showed no difference in Ti plasmid cleavage patterns when compared to plasmid DNA isolated from the tumorigenic parent strain. These results indicate that pSa does not induce detectable permanent genetic alteration of the Ti plasmid. Rather, it appears that the R plasmid suppresses some Ti plasmid function(s) necessary for tumorigenicity.     

Crystallization of transmembrane proteins in cubo: mechanisms of crystal growth and defect formation

Crystallization of membrane proteins is a major stumbling block en route to elucidating their structure and understanding their function. The novel concept of membrane protein crystallization from lipidic cubic phases, "in cubo", has yielded well-ordered crystals and high-resolution structures of several membrane proteins, yet progress has been slow due to the lack of understanding of the molecular mechanisms of protein transport, crystal nucleation, growth, and defect formation in cubo. Here, we examine at molecular and mesoscopic resolution with atomic force microscopy the morphology of in cubo grown bacteriorhodopsin crystals in inert buffers and during etching by detergent. The results reveal that crystal nucleation occurs following local rearrangement of the highly curved lipidic cubic phase into a lamellar structure, which is akin to that of the native membrane. Crystals grow within the bulk cubic phase surrounded by such lamellar structures, whereby transport towards a growing crystalline layer is constrained to within an individual lamella. This mechanism leads to lack of dislocations, generation of new crystalline layers at numerous locations, and to voids and block boundaries. The characteristic macroscopic lengthscale of these defects suggests that the crystals grow by attachment of single molecules to the nuclei. These insights into the mechanisms of nucleation, growth and transport in cubo provide guidance en route to a rational design of membrane protein crystallization, and promise to further advance the field.     

The Caenorhabditis elegans centrosomal protein SPD-2 is required for both pericentriolar material recruitment and centriole duplication

BACKGROUND: The centrosome is composed of a centriole pair and pericentriolar material (PCM). By marking the site of PCM assembly, the centrioles define the number of centrosomes present in the cell. The PCM, in turn, is responsible for the microtubule (MT) nucleation activity of centrosomes. Therefore, in order to assemble a functional bipolar mitotic spindle, a cell needs to control both centriole duplication and PCM recruitment. To date, however, the molecular mechanisms that govern these two processes still remain poorly understood. RESULTS: Here we show that SPD-2 is a novel component of the C. elegans centrosome. SPD-2 localizes to the centriole throughout the cell cycle and accumulates on the PCM during mitosis. We show that SPD-2 requires SPD-5 for its accumulation on the PCM and that in the absence of SPD-2, centrosome assembly fails. We further show that centriole duplication is also defective in spd-2(RNAi) embryos, but not in spd-5(RNAi) embryos, where PCM recruitment is efficiently blocked. CONCLUSIONS: Taken together, our results suggest that SPD-2 may link PCM recruitment and centriole duplication in C. elegans. SPD-2 shares homology with a human centrosome protein, suggesting that this key component of the C. elegans centrosome is evolutionarily conserved.     

Phosphoproteome profiling of human skin fibroblast cells in response to low- and high-dose irradiation

A hallmark of the response to high-dose radiation is the up-regulation and phosphorylation of proteins involved in cell cycle checkpoint control, DNA damage signaling, DNA repair, and apoptosis. Exposure of cells to low doses of radiation has well documented biological effects, but the underlying regulatory mechanisms are still poorly understood. The objective of this study is to provide an initial profile of the normal human skin fibroblast (HSF) phosphoproteome and explore potential differences between low- and high-dose irradiation responses at the protein phosphorylation level. Several techniques including Trizol extraction of proteins, methylation of tryptic peptides, enrichment of phosphopeptides with immobilized metal affinity chromatography (IMAC), nanoflow reversed-phase HPLC (nano-LC)/electrospray ionization, and tandem mass spectrometry were combined for analysis of the HSF cell phosphoproteome. Among 494 unique phosphopeptides, 232 were singly phosphorylated, while 262 peptides had multiple phosphorylation sites indicating the overall effectiveness of the IMAC technique to enrich both singly and multiply phosphorylated peptides. We observed approximately 1.9-fold and approximately 3.6-fold increases in the number of identified phosphopeptides in low-dose and high-dose samples respectively, suggesting both radiation levels stimulate cell signaling pathways. A 6-fold increase in the phosphorylation of cyclin dependent kinase (cdk) motifs was observed after low- dose irradiation, while high-dose irradiation stimulated phosphorylation of 3-phosphoinositide-dependent protein kinase-1 (PDK1) and AKT/RSK motifs 8.5- and 5.5-fold, respectively. High- dose radiation resulted in the increased phosphorylation of proteins involved in cell signaling pathways as well as apoptosis while low-dose and control phosphoproteins were broadly distributed among biological processes.