Aqueous two-phase polymer partitioning of lipid vesicles of defined size and composition

The kinetics of the partitioning of lipid vesicles containing acidic phospholipids in aqueous two-phase polymer systems are dependent upon the vesicle size; the larger the vesicles, the more readily they absorb to the interfaces between the two polymer phases and hence are cleared from the top phase as phase separation proceeds. The partitioning of neutral lipid vesicles is principally to the bulk interface and is the same in phase systems of both low and high electrostatic potential difference between the two phases (delta psi). The incorporation of negatively charged lipids has two effects upon partition. First, vesicles with negatively charged lipids exhibit increased bottom phase partitioning in phases of low delta psi due to an enhanced wetting of the charged lipids by the lower phase. Second, the presence of a negatively charged group on the vesicle surface results in increased partition to the interface and top phase in phase systems of high delta psi. Differences observed in the partition of vesicles containing various species of negatively charged lipid thus reflect a competition between these two opposing factors.     

Hormone epidermal growth factor interactions in development

Epidermal growth factor (EGF) is the most important member of a family of growth factors which exert their effects via a single 170,000 Mr plasma membrane receptor. Other members include transforming growth factor-alpha (TGF-alpha), amphiregulin and several viral growth factors. The receptor is widely distributed in fetal and postnatal tissues. The predominant family member in the fetus appears to be TGF-alpha. EGF production in tissues matures in the perinatal period. Activation of the receptor in the fetal and neonatal periods in rodents evokes important growth and development actions. Tissue EGF and EGF receptor concentrations are modulated by thyroid hormones, estrogen, testosterone and growth hormone, suggesting that selected growth and developmental actions of thyroid and steroid hormones may be mediated by EGF.     

Human aminoacylase-1: cloning, regional assignment to distal chromosome 3p21.1, and identification of a cross-hybridizing sequence on chromosome 18.

Aminoacylase-1 (ACY1, EC 3.5.1.14) is a cytosolic enzyme with a wide range of tissue expression and has been postulated to function in the catabolism and salvage of acylated amino acids. ACY1 has been assigned to chromosome 3p21, a region reduced to homozygosity in small-cell lung cancer and renal cell carcinoma, and has been reported to exhibit reduced or absent expression in small-cell lung cancer cell lines and tumors. Using monoclonal antibodies to human ACY1, we have isolated cDNA clones from a liver lambda gt11 cDNA library. As proof of identity, the fusion protein encoded by a putative ACY1 cDNA displayed ACY1 enzymatic activity. Additionally, it was determined that the putative ACY1 cDNA clones hybridize to an EcoR1 restriction fragment that has been mapped to chromosome 3p. Both ACY1 activity and this restriction fragment have been further demonstrated to be syntenic to distal 3p21.1 through the use of a panel of human-rodent somatic cell hybrids containing fragments of chromosome 3. An additional EcoR1 restriction fragment to which the probe hybridizes has been assigned to chromosome 18. The major mRNA species to which the ACY1 cDNA hybridizes is 0.9 kb; faint hybridization to a 4.2-kb mRNA species is also detected. These studies further refine a region of interest in the investigation of gene inactivation in small-cell lung cancer and provide a new marker on chromosome 18.     

Melanin accelerates the tyrosinase-catalyzed oxygenation of p-hydroxyanisole (MMEH)

Although pigment melanin has long been though of as "inert," recent work has attested to its chemical reactivity. In this communication, we report that either commercial synthetic melanin prepared by persulfate oxidation of tyrosine ("Sigma melanin") or sepia melanin extracted from cuttlefish markedly accelerates the in vitro oxygenation of p-hydroxyanisole (MMEH), catalyzed by mushroom or B-16 melanoma tyrosinase. Kinetics of 4-methoxy-1,2-benzoquinone formation (lambda max = 413 nm) or of molecular O2 uptake were biphasic, with an initial slow rate ("lag time") followed by a fast linear increase. The biphasic response reflects an initial slow hydroxylation followed by a fast dehydrogenation. Added melanin markedly decreased the lag time but had little effect on subsequent dehydrogenation. Similar effects were observed for tyrosine itself. A complex between MMEH and melanin appears to be the "active" species in these reactions. The results indicate that melanin acts as an electron conduit, which accepts electrons from the substrate and transfers them to tyrosinase. The magnitude of the effect depends on the type of melanin as well as on its oxidation state. Kinetic analysis indicates that both melanins are very efficient at transferring electron to tyrosinase, and that Sigma melanin is roughly threefold more efficient than sepia melanin. The qualitative similarity of reaction between the synthetic and "natural" melanins suggests that the former may serve as a first approximation to the in vivo situation. On the other hand, the observed quantitative differences and the sensitivity of these results to the chemical state of melanin suggests that this methodology might eventually be adapted as a non-destructive probe of melanin in situ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular characterization of biologically diverse envelope variants of human immunodeficiency virus type 1 derived from an individual.

The envelope genes of six viruses derived from a single sampling from an individual chronically infected with human immunodeficiency virus type 1 (RJS-4) have been analyzed. Here we present the nucleotide and predicted amino acid sequences of these variants and show a correlation between biological properties and disturbance of the envelope reading frame.     

The influence of maternal age on radiation-induced chromosome aberrations in mouse oocytes

The relative sensitivities of dictyate oocytes from young and old female mice to radiation-induced chromosome damage were examined in 2 separate experiments. Firstly, females were given either 2 or 4 Gy of X-rays and metaphase I stage oocytes collected 16.5 days later. Analysis of these cells showed dose-related increases in chromosome aberrations in both age groups. The response was significantly greater in oocytes of older females. In the second experiment, females were given 4 Gy of X-rays and metaphase I stage oocytes collected 3.5 days later. Again, a significantly larger frequency of aberrations was present in cells from older animals. Overall, these 2 experiments provide unambiguous evidence that the radiosensitivity of mouse dictyate oocytes increases with advancing maternal age.     

Human cyclin E, a new cyclin that interacts with two members of the CDC2 gene family.

A new human cyclin, named cyclin E, was isolated by complementation of a triple cln deletion in S. cerevisiae. Cyclin E showed genetic interactions with the CDC28 gene, suggesting that it functioned at START by interacting with the CDC28 protein. Two human genes were identified that could interact with cyclin E to perform START in yeast containing a cdc28 mutation. One was CDC2-HS, and the second was the human homolog of Xenopus CDK2. Cyclin E produced in E. coli bound and activated the CDC2 protein in extracts from human G1 cells, and antibodies against cyclin E immunoprecipitated a histone H1 kinase from HeLa cells. The interactions between cyclin E and CDC2, or CDK2, may be important at the G1 to S transition in human cells.     

Polyelectrolyte precipitation of proteins: II. Models of the particle size distributions

A population-balance model has been used to characterize continuous polyelectrolyte precipitation of egg white proteins. We have modeled the particle size distributions of aggregates formed under a range of mixing conditions. The models, accounting for aggregate growth (by both shear-driven and Brownian-like collisions), breakage (by hydrodynamic shear or aggregate-aggregate collisions), and birth (by the breakage of large aggregates), fit the data well. The kinetic constants show dependencies on shear rate and residence time that have not been previously theoretically predicted; these dependencies are due in part to aging effects on the aggregate. The model constants show a dominance of growth over breakage, supporting qualitative interpretations of the particle size distributions. A mechanism for growth-rate enhancement, caused by polymer extensions from the particle surfaces, produced improved model performance. A collisional breakage mechanism is supported.     

Polyelectrolyte precipitation of proteins: I. The effect of reactor conditions

Lysozyme was recovered from egg white by continuous precipitation with polyacrylic acid (molecular weight of 4 x 10(6)). Precipitator residence time and shear rate had significant effects on the size distribution of the precipitate, but no clear effects on the compositions. Precipitate mean size increased with higher shear, indicating growth phenomena predominating over breakage. Also, an enhancement of growth rate at small sizes was noted. The Camp number successfully characterized the interaction of shear rate and residence time on the particle size.