Comparative effects of a recombinant and a mutein type of granulocyte colony stimulating factor on the growth of Meth-A fibrosarcoma with 5-fluorouracil chemotherapy

The present study was designed to evaluate the effects of a recombinant human G-CSF (rhG-CSF) and a mutein G-CSF(KW-2228) on leucopenia and tumor growth in mice treated with 5-fluorouracil (5-FU). In normal mice, the number of leucocytes (white blood cell, WBC) reached the peak 12 hours after a single injection of either type of G-CSF and decreased to the normal level after 24 hours. Daily administration induced a continuous increase in the WBC count, however, administrations at intervals did not. Meth-A fibrosarcoma was subcutaneously inoculated into the backs of syngeneic BALB/c mice. The mice were treated with 5-FU alone or with G-CSFs. Chemotherapy with 5-FU alone resulted in leucopenia and an insignificant inhibition of tumor growth. The conjunctive administration of G-CSFs with 5-FU resulted in a significantly augmented inhibition of tumor growth, and leukopenia was not seen. This augmenting effect was more prominent with KW-2228.These results suggest that in 5-FU chemotherapy G-CSFs may be beneficial in restoring the number of leucocytes from leucopenic state and in augmenting the tumor inhibitory effect. Furthermore, KW-2228 may be more beneficial than the natural type rhG-CSF.     

Self diffusion and spectral modifications of a membrane protein, the Rubrivivax gelatinosus LH2 complex, incorporated into a monoolein cubic phase

The light-harvesting complex LH2 from a purple bacterium, Rubrivivax gelatinosus, has been incorporated into the Q230 cubic phase of monoolein. We measured the self-diffusion of LH2 in detergent solution and in the cubic phase by fluorescence recovery after photobleaching. We investigated also the absorption and fluorescence properties of this oligomeric membrane protein in the cubic phase, in comparison with its beta-octyl glucoside solution. In these experiments, native LH2 and LH2 labeled by a fluorescent marker were used. The results indicate that the inclusion of LH2 into the cubic phase induced modifications in the carotenoid and B800 binding sites. Despite these significant perturbations, the protein seems to keep an oligomeric structure. The relevance of these observations for the possible crystallization of this protein in the cubic phase is discussed.     

Cholesterol attenuates the interaction of the antimicrobial peptide gramicidin S with phospholipid bilayer membranes

We have investigated the effect of the presence of 25 mol percent cholesterol on the interactions of the antimicrobial peptide gramicidin S (GS) with phosphatidylcholine and phosphatidylethanolamine model membrane systems using a variety of methods. Our circular dichroism spectroscopic measurements indicate that the incorporation of cholesterol into egg phosphatidylcholine vesicles has no significant effect on the conformation of the GS molecule but that this peptide resides in a range of intermediate polarity as compared to aqueous solution or an organic solvent. Our Fourier transform infrared spectroscopic measurements confirm these findings and demonstrate that in both cholesterol-containing and cholesterol-free dimyristoylphosphatidylcholine liquid-crystalline bilayers, GS is located in a region of intermediate polarity at the polar--nonpolar interfacial region of the lipid bilayer. However, GS appears to be located in a more polar environment nearer the bilayer surface when cholesterol is present. Our (31)P-nuclear magnetic resonance studies demonstrate that the presence of cholesterol markedly reduces the tendency of GS to induce the formation of inverted nonlamellar phases in model membranes composed of an unsaturated phosphatidylethanolamine. Finally, fluorescence dye leakage experiments indicate that cholesterol inhibits the GS-induced permeabilization of phosphatidylcholine vesicles. Thus in all respects the presence of cholesterol attenuates but does not abolish the interactions of GS with, and the characteristic effects of GS on, phospholipid bilayers. These findings may explain why it is more potent at disrupting cholesterol-free bacterial than cholesterol-containing eukaryotic membranes while nevertheless disrupting the integrity of the latter at higher peptide concentrations. This additional example of the lipid specificity of GS may aid in the rational design of GS analogs with increased antibacterial but reduced hemolytic activities.     

Alternative roles for putative ice-binding residues in type I antifreeze protein

Two sets of variants of type I antifreeze protein have been synthesized to investigate the role of Leu and Asn in the activity of this 37-residue alpha-helix. Leu and Asn flank the central two of four regularly spaced ice-binding Thr in the i-1 and i + 3 positions, respectively. All three residues project from the same side of the helix to form the protein's putative ice-adsorption site and are considered in some models to act together as an "ice-binding motif". Replacement of Asn by residues with shorter side chains resulted in either a small loss (Ala) or gain (Thr) of antifreeze activity. However, substitution of Asn by its slightly larger homologue (Gln) abolished thermal hysteresis activity. The Gln-containing peptide was very soluble, largely monomeric, and fully helical. Of the three variants in which Leu was replaced by Ala, two of the three were more active than their Leu-containing counterparts, but all three variants began to precipitate as the peptide concentration increased. None of the seven variants tested showed dramatic differences in ice crystal morphology from that established by the wild type. These results are consistent with a primary role for Leu in preventing peptide aggregation at the antifreeze protein concentrations (10 mg/mL) normally present in fish serum. Similarly the role for Asn may have more to do with enhancing the solubility of these rather hydrophobic peptides than of making a stereospecific hydrogen-bonding match to the ice lattice as traditionally thought. Nevertheless, the dramatic loss of activity in the Asn-to-Gln replacement demonstrates the steric restriction on residues in or near the ice-binding site of the peptide.     

Penile vibratory stimulation in the marmoset monkey: a practical alternative to electro-ejaculation, yielding ejaculates of enhanced quality

The availability of sufficient amounts of spermatozoa of high quality is one of the main limiting factors in reproductive research and development of reproductive technologies in marmoset monkeys (Callithrix jacchus). Penile vibrostimulation (PVS) has been successfully used in semen collection in the squirrel monkey but with poor success rate in the marmoset. We report here on an improved protocol for PVS with a success rate of almost 90%. Ejaculates obtained by PVS were of enhanced quality compared with those obtained by rectal probe electro-ejaculation (RPE). PVS ejaculates contained on average three to fourfold higher numbers of total and motile spermatozoa. Assessment of sperm kinematics using computer-assisted sperm analysis indicated that there are also functional differences between spermatozoa collected by PVS and RPE. Marmoset spermatozoa in samples obtained by RPE swim in a more convoluted manner compared with those obtained by PVS.     

Isolation of Pea Thioredoxin f Precursor Protein and Characterization of its Biochemical Properties

Like many other soluble chloroplastic enzymes, thioredoxin f is nuclear-encoded and expressed as a precursor protein. After synthesis in the cytosol, it is imported into the chloroplast with subsequent cleavage of the transit sequence in the stroma. We report the expression and the partial purification of the recombinant precursor thioredoxin f protein. The prethioredoxin f was found to be located essentially in the insoluble Echerichia coli fraction, but could be renatured after urea treatment followed by dialysis. The renatured protein was active in the dithiothreitol- and thioredoxin-dependent activation of NADP malate dehydrogenase and also of fructose bisphosphatase and in the ferredoxin-thioredoxin-dependent fructose bisphosphatase activation. These data are discussed in relation with the known properties of mature thioredoxin f.     

Horned lizard (Phrynosoma) phylogeny inferred from mitochondrial genes and morphological characters: understanding conflicts using multiple approaches

The genus Phrynosoma includes 13 species of North American lizards characterized by unique and highly derived morphologies and ecologies. Understanding interspecific relationships within this genus is essential for testing hypotheses about character evolution in this group. We analyzed mitochondrial ND4 and cytochrome b gene sequence data from all species of Phrynosoma in conjunction with a previously published dataset including 12S and 16S rRNA gene sequences and morphological characters. We used multiple phylogenetic methods and diagnostic tests for data combinability and taxonomic congruence to investigate the data in separate and combined analyses. Separate data partitions resulted in several well-supported lineages, but taxonomic congruence was lacking between topologies from separate and combined analyses. Partitioned Bremer support analyses also reveals conflict between data partitions in certain tree regions. When taxa associated with well-supported clades were removed from analyses, phylogenetic signal was lost. Combined, our results initially suggest conflict between data partitions, but further tests show the data are only appropriate for phylogenetic reconstruction of those parts of the topology that were well resolved. Nonetheless, our data analyses reveal five well-supported clades: (1) Phrynosoma ditmarsi and Phrynosoma hernandesi, (2) P. ditmarsi, P. hernandesi, and Phrynosoma douglasii, (3) P. ditmarsi, P. hernandesi, P. douglasii, and Phrynosoma orbiculare, (4) Phrynosoma mcallii and Phrynosoma platyrhinos, and (5) Phrynosoma braconnieri and Phrynosoma taurus.     

Alpha 1-adrenergic and cholinergic agonists activate MAPK by separate mechanisms to inhibit secretion in lacrimal gland

Thepurpose of this study was to determine the role of p42/p44mitogen-activated protein kinase (MAPK) in₁-adrenergically and cholinergically stimulated proteinsecretion in rat lacrimal gland acinar cells and the pathways used bythese agonists to activate MAPK. Acini were isolated by collagenasedigestion and incubated with the ₁-adrenergic agonistphenylephrine or the cholinergic agonist carbachol, and activation ofMAPK and protein secretion were then measured. Phenylephrine andcarbachol activated MAPK in a time- and concentration-dependent manner.Inhibition of MAPK significantly increased phenylephrine- andcarbachol-induced protein secretion. Inhibition of EGF receptor (EGFR)with AG1478, an inhibitor of the EGFR tyrosine kinase activity,significantly increased phenylephrine- but not carbachol-inducedprotein secretion. Whereas phenylephrine-induced activation of MAPK wascompletely inhibited by AG1478, activation of MAPK by carbachol wasnot. Phenylephrine stimulated tyrosine phosphorylation of the EGFR, whereas carbachol stimulated p60^Src, and possibly Pyk2, toactivate MAPK. We conclude that, in the lacrimal gland, activation ofMAPK plays an inhibitory role in ₁-adrenergically andcholinergically stimulated protein secretion and that these agonistsuse different signaling mechanisms to activate MAPK.