The nature of phonons and solitary waves in alpha-helical proteins.

A parametric study of the Davydov model of energy transduction in alpha-helical proteins is described. Previous investigations have shown that the Davydov model predicts that nonlinear interactions between phonons and amide-I excitations can stabilize the latter and produce a long-lived combined excitation (the so-called Davydov soliton), which propagates along the helix. The dynamics of this solitary wave are approximately those of solitons described using the nonlinear Schr?dinger equation. The present study extends these previous investigations by analyzing the effect of helix length and nonlinear coupling efficiency on the phonon spectrum in short and medium length alpha-helical segments. The phonon energy accompanying amide-I excitation shows periodic variation in time with fluctuations that follow three different time scales. The phonon spectrum is highly dependent upon chain length but a majority of the energy remains localized in normal mode vibrations even in the long chain alpha-helices. Variation of the phonon-exciton coupling coefficient changes the amplitudes but not the frequencies of the phonon spectrum. The computed spectra contain frequencies ranging from 200 GHz to 6 THz, and as the chain length is increased, the long period oscillations increase in amplitude. The most important prediction of this study, however, is that the dynamics predicted by the numerical calculations have more in common with dynamics described by using the Frohlich polaron model than by using the Davydov soliton. Accordingly, the relevance of the Davydov soliton model was applied to energy transduction in alpha-helical proteins is questionable.(ABSTRACT TRUNCATED AT 250 WORDS)     

DECREASE IN NUCLEAR FEULGEN-POSITIVE MATERIAL (DNA) UPON AGING IN IN VITRO STORAGE OF BOVINE SPERMATOZOA

The Feulgen-DNA content of sperm cells from 5 bulls was studied by means of microspectrophotometry after storage at 5°C for 2, 3, 5, and 10 days in a yolk-citrate diluent permitting slow aerobic metabolism. A subsample of sperm cells from each bull was subjected to the Feulgen technique on each of the storage days selected. The cells sampled on each of these days received a standard 12 minute, 60°C hydrolysis. Absorption measurements at 546 mµof the individual cells indicated a marked progressive decrease in the Feulgen-DNA content of the stored spermatozoa. The loss of 30 per cent of the initial DNA at the end of 5 days' storage was highly significant statistically. This decrease approximately parallels the known decrease in fertility of stored sperm cells, as well as the increase in apparent embryonic mortality resulting from the use of similarly aged spermatozoa for artificial insemination.     

Expression of the v-crk oncogene product in PC12 cells results in rapid differentiation by both nerve growth factor- and epidermal growth factor-dependent pathways.

The transforming gene of the avian sarcoma virus CT10 encodes a fusion protein (p47gag-crk or v-Crk) containing viral Gag sequences fused to cellular sequences consisting primarily of Src homology regions 2 and 3 (SH2 and SH3 sequences). Here we report a novel function of v-Crk in the mammalian pheochromocytoma cell line, PC12, whereby stable expression of v-Crk induces accelerated differentiation, as assessed by induction of neurites following nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) treatment compared with the effect in native PC12 cells. Surprisingly, however, these cells also develop extensive neurite processes after epidermal growth factor (EGF) stimulation, an event which is not observed in native PC12 cells. Following EGF or NGF stimulation of the v-CrkPC12 cells, the v-Crk protein itself became tyrosine phosphorylated within 1 min. Moreover, in A431 cells or TrkA-PC12 cells, which overexpress EGF receptors and TrkA, respectively, a GST-CrkSH2 fusion protein was indeed capable of binding these receptors in a phosphotyrosine-dependent manner, suggesting that v-Crk can directly couple to receptor tyrosine kinase pathways in PC12 cells. In transformed fibroblasts, v-Crk binds to specific tyrosine-phosphorylated proteins of p130 and paxillin. Both of these proteins are also complexed to v-Crk in PC12 cells, as evidenced by their coprecipitation with v-Crk in detergent lysates, suggesting that common effector pathways may occur in both cell types. However, whereas PC12 cellular differentiation can occur solely by overexpression of the v-Src or oncogenic Ras proteins, that induced by v-Crk requires a growth factor stimulatory signal, possibility in a two-step process.     

A 31-amino-acid N-terminal extension regulates c-Crk binding to tyrosine-phosphorylated proteins.

Overproduction of v-Crk, but not of c-Crk, in chicken embryo fibroblasts results in cell transformation. The transforming activity of v-Crk mutants correlates with their ability to cause increased tyrosine phosphorylation of specific cellular proteins, a property that depends on the binding of v-Crk to phosphotyrosine residues via its SH2 domain. In this study, proteins translated in rabbit reticulocyte lysates were used to analyze interactions between Crk derivatives and tyrosine-phosphorylated proteins, particularly the epidermal growth factor (EGF) receptor. The results demonstrate that the binding affinity of c-Crk is much lower than that of v-Crk, despite the fact that both proteins contain identical SH2 domains. Moreover, a 31-amino-acid N-terminal extension of c-Crk, resulting from upstream translational initiation at a CUG codon, significantly increases the ability of the resulting protein to bind to phosphotyrosine-containing proteins. Of those 31 amino acids, 24 can be found in the 27-amino-acid region between Gag and Crk sequences in v-Crk, and removal of this region results in a protein with lower affinity toward the EGF receptor. In addition, fusion of Gag to the amino terminus of c-Crk yields a protein with a binding activity that is lower than that of v-Crk but significantly higher than that of c-Crk without the fusion. These data suggest that sequences N terminal to the Crk SH2 regulate binding activity to tyrosine-phosphorylated proteins and that the amino acids encoded immediately 5' to the c-Crk initiator AUG specifically increase binding affinity. In contrast, deletion of one or two SH3 domains of c-Crk proteins did not change their affinity for the EGF receptor. These results were confirmed in vivo by using A431-derived cell lines overproducing either the chicken c-Crk protein or c-Crk with the 31-amino-acid N-terminal extension. Furthermore, the in vivo experiments suggest that binding of Crk proteins to the stimulated EGF receptor results in Crk phosphorylation and subsequent loss of binding affinity.     

The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.     

Specificity of interferon action in protein synthesis.

Inhibitors of elongation steps in protein synthesis such as cycloheximide and anisomycin mimic interferon treatment in that they specifically inhibit the synthesis of certain viral proteins. These specific effects are seen only at very low concentrations of the antibiotics, under conditions where host cellular protein synthesis, as well as cell viability, are not severely reduced. A qualitatively as well as quantitatively close correlation between the effects of the two types of agents has been established for encephalomyocarditis virus, vesicular stomatitis virus and murine leukemia virus protein synthesis. It is concluded that one of the primary mechanisms of interferon action may be a nonspecific retardation of one or more elongation steps, and that this may be sufficient to account for its effects on the replication of certain viruses such as encephalomyocarditis and vesicular stomatitis viruses.     

A thermosensitivepdxJ mutation affecting vitamin B6 biosynthesis inEscherichia coli K-12

A temperature-sensitive mutation affecting pyridoxine biosynthesis inEscherichia coli has been isolated following nitrosoguanidine mutagenesis. This is the first report of a conditional mutation affecting that pathway. Three-point transductional analysis and an Hfr mating test indicate that the affected locus ispdxJ and that the gene order isglyA-purI-pdxJ-nadB. The data confirm the present genetic map position ofpdxJ, which had not been accurately determined. Cells carrying the mutation exhibit an absolute requirement for 2.5 μM vitamin B₆ when inenbated at temperatures of 38°C or higher.     

Ejaculate investment and attractiveness in the stalk‐eyed fly,Diasemopsis meigenii

The phenotype‐linked fertility hypothesis proposes that male fertility is advertised via phenotypic signals, explaining female preference for highly sexually ornamented males. An alternative view is that highly attractive males constrain their ejaculate allocation per mating so as to participate in a greater number of matings. Males are also expected to bias their ejaculate allocation to the most fecund females. We test these hypotheses in the African stalk‐eyed fly, Diasemopsis meigenii. We ask how male ejaculate allocation strategy is influenced by male eyespan and female size. Despite large eyespan males having larger internal reproductive organs, we found no association between male eyespan and spermatophore size or sperm number, lending no support to the phenotype‐linked fertility hypothesis. However, males mated for longer and transferred more sperm to large females. As female size was positively correlated with fecundity, this suggests that males gain a selective advantage by investing more in large females. Given these findings, we consider how female mate preference for large male eyespan can be adaptive despite the lack of obvious direct benefits.