Reactivity of essential thiols of myosin. Chemical probes of the activated state.

14C-Labeled fluorodinitrobenzene and N-ethylmaleimide have been used as chemical probes of the conformational states of myosin induced by the binding of MgADP and MgATP. The results indicate that in the high-energy conformation, MMgADP-Pi, the essential thiols are protected from modification but their diminished reactivity does not result from depletion of the reagent by reaction at nonessential thiols. The binding of MgADP to myosin exposes the essential thiols as reflected by an increased rate of their modification. The influence of the divalent cations Mg2+ and Ca2+ on the conformation of the M species has also been investigated. By monitoring the incorporation of fluorodinitrobenzene, the conformations of the M state in the presence of these cations can be clearly discerned.     

Semiquantitative paper chromatographic separation of chlorophylls a and b

[¹⁴C]Chlorophyll (chl) a has been utilized to demonstrate the contamination of chl b by (probably) oxidation products of chl a in thin-layer or paper chromatography. By circular chromatography of both chlorophylls as their pheophytins, the contamination of chl a (as pheophytin a) in chl b (as pheophytin b) may be reduced to 0.15–0.35.     

Histochemical Evidence for the Occurrence of Oligomycin-sensitive Plasmalemma ATPase in Corn Roots

A cytochemical study has been made on the localization of ATPase activity in corn (Zea mays L.) roots. Light microscopy shows washing for 4 hours to increase the general ATPase activity in the peripheral layers of the root cortex; oligomycin and N,N-dicyclohexylcarbodiimide inhibit this activity, oligomycin being more effective. Ultrastructural studies of ATPase location show oligomycin treatment to inhibit both mitochondrial and plasmalemma ATPase, but only in the epidermis and outer cortex. Studies with lipid-soluble dyes indicate that oligomycin might not penetrate very deeply into root tissue in the time span of these experiments. It is suggested that the strong inhibition of ion absorption by oligomycin without a corresponding decline in ATP content is probably due to inhibition of ion absorption in the peripheral cell layers, thus limiting the supply of ion for symplastic transport to the uninhibited tissues.     

Prolactin can stimulate general protein synthesis in human breast cancer cells (MCF-7) in long-term culture

Prolactin significantly stimulated protein synthesis rates in a human breast cancer cell line, MCF-7. Total protein per culture also increased to 148% of controls. Proteins precipitated from cell homogenates at pH 4.65 in the presence of calcium ions were resolved by sodium dodecyl sulfate- polyacrylamide gel electrophoresis. The specific radioactivity for almost every polypeptide band increased as a result of protlactin treatment. Cell-type specificity for this prolactin action was indicated by the failure of two other human cell lines (HBL-100 and HEL) to respond similarly. This report is the first demonstration that prolactin has the capability to stimulate general protein synthesis in human breast cancer cells in long-term culture.     

Environmental and Seasonal Factors Affecting the Frost-induced Stage of Cold Acclimation in Cornus stolonifera Michx

Stem tissues of red-osier dogwood (Cornus stolonifera Michx.) acclimated from −3 C to −40 or −50 C in 8 to 10 weeks under a short photoperiod (9 hours) and controlled temperature conditions. During the summer months plants did not acclimate as well as at other times. The sequence of day/night temperature regimes which induced maximum acclimation was 20/15 C for 5 to 6 weeks; 15/5 C for 2 to 3 weeks; 15/5 C plus 1 hour of frost per day for 1 week. The duration of exposure to each temperature regime influenced the rate and intensity of frost-induced acclimation. Less than 5 weeks of warm temperature preconditioning at 20/15 C reduced subsequent frost-induced acclimation. The inductive influence of frost on cold acclimation was additive over 5 days of repeated exposure, but its effects after the first exposure(s) were not immediate—requiring 1 to 4 days of 15/5 C following the frost treatments for the expression of the frost-induced acclimation to be manifest. There was a 75% increase in rRNA following 3 days of frost exposure and plants in an O₂-free atmosphere during frost exposure failed to acclimate. The results suggest that seasonal acclimation behavior was due to endogenous rhythms rather than developmental stage, and that the frost-induced phase of acclimation involves aerobic metabolic processes.     

The effect of modifications of the A5 and A19 amino acid residues on the biological activity of insulin. [Leu5-A] and [Phe19-A] sheep insulins

Two analogs of sheep insulin, both differing from the native material by a single amino acid in the A chain, have been synthesized and isolated in highly purified form by procedures developed in this laboratory. In one case, the glutamine residue in position A⁵ was replaced by leucine ([Leu⁵-A]); in the other, the tyrosine residue in position A¹⁹ was replaced by phenylalanine ([Phe¹⁹-A]). The biological behavior of these analogs was compared with natural bovine insulin inin vitro tests and in receptor-binding assays, as well as in radioimmunoassay. In the stimulation of glucose oxidation by rat adipocytes, the analogs gave relative potencies of 30% and 7.8% for [Leu⁵-A] and [Phe¹⁹-A], respectively. Receptor-binding assays in rat liver plasma membranes showed similar behavior for both analogs. In radioimmunoassay, [Leu⁵-A] displayed a relative potency of 27.9%, while [Phe¹⁹-A] showed a relative potency of 19–27%, compared with bovine insulin. At high concentration, both analogs displayed the same maximal activity as bovine insulin, and the dose-response curves are essentially parallel. It is speculated that the interaction between the glutamine residue in position 5 and the tyrosine residue in position 19 of the A chain of insulin are important in maintaining a three-dimensional structure commensurate with high biological activity. The full intrinsic activity of both analogs at high concentrations and the similarity of the potency figures in receptor-binding and glucose-oxidation assays permit the further conclusion that the reduced potency in the latter assay can be ascribed wholly to the reduced binding affinity toward insulin receptors caused by the substitutions made in the analogs. The receptor-analog complexes are fully capable of triggering the next event in the chain leading to the biological response.     

Structural implications of spectroscopic characterization of a putative zinc finger peptide from HIV-1 integrase.

The N-terminal domain of human immunodeficiency virus (HIV-1) integrase (IN) contains the sequence motif His-Xaa3-His-Xaa23-Cys-Xaa2-Cys, which is strongly conserved in all retroviral and retrotransposon IN proteins. This structural motif constitutes a putative zinc finger in which a metal ion may be coordinately bound by the His and Cys residues. A recombinant peptide, IN(1-55), composed of the N-terminal 55 amino acids of HIV-1 IN was expressed in Escherichia coli and purified. Utilizing a combination of techniques including UV-visible absorption, circular dichroism, Fourier transform infrared, and fluorescence spectroscopies, we have demonstrated that metal ions (Zn2+, Co2+, and Cd2+) are bound with equimolar stoichiometry by IN(1-55). The liganded peptide assumes a highly ordered structure with increased alpha-helical content and exhibits remarkable thermal stability. UV-visible difference spectra of the peptide-Co2+ complexes directly implicate thiols in metal coordination, and Co2+ d-d transitions in the visible range indicate that Co2+ is tetrahedrally coordinated. Mutant peptides containing conservative substitutions of one of the conserved His or either of the Cys residues displayed no significant Zn(2+)-induced conformational changes as monitored by CD and fluorescence spectra. We conclude that the N terminus of HIV-1 IN contains a metal-binding domain whose structure is stabilized by tetrahedral coordination of metal by histidines 12 and 16 and cysteines 40 and 43. A preliminary structural model for this zinc finger is presented.     

Human HIV vaccine trials: does antibody-dependent enhancement pose a genuine risk?

Antibody-dependent enhancement is a general in vitro property of enveloped viruses. In certain circumstances, antibody-dependent enhancement is a bona fide pathophysiologic mechanism in vivo. There are several examples of virus disease of humans and animals in which incomplete or partial immunity can lead to enhanced infection and/or disease. In some cases, this appears to be attributable to antibody-dependent enhancement. Conversely, there are several examples of viruses for which in vitro antibody-dependent enhancement has been demonstrated, but for which vaccines have been used safely in millions of persons for decades. Finally, antibody-dependent enhancement of HIV is a genuine concern. However, to date there is no direct clinical, experimental, or epidemiological evidence that HIV enhancement can be operative in vivo. Such evidence should be actively sought.     

Analysis of styryl-based inhibitors of the lymphocyte tyrosine protein kinase p56lck.

Several styryl-based compounds were evaluated for their capacity to act as inhibitors of the non-receptor tyrosine protein kinase p56lck. Our results demonstrate that alpha-cyanocinnamamide compounds can inhibit both the in vitro tyrosine autophosphorylation of p56lck as well as p56lck phosphorylation of exogenous substrates. Compound 67B-83-A was found to inhibit p56lck protein kinase activity with a calculated IC50 of 7 to 10 microM. This compound did not significantly inhibit the tyrosine protein kinase activity of the epidermal growth factor receptor and was found to be a less effective tyrosine protein kinase inhibitor for other members of the src family of protein kinases.