A CD determination of the alpha-helix contents of the coat proteins of four filamentous bacteriophages: fd, IKe, Pf1, and Pf3

The CD spectra of four filamentous bacteriophages--fd, IKe, Pf1, and Pf3--were analyzed to determine the alpha-helix contents of their major coat proteins. Measured spectra included the 192-nm band so that analyses could be carried out over the full wavelength range of the reference spectra for protein secondary structures available (a) from globular proteins [J.T. Yang, C.S.C. Wu, and H.M. Martinez (1986) Methods in Enzymology 130, 208-269] and (b) from poly(L-lysine) [N. Greenfield and G.D. Fasman (1960) Biochemistry 8, 4108-4116]. Extended analyses were also performed with the addition of the spectrum of a model beta-turn to the Greenfield and Fasman reference set, with the spectrum of a short alpha-helix in the Yang et al. reference set, and with an estimate of the spectrum of Trp added to both reference sets. The reference set based on the simple poly(L-lysine) polypeptide, plus a spectrum of a model beta-turn or of Trp, gave reasonably good fits to the measured spectra for all four phages and yielded the largest percentages of alpha-helix. The class I phages--fd and IKe--had large percentages of alpha-helix of 98 +/- 2 and 97 +/- 5%, respectively, while the two class II phages--Pf1 and Pf3--had similar but smaller alpha-helix contents of 83 +/- 6 and 84 +/- 2, respectively. While these alpha-helix contents were within the ranges previously reported from CD spectra of these phages in solution, they were more precise, and they indicated that the coat proteins of the intact phages have CD spectra that are probably modeled better by the reference spectra of polypeptides than by those of globular proteins.     

Basolateral plasma membrane localization of ouabain-sensitive sodium transport sites in the secretory epithelium of the avian salt gland

The distribution of Na+ pump sites (Na+-K+-ATPase) in the secretory epithelium of the avian salt gland was demonstrated by freeze-dry autoradiographic analysis of [(3)H] ouabain binding sites. Kinetic studies indicated that near saturation of tissue binding sites occurred when slices of salt glands from salt-stressed ducks were exposed to 2.2 μM ouabain (containing 5 μCi/ml [(3)H]ouabain) for 90 min. Washing with label-free Ringer's solution for 90 min extracted only 10% of the inhibitor, an amount which corresponded to ouabain present in the tissue spaces labeled by [(14)C]insulin. Increasing the KCl concentration of the incubation medium reduced the rate of ouabain binding but not the maximal amount bound. In contrast to the low level of ouabain binding to salt glands of ducks maintained on a freshwater regimen, exposure to a salt water diet led to a more than threefold increase in binding within 9-11 days. This increase paralleled the similar increment in Na+-K+-ATPase activity described previously. [(3)H]ouabain binding sites were localized autoradiographically to the folded basolateral plasma membrane of the principal secretory cells. The luminal surfaces of these cells were unlabeled. Mitotically active peripheral cells were also unlabeled. The cell-specific pattern of [(3)H]ouabain binding to principal secretory cells and the membrane-specific localization of binding sites to the nonluminal surfaces of these cells were identical to the distribution of Na+-K+-ATPase as reflected by the cytochemical localization of ouabain-sensitive and K+-dependent nitrophenyl phosphatase activity. The relationship between the nonluminal localization of Na+-K+-ATPase and the possible role of the enzyme n NaCl secretion is considered in the light of physiological data on electrolyte transport in salt glands and other secretory epithelia.     

Calcium release from rod outer segments: evidence for a cGMP-sensitive calcium binding protein.

Numerous studies investigating the cGMP-gated cation conductance in rod disk membranes have purported to measure efflux of Ca2+ entrapped in rod disk membrane vesicles. We have utilized sonication and osmotic shock as additional tests for sensitivity of cGMP- and A23187-induced Ca2+ release to elimination of the transvesicular Ca2+ gradient. We find that 1) Treatment with sonication or osmotic shock in low Ca2+ medium does not release Ca2+ from either native cGMP/Ca2(+)-loaded vesicles or solubilized, reconstituted "Ca2(+)-loaded" vesicles, 2) 70-100% of the cGMP-induced "flux" and 90-100% of the A23187-induced Ca2+ "flux" is insensitive to elimination of the Ca2+ gradient by sonication or osmotic shock in low Ca2+ medium, and 3) total amount of releasable Ca2+ is related to membrane surface area rather than vesicle entrapment volume. We conclude that 1) A23187 disrupts binding of Ca2+ to proteins and phospholipids as well as releasing entrapped Ca2+ and 2) a large fraction of the cGMP-induced release observed in rod disk vesicles is due to release of bound Ca2+.     

Intercellular communication in the rat anterior pituitary gland: an in vivo and in vitro study

The concept of "stimulus-secretion coupling" suggested by Douglas and co-workers to explain the events related to monamine discharge by the adrenal medulla (5, 7) may be applied to other endocrine tissues, such as adrenal cortex (36), pancreatic islets (4), and magnocellular hypothalamic neurons (6), which exhibit a similar ion-dependent process of hormone elaboration. In addition, they share another feature, that of joining neighbor cells via membrane junctions (12, 26, and Fletcher, unpublished observation). Given this, and the reports that hormone secretion by the pars distalis also involves a secretagogue-induced decrease in membrane bioelectric potential accompanied by a rise in cellular [Ca++] (27, 34, 41), it was appropriate to test the possibility that cells of the anterior pituitary gland are united by junctions.     

High-throughput screen identifies disulfiram as a potential therapeutic for triple-negative breast cancer cells: Interaction with IQ motif-containing factors

Triple-negative breast cancer (TNBC) represents an aggressive subtype, for which radiation and chemotherapy are the only options. Here we describe the identification of disulfiram, an FDA-approved drug used to treat alcoholism, as well as the related compound thiram, as the most potent growth inhibitors following high-throughput screens of 3185 compounds against multiple TNBC cell lines. The average IC₅₀ for disulfiram was ~300 nM. Drug affinity responsive target stability (DARTS) analysis identified IQ motif-containing factors IQGAP1 and MYH9 as direct binding targets of disulfiram. Indeed, knockdown of these factors reduced, though did not completely abolish, cell growth. Combination treatment with 4 different drugs commonly used to treat TNBC revealed that disulfiram synergizes most effectively with doxorubicin to inhibit cell growth of TNBC cells. Disulfiram and doxorubicin cooperated to induce cell death as well as cellular senescence, and targeted the ESA⁺/CD24^-/low/CD44⁺ cancer stem cell population. Our results suggest that disulfiram may be repurposed to treat TNBC in combination with doxorubicin.     

Repeated oral administration of capsaicin increases anxiety-like behaviours with prolonged stress-response in rats

This study was conducted to examine the psycho-emotional effects of repeated oral exposure to capsaicin, the principal active component of chili peppers. Each rat received 1 mL of 0.02% capsaicin into its oral cavity daily, and was subjected to behavioural tests following 10 daily administrations of capsaicin. Stereotypy counts and rostral grooming were significantly increased, and caudal grooming decreased, in capsaicin-treated rats during the ambulatory activity test. In elevated plus maze test, not only the time spent in open arms but also the percent arm entry into open arms was reduced in capsaicin-treated rats compared with control rats. In forced swim test, although swimming duration was decreased, struggling increased in the capsaicin group, immobility duration did not differ between the groups. Repeated oral capsaicin did not affect the basal levels of plasma corticosterone; however, the stress-induced elevation of plasma corticosterone was prolonged in capsaicin treated rats. Oral capsaicin exposure significantly increased c-Fos expression not only in the nucleus tractus of solitarius but also in the paraventricular nucleus. Results suggest that repeated oral exposure to capsaicin increases anxiety-like behaviours in rats, and dysfunction of the hypothalamic-pituitary-adrenal axis may play a role in its pathophysiology.     

Differential Sensitivities of Fast- and Slow-Cycling Cancer Cells to Inosine Monophosphate Dehydrogenase 2 Inhibition by Mycophenolic Acid

As uncontrolled cell proliferation requires nucleotide biosynthesis, inhibiting enzymes that mediate nucleotide biosynthesis constitutes a rational approach to the management of oncological diseases. In practice, however, results of this strategy are mixed and thus elucidation of the mechanisms by which cancer cells evade the effect of nucleotide biosynthesis restriction is urgently needed. Here we explored the notion that intrinsic differences in cancer cell cycle velocity are important in the resistance toward inhibition of inosine monophosphate dehydrogenase (IMPDH) by mycophenolic acid (MPA). In short-term experiments, MPA treatment of fast-growing cancer cells effectively elicited G0/G1 arrest and provoked apoptosis, thus inhibiting cell proliferation and colony formation. Forced expression of a mutated IMPDH2, lacking a binding site for MPA but retaining enzymatic activity, resulted in complete resistance of cancer cells to MPA. In nude mice subcutaneously engrafted with HeLa cells, MPA moderately delayed tumor formation by inhibiting cell proliferation and inducing apoptosis. Importantly, we developed a lentiviral vector–based Tet-on label-retaining system that enables to identify, isolate and functionally characterize slow-cycling or so-called label-retaining cells (LRCs) in vitro and in vivo. We surprisingly found the presence of LRCs in fast-growing tumors. LRCs were superior in colony formation, tumor initiation and resistance to MPA as compared with fast-cycling cells. Thus, the slow-cycling compartment of cancer seems predominantly responsible for resistance to MPA.