Substance P analogues function as bombesin receptor antagonists and inhibit small cell lung cancer clonal growth

Human small cell lung cancer (SCLC) produces and secretes BN/GRP (bombesin/gastrin releasing peptide). Because BN stimulates the growth of SCLC cells and these cells have receptors for BN-like peptides, it is important to define agents which disrupt this self-promoting autocrine growth cycle. Here, substance P analogues were evaluated as BN receptor antagonists using SCLC cell lines. (D-Arg¹, D-Pro², D-Trp^7,9, Leu¹¹) substance P [(APTTL)SP] was one of the more potent analogues tested in inhibiting BN-like peptide receptor binding with an IC₅₀ value of 1 μM. Micromolar concentrations of (APTTL)SP antagonized BN receptor mediated elevation of cytosolic Ca²⁺ levels and decreased the colony formation in soft agarose. These data suggest that SP analogues function as SCLC BN receptor antagonists and may be useful in disrupting the autocrine growth function of BN-like peptides.     

Gravitropism in Higher Plant Shoots : V. Changing Sensitivity to Auxin

An alternative to the Cholodny-Went, auxin-transport hypothesis of gravitropic stem bending was proposed as early as 1958, suggesting that gravistimulation induces changes in sensitivity to auxin, accounting for differential growth and bending. To test the sensitivity hypothesis, we immersed marked, decapitated sunflower (Helianthus annuus L.) hypocotyl sections in buffered auxin solutions over a wide concentration range (0, 10⁻⁸ to 10⁻² molar IAA), photographed them at half-hour intervals, analyzed the negatives with a digitizer/computer, and evaluated surface-length changes in terms of Michaelis-Menten enzyme kinetics. Bending decreases with increasing auxin concentration; above about 10⁻⁴ molar IAA the hypocotyls bend down; increasing auxin inhibits elongation growth of lower surfaces (which is high at zero or relatively low auxin levels) but promotes upper-surface growth (which is low at low auxin levels). Thus, lower surfaces have a greater K_m sensitivity to applied auxin than upper surfaces. At optimum auxin levels (maximum growth), growth of bottom surfaces exceeds that of top surfaces, so bottom tissues have a greater V_max sensitivity. V_max sensitivity of vertical controls is slightly lower than it is for either horizontal surface; K_m sensitivity is intermediate. Clearly, gravistimulation leads to significant changes in tissue sensitivity to applied auxin. Perhaps these changes are also important in normal gravitropism.     

Ultrastructure of the calcium release channel of sarcoplasmic reticulum

This study is concerned with the characterization of the morphology of the calcium release channel of sarcoplasmic reticulum (SR) from fast-twitch skeletal muscle, which is involved in excitation-contraction coupling. We have previously purified the ryanodine receptor and found it to be equivalent to the feet structures, which are involved, in situ, in the junctional association of transverse tubules with terminal cisternae of SR. The receptor is an oligomer of a single high molecular weight polypeptide and when incorporated into phospholipid bilayers, has channel conductance which is characteristic of calcium release in terminal cisternae of SR. The purified channel can be observed by electron microscopy using different methods of sample preparation, with complementary views being observed by negative staining, double staining, thin section and rotary shadowing electron microscopy. Three views can be observed and interpreted: (a) a square face which, in situ, is junctionally associated with the transverse tubule or junctional face membrane; (b) a rectangle equivalent to the side view; and (c) a diamond shape equivalent to the side view, of which the base portion appears to be equivalent to the transmembrane segment. Negative staining reveals detailed substructure of the channel. A computer averaged view of the receptor displays fourfold symmetry and ultrastructural detail. The dense central mass is divided into four domains with a 2-nm hole in the center, and is enclosed within an outer frame which has a pinwheel appearance. Double staining shows substructure of the square face in the form of parallel linear arrays (six/face). The features of the isolated receptor can be correlated with the structure observed in terminal cisternae vesicles. Sections tangential to the junctional face membrane reveal that the feet structures (23-nm squares) overlap so as to enclose smaller square spaces of approximately 14 nm/side. We suggest that this is equivalent to the transverse tubule face and that the terminal cisternae face is smaller (approximately 17 nm/face) and has larger alternating spaces as a consequence of the tapered sides of the foot structures. Image reconstruction analysis appears to be feasible and should provide the three-dimensional structure of the channel.     

The organization of taste sensibilities in hamster chorda tympani nerve fibers

Electrophysiological measurements of nerve impulse frequencies were used to explore the organization of taste sensibilities in single fibers of the hamster chorda tympani nerve. Moderately intense taste solutions that are either very similar or easily discriminated were applied to the anterior lingual surface. 40 response profiles or 13 stimulus activation patterns were considered variables and examined with multivariate statistical techniques. Three kinds of response profiles were seen in fibers that varied in their overall sensitivity to taste solutions. One profile (S) showed selectivity for sweeteners, a second (N) showed selectivity for sodium salts, and a third (H) showed sensitivity to salts, acids, and other compounds. Hierarchical cluster analysis indicated that profiles fell into discrete classes. Responses to many pairs of effective stimuli were covariant across profiles within a class, but some acidic stimuli had more idiosyncratic effects. Factor analysis of profiles identified two common factors, accounting for 77% of the variance. A unipolar factor was identified with the N profile, and a bipolar factor was identified with the S profile and its opposite, the H profile. Three stimulus activation patterns were elicited by taste solutions that varied in intensity of effect. Hierarchical cluster analysis indicated that the patterns fell into discrete classes. Factor analysis of patterns identified three common unipolar factors accounting for 82% of the variance. Eight stimuli (MgSO4, NH4Cl, KCl, citric acid, acetic acid, urea, quinine HCl, HCl) selectively activated fibers with H profiles, three stimuli (fructose, Na saccharin, sucrose) selectively activated fibers with S profiles, and two stimuli (NaNO3, NaCl) activated fibers with N profiles more strongly than fibers with H profiles. Stimuli that evoke different patterns taste distinct to hamsters. Stimuli that evoke the same pattern taste more similar. It was concluded that the hundreds of peripheral taste neurons that innervate the anterior tongue play one of three functional roles, providing information about one of three features that are shared by different chemical solutions.     

Direct localization of the tRNA--anticodon interaction site on the Escherichia coli 30 S ribosomal subunit by electron microscopy and computerized image averaging

Previous immunoelectron microscopy studies have shown that the anticodon of valyl-tRNA, photocrosslinked to the ribosomal P site at the C1400 residue of the 16 S RNA, is located in the vicinity of the cleft of the small ribosomal subunit of Escherichia coli. In this study we used single-particle image-averaging techniques to demonstrate that the 30 S-bound tRNA molecule can be localized directly, without the need for specific antibody markers. In agreement with the immunoelectron microscopy results, we find that the tRNA molecule appears to be located deep in the cleft of the 30 S subunit. We believe that the use of computer image averaging to localize ligands bound to ribosomes and other macromolecular complexes will become widespread because of the superior sensitivity, precision and objectivity of this technique compared with conventional immunoelectron microscopy.     

Nyctohemeral Rhythms of Gonadal, Thyroid and Pineal Function in the Hyperprolactinemic Male Rat

In young adult male rats bearing a donor anterior pituitary gland grafted for 3 weeks under a kidney capsule, serum prolactin (PRL) concentrations were elevated and exhibited a rhythm with the highest values in the light phase. Serum PRL in control animals did not exhibit a significant rhythm. Eutopic pituitary PRL content, manifesting a biphasic (12-hr) rhythm with crests during the day and night in controls, exhibited a similar pattern in grafted rats though an overall reduction in pituitary PRL content was seen in the grafted animals. Neither the normal biphasic serum testosterone rhythm nor the normal 24-hr rhythm (nocturnal surge) of pineal N-acetyltransferase activity and melatonin content were altered in the hyperprolactinemic rats. Serum thyroxine (T₄) and triiodothyronine (T₃) and their free indices (FT₄ I, FT₃ I) and serum thyrotropin (TSH) were highest during the day in controls and grafted rats and a 12-hr rhythmic component was detected in data for these variables. In the grafted animals, the 12-hr component was reflected in an additional peak at night detectable by testing of means. The overall serum T₄ FT₄ I, and TSH levels were lower in grafted rats though overall T₃ and FT₃I levels did not differ between grafted and controls. T₃ uptake (T₃ U) values were similar between controls and grafted rats, in both cases exhibiting a fall during the night. Changes in serum thyronines could not be explained by changes in serum binding as assessed by the T₃U₃ and thus may represent changes in thyroidal secretion of T₄. The rhythm in serum PRL of grafted rats suggests the presence of rhythmic circulating factor(s) capable of influencing ectopic lactotrophs. The reduced eutopic pituitary PRL content suggests a role for PRL in influencing eutopic lactotrophs in the pituitary-grafted hyperprolactinemic male rat model. Though circulating testosterone and pineal melatonin synthesis were not altered in this model, thyroid function appeared to be so.     

Intramolecular localization of epitopes within an oligomeric protein by immunoelectron microscopy and image processing

Three epitopes have been localized by immunoelectron microscopy on subunit Aa6 of the 4 x 6-meric hemocyanin of the scorpion Androctonus australis. Soluble immunocomplexes composed of monoclonal antibodies and of native hemocyanin were purified, negatively stained with uranyle acetate by the single-layer technique, and examined under the electron microscope (EM). The molecule images were digitized, aligned, and submitted to correspondence analysis according to the method of Van Heel and Frank (Ultramicroscopy 6:187-194, 1981). A high-precision localization of the attachment point of the Fab arm to the antigen was achieved through a careful analysis of the average images. This method easily allowed the discrimination of epitopes located in different domains (Mr 20 kDa) of the same subunit. Nonoverlapping epitopes located in the same structural domain of subunit Aa6 could be distinguished by the stain exclusion patterns of their Fab arms. The method is general and may be used for epitope mapping in any antigen producing definite EM views.     

Synthetic lethal mutations suggest interactions between U5 small nuclear RNA and four proteins required for the second step of splicing.

To investigate the function of the U5 small nuclear ribonucleoprotein (snRNP) in pre-mRNA splicing, we have screened for factors that genetically interact with Saccharomyces cerevisiae U5 snRNA. We isolated trans-acting mutations that exacerbate the phenotypes of conditional alleles of the U5 snRNA and named these genes SLU, for synergistically lethal with U5 snRNA. SLU1 and SLU2 are essential for the first catalytic step of splicing, while SLU7 and SLU4 (an allele of PRP17 [U. Vijayraghavan, M. Company, and J. Abelson, Genes Dev. 3:1206-1216, 1989]) are required only for the second step of splicing. Furthermore, slu4-1 and slu7-1 are lethal in combination with mutations in PRP16 and PRP18, which also function in the second step, but not with mutations in factors required for the first catalytic step, such as PRP8 and PRP4. We infer from these data that SLU4, SLU7, PRP18, PRP16, and the U5 snRNA interact functionally and that a major role of the U5 snRNP is to coordinate a set of factors that are required for the completion of the second catalytic step of splicing.     

Enterochromaffin-like cells in the rat stomach: effect of α-fluoromethylhistidine-evoked histamine depletion

Summary In the rat, gastric histamine is stored predominantly in the enterochromaffin-like (ECL) cells, which are located basally in the oxyntic mucosa. The functional significance of histamine in the ECL cells is a matter of speculation. In this study the effect of depletion of histamine on the properties and ultrastructure of the ECL cells was examined. Histamine synthesis was inhibited with -fluoromethylhistidine (3 mg·kg^-1·h^-1) given via osmotic minipumps over a period of 24 h. The treatment reduced the histidine decarboxylase activity (approximately 20% remaining) and histamine concentration (less than 20% remaining) in the oxyntic mucosa, as well as the intensity of histamine- and chromogranin A-immunostaining in the ECL cells, compared to control rats. The cytoplasmic (secretory) granules/vesicles were greatly reduced in number and size following -fluoromethylhistidine administration. The histamine immunostaining of the mast cells, which occurs at the mucosal surface and in the submucosa, appeared unaffected. We conclude that ECL cell histamine accounts for at least 80% of the total oxyntic mucosal histamine in the rat and that it represents a more mobile pool than mast cell histamine. The reduction in the number and size of the ECL cell granules/vesicles following histamine depletion is in accord with the idea that they represent the storage site for histamine.