Cell population changes in the intestinal mucosa of protein-depleted or starved rats. I. Changes in mitotic cycle time.

The effect of a protein-free diet and starvation on the duration of the rat ileal crypt cell cycle time was studied by Quastler's technique of labeled mitoses. Rats were fed a protein-free diet for 3, 7, or 11 wk or were starved for 7 or 10 days. Progressive protein depletion resulted in a progressive lengthening of the cycle time (GT), due primarily to a lengthening of the synthetic phase (S) of the cycle. The presynthetic gap (G(1)) was the same as the control value after 3 wk and lower, but not significantly so, due to the large variability, after 11 wk. The duration of the postsynthetic gap (G(2)) plus mitotic phase (M) was not affected by the diet. As the dietary stress became more severe, the cell cycle also became more variable. Although the GT of rats starved for as long as 10 days was only slightly different from the control, the relative duration of the components of the cycle changed significantly. S and G(2) were longer in the starved animals while G(1) was of shorter duration.

Cell population changes in the intestinal mucosa of protein-depleted or starved rats. II. Changes in cellular migration rates.

The effect of protein-free and starvation diets on the migration of cells from the crypts onto and up the villi of the rat ileum was studied. Rats starved for 3, 7, or 10 days or fed a protein-free diet (PFD) for 3, 7, or 11 wk were injected with thymidine-(3)H and sacrificed at timed intervals. The time required for the labeled cells to first appear on the villi of experimental animals was longer than in the controls. This was the result of an elongated cycle in the protein-depleted animals and a lengthening of the maturation period in both the starved and protein-depleted animals. Determination of the distance which labeled cells had migrated up the villi in control and experimental animals, after thymidine-(3)H injection, indicated that cells in animals starved for 7 days migrated more rapidly than those in the fed controls, while those of 10-day starved animals moved more slowly. The cells of animals fed PFD for 3 wk migrated up the villi more rapidly, those of animals depleted for 7 wk migrated at the same time rate, and those of 11-wk PFD animals migrated more slowly than the fed controls. There is apparently no correlation between the cell cycle time in the crypt cells and the rate of migration of cells up the villus.

Quantitation of the dynein pool in unfertilized sea urchin eggs.

A dynein-like ATPase activity has been isolated previously from soluble extracts of unfertilized sea urchin eggs. However, the use of non-quantitative isolation techniques, in particular affinity for microtubules or Ca2+/calmodulin, has precluded accurate estimates of dynein pool size. We have taken the unique approach of using dynein-like ATPase activity to quantitate the egg dynein pool. This approach is based on the isolation by anion-exchange chromatography on DEAE-Sephacel of a peak of dynein-like ATPase activity comprising 65% of soluble ATPase activity in the cytosolic extract. Identification of cytoplasmic dynein was based on dose-dependent inhibition by erythro-9-[3-(2-hydroxynonyl)]adenine and orthovanadate, low GTPase activity and a sedimentation coefficient of 12 S. Two high molecular weight polypeptides corresponding to the A- and D-bands of axonemal dynein were shown to copurify with dynein-like ATPase activity and to undergo specific photocrosslinking with [alpha-32P]ATP, suggesting that they were egg dynein catalytic polypeptides. The specific ATPase activity of these putative catalytic polypeptides was determined to be 1.2 mumol.min-1.mg-1. The specific dynein-like ATPase activity of the crude soluble extract of unfertilized sea urchin eggs was determined to be 0.004 mumol.min-1.mg-1. The concentration of putative dynein catalytic polypeptides was therefore determined from the ratio of the specific activities of crude to pure cytoplasmic dynein catalytic polypeptide to be 0.33% of soluble protein, or 99 pg per egg. This is approximately 3-fold greater than the mass of dynein catalytic polypeptides estimated to be present in cilia at the blastula stage of sea urchin embryonic development. The large amount of cytoplasmic dynein in unfertilized eggs suggests that it could act as a precursor of embryonic ciliary dynein. Three minor peaks of ATPase activity were also resolved from cytosolic extracts and shown to be dynein-like. However, their GTPase activities were 2-4-fold higher than that of cytoplasmic dynein, raising the possibility that egg cytoplasm may contain several isoforms of dynein.

Evidence that the 116 kDa component of kinesin binds and hydrolyzes ATP.

Kinesin was prepared from bovine brain as described previously for studies of translocation. A major component of kinesin, (116 kDa) was shown to undergo specific photocrosslinking with [alpha-32P]ATP, indicating it was an ATP-binding polypeptide. A low ATPase activity associated with kinesin was stimulated up to 5-fold by microtubules to a specific activity of 14 nmol . min-1 . mg-1. N-Ethylmaleimide inhibited both [alpha-32P]ATP binding to the 116 kDa polypeptide and microtubule-stimulated ATPase activity, suggesting that the 116 kDa polypeptide was the catalytic subunit of kinesin. Though the ATPase activity associated with kinesin is low, it may be sufficient to support motility assuming it is coupled to the velocity of translocation.

Aspartate mutation distinguishes ETA but not ETB receptor subtype-selective ligand binding while abolishing phospholipase C activation in both receptors.

The endothelin receptors, ETA and ETB, are G protein-coupled receptors (GPCR) that show distinctively different binding profiles for the endothelin peptides and other ligands. We recently reported that Tyr129 in the second transmembrane region (TM2) of the ETA receptor was critical for subtype-specific ligand binding [Krystek, S.R. et al. (1994) J. Biol. Chem. 269, 12383-12386]. Receptor models indicated that aspartic acids located one helical turn above (Asp133) and below (Asp126) Tyr129 in ETA had their side chains directed toward the putative binding cavity. Similarly in ETB, Asp147 and Asp154 are located one turn below and above His150, the residue that corresponds to Tyr129. Asp126 in ETA and Asp147 in ETB correspond to the highly conserved aspartate present in TM2 of many GPCR that has frequently been shown to be crucial for agonist efficacy. Mutagenesis of Asp126 of the human ETA receptor to alanine resulted in an unaltered affinity for ET-1, a 160-fold increase in ET-3 affinity and a decrease in affinity for the ETA selective naphthalenesulfonamide, BMS-182874. ET-1 activation of phospholipase C was abolished. In addition, despite the gain in binding affinity, ET-3 failed to activate phospholipase C, suggesting that Asp126 is required for signal transduction. Mutagenesis of Asp133 to alanine indicated that it was critical only for the binding of BMS-182874. In the ETB receptor, mutation of His150 to alanine or tyrosine indicated that it plays a minor role in ETB subtype-selective ligand binding; mutation of the aspartates in TM2 of ETB did not alter ligand binding. As in the Asp126 Ala ETA variant, ET-1 and ET-3 failed to increase intracellular levels of inositol phosphates in the Asp147Ala ETB mutant. Taken together, these data support the hypothesis that Asp126 and Asp133 flanking Tyr129 in TM2 of the ETA receptor play a role in defining ETA subtype-selective ligand binding but Asp147 and Asp154 that flank the His150 in TM2 of the ETB receptor do not. Furthermore, these data indicate that Asp126 in ETA and Asp147 in ETB are important for transmembrane signaling via phospholipase C.

Frequency judgements: the problem of defining a perceptual event.

In four experiments the conditions under which frequency judgments reflect the relative frequency of complex perceptual events were explored. Subjects viewed a series of 4 x 4 grids each containing seven items, which were letters and numbers in one of four typefaces. Later judgments of the relative frequency with which particular letters appeared in particular typefaces were unaffected by a warning about an upcoming frequency judgment task, but were affected by both the time available for processing the stimuli and the nature of the cover task subjects engaged in while viewing the grids. Frequency judgments were poor when exposure durations were less than 2 s and when the cover task directed subjects' attention merely to the locations of the items within the grids. Frequency judgments improved when the cover task directed subjects' attention to the identity of the stimuli, especially to the conjunction of letter and typeface. The results suggest that frequency estimation of complex stimuli may be possible only for stimuli that have been processed as phenomenal objects.

Cloning and expression of a human endothelin receptor: subtype A.

The polymerase chain reaction, employing degenerate primers specific for the intramembrane domains III and VI of G-coupled receptors, was used to generate partial clones encoding those receptors carried by cultured rat aorta smooth muscle cells. One clone, spanning the intramembrane domains IV-VI of a receptor specific for endothelin-1 (ET-R[A]), was used as a probe to screen a human placental cDNA library. The clone pL4-3, encoding a selective type of human endothelin receptor (ET-R[A]), has an open reading frame encoding a protein 427 amino acids in length, with a relative molecular weight of 48,625 daltons. The sequence analysis suggests the presence of a signal peptide, two potential sites for glycosylation in the N terminal extracellular domain, the seven transmembrane domains typical of G-protein receptors, and several potential sites for phosphorylation in the C terminal cytoplasmic domain. At the amino acid level, the human ET-R(A) shows 91% and 94% identity with the rat and bovine ET-R(A)s, respectively, and 59% similarity with the human ET-R(B). Xenopus laevis oocytes injected with the cloned cDNA express binding sites specific for endothelin-1. Expression of the message in COS 7 cells gave a membrane-bound product to which binding of the [125I]-ET-1 was inhibited by peptide analogues specific for ET-R(A).

MR imaging of the patellofemoral compartment.

This article reviews the applications of MR imaging of the patellofemoral compartment. Axial plane images are the most informative for abnormalities of this compartment. The role of MR imaging in the evaluation of the medial synovial plica and in the detection of chondromalacia is discussed. MR imaging can reliably detect and delineate the complex of injuries associated with patellar dislocations and valgus hyperextension.

Omphalitis and peritonitis in a young West Indian manatee (Trichechus manatus).

Mortality data for the West Indian manatee (Trichechus manatus) indicates that from 1979 to 1984 16% of the recorded deaths involved young juveniles. Necropsy of a young manatee from the west coast of Florida revealed an active infection of the umbilical area (omphalitis) extending down the umbilical artery and veins. A generalized peritonitis was present. Bacterial cultures revealed Streptococcus faecium, Plesiomonas shigelloides, Pseudomonas putrefaciens and Escherichia coli.

Chondromalacia patellae: fat-suppressed MR imaging.

PURPOSE: To evaluate the accuracy of fat-suppressed magnetic resonance (MR) imaging in diagnosing chondromalacia patellae. MATERIALS AND METHODS: Seventy-one patients underwent fat-suppressed MR imaging and arthroscopy of the patellofemoral compartment. Findings were classified as early or advanced chondromalacia or as normal and were correlated with arthroscopic findings. RESULTS: Early and advanced stages of chondromalacia patellae were reliably detected, with positive predictive values of 85% and 92%, respectively. Specificity in early stages was 94% and in late stages was 98%. However, the overall accuracies did not differ substantially from those reported in studies that did not use fat-suppressed imaging. CONCLUSION: Axial, fat-suppressed MR imaging accurately depicts changes caused by chondromalacia patellae. Early stages can be seen as intrasubstance changes of increased signal intensity. Results of this study suggest a high degree of specificity in excluding both early and advanced changes.

Endothelins stimulate mitogen-activated protein kinase cascade through either ETA or ETB.

Endothelin (ET) has been shown to activate mitogen-activated protein kinase (MAPK). However, it has been unclear which of the ET receptors is coupled to MAPK activation. In the present study, we conducted experiments to determine which ET receptor is linked to MAPK activation. We found that both human ETA and ETB were coupled to the MAPK cascade in ETA or ETB cDNA-transfected Chinese hamster ovary cells. ET-1 was more potent than ET-3 in the activation of p42 MAPK, induction of MAPK kinase (MAPKK) gel retardation and uptake of [3H]thymidine in ETA-transfected cells, whereas sarafotoxin (S6c) showed no stimulatory effect on the kinases and [3H]thymidine uptake. ET-1, ET-3, and S6c had approximately the same potency to activate p42 MAPK, MAPKK gel retardation, and [3H]thymidine uptake in ETB-transfected cells. These data suggest that 1) ET isopeptides, through either ETA or ETB receptors, induce the MAPK cascade as well as cell proliferation; and 2) the different potencies of ET isopeptides for activation of the MAPK cascade and induction of cell growth are mainly due to their different affinities toward ETA and ETB.

Mental images can be ambiguous: reconstruals and reference-frame reversals.

Philosophers and psychologists have debated whether or not mental images of ambiguous figures are reversible as pictures of such figures are. Previously, empirical evidence both pro (Finke, Pinker, & Farah, 1989) and con (Chambers & Reisberg, 1985) has been obtained. In a series of four experiments, we identify the conditions under which images of classic ambiguous figures like the duck/rabbit and the snail/elephant are reversible. We distinguish between two types of reversal: those that entail a change in reference-frame specification as well as a reconstrual of image components (reference-frame realignments) and those that entail reconstruals only (reconstruals). We show that reference-frame realignments can occur in imagery, particularly if observers are given an explicit or an implicit suggestion; and that reconstruals of images occur commonly, regardless of experimental conditions. In addition, we show that images constructed from good parts are more likely to reverse than images constructed from poor parts. On the basis of these results, we propose a functional organization of shape memory that is consistent with shape recognition findings as well as with our reversal findings.

Endothelin-1 stimulates cytosolic phospholipase A2 in Chinese hamster ovary cells stably expressing the human ETA or ETB receptor subtype.

The ability of endothelin-1 (ET-1) to activate cytosolic phospholipase A2 (cPLA2) has been studied in Chinese Hamster ovary (CHO) cells stably expressing either the human ETA (CHO/ETA) or the human ETB (CHO/ETB) receptor subtype. ET-1 dose-dependently increased a dithiothreitol-insensitive cPLA2 activity in both cell types. In CHO/ETA cells, BQ-123, an ETA-selective antagonist, completely blocked ET-1-induced cPLA2 stimulation. In CHO/ETB cells, the ET-1 response was mimicked by 4AlaET-1 which could be blocked partially by PD 145065, a nonselective antagonist of ETA and ETB. As expected, ET-1 stimulated PGE2 synthesis in CHO cells transfected with ET receptors. We conclude that ET-1 can stimulate cPLA2 via both the human ETA and ETB receptor subtype.

Extracellular activities of human granzyme A. Monocyte activation by granzyme A versus alpha-thrombin.

Granzymes, serine proteases located in the granules of cytotoxic T cel ls and NK cells, are essential for induction of target cell apoptosis. However, since cytotoxic cells constitutively secrete a portion of their synthesized granzymes, these proteases could mediate extracellular functions independent of their role in the lytic event. Thrombin, another serine protease, can induce cytokine production in a number of different cell types. In this study, we test the hypothesis that granzymes, like thrombin, can regulate cell-mediated immunity by inducing the production of different cytokines. We show that granzyme A (GA) stimulates IL-6, IL-8, and TNF-alpha production by human PBMC and purified monocytes. In contrast, monocytes exposed to thrombin had enhanced IL-8 production with no induction of IL-6 or TNF-alpha production. However, monocytes exposed to either GA or thrombin had enhanced phagocytic activity. The enzymatic activity of GA and thrombin was required for the induction of cytokine production and for the enhancement of phagocytic activity. The induction of different cytokine profiles by GA vs thrombin suggested that GA activates monocytes via a receptor that was different from the thrombin receptor. This conclusion was strengthened by the fact that GA was incapable of inducing Ca2+ mobilization in insect cells transfected with the thrombin receptor. These results suggest that enzymatically active GA mediates important immunoregulatory functions through signaling pathways that does not involve thrombin receptor activation.

Expression of the murine interferon gamma receptor in Xenopus laevis oocytes.

This study describes the isolation of mRNA for the murine interferon gamma receptor and its expression in frog oocytes. The binding properties and apparent molecular weight of the murine interferon gamma receptor protein synthesized in frog oocytes is similar to that found on mouse cells. This is the first report of a functional receptor for a polypeptide ligand (interferon gamma) expressed in and directly assayed on frog oocytes.

Endothelin receptor B expression in the rat and rabbit lung as determined by in situ hybridization using nonisotopic probes.

Endothelins are a family of potent vasoactive peptides. Full-length cDNA clones to human endothelin receptor B (ETB) mRNA were random prime-labeled with nucleotides conjugated to digoxigenin for in situ hybridization. The labeled cDNA was used to probe frozen sections of rat and rabbit lung. Detection of the digoxigenin-labeled probe was accomplished by an antibody-enzyme conjugate, anti-digoxigenin alkaline phosphatase. The location of the antibody-antigen complex was visualized as an enzyme-linked color reaction. The hybridization, washings, and detection steps were performed under stringent conditions. The following cell types of the rat and rabbit lung had abundant positive reaction product to the ETB probe: bronchiolar and bronchial epithelium, endothelium of smooth-muscle--walled vessels, and bronchial and bronchiolar-associated lymphoid tissue. Abundant positive reaction product was also observed in cell populations in the lung parenchyma. Additional studies are being performed to identify those populations. The results of this study suggest that in addition to vasoactivity, endothelins play other important roles in the lung.

Transvaginal ultrasonographic findings in surgically verified ectopic pregnancy.

OBJECTIVE: Our purpose was to evaluate transvaginal ultrasonographic findings in ectopic pregnancies for positive ultrasonographic sign(s). STUDY DESIGN: Eighty-nine patients admitted with an ectopic pregnancy from September 1987 through September 1989 were retrospectively reviewed. Sixty-nine had undergone transvaginal ultrasonography within 10 days before surgery. The ultrasonographic examinations were reviewed by four radiologists. RESULTS: Ultrasonography revealed adnexal masses in 54 patients (78%). Thirty-six masses had an appearance consistent with an adnexal ring. Twenty-four adnexal rings demonstrated a thin sonolucent area surrounding the ring, a "halo sign" (67%). A control group of 116 intrauterine pregnancies were evaluated by ultrasonography. Forty-one women had adnexal cysts. Twenty-seven of these had an adnexal ring; only two of these had halos. CONCLUSION: The halo sign is presumptive evidence of a living ectopic pregnancy and, when identified, may allow earlier diagnosis and intervention.

BMS-182874 is a selective, nonpeptide endothelin ETA receptor antagonist.

BMS-182874 [5-(dimethylamino)-N-(3,4-dimethyl-5-isoxazolyl)-1-naphthalene sulfonamide] is a recently discovered, low molecular weight, nonpeptide endothelin (ET) receptor antagonist. BMS-182874 competitively inhibited the binding of [125I]ET-1 to ETA receptors in rat vascular smooth muscle A10 (VSM-A10) cell membranes (Ki = 61 nM) and in CHO cells stably expressing the human ETA receptor (Ki = 48 nM), but was a weak inhibitor at ETB receptors (Ki > 50 microM) and non-ET receptors. BMS-182874 inhibited ET-1-stimulated inositol phosphate accumulation (KB = 75 nM) and calcium mobilization (KB = 140 nM) without suppressing the maximal responses in VSM-A10 cells. BMS-182874 was a competitive antagonist of force development elicited by stimulation of ETA, but not other, receptors in isolated blood vessels such as the rabbit carotid artery (KB = 520 nM). The apparent discrepancy between efficacy in cell and tissue models was likely related to the high degree of protein binding exhibited by BMS-182874. When administered either orally (ED50 = 30 mumol/kg) or intravenously (ED50 = 24 mumol/kg) to conscious, normotensive rats, BMS-182874 blunted the pressor response to exogenous ET-1. These data demonstrate that BMS-182874 is a competitive, selective and orally active ETA receptor antagonist that will be useful in understanding the role of ET in normal and disease states.

Cloning and functional expression of a cDNA encoding a human type 2 neuropeptide Y receptor.

Neuropeptide Y (NPY) is a 36-amino acid polypeptide that is widely distributed in the central nervous system and periphery. Pharmacological studies have suggested that there are at least three receptor subtypes, Y1, Y2, and Y3. Cloning of the Y1 subtype has been reported previously. Here we report the isolation by expression cloning of a cDNA encoding a human NPY receptor displaying a pharmacology typical of a Y2 receptor. COS-7 cells transfected with the cDNA express high affinity binding sites for NPY, peptide YY, and NPY13-36, whereas [Leu31,Pro34]NPY binds with lower affinity. The receptor is 381 amino acids in length and has seven putative transmembrane regions typical of G-protein-coupled receptors. Comparison of the amino acid sequence of this Y2 receptor to that of the human Y1 receptor indicates that the two receptors are 31% identical at the amino acid level. Northern blot analyses reveal a single 4-kilobase mRNA species and indicate that the messenger RNA is present in many areas of the central nervous system. NPY induced calcium mobilization and inhibited forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells that stably express the Y2 receptor cDNA, indicating that the recombinant Y2 receptor is functionally coupled to second messenger systems.