Species differences in the immunoreactive patterns of calcitonin gene-related peptide in the pancreas. In the pancreas, calcitonin gene-related peptide (CGRP) immunoreactivity has been described in nerve fibers and in distinct types of islet cells. This unique, apparently species-specific cell-type expression prompted the present investigation to clarify further the pattern of CGRP immunoreactivity in different mammalian species (i.e., different strains of rats, mice, guinea pigs, rabbits, cats, dogs, pigs, and humans) commonly used for functional and anatomical studies of the pancreas by means of immunohistochemistry using three different CGRP antibodies. In each species, CGRP-immunoreactive neurites innervate the exocrine and endocrine compartments, the vasculature, and the intrapancreatic ganglia, where they form dense networks encircling unstained cell bodies. The only exception is the pig pancreas, where the islets appear to be devoid of immunoreactive fibers. The overall density of immunoreactive pancreatic axons in different species is as follows: rat, mouse, and rabbit greater than guinea pig greater than or equal to pig and cat much greater than dog and human. CGRP-immunoreactive endocrine cells appear to be restricted to the rat pancreas, where they form a subpopulation of somatostatin-containing D cells. In contrast, in mouse, guinea pig, cat, dog, and human pancreas, a homogeneous staining of the core of the islets, where insulin-producing B cells are located, was visualized in sections incubated with the rabbit CGRP antiserum at 4 degrees C, but not at 37 degrees C (an incubation temperature that does not affect the islet cell staining in the rat nor the fiber labeling in any species). Furthermore, the staining of islet B cells was not reproducible with all the CGRP antibodies used, all of which comparably stain nerve fibers in each species, and islet D cells in the rat. Immunoreactive islet cells were not visualized in pig and rabbit pancreas. These results are consistent with the hypothesis that the expression of CGRP in nerve fibers is a common feature of mammalian pancreas, whereas its expression in endocrine cells appears to be restricted to the D cells of the rat pancreas.


Segmental peptidergic innervation of abdominal targets in larval and adult dipteran insects revealed with an antiserum against leucokinin I. An antiserum against the cockroach neuropeptide leucokinin I (LKI) was used to study peptidergic neurons and their innervation patterns in larvae and adults of three species of higher dipteran insects, the flies Drosophila melanogaster, Calliphora vomitoria, and Phormia terraenovae, as well as larvae of a primitive dipteran insect, the crane fly Phalacrocera replicata. In the larvae of the higher dipteran flies, the antiserum revealed three pairs of cells in the brain, three pairs of ventro-medial cells in the subesophageal ganglion, and seven pairs of ventro-lateral cells in the abdominal ganglia. Each of these 14 abdominal leucokinin-immunoreactive (LKIR) neurons innervates a single muscle of the abdominal body wall (muscle 8), which is known to degenerate shortly after adult emergence. Conventional electron microscopy demonstrates that this muscle is innervated by at least one axon containing clear vesicles and two axons containing dense-cored vesicles. Electron-microscopical immunocytochemistry shows that the LKIR axon is one of these two axons with dense-cored vesicles and that it forms terminals on the sarcolemma of its target muscle. The abdominal LKIR neurons appear to survive metamorphosis. In the adult fly, the efferent abdominal LKIR neurons innervate the spiracles, the heart, and neurohemal regions of the abdominal wall. In the crane fly larva, dorso-medial and ventrolateral LKIR cell bodies are located in both thoracic and abdominal ganglia of the ventral nerve cord. As in the larvae of the other flies, the abdominal ventrolateral LKIR neurons form efferent axons. However, in the crane fly larva there are two pairs of efferent LKIR neurons in each of the abdominal ganglia and their peripheral targets include neurohemal regions of the dorsal transverse nerves. An additional difference is that in the crane fly, a caudal pair of LKIR axons originating from the penultimate pair of dorso-median LKIR cells in the terminal ganglion innervate the hind-gut.


Distribution of calcitonin gene-related peptide immunoreactivity in the central nervous system of the frog, Rana esculenta. The distribution of calcitonin gene-related peptide (CGRP), immunoreactive structures in the central nervous system of the frog, Rana esculenta, was studied using the peroxidase immunohistochemical method. Immunoreactive perikarya were found in all major parts of the brain. In the forebrain, neurons of the septohippocampal formation, the amygdala, the ventromedial and posterocentral thalamic nuclei, and the cerebrospinal fluid contacting neurons in the diencephalic periventricular organ showed immunoreactivity. The pear-shaped neurons of the optic tectum, and perikarya of the oculomotor nucleus in the midbrain were also immunoreactive. In the hindbrain, neurons of the cranial nerve motor nuclei, neurons of the superior vestibular nucleus, giant cells of the reticular formation, and preganglionic parasympathetic neurons of the superior salivatory nucleus were stained. Motoneurons presented immunostaining also in the spinal cord. Immunoreactive fibers were shown to occur in the olfactory tract, the striatum, the tegmentum and the basis mesencephali, the descending tract of the trigeminal nerve, the solitary tract, Lissauer's tract, and the dorsal horn of spinal cord. A comparison of the distribution of CGRP immunoreactivity in the mammalian and amphibian central nervous system revealed that, in relation to the size of the brain, CGRP is more extensively distributed in the amphibian than in the mammalian limbic system.


Tunicamycin, puromycin and brefeldin A influence the subcellular distribution of neuropeptides in hypothalamic magnocellular neurones of rat. Magnocellular neurones in the supraoptic nuclei of normal Long Evans and homozygous Brattleboro rats were examined electron-microscopically after intracisternal injections of tunicamycin, puromycin, or brefeldin A. Moderate (50 micrograms) or high (200 micrograms) doses of tunicamycin caused the formation of electron-dense filamentous accretions in the endoplasmic reticulum (ER) cisterns of vasopressin neurones, but only the high dose of tunicamycin also caused accretions to form in the ER of some oxytocin neurones. Immunogold labelling of ultrathin sections from tunicamycin-treated rats revealed that, in about 5% of vasopressin neurones, the accretions could be immunogold-labelled for vasopressin and its associated neurophysin. However, in the majority of vasopressin neurones, the sections required trypsinisation before immunolabelling of the accretions could be detected. Small accretions in the ER of oxytocin neurones did not label for oxytocin or its neurophysin without prior trypsinisation, whereas larger accretions in other oxytocin cells could be labelled without prior trypsin treatment. Administration of puromycin resulted in the formation of small ER accretions in both vasopressin and oxytocin neurones. These accretions were immunolabelled with antisera, respectively, to vasopressin and oxytocin, but neurophysin-immunoreactivity was in most cases absent and was not revealed by treatment with trypsin, suggesting that neurophysin-immunoreactive epitopes were absent from truncated peptides forming the accretions. Brefeldin A caused dilatation of ER cisterns and disruption of the Golgi apparatus in both oxytocin and vasopressin neurones, but did not cause accretions to form in the ER.


Immunogold labelling of the cytoplasmic estradiol receptor in resting porcine endometrium. Serial sections of resting porcine endometrium were analyzed with the monoclonal antibody 13H2 using goat antimouse IgG/5 nm gold as secondary reagent or with either polyclonal antibodies from goat #402 or the rat monoclonal antibody H222, both in combination with protein G/12 nm gold. A modestly higher labelling of nuclei than of cytoplasm was seen only with the monoclonal antibody H222. Polyclonal #402 and monoclonal 13H2 showed fewer attachments over nuclear than over cytoplasmic areas. The highest densities of attachment and of predominantly cytoplasmic labelling were obtained with the monoclonal antibody 13H2. The results confirm the earlier assumption of a restricted accessibility of estradiol receptor in the cytoplasm of resting cells for immunoreagents.


Ultrastructural and cytochemical studies of the effects of prolactin on the lateral prostate and the seminal vesicle of the castrated guinea pig. Administration of ovine prolactin to castrated guinea pigs for 2 weeks induced hypertrophy of secretory cells in the lateral prostate when compared with the castrated controls. This was accompanied by an apparent increase in the number of profiles of granular endoplasmic reticulum and well developed Golgi complexes with dilated cisternae. An increase in the number of low-contrast electron-dense secretory granules was observed 4 weeks after prolactin treatment. In the seminal vesicle, dilatation and degranulation of granular endoplasmic reticulum and an apparent decrease in the number of secretory granules were observed 4 weeks after prolactin administration. Following castration and 2 weeks after prolactin treatment, thiamine pyrophosphatase (TPPase)-reaction product was mainly confined to 1-2 trans cisternae of the Golgi complexes in secretory cells of the lateral prostate and the seminal vesicle. In both glands, a reduction of TPPase activity was observed 2 weeks following prolactin administration, and the reaction product was totally absent after prolonged treatment for 4 weeks. The present study has provided morphological evidence that prolactin is capable of stimulating the secretory function of the lateral prostate while exerting some inhibitory effects on the seminal vesicle of the castrated guinea pig. In both glands, TPPase activity, and hence the process of glycosylation was inhibited after prolactin administration. The results from radioimmunoassay indicated that the action of prolactin on these glands could be a direct effect and not mediated through testosterone.


Allatostatin-immunoreactive neurons projecting to the corpora allata of adult Diploptera punctata. A monoclonal antibody against allatostatin I was used to demonstrate the allatostatin-immunoreactive pathways between the brain and the corpus cardiacum-corpus allatum complex in the adult cockroach Diploptera punctata. The antibody was two to three orders of magnitude more sensitive to allatostatin I than to the other four known members of the allatostatin family. Whole and sectioned brains in which immunoreactivity was localized with horseradish peroxidase-H2O2-diaminobenzidine reaction showed strongly immunoreactive cells in the pars lateralis of the brain with axons leading to and arborizing in the corpus cardiacum and the corpus allatum. Although many neurosecretory cells of the pars intercerebralis project to the corpora allata only, four strongly immunoreactive cells were evident here (two pairs on either side), and these did not project to the corpus cardiacum and corpus allatum but rather terminated within the protocerebrum in areas in which lateral cells also formed arborizations. Immunoreactivity was found in many other cells in the brain, especially in the tritocerebrum.


Immuno-electron-microscopic localization of laminin and collagen type IV in normal and denervated tooth pulp of the cat. The distribution of laminin-like immunoreactivity in adult normal and denervated cat mandibular tooth pulps was studied by the use of fluorescence microscopy and pre-embedding immunogold electron microscopy. Immunoreactivity to collagen IV was also assessed in order to distinguish basement membranes. In normal pulps, light-microscope laminin-like immunoreactivity was strong along blood vessels and Schwann cell sheaths, and a faint immunoreactivity was seen also in the odontoblast layer. Electron microscopy confirmed the laminin-like immunoreactivity of endothelial and Schwann cell basement membranes at all pulpal levels. In the odontoblast layer and the predentine, nerve-like structures lacking basement membranes but possessing strong membrane laminin-like immunoreactivity were encountered. In addition, a clear-cut laminin-like immunoreactivity of plasma membranes of the somata and processes of odontoblasts was seen. Observations on denervated pulps as well as pulps in which nerve regeneration had taken place did not reveal any changes in the pattern of laminin-immunoreactivity in basement membranes or odontoblasts. Distribution of collagen IV-like immunoreactivity was very similar to laminin-like immunoreactivity in basement membranes of blood vessels and Schwann cells, and appeared unaffected by denervation. The odontoblasts and nerve-like profiles in the odontoblast layer were devoid of collagen IV-like immunoreactivity. We propose that odontoblast-associated laminin could be of significance as guidance for regenerating terminal pulpal nerve fibers to appropriate targets.


The cytoarchitecture of the medial layer in rat thoracic aorta: a scanning electron-microscopic study. The cytoarchitecture of the medial layer of rat thoracic aorta was examined by scanning electron microscopy after removal of the connective tissue. The outermost lamella showed a lattice-like structure of muscle bundles of closely apposed smooth muscle cells (SMCs), whereas the inner lamellae consisted of more-or-less continuous muscle sheets of vaguely defined subgroups of parallel SMCs. Longitudinal rows of ridges ran along the adventitial surface of these muscle bundles and sheets. The SMCs of the outermost lamella, were 5.1 microns wide, and varied in shape, whereas those of the inner lamellae, were 52.7 microns long, 2.6 microns wide and 4.1 microns thick, and were elongated, spindle-shaped cells with serrated outlines. These latter SMCs extended obliquely, and partially overlapped each other. The surface of the SMCs in the outermost lamella exhibited a rugged texture, with nodular protrusions and oblique and longitudinal laminar folds, while the inner lamellar cells showed longitudinal laminar folds and finger-like processes on both sides of the ridges, pointing in opposite directions to the ridges. The angle of deviation from the transverse axis of the vessel, of the muscle bundles and subgroups in the outermost lamella, was 33.6 degrees, in the second and third lamellae, 22.5 degrees, and in the innermost lamellae, 12.8 degrees. The mean angle of the muscle bundle and subgroup arrangement, with respect to the long axis of the vessel, however, was basically 90 degrees in all lamellae.


Differentiation of human skeletal muscle cells in culture: maturation as indicated by titin and desmin striation. This report describes a phenotyping study of differentiating human skeletal muscle cells in tissue culture. Satellite cells (adult myoblasts), isolated from biopsy material, showed a proliferative behaviour in high-nutrition medium, but fused to form myotubes when grown in low-nutrition medium. The expression and structural organization of the intermediate filament proteins desmin and vimentin as well as the sarcomeric constituents alpha-actin, alpha-actinin, nebulin, myosin and especially titin during myofibrillogenesis in vitro, were studied by means of indirect immunofluorescence assays. The proliferating myoblasts contained both desmin and vimentin, alpha-actinin and the filamentous form of actin. Shortly after the change of medium, expression of titin, sarcomeric myosin and skeletal muscle alpha-actin was found in mononuclear cells in a diffuse, filamentous (titin, myosin, alpha-actin) or punctate (titin, myosin) pattern. Four to 10 days after the medium change, mature myotubes showed desmin, titin, alpha-actinin, nebulin, sarcomeric myosin and actin cross-striations, while vimentin was no longer detected. We conclude that human skeletal muscle cell cultures are an appropriate model system to study the molecular basis of myofibrillogenesis. Especially the presence of desmin in a striated fashion points to a high degree of maturation of the muscle cell cultures.


Evidence for the coexistence of serotonin and calcitonin gene-related peptide at the subcellular level in neuroepithelial bodies in the lung of a marsupial, Isoodon macrourus. The coexistence of serotonin and calcitonin gene-related peptide (CGRP) in neuroepithelial bodies of the bandicoot, Isoodon macrourus, has been examined using immunocytochemistry at the light- and electron-microscope levels. The avidin-biotin technique of antigen localisation was used initially to identify serotonin-like and CGRP-like immunoreactivity (-LI). Serotonin-LI and CGRP-LI were found in neuroepithelial cells in the lungs of 30-day-old bandicoots. CGRP-LI could also be demonstrated in nerve fibres associated with some neuroepithelial bodies. The protein A-gold technique of antigen localisation was used to label neuroepithelial cells and nerve fibres at the subcellular level. Serotonin-LI and CGRP-LI were observed in the same dense-cored vesicles of most neuroepithelial cells; however, some neuroepithelial cells were shown to possess serotonin-LI without CGRP-LI. Nerve fibres immediately adjacent to neuroepithelial bodies exhibited mainly CGRP-LI. These results show that serotonin-LI and CGRP-LI are present in neuroepithelial cells of the bandicoot in the same secretory vesicles. This pattern of co-localisation may reflect co-ordinated or synergistic actions of these two neuroactive substances.


Activity labeling patterns in the medulla of Drosophila melanogaster caused by motion stimuli. We quantitatively describe 2-deoxyglucose (2-DG) neuronal activity labeling patterns in the first and second visual neuropil regions of the Drosophila brain, the lamina and the medulla. Careful evaluation of activity patterns resulting from large-field motion stimulation shows that the stimulus-specific bands in the medulla correspond well to the layers found in a quantitative analysis of Golgi-impregnated columnar neurons. A systematic analysis of autoradiograms of different intensities reveals a hierarchy of labeling in the medulla. Under certain conditions, only neurons of the lamina are labeled. Their characteristic terminals in the medulla are used to differentiate among the involved lamina monopolar cell types. The 2-DG banding pattern in the medulla marks layers M1 and M5, the input layers of pathway p1 (the L1 pathway). Therefore, activity labeling of L1 by motion stimuli is very likely. More heavily labeled autoradiograms display activated cells also in layers M2, M9, and M10. The circuitry involved in the processing of motion information thus concentrates on pathways p1 and p2. Layers M4 and M6 of the distal medulla hardly display any label under the stimulus conditions used. The functional significance of selective activity in the medulla is discussed.


Total numbers of glomeruli and individual glomerular cell types in the normal rat kidney. Alterations in numbers of glomeruli and glomerular cells occur in various renal disorders. Although values for these parameters have previously been reported for several species, the estimates have often been biased due to assumptions regarding glomerular and/or nuclear size and shape. Other studies have used tedious serial-section reconstruction methods. In the present study, unbiased stereological methods were used to estimate total numbers of glomeruli and individual glomerular cell types in normal rats. The kidneys of seven adult Sprague-Dawley rats were perfused with 4% paraformaldehyde and 1% glutaraldehyde in phosphate buffer and embedded in either glycol-methacrylate (for light microscopy, LM) or Epon/Araldite (for transmission electron microscopy, TEM). Total glomerular number was estimated using an LM physical disector/fractionator combination; the total number of cells per average glomerulus was estimated using an LM optical disector/Cavalieri combination; and TEM physical disectors were used to count individual cell types. The normal rat kidney was found to contain 31,764 +/- 3667 (mean +/- SD) glomeruli. An average glomerulus contained 674 +/- 129 cells, of which 181 +/- 53 were epithelial cells (podocytes), 248 +/- 53 were endothelial cells, and 245 +/- 45 were mesangial cells. An average renal corpuscle contained 117 +/- 27 parietal epithelial cells. Following sectioning and staining, less than 6.5 h was needed to obtain the above estimates for a single animal, with coefficients of variation (SD as a percent of the mean) ranging from 10% to 25%. The unbiased stereological methods used in the present study constitute an unbiased, precise and cost-efficient set of quantitative tools for assessing glomerular morphology in health and disease.


Ultrastructural evidence for multiple mucous domains in frog olfactory epithelium. This study showed that the olfactory mucus is a highly structured extracellular matrix. Several olfactory epithelial glycoconjugates in the frog Rana pipiens were localized ultrastructurally using rapid-freeze, freeze-substitution and post-embedding (Lowicryl K11M) immunocytochemistry. Two of these conjugates were obtained from membrane preparations of olfactory cilia, the glycoproteins gp95 and olfactomedin. The other conjugates have a carbohydrate group which in the olfactory bulb appears to be mostly on neural cell-adhesion molecules (N-CAMs); in the olfactory epithelium this carbohydrate is present on more molecules. Localization of the latter conjugates was determined with monoclonal antibodies 9-OE and 5-OE. Ultrastructurally all antigens localized in secretory granules of apical regions of frog olfactory supporting cells and in the mucus overlying the epithelial surface, where they all had different, but partly overlapping, distributions. Monoclonal antibody 18.1, to gp95, labeled the mucus throughout, whereas poly- and monoclonal anti-olfactomedin labeled a deep mucous layer surrounding dendritic endings, proximal parts of cilia, and supporting cell microvilli. Labeling was absent in the superficial mucous layer, which contained the distal parts of the olfactory cilia. Monoclonal antibody 9-OE labeled rather distinct areas of mucus. These areas sometimes surrounded dendritic endings and olfactory cilia. Monoclonal antibody 5-OE labeled membranes of dendritic endings and cilia, and their glycocalyces, and also dendritic membranes.


Quantitative changes in the frequency and distribution of the C-cell population in the rat thyroid gland with age. The development of calcitonin cells (C-cells) was investigated in rat thyroid glands from birth to 120 days, using an immunoperoxidase technique and a point-counting method. The proportion of C-cells to follicular cells was 4.5% on the day of birth and increased progressively to 10.4% by 120 days. The highest density of C-cells was noted in the mid-region of the lobes along a longitudinal axis. The caudal and cephalic regions of the lobes contained smaller numbers of C-cells. The C-cells tended to be more numerous in the posterior aspects of the lobes. Although the numbers of C-cells in 120-day-old animal were markedly increased as compared to animals at the time of birth, the cell distributions within the glands were similar at all ages.


Immunohistochemical localization of the eosinophil major basic protein in the uterus horn and cervix of the rat at term and after parturition. Distribution of the eosinophil major basic protein (MBP) was studied in the rat uterus horn and cervix by means of immunohistochemistry using an antiserum raised against rat MBP. Various hormonal contexts were investigated: pre- and post-parturition, the estrous cycle, and ovariectomy followed by hormonal treatment or without treatment. MBP was detectable in the cervix as early as 12 h post-partum, appearing in the stroma close to the myometrium. The MBP had spread throughout the stroma toward the luminal epithelium after a few days. In contrast, no MBP was seen in sections of the corresponding pre- and post-partum uteri and in the pre-partum cervix. In cycling rats, MBP was distributed equally in the cervix and uterus and was more abundant during proestrus and estrus. In ovariectomized rats subsequently treated with progesterone, no MBP was detected in the cervix or uterus. In the cervix of ovariectomized rats treated with estradiol, MBP first appeared in the muscle layer situated between the two cervical lumina and then reached the stroma; within a few days only the stroma was stained. Inversely, in the uterus MBP-staining first appeared in the stroma. In conclusion, analysis of the distribution of MBP in rat uterus revealed a marked difference in the response of the cervix and horn to a hormonal environment.


A novel principle for quantitation of fast intracellular calcium changes using Fura-2 and a modified image processing system--applications in studies of neutrophil motility and phagocytosis. A new principle is described for imaging intracellular free calcium [Ca2+]i changes in single, living cells utilizing the fluorescent probe Fura-2. It is based upon video color mixing in real time and allows high-speed visualization, at maximum image resolution, of [Ca2+]i changes without digital image ratioing. The epifluorescence images produced by 340 and 380 nm excitations are stored in two memory buffers of a personal computer-based image processing system. Two video signals are generated independently from each buffer and connected to the red and green inputs of a video display. An image is this way created, in which [Ca2+]i shows up as a specific hue, whereas changes in dye concentration, light intensity, cell thickness show up as variations in brightness of the imaged cells. The method has advantages over conventional ratio imaging, notably simplicity and speed, since no calculations are made. Yet it can be combined with traditional digital image processing. The imaging technique allows monitoring of [Ca2+]i changes in rapidly moving cells, like neutrophils. It is demonstrated that during random locomotion on serum-coated glass surfaces, [Ca2+]i levels appeared to oscillate and that the frequency of the oscillations are related to locomotive activity. Furthermore, in Ca2+ free medium, the cells continue to move and phagocytose in the presence of Ca2+ ionophore (ionomycin) and 2 mM EGTA. In the presence of 1 mM extracellular Ca2+, ionomycin-treated cells were not able to move or phagocytose.


Thapsigargin reveals evidence for fMLP-insensitive calcium pools in human leukocytes. To investigate the relationship between different intracellular Ca2+ pools, cytosolic free calcium ([Ca2+]i) was surveyed by means of a Fura-2 fluorescence ratio method on single isolated human leukocytes. Both monocytes and neutrophilic granulocytes (PMN) displayed long lasting spontaneous [Ca2+]i transient changes (1-2 min). In PMN stimulated with the bacterial peptide fMLP we observed transients with shorter duration (10-30 s) and smaller amplitude often superimposed on the long lasting transients. The time course of changes in [Ca2+]i was recorded in a large number (149) of single leukocytes prestimulated for 5 min with fMLP and then challenged with thapsigargin (a blocker of Ca2+ uptake in intracellular pools). Statistical analysis of [Ca2+]i responses revealed that fMLP-sensitive pools contributed to the long lasting [Ca2+]i transients seen in both leukocyte types. However, the existence of fMLP-insensitive calcium pools may explain the superimposed transients seen in PMN. Thapsigargin was also added together with EGTA (to impede contribution from extracellular Ca2+) to 198 fMLP prestimulated and 153 unstimulated PMN. Based on Ca2+ registrations in these cells and a mathematical model (supposing two separate first order responses) the amount of Ca2+ stored in the various pools and their release kinetics were estimated. The results indicate that fMLP-insensitive calcium pools exist in PMN but not in monocytes. Since the digital imaging technique also depicts cellular motility, an additional finding was that the leukocyte's ability to sequestrate the Ca2+ from the cytosol seemed important to locomotion.


Mechanically induced electrical and intracellular calcium responses in normal and cancerous mammary cells. Mechanically induced channel activities and increase of intracellular calcium ([Ca2+]i) in normal and cancerous murine mammary cells (MMT 060562) were investigated using the patch clamp technique and Fura-2 fluorescence. Both cell types showed similar properties. Upon mechanical stimulation, activation of the Ca(2+)-dependent K+ channel or outward membrane current was recorded in cells which were several cells distant from the stimulated cell. Mechanical stimulation also induced an increase of [Ca2+]i in the touched cell, and this increase of [Ca2+]i spread to the surrounding cells. The [Ca2+]i signal travelled a distance of 100-200 microns within 20-40 s and then diminished. The presence of cell-to-cell communication between adjacent mammary cells through gap junction was indicated by injection of lucifer yellow and measurements of electrical coupling (coupling constant = 0.2-0.3). The mechanically induced increase of the [Ca2+]i signal spread to adjacent cells even when the stimulated cell had no physical contact with them. In the absence of fluid movement, the pattern of the spread of the [Ca2+]i signal was a concentric circle. However, in the presence of fluid movement, the pattern changed to elongate to the direction of the flow. These findings suggested that a certain factor was released from the mechanically stimulated cell to the extracellular space, and this factor induces the increase of [Ca2+]i in surrounding cells.


The effects of halothane and isoflurane on intracellular Ca2+ regulation in cultured cells with characteristics of vascular smooth muscle. The Ca(2+)-sensitive photoprotein aequorin was used to monitor changes in intracellular [Ca2+] within cultured cells with characteristics of vascular smooth muscle. Two cell lines were investigated: they were A10 cells, which are transformed cells originally derived from rat aorta, and BC3H1 cells obtained from mouse brain neoplasm. Transient increases in intracellular [Ca2+] were induced following exposure to two different volatile anaesthetics (halothane and isoflurane) and various vasoactive substances (acetylcholine, endothelin, histamine, serotonin and vasopressin). The amplitude of the transients induced by isoflurane were more dependent on the presence of extracellular Ca2+ than those induced by halothane, thus the modes and/or locations of action of these two anesthetics are somewhat different. The response of the two cell lines to the vasoactive substances are unique. Receptor activated changes in [Ca2+]i by various agonists were diminished in the presence and absence of either anesthetic. These data suggest that, although the receptor populations within each cell line were slightly different, the prior application of a volatile anesthetic in a clinically-relevant dose induced a transient increase in [Ca2+]i that could subsequently diminish agonist responses.


Transient but not oscillating component of the calcium mobilizing response to gonadotropin-releasing hormone depends on calcium influx in pituitary gonadotrophs. Gonadotropin-releasing hormone (GnRH)-stimulated changes in the cytosolic free Ca2+ concentration ([Ca2+]i) were studied in gonadotrophs cultured from 3-week ovariectomized rat pituitaries. One animal was used per cell preparation. [Ca2+]i was monitored in individual gonadotrophs by dual emission microspectrofluorimetry, using Indo-1 as the intracellular fluorescent Ca2+ probe. A short stimulation with GnRH evoked a complex concentration-dependent Ca2+ response in individual gonadotrophs. 0.1-1 nM GnRH triggered a series of sinusoidal-like [Ca2+]i oscillations superimposed upon a modest slow [Ca2+]i rise--the oscillating response mode--while 10-100 nM GnRH caused a biphasic increase in [Ca2+]i consisting of a monophasic transient and oscillations--the transient/oscillating response mode. Despite the consistency of Ca2+ responses, an inter-preparation heterogeneity of [Ca2+]i oscillations frequency was noticed. Moreover, we observed that, within a given cell preparation, the frequency of [Ca2+]i oscillations was independent of GnRH concentration whereas both peak [Ca2+]i and area under the [Ca2+]i versus time curve were concentration-dependent. Thus, in gonadotrophs, the presence of the GnRH signal would lead to [Ca2+]i oscillations, while the amplitude of the [Ca2+]i responses would code for the concentration of agonist. Both transient and oscillating components of GnRH responses depended on releasing activity of Ca(2+)-sequestering pools in as much as GnRH responses were unaffected by brief removal of external Ca2+, but suppressed by chelating intracellular free Ca2+ with BAPTA. However, prolonged exposure to a Ca(2+)-free medium suppressed the transient component while leaving the oscillating component unaffected. We therefore propose that gonadotrophs employ Ca(2+)-sequestering pools, whose maintenance depends on a slow Ca(2+)-entry, to give an amplitude-coded Ca2+ rise in response to a short GnRH stimulation.


[Multilocus enzyme electrophoretic types of Neisseria meningitidis serogroup B and their epidemiologic significances]. In order to expand the epidemiologic relationships between the strains of Neisseria meningitidis serogroup B isolated from patients and carriers, 57 cases and 45 carrier isolates were collected in 11 provinces and 2 municipalities of China since the 1970s and their multilocus enzyme electrophoretic types and clonal population structures were studied by multilocus enzyme electrophoresis. It was primarily found that the above strains could be divided into 69 electrophoretic types (ET) and 13 clones. Among others, the clone I was the most important one, because the clone I represented 63.7% of all strains tested and 77.2% of the case isolates and its district distributions were quite wide since the 1970s. In addition, it was an obvious tendency that more and more of the case isolates gathered up the clone I since 1984 and the predominant ETs also occurred in the complex ET 1 and ET 24 of the clone I. As compared with the case isolates, the above carrier isolates displayed more heterogenetic types. Only 40% of all carrier isolates belonged to the above clone I and predominant ETs.


[A study on the relationship between maximal titers of anti-HBs antibodies and persistence of protective level after vaccination]. 300 recipients with negative for HBsAg anti-HBs, anti-HBc were vaccinated by 3 kinds of hepatitis B vaccine at 0, 1, 6 months. 266 students with maximal titers of anti-HBs detected after the 3rd immunization were divided into 4 groups: 10-100, 101-500, 501-1,000, > 1000 mlU/ml. A study on the relationship between maximal titers of anti-HBs and the protective time was carried out. The results showed that in the 1st group the titers of anti-HBs of 52.6%, 84.2% and 94.7% recipients decreased to or below 10 mIU/ml when detected one, two and three years after vaccination respectively; the titers of 5.2%, 31% and 56.9% decreased to or below 10 mIU/ml when detected. 1, 2 and 3 years after vaccination respectively in the 2nd group, while the titers of only 0.7% recipients decreased to or below 10 mIU/ml when detected 3 years after vaccination in the 1st group. The decreasing rate of titers of Anti-HBs was similar in 4 groups. The titers of anti-HBs were 90% reduction after 1st immunization in the first 24 months. Then the decreasing rate became slower. Therefore, we suggested that peoples should be revaccinated according to their maximal titers of anti-HBs after completed course of vaccination.


[A study on measles vaccination rate and its antibody level in children aged 12-24 months in Guangxi, 1989]. This report described the measles vaccination rate (MVR) as well as its HI antibodies of 1842 children aged from 12 to 24 months. The MVR was 99.57% but HI positive rate was only 83.50%. HI of 298 target children still remained negative. Among them, 6 children were unvaccinated and others vaccinated. The HI negative rate reached 16.50% The HI positive rate was higher in the target children who lived in the area with convenient transportation and relatively low atmospheric temperature, and who received measles vaccine at the age of 8 months or more. Otherwise, the HI positive rate was lower. So some suggestions were proposed that the vaccination season, age of vaccine, increasing vaccine dosage should be considered carefully before measles vaccination session had arranged.


[A study on the interference between smooth and rough species of brucella in mice]. Smooth-B. abortus 104M. B.melitensis Rev-1 and B.suis S2 mixed with rough-B.canis RM 6/66 respectively were injected into mice. The results demonstrated that s-species of Brucella suppressed R-species of B.canis RM6/66 in mice. The epidemiological phenomenon that R-species of Brucella were difficulty isolated in focus of s-species should be explained on the bases of the study.


[Use of Cox's model in long-term survival analysis of cerebrovascular disease]. A long-term survival study on 885 cases of cerebrovascular disease was carried out. The results showed that after follow-up for 3, 5, and 8 years, the survival rates were 86.98%, 75.70% and 64.26% respectively, and all were lower obviously than that of the general population in the same district. The results of Cox's model analysis showed that recurrence, hypertension and age were the risk factors influencing the long-term survival of cerebrovascular disease, and the relative risks were 3.29, 2.17 and 1.65 respectively.


[Cluster and multiple regression analysis of leptospirosis epidemic factors]. Ten-year surveillance of Rice-field-typed leptospirosis was carried out in high-infected foci. Twenty two parameters of epidemic data were divided into 3 categories and cluster-analyzed. 5 typical variables were selected for multiple regression equation. The equation contained four factors, i.e. rodents density, carriage rates of main animal hosts, GMT of population antibody against leptospira and quantity of rainfall in August. The expected morbidity of leptospirosis calculated by the equation were roughly identical to the real morbidity.


[A prospective study on the relationship between abnormal pregnant outcome and Toxoplasma gondii infection]. 2821 pregnant women were divided into three groups according to the phenomena of Toxoplasma gondii infection, and the pregnancy outcomes were followed up. Results show that the total incidence rates of spontaneous abortion, premature birth, still birth and congenital defects are 0.67%, 3.47%, 0.85% and 0.96% respectively. These four rates in the group serodiagnosed as recently or actively infected are significantly higher than that diagnosed as no evidence of infections and the relative risk estimates (ORmh) are 7.38, 3.52, 8.68 and 9.44 respectively. Differences of the four rates between group diagnosed as past infection and that of no evidence of infection are not statistically significant. Analysis between the time of serodiagnosis and the incidence of abnormal pregnant outcome shows that congenital defects is statistically related with infection in the first trimester of gestation.


[Hygienic epidemiological assessment of electrodialysis drinking water]. It is first reported the hygienic epidemiological assessment of electrodialysis drinking water with multidiscipline methods including environmental epidemiology, toxicology, chemistry and clinical medicine. The results showed that the occurrence of malignant tumours in residents drinking electrodialysis water did not directly associate with their drinking water, we also did not find that there was any influence of electrodialysis water on residents' liver and gastrointestinal function, and the rate of thyroid enlargement, prevalence rates of dental fluorosis and dental caries as well as the level of saliva lysozyme in children. However, the morbidity rate of hypertension in the residents drinking electrodialysis water was higher than that in those drinking non-electrodialysis water.


[The economic loss of poliomyelitis outbreak in Pi County and economic beneficial analysis on EPI]. In 1989, there was a poliomyelitis outbreak in Pi county, Jiangsu province. The total case number was 597 in which 52 died. It caused the patients' families and national economic loss 2,713,107.32 yuan and lessened creating value of 18,583,942.35 yuan for the country. The outbreak was mainly caused by the fact that many children did not set vaccination card, missed vaccination chance and lacked in immunity to polio. So the ratio of cost-benefit will be expected to reach 1:6.85, and the net benefit of 2,317,268.16 yuan can be gained if the task of EPI is implemented well to avoid the occurrence of outbreak.


[The monitor method of immunization coverage]. A comprehensive monitor method of immunization coverage is suggested to be applied to the expanded program on immunization. The routine coverage reporting system should be set up combined with surveys in selected townships. The criteria for doing survey is expressed in the flow chart. Two sampling methods are presented based on the principle of lot quality assurance sampling (LQAS) and coverage rate could be estimated after the surveys.


Stress and psychological disorders in Zimbabwe: a report on patients seen in private practice. This report is based on the analysis of 326 case files of patients that I saw in part-time private practice over a period of five years. The aim of the investigation was to establish the stressful life events that are associated with psychological disorder. Thirteen categories of stressful events were identified. Statistical analyses revealed that these stresses were significantly associated with a number of demographic variables.


The effects of established and gestational diabetes on pregnancy outcome at Harare Maternity Hospital. A study was carried out to determine the effect of established and gestational diabetes on pregnancy outcome over a period of two years at Harare Maternity Hospital, Harare, Zimbabwe. During the period, 51 patients with established diabetes mellitus and 70 patients with gestational diabetes were treated. The perinatal mortality was higher among this group (124 per 1,000) compared with the rest of the total hospital population (44 per 1,000) who delivered during the same period.


Reference values for glucose tolerance test in the urban Zimbabwean pregnant woman. A 50 g oral glucose tolerance test (OGTT) was performed on normal pregnant urban Zimbabwean women at 26-28 weeks (n = 65) and 36-40 weeks (n = 72) gestation. Women with factors predisposing to impaired glucose tolerance were excluded. The fasting, one hour and two hours values were compared to the North American standards as proposed by O'Sullivan and Mahan which are currently in use. The mean fasting levels were significantly higher and those at one and two hours significantly lower than the standards. The study also failed to demonstrate impairment of glucose tolerance with advancing gestation. These may have implications in the screening for impaired glucose tolerance in our African population.


Cancer of the oesophagus in Zimbabwe.

Cancer of the oesophagus in Zimbabwe. Data for oesophageal cancers were requested from the Cancer Registry of Zimbabwe for the three years 1986-1988. There were 437 cases notified compared with 783 cases recorded in the Government's Statistical returns. There was a ratio of 6.9 males to one female accounting for 13.2 pc of all reported cancers in males and 1.7 pc of cancers in females. Prevalence increased with age to a rate of around 83 per hundred thousand in men aged 55 and older and 19 per hundred thousand in women over 65 years old. Notifications are only received from the Central Hospitals in Harare and Bulawayo. Addresses were not available for 31 pc of patients so that geographical variations could not be determined accurately. However, higher rates occurred in Harare and in the Mashonaland Provinces. It is recommended that cancer notifications should be obtained from all Government, Mission and Private hospitals in Zimbabwe.


Skin sensitivity patterns to inhalant allergens in Nigerian asthmatic patients. The pattern of skin sensitivity response to inhalant allergens in 746 Nigerian asthmatics and 92 normal controls tested over 10 years period is reported. Significant differences in reactions were observed in seven of the 16 allergens routinely used in the study. House dust mite--dermatophagoides pteronyssinus--gave the highest incidence of positive skin reaction in 58 pc asthmatics and 4.3 pc controls (P = 0.002). Other significant allergens were house dust, 51.7 pc, feathers; 24.4 pc, dog hair, 12.9 pc, cat fur, 11.9 pc; grass pollen, 6.8 pc and flower pollen, 6.3 pc. More males than females reacted to most of the allergens but this was statistically significant only for feathers (p less than 0.02). There was good apparent association between history and skin sensitivity for most allergens. Eighty-two comma three percent of patients with a positive skin test to D. pteronyssinus gave a positive history of dust induced asthmatic attacks. Similar results were obtained for pollens; (grass, 86.3 pc, flower, 83 pc and feathers 57.7 pc.) The association was, however, poor in respect of animal allergens (dog, 30.6 pc; cat, 36 pc). From the findings of this study, there may be no point in routinely testing patients with a battery of allergens most of which may have no importance in the tropics. Attempts are, however, underway to incorporate local substances in skin testing materials. It is hoped that the results of this study will assist doctors who may not have access to skin test reagents in management of their asthmatic patients.


Evaluation of Pathstrips for semi-quantitative determination of proteinuria. Pathstrips semi-quantitatively determine concentration of protein in urine. The Pathstrip has a protein detection limit of 0.3 g/l and is stable at high temperature (40 degrees Celsius). Pathstrip was found favourably comparable to currently available Reagent strips for protein detection in urine (e.g. Ames Reagent strips). The Pathstrip is easy to use and add in the range of reagent strips for detection of urine protein as a test near the patient. No significant difference was obtained in semi-quantitation of urine proteins between Pathstrips and other currently available Reagent strips (e.g. Ames and BM Test).


Carcinoma of the cervix and cervical cytology--short epidemiological review. Carcinoma of the cervix is an important disease of well-documented epidemiology but uncertain cause. It causes appreciable morbidity and mortality in all countries, including Zimbabwe, with a significant load on curative services. Epidemiology and the role of cytology are reviewed. Cytology screening programmes have suffered from an inability to cover whole populations, particularly less affluent and/or socially disadvantaged groups, which are most at risk. Despite this difficulty, the magnitude of the problem makes it necessary to continue its study. In particular, efforts are needed in developing countries to study incidence, to better define high risk groups and to devise economical ways of detecting more cases in the earlier stages.


Adult intussusception in northern Nigeria: a changing disease? Intussusception in the tropics and sub-tropics had always been regarded as mainly a disease of adults. This paper reports on both a decrease in frequency of the disease in the adult as well as a reduction in the frequency of large bowel involvement. Sixteen adults were treated for intussusception in Ahmadu Bello University Teaching Hospital, Zaria in 10 years. The correct diagnosis was made in one patient prior to operation. Eight intussusceptions were of small intestinal origin and eight were colonic. One patient in the enteric group and three in the colonic group had benign tumours. One colonic tumour was malignant. Eleven intussusceptions were "idiopathic". Four patients in the enteric group had resection; one for neoplasm and three for irreducibility. Four patients in the colonic group had resection, all for neoplasm. Morbidity was minimal and there was no death. A treatment strategy at variance with that advocated in the Western world is suggested.


Headache in ischemic cerebrovascular disease. Part I: Clinical features. Headache is a common, although under-emphasized, feature of ischemic cerebrovascular disease. In this review, we summarize the literature on the clinical features of headache in ischemic cerebrovascular disease (Part I) and elaborate upon the underlying mechanisms of the head pain (Part II--December issue).


Reduction of calcitonin gene-related peptide in jugular blood following electrical stimulation of rat greater occipital nerve. Although it is known that pain in the forehead may be induced by neck abnormalities, the actual neck-head connections responsible for development of pain in trigeminal areas are poorly understood. Vasoactive neuropeptides released from sensory fibres, such as substance P (SP) and calcitonin gene-related peptide (CGRP), have been considered as important elements in headache pathophysiology. The levels of CGRP-like immunoreactivity (LI) were measured bilaterally in the jugular blood (52 rats) and intraocular aspirates (66 rats) following electrical stimulation of the left greater occipital nerve, and in the jugular blood of 13 control animals. One-third of the stimulated rats had varying combinations of conjunctival injection, tearing, diminished eye aperture and miosis or mydriasis on the stimulated side. The other two-thirds exhibited no ocular signs. Significantly lower levels of CGRP-LI were present in the jugular blood on the stimulated side in comparison with control rats. There was comparatively lower CGRP-LI on the non-stimulated side as well, but to a lesser extent. Significant differences between the stimulated and the non-stimulated side were present, particularly in the tearing/diminished eye cleft group. It is proposed that stimulation of the rat GON inhibits the trigeminal system (reduction of CGRP-LI) and possibly activates parasympathetic fibres (ocular changes).


The influence of acetazolamide on cerebral low-flow regions in migraine--an interictal 99mTc-HMPAO SPECT study. Acetazolamide, a carbonic anhydrase inhibitor, has proved to be useful in the assessment of "vasodilatory capacity" in cerebrovascular disease. To obtain further information on the nature of interictal low-flow regions in migraine, we reinvestigated 20 asymptomatic patients suffering from migraine with aura (n = 15) or without aura (n = 5) and who had either minor (n = 12) or marked (n = 8) regional hypoperfusion when examined in a previous 99mTc-HMPAO SPECT investigation. These patients received acetazolamide IV prior to tracer application. In 14/20 cases regional hypoperfusion resolved. Three patients with migraine with aura had less pronounced regional hypoperfusion compared to baseline. No change in baseline hypoperfusion was detectable in three older patients. No further decreases in flow were measured. In contrast to patients with cerebrovascular ischemia, in whom acetazolamide usually enhances low-flow regions, vasodilatory capacity appears intact in most migraine patients with interictal regional hypoperfusion. Thus, the "acetazolamide test" might be useful in the differential diagnosis of migraine with aura from transient cerebrovascular ischemia.


The blink reflex in migraine.

The blink reflex in migraine. The blink reflex is an objective and useful method to study the trigeminal system. It was recorded in 43 migraine patients and the findings compared with those of 31 healthy controls. The latencies of the R1 component were in the normal range in both groups. The R2 latencies ranged between 30 and 32 ms in the control group. In contrast, more than half of the patients with migraine had R2 latencies between 32 and 35 ms in the migraine group. Some migraine patients had latencies above 35 ms. The R2 latency was statistically significantly different between controls and migraineurs (p < 0.0001). Our findings indicate that trigeminal afferents and/or polysynaptic pathway in brainstem may be altered in migraine.


Transcranial Doppler ultrasonic features in episodic tension-type headache. In a pilot study we used transcranial Doppler ultrasound (TCD) to measure cerebral blood flow velocities in 21 headache-free episodic tension-type headache sufferers and in the same number of age- and sex-matched control subjects. We found increased blood flow velocities in the anterior, middle and posterior cerebral arteries and a decreased pulsality index in the middle and posterior cerebral arteries in tension-type headache sufferers compared to controls. Vascular bruits were significantly more frequent in the basal cerebral arteries of the patients compared to controls. There were no significant asymmetries of blood flow velocities in corresponding arteries. The findings suggest a multifactorial pathogenesis in episodic tension-type headache including vascular abnormalities.


The optimal length of headache recording in juvenile migraine patients. The representativeness of headache recording periods of one, two, three, four and five weeks for juvenile migraine was examined in 41 juvenile patients (suffering from migraine attacks at least twice a month). At pre-treatment and at follow-up, on average a three-week recording period was found to be adequate. On the basis of these findings a longer data-collection period is recommended for juvenile migraineurs than for adult migraine patients at follow-up.


Headache and Chiari type I malformation: occurrence in female monozygotic twins and first-degree relatives. The Chiari type I malformation has recently been reported in three generations of a family, including female monozygotic twins. Headache was the main symptom in several family members. Various headache patterns consistent with migraine without aura, cough headache, tension headache and hypoliquorrhoeic headache were present, and different patterns might coexist in the same patient. In the twins and their mother, who all had a headache consistent with migraine without aura, there were atypical features such as occipital pain location, lack of side-shift of the pain, lack of ergotamine effect and precipitation of attacks by Valsalva-like manoeuvres. The headache was significantly relieved by operation in the one twin with major herniation and the most marked headache. We suggest that in these patients the migraine-like headache mimics true migraine but that there is a causal relationship between the different headache types reported and the malformation.


Pain as the only manifestation of internal carotid artery dissection. Internal carotid artery dissection is a major cause of ischemic stroke in the young. Pain is the leading symptom and is associated with other focal signs such as Horner's syndrome and painful tinnitus or with signs of cerebral or retinal ischemia. We report two patients with angiographically confirmed extracranial internal carotid artery dissection presenting with cephalic pain as the only manifestation. The first patient had a diffuse headache and a latero-cervical pain lasting for 12 days, reminiscent of carotidynia. The second patient experienced an exploding headache suggestive of subarachnoid hemorrhage, which was ruled out by computed tomography of the head and cerebrospinal fluid study. These patients demonstrate that recognition of carotid artery dissection as a cause of carotidynia and headache suggestive of subarachnoid hemorrhage may permit an earlier diagnosis and possibly the prevention of a stroke through the use of anticoagulation.


Ipsilateral cluster headache and chronic paroxysmal hemicrania: two case reports. We report two patients with ipsilateral attacks of cluster headache and chronic paroxysmal hemicrania. The first patient, a 33-year-old man, started having attacks of chronic cluster headache at the age of 27. At 33, they were replaced by typical attacks of ipsilateral chronic paroxysmal hemicrania which showed a dramatic improvement with indomethacin 150 mg daily. After two days of complete remission, cluster headache attacks reappeared and persisted until verapamil, 360 mg a day, was added to indomethacin. The second patient, a 45-year-old man, first developed attacks of episodic cluster headache at the age of 35. At 44, he experienced ipsilateral typical attacks of chronic paroxysmal hemicrania, and two months later attacks of cluster headache. Under verapamil 240 mg daily, attacks of cluster headache disappeared, but those of chronic paroxysmal hemicrania increased in frequency until indomethacin 150 mg daily was added. These observations suggest a close relationship but not a similarity between cluster headache and chronic paraoxysmal hemicrania, and show the practical therapeutic interest of maintaining this distinction.


Platelet size and volume distribution measured by automated platelet analyzer. Thirty migraine without aura patients between attacks, 10 other during a migraine without aura attack and 30 normal subjects without headache were studied for platelet size and volume distribution using a new quantitative automated hematology analyzer (Coulter STKS). Platelet histograms, platelet counts and mean platelet volume were not significantly different in the three populations.


A double-blind controlled study of nifedipine as an abortive treatment in acute attacks of migraine with aura. Patients with migraine with aura treated each of six acute attacks with either nifedipine or vehicle administered in double-blind, randomized form. Two modes of administration were studied. Both increased the intensity of headaches compared to vehicle. We conclude that nifedipine is not useful as an abortive treatment of migraine with aura.


Salvage angioplasty: an alternative to high risk surgery for unstable angina? This prospective, Human Subjects Committee and Ethics Committee approved investigation was performed to determine if coronary angioplasty (PTCA) might be a reasonable alternative revascularization method for unstable angina patients thought to be at high risk for operative (CABG) mortality. Between March 1990 and October 1991, thirty-four consecutive patients with medically refractory rest angina were deamed to have high risk of surgical mortality and underwent PTCA without surgical backup. Predicted operative mortality was calculated for each patient based upon the VA Surgical Risk Assessment model. Angioplasty of 52 vessels was attempted. Reduction in lumenal narrowing to < 50% and improved angiographic flow was obtained in 47 vessels. There were four complicating infarctions. One death occurred in the lab, and three patients with unsuccessful angioplasty died within 30 days of pump failure. Relief of angina occurred in 30/34. Thirty patients were discharged home. In follow-up from 1 to 12 months, there have been 2 late sudden deaths at 4 months and 9 months, 1 death from lung cancer; 4 patients have stable exertional angina; 2 are awaiting heart transplant but are pain free, and one patient who had PTCA during cardiogenic shock from acute myocardial infarction had elective coronary artery bypass surgery. There have been no late myocardial infarctions. The observed angioplasty 30-day mortality of 11.8% (95% confidence limit 1% to 22.6%) compares favorably with the predicted operative mortality of 23.8% for this group. This prospective but non-randomized series supports the concept that balloon angioplasty may be a reasonable alternative to surgical intervention in some patients with unstable angina and high risk for surgery. A prospective randomized trial is warranted.


Balloon catheter requirements during contemporary percutaneous transluminal coronary angioplasty. In order to examine the outcome of utilizing multiple balloon catheters during the percutaneous transluminal coronary angioplasty (PTCA), 300 consecutive PTCAs were analyzed. PTCA was performed in a single lesion in 163 patients (54%), in multiple lesions in 52 patients (18%), and in multiple vessels in 85 patients (28%). A total of 488 balloon catheters were used (1.6/case, range 1-9) and in 112 cases (37%) > or = 2 catheters were used. Multiple balloon catheters were needed in 27 single lesion (17%), 24 multilesion (48%), and 61 multivessel (72%) cases (p < .05). There was no difference in the angiographic success rate in cases completed with 1 balloon catheter (238/251, 95%) compared to those requiring > or = 2 catheters (260/274, 95%). Emergency bypass was required in 5 patients (1.7%) and there were 2 deaths (0.6%). Thus, although contemporary PTCA frequently requires multiple balloon catheters for completion, success rates remain high and appear to offset the increased procedural expenses.


Changing incidence and management of abrupt closure following coronary intervention in the new device era. Abrupt closure of the dilated segment occurs in approximately 4 to 7% of cases following conventional percutaneous coronary angioplasty. Additional balloon angioplasty reverses roughly 40% of these closures, to yield an overall 1.6 to 3.4% rate of emergent surgery. The impact of new devices on the incidence and reversal rate of abrupt closure has not been examined. Abrupt closure occurred in 80 (4.2%) of 1,919 consecutive coronary angioplasties performed in our single center, 389 (20%) of which were performed using newer interventions (208 Palmaz-Schatz stents, 170 directional coronary atherectomies, and 11 elective laser balloon angioplasties). Abrupt closure was less frequent following newer coronary interventions (1.8%) compared to standard balloon angioplasty (4.9%, P < 0.01), possibly reflecting case selection. When abrupt closure did occur, percutaneous rescue was successful in 53 (66%) patients, including 42 (53%) who were rescued using standard or perfusion balloon angioplasty, and 11 (13%) who were rescued using laser balloon balloon angioplasty after failure of additional angioplasty attempts. Medical therapy alone was used in 8 (10%), while emergent coronary bypass surgery was performed in 18 (23%), yielding an overall emergent surgery rate of 0.9%. Q-wave myocardial infarction was significantly less frequent (0.2%) following percutaneous rescue, compared to either medical therapy (25%) or emergent surgery (33%, P < 0.001). In our catheterization laboratory, use of these 3 new coronary interventional devices coincides with a trend towards a lower incidence of abrupt closure, a higher percutaneous rescue rate with reduced incidence of myocardial infarction, and a lower emergent bypass surgery rate.


Coronary angioplasty of bifurcational lesions without protection of large side branches. To assess the risk and clinical relevance of side branch (SB) occlusion during angioplasty (PTCA) we attempted PTCA of major branches (MB) without protection of lesion-associated large (> or = 1.8 mm) SBs in 67 patients (50 men). There were 32 patients with unstable angina and 35 with stable angina. Their mean age was 55 years (range 31-77). There were 69 SBs: 43 with severe ostium stenosis (type A); 6 with severe non ostial stenosis (type B); and 20 with no or slight nonostial stenosis (type C). PTCA of the MB was successful in all but one patient who underwent acute bypass surgery. After MB PTCA occlusion occurred in 10 SBs (7A, 1B, 2C) and was asymptomatic in 5. Recanalization and dilatation was successful in 4 out of 5 symptomatic SB occlusions. A single patient developed a non-q wave myocardial infarction. PTCA was also attempted in 21 diseased SBs and failed in one. 21 SBs remained severely stenotic and 6 occluded. During follow-up symptomatic MB restenosis occurred in 12 patients, associated with restenosis in 4 out of 6 dilated SBs. Four patients underwent bypass surgery and 8 repeat successful PTCA. The SB was redilated in 2 cases and occluded silently in one. Angiography in 16 asymptomatic patients showed moderate MB restenosis in 3 and SB occlusion in 2. At 2.2 years follow-up 60 (89%) patients were asymptomatic with a normal exercise test and/or maintained angiographic result. Angioplasty of bifurcational lesions without SB protection can be effectively performed with a low rate of complications and a favourable long-term outcome.(ABSTRACT TRUNCATED AT 250 WORDS)


Measurements of central blood vessels in infants and children: normal values. In order to provide physicians interpreting vascular radiographic studies with normal data regarding central blood vessel size in children and to facilitate the design and adaptation of intravascular devices for pediatric use, we measured lengths and diameters of central blood vessels in 141 radiographic studies in 136 children. The diameters of the following vessels were determined: right and left internal jugular veins and common carotid arteries; the inferior vena cava and the descending thoracic aorta; right and left iliac veins; and right and left femoral veins and arteries. In addition, the lengths of the inferior vena cava and the descending aorta were also determined. Blood vessel dimensions were highly correlated with age, height, weight, and body surface area. The linear regression equations for each measured dimension against age, weight, height, and surface area are provided, along with a table of predicted vessel size as a function of age.


Excimer laser ablation before autoperfusion balloon inflation: a novel therapeutic approach to high grade stenoses in vessels supplying substantial myocardium at risk. The utility of a novel approach employing excimer laser ablation to form a channel for subsequent autoperfusion balloon angioplasty is presented. Two important advantages of this strategy are highlighted: (1) applicability to severe stenoses in vessels supplying substantial myocardium at risk and (2) ability to allow prolonged inflation time to minimize procedure related ischemia and optimize revascularization of the heart. We prospectively selected and studied five patients and performed excimer laser coronary angioplasty using either a 1.3 mm or 1.6 mm laser catheter followed by autoperfusion balloon dilatation. Procedural success was documented by a significant reduction in mean percent diameter stenosis from 89 +/- 4% (S.D.) to 53 +/- 4% after laser angioplasty (p < 0.0001) and subsequently to 20 +/- 4% after autoperfusion balloon angioplasty (p < 0.0001). Clinical success was attained and characterized by resolution of anginal symptoms for at least 4 weeks after hospital discharge. There were no major acute complications encountered; however, restenosis has occurred in 2 out of 5 patients. We therefore recommend this novel pre-dilation strategy with excimer laser followed by autoperfusion balloon angioplasty in selected patients with an extensive amount of myocardium at risk.


Shearing of the plastic coating of a hydrophilic guide wire in a right femoropopliteal bypass graft: removal from the contralateral side. The plastic coating of a hydrophilic guide wire tore off during introduction through a beveled single wall arterial entry needle in a 75 year old male. The sheared piece of coating got stuck in his femoropopliteal bypass. It was removed by means of a modified arterial filter set from the contralateral side. The technique of its removal is described.


Budd-Chiari syndrome treated successfully by percutaneous transluminal balloon angioplasty: two cases followed-up for 6 years. We report 2 cases of Budd-Chiari syndrome that are due to complete membranous obstruction between the junction of the inferior vena cava and the right atrium. Both cases were treated successfully by King's bioptome breakthrough followed by balloon dilatation of the membrane. The patients showed remarkable symptomatic improvement and promising hemodynamic and angiographic results immediately after the procedure and 6 years later. We conclude that balloon angioplasty is a safe and effective method for treating this rare disease in selected cases.


Anomalous origin of the left coronary artery from the pulmonary artery: direct assessment of anomalous and collateral coronary flow by pulsed Doppler echocardiography. We directly assessed the anomalous and collateral coronary flow profiles of a 58 year-old-man with Bland-White-Garland syndrome using pulsed Doppler echocardiography. Doppler recordings in this patient document the utility of pulsed Doppler echocardiography in the assessment of a cardiac shunt associated with a congenital coronary anomaly in an adult.


Endomyocardial biopsy: advantages of adding a posterior bend to the bioptome. A modification of the distal end of a flexible bioptome used for endomyocardial biopsy is described which facilitates the approach to the interventricular septum. Contact with the right ventricular free wall is avoided.


Peripheral stent recovery after failed intracoronary delivery. The Palmaz-Schatz stent can be successfully deployed in most patients. However, in a small percentage of instances a systemic embolism of the stent has been reported. In the present article we describe an easy and fast method of stent capture when detachment from the balloon has happened (pulling back the stent-loaded balloon into the guiding catheter or femoral sheath). In this situation we propose the use of the coronary guide-wire "as a guide" to capture coaxially the lost stent. This method allows for continuing the procedure without removal of the femoral sheath.


Long-tip guiding catheter: successful and safe for left coronary artery angioplasty. A long-tip guiding catheter was designed for angioplasty of the left coronary artery. Principal factors of guiding catheter function were identified, and the catheter's shape was designed to utilize them efficiently. Emphasis was placed on an overbent secondary curve (150-180 degrees) for more precise catheter control. The distal tip of the catheter is 2 cm long in the 4.0 size and the primary bend is shallow, approximately 20%. A 1.5 cm long segment between the secondary and tertiary curves enhances stability and support. Catheter performance was studied during procedures on 90 patients; 89 patients underwent coronary artery angioplasty and one patient underwent diagnostic angiography. The success rate for angioplasty was 95% with no major complications. Mild pressure damping occurred in 18 patients, and mild catheter displacement from the left main coronary artery occurred in 24 patients. Catheter support was judged as excellent to very good in 82 patients. Judkins or Amplatz catheters were not required during this study. The observed disadvantages of the long-tip catheter were the risk of catheter buckling up during advancement into the left main coronary artery and, perhaps, a higher risk of pressure damping. Superselective engagement of the catheter in the left anterior descending or circumflex arteries may be a problem when the left main coronary artery is very short. This study showed the long-tip catheter to be safe and highly successful for angioplasty of the left coronary artery.


Molecular signals in the interactions between plants and microbes. The field of plant-microbe interactions has witnessed several recent breakthroughs, such as the molecular details of vir gene induction, identification of Nod factors, and the cloning and characterization of avr genes. Other breakthroughs, such as the cloning and characterization of R genes, appear imminent. Parallels to mammalian systems are emerging in the world of plant-microbe interactions, for example, ion channels formed by Rhizobium proteins, similarities of hrp genes to pathogenicity genes of mammalian pathogens, and plant signal transduction via calcium and protein phosphorylation. We remain, however, largely ignorant of many facets of signaling in plant-microbe interactions. We know little about how microbial signals are perceived by plants or how subsequent signal transduction occurs within plant cells and are probably unaware of many of the microbe-generated signals to which plants respond or of plant-generated signals to which bacteria and fungi respond. Contributions from those working on the genetics, molecular biology, and physiology of bacteria, fungi, and plants will be required to address these questions. The many nonpathogenic plant-microbe interactions in addition to the Rhizobium-plant interaction remain relatively unexplored. Genetic and molecular approaches are being initiated to investigate the signaling that is likely to underlie interactions such as those between mycorrhizal fungi and plant roots and between epiphytic bacteria and plant leaf surfaces. The importance of these interactions to plant growth and development makes it likely that they will figure more prominently at future symposia.(ABSTRACT TRUNCATED AT 250 WORDS)


Protein splicing in the maturation of M. tuberculosis recA protein: a mechanism for tolerating a novel class of intervening sequence. The M. tuberculosis recA locus comprises an 85 kd open reading frame but produced 38 kd RecA and 47 kd products in E. coli. No RNA processing was detected; rather, an 85 kd precursor protein was spliced, releasing a 47 kd spacer protein, and joining its terminal fragments to form mature RecA protein. "Spacer" protein was also produced in M. tuberculosis and from a hybrid spacer-LacZ alpha fusion molecule. Mutagenesis at codon wobble positions at one splice junction showed that protein rather than nucleotide sequence determined splicing activity. Other mutants defined additional regions needed for splicing and allowed processing to be followed. Splicing was essential for RecA activity in E. coli. The possibility that splicing is a manifestation of a novel class of genetic element is discussed.


PCF4 encodes an RNA polymerase III transcription factor with homology to TFIIB. A dominant mutation in the PCF4 gene of S. cerevisiae was isolated as a suppressor of a tRNA gene A block promoter mutation. In vitro studies indicate that PCF4 is a stoichiometrically-required RNA polymerase III (pol III) transcription initiation factor. We show that the PCF4-1 mutation increases the number of transcriptionally competent preinitiation complexes by affecting a limiting activity in yeast cell extracts that is squelched by excess TFIIIC. The PCF4 gene encodes a TFIIB homolog whose size, biochemical, and genetic properties are consistent with those of the 70 kd subunit of TFIIIB. The TFIIB homology of PCF4 suggests a means for determining the polymerase specificity of a gene.


A suppressor of TBP mutations encodes an RNA polymerase III transcription factor with homology to TFIIB. The TDS4 gene of S. cerevisiae was isolated as an allele-specific high copy suppressor of mutations within the basic region of the TATA-binding protein (TBP). The gene is essential for viability and encodes a 596 aa protein. The first 300 aa of the TDS4 protein exhibit significant sequence similarity to the RNA polymerase II transcription factor TFIIB. However, TDS4 is required for RNA polymerase III transcription in vivo and in vitro. Antibodies specific for TDS4 or TBP react with the TFIIIB complex, indicating that both proteins are components of the RNA polymerase III initiation complex. These findings suggest that the RNA polymerase II and III initiation mechanisms are extremely similar, and they explain how the TATA-binding protein can function in both systems.


A novel B cell-derived coactivator potentiates the activation of immunoglobulin promoters by octamer-binding transcription factors. A novel B cell-restricted activity, required for high levels of octamer/Oct-dependent transcription from an immunoglobulin heavy chain (IgH) promoter, was detected in an in vitro system consisting of HeLa cell-derived extracts complemented with fractionated B cell nuclear proteins. The factor responsible for this activity was designated Oct coactivator from B cells (OCA-B). OCA-B stimulates the transcription from an IgH promoter in conjunction with either Oct-1 or Oct-2 but shows no significant effect on the octamer/Oct-dependent transcription of the ubiquitously expressed histone H2B promoter and the transcription of USF- and Sp1-regulated promoters. Taken together, our results suggest that OCA-B is a tissue-, promoter-, and factor-specific coactivator and that OCA-B may be a major determinant for B cell-specific activation of immunoglobulin promoters. In light of the evidence showing physical and functional interactions between Oct factors and OCA-B, we propose a mechanism of action for OCA-B and discuss the implications of OCA-B for the transcriptional regulation of other tissue-specific promoters.


The precursor of NF-kappa B p50 has I kappa B-like functions. The C-terminal half of the p105 precursor of the NF-kappa B p50 subunit contains ankyrin-like repeats similar to those in I kappa B molecules, which are known to retain NF-kappa B complexes in the cytoplasm. We demonstrate that in various cell lines p105 is found associated with either c-rel or p65 in the cytoplasm and serves I kappa B-like functions. p105 retains c-rel or p65 in the cytoplasm in cotransfection experiments in COS cells. It also inhibits DNA binding by c-rel in gel retardation assays. Stable interaction of p105 with c-rel or p65 requires the putative dimerization domain in the conserved rel homology region of p105, as well as a second contact with the I kappa B-related C-terminal part of p105. Pulse-chase experiments indicate that cytoplasmic complexes of p105 with c-rel or p65 give rise to cytoplasmic as well as nuclear p50-c-rel and p50-p65, respectively, probably through processing of p105. Thus, p105, like the I kappa Bs, controls the subcellular localization and hence the transcriptional activity of at least two other members of the rel/NF-kappa B family.


The chromatin-associated protein H-NS interacts with curved DNA to influence DNA topology and gene expression. H-NS is an abundant structural component of bacterial chromatin and influences many cellular processes, including recombination, transposition, and transcription. We have studied the mechanism of action of H-NS at the osmotically regulated proU promoter. The interaction of H-NS with a curved DNA element located downstream of the proU promoter is required for normal regulation of expression. Heterologous curved sequences can replace the regulatory role of the proU curve. Hence, the luxAB and lacZ reporter genes, which differ in the presence or absence of a curve, can indicate very different patterns of transcription. H-NS interacts preferentially with these curved DNA elements in vitro. Furthermore, in vivo the interaction of H-NS with curved DNA participates in the control of plasmid linking number. The data suggest that H-NS-dependent changes in DNA topology play a role in the osmoregulation of proU expression.


The arrest of replication forks in the rDNA of yeast occurs independently of transcription. Replication forks, moving opposite to the direction of transcription, are arrested at the 3' ends of the 35S transcription units in the rDNA locus of S. cerevisiae. Because of its position and polarity, we tested the hypothesis that this replication fork barrier (RFB) results from the act of transcription. Three results contradict this hypothesis. First, the RFB persists in a strain containing a disruption of the gene for the 135 kd subunit of RNA polymerase I. Second, the RFB causes a polar arrest of replication forks when transplanted to a plasmid. Third, transcription by RNA polymerase II of a plasmid copy of the 35S transcription unit lacking the RFB does not generate a barrier. We propose that replication forks are arrested in a directional manner through the binding of one or more proteins to two closely spaced sites in the RFB.


Localization of nanos RNA controls embryonic polarity. Anterior-posterior polarity of the Drosophila embryo is initiated during oogenesis through differential maternal RNA localization. The RNA of the anterior morphogen bicoid is localized to the anterior pole of the embryo, where bicoid protein controls head and thorax development. The RNA of the posterior morphogen nanos is localized to the posterior pole, where nanos protein is required for abdomen formation. Here we show that the nanos 3' untranslated region, like that of the bicoid RNA, is sufficient for RNA localization. We have used the bicoid RNA localization signal to mislocalize nanos, producing embryos with two sources of nanos protein. Such embryos form two abdomens with mirror image symmetry. Embryos with nanos RNA localized only to the anterior have greater nanos gene activity than embryos with nanos RNA localized posteriorly. We propose a role for RNA localization in regulating nanos activity.


Cytotoxicity with target DNA breakdown by rat basophilic leukemia cells expressing both cytolysin and granzyme A. The noncytotoxic rat mast cell tumor line RBL was transfected with genes for the cytotoxic lymphocyte granule proteins cytolysin (perforin) and granzyme A, giving transfectants with mRNA and protein expression levels comparable with cloned cytotoxic T lymphocytes. Both RBL-cytolysin and RBL-cytolysin-granzyme A transfectants showed extremely potent killing of red cell targets and lysed 20%-60% of EL4 lymphoma targets at an effector-to-target ratio of 30. RBL transfectants expressing only granzyme A were not cytotoxic. Significant EL4 DNA breakdown accompanying lysis was observed only with RBL that was transfected with both cytolysin and granzyme A. These results support the granule-exocytosis model for lymphocyte cytotoxicity and show that effector granzyme A plays a role in target cell DNA breakdown.


Identification and properties of an atypical catalytic subunit (p34PSK-J3/cdk4) for mammalian D type G1 cyclins. Murine D type cyclins associate with a catalytic subunit (p34PSK-J3) with properties distinct from known cyclin-dependent kinases (cdks). Mouse p34PSK-J3 shows less than 50% amino acid identity to p34cdc2, p33cdk2, and p36cdk3, lacks a PSTAIRE motif, and does not bind to p13suc1. Cyclin D1-p34PSK-J3 complexes accumulate in macrophages during G1 and decline in S phase, whereas complexes involving cyclins D2 and D3 form in proliferating T cells. Although histone H1 kinase activity is not detected in cyclin D or PSK-J3 immunoprecipitates, cyclin D-p34PSK-J3 complexes assembled in vitro stably bind and phosphorylate the retinoblastoma gene product (pRb) and an Rb-like protein (p107) but do not interact with pRb mutants that are functionally inactive. Thus, p34PSK-J3 is a cyclin D-regulated catalytic subunit that acts as an Rb (but not H1) kinase.


Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells. apoE-deficient mice have been created by homologous recombination in ES cells. On a low fat, low cholesterol chow diet these animals have plasma cholesterol levels of 494 mg/dl compared with 60 mg/dl in control animals, and when challenged with a high fat Western-type diet, these animals have plasma cholesterol levels of 1821 mg/dl compared with 132 mg/dl in controls. This marked hypercholesterolemia is primarily due to elevated levels of very low and intermediate density lipoproteins. At 10 weeks of age, apoE-deficient mice have already developed atherosclerotic lesions in the aorta and coronary and pulmonary arteries. apoE-deficient mice are a promising small animal model to help understand the role of apoE in vivo and the genetic and environmental determinants of atherosclerosis.


Targeted inactivation of the muscle regulatory gene Myf-5 results in abnormal rib development and perinatal death. The Myf-5 gene, a member of the myogenic basic HLH factor family, has been inactivated in mice after homologous recombination in ES cells. Mice lacking Myf-5 were unable to breathe and died immediately after birth, owing to the absence of the major distal part of the ribs. Other skeletal abnormalities, except for complete ossification of the sternum, were not apparent. Histological examination of skeletal muscle from newborn mice revealed no morphological abnormalities. Northern blot analysis demonstrated normal levels of muscle-specific mRNAs including MyoD, myogenin, and Myf-6. However, the appearance of myotomal cells in early somites was delayed by several days. These results suggest that while Myf-5 plays a crucial role in the formation of lateral sclerotome derivatives, Myf-5 is dispensable for the development of skeletal muscle, perhaps because other members of the myogenic HLH family substitute for Myf-5 activity.


Fertility in mice requires X-Y pairing and a Y-chromosomal "spermiogenesis" gene mapping to the long arm. There is accumulating evidence that the mammalian Y chromosome, in addition to its testis-determining function, may have other male limited functions, particularly in spermatogenesis. We have previously shown that the short arm of the mouse Y carries information needed for spermatogonial proliferation. This information, together with the testis-determining gene Sry, is contained within the Y-derived sex reversal factor Sxra. XO males carrying a copy of Sxra attached to the X chromosome are nevertheless sterile owing to an almost complete arrest during the meiotic metaphase stages. Here we show that this meiotic block can be overcome by providing a meiotic pairing partner (with no Y-specific DNA) for the XSxra chromosome. However, this does not restore fertility because the sperm produced all have abnormal heads. It is concluded that the Y-specific region of the mouse Y chromosome long arm includes information essential for the normal development of the sperm head.


LDL receptor-related protein internalizes and degrades uPA-PAI-1 complexes and is essential for embryo implantation. The low density lipoprotein receptor-related protein (LRP) is a large multifunctional clearance receptor that has been implicated in the hepatic uptake of chylomicron remnants and in the removal of protease-inhibitor complexes from the circulation and from the extracellular space. Disruption of the LRP gene in mice blocks development of LRP-/- embryos around the implantation stage. The expression pattern of LRP in the postimplantation stage embryo is identical to that of urokinase, a plasminogen activator that confers invasive properties to migrating cells. We demonstrate that LRP mediates uptake and degradation of urokinase-type plasminogen activator-plasminogen activator inhibitor 1 complexes and propose that the inability of the giant cells to remove the inactive protease complexes from their surfaces interferes with implantation of the embryo.


Differentiation of an immature T cell line: a model of thymic positive selection. Thymocyte differentiation is dependent upon recognition of major histocompatibility complex (MHC) molecules on thymic stroma, a process called positive selection. Here we describe an immature CD4+8+ T cell line derived from a TCR transgenic mouse that differentiates into CD4+8- cells in response to antigen and nonthymic antigen-presenting cells. When injected intrathymically, these cells differentiate in the absence of antigen. The ability of immature T cells to recognize MHC molecules in the absence of foreign antigen in the thymus can thus be attributed to a unique property of thymic antigen-presenting cells. These studies also demonstrate the phenotypic and functional changes associated with TCR-mediated T cell maturation and establish an in vitro model system of positive selection.


Decapentaplegic acts as a morphogen to organize dorsal-ventral pattern in the Drosophila embryo. Zygotic expression of the Drosophila TGF beta family member decapentaplegic (dpp) is required for the development of the dorsal embryonic structures. By injecting dpp transcripts into young embryos, we find that 2- to 4-fold increases in the concentration of injected RNA elicit progressively more dorsal cell fates: only low levels of dpp permit development of ventral ectoderm, intermediate dpp levels drive dorsal epidermal development, and high dpp levels drive cells to differentiate as the most dorsal pattern element, the amnioserosa. Localized dpp RNA injections into embryos that lack all known maternal and zygotic dorsal-ventral polarity indicate that dpp can both define embryonic polarity and organize detailed patterning within the ectoderm. We infer that dpp acts as an extracellular morphogen and that the graded activity of dpp specifies the pattern of ectodermal cell fates in the Drosophila embryo.


SHR3: a novel component of the secretory pathway specifically required for localization of amino acid permeases in yeast. Mutations in SHR3 block amino acid uptake into yeast by reducing the levels of multiple amino acid permeases within the plasma membrane. SHR3 is a novel integral membrane protein component of the endoplasmic reticulum (ER). shr3 null mutants specifically accumulate amino acid permeases in the ER; other plasma membrane proteins, secretory proteins, and vacuolar proteins are processed and targeted correctly. Our findings suggest that SHR3 interacts with a structural domain shared by amino acid permeases, an interaction required for permease-specific processing and transport from the ER. Even in the presence of excess amino acids, shr3 mutants exhibit starvation responses. shr3 mutants constitutively express elevated levels of GCN4, and mutant shr3/shr3 diploids undergo dimorphic transitions that result in filamentous growth at enhanced frequencies.


The reovirus cell attachment protein possesses two independently active trimerization domains: basis of dominant negative effects. The reovirus cell attachment protein, sigma 1, is a homotrimer with an N-terminal fibrous tail and a C-terminal globular head. By cotranslating full-length and various truncated sigma 1 proteins in vitro, we show that the N- and C-terminal halves of sigma 1 possess independent trimerization and folding domains. Trimerization of sigma 1 is initiated at the N-terminus by the formation of a "loose," protease-sensitive, three-stranded, alpha-helical coiled coil. This serves to bring the three unfolded C-termini into close proximity to one another, facilitating their subsequent trimerization and cooperative folding. Concomitant with, but independent of, this latter process, the N-terminal fiber further matures into a more stable and protease-resistant structure. The coordinated folding of sigma 1 trimers exemplifies the dominant negative effects of mutant subunits in oligomeric complexes.


A mammalian homolog of SEC61p and SECYp is associated with ribosomes and nascent polypeptides during translocation. SEC61p is essential for protein translocation across the endoplasmic reticulum membrane of S. cerevisiae. We have found a mammalian homolog that shows more than 50% sequence identity with the yeast protein. Moreover, several regions of SEC61p have significant similarities with corresponding ones of SecYp of bacteria, indicating a strong evolutionary conservation of the mechanism of protein translocation. Mammalian Sec61p, like the yeast protein, is located in the immediate vicinity of nascent polypeptides during their membrane passage. It is tightly associated with membrane-bound ribosomes, suggesting that the nascent chain passes directly from the ribosome into a protein-conducting channel. These results define Sec61p as a ubiquitous key component of the protein translocation apparatus.


The product of the mouse Xist gene is a 15 kb inactive X-specific transcript containing no conserved ORF and located in the nucleus. The Xist gene maps to the X inactivation center region in both mouse and human, and previous analysis of the 3' end of the gene has demonstrated inactive X-specific expression, suggesting a possible role in X inactivation. We have now analyzed the entire mouse Xist gene. The mature inactive X-specific transcript is 15 kb in length and contains no conserved ORF. The Xist sequence contains a number of regions comprised of tandem repeats. Comparison with the human XIST gene demonstrates significant conservation of sequence and gene structure. Xist RNA is not associated with the translational machinery of the cell and is located almost exclusively in the nucleus. Together with conservation of inactive X-specific expression, these findings support a role for Xist in X inactivation, possibly as a functional RNA or as a chromatin organizer region.


The human XIST gene: analysis of a 17 kb inactive X-specific RNA that contains conserved repeats and is highly localized within the nucleus. X chromosome inactivation in mammalian females results in the cis-limited transcriptional inactivity of most of the genes on one X chromosome. The XIST gene is unique among X-linked genes in being expressed exclusively from the inactive X chromosome. Human XIST cDNAs containing at least eight exons and totaling 17 kb have been isolated and sequenced within the region on the X chromosome known to contain the X inactivation center. The XIST gene includes several tandem repeats, the most 5' of which are evolutionarily conserved. The gene does not contain any significant conserved ORFs and thus does not appear to encode a protein, suggesting that XIST may function as a structural RNA within the nucleus. Consistent with this, fluorescence in situ hybridization experiments demonstrate localization of XIST RNA within the nucleus to a position indistinguishable from the X inactivation-associated Barr body.


Pleiotropic effects of a null mutation in the c-fos proto-oncogene. The c-fos proto-oncogene has been implicated as a central regulatory component of the nuclear response to mitogens and other extracellular stimuli. Embryonic stem cells targeted at the c-fos locus have been used to generate chimeric mice that have transmitted the mutated allele through the germline. Homozygous mutants show reduced placental and fetal weights and significant loss of viability at birth. Approximately 40% of the homozygous mutants survive and grow at normal rates until severe osteopetrosis, characterized by foreshortening of the long bones, ossification of the marrow space, and absence of tooth eruption, begins to develop at approximately 11 days. Among other abnormalities, these mice show delayed or absent gametogenesis, lymphopenia, and altered behavior. Despite these defects, many live as long as their wild-type or heterozygous littermates (currently 7 months). These data indicate that c-fos is not required for the growth of most cell types but is involved in the development and function of several distinct tissues.


A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell cycle checkpoints can enhance cell survival and limit mutagenic events following DNA damage. Primary murine fibroblasts became deficient in a G1 checkpoint activated by ionizing radiation (IR) when both wild-type p53 alleles were disrupted. In addition, cells from patients with the radiosensitive, cancer-prone disease ataxia-telangiectasia (AT) lacked the IR-induced increase in p53 protein levels seen in normal cells. Finally, IR induction of the human GADD45 gene, an induction that is also defective in AT cells, was dependent on wild-type p53 function. Wild-type but not mutant p53 bound strongly to a conserved element in the GADD45 gene, and a p53-containing nuclear factor, which bound this element, was detected in extracts from irradiated cells. Thus, we identified three participants (AT gene(s), p53, and GADD45) in a signal transduction pathway that controls cell cycle arrest following DNA damage; abnormalities in this pathway probably contribute to tumor development.


The ts41 mutation in Chinese hamster cells leads to successive S phases in the absence of intervening G2, M, and G1. The ts41 mutation of Chinese hamster cells was first isolated and characterized by Hirschberg and Marcus (1982) who showed that at nonpermissive temperature, cells accumulate up to 16C equivalents of DNA. Here we show that the mutation is recessive and at nonpermissive temperature, cells replicate their genome normally, but instead of going on into G2, M, and G1, they pass directly into a second S phase. Entry into a second S phase does not require serum nor is it inhibited by G2 checkpoints or mitotic inhibitors. Temperature-shift experiments suggest that the ts41 gene product participates in two functions in the cell cycle: entry into mitosis and inhibition of entry into S phase. The ts41 mutation seems to define a class of cell cycle mutant that couples the sequential events of DNA replication and mitosis.


cactus, a gene involved in dorsoventral pattern formation of Drosophila, is related to the I kappa B gene family of vertebrates. Among the maternally active genes of Drosophila, cactus is the only one whose loss of function mutations specifically produce ventralized embryos. Its product inhibits nuclear translocation of the dorsal morphogen in the dorsal region of the embryo. Here we report the cloning of cactus and the sequencing of its maternal transcript. The identity of our clones was verified by induction of phenocopies with antisense RNA and rescue of the mutant phenotype with sense RNA. cactus is predicted to encode an acidic, cytoplasmic protein with seven ankyrin repeats. The sequence has similarity to the I kappa B proteins that inhibit the vertebrate transcription factor NF-kappa B. In analogy to results obtained with I kappa B and NF-kappa B, bacterially expressed cactus protein can inhibit DNA binding of dorsal protein in vitro.


Characterization of the Drosophila cactus locus and analysis of interactions between cactus and dorsal proteins. An oligonucleotide based on the cdc 10/SWI6 repeats of the Drosophila Notch protein was used to isolate other Drosophila genes with these repeats. One of these genes is the cactus locus, 1 of 11 genes required maternally for the establishment in embryos of a gradient of nuclear localization of dorsal protein, a rel-like transcription factor. Previous work showed that in cactus mutants more dorsal protein enters the nucleus in dorsal regions, resulting in a ventralized phenotype. It is now shown that the cactus locus produces two proteins that differ at their carboxy termini; both contain six cdc 10/SWI6 repeats that are sufficient for binding to dorsal and for inhibiting the ability of dorsal to bind DNA. The site on dorsal to which cactus binds was localized to the rel homology domain, where it overlaps with, or is adjacent to, the nuclear localization signal. In vivo the bulk of the cactus protein associated with dorsal is phosphorylated. This, or the association with dorsal, appears to stabilize the maternal cactus protein.