Vitamin A deficiency: effect on retinal structure of the moth Manduca sexta.

Sphingid moths (Manduca sexta) were reared for several generations on an artificial diet deficient in vitamin A and its precursors. Retinal tissue from depleted moths was removed for histological examination. There was extensive histolysis in the retinal epithelium and underlying nervous and connective tissues. This pathology correlated with severe visual impairment, even though normal growth, metamorphosis, and reproduction occurred. In the adult this pathology could be reversed when the larvae were reared only on tobacco (its usual host) or on the artificial diet supplemented with beta-carotene or vitamin A palmitate.

Temperature-sensitive paralytic mutations demonstrate that synaptic exocytosis requires SNARE complex assembly and disassembly.

The neuronal SNARE complex is formed via the interaction of synaptobrevin with syntaxin and SNAP-25. Purified SNARE proteins assemble spontaneously, while disassembly requires the ATPase NSF. Cycles of assembly and disassembly have been proposed to drive lipid bilayer fusion. However, this hypothesis remains to be tested in vivo. We have isolated a Drosophila temperature-sensitive paralytic mutation in syntaxin that rapidly blocks synaptic transmission at nonpermissive temperatures. This paralytic mutation specifically and selectively decreases binding to synaptobrevin and abolishes assembly of the 7S SNARE complex. Temperature-sensitive paralytic mutations in NSF (comatose) also block synaptic transmission, but over a much slower time course and with the accumulation of syntaxin and SNARE complexes on synaptic vesicles. These results provide in vivo evidence that cycles of assembly and disassembly of SNARE complexes drive membrane trafficking at synapses.

synaptotagmin mutants reveal essential functions for the C2B domain in Ca2+-triggered fusion and recycling of synaptic vesicles in vivo.

Synaptotagmin has been proposed to function as a Ca(2+) sensor that regulates synaptic vesicle exocytosis, whereas the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is thought to form the core of a conserved membrane fusion machine. Little is known concerning the functional relationships between synaptotagmin and SNAREs. Here we report that synaptotagmin can facilitate SNARE complex formation in vitro and that synaptotagmin mutations disrupt SNARE complex formation in vivo. Synaptotagmin oligomers efficiently bind SNARE complexes, whereas Ca(2+) acting via synaptotagmin triggers cross-linking of SNARE complexes into dimers. Mutations in Drosophila that delete the C2B domain of synaptotagmin disrupt clathrin AP-2 binding and endocytosis. In contrast, a mutation that blocks Ca(2+)-triggered conformational changes in C2B and diminishes Ca(2+)-triggered synaptotagmin oligomerization results in a postdocking defect in neurotransmitter release and a decrease in SNARE assembly in vivo. These data suggest that Ca(2+)-driven oligomerization via the C2B domain of synaptotagmin may trigger synaptic vesicle fusion via the assembly and clustering of SNARE complexes.

Fine structure and blood-brain barrier properties of the central nervous system of a dipteran larva.

Using scanning and transmission electron microscopy, we studied basic ultrastructure, membrane specializations, and blood-brain barrier properties of the ventral ganglion and abdominal nerves of the last (third) instar larva of a dipteran fly, Delia platura. Both ganglion and nerves are covered with a non-cellular neural lamella. A monolayer of flattened perineurial cells lies beneath the neural lamella. Perineurial cells contain stores of metabolites and nutrients and these cells extensively interdigitate with one another. An extensive extracellular series of channels pervades perineurial cells. Glial cells beneath the perineurium envelope but do not entwine axons. In a minority of cases, adjacent axons in nerve and neuropil appear to be contiguous without glial intervention. Extensive (pleated) septate junctions with triangular septa are present between perineurial cells. Hemidesmosomes, half desmosomes (a first report for invertebrates), and desmosomes were also observed. Although no tight junctions were discovered, an effective blood-brain barrier exists, and tracer (ionic lanthanum) in no case reached neuronal surfaces. Extracellular tracer halted within the extensive septate junctions between perineurial cells. We postulate that in the absence of tight junctions the functional blood-brain barrier is effected by the septate junctions in the central nervous system of the Delia larva.

Effect of dietary egg on serum cholesterol and triglyceride of human males.

One hundred fourteen male volunteers (mean age 44.6 years) consumed one whole egg daily in their customary diets for 3 months. Their final serum cholesterol (SCHOL) and triglycerides (STG) levels were compared with their initial levels on customary free choice diets and also with their levels after a 3-month elimination of dietary whole eggs. All subjects had previously confirmed normal serum lipid levels and no history of heart disease. Four-day food records were kept during both experimental dietary periods. A Latin square design allowed analysis for seasonal effects on lipid levels. No significant change in mean SCHOL on either diet was found; there was a seasonal effect on mean STG. Significant linear associations of fat intake and of energy intake were found. There was no significant association of dietary cholesterol intake with either SCHOL or STG.

Scanning electron microscopy of normal and malignant human urothelium.

Scanning electron microscopy (SEM) was used to evaluate and compare normal bladder mucosa to tumor epithelium as well as to normal-appearing mucosa in patients with diagnosed bladder tumor. Bladder urothelium adjacent to tumors exhibits a characteristic pattern of fine structural relief which is distinct from normal uroepithelium. These changes in microcontour may be guides to the identification of early malignancy, and thus the technique will be helpful in deciding how aggressive the treatment of bladder tumor should be.

Cardiovascular reactivity and interpersonal influence: active coping in a social context.

Previous studies have demonstrated that effortful attempts to secure positive outcomes or avoid negative outcomes produce significant increases in systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate (HR). Although these effects of active coping on cardiovascular reactivity are central in current psychosomatic theories, virtually all of the research to date has used impersonal, asocial tasks. Our two studies examined the cardiovascular effects of effortful attempts to influence other people. In Study 1, male subjects attempting to influence the opinions of their discussion partner to improve their own chances of winning money displayed significantly greater SBP, DBP, and HR reactivity. In Study 2, we obtained similar effects on SBP and DBP reactivity in men and women, while both preparing an influence attempt and making that attempt. Furthermore, reactivity levels were larger as the magnitude of incentive for successful persuasion increased. Implications of this interpersonal equivalent of active coping for the development of cardiovascular disease are discussed.

Enteral nutritional support management in a university teaching hospital: team vs nonteam.

Current hospital cost containment pressures have prompted a critical evaluation of whether nutritional support teams render more clinically effective and efficient patient care than nonteam management. To address this question with regard to enteral feeding, 102 consecutive hospitalized patients who required enteral nutritional support (ENS) by tube feeding during a 3 1/2-month period were prospectively studied. Fifty patients were managed by a nutritional support team; the other 52 were managed by their primary physicians. Choice of enteral formula, formula modifications, frequency of laboratory tests, and amounts of energy and protein received were recorded daily. In addition, each patient was monitored for pulmonary, mechanical, gastrointestinal, and metabolic abnormalities. Team-managed (T) and nonteam-managed (NT) patients received ENS for 632 and 398 days, respectively. The average time period for ENS was significantly longer in the team-managed patients (12.6 +/- 12.1 days vs 7.7 +/- 6.2 days, p less than 0.01). Significantly more of the team patients attained 1.2 X basal energy expenditure (BEE) (37 vs 26, p less than 0.05). Total number of abnormalities in each group was similar (T = 398, NT = 390); however, the abnormalities per day were significantly lower in the team group (T = 0.63 vs NT = 0.98, p less than 0.01). Mechanical (T = 0.05 vs NT = 0.11, p less than 0.01), gastrointestinal (T = 0.99 vs NT = 0.14, p less than 0.05), and metabolic (T = 0.49 vs NT = 0.72, p less than 0.01) abnormalities per day all were significantly lower in the team-managed patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Blue and ultraviolet light induced damage to the Drosophila retina: ultrastructure.

Intense ultraviolet (UV) and blue stimulation photolyses rhodopsin through a fluorescent metarhodopsin (M') in the predominant photoreceptor type, R1-6, of the compound eye of white eyed Drosophila melanogaster. We investigated the associated retinal degeneration using High Voltage Electron Microscopy (HVEM). The threshold for UV induced damage was about 19 log quanta/cm2 while for blue, the threshold was about 20. These intensities are toward the upper level of the dynamic range for rhodopsin photolysis. Thus, there is a sensitization for near UV induced degeneration as had been found for photolysis of the visual pigment. Vitamin A deprivation protects against light elicited retinal degeneration, particularly in the UV. Since vitamin A deprivation eliminates the blue absorbing rhodopsin and a UV sensitizing pigment in R1-6, the degeneration is likely mediated through quantal absorption through these photoexcitation pigments. Intense light converts the microvilli of the rhabdomeres (the photopigment containing organelles) into dense strands and the cytoplasm fills with a dense reticulum. Such damage is elicited shortly after stimulation and is permanent. Under most conditions, the second order interneurons are spared. These results are discussed in the context of other animal models of intense light retinal degeneration.

Membrane specializations in the first optic neuropil of the housefly, Musca domestica L. I. Junctions between neurons.

Thin section and freeze-fracture replicas of the first optic neuropil (lamina ganglionaris) of the fly Musca were studied to determine the types, extent and location of membrane specializations between neurons. Five junctional types are found, exclusive of chemical synapses. These are gap, tight and septate junctions, close appositions between retinular (R) axons and capitate projections (in which an epithelial glial cell invaginates into an R axon). Junctional types and their cellular associations follow: gap junctions, between lamina (L) interneurons, L1-L2; tight junctions, between L1-L2; L3-L4; L4-epithelial glial cell; and R7-R8. Septate junctions, between L1-L2, L3-L4, L3-beta, L4-beta, alpha-beta, and an unidentified fibre making septate junctions with L1 and L2. Close appositions are found between R axons in the distal portion of the optic cartridges of this neuropil prior to extensive R chemical synapses with L1, L2. These loci (seen in freeze-fracture replicas) have rhomboidal patches of hexagonally arrayed P face particles. Intermembranous clefts between R axons are about 50 A and are invariably electron lucent. These points of near contact between R terminals are probably the sites of low electrical resistance measured by Shaw (1979). Capitate projections are for the first time revealed in freeze fracture surfaces. Here epithelial glia send many, short, mushroom-shaped processes invaginating into R axons forming a tenacious structural bond. All four membrane leaflets (P and E faces of R axon and glial membrane) in the capitate projection possess particles in higher densities than in the surrounding nonspecialized regions. The known, general functions of each membrane specialization were correlated with the functional capacities of those lamina neurons possessing them in an effort to interpret better the integrative capacity of this neuropil. These data provide some fine structural bases for a putative 'blood-brain' barrier between lamina and haemolymph, between lamina and peripheral retina, and possibly between lamina and second optic neuropil.

Membrane specializations in the first optic neuropil of the housefly, Musca domestica L. II. Junctions between glial cells.

Membrane specializations between the three types of glial cells in the first optic neuropil (lamina ganglionaris) of the housefly were determined from thin sections and freeze-fracture replicas. Three strata of glia cells are present in the lamina. A relatively thin layer of satellite glia covers the distal (perikaryal) rind of the lamina and these cells wrap retinular axons that enter the lamina. The central synaptic fields of the lamina neurons are enclosed by epithelial glia, while the proximal surface of the lamina is capped by marginal glial cells. Satellite glia bond to each other via desmosomes, septate and gap junctions. Freeze-fracture replicas show gap junctions as aggregations of E face particles and P face pits on the intramembranous surfaces. Parallel rows of P face particles are indicative of septate junctions. Angulated, intersecting, P face particle ridges are arranged in circumferential bands around retinular axons at the glia-axon interface. Thin section correlates of these junctions are presented. Epithelial glia are characterized by elaborate series of parallel membranes which appear to be suspended in the cytoplasm but may be the invaginated plasma membranes of a neighbouring glial cell. An intermembranous cleft of 40-50 A is noted and this area has an appreciable electron density which give the appearance of a gap junction. When cleaved, these membranes show plaques of particles on the P face. The marginal glial cells are relatively large and are joined by a newly discovered junction which is characterized (from freeze-fracture data) by numerous, undulating, uninterrupted, parallel P face ridges which sometimes become circular and form enclosures. In thin section, electron-dense material fills the membrane appositional areas and in tangential sections faint diffuse parallel striae are seen. This specialized cell contact may be a variant of a continuous junction although, based on fracture replicas, there are obvious similarities to tight junctions. These membranes specializations are related, in the three dimensions of the optic cartridges, to functions in a possible blood-eye barrier system.

Lanthanum and freeze fracture studies on the retinular cell junction in the compound eye of the housefly.

The retinular (R) cell junction between adjacent photoreceptor cells in the house-fly ommatidium was characterized by freeze fracture, thin section and tracer (lanthanum) studies. Focal tight junctions occur between cells, and some P face ridge-E face groove correspondences are present in this intramembranal area. When colloidal lanthanum was introduced into the extracellular space (ECS) of the peripheral retina of the housefly, this electron-dense tracer moved from the ECS (extra-ommatidial space), through the R-cell junctions and belt desmosomes, into the ommatidial cavity (OC = intrarhabdomal space) of each ommatidium. In the OC, lanthanum outlined a meshwork structure that pervaded this space. The evidence of this tracer movement suggests that there may be ionic continuity between the "traditional" ECS and the fluid bathing the individual rhabdomeres. The volume of the OC is calculated and we suggest that this space is part of the ECS. The functional implications of this postulate are considered in the light of: (1) the different functions of the peripheral and central cells; (2) the dissimilarity of rhabdomal membrane surface facing the OC compared to the "unmodified" plasma membrane of the photoreceptor cell facing the extra-ommatidial cavity; (3) the permeability properties of the R cell junction; and (4) the total ECS containing an ion store capable of sustaining current for the generator potential.

The perineurium of the adult housefly: ultrastructure and permeability to lanthanum.

The ultrastructure of the perineurial cells of Musca overlying the first optic neuropile was examined by transmission electron microscopy. These cells are somewhat similar to those of other insects but cytoplasmic flanges seem to be absent, and mitochondria are relatively large and sinuous. The intercellular channel system on the lateral border of the cells is relatively spacious and highly meandering. Perineurial cells are joined by septate, gap, and tight junctions, hemidesmosomes, and desmosomes. Tight and septate junctions bond perineurial cells and glial cells. These data are evaluated on the basis of tracer studies with lanthanum. This material penetrates the extracellular space between perineurium and underlying glial and nerve cells, between epithelial glial cells and retinular axon terminals (capitate projections), and between the alpha-beta fiber pair in the optic cartridge (gnarls). If no damage occurs to the perineurial cells during tissue preparation, this passage of lanthanum to neuronal surfaces indicates that the blood brain barrier is incomplete in this restricted area. Supportive evidence for such permeance is based on electrophysiological data, considerations of membrane specializations in the optic neuropile, and Na+/K+ ratios of dipteran hemolymph.

Ultrastructure of capitate projections in the optic neuropil of Diptera.

Photoreceptor axons in the first optic neuropil of the dipteran flies Musca domestica and Drosophila melanogaster was examined with electron microscopy. The objective was to determine ultrastructure, persistence and glial source of the capitate projections found within these neurons. Capitate projections are simple or compound processes of epithelial glial cells which profusely insert into form-fitting folds of axon terminals of the peripheral retinular cells (R1-6) in the synaptic plexus portion of the first optic neuropil. These neuro-glial junctions may be simple indentations, have a head with a single stalk, or possess a single, circular stalk from which 3 or 4 bulbous (glial) heads are elaborated. Using serial thick sections of Drosophila neuropil for HVEM we were able to observe that the stalks connecting nearly all capitate projections led directly to a glial cell. Thus no disembodied heads were found suspended in axoplasm. Capitate projections appeared to be persistent structures, present in young as well as senescent adults. No evolution of form was found; thus 3 distinct expressions of these glial processes (without transitional forms) are present. From freeze-fracture replicas and serial HVEM sections it was determined that there were approximately 3 capitate projections per micron 2 in Drosophila and Musca, respectively. About 800 capitate projections exist per Musca axon terminal or about 5 times the number of chemical synapses. Cp's were slightly larger in Drosophila than in Musca, although the Musca retinular axon has twice the diameter and length of that of the fruit fly. The evidence was reviewed in light of the likely supportive function of capitate projections on the R1-6 terminals.

Analog of vertebrate anionic sites in blood-brain interface of larval Drosophila.

The blood-brain barrier ensures brain function in vertebrates and in some invertebrates by maintaining ionic integrity of the extraneuronal bathing fluid. Recent studies have demonstrated that anionic sites on the luminal surface of vascular endothelial cells collaborate with tight junctions to effect this barrier in vertebrates. We characterize these two analogous barrier factors for the first time on Drosophila larva by an electron-dense tracer and cationic gold labeling. Ionic lanthanum entered into but not through the extracellular channels between perineurial cells. Tracer is ultimately excluded from neurons in the ventral ganglion mainly by an extensive series of (pleated sheet) septate junctions between perineurial cells. Continuous junctions, a variant of the septate junction, were not as efficient as the pleated sheet variety in blocking tracer. An anionic domain now is demonstrated in Drosophila central nervous system through the use of cationic colloidal gold in LR White embedment. Anionic domains are specifically stationed in the neural lamella and not noted in the other cell levels of the blood-brain interface. It is proposed that in the central nervous system of the Drosophila larva the array of septate junctions between perineurial cells is the physical barrier, while the anionic domains in neural lamella are a "charge-selective barrier" for cations. All of these results are discussed relative to analogous characteristics of the vertebrate blood-brain barrier.

The distal ommatidium of the compound eye of the housefly (Musca domestica): a scanning electron microscope study.

The distal aspect of the housefly ommatidium was surveyed by the scanning electron microscope. Attention was directed to the somal eminence of the superior central cell and the lens to large pigment cell junction. The underside of each lens facet exhibits six hexagonally arranged incisures. Into each of these indentations are fitted several large pigment cells. This hexagonal indentation appears to be a tenacious anchorage. Two corneal pigment cells laterally encircle the pseudocone and at their proximal extension they enclose the Semper cells and neck of the retinula. The somal eminence of the superior central cell is about 10 mum from the base of the corneal pigment cell enclosure. Micrographs were used to construct a diagram of the ommatidium above the basement membrane. Suggestions are made as to the functional correlates of the observed ommatidial structures.

Ultrastructural pathology of the compound eye and optic neuropiles of the retinal degeneration mutant (w rdg BKS222) Drosophila melanogaster.

The compound eye and the two most distal optic neuropils (lamina ganglionaris and medulla externa) of the Drosophila mutant w rdg BKS222 were examined with transmission electron microscopes at conventional (60 kV) and high (0.8-1 MV) voltages. Eye tissue was sampled in the newly emerged and at 3, 7, and 21 days following eclosion. This mutant is known to show a light-induced degeneration of the peripheral retinular cells (R1-6); the spectral sensitivity is altered and the threshold is increased reflecting the function of the central cells (R7, 8) which do not degenerate. A totally normal appearing visual system (peripheral retina and optic neuropiles) was found in newly emerged adults. After 3 days the somata of some of the peripheral retinal cells are affected and all of their axons show degeneration. At one week the R1-6 pathology is well advanced in both somal and axonal regions. In affected cells the cytoplasm is more or less uniformly electron dense and contains liposomes, lysosome-like bodies, myeloid figures and vacuoles suggesting autophagy. Such cytoplasm (noted at 3 and 7 days post-eclosion) exhibits an electron dense reticulum and degenerate mitochondria. Microvilli become more electron dense. Retinular axon terminals are electron opaque and lack synaptic vesicles with few if any presynaptic structures. Mitochondrial remains are barely recognizable. Transsynaptic degeneration was not found. After 3 weeks, the structure of R1-6 in the peripheral retina (somata and rhabdomeres) is greatly reduced or lost while R7 and R8 and higher order neurons are not affected. The debris from cell bodies and axon terminals or R1-6 seems diminished, so that some phagocytosis probably takes place along with gliosis in the lamina.