Methods for studying the distribution of pigment in malpighian tubules of Drosophila melanogaster.

The pigmented cells in Malpighian tubules of Drosophila melanogaster contain yellow globules which are blackened by osmium tetroxide. Permanent preparations of the tubules showing pigmentation can thus be made. Following prestaining acid hydrolysis, osmium-fixed tubules can be stained with orcein. For observations on pigment distribution and Feulgen-DNA content in the same preparation, tubules are fixed in mercuric chloride. Color remains visible for several hours in unfixed tubules mounted in modified buffered Ringer solution.     

Advantages and limitations of the use of isolated kidney tubules in pharmacotoxicology

Among the cellular models used in in vitro renal pharmacotoxicology, isolated kidney tubules, used as suspensions mainly of proximal tubules, offer important advantages. They can be prepared in large amounts under nonsterile conditions within 1–2 h; thus, it is possible to employ a great number of experimental conditions simultaneously and to obtain rapidly many experimental results. Kidney tubules can be prepared from the kidney of many animal species and also from the human kidney; given the very limited availability of healthy human renal tissue, it is therefore possible to choose the most appropriate species for the study of a particular problem encountered in man. Kidney tubules can be used for screening and prevention of nephrotoxic effects and to identify their mechanisms as well as to study the renal metabolism of xenobiotics. When compared with cultured renal cell, a major advantage of kidney tubules is that they remain differentiated. The main limitations of the use of kidney tubules in pharmacotoxicology are (1) the necessity to prepare them as soon as the renal tissue sample is obtained; (2) their limited viability, which is restricted to 2–3 h; (3) the inability to expose them chronically to a potential nephrotoxic drug; (4) the inability to study transepithelial transport; and (5) the uncertainty in the extrapolation to man of the results obtained using animal kidney tubules. These advantages and limitations of the use of human and animal kidney tubules in pharmacotoxicology are illustrated mainly by the results of experiments performed with valproate, an antiepileptic and moderately hyperammonemic agent. The fact that kidney tubules, unlike cultured renal cells, retain key metabolic properties is also shown to be of the utmost importance in detecting certain nephrotoxic effects.     

Relationship between age and deposit of peritubular dentine

Peritubular dentine is a mineralised deposit formed centripetally in the dentine tubules with advancing age, so that the tubular diameter is smaller in teeth from older persons. The aim of the present study was to investigate the relationship between age in humans and the amount of peritubular dentine and the extent of the consequent obliteration of the tubules, and to find out whether this relationship was strong enough to be used as a parameter for age estimation. Fifty mandibular central and lateral human incisors were ground on the lingual aspect of the root and examined in a scanning electron microscope (SEM). The number of open tubules was counted and the diameter of the tubules measured both before and after etching with 35% orthophosphoric acid. The difference in the number of tubules in unetched and etched specimens was taken to be the number of occluded tubules, and the difference in radii before and after etching to be the thickness of peritubular dentine. The results did not demonstrate a significant relationship between age and the reduction in the number of tubules. One explanation might be that a certain age has to be reached before obliterated dentine tubules can be observed. The correlation between age and the thickness of peritubular dentine was not significant in teeth extracted because of periodontal disease, so these teeth were excluded from the regression analysis with age as the dependent variable. Only the thickness of peritubular dentine was included in the regression (r=0.69); this factor was a better indicator of age than the tubular diameter, but not so closely related to age that it can be recommended for general use in forensic and archaeological age estimations.     

Fluid secretion by in vitro preparations of the Malpighian tubules of the desert locust Schistocerca gregaria

In vitro preparations of locust Malpighian tubules can conveniently be made by a new technique in which the alimentary canal to which the tubules attach is removed from the insect and set up in Ringer's solution under liquid paraffin. Such Malpighian tubules will secrete a fluid iso-osmotic to the bathing fluid at a steady rate of about 1 to 2 nl min^?1 for some hours. The secreted fluid is rich in potassium ions, the lumen is at a potential positive to that of the bathing solution, and the rate of secretion can be controlled by changing the potassium concentration of the bathing fluid. It seems likely, therefore, that an active transport of potassium drives secretion ny locust Malpighian tubules. The secreted fluid contains an elevated concentration of phosphate ions. The Malpighian tubules will secrete at a high rate in a chloride-free phosphate-based solution. The rate of fluid secretion can be increased by treatment with cyclic AMP but 5-hydroxytryptamine has no such effect.     

Probing the structure of diacetylenic phospholipid tubules with fluorescent lipophiles.

Novel lipid structures called tubules can be prepared from diacetylenic phospholipids. We have prepared fluorescent tubules from mixtures of 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphatidylcholine and 1 mol% fluorescent lipophiles to study the characteristics of the tubule lipid matrix. We have found that once formed, tubules do not incorporate lipophiles from the aqueous phase into their lipid matrix. The spectral characteristics of the fluorophore laurodan in tubules, and the lack of diffusion of N-nitrobenzoxydiazol phosphatidylethanolamine in tubules, have allowed us to characterize the microenvironment of these structures as being extremely rigid and tightly packed. Despite their rigid characteristic, tubules are formed from intact liposomes as demonstrated by the formation of doubly labeled tubules from two populations of liposomes, each of which contained a different nonexchangeable fluorescent lipophile.     

3-D reconstruction of bluetongue virus tubules using cryoelectron microscopy.

Bluetongue virus (BTV) forms tubules in infected mammalian cells. These tubules are virally encoded entities which can be formed with only one protein, NS1. The NS1 protein does not form a part of virus particles, and its function in viral infection is uncertain. Expression of the NS1 gene in insect cells by recombinant baculovirus yields high amounts of NS1 tubules (ca. 50% of cellular proteins) which are morphologically and immunologically similar to authentic BTV NS1 and can be isolated to about 90% purity. The structure of these synthetic NS1 tubules was investigated by cryoelectron microscopy. NS1 tubules are on average 52.3 nm in diameter and up to 100 nm long. The structure of their helical surface lattice has been determined using computer image processing to a resolution of 40 A. The NS1 protein is about 5.3 nm in diameter and forms a dimer-like structure, so that the tubules are composed of helically coiled ribbons of NS1 "dimers," with 21 or 22 dimers per turn. The surface lattice displays P2 symmetry and forms a one-start helix with a pitch of 9.1 nm. The NS1 tubules exist in two slightly different pH-dependent conformational states.     

Intercellular transport of steroids in the infused rabbit testis

Tritiated-pregnenolone and tritiated-testosterone were infused via the testicular artery into the rabbit testis in situ, in order to determine if steroids can pass the "blood-testis barrier". After various periods of infusion (5-60 minutes) the testis were frozen cryostat sections were cut and freeze-dried. Interstitium and tubules were isolated by micro dissection and radioactivity per mcg dry weight was measured in both tissue compartments. Radioactive steroids could be isolated from the interstitial tissue as well as from the seminiferous tubules. Levels of radioactivity in the testes after infusion of tritiated-pregnenolone varied from 4 to 50% of the infused dose and were found to be dependent on the type of perfusion medium and the duration of the perfusion. Separation and identification of the radioactive steroids after infusing tritiated prognenolone showed that pregnenolone and testosterone were the major radioactive steroids in both interstitium and seminiferous tubules. After infusion with tritiated-testosterone, both tritiated-testosterone (77%) and tritiated-androstenedione (23%) were dound in the seminiferous tubules. It is concluded that steroids can be transported from the Leydig cells to the seminiferous tubules.     

Effects of Oil Type on Sterol-Based Organogels and Emulsions

The present study investigates the effect of oil type on the formation, morphology and mechanical properties of phytosterol-based organogels. The formation of organogels can be satisfactorily predicted with a criterion based on Hansen Solubility Parameters (HSPs), provided that the sterol and sterol ester in these systems assemble as tubules. When structures other than tubules are formed, the predictability of the HSP-based criterion becomes void. In cases where organogelling occurred, the morphology and mechanical properties of the tubular network of the gels and water-in-oil emulsions were investigated. The findings revealed that the structure of the tubular network formed in oils with different compositions, could be grouped based on the dielectric constants of the oils. Curly and bundled tubules which formed networks, were observed in gels prepared with low dielectric constant oils (i.e. decane and limonene). For oils with a moderate dielectric constant (i.e. castor oil and sunflower oil), the tubules became less curly and straighter. Upon increasing the dielectric constant of the oil (eugenol), individual tubules were observed next to the bundled tubules. The results showed that straighter, bundled tubules are associated with firmer gels, whereas less straight (i.e. curly) tubules rendered weaker gels. The tubular network of the water-in-oil emulsions obtained for oils with a low dielectric constant appeared more open with straighter tubules. For oils with relatively high dielectric constant, the water-in-oil emulsions lost most of their tubular structure and only a few tubules could be observed. In the presence of emulsion droplets fewer tubules are formed, resulting in weaker networks.

     

Grab a Golgi: Laser Trapping of Golgi Bodies Reveals in vivo Interactions with the Endoplasmic Reticulum

In many vacuolate plant cells, individual Golgi bodies appear to be attached to tubules of the pleiomorphic cortical endoplasmic reticulum (ER) network. Such observations culminated in the controversial mobile secretory unit hypothesis to explain transport of cargo from the ER to Golgi via Golgi attached export sites. This proposes that individual Golgi bodies and an attached‐ER exit machinery move over or with the surface of the ER whilst collecting cargo for secretion. By the application of infrared laser optical traps to individual Golgi bodies within living leaf cells, we show that individual Golgi bodies can be micromanipulated to reveal their association with the ER. Golgi bodies are physically attached to ER tubules and lateral displacement of individual Golgi bodies results in the rapid growth of the attached ER tubule. Remarkably, the ER network can be remodelled in living cells simply by movement of laser trapped Golgi dragging new ER tubules through the cytoplasm and new ER anchor sites can be established. Finally, we show that trapped Golgi ripped off the ER are ‘sticky’ and can be docked on to and attached to ER tubules, which will again show rapid growth whilst pulled by moving Golgi.     

Tension in tubulovesicular networks of Golgi and endoplasmic reticulum membranes

The endoplasmic reticulum (ER) and Golgi have robust bidirectional traffic between them and yet form distinct membrane compartments. Membrane tubules are pulled from large aggregates of ER or Golgi by microtubule motors to form ER tubulovesicular networks or Golgi tubules both in vivo and in vitro. The physical properties of membranes are critical for membrane traffic and organelle morphology. For example, tension applied to membranes can create tethers, drive membrane flow, and set the diameter of the tubules. Here, we formed ER and Golgi membrane networks in vitro and used optical tweezers to measure directly, for the first time, the membrane tensions of these organelles to clarify the possible role of tension in membrane flow. We report that higher forces are needed to form tethers from ER (18.6 +/- 2.8 pN) than from Golgi (11.4 +/- 1.4 pN) membrane tubules in vitro. Since ER tubules are smaller in diameter than Golgi tubules, it follows that Golgi networks have a lower tension than ER. The lower tension of the ER could be an explanation of how Golgi tubules can be rapidly drawn into the ER by tension-driven flow after fusion, as is observed in vivo.     

Isolation and staging of horse seminiferous tubules by transillumination

Stages of the spermatogenic cycle in the horse were determined by trans-illumination of enzymically isolated, seminiferous tubules and were verified by whole-mounted tubules observed by Nomarski optics and by conventional histology. Isolated tubules were obtained from young (less than 2 years) and adult (4-10 years) horses by enzymic digestion. Dispersed tubules were separated into three different groups based on the presence, size, and intensity of a dark region in the centre of the tubules: (1) pale--homogeneously light, (2) spotty--light on the periphery with a wide spotty region in the central two-thirds, or (3) dark--an intensely dark, narrow region through the central one-third. Seminiferous tubules from young stallions separated easily, but were only of the homogeneously light pattern as they lacked mature spermatids. After observation by Nomarski optics and bright-field microscopy, pale tubules under transillumination largely contained Stages I and II, spotty tubules contained Stages V and VI, and dark tubules contained Stages VII and VIII of the spermatogenic cycle. In-vitro incorporation of [3H]thymidine in spermatogonia and preleptotene/leptotene primary spermatocytes of these tubules confirmed the viability of germ cells in isolated tubules, and ultrastructural analysis confirmed excellent preservation of normal structure of seminiferous epithelium in isolated tubules. Hence, segments of seminiferous tubules in specific stages of the spermatogenic cycle can be obtained from enzymically digested horse testes when viewed by transillumination.     

Evidence for a decreased membrane recycling in the cells of renal proximal tubules exposed to high concentrations of ferritin

Summary The present study was performed to investigate whether membrane recycling via the dense apical tubules in cells of renal proximal tubules could be modified after exposure to large amounts of cationized ferritin. Proximal tubules in the rat kidney were microinfused in vivo with cationized ferritin for 10 or 30 min and then fixed with glutaraldehyde by microinfusion, or proximal tubules were microinfused with ferritin for 30 min and then fixed 2 h thereafter. The tubules were processed for electron microscopy, and the surface density and the volume density of the different cell organelles involved in endocytosis were determined by morphometry. The morphometric analyses showed that after loading of the endocytic vesicles with ferritin the surface density of dense apical tubules decreased to about 50% of the original value. However, 2 h later when ferritin had accumulated in the lysosomes the surface density of dense apical tubules had returned to control values. Furthermore, cationized ferritin was virtually absent from the Golgi region, indicating that the Golgi apparatus in these cells does not participate in membrane recycling. In conclusion, the present study shows that membrane recycling in renal proximal tubule cells can in part be inhibited by loading the endocytic vacuoles with ferritin.     

Tubulation of class II MHC compartments is microtubule dependent and involves multiple endolysosomal membrane proteins in primary dendritic cells

Immature dendritic cells (DCs) capture exogenous Ags in the periphery for eventual processing in endolysosomes. Upon maturation by TLR agonists, DCs deliver peptide-loaded class II MHC molecules from these compartments to the cell surface via long tubular structures (endolysosomal tubules). The nature and rules that govern the movement of these DC compartments are unknown. In this study, we demonstrate that the tubules contain multiple proteins including the class II MHC molecules and LAMP1, a lysosomal resident protein, as well as CD63 and CD82, members of the tetraspanin family. Endolysosomal tubules can be stained with acidotropic dyes, indicating that they are extensions of lysosomes. However, the proper trafficking of class II MHC molecules themselves is not necessary for endolysosomal tubule formation. DCs lacking MyD88 can also form endolysosomal tubules, demonstrating that MyD88-dependent TLR activation is not necessary for the formation of this compartment. Endolysosomal tubules in DCs exhibit dynamic and saltatory movement, including bidirectional travel. Measured velocities are consistent with motor-based movement along microtubules. Indeed, nocodazole causes the collapse of endolysosomal tubules. In addition to its association with microtubules, endolysosomal tubules follow the plus ends of microtubules as visualized in primary DCs expressing end binding protein 1 (EB1)-enhanced GFP.     

Albumin is a major protein component of transverse tubule vesicles isolated from skeletal muscle

Despite multiple procedures used to isolate transverse tubule vesicles from rabbit skeletal muscle, few proteins have been identified and shown to be specific to transverse tubule vesicles. Markers for purified transverse tubules have included high affinity dihydropyridine binding, cholesterol content, Mg2+-ATPase activity, (Na+,K+)-ATPase activity, and [3H] ouabain binding. Despite these markers, few proteins from purified transverse tubules can be unequivocally identified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In this report we have biochemically and immunologically identified rabbit albumin as a major component of purified transverse tubule membranes from rabbit skeletal muscle. Albumin composed between 5.1 and 9.8% (n = 4) of the total protein in purified transverse tubules based on scans of SDS-PAGE. Furthermore, albumin and other serum proteins are present in preparations of transverse tubules and triads but not in light sarcoplasmic reticulum. Extraction of triads with low concentrations of saponin or sodium dodecyl sulfate completely removes albumin without removing intrinsic membrane proteins. Our results suggest that albumin and other serum proteins are present in the lumen of preparations of transverse tubules and albumin may be used as a marker for the transverse tubules when analyzed on SDS gels.     

STRUCTURE OF THE MITOTIC SPINDLE IN L STRAIN FIBROBLASTS

The mitotic spindle of L strain fibroblasts, fixed with glutaraldehyde followed by osmium tetroxide, contains many 150- to 180-A tubules. They appear first in the cytoplasm. They extend from the centrospheres to the kinetochores, and from one centrosphere to the other. Only occasionally can points of continuity between the spindle tubules and the tubules of the centrioles be observed. The chromosomal insertion is by a means of a thin dense plate of the kinetochore. The total number of continuous spindle tubules is between 500 and 600. Occasionally, tubules appear paired. At anaphase, short lengths of individual spindle tubules possess a coating of a substance of high density midway between the poles. These parts of the spindle tubules aggregate to form irregular groups, comprising the stem-body, and, by becoming aligned into a plate, they form the mid-body.     

The Plaque Matrix (PQM) and Tubules at the Surface of Intramuscular Parasite, Trachipleistophora hominis

ABSTRACT. Surface plaque matrix (PQM) and a tubular arrangement of filaments border Trachipleistophora hominis parasites during growth within host muscle. The PQM at the parasite surface forms a network of processes which can be associated with filamentous tubules. Peroxidase tracer delineated the PQM and showed apparent connections with the tubules. The tubules at the interface of T. hominis -infected cells are structurally similar to the extrasporular tubules of the microsporidian, Ameson michaelis . The extrasporular tubules of A. michaelis and the proteins from T. hominis -infected muscle reacted to keratin antibodies, K8.13, K4 and K13. Conversely, antibodies produced to T. hominis -infected muscle, reacted with the extrasporular tubular proteins of A. michaelis . The PQM and tubular elements are thought to play an important role in affecting molecular traffic between the host and parasite.     

Novel actin cytoskeleton: actin tubules

In spores of Dictyostelium discoideum three actin filaments are bundled to form a novel tubular structure and the tubules are then organized into rods. These tubular structures we will term actin tubules. Actin tubules are reconstructed from the supernatant of spore homogenates, while the usual actin filaments were bundled after incubation of supernatants from growing cells. Alpha-actinin, ABP-120 and EF-1alpha are not essential for rod formation. Cofilin is a component of the cytoplasmic rods but few cofilin molecules are included in the nuclear rods. The viability of spores lacking actin rods is very low, and the spore shape is round instead of capsular. The rods can be fragmented by pressure, indicating that the rods may be effective in absorbing physical pressure. The complex organization of actin filaments, actin tubules and rods may be required for spores to achieve complete dormancy and maintain viability.     

Structure of ivory

Profiles with all orientations have been used to visualize the 3D structure of ivory from tusks of elephant, mammoth, walrus, hippopotamus, pig (bush, boar, and warthog), sperm whale, killer whale, and narwhal. Polished, forming, fractured, aged, and stained surfaces were prepared for microscopy using epi-illumination. Tusks have a minor peripheral component, the cementum, a soft derivative of the enamel layer, and a main core of dentine=ivory. The dentine is composed of a matrix of particles 5-20 microm in diameter in a ground substance containing dentinal tubules about 5 microm in diameter with a center to center spacing of 10-20 microm. Dentinal tubules may be straight (most) or curly (pigs). The main findings relate to the way that dentinal tubules align in sheets to form microlaminae in the length of the tusk. Microlaminae are sheets of laterally aligned dentinal tubules. They are axial but may be radial (most), angled to the forming face (pigs and hippopotamus canines), or radial but helical (narwhals). Within the microlaminae the dentinal tubules may be radial, angled to the axis (whales, walrus, and pigs), or may change their orientation from one microlamina to the next in helicoids (canines of hippopotamuses, incisors of proboscidea). In the nonbanded, featureless ivories from the hippopotamus incisors, the dentinal tubules form radial microlamina from which the arrangements in other ivories can be derived. In the canines of hippopotamuses and incisors of proboscidea, the dentinal tubule orientation changes incrementally from one microlamina to the next in a helicoid, a stack of dentinal tubules that change their orientation by 180 degrees anticlockwise. Dentinal tubules having different orientations are laid down concurrently, not layer by layer as in most examples of helicoidal architecture (e.g., insect cuticle). In proboscidean ivory, the microlaminae are radial, normal to the banding of growth layers marking the plane of deposition. They form radial segments with each 180 degrees turn in the orientation of their constituent dentinal tubules. Below the cementum they are almost complete 180 degrees helicoids, but nearer to the core they become narrower with the loss of radially oriented dentinal tubules. These truncated helicoidal patterns appear in longitudinal profile as VVVV feather patterns rather than intersection intersection intersection intersection, each V or intersection being the side view of a partial or complete helicoid. The Schreger pattern in proboscidean ivory consists of these helicoids divided tangentially into columns in the length of the tusk. Narwhals have the most abundant matrix particles with their radial/helical dentinal tubules having a twist opposite to that in the cementum.     

Evidence for a species-specific barrier to sperm transport within the vagina of the chicken hen

Following their insemination into the vagina of chicken hens, turkey spermatozoa did not appear to reach the ovum within the upper magnum or infundibulum and were only occasionally found within the sperm storage tubules at the uterovaginal junction. Turkey spermatozoa were able to populate chicken uterovaginal sperm storage tubules as (or more) efficiently as fowl spermatozoa in uterovaginal junction tissue in vitro. They also populated uterovaginal junction sperm storage tubules in vivo after insemination directly into the uterovaginal region. Thus, a barrier to foreign spermatozoa appears to exist within the vagina of the chicken and not at the level of the uterovaginal junction sperm storage tubules. The nature of this barrier is not known; however it can be shown that while chicken and turkey spermatozoa have similar morphological features and motility characteristics, they have distinct surface antigenicity. Recognition of surface antigenicity by a localised immunological mechanism may be the basis of sperm selection within the hens vagina.     

Spermatogonial stem cell transplantation, cryopreservation and culture.

Testis cells of a fertile male mouse can be transplanted to the seminiferous tubules of an infertile male, where the donor spermatogonial stem cells will establish spermatogenesis and produce spermatozoa that transmit the donor haplotype to progeny. In addition, stem cells can be cryopreserved for long periods, thereby making male germ lines immortal. Recently, mouse testis cells have been cultured for longer than 3 months and, following transplantation, produced spermatogenesis. These techniques are likely to be applicable to many species, since rat testis cells can be cryopreserved and generate spermatogenesis in the seminiferous tubules of immunodeficient mice.