Fine structure of the giant aflagellate spermatozoon in pseudostomum quadrioculatum (leuckart) (platyhelminthes,prolecithophora)

The giant aflagellate spermatozoa of P. quadrioculatum are composed of two different parts: a thicker head piece and a more slender tail piece. In the head there exist a large elongated nucleus and an elongated mitochondrial derivative situated in a groove-like cavity of the nucleus. In mature spermatozoa the nuclear material is arranged in many small membrane bounded areas. Both structures, nucleus and mitochondrial derivative, are spirally coiled. The outer part of the membrane in the mitochondrial derivative forms many loop-like foldings. Both organelles continue to the tail in form of two small, helically coiled ribbons; the nucleus is anchored within the mitochondrial derivative by an electron-opaque process. A sheath of spirally-orientated cortical microtubules starting from the tip of the head runs to the tip of the tail under the cell membrane. In addition, a second sheath of tubules occurs in the tail region, these tubules also run parallel to each other, but in the opposite direction to the microtubules of the outer sheath.The possible relations between the structures observed and the motility of the spermatozoa are discussed; in addition, some phylogenetic comments are attempted.Abbreviations c — cerebrum - com — cortical microtubules - cop — copulatory organ - fm — foldings of the mitochondrial membrane - l — lattice - mid — mitochondrial derivative - mt — microtubules - n — nucleus - ne — nuclear envelope - ph — pharynx - pn — protonephidium - rp — ribbon-like nuclear process - te — testis - tt — testis - tt — tip of the tail - vi — vitellarium - vs — vesicula seminalis     

Cycloheptaamylose-dansyl chloride complex as a fluorescent label of surface membranes in living ciliates

Summary Labelling of surface membrane of living ciliates: Paramecium aurelia and Tetrahymena pyriformis with fluorescent compound — cycloheptaamylose-dansyl chloride complex (CDC) has been achieved. Fluorescence micrographs of the dried samples showed specific localization of CDC on the cell membrane without any intracellular penetration. On the contrary the ciliates which have been dead during labelling revealed a non-specific fluorescence of their whole bodies. Microspectrofluorimetric analysis of labelled Paramecium cells was performed with Leitz microspectrograph. Spectrum of fluorescence emission measured over the cell membrane level had maximum at 450 nm. Strikingly, the emission maximum of the cells dead at the moment of labelling was shifted 10 nm to a longer wavelength. The rate of photofading measured in this case was almost 3-fold higher than for the ciliates labelled as living ones. Fluorescence excitation spectra did not show any difference in the peak position. Thus CDC staining appears to be an useful method of supravital labelling of cell surface enabling also to distinguish — on the basis of spectral characteristics — the ciliates being alive from those dead at the moment of fluorochrome binding.     

Molecular aspects of the interaction between amphotericin B and a phospholipid bilayer: molecular dynamics studies

Amphotericin B (AmB) is a polyene macrolide antibiotic used to treat systemic fungal infections. The molecular mechanism of AmB action is still only partly characterized. AmB interacts with cell-membrane components and forms membrane channels that eventually lead to cell death. The interaction between AmB and the membrane surface can be regarded as the first (presumably crucial) step on the way to channel formation. In this study molecular dynamics simulations were performed for an AmB–lipid bilayer model in order to characterize the molecular aspects of AmB–membrane interactions. The system studied contained a box of 200 dimyristoylphosphatidylcholine (DMPC) molecules, a single AmB molecule placed on the surface of the lipid bilayer and 8,065 water molecules. Two molecular dynamics simulations (NVT ensemble), each lasting 1 ns, were performed for the model studied. Two different programs, CHARMM and NAMD2, were used in order to test simulation conditions. The analysis of MD trajectories brought interesting information concerning interactions between polar groups of AmB and both DMPC and water molecules. Our studies show that AmB preferentially took a vertical position, perpendicular to the membrane surface, with no propensity to enter the membrane. Our finding may suggest that a single AmB molecule entering the membrane is very unlikely.Figure The figure presents the whole structure of the system simulated—starting point. AmB is presented as a space-filling model, DMPC molecules—green sticks, water molecules—red sticks     

Cell structure and reproduction process in the Blue-Green algaChamaesiphon

Ultrathin sections were studied in 2 strains and 2 samples from the nature of the genusChamaesiphon, representing 4 different species. Thylakoids are distributed mainly on the periphery of the cells, the cell-wall is probably 2-layered, and variable multilayered mucilaginous envelopes are developed around the cells. The cell division starts, as well as in otherCyanophyceae, by the invagination of the cytoplasmic membrane and of cell-wall layers into the protoplast; the mucilaginous envelopes—pseudovaginae—do not participate in this process but they form only the firm sheaths around the cells. The way of reproduction is, therefore, essentially the same as that described in other chroococcal Blue-Green algae (e.g.,Synechococcus), and the main difference is the polarized growth of theChamaesiphon cells. The taxonomical position of chamaesiphonoid algae is not as isolated as it was earlier supposed, the similarity withEntophysalidaceae is evident.     

In situ analysis of normal and abnormal patterns of the mitotic apparatus in cultured rat-kangaroo cells

Dividing cells in monolayers of the rat-kangaroo (Potorous tridactylis) cell line Pt-K₁ have large spindles and are flat, thus making possible studies of interactions between the achromatic and chromatic parts of the mitotic apparatus during the cell cycle. At prophase, asters and centrioles seem to exert pressure on the nuclear membrane leading to its rupture and penetrance of the centrioles. Apparently, the long axis of the spindle is shorter than the nuclear diameter. What appears as persistent, large portions of the nuclear membrane were observed in some metaphase and anaphase cells. Such a condition might also indicate an arrested mitosis. The midbody, which was often bipartite, was found to be of a ribonucleoprotein nature. — Three-group metaphases were of common occurrence and might represent early stages of chromosome orientation preceding the final alignment of the chromosomes on the equatorial plate. They could also be an expression of an anomalous condition as a result of mitotic arrest during prometaphase owing to spindle inactivation or breakage, errors in centromere-spindle attachments, interference with chromosome movement, or a duplicated centriolar constitution. Most of these aberrations could be attributed to the flatness of dividing cells, which might also bring about the failure of centriole separation and spindle organization in prometaphase stages, as well as multipolar mitosis.De novo organization of half spindles might take place in cells with ruptured spindles. Anaphase cells showing signs of a previous three-group orientation were rare. — Multipolar mitoses were prevalent mainly in cells with high chromosome numbers. They were often star-shaped with the chromosomes oriented between opposite and adjacent poles, and rarely as end-to-end associations of spindles. Apparently, one or more centrioles might share a common polar region. Multipolar configurations have either a mono- or multinuclear origin. Nuclei usually enter division synchronously in binucleate cells and the spindles become organized between centrioles associated with individual or different nuclei.     

Immunogenetic polymorphism of lipoproteins in swine

Five new antigenic markers (allotypes) of swine serum lipoproteins are described. Specific antiallotype reagents were obtained from alloimmune precipitating sera. Identification studies and genetic analysis indicate that the five serum alloantigens—designated Lpp6, Lpp11, Lpp12, Lpp13, and Lpp14—are markers of low-density lipoproteins (LDL),d 1.002–1.075 g/ml, and are members of a previously described Lpp system. The Lpp6 allotype belongs to the group of individual markers and is determined by a new codominant allelic gene,Lpp ⁶, whereas the remaining four antigens—Lpp11, Lpp12, Lpp13, and Lpp 14—named common specificities, behave as alternative variants to Lpp1, Lpp2, Lpp3, and Lpp4, respectively, forming pairs of mutually exclusive alloantigens. Each Lpp gene in a heterozygous animal expresses itself independently on separate molecules and each haplotype carries one individual and at least four common specificities. The relationship between common and individual specificities, together with their number in the complex haplotypes, seems to shed some light on evolution of Lpp genes. It is proposed in this concept that the original gene for low-density lipoproteins in swine, and also in rhesus monkeys and the human, consisted of genetic information for common specificities only, the individual specificities evolving later as a result of point mutations.     

Brownian dynamics simulation of the lateral distribution of charged membrane components

Brownian dynamics simulations were performed to study the contribution of electric interactions between charged membrane components to their lateral distribution in a two-dimensional viscous liquid (bilayer lipid membrane). The electrostatic interaction potential was derived from an analytical solution of the linearized Poisson-Boltzmann equation for point charges in an electrolyte solution — membrane — electrolyte solution system. Equilibrium as well as dynamic quantities were investigated. The lateral organization of membrane particles, modelled by mobile cylinders in a homogeneous membrane separating two electrolyte solutions was described by spatial distribution functions, diffusion coefficients and cluster statistics. Disorder, local order and crystal-like arrangements were observed as a function of the particle charge, the closest possible distances between the charges and the particle density. The simulations revealed that the system is very sensitive to the position of the charges with respect to the electrolyte solution — membrane interface. Electrostatic interactions of charges placed directly on the membrane surface were almost negligible, whereas deeper charges demonstrated pronounced interaction. Biologically relevant parameters corresponded at most to local and transient ordering. It was found that lateral electric forces can give rise to a preferred formation of clusters with an even number of constituents provided that the closest possible charge-charge distances are small. It is concluded that lateral electrostatic interactions can account for local particle aggregations, but their impact on the global arrangement and movement of membrane components is limited. Correspondence to: D. Walther     

The grasshopper X chromosome

The X chromosome can be identified with the light microscope throughout all stages of the gonial cell cycle (including interphase) in the grasshopper Brachystola magna. At gonial mitotic stages the X chromosome gives the appearance of being undercondensed or negatively heteropycnotic. At interphase the X projects out from the body of the nucleus. — Examination with the electron microscope reveals that the X is compartmentalized at least two gonial cell cycles prior to the entry of the cells into meiotic prophase. The membrane layers that envelope the X chromatin at interphase remain associated with the X chromosome throughout gonial mitotic stages providing the ultrastructural basis for the apparent negative heteropycnosis observed with the light microscope. — The X chromosome is inactive in RNA synthesis during gonial mitotic stages but is hyperactive in RNA synthesis when compared to autosomes at gonial interphase. — X chromosome condensation which reaches its maximum at premieotic interphase is initiated at or prior to the pre-pentultimate gonial division.     

Barrier membranes at the outer surface of the brain of an elasmobranch,Raja erinacea

Summary This report gives the results of the first electron-microscopic examination of the cell layers covering the outer brain surface and the inner surface of the cartilaginous skull in the skate, Raja erinacea. The perivascular glial blood-brain barrier — a characteristic of elasmobranchs — extends to the outer surface of the brain. This outer barrier layer is surrounded, in turn, by a subarachnoid compartment (depth: 30–40 m), containing loose connective tissue and blood vessels; by an arachnoid-like epithelium (10–15 cell layers), impermeable to horseradish peroxidase; and, by perimeningeal fluid, a fluid with a slow turnover rate and a protein composition different from plasma. The inside of the skull, facing the perimeningeal fluid, is covered by a multilayered (10–15 layers) cuboidal epithelium, also impermeable to horseradish peroxidase. Closely apposed cells in the luminal layer of this epithelium have apical microvilli and numerous vesicular profiles, containing material of moderate electron density. These observations may explain, in terms of structure, the regulated protein content of perimeningeal fluid and the restricted exchange of solutes between brain and perimeningeal fluid in elasmobranchs.     

Isolation and protein composition of the outer membrane ofErwinia amylovora

The outer membrane (OM) ofErwinia amylovora was separated from the cytoplasmic membrane either by isopycnic sucrose density gradient centrifugation or by treating the envelope preparation with sodium lauroylsarcosine. Outer membranes, prepared by using either method, were similar in the content of the major proteins as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The wild-type strains ofE. amylovora (E9, E8, EA178, EA198, EA225, EA273) had—in common in the OM—two major protein bands of apparent molecular weights of 15,800 (protein z) and 38,000 (protein y). The OM protein profiles of the virulent wild-type (E9) and the avirulent mutant (E8) were identical but differed from otherE. amylovora strains by the presence of an additional major protein band of approximately 40,000 (protein x). In strain E9, protein x appeared to be associated with the peptidoglycan, whereas proteins y and z were apparently not peptidoglycan-bound.     

Immobilization of acetylcholinesterase on new modified acrylonitrile copolymer membranes

The three new dual-layer matrices (polyacrylonitrile (PAN) membranes coated with physically bound chitosan (CHI)—PANCHI-A and chemically bound chitosan—PANCHI-B and PANCHI-C) for immobilization of acetylcholinesterase (AChE) were obtained. The chemical-modified PAN membrane (PAN-NaOH + ethylenediamine (EDA)) was used as a base for the prepared dual-layer membranes. For chemical chitosan bound membrane, chitosan was tethered onto the membrane surface to form a dual-layer biomimetic membrane in the presence of glutaraldehyde (GA). The basic characteristics (amount of amino groups, hydrophilicity and transport characteristics) of the chitosan-modified membranes were investigated. The SEM analyses were shown essential morphology change in the different chitosan membranes.The relative activities and V_max of the covalently immobilized enzyme on PANCHI-B and PANCHI-C membranes were higher than that on PANCHI-A membrane and chemical-modified membrane with NaOH + EDA. K_m values for the different modified membranes are lower for the chitosan-treated membranes. The pH and temperature optimum of immobilized enzyme were determined. The bound enzymes on PANCHI-B and PANCHI-C have higher thermal and storage stability in comparison with AChE on PANCHI-A membrane and free enzyme.     

Characteristics of oviposition behaviour of the pollen beetle, Meligethes aeneus on four different host plants

Oviposition behaviour of Meligethes aeneus F. (Coleoptera: Nitidulidae) is characterised and quantified on four different plant species. Six behavioural components are identified: W—walking, WA—walking with abdomen on surface, R—resting, B—biting, AOH—placing abdomen over the bite hole and OVI—oviposition. Comparison of host acceptance behaviours on Brassica napus L., Brassica juncea (L.) Czern, Brassica nigra (L.) Koch and Sinapis alba L. showed that S. alba was accepted as a host only after a long exposure to the plant. Behaviour on the Brassica species was similar, however on B. nigra beetles spent a high proportion of time actually ovipositing. We conclude that important cues for oviposition are located both on the bud surface and inside the bud.     

GEFs, GAPs, GDIs and effectors: taking a closer (3D) look at the regulation of Ras-related GTP-binding proteins

Cell biology depends on the interactions of macromolecules, such as protein—DNA, protein—protein or protein—nucleotide interactions. GTP-binding proteins are no exception to the rule. They regulate cellular processes as diverse as protein biosynthesis and intracellular membrane trafficking. Recently, a large number of genes encoding GTP-binding proteins and the proteins that interact witht these molecular switches have been cloned and expressed. The 3D structures of some of these have also been elucidated     

Bone and Joint Disorders in Wild Japanese Macaques from Nagano Prefecture,Japan

I studied bone and joint disorders in wild Japanese macaques (Macaca fuscata fuscata) in order to discern some aspects of their life history from the skeletal material. The specimens comprise 107 nearly complete skeletons of subadults and adults that were killed as crop-raiding monkeys between 1997 and 1998 in Nagano Prefecture, Japan. The most frequent disorder is angular deformity due to fractures: 80 healed fractures in 31 of 52 males and 71 healed fractures in 26 of 55 females. Secondary osteoarthritis due to fractures is rare. Two males have osteochondritis dissecans bilaterally on the posterior surface of the lateral femoral condyles. Degenerative changes are common in the aged individuals. Fractures of the trunk—clavicle, scapula, vertebrae, ribs or hip—are frequent in the males. Contrarily, the majority of fractures in females are in the hands and feet. While most fractures in males appear to have occurred during adulthood, those in females occurred during childhood and senescence. Interindividual violence should not be regarded as a principal cause for fractures in males and females because there is no bite wound except perhaps for one case of an amputated digit. Fractures of the trunk in males were probably caused by impact forces against their shoulders or hips or both caused by rolling down a steep slope or falling out of trees, perhaps during intertroop transfers.     

Human keratinocytes cultured on collagen gels form an epidermis which synthesizes bullous pemphigoid antigens and α2β1 integrins and secretes laminin, type IV collagen, and heparan sulfate proteoglycan at the basal cell surface

Single cell suspensions of human keratinocytes when seeded onto floating three-dimensional gels constructed with type I collagen form a tissue resembling epidermis. These morphogenetic events occur in a serum-free environment in the absence of fibroblasts. Light and transmission electron microscopy show that cells form a basal layer plus suprabasilar cell layers corresponding to the stratum spinosum, stratum granulosum, and stratum corneum. The suprabasilar keratinocyte layers show morphologies which resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-fillagrin granules. The basal cell layer differs from skin in vivo in that there is no connection to a basement membrane via hemidesmosomes. Cells in the basal layers are polarized as evidenced by the secretion of type IV collagen, heparan sulfate proteoglycans, and laminin at the cell membrane interface with the collagen gel. These proteins are not organized into a cytological basement membrane. Bullous pemphigoid antigen, a protein component of hemidesmosomes, is synthesized by basal keratinocytes, but like the basement membrane proteins it is not incorporated into a definable cytological structure. Keratinocytes in the basal and suprabasilar layers also synthesize α2β1 integrins. The mechanisms of keratinocyte adhesion to the gel may be through the interactions of this cell surface receptor with laminin and type IV collagen synthesized by the cell and/or direct interactions between the receptor and type I collagen within the gel. This in vitro experimental system is a useful model for defining the molecular events which control the formation and turnover of basement membranes and the mechanisms by which keratinocytes adhere to type I collagen when sheets of keratinocytes are used clinically for wound coverage.     

Quantitative analysis of phospholipid peroxidation and antioxidant protection in live human epidermal keratinocytes

To characterize oxidative stress in phospholipids of normal human epidermal keratinocytes we metabolically labeled their membrane phospholipids with a natural oxidation-sensitive fluorescent fatty acid, cis-parinaric acid, and exposed the cells to two different sources of oxidants—a lipid-soluble azo-initiator of peroxyl radicals, 2,2'-azobis(2,4-dimethyl-valeronitrile), AMVN, and a superoxide generator, xanthine oxidase/xanthine. We demonstrated that both oxidants induced pronounced oxidation of four major classes of cis-parinaric acid-labeled phospholipids—phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol—in normal human epidermal keratinocytes that was not detectable as any significant change of their phospholipid composition. Vitamin E was effective in protecting the cells against phospholipid peroxidation. Since viability of normal human epidermal keratinocytes was not changed either by labeling or exposure to oxidants the labeling protocol and oxidative stress employed are compatible with the quantitative analysis of phospholipid peroxidation in viable cells.     

A new Escherichia coli gene, fdrA, identified by suppression analysis of dominant negative FtsH mutations

An Escherichia coli membrane protein, FtsH, has been implicated in several cellular processes, including integration of membrane proteins, translocation of secreted proteins, and degradation of some unstable proteins. However, how it takes part in such diverse cellular events is largely unknown. We previously isolated dominant negative ftsH mutations and proposed that FtsH functions in association with some other cellular factor(s). To test this proposal we isolated multicopy suppressors of dominant negative ftsH mutations. One of the multicopy suppressor clones contained an N-terminally truncated version of a new gene that was designated fdrA. The FdrA fragment suppressed both of the phenotypes — increased abnormal translocation of a normally cytoplasmic domain of a model membrane protein and retardation of protein export — caused by dominant negative FtsH proteins. The intact fdrA gene (11.9 min on the chromosome) directed the synthesis of a 60 kDa protein in vitro.     

Lutein-based plumage coloration in songbirds is a consequence of selective pigment incorporation into feathers

Many birds obtain colorful carotenoid pigments from the diet and deposit them into growing tissues to develop extravagant red, orange or yellow sexual ornaments. In these instances, it is often unclear whether all dietary pigments are used as integumentary colorants or whether certain carotenoids are preferentially excluded or incorporated into tissues. We examined the carotenoid profiles of three New World passerines that display yellow plumage coloration—the yellow warbler (Dendroica petechia), common yellowthroat (Geothlypis trichas) and evening grosbeak (Coccothraustes vespertinus). Using high-performance liquid chromatography, we found that all species used only one carotenoid—lutein—to color their plumage yellow. Analyses of blood carotenoids (which document those pigments taken up from the diet) in two of the species, however, revealed the presence of two dietary xanthophylls—lutein and zeaxanthin—that commonly co-occur in plants and animals. These findings demonstrate post-absorptive selectivity of carotenoid deposition in bird feathers. To learn more about the site of pigment discrimination, we also analyzed the carotenoid composition of lipid fractions from the follicles of immature yellow-pigmented feathers in G. trichas and D. petechia and again detected both lutein and zeaxanthin. This suggests that selective lutein incorporation in feathers is under local control at the maturing feather follicle.     

Fossil Humankind and Other Anthropoid Primates of China

More than 70 sites have yielded human fossils in China. They are attributed to Homo sapiens erectus and Homo sapiens sapiens. The earliest one is possibly about 1.7 Ma. A series of common morphological features, including shovel-shaped incisors and flatness of the face, characterize them. There is a morphological mosaic between H. s. erectus and H. s. sapiens in China. The existence of common features and the morphological mosaic suggest continuity of human evolution in China. That there are a few features which are more commonly seen in the Neanderthal lineage, occurring in a few Chinese fossil skulls, probably suggests gene flow between China and the West. Based on them, in 1998 I proposed an hypothesis—continuity with hybridization—for human evolution in China. The hypothesis is supported by paleolithic archeology, and it supports the multiregional evolution hypothesis of modern human origins. The anatomically modern humans of East Asia originated most probably in China. Although some nonhuman anthropoid primates of China—Gigantopithecus, Sivapithecus, Ramapithecus and Lufengpithecus—have been suggested as the direct ancestors of human beings, the discovery of more specimens and further studies do not support these suggestions. Therefore, it is most probable that the transition between apes and humans did not occur in China.     

Seasonal variation in the growth and nutrient content of yellow-birch replacement roots

Summary Modified air layers were established on lateral long roots of 9 yellow birch (Betula alleghniensis Britton) trees, and all replacement roots >. 5 cm long were harvested periodically during the 1971 and 1972 growing seasons. The first replacement roots grew 6 weeks after layer establishment. Root layers were inactive from 29 Oct. 71 to 5 May 72. Active root layers produced an average of 208 mg per tree during the first season and 198 mg per tree during the second season. Concentrations of N, P, K, Ca, Mg, Fe, Mn, Zn, and Al all varied within growing season, and average concentration of some elements—Ca in particular—varied between growing seasons. This technique shows promise for studying the nutrient status of root systems of forest trees.