Attachment of Algal Cells to Zwitterionic Self-Assembled Monolayers Comprised of Different Anionic Compounds.

The influence of zwitterionic self-assembled monolayers on settlement and removal of algae was studied. The monolayers were constructed either from zwitterionic thiols or from solutions of positively and negatively charged thiols. The cationic component was composed of quaternary ammonium terminated thiols and the anionic component contained sulfate or carboxylate termination. During assembly, all surfaces showed a strong tendency for equilibration of the surface charge. Settlement and adhesion assays with zoospores of Ulva linza and the diatom Navicula incerta, and field tests of the initial surface colonization revealed the relevance of charge equilibration for the biological inertness of the prepared surfaces.

Resistance of Amphiphilic Polysaccharides against Marine Fouling Organisms.

Amphiphilic coatings are promising candidates for fouling-release applications. As hydrophilic components, polysaccharides are interesting and environmentally benign building blocks. We used covalently coupled alginic acid (AA) and hyaluronic acid (HA) and postmodified them with a hydrophobic fluorinated amine. The surfaces showed good stability under marine conditions and fluorination led to a decreased uptake of Ca(2+) ions after modification. In single species settlement assays (bacteria, diatoms, barnacle cypris larvae), the modification decreased the settlement density and/or the adhesion strength of many of the tested species. Field studies supported findings of the laboratory experiments, as hydrophobic modification of AA and HA decreased diatom colonization.

The Bone Sialoprotein RGD Domain Modulates and Maintains Periodontal Development.

Bone sialoprotein (gene: Ibsp; protein: BSP) is a multifunctional extracellular matrix protein present in bone, cementum, and dentin. Accumulating evidence supports BSP as a key regulator of mineralized tissue formation via evolutionarily conserved functional domains, including a C-terminal integrin-binding Arg-Gly-Asp (RGD) domain implicated in extracellular matrix-cell signaling. Ablation of Ibsp in mice (Ibsp-/-) results in impaired bone growth and mineralization and defective osteoclastogenesis, with effects in the craniofacial region including reduced acellular cementum formation, detachment of the periodontal ligament (PDL), alveolar bone hypomineralization, and severe periodontal breakdown. We hypothesized that BSP-RGD plays an important role in cementum and alveolar bone formation and mineralization, as well as periodontal function. This hypothesis was tested by replacing the RGD motif with a nonfunctional Lys-Ala-Glu (KAE) sequence in (IbspKAE/KAE) mice and OCCM.30 murine (IbspKAE) cementoblasts. The RGD domain was not critical for acellular or cellular cementum formation in IbspKAE/KAE mice. However, PDL volume and thickness were increased, and significantly more tartrate-resistant acid phosphatase-positive osteoclasts were found on alveolar bone surfaces of IbspKAE/KAE mice versus wild type mice. PDL organization was disrupted as indicated by picrosirius red stain, second harmonic generation imaging, dynamic mechanical analysis, and decreased asporin proteoglycan localization. In vitro studies implicated RGD functions in cell migration, adhesion, and mineralization, and this was confirmed by an ossicle implant model where cells lacking BSP-RGD showed substantial defects as compared with controls. In total, the BSP-RGD domain is implicated in periodontal development, though the scale and scope of changes indicated by in vitro studies indicate that other factors may partially compensate for and reduce the phenotypic severity of mice lacking BSP-RGD in vivo.

Changes in architecture of the Golgi complex and other subcellular organelles during myogenesis.

Myogenesis involves changes in both gene expression and cellular architecture. Little is known of the organization, in muscle in vivo, of the subcellular organelles involved in protein synthesis despite the potential importance of targeted protein synthesis for formation and maintenance of functional domains such as the neuromuscular junction. A panel of antibodies to markers of the ER, the Golgi complex, and the centrosome were used to localize these organelles by immunofluorescence in myoblasts and myotubes of the mouse muscle cell line C2 in vitro, and in intact single muscle fibers from the rat flexor digitorum brevis. Antibodies to the ER stained structures throughout the cytoplasm of both C2 myoblasts and myotubes. In contrast, the spatial relationship between nucleus, centrosome, and Golgi complex was dramatically altered. These changes could also be observed in a low-calcium medium that allowed differentiation while preventing myoblast fusion. Muscle fibers in vivo resembled myotubes except that the ER occupied a smaller volume of cytoplasm and no staining was found for one of the Golgi complex markers, the enzyme alpha-mannosidase II. Electron microscopy, however, clearly showed the presence of stacks of Golgi cisternae in both junctional and extrajunctional regions of muscle fibers. The perinuclear distribution of the Golgi complex was also observed in live muscle fibers stained with a fluorescent lipid. Thus, the distribution of subcellular organelles of the secretory pathway was found to be similar in myotubes and muscle fibers, and all organelles were found in both junctional and extrajunctional areas of muscle.

Restricted distribution of mRNA produced from a single nucleus in hybrid myotubes.

Although the proteins encoded by a single nucleus in multinucleated myotubes have a wide range of distributions within the myofiber, little is known about the distributions of their mRNAs. We have used hybrid myotubes in which one or a few nuclei are derived from myoblasts that express nonmuscle proteins to investigate this question. We find that three different mRNAs, encoding proteins that are, respectively, nuclear, cytoplasmic, and targeted to the ER, have similar distributions within myotubes. Each is confined to an area within approximately 100 microns of the nucleus that expresses it.

Intracellular and surface distribution of a membrane protein (CD8) derived from a single nucleus in multinucleated myotubes.

We have investigated the contribution of an individual nucleus to intracellular and surface membranes in multinucleated muscle fibers. Using a retroviral vector, we introduced the gene encoding the human T-lymphocyte antigen CD8 into C2 mouse muscle cells to form a stable line expressing the human protein on its surface. The intracellular and surface distributions of the protein were then investigated by immunocytochemistry in hybrid myotubes containing a single nucleus expressing CD8. We show that the intracellular distribution of CD8 is limited to a local area surrounding the nucleus encoding it and several neighboring nuclei. On the cell surface, however, the protein is distributed over the entire myotube. Widespread distribution of a surface membrane protein in multinucleated myotubes can thus result from localized synthesis and processing.

Acetylcholine receptor in a C2 muscle cell variant is retained in the endoplasmic reticulum.

We have examined the properties and intracellular localization of acetylcholine receptors in the C2 muscle cell line and in a variant (T-) that accumulates AChR intracellularly. On immunoblots, the subunit structures of the AChR from wild-type and T- cells were similar except that the gamma and delta subunits of the variant AChR had altered mobilities. Digestion with endoglycosidases H and F demonstrated that this difference results from a failure of high-mannose N-linked oligosaccharides on AChR subunits to be processed to complex forms in the variant. N-linked glycosylation of other proteins in the variant was normal. When examined by immunocytochemistry, the distribution of internal AChR in wild-type cells was consistent with a location both in the endoplasmic reticulum and in the Golgi. Variant cells, however, showed no evidence of Golgi staining. Subcellular fractionation experiments also demonstrated AChR in the Golgi fractions of wild-type cells, but not in those derived from T- cells. We conclude that in T- myotubes most of the AChR fails to be transported out of the endoplasmic reticulum.

Analysis of GLUT4 distribution in whole skeletal muscle fibers: identification of distinct storage compartments that are recruited by insulin and muscle contractions.

The effects of insulin stimulation and muscle contractions on the subcellular distribution of GLUT4 in skeletal muscle have been studied on a preparation of single whole fibers from the rat soleus. The fibers were labeled for GLUT4 by a preembedding technique and observed as whole mounts by immunofluorescence microscopy, or after sectioning, by immunogold electron microscopy. The advantage of this preparation for cells of the size of muscle fibers is that it provides global views of the staining from one end of a fiber to the other and from one side to the other through the core of the fiber. In addition, the labeling efficiency is much higher than can be obtained with ultracryosections. In nonstimulated fibers, GLUT4 is excluded from the plasma membrane and T tubules. It is distributed throughout the muscle fibers with approximately 23% associated with large structures including multivesicular endosomes located in the TGN region, and 77% with small tubulovesicular structures. The two stimuli cause translocation of GLUT4 to both plasma membrane and T tubules. Quantitation of the immunogold electron microscopy shows that the effects of insulin and contraction are additive and that each stimulus recruits GLUT4 from both large and small depots. Immunofluorescence double labeling for GLUT4 and transferrin receptor (TfR) shows that the small depots can be further subdivided into TfR-positive and TfR-negative elements. Interestingly, we observe that colocalization of TfR and GLUT4 is increased by insulin and decreased by contractions. These results, supported by subcellular fractionation experiments, suggest that TfR-positive depots are only recruited by contractions. We do not find evidence for stimulation-induced unmasking of resident surface membrane GLUT4 transporters or for dilation of the T tubule system (Wang, W., P.A. Hansen, B.A. Marshall, J.O. Holloszy, and M. Mueckler. 1996. J. Cell Biol. 135:415-430).

Transfer of a protein encoded by a single nucleus to nearby nuclei in multinucleated myotubes.

Specialized regions of muscle fibers may result from differential gene expression within a single fiber. In order to investigate the range of action of individual nuclei in multinucleated myotubes, C2 myoblasts were transfected to obtain stable cell lines that express a reporter protein that is targeted to the nucleus. Hybrid myotubes were then formed containing one or a few transfected nuclei as well as a large number of nuclei from the parental strain. In order to determine how far the products of a single nucleus extend, transfected nuclei were labeled with [3H]thymidine before fusion and the myotubes were stained to identify the reporter protein. In such myotubes the fusion protein was not confined to its nucleus of origin, but was restricted to nearby nuclei.

A single genetic unit specifies two transposition functions in the maize element activator.

The self-mobile maize transposable element Ac (Activator) displays two trans-acting genetic functions: it induces transposition of the element Ds (Dissociation) but, as its dosage is increased, it also inhibits transposition. Previous work has shown that the 4563 base pair (bp)-long Ac element contains three open reading frames (ORF's) and that a deletion in ORF 1 in wx-m9(Ds), a Ds derivative from Ac isolated at the wx (waxy) locus, results in loss of transposition. The Ds element in the bronze allele bz-m2(DI) is shown to have arisen from Ac by a 1312-bp deletion that is located almost entirely within ORF 2 and does not affect ORF 1. The Ds elements in wx-m9(Ds) and bzm2(DI), defective in ORF 1 and ORF 2, respectively, do not complement genetically to restore the transposition function of Ac; therefore, this function must be specified jointly by ORF's 1 and 2. Furthermore, since bz-m2(DI) does not contribute to Ac's inhibitory dosage effect, both Ac properties result from the expression of the same genetic functional unit.

Detection and imaging of non-contractile inclusions and sarcomeric anomalies in skeletal muscle by second harmonic generation combined with two-photon excited fluorescence.

The large size of the multinucleated muscle fibers of skeletal muscle makes their examination for structural and pathological defects a challenge. Sections and single fibers are accessible to antibodies and other markers but imaging of such samples does not provide a three-dimensional view of the muscle. Regrettably, bundles of fibers cannot be stained or imaged easily. Two-photon microscopy techniques overcome these obstacles. Second harmonic generation (SHG) by myosin filaments and two-photon excited fluorescence (2PEF) of mitochondrial and lysosomal components provides detailed structural information on unstained tissue. Furthermore, the infrared exciting light can penetrate several layers of muscle fibers and the minimal processing is particularly valuable for fragile biopsies. Here we demonstrate the usefulness of SHG, combined with 2PEF, to reveal enlarged lysosomes and accumulations of non-contractile material in muscles from the mouse model for the lysosomal storage disorder Pompe disease (PD), and in biopsies from adult and infant PD patients. SHG and 2PEF also detect sarcomeric defects that may presage the loss of myofibrils in atrophying muscle and signify loss of elasticity. The combination of SHG and 2PEF should be useful in the analysis and diagnosis of a wide range of skeletal muscle pathologies.

Variable Patterns of Transposition of the Maize Element Activator in Tobacco.

The strategy to be followed in a transposon tagging experiment will be determined largely by the transposition pattern of the transposon in question. With a view to utilizing the maize element Activator (Ac) as a transposon tag in heterologous systems, we investigated the pattern of Ac transposition from six different loci in transgenic tobacco. We isolated germinal revertants from plants carrying mutable alleles of the antibiotic-resistant gene streptomycin phosphotransferase (SPT) and mapped the location of the transposed Ac (trAc) elements relative to the donor SPT gene. A comparison of the distributions of trAcs among the six loci revealed that, although the receptor sites for trAcs tend to be linked to the donor locus, the pattern of Ac transposition in tobacco displays surprising locus-to-locus variation. Some trAc distributions showed the same tight clustering around the donor locus previously seen in maize, whereas others were more dispersed. The possible meaning of these findings and their implication for transposon tagging in heterologous systems are discussed.

Tagging and Cloning of a Petunia Flower Color Gene with the Maize Transposable Element Activator.

We report here the use of the maize transposable element Activator (Ac) to isolate a dicot gene. Ac was introduced into petunia, where it transposed into Ph6, one of several genes that modify anthocyanin pigmentation in flowers by affecting the pH of the corolla. Like other Ac-mutable alleles, the new mutation is unstable and reverts to a functional form in somatic and germinal tissues. The mutant gene was cloned using Ac as a probe, demonstrating the feasibility of heterologous transposon tagging in higher plants. Confirmation that the cloned DNA fragment corresponded to the mutated gene was obtained from an analysis of revertants. In every case examined, reversion to the wild-type phenotype was correlated with restoration of a wild-type-sized DNA fragment. New transposed Acs were detected in many of the revertants. As in maize, the frequency of somatic and germinal excision of Ac from the mutable allele appears to be dependent on genetic background.

Golgi complex reorganization during muscle differentiation: visualization in living cells and mechanism.

During skeletal muscle differentiation, the Golgi complex (GC) undergoes a dramatic reorganization. We have now visualized the differentiation and fusion of living myoblasts of the mouse muscle cell line C2, permanently expressing a mannosidase-green fluorescent protein (GFP) construct. These experiments reveal that the reorganization of the GC is progressive (1-2 h) and is completed before the cells start fusing. Fluorescence recovery after photobleaching (FRAP), immunofluorescence, and immunogold electron microscopy demonstrate that the GC is fragmented into elements localized near the endoplasmic reticulum (ER) exit sites. FRAP analysis and the ER relocation of endogenous GC proteins by phospholipase A2 inhibitors demonstrate that Golgi-ER cycling of resident GC proteins takes place in both myoblasts and myotubes. All results support a model in which the GC reorganization in muscle reflects changes in the Golgi-ER cycling. The mechanism is similar to that leading to the dispersal of the GC caused, in all mammalian cells, by microtubule-disrupting drugs. We propose that the trigger for the dispersal results, in muscle, from combined changes in microtubule nucleation and ER exit site localization, which place the ER exit sites near microtubule minus ends. Thus, changes in GC organization that initially appear specific to muscle cells, in fact use pathways common to all mammalian cells.

The organization of the Golgi complex and microtubules in skeletal muscle is fiber type-dependent.

Skeletal muscle has a nonconventional Golgi complex (GC), the organization of which has been a subject of controversy in the past. We have now examined the distribution of the GC by immunofluorescence and immunogold electron microscopy in whole fibers from different rat muscles, both innervated and experimentally denervated. The total number of GC elements, small polarized stacks of cisternae, is quite similar in all fibers, but their intracellular distribution is fiber type-dependent. Thus, in slow-twitch, type I fibers, approximately 75% of all GC elements are located within 1 micrometer from the plasma membrane, and each nucleus is surrounded by a belt of GC elements. In contrast, in the fast-twitch type IIB fibers, most GC elements are in the fiber core, and most nuclei only have GC elements at their poles. Intermediate, type IIA fibers also have an intermediate distribution of GC elements. Interestingly, the distribution of microtubules, with which GC elements colocalize, is fiber type-dependent as well. At the neuromuscular junction, the distribution of GC elements and microtubules is independent of fiber type, and junctional nuclei are surrounded by GC elements in all fibers. After denervation of the hindlimb muscles, GC elements as well as microtubules converge toward a common pattern, that of the slow-twitch fibers, in all fibers. Our data suggest that innervation regulates the distribution of microtubules, which in turn organize the Golgi complex according to muscle fiber type.

Golgi complex, endoplasmic reticulum exit sites, and microtubules in skeletal muscle fibers are organized by patterned activity.

The Golgi complex of skeletal muscle fibers is made of thousands of dispersed elements. The distributions of these elements and of the microtubules they associate with differ in fast compared with slow and in innervated compared with denervated fibers. To investigate the role of muscle impulse activity, we denervated fast extensor digitorum longus (EDL) and slow soleus (SOL) muscles of adult rats and stimulated them directly with patterns that resemble the impulse patterns of normal fast EDL (25 pulses at 150 Hz every 15 min) and slow SOL (200 pulses at 20 Hz every 30 sec) motor units. After 2 weeks of denervation plus stimulation, peripheral and central regions of muscle fibers were examined by immunofluorescence microscopy with regard to density and distribution of Golgi complex, microtubules, glucose transporter GLUT4, centrosomes, and endoplasmic reticulum exit sites. In extrajunctional regions, fast pattern stimulation preserved normal fast characteristics of all markers in EDL type IIB/IIX fibers, although inducing changes toward the fast phenotype in originally slow type I SOL fibers, such as a 1.5-fold decrease of the density of Golgi elements at the fiber surface. Slow pattern stimulation had converse effects such as a 2.2-fold increase of the density of Golgi elements at the EDL fiber surface. In junctional regions, where fast and slow fibers are similar, both stimulation patterns prevented a denervation-induced accumulation of GLUT4. The results indicate that patterns of muscle impulse activity, as normally imposed by motor neurons, play a major role in regulating the organization of Golgi complex and related proteins in the extrajunctional region of muscle fibers.

Lysophosphatidylcholine in liposomal membranes: enhanced permeability but little effect on transfer of a water-soluble fluorescent marker into human lymphocytes.

In an attempt to enhance delivery of liposome contents into cells, we tested the effect of lysophosphatidylcholine on transfer of the fluorescent dye, carboxyfluorescein, from small unilamellar and large multilamellar vesicles to human lymphocytes. Dioleoyl phosphatidylcholine and dioleoyl phosphatidylcholine-lysophosphatidylcholine small unilamellar vesicles with varying lipid ratios were prepared and characterized. In the presence of lysophosphatidylcholine, small unilamellar vesicles were slightly smaller and more leaky than those made without lysophosphatidylcholine. Lysophosphatidylcholine induced less leakage in large multilamellar vesicles. It did not show any appreciable effect on transfer of liposome contents, whether included as part of the liposomal bilayer (of unilamellar or multilamellar vesicles) or added exogenously together with small unilamellar dioleoyl phosphatidylcholine vesicles.

Sequence of three bronze alleles of maize and correlation with the genetic fine structure.

The genomic sequences of three bronze alleles from Zea mays, Bz-McC, Bz-W22 and bz-R, are presented together with their flanking sequences. The bronze locus encodes UDPglucose flavonoid glucosyl-transferase (UFGT), an anthocyanin biosynthetic enzyme. The wild-type alleles Bz-McC and Bz-W22 condition purple phenotypes in the seed and plant, while bz-R conditions a bronze color. A full length cDNA corresponding to the Bz-McC allele was cloned and sequenced. Primer extension and RNase protection experiments were used to verify the 5' end of the bronze transcript. The Bz-McC allele has a 1416-bp coding region, a 100-bp intron and an approximately 83-bp 5' leader. Upstream of the message initiation site the sequences CTAACT and AATAAA occupy the positions where the eukaryotic consensus CCAAT and TATA boxes are normally found. The alleles Bz-McC and bz-R each have different large insertions with characteristics of transposable elements in their 5' flanking regions. The bz-R allele is distinguished by a 340-bp deletion starting within the intron and including 285 bp of the second exon. The Bz-McC and Bz-W22 isoalleles are known to differ in two genetically defined locations. The uts and uqv sites from the Bz-McC allele condition, respectively, lowered thermostability for the UFGT enzyme and increased amount of UFGT activity when compared with the corresponding sites in the Bz-W22 allele. The uts site maps to a region of the gene encoding two adjacent amino acid differences, either or both of which might alter the thermostability of the UFGT enzyme. The difference in UFGT levels conditioned by the uqv site is shown here to be correlated with variation in the bronze mRNA level. A likely cause of this decreased bronze mRNA level in Bz-W22 is a 6-bp duplication near the sequence CTAACT located 74 bp upstream of the bronze message initiation site. This region is therefore tentatively identified as the uqv site.

Distribution of unlinked receptor sites for transposed Ac elements from the bz-m2(Ac) allele in maize.

We have shown before that the Ac element from the maize bz-m2(Ac) allele, located in the short arm of chromosome 9 (9S), transposes preferentially to sites that are linked to the bz donor locus. Yet, about half of the Ac transpositions recovered from bz-m2(Ac) are in receptor sites not linked to the donor locus. In this study, we have analyzed the distribution of those unlinked receptor sites. Thirty-seven transposed Ac (trAc) elements that recombined independently of the bz locus were mapped using a set of wx reciprocal translocations. We found that the distribution of unlinked receptor sites for trAs was not random. Ten trAcs mapped to 9L, i.e., Ac had transposed to sites physically, if not genetically, linked to the donor site. Among chromosomes other than 9, the Ac element of bz-m2(Ac) appeared to have transposed preferentially to certain chromosomes, such as 5 and 7, but infrequently to others, such as 1, the longest chromosome in the maize genome. The seven trAc elements in chromosome 5 were mapped relative to markers in 5S and 5L and localized to both arms of 5. We also investigated the transposition of Ac to the homolog of the donor chromosome. We found that Ac rarely transposes from bz-m2(Ac) to the homologous chromosome 9. The clustering of Ac receptor sites around the donor locus has been taken to mean that a physical association between the donor site and nearby receptor sites occurs during transposition. The preferential occurrence of 9L among chromosomes harboring unlinked receptor sites would be expected according to this model, since sites in 9L would tend to be physically closer to 9S than sites in other chromosomes. The nonrandom pattern seen among the remaining chromosomes could reflect an underlying nuclear architecture, i.e., an ordering of the chromosomes in the interphase nucleus, as suggested from previous cytological observations.

The ability of polymorphonuclear leukocyte priming agents to overcome influenza A virus-induced cell dysfunction.

The major mortality and morbidity resulting from influenza virus infections are due to secondary bacterial infections which occur in association with virus-induced inhibition of polymorphonuclear leukocyte (PMNL) function. The present study was undertaken to determine if compounds which prime PMNL function to subsequent stimulation with N-formylmethionyl-leucylphenylalanine (FMLP) or phorbol 12-myristate 13-acetate (PMA) can overcome influenza A virus (IAV)-induced inhibition of the PMNL chemiluminescence response to these stimuli. Granulocyte-macrophage colony stimulating factor (GM-CSF), guanosine triphosphate (GTP), and 1-oleoyl-2-acetylglycerol (OAG) were able to prime the PMNL response to FMLP and/or PMA and totally or partially overcome IAV-induced PMNL dysfunction in cells stimulated with FMLP or PMA. A direct correlation was found between the extent of PMNL priming due to GM-CSF, GTP, and OAG and the capacity of these compounds to overcome virus-induced PMNL dysfunction. The implications of these findings in regard to the mechanism by which priming agents overcome IAV-induced cell dysfunction and the potential of these compounds as therapeutic agents to treat secondary bacterial infections are discussed.