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Objective A new technique for producing splints for orthognathic surgery using a 3D printer is presented. Study design After 3-dimensional (3D) data acquisition by computerized tomography (CT) or cone-beam computerized tomography (CBCT) from patients with orthognathic deformations, it is possible to perform virtual repositioning of the jaws. To reduce artifacts, plaster models were scanned either simultaneously with the patient during the 3D data acquisition or separately using a surface scanner. Importing and combining these data into the preoperative planning situation allows the transformation of the planned repositioning and the ideal occlusion. Setting a virtual splint between the tooth rows makes it possible to encode the repositioning. After performing a boolean operation, tooth impressions are subtracted from the virtual splint. The “definitive” splint is then printed out by a 3D printer. Conclusion The presented technique combines the advantages of conventional plaster models, precise virtual 3D planning, and the possibility of transforming the acquired information into a dental splint.

We investigate the gain formation properties of In x Ga 1-x N laser diodes emitting at 404 nm and 470 nm (sample A and B, respectively). Employing the Hakki-Paoli method, their optical gain spectra were measured at 298 K and 333 K. Since spatial fluctuations of the well width and In composition are considerable for long-wavelength laser diodes, broadening of the optical gain and luminescence spectra are clearly enhanced in sample B when compared with sample A. Moreover, population inversion occurs in at least two discrete energy levels for the gain spectra of sample B. It was found that only the modal gain of the higher gain peak can overcome the mirror loss, therefore the lower one does not contribute to lasing action. We also estimate internal loss of A and B to be 25 cm -1 and 30 cm -1 , respectively. The maximum gain value of A increases linearly with increasing injected current even at 333 K, while that of B grows sub-linearly.

We study the lateral diffusion of photogenerated carriers within InGaN/GaN quantum wells by scanning in the focal plane of our confocal microscope. The photoluminescence (PL) images are analyzed at varying excitation densities in the temperature range between 4 K and room temperature. The results of a blue and a green emitting sample are discussed in context of reported diffusion lengths for InGaN/GaN quantum wells. (© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

During the development of the central nervous system (CNS), the correct wiring of outgrowing neurites is mediated by antagonistic mechanisms. Aberrant growth is prevented by repulsive factors such as semaphorins. Expression of the ligands Sema3A and -3E and the receptors neuropilin Npn-1, -2a and -2b in the chick visual system were analyzed by RT-PCR. Whereas Sema3A and its major receptor Npn-1 were abundant, Sema3E and Npn-2 isoform expression was highly restricted and developmentally regulated. Peak expression occurred during retinal axon innervation of the tectum. Functional in vitro assays with recombinant proteins revealed a topography-specific growth cone collapsing activity of Sema3A for tectal axons. Interestingly, whereas tectal axons collapsed in a topographic-specific manner only in the presence of Sema3A, retinal axons responded only to Sema3E. The collapsing activity was intracellularly mediated by cGMP. For a detailed analysis of neuronal responses to sempahorins, time lapse video recording was performed. When tectal and retinal axons were pre-exposed to brain-derived neurotrophic factor (BDNF), a protective effect was evident only in the case of retinal axons. Our results suggest a molecular mechanism whereby ingrowth of retinal axons into the tectum can be regulated by Sema3E/BDNF modulation without disturbing tectal axon growth out of the tectum mediated by Sema3A.

We have calculated the propagation dynamics of an initial off-axis vortex with topological charge 1 in Laguerre–Gaussian background beams ( LG 1 0 and LG 7 0 ) , which are examples of background beams with non-generic dislocation surfaces, on which the real and imaginary parts of the light field are zero. When initially a vortex with broad core (e.g., r-vortex) is embedded in the background beam, the dislocation surfaces are destroyed during propagation and two vortices with opposite charge are created per dislocation surface in planes perpendicular to the propagation direction. For a vortex with narrow core (e.g., point vortex) diffraction is important and leads to the birth of more than two vortices per dislocation surface. These results are also valid for other background beams with dislocation surfaces, e.g., Hermite–Gaussian and Ince–Gaussian beams. We investigated experimentally the spatial evolution of the intensity distribution of an initial off-axis vortex with narrow core and topological charge 1 in LG 1 0 and LG 7 0 background beams. The experimental results are in good agreement with the calculated intensity distributions.

Neuroembryogenesis critically depends on signaling molecules that modulate cell proliferation, differentiation, and the formation of neural networks. In an attempt to identify potential morphogenetic active components that are distributed in a graded fashion in the developing nervous system, we generated substraction libraries of the embryonic nasal and temporal chick retina. Selected clones were analyzed by sequencing, Northern and Western blotting, in situ hybridization, and immunocytochemistry. Retinal fatty acid-binding protein (R-FABP) mRNA displayed the most pronounced topographic gradient. R-FABP was most strongly expressed in nasal retina, though topographic differences were not evident on the protein level. R-FABP expression was subject to a pronounced spatio-temporal regulation. Peak expression was at the period of cell generation/migration and differentiation. To identify the cell types involved in R-FAPB synthesis, ganglion cells as the only retinal projection neurons were enriched by enzymatic delayering. Cell somata, axons, and growth cones were R-FABP immunoreactive. Most interestingly, R-FABP immunoreactivity was critically dependent on the growth substratum. It was abrogated when axons grew on isolated glial endfeet. Radial glia purified by complement-mediated cytolysis also expressed R-FABP at moderate levels. The expression level was significantly increased during mitosis and dropped down again in postmitotic cells. Further on, transient loss of cell-cell and substratum contact induced a subcellular redistribution of R-FABP. In conjunction with the morphogen-binding activity of other FABP family members and their impact on cell migration and tissue differentiation, R-FABP characteristics suggest a regulatory function during retinal histogenesis but not during topographic map formation.

This study is the first attempt to characterise the chemical composition of the secretion of the smooth pads of the locust Locusta migratoria and to relate this to the composition of the cuticle coverage of the pads and the wings. Gas-chromatography and mass-spectrometry (GC-MS) were the principal techniques used for the characterization of these materials. Secretion droplets were visualised and quantified with the aid of diverse microscopic techniques. The chemical composition of prints is shown to differ from the cuticle coverage, in particular, with respect to the fatty acid distribution: in the secretion, saturated and unsaturated fatty acids with chain lengths between C 16 and C 20 in both the free form and as glycerides predominate, whereas cuticle coverage contains waxes of long-chained fatty-acids bound to long-chain primary alcohols. The second important difference is the significant amount of glucose and other saccharides found in methanolyzates of the pad fluid. A considerable amount of the amino acids (up to 53%) was detected in the non-volatile portion of the fluid. Data obtained from the shock-freezing, carbon-platinum coating and replica preparation show that the secretory droplets contain nano-droplets on their surfaces. The results lead us to suggest that the pad secretion is an emulsion consisting of lipidic nano-droplets dispersed in an aqueous liquid. According to the chemical composition of the secretion, a high-viscosity of the fluid may be suggested. Presumably, the fluid is a kind of a coupling agent, promoting and strengthening adhesion between otherwise incompatible materials by providing the proximity of contact for intermolecular forces.

Design of attachment devices in insects varies enormously in relation to different functional loads. Many systems, located on different parts of the body, involve surfaces with particular frictional properties. Such systems evolved to attach parts of the body to each other, or to attach an insect to the substratum by providing fast and reversible attachment/detachment. Among these systems, there are some that deal with predefined surfaces, and others, in which one surface remains unpredictable. The first type of system occurs, for example, in wing-locking devices and head-arresting systems and is called probabilistic fasteners. The second type is mainly represented by insect attachment pads of two alternative designs: hairy and smooth. The relationship between surface patterns and/or mechanical properties of materials of contact pairs results in two main working principles of the frictional devices: mechanical interlocking, or maximization of the contact area. We give an overview of the functional design of two main groups of friction-based attachment devices in insects: probabilistic fasteners and attachment pads.

SUMMARYThis paper studies slide-resisting forces generated by claws in the free-walking beetle Pachnoda marginata (Coleoptera, Scarabaeoidea)with emphasis on the relationship between the dimension of the claw tip and the substrate texture. To evaluate the force range by which the claw can interact with a substrate, forces generated by the freely moving legs were measured using a load cell force transducer. To obtain information about material properties of the claw, its mechanical strength was tested in a fracture experiment, and the internal structure of the fractured claw material was studied by scanning electron microscopy. The bending stress of the claw was evaluated as 143.4-684.2 MPa, depending on the cross-section model selected. Data from these different approaches led us to propose a model explaining the saturation of friction force with increased texture roughness. The forces are determined by the relative size of the surface roughness Ra (or an average particle diameter) and the diameter of the claw tip. When surface roughness is much bigger than the claw tip diameter, the beetle can grasp surface irregularities and generate a high degree of attachment due to mechanical interlocking with substrate texture. When Ra is lower than or comparable to the claw tip diameter, the frictional properties of the contact between claw and substrate particles play a key role in the generation of the friction force.

The highly conserved domain of unknown function in the cyanobactin superfamily has a novel fold. The protein does not appear to bind the most plausible substrates, leaving questions as to its role., Patellamides are members of the cyanobactin family of ribosomally synthesized and post-translationally modified cyclic peptide natural products, many of which, including some patellamides, are biologically active. A detailed mechanistic understanding of the biosynthetic pathway would enable the construction of a biotechnological ‘toolkit’ to make novel analogues of patellamides that are not found in nature. All but two of the protein domains involved in patellamide biosynthesis have been characterized. The two domains of unknown function (DUFs) are homologous to each other and are found at the C-termini of the multi-domain proteins PatA and PatG. The domain sequence is found in all cyanobactin-biosynthetic pathways characterized to date, implying a functional role in cyanobactin biosynthesis. Here, the crystal structure of the PatG DUF domain is reported and its binding interactions with plausible substrates are investigated.

A method has been explored for the production of low background polyacrylamide gradient gel-filled capillaries. The main idea is to prepare the capillaries by filling them with short steps of gelling solutions, including a long section of buffer for detection. This method is demonstrated to be practicable by scanning the filled capillaries. Smooth gradients can be formed at a step-length of 1.5 cm if the relevant capillaries are filled with solutions in a step-down order. By using this method, the desired capillaries were prepared with a success rate of >90% and gradient position error of ±0.62 mm. As shown by capillary gradient gel electrophoresis, this new type of capillary can be used for fast and efficient separation of polyaspartate and murein oligosaccharides (from E. coli ). They also allow the use of any available UV wavelength for high detection sensitivity. The unlabelled oligosaccharides at a total concentration of 1 mg ml −1 were directly detected at 200 nm.

Monoclonal antibody 3CB2 recognizes an antigen expressed in discrete cell types derived from ectoderm and mesoderm. Biochemical and immunohistochemical studies indicate that the antigen recognized by the antibody is a 55 kDa cytoplasmic protein that may be an intermediate filament associated protein (IFAP). Developmental studies show that 3CB2 antigen is intensely expressed very early in the chick embryo, in the neural tube, myotomes, and in mesenchymal cells of visceral arches and the presumptive facial area. As development proceeds, antigen expression becomes restricted to astrocytes and radial glia cells throughout the brain. A detailed immunohistochemical study of the developing chick retina shows that the expression of 3CB2 antigen at embryonic day 8 (E8) is restricted to Müller cells, with the pattern of expression closely related to their degree of differentiation. We show, by immunocytochemistry labeling of entire Müller cells dissociated from retinas of E16-E20, that 3CB2 monoclonal is labeling the whole cell. 3CB2 selectively labels Müller cells in the rat and chameleon, but not their retinal horizontal cell axons. 3CB2 monoclonal is a very sensitive marker for early differentiating Müller cells. Our results provide evidence that 3CB2 antigen is a cytoskeletal component which may be involved in the morphogenesis of these cells, and also perhaps in the outgrowth of some axons.

Capillary coupling techniques are suggested for the connection of a non-flow buffer-filled capillary to gel-filled capillaries as a low-background detection cell. The UV detection sensitivity of the resulting capillaries is improved at least fivefold, as demonstrated by using polyamino acids as test samples. The detection sensitivity can be further improved by using a low-background running buffer. Several ways are suggested to overcome the problems of resolution decrease and baseline drif which were observed after coupling.

A method was established for the preparation of capillaries filled partially with highly condensed polyacrylamide gels (including step gradient gels) used for separation and partially with a buffer used for detection. These novel capillaries combine the high resolution of gel-filled capillaries and the low background of buffer-filled capillaries. They can be used for more than 1 week or for more than 50 injections, as demonstrated by the separation of poly-(α,β)- d.l -aspartate. The detection limit tested with diaspartate is about two orders of magnitude lower than that with the totally gel-filled capillaries. Some problems such as baseline drifting arise when using these capillaries but can be overcome by pre-running the capillaries several times with highly concentrated samples.

A fairly quick method has been established for the production of highly condensed polyacrylamide gel-filled capillaries. Void-free capillaries with inner diameters as small as 25 μm and monomer concentrations of up to over 30% T + 5% C can successfully be prepared within 5 h. These capillaries can be used for over 20 injections of poly-(α,β)- d , l -aspartate at 200 V/cm and 25°C, with gels immobilized at the capillary tips after polymerization, and for more than 70 injections with gels immobilized, during polymerization, over a longer section of at least 0.5 cm. The important polymerization conditions in this method are the application of a slight pressure, controlled polymerization directions and the selections of buffer components and/or the concentrations of radicals and catalyst.

F11 and Nr-CAM/Bravo are two axon-associated glycoproteins belonging to different subgroups of the immunoglobulin superfamily. In this report we have investigated the interaction of both proteins using neurite outgrowth and binding assays. Antibody blocking experiments demonstrate that neurite extension of tectal cells on immobilized F11 is mediated by Nr-CAM/Bravo. Binding studies further reveal a direct heterophilic interaction between F11 and Nr-CAM/Bravo. This activity can be mapped to the amino-terminal second or third immunoglobulin-like domain within F11 with domainspecific monoclonal antibodies and deletion mutant proteins expressed on COS cells. Furthermore, perturbation experiments with domain-specific monoclonal antibodies demonstrate that this region is required for adhesion and neurite extension.

The tectobulbar tract is the first long-distance projecting fiber pathway to appear during the development of the avian optic tectum (dorsal half of the mesencephalon). Immunologically stained wholemounts of the E3 mesencephalon reveal that the early tectobulbar axons course in a dorsal-to-ventral direction and abruptly turn in a caudal direction shortly before reaching the ventral midline. During subsequent development, more tectobulbar axons are generated that form a parallel array of thick fascicles coursing ventrally within the mesencephalon. At this later stage of development, the tectobulbar tract bifurcates into an ipsilateral and contralateral pathway, both growing in a caudal direction near the mesencephalic ventral midline. Bifurcation and change in direction of growth is accompanied by a complete loss of the fasciculated growth pattern. Each tectobulbar axon is thus divided into a proximal fasciculated and a distal unfasciculated segment. Tectobulbar fascicles occupy the most superficial surface layer of the mesencephalon at early stages and are displaced deeper into the tissue beginning at embryonic day 5. In both of these locations, tectobulbar axons express molecules involved in axon-axon and axon-substrate interactions like the G4 antigen, neural cell adhesion molecule (N-CAM), neurofascin, and T61 antigen as revealed by immunohistochemistry and immunoblotting. Stripes of the mesencephalon explanted onto a basal lamina substratum show vigorous outgrowth of neurites. These processes grow in fascicles at a growth rate of 40 microns/h. Staining of the neurites with specific antibodies, as well as the position of the retrogradely labeled cell bodies, is in agreement with these processes being tectobulbar axons. This in vitro explant system was used to investigate the expression and possible functional involvement of N-CAM, neurofascin, G4 protein, and T61 antigen in the growth of these axons. The presence of antigen- binding fragments of polyclonal anti-G4 antibodies completely blocks fasciculation of the neurites but has no influence on their rate of elongation. Antibodies against N-CAM and neurofascin have no detectable effects. The number and length of the in vitro growing axons are reduced by the monoclonal T61 antibody. This effect is reversible. The elucidation of the exact course in vivo and the accessibility to the axons growing in vitro make the tectobulbar tract an excellent model system for the investigation of the role of these and other proteins in axonal growth and guidance during the development of the CNS.

We describe a method for the selective labelling, isolation and electrophoretic analysis of cell-surface molecules and extracellular matrix components. Intact tissues are reacted with activated esters of biotin and the labelled surface molecules identified on Western blots with horseradish-peroxidase-coupled or 35S-labelled streptavidin. Alternatively, the biotinylated proteins can be purified from tissue homogenates by affinity chromatography on an avidin-agarose column. Evidence is presented to show that this method is indeed specific for membrane and matrix components. Its practical application to embryonic neural tissues is demonstrated.

The internal quantum efficiency as a function of the internal electric field was studied in InGaN/GaN based quantumwell heterostructures. Most striking, we find the IQE to be independent of the electron hole overlap for a standard green-emitting single quantum-well LED structure. In standard c-plane grown InGaN quantum wells, internal piezo-fields are responsible for a reduced overlap of electron and hole wavefunction. Minimization of these fields, for example by growth on non-polar m- and a-planes, is generally considered a key to improve the performance of nitride-based light emitting devices. In our experiment, we manipulate the overlap by applying different bias voltages to the standard c-plane grown sample, thus superimposing a voltage induced band-bending to the internal fields. In contrast to the IQE measurement, the dependence of carrier lifetime and wavelength shift on bias voltage could be explained solely by the internal piezo-fields according to the quantum confined Stark effect. Measurements were performed using temperature and bias dependent resonant photoluminescence, measuring luminescence and photocurrent simultaneously. Furthermore, the doping profile in the immediate vicinity of the QWs was found to be a key parameter that strongly influences the IQE measurement. A doping induced intrinsic hole reservoir inside the QWs is suggested to enhance the radiative exciton recombination rate and thus to improve saturation of photoluminescence efficiency.

The genomes of the related crenarchaea Pyrobaculum aerophilum and Thermoproteus tenax lack any obvious gene encoding a single-stranded DNA binding protein (SSB). SSBs are essential for DNA replication, recombination, and repair and are found in all other genomes across the three domains of life. These two archaeal genomes also have only one identifiable gene encoding a chromatin protein (the Alba protein), while most other archaea have at least two different abundant chromatin proteins. We performed a biochemical screen for novel nucleic acid binding proteins present in cell extracts of T. tenax . An assay for proteins capable of binding to a single-stranded DNA oligonucleotide resulted in identification of three proteins. The first protein, Alba, has been shown previously to bind single-stranded DNA as well as duplex DNA. The two other proteins, which we designated CC1 (for crenarchaeal chromatin protein 1), are very closely related to one another, and homologs are restricted to the P. aerophilum and Aeropyrum pernix genomes. CC1 is a 6-kDa, monomeric, basic protein that is expressed at a high level in T. tenax . This protein binds single- and double-stranded DNAs with similar affinities. These properties are consistent with a role for CC1 as a crenarchaeal chromatin protein.

Gram-positive pathogens are a major concern to global health, with increasing resistance to antimicrobials and the lack of preventative therapeutics. Understanding how these bacteria interact with host cells is vital for the development of novel strategies to combat disease. One of the most crucial steps in infection is adhesion to the host cell. The discovery of complex cell-surface associated proteins, such as pili, has advanced our knowledge of this interaction, however the precise molecular mechanisms underlying this process remain unclear. Structural studies of pili revealed the presence of highly unusual intramolecular covalent bonds between amino acid side chains. These include isopeptide bonds between Lys and Asp/Asn residues, conferring mechanical strength, thermal stability and resistance to proteases [1,2]. In Streptococcus pyogenes pili, the adhesin Spy0125 (or Cpa) interacts with the host cell. It comprises three domains, two of which contain stabilising isopeptide bonds [2,3]. Intriguingly, the third domain contains an extremely rare thioester bond, between a Cys and a Gln residue. A Cys to Ala mutation results in a 75% reduction in adhesion, suggesting that this internal linkage may mediate direct attachment [3]. We have now discovered putative thioester domains (TEDs) in cell-surface proteins of several clinically important pathogens. The only other example of an internal thioester is found in complement proteins, where the reactive bond enables the formation of covalent attachment to pathogens. The presence of these bonds in bacterial proteins suggests the possibility of an as-yet uncharacterised, conserved mechanism of covalent host cell attachment. For a selection of pathogens, we have used mass spectrometry and crystallography to confirm the presence of the covalent link between the Cys and Gln residues within the TEDs. Furthermore, we have identified putative host cell targets of TEDs and confirmed covalent linkages between the TED and the target.

We studied mechanisms underlying the generation of topographic order within the developing chick retinotectal connection by combining the recently introduced stripe assay with a novel membrane protein fractionation technique. Our experiments show a preference of temporal and nasal retinal fibers for growing on cell membranes prepared from their proper target area. In addition, membrane preparations from posterior tectum were found to prolong substantially the survival of nasal neurites in vitro. We conclude that tropic as well as trophic interactions contribute to the generation of topographic maps during embryogenesis, in our case to the homing of nasal axons within the posterior tectum.

Anthony van Leeuwenhoek, in a letter to the Royal Society, described the little animals he saw in his microscope. In his note he specifically mentioned that these little creatures were bonded by some sort of structure and expressed his hope to resolve the question of what held them together (from Salton, 1960). It was about 200 years later, in 1887, that the first publication appeared on the biochemical properties of a structure which the author Livio Vincenzi, believed to be the cell wall of the “fission yeast” Bacillus subtilis (Vincenzi, L., 1887). It took almost another 70 years until isolated bacterial envelopes and finally the chemically pure shape maintaining structure of the bacterial cell wall, the sacculus, were available for structural and chemical characterization (Review: Salton, M.R.J., 1960)

Monoclonal antibodies provide a powerful tool for the identification and analysis of novel cell-surface molecules. We present here a method for antigen preparation and an immunization protocol that facilitates generation of mAb reactive with cell-surface molecules of low abundance and/or low antigenicity. The procedure involves isolation and extensive fractionation of cell-surface and detergent-soluble extracellular-matrix molecules prior to immunization. Cell-surface proteins on intact tissue are biotin-labeled using a reagent that does not penetrate cells. Avidin affinity chromatography is then used to purify these biotinylated molecules. Size-exclusion HPLC is used to separate these surface molecules on the basis of apparent molecular mass. Finally, immunization with antigen coupled to keyhole-limpet hemocyanin is combined with long-term booster immunizations to generate a hyperimmune response resulting in high-affinity IgG. A test application of this approach was aimed at the generation of mAb against cell-surface molecules of approximately 135 kDa in the developing chicken retinotectal system. Immunochemical analyses using antibodies produced by this approach which showed restricted patterns of tissue staining reveal that mAb were generated against all previously identified immunoglobulin superfamily molecules of this size in this system. Furthermore, we produced many additional antibodies that labeled retinotectal tissue in novel staining patterns. In the two cases analyzed in detail, these new patterns reflect the distributions of previously uncharacterized members of the immunoglobulin superfamily. The success of this initial study suggests that this method may represent a broadly applicable approach towards the preparation of extensive libraries of antibodies against cell-surface molecules expressed on cells from numerous sources.

Escherichia coli has two murein transglycosylases, which are found in the soluble and the particulate fraction, respectively. The enzymes have been purified and have been shown to differ in some of their molecular properties [Mett, H., Keck, W., Funk, A. & Schwarz, U. (1980) J. Bacteriol. 144, 45–52]. We improved and simplified the purification procedure for the membrane-derived transglycosylase and characterized the two enzymes in more detail by peptide mapping and by immunological procedures. The peptide pattern obtained after tryptic digestion of the purified enzymes differed for the two enzymes. Antisera to the transglycosylases reacted only with their own antigen as shown by specific inhibition of the enzymatic activity, double immunodiffusion and by immunochemical staining of protein blots on nitrocellulose filters. Thus we conclude that the transglycosylases are two distinct proteins and that the one is not a precursor of the other.

Retinotopic analysis of the pathways of normal and aberrant retinal axons within the tectum of developing chick embryos was performed by selective labeling of retinal axons with a fluorescent dye, rhodamine-B isothiocyanate. To produce aberrant retinal axons, the presumptive optic chiasma was surgically disorganized at the 3rd day of incubation. At the 11th and 13th days of incubation, more than half of the operated embryos exhibited several aberrant retinal axons which reached ectopic parts of the tectum. The pathways of these aberrant axons within the tectum depended on the position of their initial invasion into the tectum at the diencephalotectal junction, and not on their position of origin within the retina. The aberrant retinal axons did not show any sign of correction of their pathways toward their normal sites of innervation within the tectum. As development proceeded, elimination of the aberrant retinal axons occurred. By the 16th day of incubation, almost all operated embryos lacked aberrant retinal axons and although the total number of axons often appeared reduced, a nearly normal topography of retinotectal projections was established. These findings indicate that the initial invasion of the retinal axons into the tectum is conducted predominantly by nonspecific mechanisms and, thereafter, a selective maintenance of appropriate retinal axons occurs.

SUMMARY: The morphogenetic capacity of E. coli was studied by converting the rod-shaped cells into spheres and then determining whether these spheres could revert to rods. The morphogenesis of cells was followed by immobilizing them in a viscous Methocel-containing medium. Two different types of spheres were prepared: cells which retained a mechanically intact sacculus, and osmotically sensitive sphaero-plasts lacking a sacculus. The sphaeroplasts were not able to revert to rods although they were able to synthesize a new sacculus. In contrast, spheres which had retained an intact sacculus were able to reshape themselves into rods. They were also able to form new ends at (or near) the sites of the ends on the original rods.

The intermediates in the biosynthetic pathway of murein were examined in two strains of Escherichia coli to determine whether they synthesized oligomeric precursors in vivo. No oligomeric precursors could be detected; the only intermediates found were the previously described UDP-N-acetylmuramyl peptides, and the two lipid-linked compounds, N-acetylglucosamyl-N-acetylmuramyl-(pentapeptide)-pyrophosphoryl-undecaprenol and N-acetylmuramyl-(pentapeptide)-pyrophosphoryl-undecaprenol. It was concluded that lipid-linked monomers are directly incorporated into the murein sacculus in vivo and do not pass through an oligomeric stage.

Minicells and filaments of Escherichia coli strain P678–54 provide a convenient means of preparing cell-envelope fractions that are, respectively, greatly enriched or reduced in material derived from the cell poles. The inner (cytoplasmic) membrane, outer membrane, and sacculus were prepared both from minicells and from filaments purified from a single culture. One protein component and phosphatidylglycerol were found to be enriched in minicell inner membrane.

Properties of murein hydrolases, assumed to play an essential role in bacterial morphogenesis, are described. Evidence is presented that in the intact cell these enzymes are prevented from uncontrolled reaction. It is shown that the “barrier” between the enzymes and their substrate sacculus, a shape-maintaining macromolecule within the cell envelope, can be artificially broken. Assay systems to measure murein hydrolase activity in the intact cell, in isolated cell envelopes and after solubilization are described. A combination of Triton X-100 and sodium chloride is shown to liberate membrane-bound hydrolases effectively.

Escherichia coli W7 cells were found to release three different muropeptides into the culture medium: tetrapeptide (L-Ala-D-Glu-meso-diaminopimelic acid-D-Ala), tripeptide (L-Ala-D-Glu-meso-diaminopimelic acid), and a previously undescribed dipeptide (meso-diaminopimelic acid-D-Ala). From the rate of release of these three peptides, it was calculated that 6 to 8% of the murein in the sacculus was lost per generation.

High-pressure liquid chromatography of a muramidase digest of murein sacculi from Caulobacter crescentus showed that the absence of D-alanine carboxypeptidase activity in the cells was reflected by a very high content of pentapeptide in the murein. Approximately half of the pentapeptide side chains were shown to contain glycine, which replaced D-alanine as the terminal amino acid.

In Escherichia coli, peptide cross bridges in the murein undergo turnover after they are synthesized. Peptide cross bridges formed in the presence of [3H]diaminopimelic acid were found to lose 3H label from their donor peptides after the [3H]diaminopimelic acid was removed from the growth medium. There was a corresponding increase in the amount of 3H label in acceptor peptides so that the total amount of label in the peptide cross bridges remained constant. Our explanation of this observation is that the cross bridges are cleaved by the cell, and the original 3H-labeled donor peptides are incorporated into new cross bridges. Since these 3H-labeled peptides are now only tetrapeptides, they can only be used as acceptors when new cross bridges are formed.

Derivatives of the Escherichia coli penicillin-binding protein 5 (PBP5) with truncated carboxyl terminals were obtained by altering the carboxyl-coding end of the dacA gene. After cloning the modified dacA gene into a runaway-replication-control plasmid, one clone that overproduced and excreted the desired protein into the periplasm was used as a source for the isolation of a water-soluble PBP5 (i.e. PBP5S). In PBP5S the carboxyl-terminal 21-amino-acid region of the wild-type protein was replaced by a short 9-amino-acid segment. Milligram amounts of PBP5S were purified by penicillin affinity chromatography in the absence of detergents or of chaotropic agents. PBP5S was stable and possessed DD-carboxypeptidase activity without added Triton X-100. Upon reaction with [14C]benzylpenicillin it was converted into a rather short-lived acyl-enzyme complex, as observed with PBP5. Both PBP5 and PBP5S were crystallized. In contrast to PBP5, PBP5S yielded enzymatically active, well-formed prismatic crystals suitable for X-ray analysis.

In vivo studies of murein biosynthesis show that newly synthesized murein unexpectedly differs in its chemical composition from preexisting murein. New murein is loosely crosslinked with the preexisting sacculus; in a maturation process involving further transpeptidation, the final stage of crosslinkage in murein is achieved. Newly inserted murein initially carries pentapeptide subunits, which are the donor of the secondary transpeptidation reaction. In mutants with defective penicillin-binding protein 4 the secondary transpeptidation step is abolished. Uncoupling of the secondary transpeptidation reaction from crosslink formation during the initial insertion of new murein was also found in a mutant with a defect in lipoprotein biosynthesis. We conclude that the initial transpeptidation of murein and crosslink formation during the maturation of newly inserted murein are catalyzed by two different enzyme systems.

Two mutants of Escherichia coli that are deficient in the penicillin-insensitive DD-endopeptidase have been isolated. The strain JE10874 (mepA) has about 10%-20% of the residual activity and another strain, JE10368 (mepB) has 40%-50% of the activity found in the wild-type, parental strain, PA3092. The penicillin-insensitive endopeptidase is a periplasmic enzyme. Genetic mapping studies show that the mutation mepA is located close to aroC (50 min) and the other mutation, mepB, is very close to malE (91 min) on the chromosome. These mutants grow normally under a wide range of growth conditions; other phenotypic properties of the mutants are very similar to those of the parent strain. A double mutant (mepA mepB), and a triple mutant (mepA mepB dacB), deficient in both penicillin-insensitive and penicillin-sensitive endopeptidases, were constructed. Again, these mutants grew normally. We conclude that either the very low level of residual enzyme activity in the mutants is enough for their survival or that the penicillin-insensitive endopeptidase is not essential for survival under laboratory conditions.

A micro method for the isolation and characterization of the penicillin-binding sites in penicillin-binding proteins (PBPs) was developed. Only 10 nmol of a pure PBP are required for the whole procedure which is based on high-pressure liquid chromatography (HPLC). We showed that serine 44 in PBP 5 from Escherichia coli binds penicillin covalently.

Both the polarity of the axonal growth and the formation of the optic fiber pattern early in retinal morphogenesis were studied in silver stained whole mounts of embryonic chick, quail, and pigeon retinae. The surface area of the retina and of the optic fiber layer increases in size exponentially, the optic fiber layer expanding faster than the retina. The optic fiber layer covers the retinal surface at E5 in quail and at E6 in chick and pigeon. In all species studied, the retinal fiber layer does not expand homogeneously with the optic nerve head as the center. Instead, the retinal fiber layer enlarges with polarities in the dorsal to ventral and nasal to temporal direction. The very first axon bearing ganglion cells appear at stage 16 in the dorsal and central portion of the retina and grow ventrally to merge at the optic disk. From stage 23 on, the optic fiber layer expands faster in the temporal than in the nasal side. Measurements on the initial polarization of young axonal processes show that the axonal growth is directed toward the optic fissure and the optic nerve head. This growth polarization is found at the onset of growth cone formation and in axons far from the nearest ganglion cells or ganglion cell axons. Therefore axon-axon interaction cannot be involved in the initial axon orientation early in retinal morphogenesis.

Polar cap formation has been studied in synchronized Escherichia coli cells. It is dependent on a signal given after completion of a round of DNA replication. A 20 min time interval between the release of this signal and physical cell separation is probably the time required for the completion of polar caps. During this time murein is synthesized at an increased rate and cells are especially sensitive to penicillin.

The shape of most bacteria is determined by a defined macromolecule which encloses the cell completely. Therefore, in this case, morphogenesis can be studied by analyzing the biosynthesis of this defined type of molecule, a sacculus, which is made up from the polymer murein. The sacculus can be isolated in pure form and can be examined under the electron microscope. We took advantage of this fact to study some topological aspects of murein biosynthesis in Escherichia coli. The growth of the structural determinant of the cell wall, the sacculus, was found to be carried out by several functionally different systems. One is involved in cell elongation, others in cell division; these systems were revealed by their differential sensitivity to penicillin. By use of this antibiotic, we were able to localize the site of action of murein hydrolases which are directly involved in bacterial morphogenesis; the topology and timing of their action are correlated with DNA replication. This procedure revealed zonal growth of the sacculus during cell division. Thus, this structural element of the cell wall is enlarged exactly as predicted by the replicon hypothesis.

The characteristic shape of bacterial cells is maintained by the strength and form of the murein layer of the cell wall1. Thus, rod-shaped bacteria need to synthesize a rod-shaped murein. The shape of the murein may be determined by the properties of the penicillin-binding proteins (PBPs) that catalyse the insertion of murein precursors into the cell wall2. In Escherichia coli inhibition of PBPs produces characteristic effects on bacterial morphology. For example, inactivation of PBP 2 results in an inability to synthesize cylindrical murein during cell elongation and the growth of E. coli as spherical cells2. We report here that osmotically stable spherical cells of E. coli can also be produced by an increase in the level of PBP 5, a D-alanine carboxypeptidase3, and show that these cells, and those produced by inactivation of PBP 2, have the same abnormality in the structure of the newly inserted murein.

The ability of human milk galactosyltransferase to attach D-galactose residues quantitatively to the C-4 of N-acetylglucosamine moieties at the ends of oligosaccharides has been utilized for the specific labeling and quantitative determination of the chain length of the glycan moiety of the bacterial cell wall. The average polysaccharide chain length of the soluble, uncrosslinked peptidoglycan secreted by Micrococcus luteus cells on incubation with penicillin G was studied with this technique and found to be approximately 70 hexosamines long. Furthermore, the peptidoglycan chain length of Escherichia coli sacculi of different cell shapes and dimensions was determined both in rod-shaped cells and in filaments induced by temperature shift of a division mutant or by addition of cephalexin or nalidixic acid. The average chain length found in most of these sacculi was between 70 and 100 hexosamines long. Small spherical 'mini' cells had chain lengths similar to those of the isogenic rod-like cells.

The cell adhesion molecule isolated from neural tissue (N-CAM) is a membrane glycoprotein which is directly involved in calcium-independent adhesion among nerve cells and their processes (for review see refs 1,2). N-CAM has an unusual carbohydrate moiety containing a large and variable amount of sialic acid, the variation reflecting both the type of tissue and its developmental age3,4. N-CAM is believed to be a ligand in the formation of cell-cell bonds5 and a decrease in sialic acid content from 30% to 10% is associated with a marked enhancement of the molecule's binding activity6–8. Antibodies to N-CAM block its function and inhibit or alter bundling of nerve fibres9, retinal cell development10–12 and nerve-muscle interaction13,14. Here we use micro-gel elec-trophoresis15 to compare N-CAM from several parts of the developing chick visual system. The results indicate that N-CAM from the retina of 5–10-day-old embryos already exists in a relatively sialic acid-poor form, whereas the tectum and optic nerve beyond the eye contain sialic acid-rich N-CAM until much later in development. These studies suggest that the perikaryon and proximal axon shaft of retinoganglion cells have N-CAM with a lower sialic acid content than the distal portion of the axons, and that resulting differences in neurite adhesivity may be an important factor in the formation of the optic system.