Fructose production by Zymomonas mobilis in fed-batch culture with minimal sorbitol formation

Summary Fed-batch cultures of Zymomonas mobilis (UQM 2864), a mutant unable to metabolise fructose, grown on diluted sugar cane syrup (200 g/l sucrose) achieved yields of 90.5 g/l fructose and 48.3 g/l ethanol with minimal sorbitol formation and complete utilization of the substrate. The effect of inoculum size on sorbitol formation in the batch stage of fed-batch fermentation are reported. Fermentation of sucrose (350 g/l) supplemented with nutrients yielded 142 g/l fructose and 76.5 g/l ethanol. Some fructose product loss at high fructose concentrations was observed. The fed-batch fermentation process offers a method for obtaining high concentrations of fructose and ethanol from sucrose materials.     

Expression in Bacillus subtilis of the gene for human urogastrone using synthetic ribosome binding sites

Summary A chemically synthesised gene coding for human urogastrone which was earlier cloned in E. coli (Smith et al. 1982) has now been cloned into expression vectors for Bacillus subtilis Two types of constructs have been made, one giving production of methionylurogastrone and the other giving rise to a methionyl-urogastrone- galactosidase fusion polypeptide facilitating quantification of expression levels.The ribosome binding sites used in the expression plasmids are synthetically made oligonucleotides residing on short restriction fragments to allow easy replacement by other ribosome binding sites.Using shuttle vectors and constitutive promoters from Bacillus phages 105 and SPP1, we were able to detect levels of expression amounting to a few thousand molecules per cell during logarithmic growth in both E. coli and B. subtilis.     

Localization of glycoprotein glycosyltransferases in the Golgi apparatus of rat and mouse testis

Golgi fractions prepared from rat testis have been shown to be enriched in the following glycoprotein glycosyltransferases: N-acetylglucosaminyltransferase, 47-fold, galactosyltransferase, 33-fold, and N-acetylglucosaminide fucosyltransferase, 15-fold. Appreciably lower transferase levels were obtained in other subcellular fractions. In the mouse, Golgi fractions were prepared from testis homogenates, testis cell suspensions and partially purified testis germinal cells; these fractions were also enriched in the above glycoprotein glycosyltransferases. Electron microscopic analysis indicated that a major portion of the total transferase activity was located in the Golgi apparatus of both rat and mouse testis although these experiments could not rule out the possible presence of some transferase activity in other organelles.     

GENETIC DIVERSITY IN THE MONOECIOUS HYDROPHILE CERATOPHYLLUM (CERATOPHYLLACEAE)

This study surveys genetic variation in two clonal, monoecious, water-pollinated species that differ in their extent of sexuality and distributional range. Electrophoresis was used to quantify allozyme variability in 12 Wisconsin populations of the widespread Ceratophyllum demersum and the rare C. echinatum. Electrophoretic data indicate that populations of both species have low levels of sexual recombination, low levels of variation, and are structured genetically like inbreeding terrestrial plants. Ceratophyllum populations differ from “typical” clonal terrestrial plants by lower genetic diversity, lower proportions of multiclonal populations, and fewer genotypes per population. In two populations where sexual recombination is documented, heterozygosity is low with significant deficiencies. Monoecy in Ceratophyllum may be related to historical evolutionary factors, whereas vegetative reproduction has a greater influence on the genetic population structure of extant populations. The low genetic identity between C. demersum and C. echinatum supports their recognition as distinct species.     

Effects of Exogenous Androgen and Antiandrogen on Territorial and Nonterritorial Red-winged Blackbirds (Aves: Icterinae)

We investigated the role of testosterone (T) in territory establishment and maintenance in male red-winged blackbirds (Agelaius phoeniceus) by implanting exogenous T or pharmacological agents that block the action of T in nonterritorial floaters and territory owners. Floaters with artificially elevated plasma T levels were unable to obtain territories. Territory owners implanted with T did not expand their territories, although they engaged in more aggressive behavior than did control males. Flutamide, which binds to T receptors in target areas, had no effect on territoriality. However, most territory owners given a combination of flutamide and ATD, an aromatization blocker, lost portions of their territories even though they actively defended them. Our results 1) suggest that, although T influences aggressive behavior, elevated plasma levels alone are insufficient to overcome previously-established social relationships between territory owners or between owners and floaters; 2) indicate that impairing the action of T leads to reduced abilities of territory owners to maintain territories against vigorous challenges: and 3) support recent findings that T acts on reproductive behavior in birds through both androgenic and estrogenic metabolites.     

Neurotrophin-3–enhanced Nerve Regeneration Selectively Improves Recovery of Muscle Fibers Expressing Myosin Heavy Chains 2b

The purpose of this study was to evaluate the effect of neurotrophin 3 (NT-3) enhanced nerve regeneration on the reinnervation of a target muscle. Muscle fibers can be classified according to their mechanical properties and myosin heavy chain (MHC) isoform composition. MHC1 containing slow-type and MHC2a or 2b fast-type fibers are normally distributed in a mosaic pattern, their phenotype dictated by motor innervation. After denervation, all fibers switch to fast-type MHC2b expression and also undergo atrophy resulting in loss of muscle mass. After regeneration, discrimination between fast and slow fibers returns, but the distribution and fiber size change according to the level of reinnervation. In this study, rat gastrocnemius muscles (ipsilateral and contralateral to the side of nerve injury) were collected up to 8 mo after nerve repair, with or without local delivery of NT-3. The phenotype changes of MHC1, 2a, and 2b were analyzed by immunohistochemistry, and fiber type proportion, diameter, and grouping were assessed by computerized image analysis. At 8 mo, the local delivery of NT-3 resulted in significant improvement in gastrocnemius muscle weight compared with controls (NT-3 group 47%, controls 39% weight of contralateral normal muscle; P < 0.05). NT-3 delivery resulted in a significant increase in the proportion (NT-3 43.3%, controls 35.7%; P < 0.05) and diameter (NT-3 87.8 μm, controls 70.8 μm; P < 0.05) of fast type 2b fibers after reinnervation. This effect was specific to type 2b fibers; no normalization was seen in other fiber types.This study indicates that NT-3–enhanced axonal regeneration has a beneficial effect on the motor target organ. Also, NT-3 may be specifically affecting a subset of motoneurons that determine type 2b muscle fiber phenotype. As NT-3 was topically applied to cut nerves, our data suggest a discriminating effect of the neurotrophin on neuro–muscular interaction. These results would imply that muscle fibers may be differentially responsive to other neurotrophic factors and indicate the potential clinical role of NT-3 in the prevention of muscle atrophy after nerve injury.There has been much recent interest in the use of growth factors to augment peripheral nerve regeneration. A family of growth factors collectively known as the neurotrophins are now considered critical for the development, maintenance, and regeneration of the nervous system. The neurotrophin family includes NGF, brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3),¹ and neurotrophin-4/5 (NT-4/5) (Lewin and Barde, 1996; Lindsay, 1996). Little is known of their effect on regeneration of the peripheral nervous system.NT-3 has been shown to act on a subpopulation of muscle sensory neurons innervating muscle spindles and Golgi tendon organs, and there is also evidence of its effect on a subpopulation of cutaneous afferents (Ernfors et al., 1994; Tessarolo et al., 1994; Airaksinen et al., 1996). NT-3 has shown various effects on motor nerve regeneration, including differentiation of motoneurons from avian neural tube progenitor cells (Averbuch-Heller et al., 1994) and survival of neonatal and adult motoneurons in vitro (Hughes et al., 1993) and of neonatal motoneurons in vivo (Li et al., 1994; Vejsada et al., 1995), although the evidence is sometimes contradictory (Eriksson et al., 1994). In cocultures of adult muscle and embryonic motoneurons, NT-3 enhances the number and length of neurite outgrowths, the density of endplates per muscle fiber, and the amount of muscle innervation (Braun et al., 1996). NT-3 also plays a role in functional maturation of neuromuscular synapses (Lohoff et al., 1993; Wang et al., 1995) and regulates the cholinergic phenotype of developing motoneurons (Wong et al., 1993; Kato and Lindsay, 1994). NT-3 knockout mice show a loss of all muscle spindle afferent innervation and fusimotor neurons to the muscle but lose only few skeletomotor nerve fibers (Kucera et al., 1995a ). About 80% of adult motoneurons express the NT-3–specific trkC receptor (Henderson et al., 1993; Griesbeck et al., 1995), and NT-3 is the predominant neurotrophin expressed in skeletal muscle (Griesbeck et al., 1995). Furthermore, NT-3 is internalized and retrogradely transported from the periphery to motoneuron cell bodies (Di Stefano et al., 1992). Thus, there is experimental and circumstantial evidence to suggest that NT-3 may play a role in adult motoneurons, although in vivo data on the survival effect of NT-3 on adult motoneurons is still lacking. Furthermore, there is no evidence of an NT-3–dependent effect on neuro–muscular interaction.When a skeletal muscle is denervated and subsequently reinnervated, a characteristic sequence of events ensues. The muscle rapidly loses weight as the muscle fibers atrophy (Pellegrino and Franzini, 1963), but after reinnervation, it gradually recovers mass to a variable extent, depending upon the degree of reinnervation (Bertelli and Mira, 1995) and correlating with the maximum force of contraction (Gillespie et al., 1987). The fibers within an individual skeletal muscle do not exist as a homogenous population but can be classified according to their different metabolic and contractile properties (Burke et al., 1971; Peter et al., 1972). Also, they can be identified morphologically according to differential expression of specific myosin heavy chain isoforms. Slow, oxidative type 1 muscle fibers contain myosin heavy chain 1 (MHC 1), fast oxidative glycolytic type 2a fibers contain myosin heavy chain 2a (MHC 2a), while fast glycolytic type 2b fibers contain myosin heavy chain 2b (MHC 2b) (Bar and Pette, 1988). Muscle fiber phenotype is conferred by its innervation, and changes of neuro–muscular interaction lead to alteration of muscle fiber phenotype (Romanul and Van der Meulen, 1966; Fex and Sonneson, 1970; Salmons and Sreter, 1975). The relative proportions of fiber types vary with age, sex, strain, species, and muscle type (Maltin et al., 1989). Generally, there is a high proportion of type 1 fibers in postural muscles (e.g., soleus) and of type 2 fibers in fast muscles (e.g., extensor digitorum longus), while in mixed muscles (e.g., gastrocnemius) there are varying proportions of each type. Muscle fiber type proportion also varies dynamically with physiological and pathological parameters (Jansson et al., 1978; Green et al., 1983; Izumo et al., 1986; Goldspink et al., 1992; Pette and Vrbova, 1992). For example, the distribution of fiber types in normal muscle is dispersed in a “mosaic pattern,” but after denervation and reinnervation of the muscle there is a shift to grouping (Karpati and Engel, 1968). Also, there is a change in the proportions of fiber types, and in the rat the majority of fibers become initially fast with denervation, with subsequent fiber specialization being dictated by patterns of reinnervation (Fields and Ellisman, 1986). This plastic nature of muscle makes it an interesting model to investigate reinnervation changes that may occur after NT-3 administration.We have recently demonstrated that the local delivery of NT-3 to rat sciatic nerve enhances the rate and amount of nerve regeneration, and at 8 mo postoperative, there was a 40% increase in the myelinated fiber count (Sterne et al., 1997). However, enhanced regeneration by itself is not necessarily indicative of a beneficial effect on the target muscles, such as reacquisition of more normal physiological function. Therefore, the aim of this study was to assess whether NT-3–enhanced nerve regeneration resulted in biochemical or morphological changes in a target muscle (gastrocnemius), which would be suggestive of significant improvement of physiological function above that seen after nerve repair without administration of neurotrophin. Immunohistochemistry, in conjunction with computerized quantification and morphometrical analysis, was used to analyze the number, size, and pattern of distribution of the MHC fiber types after denervation and reinnervation of the gastrocnemius muscle.     

Cell envelope associations of Aquaspirillum serpens flagella.

Specific regions of the cell envelope associated with the flagellar basal complex of the gram-negative bacterium Aquaspirillum (Spirillum) serpens were identified by studying each of the envelope layers: outer membrane, mucopeptide, and plasma membrane. The outer membrane around the flagella insertion site was differentiated by concentric membrane rings and central perforations surrounded by a closely set collar. The perforations in both the outer membrane and the isolated mucopeptide layer were of a size accomodating the central rod of the basal complex but smaller than either the L or the P disks. The P disk of the complex may lie between the mucopeptide and the outer membrane. Electron microscopy of intact, spheroplasted, or autolyzed preparations did not adequately resolve the location of the inner pair of disks of the basal complex. Freeze-etching, however, revealed differentiation within the plasma membrane that appeared to be related to the basal complex. The convex fracture face showed depressions which are interpreted as impressions of a disk surrounded by a set of evenly spaced macromolecular studs and containing a central "plug" interpreted as the central rod. In thin sections, blebs, which appear to be associated with the flagellar apparatus, were seen on the cytoplasmic side of the plasma membrane. Superimposing the dimensions of the flagellar basal complex and the spacings of the cell envelope layers and using the position of the L disk within the outer membrane for reference, showed that the S disk might be within and the M disk beneath the plasma membrane. A tentative model was developed for comparison with that based on the structure of the Escherichia coli basal complex.