Abnormal bone growth and selective translational regulation in basic fibroblast growth factor (FGF-2) transgenic mice.

Basic fibroblast growth factor (FGF-2) is a pleiotropic growth factor detected in many different cells and tissues. Normally synthesized at low levels, FGF-2 is elevated in various pathologies, most notably in cancer and injury repair. To investigate the effects of elevated FGF-2, the human full-length cDNA was expressed in transgenic mice under control of a phosphoglycerate kinase promoter. Overexpression of FGF-2 caused a variety of skeletal malformations including shortening and flattening of long bones and moderate macrocephaly. Comparison by Western blot of FGF-2 transgenic mice to nontransgenic littermates showed expression of human FGF-2 protein in all major organs and tissues examined including brain, heart, lung, liver, kidney, spleen, and skeletal muscle; however, different molar ratios of FGF-2 protein isoforms were observed between different organs and tissues. Some tissues preferentially synthesize larger isoforms of FGF-2 while other tissues produce predominantly smaller 18-kDa FGF-2. Translation of the high molecular weight isoforms initiates from unconventional CUG codons and translation of the 18-kDa isoform initiates from an AUG codon in the FGF-2 mRNA. Thus the Western blot data from the FGF-2 transgenic mice suggest that tissue-specific expression of FGF-2 isoforms is regulated translationally.     

Process intensification strategies toward cell culture-based high-yield production of a fusogenic oncolytic virus

We present a proof-of-concept study for production of a recombinant vesicular stomatitis virus (rVSV)-based fusogenic oncolytic virus (OV), rVSV-Newcastle disease virus (NDV), at high cell densities (HCD). Based on comprehensive experiments in 1 L stirred tank reactors (STRs) in batch mode, first optimization studies at HCD were carried out in semi-perfusion in small-scale cultivations using shake flasks. Further, a perfusion process was established using an acoustic settler for cell retention. Growth, production yields, and process-related impurities were evaluated for three candidate cell lines (AGE1.CR, BHK-21, HEK293SF)infected at densities ranging from 15 to 30 × 10⁶ cells/mL. The acoustic settler allowed continuous harvesting of rVSV-NDV with high cell retention efficiencies (above 97%) and infectious virus titers (up to 2.4 × 10⁹ TCID₅₀/mL), more than 4–100 times higher than for optimized batch processes. No decrease in cell-specific virus yield (CSVY) was observed at HCD, regardless of the cell substrate. Taking into account the accumulated number of virions both from the harvest and bioreactor, a 15–30 fold increased volumetric virus productivity for AGE1.CR and HEK293SF was obtained compared to batch processes performed at the same scale. In contrast to all previous findings, formation of syncytia was observed at HCD for the suspension cells BHK 21 and HEK293SF. Oncolytic potency was not affected compared to production in batch mode. Overall, our study describes promising options for the establishment of perfusion processes for efficient large-scale manufacturing of fusogenic rVSV-NDV at HCD for all three candidate cell lines.     

Electric Currents around Growing Trichoderma Hyphae, before and after Photoinduction of Conidiation

Electric currents were measured around Trichoderma harzianum (Rifai) hyphae using an extracellular vibrating electrode. A steady current enters growing hyphal tips and along the side of the apical millimeter. In addition, outward currents were detected at about one-ninth of the locations tested, 60 to 150 minutes after illumination but not in dark controls. This sporadic, localized outward current pattern might be an early biophysical response to blue light.     

Effect of culture conditions on synthesis of L-asparaginase by Escherichia coli A-1.

The nutritional requirements and culture conditions affecting biosynthesis of L-asparaginase in a mutant of Escherichia coli HAP designated strain A-1 were studied. Asparaginase activity was increased by the addition of L-glutamic acid, L-glutamine, or commercial-grade monosodium glutamate. The rate of enzyme synthesis was dependent on the interaction between the pH of the culture and the amount of oxygen dissolved in the medium. A critical oxygen transfer rate essential for asparaginase formation was identified, and a fermentation procedure is described in which enzyme synthesis is controlled by aeration rate. Enhancement of L-asparaginase activity by monosodium glutamate was inhibited by the presence of glucose, culture pH, chloramphenicol, and oxygen dissolved in the fermentation medium.     

Critical Reactions in Fluorobenzoic Acid Degradation by Pseudomonas sp. B13

3-Chlorobenzoate-grown cells of Pseudomonas sp. B13 readily cometabolized monofluorobenzoates. A catabolic pathway for the isomeric fluorobenzoates is proposed on the basis of key metabolites isolated. Only 4-fluorobenzoate was utilized and totally degraded after a short period of adaptation. The isoenzymes for total degradation of chlorocatechols, being found during growth with 3-chlorobenzoate or 4-chlorophenol, were not induced in the presence of fluorobenzoates. Correspondingly, only the ordinary enzymes of the benzoate pathway were detected in 4-fluorobenzoate-grown cells. Ring cleavage of 3-fluorocatechol was recognized as a critical step in 3-fluorobenzoate degradation. 2-Fluoro-cis,cis-muconic acid was identified as a dead-end metabolite from 2- and 3-fluorobenzoate catabolism. During 2-fluorobenzoate cometabolism, fluoride is eliminated by the initial dioxygenation.     

Natural H Currents Traverse Growing Roots and Root Hairs of Barley (Hordeum vulgare L.)

With the aid of an extracellular vibrating electrode, natural electric fields were detected and measured in the medium near growing roots and root hairs of barley seedlings. An exploration of these fields indicates that both the root as a whole, as well as individual root hairs, drive large steady currents through themselves. Current consistently enters both the main elongation zone of the root as well as the growing tips of elongating root hairs; it leaves the surface of the root beneath the root hairs. These currents enter with a density of about 2 microamperes per square centimeter, leave with a density of about 0.5 to 1 microampere per square centimeter, and total about 30 nanoamperes.     

A new species of Penstemon (Scrophulariaceae) from Wyoming

A new species from Wyoming,Penstemon gibbensii, is described and contrasted to related species.     

Response properties of thick myelinated group II afferents in the medial articular nerve of normal and inflamed knee joints of the cat.

This study investigated the responses to innocuous and noxious mechanical stimuli of joint mechanoreceptors with thick myelinated articular afferents (conduction velocities 21-65 m/sec) in the medial articular nerve of the cat's knee. In nine experiments, we examined whether acute arthritis would modify the discharge properties. The vast majority of the group II afferents were excited by gentle local stimuli and by movements in the working range of the knee. Although they encoded pressure and particular movement stimuli up to the noxious range, their responses were more closely related to the particular type of stimulus (e.g., a movement in a specific direction) than to its intensity (innocuous vs. noxious). In inflamed joints, the response patterns of group II units were similar with regard to local mechanical thresholds, thresholds for passive movements, and patterns of responses to passive movements. In both situations, most units had no resting activity. These results suggest that articular group II afferents do not play a significant role in nociception. Rather, they subserve proprioceptive functions such as deep pressure sensation and kinesthesia in normal as well as inflamed joints.     

Insulin-like immunoreactivity in the human brain

Summary The localization and regional distribution of insulin-like immunoreactivity (IRI) was studied in human brain autopsy material using the indirect immunofluorescence technique. A positive reaction for IRI could be observed in many neurons of the hypothalamus, the hippocampus, corpus amygdaloideum, medulla oblongata (especially within the nuclei of cranial nerves IX, X and XII), and the cerebral cortex, whereas the cerebellar cortex was lacking in immunohistochemically detectable insulin-like material. No nerve fibres containing polypeptides could be revealed. Additionally, the inuslin content of various brain regions was estimated by radioimmunosassay. Insulin concentrations in human nervous tissue were found to be elevated in comparison to blood plasma levels.The present investigation was supported by the HFR Neurobiologie und Hirnforschung and the HFR Diabetes mellitus und Fettstoffwechselstörungen of the GDR (Ministries of Higher Education and Health respectively) and the Finnish Ministry of Education