Sorbitol production by Zymomonas mobilis

Summary High resolution ¹³C Nuclear Magnetic Resonance (NMR) spectroscopy has been employed to determine the chemical composition of the unknown major products in a sucrose or fructose plus glucose fermentation to ethanol by the bacterium Zymmonas mobilis. When grown on these sugars Z.mobilis was found to produce significant amounts of sorbitol, up to 43 g·l^-1 for strain ZM31 when grown on 250 g·l^-1 sucrose.The production of sorbitol and decrease of glucose, fructose, or sucrose was followed throughout batch fermentations by NMR and HPLC. Sorbitol was shown to be derived only from fructose by [¹⁴C]-feeding experiments. Additionally ³¹P NMR spectroscopy was utilized to determine the concentrations of both glucose 6-phosphate and fructose 6-phosphate relative to their respective concentrations in Z.mobilis cells fermenting glucose or fructose alone.It is suggested that free glucose inside the cell inhibits fructokinase. Free intracellular fructose may then be reduced to sorbitol via a dehydrogenase type enzyme. Attempts to grow Z.mobilis on sorbitol were unsuccessful, as were experiments to induce growth via mutagenesis.This work was supported in part by the National Energy Research, Development and Demonstration Council of Australia     

A novel CSP C-terminal epitope targeted by an antibody with protective activity against Plasmodium falciparum

Potent and durable vaccine responses will be required for control of malaria caused by Plasmodium falciparum (Pf). RTS,S/AS01 is the first, and to date, the only vaccine that has demonstrated significant reduction of clinical and severe malaria in endemic cohorts in Phase 3 trials. Although the vaccine is protective, efficacy declines over time with kinetics paralleling the decline in antibody responses to the Pf circumsporozoite protein (PfCSP). Although most attention has focused on antibodies to repeat motifs on PfCSP, antibodies to other regions may play a role in protection. Here, we expressed and characterized seven monoclonal antibodies to the C-terminal domain of CSP (ctCSP) from volunteers immunized with RTS,S/AS01. Competition and crystal structure studies indicated that the antibodies target two different sites on opposite faces of ctCSP. One site contains a polymorphic region (denoted α-ctCSP) and has been previously characterized, whereas the second is a previously undescribed site on the conserved β-sheet face of the ctCSP (denoted β-ctCSP). Antibodies to the β-ctCSP site exhibited broad reactivity with a diverse panel of ctCSP peptides whose sequences were derived from field isolates of P. falciparum whereas antibodies to the α-ctCSP site showed very limited cross reactivity. Importantly, an antibody to the β-site demonstrated inhibition activity against malaria infection in a murine model. This study identifies a previously unidentified conserved epitope on CSP that could be targeted by prophylactic antibodies and exploited in structure-based vaccine design.     

Tomato golden mosaic virus A component DNA replicates autonomously in transgenic plants

Phenotypically normal petunia plants carrying chromosomal inserts of either the tomato golden mosaic virus (TGMV) A or the B component DNA, as single or tandem inserts, were obtained using an Agrobacterium tumefaciens Ti plasmid-based transformation system. Southern hybridization analysis revealed that the tandem, direct-repeat A plants contained free single and double stranded A component DNAs. No free B component DNA was detected in plants carrying tandem repeats of the B component. Progeny of self-fertilized plants appeared normal. In contrast, one-quarter of the progeny from tandem A by tandem B plant crosses showed chlorotic lesions on their leaves similar to virus symptoms. The significance of these results and the use of this method for the study of virus functions involved in TGMV replication and symptom production are discussed.     

Sample preparation for gas chromatography with electron capture detection: determination of total and free thyroxin in serum

Relatively clean gas chromatography with electron capture detection (GC-ECD) chromatograms are obtained for both total and free thyroxin (T4) in serum by improving sample preparation. This is based on establishing a sequence of steps that cumulatively overcome two classes of interference: those present in the initial sample and those introduced by the procedure. The main source for the latter contaminants is the derivatization step, a problem that was largely overcome by employing HPLC after this step. Also it is helpful to use ion-exchange columns early in the procedure under fast-flow conditions with intermediate flows of air to speed up and enhance their reliability. The work establishes some guidelines for future applications of GC-ECD to the determination of sub-nanogram analytes requiring derivatization, an area in which GC-ECD has been remiss in the past. As a side benefit, total T4 in serum is determined by HPLC for the first time with uv detection.     

Hypothalamic paraventricular nucleus stimulation-induced gastric acid secretion and bradycardia suppressed by oxytocin antagonist

Unilateral microstimulation of the medial parvocellular division of the hypothalamic paraventricular nucleus (PVNmp) elicits significant increases in gastric acid secretion and bradycardia. An injection of 25 picomoles of the oxytocin antagonist dET2Tyr(Et)Orn8 Vasotocin (ETOV), suspended in 5 nanoliters of artificial of cerebrospinal fluid (CSF), into the dorsal motor nucleus of the vagus (DMN) immediately preceding microstimulation of the PVNmp suppresses this change in gastric acid secretion and heart rate. The injection of an equal volume (5 nanoliters) of artificial CSF vehicle solution into this region of the DMN, prior to PVNmp microstimulation, has no effect on either the subsequent stimulation-evoked changes in acid secretion or cardiac activity. This suppression of PVNmp stimulation-evoked changes in gastric acid levels and heart rate by the presence of the oxytocin antagonist, ETOV, within the DMN supports the hypothesis that oxytocin may be a neurotransmitter used for descending communication from the PVNmp to neurons within the DMN that regulates these two functions.     

GABA-Mediated Synaptic Interaction Between the Visual and Vestibular Pathways of Hermissenda

Abstract: The synaptic convergence of the eyes and the vestibular hair cells in the nudibranch mollusc Hermissenda has been shown previously to mediate the learning of simple visual-vestibular associations. The neurotransmitter mediating this interaction between the visual and vestibular organs was characterized. HPLC chromatography, confirmed by mass spectroscopic analysis, demonstrated endogenous GABA in the statocysts, in a concentration approximately 150 times greater than in the whole CMS. Additional confirmation was provided by immunocytochemical localization of GABA in hair cell axons and branches that converge with photoreceptor terminal branches. Depolarization of the hair cells in the caudal region of the statocyst in response to positive current injection or vibratory stimulation caused a hyperpolarization and a cessation of the type B photoreceptor impulse activity. The inhibition of the B cell was unaffected by addition to the artificial sea water bath of the adrenergic antagonist yohimbine (250 μM), the cholinergic antagonist atropine (250 μM), and the serotonergic antagonist imipramine (50 μM). In contrast, the GABA_A antagonist bicuculline (250 μM) significantly reduced the inhibitory interaction. Moreover, the GABA reuptake inhibitor guvisine (250 μM)M) increased the hyperpolarization. Pressure microapplication of GABA (12.5 or 25 μM) onto the terminal branches of the B cell resulted in a concentration-dependent hyperpolarization and cessation of spikes in the B cell. Depolarization of the caudal hair cell, or direct GABA application, decreased input resistance across the B cell soma membrane. Moreover, removal of chloride from the extracellular solution reduced inhibition of the B cell induced by GABA application or hair cell stimulation. Furthermore, application of the GABA_B agonist baclofen hyperpolarized the type B cell and reduced or eliminated spontaneous impulse activity at the resting membrane potential. The reversal potentials for inhibition induced in all three procedures ranged from ?70 to ?80 mV and were consistent with mixed Cl^- and K⁺ conductances. These results implicate GABA as the endogenous neurotransmitter mediating visual-vestibular interactions in this animal, and suggest a possible role of GABA in visual-vestibular associative learning.     

CMT3 alters mitochondrial function in murine osteoclast lineage cells

Chemically modified tetracyclines (CMTs 1-10) were developed as non-antibiotic inhibitors of matrix metalloproteinases (MMPs). We previously demonstrated that MMP inhibition alone is insufficient to explain the pro-apoptotic action of CMTs in osteoclast lineage cells and we have explored additional mechanisms of action. We compared the characteristics of apoptosis in RAW264.7 murine monocyte and osteoclast cultures treated with pharmacologically relevant concentrations of CMT3 or the bisphosphonate alendronate, which induces osteoclast apoptosis through inhibition of farnesyl diphosphate synthase. CMT3 induced apoptosis rapidly (2-3 h), whereas alendronate-induced apoptosis was delayed (>12 h). CMT3-treated cells did not accumulate unprenylated Rap1A in contrast to cells treated with alendronate. Importantly, CMT3 induced a rapid loss of mitochondrial stability in RAW264.7 cells measured by loss of Mitotracker^® Red fluorescence, while bongkrekic acid protected polykaryons from CMT3-induced apoptosis. Modulation of mitochondrial function is therefore a significant early action of CMT3 that promotes apoptosis in osteoclast lineage cells.