Evaluation of monitoring approaches and effects of culture conditions on recombinant protein production in baculovirus-infected insect cells

The baculovirus infection process ofSpodoptera frugiperda (Sf9) insect cells in oxygen-controlled bioreactors in serum-free medium was investigated using a recombinantAutographa californica (AcNPV) virus expressing -galactosidase enzyme as a model system. A variety of monitoring techniques including trypan blue exclusion, fluorescent dye staining, oxygen uptake rate (OUR) measurements, and glucose consumption were applied to infected cells to determine the best way of evaluating cell integrity and assessing the course of baculovirus infection. The metabolism of newly-infected cells increased 90% during the first 24 hours, but as infection proceeded, and cells gradually succumbed to the baculovirus infection, the cytopathic effect of the baculovirus on the cells became evident. Oxygen and glucose uptake rate measurements appeared to more accurately assess the condition of infected cells than conventional trypan blue staining, which tended to overestimate cell viability in the mid stages of infection. The optimal harvest time varied, depending on which technique — SDS-PAGE, chromogenic (ONPG) or fluorometric (C₁₂FDG) — was used to monitor -galactosidase production. Specific -galactosidase production was found to be insensitive to a wide range of culture dissolved oxygen tensions, whereas resuspending cells in fresh medium prior to infection increased volumetric productivity approximately two-fold (800,000 units -galactosidase/ml) compared to cultures infected in batch mode and allowed successful infections to occur at higher cell densities.Abbreviations ONPG ortho-phenyl 2--D-galactopyranoside - OUR oxygen uptake rate (-mol O₂/liter/hour) - q_glucose specific glucose uptake rate (mg glucose/10⁶cell/hour) - q_glutamine specific glutamine uptake rate (mg glutamine/10⁶cell/hour) - qO₂ specific oxygen uptake rate (-mol O₂/10⁶cell/hour) - MOI virus multiplicity of infection (viral plaque forming units/cell)     

Bacterial toxins induce heat shock proteins in human neutrophils

We studied the influence of different bacterial toxins (alveolysin; toxic shock syndrome toxin 1, TSST-1 and erythrogenic toxin A, ETA) on the expression of heat shock proteins (hsps) in isolated human polymorphonuclear granulocytes (PMNs). As was shown by Western blotting (anti-hsp72) ETA and TSST-1 were potent inducers of hsps at low toxin concentrations (10 ng/ml). Alveolysin led to the expression of hsps at hemolytic concentrations (1 HU; 700 ng/ml) whereas at subhemolytic concentrations (7 ng/ml) no heat shock response was observed. The induction of heat shock proteins was also accompanied by increased mRNA levels for hsp70 as was determined by PCR-analysis.     

On the reporting and review requirements of ISO 14044

The International Journal of Life Cycle Assessment -     

Activity of neck-muscle motoneurones during eye cleaning behaviour in the cricket Gryllus campestris

The activity of neck-muscle motoneurones which control head movements during eye cleaning behaviour was recorded from motor nerves with chronically implanted electrodes in unrestrained crickets. We show that motoneurones of the dorso-ventral muscles displayed strong activity differences between both sides of the neck, with higher discharge frequencies either ipsi- or contralateral to the direction of the head movement. Motoneurones innervating dorsal-longitudinal muscles were equally active on both sides. A single excitatory motoneurone of one dorso-ventral muscle showed a discharge pattern unequivocally related to eye cleaning. Lesions of connectives revealed that this motoneurone is monitored by interneuronal pathways from the suboesophageal ganglion although the primary sensory axons eliciting eye cleaning, project into the prothoracic ganglion.     

Serotonin transporter function, but not expression, is dependent on brain-derived neurotrophic factor (BDNF): in vivo studies in BDNF-deficient mice

In the present study, we used high-speed chronoamperometry to examine serotonin (5-HT) transporter (5-HTT) function in vivo in 2-, 5-, and 10-month-old brain-derived neurotrophic factor (BDNF)+/- mice. The rate of clearance of exogenously applied 5-HT was measured in CA3 region of hippocampus. In 2-month-old mice, the rate of 5-HT clearance did not differ between BDNF+/+ and BDNF+/- mice. In BDNF+/+ mice, 5-HT clearance rate (Tc) increased markedly with age. In contrast, Tc remained relatively static in BDNF+/- mice across 2-, 5-, and 10-month age groups. At 5 months of age, female BDNF+/+ mice had a lower maximal velocity (Vmax) for 5-HT clearance than male BDNF+/+ mice. There was a similar trend in 5-month-old BDNF+/- mice, but this did not reach statistical significance. There was an age-dependent increase in KT value for 5-HT clearance (i.e., decreased in vivo affinity of 5-HTT), but no significant effect of genotype or gender. 5-HTT density, as measured by [3H]cyanoimipramine binding, was not different between BDNF+/+ and BDNF+/- mice, although there was a significant increase in 5-HTT binding with age. The selective 5-HT reuptake inhibitor fluvoxamine (50 and 100 pmol) significantly decreased 5-HT clearance in BDNF+/+ mice, but not in BDNF+/- mice. Our data suggest that the profoundly reduced ability of 5- and 10-month-old BDNF+/- mice to clear 5-HT is not because of a decrease in the total number of 5-HTTs, but may be due to functional deficits in the 5-HTT, e.g., in the machinery/signaling required for insertion of 5-HTTs into the plasma membrane and/or activation of the 5-HTT once it is positioned to take up 5-HT from extracellular fluid.     

Group B streptococcal pilus proteins contribute to adherence to and invasion of brain microvascular endothelial cells

Surface filamentous structures known as pili have been discovered recently in the gram-positive streptococcal pathogens that cause invasive disease in humans, including group B Streptococcus (GBS). We show that two GBS proteins involved in pilus formation, encoded by pilA and pilB, also facilitate the interaction of this important agent of central nervous system infection with endothelial cells of the human blood-brain barrier.     

Fijimycins A-C, three antibacterial etamycin-class depsipeptides from a marine-derived Streptomyces sp

Three new depsipeptides, fijimycins A-C (1-3), together with the known etamycin A (4), were isolated and identified from the fermentation broth of strain CNS-575, a Streptomyces sp. cultured from a marine sediment sample collected off Nasese, Fiji. The planar structures of the new fijimycins were assigned by combined interpretation of NMR and MS/MS spectroscopic data. These assignments were complicated by the fact that 1-3 occurred as complex amide conformational mixtures. The absolute configurations of the component amino acids were established using the Marfey's method. Fijimycins A-C, and etamycin A, were shown to possess significant in vitro antibacterial activity against three methicillin-resistant Staphylococcus aureus (MRSA) strains with MIC(100) values between 4 and 16 μg mL(-1).     

DC-SIGN is a receptor for human herpesvirus 8 on dendritic cells and macrophages

Human herpesvirus 8 (HHV-8) causes Kaposi's sarcoma and pleural effusion lymphoma. In this study, we show that dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN; CD209) is a receptor for HHV-8 infection of myeloid DCs and macrophages. DC-SIGN was required for virus attachment to these cells and DC-SIGN-expressing cell lines. HHV-8 binding and infection were blocked by anti-DC-SIGN mAb and soluble DC-SIGN, and mannan, a natural ligand for DC-SIGN. Infection of DCs and macrophages with HHV-8 led to production of viral proteins, with little production of viral DNA, similar to HHV-8 infection of vascular endothelial cells. Infection of DCs resulted in down-regulation of DC-SIGN, a decrease in endocytic activity, and an inhibition of Ag stimulation of CD8+ T cells. We propose that DC-SIGN serves as a portal for immune dysfunction and oncogenesis caused by HHV-8 infection.     

Serotonin-1A receptor function in the dorsal raphe nucleus following chronic administration of the selective serotonin reuptake inhibitor sertraline

Serotonin-1A (5-HT_1A) receptors in the dorsal raphe nucleus (DRN) function as somatodendritic autoreceptors, and therefore play a critical role in controlling serotonergic cell firing and serotonergic neurotransmission. We hypothesized that a decrease in the capacity of 5-HT_1A receptors to activate G proteins was a general mechanism by which 5-HT_1A receptors in the DRN are desensitized following chronic administration of selective serotonin reuptake inhibitors (SSRIs). Using in vivo microdialysis, we found that the ability of the 5-HT_1A receptor agonist 8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT) (0.025 mg/kg, s.c.) to decrease extracellular 5-HT levels in striatum was attenuated following chronic treatment of rats with the SSRIs sertraline or fluoxetine. This apparent desensitization of somatodendritic 5-HT_1A autoreceptor function was not accompanied by a decrease in 5-HT_1A receptor sites in the coupled, high-affinity agonist state as measured by the binding of [3H]8-OH-DPAT. In marked contrast to what was observed following chronic administration of fluoxetine, 5-HT_1A receptor-stimulated [³⁵S]GTPγS binding in the DRN was not altered following chronic sertraline treatment. Thus, desensitization of 5-HT_1A somatodendritic autoreceptor function following chronic sertraline administration appears not to be due to a decrease in the capacity 5-HT_1A receptors to activate G proteins in the DRN. Our findings suggest that the SSRIs may not be a homogeneous class of antidepressant drug with regard to the mechanism by which the function of somatodendritic 5-HT_1A autoreceptors is regulated.