Fed-batch mode in shake flasks by slow-release technique

Most industrial production processes are performed in fed-batch operational mode. In contrast, the screenings for microbial production strains are run in batch mode which results in completely different physiological conditions than relevant for production conditions. This may lead to wrong selections of strains. Silicone elastomer discs containing glucose crystals were developed to realize fed-batch fermentation in shake flasks. No other device for feeding was required. Glucose was fed in this way to Hansenula polymorpha cultures controlled by diffusion. Two strains of H. polymorpha were investigated in shake flasks: the wild-type strain (DSM 70277) and a recombinant strain pC10-FMD (P(FMD)-GFP). The oxygen transfer rate (OTR) and respiratory quotient (RQ) of the cultures were monitored online in shake flasks with a Respiration Activity Monitoring System (RAMOS). Formation of biomass and green fluorescent protein (GFP), pH-drift and the metabolite dynamics of glucose, ethanol and acetic acid were measured offline. With the slow-release technique overflow metabolism could be reduced leading to an increase of 85% in biomass yield. To date, 23.4 g/L cell dry weight of H. polymorpha could be achieved in shake flask. Biomass yields of 0.38-0.47 were obtained which are in the same magnitude of laboratory scale fermentors equipped with a substrate feed pump. GFP yield could be increased by a factor of 35 in Syn6-MES mineral medium. In fed-batch mode 88 mg/L GFP was synthesized with 35.9 g/L fed glucose. In contrast, only 2.5 mg/L with 40 g/L metabolized glucose was revealed in batch mode. In YNB mineral medium over 420-fold improvement in fed-batch mode was achieved with 421 mg/L GFP at 41.3 g/L fed glucose in comparison to less than 1 mg/L in batch mode with 40 g/L glucose.     

Bradykinin activates ADP-ribosyl cyclase in neuroblastoma cells: intracellular concentration decrease in NAD and increase in cyclic ADP-ribose

ADP-ribosyl cyclase activity in the crude membrane fraction of neuroblastomaxglioma NGPM1-27 hybrid cells was measured by monitoring [(3)H] cyclic ADP-ribose (cADPR) formation from [(3)H] NAD(+). Bradykinin (BK) at 100nM increased ADP-ribosyl cyclase activity by about 2.5-fold. Application of 300nM BK to living NGPM1-27 cells decreased NAD(+) to 78% of the prestimulation level at 30s. In contrast, intracellular cADPR concentrations were increased by 2-3-fold during the period from 30 to 120s after the same treatment. Our results suggest that cADPR is one of the second messengers downstream of B(2) BK receptors.     

Activation of pannexin 1 channels by ATP through P2Y receptors and by cytoplasmic calcium

The ability for long-range communication through intercellular calcium waves is inherent to cells of many tissues. A dual propagation mode for these waves includes passage of IP3 through gap junctions as well as an extracellular pathway involving ATP. The wave can be regenerative and include ATP-induced ATP release via an unknown mechanism. Here, we show that pannexin 1 channels can be activated by extracellular ATP acting through purinergic receptors of the P2Y group as well as by cytoplasmic calcium. Based on its properties, including ATP permeability, pannexin 1 may be involved in both initiation and propagation of calcium waves.     

Pathognomonic genetic expression profile within peripheral blood mononuclear cells of rheumatic heart disease patients

In this study we have tested the effects of d-propranolol (D-Pro) on the iron uptake, iron release and oxidative response of iron-loaded cells in a cellular model of iron-overload using isolated rat peritoneal macrophages incubated with iron-dextran (Fe-D). Pretreatment of macrophages with D-Pro (5–200 μ M) prior to Fe-D exposure decreased the cellular iron content and partially prevented iron release from latex-activated macrophages. Release of reactive oxygen species from activated cells was detected by dichlorodihydrofluorescein (DCDHF, 5 μ M) oxidation. We found that loading cells with Fe-D increased their response to latex, which was prevented by the lysosomotropic antioxidant agent D-Pro (10 μ M).     

Class-1 release factor eRF1 promotes GTP binding by class-2 release factor eRF3

In eukaryotes, termination of mRNA translation is triggered by the essential polypeptide chain release factors eRF1, recognizing all three stop codons, and eRF3, a member of the GTPase superfamily with a role that has remained opaque. We have studied the kinetic and thermodynamic parameters of the interactions between eRF3 and GTP, GDP and the non-hydrolysable GTP analogue GDPNP in the presence (K(D)(GDP)=1.3+/-0.2 muM, K(D)(GTP) approximately 200 muM and K(D)(GDPNP)>160 muM) as well as absence (K(D)(GDP)=1.9+/-0.3 muM, K(D)(GTP) 0.7+/-0.2 muM and K(D)(GDPNP) approximately 200 muM) of eRF1. From the present data we propose that (i) free eRF3 has a strong preference to bind GDP compared to GTP (ii) eRF3 in complex with eRF1 has much stronger affinity to GTP than free eRF3 (iii) eRF3 in complex with PABP has weak affinity to GTP (iv) eRF3 in complex with eRF1 does not have strong affinity to GDPNP, implying that GDPNP is a poor analogue of GTP for eRF3 binding.     

Vesicular Ca(2+) -induced secretion promoted by intracellular pH-gradient disruption

The actions of the protonophore CCCP on intracellular Ca2+ regulation and exocytosis in chromaffin cells have been examined. Simultaneous fura-2 imaging and amperometry reveal that exposure to CCCP not only perturbs mitochondrial function but that it also alters vesicular storage of Ca2+ and catecholamines. By disrupting the pH gradient of the secretory vesicle membrane, the protonophore allows both Ca(2+) and catecholamine to leak into the cytosol. Unlike the high cytosolic Ca2+ concentrations resulting from mitochondrial membrane disruption, Ca2+ leakage from secretory vesicles may initiate exocytotic release. In conjunction with previous studies, this work reveals that catalytic and self-sustained vesicular Ca(2+) -induced exocytosis occurs with extended exposure to weak acid or base protonophores.     

Wet-season dormancy release in seed banks of a tropical leguminous shrub is determined by wet heat

BACKGROUND AND AIMS: Hard-seeded (physical) dormancy is common among plants, yet mechanisms for dormancy release are poorly understood, especially in the tropics. The following questions are asked: (a) whether dormancy release in seed banks of the tropical shrub Parkinsonia aculeata (Caesalpiniaceae) is determined by wet heat (incubation under wet, warm to hot, conditions); and (b) whether its effect is modified by microclimate. METHODS: A seed burial trial was conducted in the wet-dry tropics (northern Australia) to compare dormancy release across different habitats (open, artificial cover, ground cover and canopy cover), burial depths (0, 3 and 20 cm) and burial durations (2, 6 and 14 weeks). Results were compared with predictions using a laboratory-derived relationship between wet heat and dormancy release, and microclimate data collected during the trial. KEY RESULTS: Wet heat (defined as the soil temperature above which seeds were exposed to field capacity or higher for a cumulative total of 24 h) was 43.6 degrees C in the 0 cm open treatment, and decreased with increasing shade and depth to 29.5 degrees C at 20 cm under canopy cover. The dormancy release model showed that wet heat was a good predictor of the proportion of seeds remaining dormant. Furthermore, dormancy release was particularly sensitive to wet heat across the temperature range encountered across treatments. This resulted in a 16-fold difference in dormancy levels between open (<5 % of seeds still dormant) and covered (82 %) microhabitats. CONCLUSIONS: These results demonstrate that wet heat is the principal dormancy release mechanism for P. aculeata when conditions are hot and wet. They also highlight the potential importance of microclimate in driving the population dynamics of such species.     

Transporter mediated GABA release in the retina: role of excitatory amino acids and dopamine

In general, the release of neurotransmitters in the central nervous system is accomplished by a calcium-dependent process which constitutes a common feature of exocytosis, a conserved mechanism for transmitter release in all species. However, neurotransmitters can also be released by the reversal of their transporters. In the retina, a large portion of GABA is released by this mechanism, which is under the control of neuroactive agents, such as excitatory amino acids and dopamine. In this review, we will focus on the transporter mediated GABA release and the role played by excitatory amino acids and dopamine in this process. First, we will discuss the works that used radiolabeled GABA to study the outflow of the neurotransmitter and then the works that took into consideration the endogenous pool of GABA and the topography of GABAergic circuits influenced by excitatory amino acids and dopamine.     

Uncoupled IP3 receptor can function as a Ca2+-leak channel: cell biological and pathological consequences

Ca(2+) release via intracellular release channels, IP(3)Rs (inositol 1,4,5-trisphosphate receptors) and RyRs (ryanodine receptors), is perhaps the most ubiquitous and versatile cellular signalling mechanism, and is involved in a vast number of cellular processes. In addition to this classical release pathway there is limited, but yet persistent, information about less well-defined Ca(2+)-leak pathways that may play an important role in the control of the Ca(2+) load of the endo(sarco)plasmic reticulum. The mechanisms responsible for this 'basal' leak are not known, but recent data suggest that both IP(3)Rs and RyRs may also operate as Ca(2+)-leak channels, particularly in pathological conditions. Proteolytic cleavage or biochemical modification (such as hyperphosphorylation or nitrosylation), for example, occurring during conditions of cell stress or apoptosis, can functionally uncouple the cytoplasmic control domains from the channel domain of the receptor. Highly significant information has been obtained from studies of malfunctioning channels in various disorders; for example, RyRs in cardiac malfunction or genetic muscle diseases and IP(3)Rs in neurodegenerative diseases. In this review we aim to summarize the existing information about functionally uncoupled IP(3)R and RyR channels, and to discuss the concept that those channels can participate in Ca(2+)-leak pathways.