Neural substrate and allatostatin-like innervation of the gut of Locusta migratoria

Allatostatin-like immunoreactivity (ALI) is widely distributed in processes and varicosities on the fore-, mid-, and hindgut of the locust, and within midgut open-type endocrine-like cells. ALI is also observed in cells and processes in all ganglia of the central nervous system (CNS) and the stomatogastric nervous system (SNS). Ventral unpaired median neurons (VUMs) contained ALI within abdominal ganglia IV-VII. Neurobiotin retrograde fills of the branches of the 11th sternal nerve that innervate the hindgut revealed 2-4 VUMs in abdominal ganglia IV-VIIth, which also contain ALI. The VIIIth abdominal ganglion contained three ventral medial groups of neurons that filled with neurobiotin and contained ALI. The co-localization of ALI in the identified neurons suggests that these cells are the source of ALI on the hindgut. A retrograde fill of the nerves of the ingluvial ganglia that innervate the foregut revealed numerous neurons within the frontal ganglion and an extensive neuropile in the hypocerebral ganglion, but there seems to be no apparent co-localization of neurobiotin and ALI in these neurons, indicating the source of ALI on the foregut comes via the brain, through the SNS.     

Quantifying conversion of linoleic to arachidonic and other n-6 polyunsaturated fatty acids in unanesthetized rats

Isotope feeding studies report a wide range of conversion fractions of dietary shorter-chain polyunsaturated fatty acids (PUFAs) to long-chain PUFAs, which limits assessing nutritional requirements and organ effects of arachidonic (AA, 20:4n-6) and docosahexaenoic (DHA, 22:6n-3) acids. In this study, whole-body (largely liver) steady-state conversion coefficients and rates of circulating unesterified linoleic acid (LA, 18:2n-6) to esterified AA and other elongated n-6 PUFAs were quantified directly using operational equations, in unanesthetized adult rats on a high-DHA but AA-free diet, using 2 h of intravenous [U-¹³C]LA infusion. Unesterified LA was converted to esterified LA in plasma at a greater rate than to esterified γ-linolenic (γ-LNA, 18:3n-6), eicosatrienoic acid (ETA, 20:3n-6), or AA. The steady-state whole-body synthesis-secretion (conversion) coefficient to AA equaled 5.4 × 10⁻³ min⁻¹, while the conversion rate (coefficient × concentration) equaled 16.1 μmol/day. This rate exceeds the reported brain AA consumption rate by 27-fold. As brain and heart cannot synthesize significant AA from circulating LA, liver synthesis is necessary to maintain their homeostatic AA concentrations in the absence of dietary AA. The heavy-isotope intravenous infusion method could be used to quantify steady-state liver synthesis-secretion of AA from LA under different conditions in rodents and in humans.     

Pitfalls and solutions in assaying anandamide transport in cells

Nonspecific binding of anandamide to plastic exhibits many features that could be mistaken as biological processes, thereby representing an important source of conflicting data on the uptake and release of this lipophilic substance. Herein, we propose an improved method to assay anandamide transport, by using glass slides (i.e., coverslips) as physical support to grow cells. Although the results obtained using plastic do not differ significantly from those obtained using glass, the new procedure has the advantage of being faster, simpler, and more accurate. In fact, the lack of aspecific adsorption of anandamide to the glass surface yields a lower background and a higher precision and accuracy in determining transport kinetics, especially for the export process. Remarkably, the kinetic parameters of anandamide uptake obtained with the old and the new procedures may be similar or different depending on the cell type, thus demonstrating the complexity of the interference of plastic on the transport process. In addition, the novel procedure is particularly suitable for visualization and measurement of anandamide transport in intact cells by using a biotinylated derivative in confocal fluorescence microscopy.     

The Solution Structure of the C-Terminal Ig-like Domain of the Bacteriophage λ Tail Tube Protein

Immunoglobulin (Ig)-like domains are found frequently on the surface of tailed double-stranded DNA bacteriophages, yet their functional role remains obscure. Here, we have investigated the structure and function of the C-terminal Ig-like domain of gpV (gpV_C), the tail tube protein of phage λ. This domain has been predicted through sequence similarity to be a member of the bacterial Ig-like domain 2 (Big_2) family, which is composed of more than 1300 phage and bacterial sequences. Using trypsin proteolysis, we have delineated the boundaries of gpV_C and have shown that its removal reduces the biological activity of gpV by 100-fold; thus providing a definitive demonstration of a functional role for this domain. Determination of the solution structure of gpV_C by NMR spectroscopy showed that it adopts a canonical Ig-like fold of the I-set class. This represents the first structure of a phage-encoded Ig-like domain and only the second structure of a Big_2 domain. Structural and sequence comparisons indicate that the gpV_C structure is more representative of both the phage-encoded Big_2 domains and Big_2 domains in general than the other available Big_2 structure. Bioinformatics analyses have identified two conserved clusters of residues on the surface of gpV_C that may be important in mediating the function of this domain.     

A putative kinase-related protein (PKRP) from Plasmodium berghei mediates infection in the midgut and salivary glands of the mosquito

The completion of the Plasmodium (malaria) life cycle in the mosquito requires the parasite to traverse first the midgut and later the salivary gland epithelium. We have identified a putative kinase-related protein (PKRP) that is predicted to be an atypical protein kinase, which is conserved across many species of Plasmodium. The pkrp gene encodes a RNA of about 5300 nucleotides that is expressed as a 90 kDa protein in sporozoites. Targeted disruption of the pkrp gene in Plasmodium berghei, a rodent model of malaria, compromises the ability of parasites to infect different tissues within the mosquito host. Early infection of mosquito midgut is reduced by 58-71%, midgut oocyst production is reduced by 50-90% and those sporozoites that are produced are defective in their ability to invade mosquito salivary glands. Midgut sporozoites are not morphologically different from wild-type parasites by electron microscopy. Some sporozoites that emerged from oocysts were attached to the salivary glands but most were found circulating in the mosquito hemocoel. Our findings indicate that a signalling pathway involving PbPKRP regulates the level of Plasmodium infection in the mosquito midgut and salivary glands.     

Collapse temperature of solutions important for lyopreservation of living cells at ambient temperature

In this study, the collapse temperature was determined using the freeze‐drying microscopy (FDM) method for a variety of cell culture medium‐based solutions (with 0.05–0.8 M trehalose) that are important for long‐term stabilization of living cells in the dry state at ambient temperature (lyopreservation) by freeze‐drying. Being consistent with what has been reported in the literature, the collapse temperature of binary water‐trehalose solutions was found to be similar to the glass transition temperature (T′_g ～ ?30°C) of the maximally freeze‐concentrated trehalose solution (～80 wt% trehalose) during the freezing step of freeze‐drying, regardless of the initial concentration of trehalose. However, the effect of the initial trehalose concentration on the collapse temperature of the cell culture medium‐based trehalose solutions was identified to be much more significant, particularly when the trehalose concentration is less than 0.2 M (the collapse temperature can be as low as ?65°C). We also determined that cell density from 1 to 10 million cells/mL and ice seeding at high subzero temperatures (?4 and ?7°C) have negligible impact on the solution collapse temperature. However, ice seeding does significantly affect the ice crystal morphology formed during the freezing step and therefore the drying rate. Finally, bulking agents (mannitol) could significantly affect the collapse temperature only when trehalose concentration is low (<0.2 M). However, improving the collapse temperature by using a high concentration of trehalose might be preferred to the addition of bulking agents in the solutions for freeze‐drying of living cells. We further confirmed the applicability of the collapse temperature measured with small‐scale (2 µL) samples using the FDM system to freeze‐drying of large‐scale (1 mL) samples using scanning electron microscopy (SEM) data. Taken together, the results reported in this study should provide useful guidance to the development of optimal freeze‐drying protocols for lyopreservation of living cells at ambient temperature for easy maintenance and convenient wide distribution to end users, which is important to the eventual success of modern cell‐based medicine. Biotechnol. Bioeng. 2010;106: 247–259. © 2010 Wiley Periodicals, Inc.     

Model‐based high cell density cultivation of Rhodospirillum rubrum under respiratory dark conditions

The potential of facultative photosynthetic bacteria as producers of photosynthetic pigments, vitamins, coenzymes and other valuable products has been recognized for decades. However, mass cultivation under photosynthetic conditions is generally inefficient due to the inevitable limitation of light supply when cell densities become very high. The previous development of a new cultivation process for maximal expression of photosynthetic genes under semi‐aerobic dark conditions in common bioreactors offers a new perspective for utilizing the facultative photosynthetic bacterium Rhodospirillum rubrum for large‐scale applications. Based on this cultivation system, the present study aimed in determining the maximal achievable cell density of R. rubrum in a bioreactor, thereby providing a major milestone on the way to industrial bioprocesses. As a starting point, we focus on aerobic growth due to higher growth rates and more facile process control under this condition, with the option to extend the process by an anaerobic production phase. Process design and optimization were supported by an unstructured computational process model, based on mixed‐substrate kinetics. Key parameters for growth and process control were determined in shake‐flask experiments or estimated by simulation studies. For fed‐batch cultivation, a computer‐controlled exponential feed algorithm in combination with a pH‐stat element was implemented. As a result, a maximal cell density of 59 g cell dry weight (CDW) L^?1 was obtained, representing so far not attainable cell densities for photosynthetic bacteria. The applied exponential fed‐batch methodology therefore enters a range which is commonly employed for industrial applications with microbial cells. The biochemical analysis of high cell density cultures revealed metabolic imbalances, such as the accumulation and excretion of tetrapyrrole intermediates of the bacteriochlorophyll biosynthetic pathway. Biotechnol. Bioeng. 2010. 105: 729–739. © 2009 Wiley Periodicals, Inc.