The production of Pseudomonas putida for the hydroxylation of toluene to its cis-glycol

A constitutive blocked mutant (UV4) of Pseudomonas putida was grown in a 2.5-l fermentor on a mineral salts medium. Glucose was fed normally over an 18-h period. Gluconate reached about 5 gl^–1 in the medium and then fell to zero as it was utilised. The maximum toluene dioxygenase specific activity (2 g g^–1 h^–1) was obtained over the last 6 h of the fermentation when the pH was fully controlled. In fermentations done at low dissolved O₂ tension (DOT) values there was an overall reduction in the cellular enzyme level. When stored at 4°C in phosphate buffer, pH 7.0, harvested bacteria lost half their activity in about 90 h.     

Deacylation of benzylpenicillin by immobilized penicillin acylase in a continuous four-stage stirred-tank reactor

Immobilized penicillin acylase has been used for the deacylation of benzylpenicillin at 37°C in a continuous reactor consisting of four 1 liter stirred tanks connected in series. There was good agreement between the predicted and actual conversions obtained in each tank under steady-state conditions. The operational stability of the immobilized enzyme in the tanks depended on the pH and the rate of addition and concentration of alkali needed to neutralize the acid produced during the reaction. At pH 7 with the addition of 2M NaOH, the half-life for enzyme stability was greater than 400 hr in all tanks. This was over half the value for the immobilized enzyme when stored at 37°C and pH 7.     

The effect of agitation on the morphology and penicillin production of Penicillium chrysogenum

Penicillium chrysogenum strain P1 was grown on complex media in 10 and 100 L agitated fermenters at various aeration rates and stirrer speeds. Samples were removed at intervals for measurements of the culture morphology. At high stirrer speeds (1000 and 1200 rpm) in 10-L fermentations the rate of decrease in the mean effective hyphal length was faster and the rate of penicillin production was lower than fermentations done at 800 rpm. At similar power inputs per unit volume in 100-L fermentations, the change in mean effective hyphal length was less and higher penicillin production rates were observed. This work comparing the results at two scales has shown that neither of the concepts of impeller tip speed or the dissipation rate of turbulence have general validity as a measure of hyphal damage. Our results are reasonaby well correlated by groups similar to circulation rate (ND(i) (3)/V) with lower circulation rates being beneficial. An adaptation of the van Suijdam and Metz relationship, expressed as P/D(i) (3)t(c), was most successful. Our data are insufficient to demonstrate the generality of the relationship but do support the concept of a dispersion zone around the impellers in which mycelia may be damaged. The greater the frequency of circulation of mycelia through the zone the greater the damage and the lower the rate of penicillin synthesis by the culture.     

The large-scale immobilization of Penicillium chrysogenum: batch and continuous operation in an air-lift reactor

A temperature-sensitive cell division cycle mutant of Penicillium chrysogenum P2 has been immobilized on Celite and grown in a 250-320-L working volume air-lift fermenter. The ability to uncouple growth and penicillin synthesis by raising the temperature to 30 degrees C also overcame the problem of the free cell mass which appeared after 300 h operation with the parent organism. After 500 h operation, penicillin and ACV dimer were still being synthesized.     

Trait-like immunological and hematological measures in female rhesus across varied environmental conditions

In this 2-year longitudinal study, 45 2-year-old female rhesus were observed as they were captured and removed from a free-ranging setting (Phase I), single caged for 1 year (Phase II), and housed in small, stable social groups for an additional year (Phase III). During the study, eight blood samples were taken, and hematological, immunological, and hormonal variables were assayed to determine whether 1) any of the measures would exhibit trait-like, inter-individual longitudinal stability, despite fluctuations in population means induced by Phases I, II, and III; 2) plasma concentrations of cortisol, prolactin, and norepinephrine would be lowest in Phase III, and elevated during the periods of acute and chronic stress associated with Phases I and II; and 3) there would be any evidence of immunosuppression associated with Phases I and II. The results suggest that the majority of hematological/immunological variables were trait-like throughout the study in contrast to plasma cortisol, prolactin, and norepinephrine concentrations. Thus, red blood cells, hemoglobin, hematocrit, platelets, mean corpuscular volume and hemoglobin, as well as white blood cells, the absolute number of CD4+ (T-helper/inducer) cells, the absolute number of CD8+ (T-suppressor/cytotoxic) cells, total T cells (CD2+%), total B cells (CD20+%), and the ratio of CD4+/CD8+ cells were trait-like. The hematological measures were changed dramatically by capture and the subsequent single caging, with most not recovering to presumed baselines until after 12–28 weeks. The immune measures were depressed at capture (excepting B cells), and during 7 months of single caging failed to return to normal levels associated with social housing. We thus conclude that single housing can produce significant, long-term features of immunosuppression. Capture produced significant increases in plasma cortisol, prolactin, and norepinephrine concentrations. Long periods of single caging produced significant increases in plasma prolactin concentrations, indicative of stress-induced anxiety, and may also have been associated with down-regulation of plasma norepinephrine and cortisol concentrations. Am. J. Primatol. 48:197–223, 1999. © 1999 Wiley-Liss, Inc.     

ATP-Dependent Formation of Phosphatidylserine-Rich Vesicles from the Endoplasmic Reticulum of Leek Cells

Leek (Allium porrum) plasma membrane is enriched in phosphatidylserine (PS) by the vesicular pathway, in a way similar to that already observed in animal cells (B. Sturbois-Balcerzak, D.J. Morré, O. Loreau, J.P. Noel, P. Moreau, C. Cassagne [1995] Plant Physiol Biochem 33: 625–637). In this paper we document the formation of PS-rich small vesicles from leek endoplasmic reticulum (ER) membranes upon addition of ATP and other factors. The omission of ATP or its replacement by ATPγ-S prevents vesicle formation. These vesicles correspond to small structures (70–80 nm) and their phospholipid composition, characterized by a PS enrichment, is compatible with a role in PS transport. Moreover, the PS enrichment over phosphatidylinositol in the ER-derived vesicles is the first example, to our knowledge, of phospholipid sorting from the ER to ER-derived vesicles in plant cells.     

Brain structure variation in great apes, with attention to the mountain gorilla (Gorilla beringei beringei)

This report presents data regarding the brain structure of mountain gorillas (Gorilla beringei beringei) in comparison with other great apes. Magnetic resonance (MR) images of three mountain gorilla brains were obtained with a 3T scanner, and the volume of major neuroanatomical structures (neocortical gray matter, hippocampus, thalamus, striatum, and cerebellum) was measured. These data were included with our existing database that includes 23 chimpanzees, three western lowland gorillas, and six orangutans. We defined a multidimensional space by calculating the principal components (PCs) from the correlation matrix of brain structure fractions in the well-represented sample of chimpanzees. We then plotted data from all of the taxa in this space to examine phyletic variation in neural organization. Most of the variance in mountain gorillas, as well as other great apes, was contained within the chimpanzee range along the first two PCs, which accounted for 61.73% of the total variance. Thus, the majority of interspecific variation in brain structure observed among these ape taxa was no greater than the within-species variation seen in chimpanzees. The loadings on PCs indicated that the brain structure of great apes differs among taxa mostly in the relative sizes of the striatum, cerebellum, and hippocampus. These findings suggest possible functional differences among taxa in terms of neural adaptations for ecological and locomotor capacities. Importantly, these results fill a critical gap in current knowledge regarding great ape neuroanatomical diversity.     

siRNA relieves chronic neuropathic pain

Double stranded, short interfering RNAs (siRNA) of 21–22 nt length initiate a sequence-specific, post-trancriptional gene silencing in animals and plants known as RNA interference (RNAi). Here we show that RNAi can block a pathophysiological pain response and provide relief from neuropathic pain in a rat disease model by down regulating an endogenous, neuronally expressed gene. Rats, intrathecally infused with a 21 nt siRNA perfectly complementary to the pain-related cation-channel P2X₃, showed diminished pain responses compared to missense (MS) siRNA-treated and untreated controls in models of both agonist-evoked pain and chronic neuropathic pain. This form of delivery caused no adverse effects in any of the animals receiving P2X₃ siRNA, MS siRNA or vehicle. Molecular analysis of tissues revealed that P2X₃ mRNA expressed in dorsal root ganglia, and P2X₃ protein translocated into the dorsal horn of the spinal cord, were significantly diminished. These observations open a path toward use of siRNA as a genetic tool for drug target validation in the mammalian central nervous system, as well as for proof of concept studies and as therapeutic agents in man.