Functional Expression of the Alkane-Inducible Monooxygenase System of the Yeast: Candida tropicalis IN Saccharomyces cerevisiae

The genes for the alkane-inducible monooxygenase system of the yeast Candida tropicalis, namely a cytochrome P450_alk (P450_alk) and a NADPH cytochrome P450 oxidoreductase (NCPR) gene, were transferred in Saccharomyces cerevisiae. The P450_alk gene was expressed in this host with the help of the yeast alcohol dehydrogenase I (ADHI) promoter and terminator, whereas the NCPR gene could be expressed with its own structural elements. The presence of P450_alk in S. cerevisiae microsomal fractions resulted in a new acquired lauric acid terminal hydroxylation activity. Moreover, the same activity, coupled with the appearance of 12-hydroxylauric acid derivatives, could be obtained by the addition of lauric acid to intact cells expressing P450_alk. The coordinate expression of the P450_alk and NCPR genes in S. cerevisiae elevated the turnover rate of the P450_alk monooxygenase activity about 2-fold.     

Cellular source of soluble interleukin 2 receptors in serum of mice after recombinant interleukin 2 administration.

Previous studies have shown that peripheral blood mononuclear cells activated in vitro not only express cell-associated interleukin 2 receptors (IL2R) but also release a soluble form of this receptor. In this study, we demonstrate that administration of human recombinant IL 2 (rIL 2) to mice results in increased spleen weights, splenic natural killer (NK) cell cytolytic activity, and serum levels of soluble IL2R. However, compared with rIL 2-treated heterozygote controls, beige mice treated with rIL 2 displayed similar elevations in serum soluble IL2R but significantly less splenic NK activity. Likewise, administration of anti-asialo GM1 antiserum to rIL 2-treated mice resulted in a dramatic reduction in splenic NK cytolytic activity, but no reduction in serum soluble IL2R. Conversely, while rIL 2 treatment of BALB/c mice produced increased splenic NK activity and serum soluble IL2R, similar treatment of BALB/c nude mice resulted in elevation of only splenic NK activity. These studies demonstrate that administration of rIL 2 to normal mice can elevate both serum IL2R levels and splenic NK cytolytic activity. However, the results suggest that T cells are likely to be the source of elevated serum IL2R after rIL 2 administration.     

Effect of common dead space on VA/Q distribution in the dog

Several previous studies have shown worsening ventilation-perfusion (VA/Q) relationships in humans during heavy exercise at sea level. However, the mechanism of this deterioration remains unclear because of the correlation with ventilatory and circulatory variables. Our hypothesis was that the decrease in the series dead space-to-tidal volume ratio during exercise might be partly responsible because mixing in the common dead space can reduce apparent inequality. We tested this notion in 10 resting anesthetized normocapnic dogs passively hyperventilated by increase tidal volume and a) inspired CO2 or b) external dead space. We predicted less apparent VA/Q inequality in condition b because of mixing in the added dead space. After base-line measurements, conditions a and b were randomly assigned, and after a second set of base-line measurements they were repeated in the reverse order in each dog. VA/Q inequality was measured by the multiple inert gas elimination technique. Comparison of conditions a and b demonstrated that additional external dead space improved (P less than 0.001) the blood flow distributions as hypothesized [log standard deviation of perfusion = 0.49 +/- 0.02 (SE) in condition b and 0.61 +/- 0.03 in condition a with respect to 0.52 +/- 0.03 at base line]. This study suggests that the increased tidal volume during exercise could uncover VA/Q inequality not evident at rest because of the higher ratio of common dead space to tidal volume at rest.     

Pulmonary microcirculatory kinetics of neutrophils deficient in leukocyte adhesion-promoting glycoproteins

The mechanism that causes neutrophils to sequester in the pulmonary circulation is unknown. Because the CD11/CD18 glycoprotein family on the surface membrane of neutrophils participates in many adhesive interactions with the endothelium, we investigated the role of these proteins in the intravascular sequestration of pulmonary neutrophils. Neutrophils were isolated from normal dogs and from the only living dog known to have leukocyte adhesion deficiency disease, an inherited deficiency of the CD11/CD18 adhesion family. The neutrophils were labeled with fluorescein dye, injected into normal recipient dogs, and their passage through the pulmonary microcirculation was recorded by in vivo videofluorescence microscopy through a transparent thoracic window. Transit times for normal and deficient neutrophils were similar over a wide range of hemo-dynamic conditions. Activation by zymosan-activated plasma, which increases the surface membrane expression of CD11/CD18, prolonged the transit of normal neutrophils but did not alter the transit time of the deficient neutrophils. These results indicate that neutrophil CD11/CD18 adhesion-promoting glycoproteins are not involved in the normal pulmonary sequestration of neutrophils but have a significant role in the arrest of activated neutrophils in the pulmonary capillaries.     

Low synonymous site variation at thelacY locus inEscherichia coli suggests the action of positive selection

We have determined the nucleotide sequences of sevenlacY alleles isolated from natural isolates ofEscherichia coli. Nucleotide heterozygosity estimates for this locus were compared to those obtained from previous studies of intraspecific variation at chromosomal loci, revealing thatlacY has unusually low synonymous site variation. The average pairwise heterozygosity of synonymous sites (Ks=0.0112+/-0.0100) is the second lowest reported and the lowest for loci that have an equivalent level of nonsynonymous variation. We consider several hypotheses to explain how different forces in evolution could act to create the observed pattern of polymorphism, including selection for translational efficiency and positive selection. Our analysis most strongly supports the hypothesis that positive selection has acted on thelacY locus inE. coli.     

Emmetropization and optical development of the eye of the barn owl (Tyto alba)

1. We have studied the development of the refractive state in young barn owls (Tyto alba pratincola). Strikingly, the eyes had severe refractive errors shortly after lid opening (which occurred around day 14 after hatching; average from 6 owls: 13.83 ± 1.47 days). Refractive errors vanished in the subsequent one or two weeks (Fig. 1, Fig. 2).
2. Refractive errors did not differ by more than 1 diopter (D) in both eyes of an individual (Fig. 2). Thus, non-visual control of eye growth was sufficient to produce non-random refractions. However, visual input was finally required to adjust the optical system to emmetropia.
3. Using in-vivo A-scan ultrasonography of ocular dimensions (Fig. 4A), photokeratometric measurements of corneal radius of curvature (Fig. 4B), and frozen sections of excised eyes (Fig. 3), we developed paraxial schematic eye models which described age-dependent changes in ocular parameters and were applicable through the ages from lid opening to fledging (Table 1). A schematic eye for the adult barn owl (European subspecies: Tyto alba alba) is also provided. Eye sizes in an adult owl of the American (Tyto alba pratincola) and the European subspecies (T. alba alba) were similar despite of different body weights (500 g and 350 g, respectively).
4. The schematic eyes were used to test which ocular parameters might have caused the recovery from refractive errors. However, none of the ocular dimensions measured underwent obvious changes in their growth curves as visual input became available. Apparently, coordinated growth of several ocular components produced emmetropia.
5. From the schematic eye model, the developmental changes in image brightness and image magnification were calculated (Fig. 5). In barn owl eyes, image size was not quite as extreme as in the tawny owl or the great horned owl. However, the image was larger and the f/number was lower than in diurnal birds of comparable weight (pigeon, chicken). The observation supports a conclusion that image size is maximised in owls to permit a higher degree of photoreceptor convergence for higher light sensitivity at dusk while spatial acuity remains comparable to diurnal birds with smaller eyes.

     

Initial steps of sophoroselipid biosynthesis by Candida bombicola ATCC 22214 grown on glucose

 Candida bombicola ATCC 22214 produces the glycolipid sophoroselipid when cultivated on a medium with glucose as the sole carbon source. Under phosphate-limiting conditions the product yield rises from 0.033 to 0.143 and the specific product formation rate rises from 0.004 h^-1 to 0.007 h^-1. Enhanced sophoroselipid synthesis is initiated by the decline of the specific activities of NAD- and NADP-dependent isocitrate dehydrogenase (EC 1.1.1.41 and 1.1.1.42) to 2% and 0% of the initial activities respectively. Constantly high specific activity of citrate synthase (EC 4.1.3.7) causes an accumulation of isocitrate and citrate in the mitochondria. Both acids are transported into the cytosol where citrate is cleaved by ATP: citrate lyase (EC 4.1.3.8) giving rise to acetyl-CoA, the precursor of fatty acid synthesis. The ATP: citrate lyase is unaffected by different energy charges; the apparent K _m values for coenzyme A, ATP and citrate are 23 μM, 250 μM and 256 μM respectively. NADPH for fatty acid synthesis might be generated by further metabolism of oxaloacetate, the other product of the citrate-cleaving reaction, by oxidation of the isocitrate by the cytosolic NADP-dependent isocitrate dehydrogenase or via the hexose monophosphate shunt. A possible explanation for sophoroselipid formation during exponential growth is given. Received: 7 November 1995/Received revision: 19 March 1996/Accepted: 25 March 1996     

Characterization of bacterial communities from activated sludge: Culture-dependent numerical identification versus in situ identification using group- and genus-specific rRNA-targeted oligonucleotide probes

The structures of bacterial communities were studied in activated sludge samples obtained from the aerobic and anaerobic zones of a wastewater treatment plant showing enhanced phosphorous removal. Samples were analyzed by in situ hybridization with oligonucleotide probes complementary to selected regions of the 16S and 23S ribosomal RNA (rRNA) characteristic for defined phylogenetic entities (genera and larger groups). The microbial community structures revealed by molecular techniques were compared with the compositions of culturable bacterial communities, obtained from the characterization of 255 isolates from tryptone-soy (TS) agar and R2A agar. These isolates were characterized by 89 physiological tests and their cellular fatty acid patterns, and identified. Culture-dependent techniques indicated the following distribution: different Aeromonas spp. (2.7–8.3% on R2A agar; 45.0–63.7% on TS agar), Acinetobacter spp. (5.4–9.0% on R2A agar; 5.0–9.1% on TS agar), Pseudomonas spp. (up to 10% on R2A agar) and Shewanella putrefaciens (up to 3.0% on R2A agar), all members of the gamma subclass of Proteobacteria, were isolated most frequently. The relatively rare isolates of the beta subclass were identified as Acidovorax spp., Alcaligenes spp., and Comamonas spp., The Gram-positive bacteria (high DNA G+C) were assigned mainly to Arthrobacter spp., Microbacterium spp., and Mycobacterium phlei. In order to assess the in situ abundance of the most frequently isolated genus, Aeromonas, two rRNA-targeted oligonucleotide probes were developed. The two gamma proteobacterial genera Aeromonas and Acinetobacter constituted less than 5% of all bacteria. In situ, Proteobacteria belonging to the beta subclass and high G+C Gram-positive bacteria were dominant. From filamentous bacteria, Sphaerotilus spp. and Leptothrix spp. could be detected occasionally. In addition, one sample contained a high proportion of the morphologically distinct filaments of Microthrix parvicella.As for the genus Acinetobacter, the relative abundance of the most frequently gamma-proteobacterial genus Aeromonas was overestimated by the intrinsic selectivity of cultivation. Cultivation on nutrient-rich medium (TS-agar) especially supported an enhanced isolation of bacteria belonging to these two genera. Correspondence to: P. Kämpfer.