Phylogenetic relationships within the class Oligohymenophorea,phylum Cilophora,inferred from the complete small subunit rRNA gene sequences ofColpidium campylum,Glaucoma chattoni,andOpisthonecta henneguyi

Summary Phylogenetic relationships within the class Oligohymenophorea, phylum Ciliophora, were investigated by determining the complete small subunit rRNA (SSrRNA) gene sequences for the hymenostomesColpidium campylum, Glaucoma chattoni, and the peritrichOpisthonecta henneguyi. The affiliations of the oligohymenophoreans were assessed using both distance matrix (DM) and maximum parsimony (MP) analyses. Variations do exist in the phylogenies created by the two methods. However, the basic tree topologies are consistent. In both the DM and MP analyses the hymenostomes (C. campylum, G. chattoni, and the tetrahymenas) all form a very tight group associated with the peritrichO. henneguyi. TheTetrahymena lineage was monophyletic whereasColpidium andGlaucoma were more closely related to each other than either was to the tetrahymenas. The monophyly of the genusTetrahymena in the present analysis supports the phylogenies determined from morphological data and molecular sequence data from the histone H3II/H4II region of the genome. The perplexing and controversial phylogenetic position of the peritrichs is once again depicted in the present analysis. The distinctiveness of the peritrichOpisthonecta from both hymenostome and nassophorean ciliates based on evolutionary distances suggests that the elevation of the peritrichs to a higher taxonomic rank should be reconsidered.     

Identification of pristanoyl-CoA oxidase as a distinct, clofibrate non-inducible enzyme in rat liver peroxisomes.

In this paper we describe the identification of pristanoyl-CoA oxidase activity in rat liver peroxisomes. This activity was not stimulated by clofibrate feeding. Furthermore, the activity was found in multiple tissues. These results show that pristanoyl-CoA oxidase is different from any of the known oxidases which include a clofibrate-inducible acyl-CoA oxidase and the recently identified cholestanoyl-CoA oxidase. Gelfiltration and chromatofocusing experiments provide conclusive evidence that we are dealing with a novel acyl-CoA oxidase with a unique function in peroxisomal beta-oxidation.     

Interleukin-6 is used as a growth factor by virulent Mycobacterium avium: presence of specific receptors.

In this paper, we examined the contribution of the lymphokine interleukin-6 (IL-6) to the growth of four virulent strains of Mycobacterium avium and the nature of the binding moieties on the mycobacteria. First, we showed that human or mouse recombinant interleukin-6 are potent growth factors for four strains of virulent M. avium. This was shown to occur in tissue culture medium, which does not support maximal growth of M. avium. Bioactive IL-6 was required, inasmuch as heat-activating IL-6 or adding an antibody against IL-6 blocked this growth-enhancing ability. The rapid uptake of IL-6 by M. avium was indicated by the fact that the incubation of IL-6 with the four M. avium strains led to a rapid removal of the bioactivity from the culture medium and a rapid removal of radiolabeled IL-6. Scatchard analysis of receptor interaction showed that the M. avium strains had a single receptor species with a Kd of 50 nM and the number of receptor sites was approximately 15,000 bacterium. Blocking experiments showed that the binding of radiolabeled IL-6 was fully displaceable with cold IL-6, but not with other lymphokines. These data suggest that IL-6 may play an important role in the pathogenesis of M. avium infections, notably by promoting growth of M. avium, and that some virulent M. avium strains bind IL-6 in a specific manner.     

Sequence of an oleocin cDNA from Brassica napus

Antibodies raised against purified rapeseed 19 kDa oleosin protein were used to screen an embryo-derived gt11 expression library from Brassica napus. A near full-length cDNA clone, BnV, was isolated. The 781 bp cDNA contained an open reading frame of 549 bp followed by an untranslated region of 222 pb and a poly(A) region of 10 bp. Comparisons between this cDNA and a different oleosin cDNA previously isolated from the same library showed high degrees of sequence similarity in the central domain region and in the 3 untranslated region. Sequence similarities between the derived protein sequence of this cDNA and all other known oleosin protein sequences are discussed.     

Some factors influencing the proportion of periplasmic hepatitisB virus pre-S2 antigen in the recombinant yeastHansenula polymorpha

Summary A central composite design (CCD) was used to evaluate, for the purpose of future process optimization, the influence of pH, yeast extract and ammonium chloride concentrations on the proportion of periplasmic hepatitisB pre-S2 antigen in the recombinant yeastHansenula polymorpha. Each factor was tested at five levels, and a second order polynomial model for the proportion of periplasmic antigen was fitted to the treatment combinations. pH showed the greatest effect: the proportion of periplasmic antigen was greatly increased at the higher pH levels. At the higher pH levels used, the proportion of periplasmic antigen was enhanced by a high concentration of ammonium chloride. Additional experiments have confirmed both the validity of the selected model and the optimal conditions found. A significant correlation was found between the proportion of periplasmic antigen and the total yield of antigen. These results indicated that is should be possible to modulate the distribution of the pre-S2 antigen between the periplasm and the cytoplasm of the yeast.     

The activity of one soluble component of the cell-free NADPH:O2 oxidoreductase of human neutrophils depends on guanosine 5'-O-(3-thio)triphosphate

Neutrophil NADPH:O2 oxidoreductase activity, essential in the killing of bacteria by neutrophils, can be elicited in a cell-free system that requires plasma membranes, cytosol and sodium dodecyl sulfate. In addition, GTP or its nonhydrolyzable analog guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) enhances NADPH oxidase activity. We investigated the mechanism of this effect of GTP gamma S in the cell-free system. Cytosol from human neutrophils was separated in three different soluble oxidase components (SOC I, SOC II, and SOC III). Previously we (Bolscher, B. G. J. M., Van Zwieten, R., Kramer, I. J. M., Weening, R. S., Verhoeven, A. J., and Roos, D. (1989) J. Clin. Invest. 83, 757-763) reported that the cytosol contains two components which act synergistically. We now report that one component (previously labeled SOC II) contains two different components that can be separated by ion exchange chromatography. Immunoblotting with antiserum B-1 (Volpp, B. D., Nauseef, W. M., and Clark, R. A. (1988) Science 242, 1295-1297), directed against a cytosolic complex capable of activating latent membranes in the cell-free system, showed a 47-kDa protein in SOC II and a 67-kDa protein in SOC III. SOC II also contains the 47-kDa phosphoprotein, which indicates that this phosphoprotein and the protein recognized by the antiserum are identical. Low rates of NADPH-dependent O2 consumption can be elicited by SOC II and SOC III in the absence of SOC I. This activity is independent of GTP gamma S. Addition of SOC I increases this activity 3-4-fold, only when GTP gamma S is present. Plasma membranes, incubated with SOC I plus GTP gamma S and re-isolated, showed a similar 3-4-fold enhanced O2 consumption with SOC II and SOC III. The GTP gamma S effect is exerted primarily at the level of the plasma membrane. The concentration of GTP gamma S that causes a half-maximal stimulation was 0.4 mu M. It is concluded that SOC I is a functional component of the NADPH oxidase.     

Isolation and characterization of cytochrome b5 from Candida tropicalis grown on alkane

Cytochrome b₅ from Candida tropicalis grown on alkane has been solubilized in three different ways (sodium cholate, trypsin, osmotic wash). After solubilization of the microsomal membrane with sodium cholate, the purification of cytochrome b₅ was achieved by DEAE-cellulose chromatography, hydroxylapatite chromatography, a second DEAE-cellulose chromatography and a Sephadex G-75 gel filtration. The purified protein had an apparent molecular weight of 16 000 ± 1 000. After solubilization by trypsin treatment or osmotic wash, the purification procedure yielded a protein with an apparent molecular weight of 12 000 ± 1 000. Though the purified proteins presented molecular weights depending on the technique of solubilization, they exhibited identical optical properties, a great stability with respect to temperature and pH, and were all autooxidable. Redox titrations revealed differences in their midpoint potential values, which were 35 ± 5 mV for the b₅ purified after cholate solubilization, —59 ± 5 mV for the b₅ purified after trypsin treatment and —65 ± 5 mV for the b₅ purified after osmotic wash.     

Relationship Between Plasma and Brain Tryptophan in Pigs During Experimental Hepatic Coma Before and After Hernodialysis with Selective Membranes

Experimental acute liver ischemia in pigs induces an increment in plasma free tryptophan with decreased total tryptophan. Brain tryptophan is elevated in all brain areas. A slight, but significant increase of brain serotonin is demonstrated in the striatum only, while 5-HIAA (5-hydroxyindoleacetic acid) is significantly lower in the hypothalamus. Other brain areas do not show significant changes in serotonin and 5-HIAA levels. Neither the high plasma free tryptophan levels, nor the decreased sum of neutral competitive amino acids are consistent with such an elevation of brain tryptophan. Hemodialysis was carried out with two different kinds of membranes: cuprophan (with an efficient removal of molecules up to molecular weight 1300) and AN 69 polyacrylonitrile (efficient removal up to 15,000). Ammonia and aminoacid clearance are similar for both membranes. After AN 69, plasmatic free tryptophan and brain tryptophan are lower than after liver devascularization, but still higher than normal. Serotonin significantly increases in the cortex, midbrain and hypothalamus without concomitant rise of 5-HIAA levels. After cuprophan hemodialysis, plasma total tryptophan is lower than in normal and even comatose animals, whereas free tryptophan is normal. Intracerebral tryptophan is similar to AN 69 dialysed animals, but in the hypothalamus it is similar to nondialysed animals. Brain serotonin levels are not modified. 5-HIAA decreases in the hypothalamus. This finding suggests that middle molecules (which are not cleared out with cuprophan hemodialysis) are involved in the intracerebral transfer of tryptophan and the metabolism of serotonin, mainly in the hypothalamus.     

BCG-induced granulomatous pulmonary inflammation and splenomegaly in mice: A T-cell-dependent response

When C57BL/6 mice were injected iv with BCG in an oil-in-saline emulsion, they developed intense pulmonary granulomatous inflammation (PGI) and splenomegaly as well as chemotactic activity for macrophages and angiotensin-converting enzyme (ACE) in their lung fluids. PGI, splenomegaly, and levels of chemotactic activity and ACE were markedly reduced in T-cell-deficient “B” mice. The capacity to develop PGI was fully restored and splenomegaly was partially restored in “B” mice by the provision of syngeneic thymocytes, spleen cells, or purified T cells. These results indicate that the full expression of BCG-induced PGI is dependent upon thymus-derived cells and is associated with high levels of chemotactic activity for macrophages and ACE in the lung lavage fluid. Although BCG-induced splenomegaly appears to be T cell dependent, it did not reach its full magnitude in reconstituted “B” mice.