Archaeal diversity in two thermophilic chalcopyrite bioleaching reactors

This study used a culture-independent molecular approach to investigate the archaeal community composition of thermophilic bioleaching reactors. Two culture samples, MTC-A and MTC-B, grown with different concentrations of chalcopyrite (CuFeS₂), a copper sulfidic ore, at a temperature of 78°C and pH 1.6 were studied. Phylogenetic analysis of the 16S rRNA genes revealed that both cultures consisted of Archaea belonging to the Sulfolobales . The 16S rRNA gene clone library of MTC-A grown with 4% (w/v) chalcopyrite was dominated by a unique phylotype related to Sulfolobus shibatae (69% of total clones). The remaining clones were affiliated with Stygiolobus azoricus (11%), Metallosphaera sp. J1 (8%), Acidianus infernus (2%), and a novel phylotype related to Sulfurisphaera ohwakuensis (10%). In contrast, the clones from MTC-B grown with 12% (w/v) chalcopyrite did not appear to contain Sulfolobus shibatae -like organisms. Instead the bioleaching consortium was dominated by clones related to Sulfurisphaera ohwakuensis (73.9% of total clones). The remaining microorganisms detected were similar to those found in MTC-A.     

HFE association with transferrin receptor 2 increases cellular uptake of transferrin-bound iron

Mutations in either HFE or transferrin receptor 2 (TfR2) cause decreased expression of the iron regulatory hormone hepcidin and hemochromatosis. HFE and TfR2 were recently discovered to form a stable complex at the cell membrane when co-expressed in heterologous cell lines. We analyzed the functional consequences of the co-expression of these proteins using transfected TRVb cells, a Chinese hamster ovary derived cell line without endogenous HFE or transferrin receptor. The co-expression of TfR2 in TRVb cells expressing HFE led to accelerated HFE biosynthesis and late-Golgi maturation, suggesting interaction prior to cell surface localization. The co-expression of HFE in cells expressing TfR2 led to increased affinity for diferric transferrin, increased transferrin-dependent iron uptake, and relative resistance to iron chelation. These observations indicate that HFE influences the functional properties of TfR2, and suggests a model in which the interaction of these proteins might influence signal transduction to hepcidin.     

Deficiency of SP-B reveals protective role of SP-C during oxygen lung injury.

Although the surface properties of surfactant protein (SP)-B and SP-C are similar, the contributions that either protein may make to lung function have not been identified in vivo. Mutations in SP-B cause lethal respiratory failure at birth; however, SP-B null mice are deficient in both SP-B and SP-C. To identify potential contributions of SP-C to lung function in vivo, the following transgenic mice were generated and exposed to 95% O(2) for 3 days: (SP-B(+/+),SP-C(+/+)), (SP-B(+/+), SP-C(-/-)), (SP-B(+/-),SP-C(+/+)), (SP-B(+/-),SP-C(+/-)), and (SP-B(+/-),SP-C(-/-)). Hyperoxia altered pressure-volume curves in mice that were heterozygous for SP-B, and these values were further decreased in (SP-B(+/-),SP-C(-/-)) mice. Likewise, alveolar interleukin (IL)-6 and IL-1 beta were maximally increased by O(2) exposure of (SP-B(+/-),SP-C(-/-)) mice compared with the other genotypes. Lung hysteresivity was lower in the (SP-B(+/-),SP-C(-/-)) mice. Surfactant isolated from (SP-B(+/+),SP-C(-/-)) and (SP-B(+/-),SP-C(-/-)) mice failed to stabilize the surface tension of microbubbles, showing that SP-C plays a role in stabilization or recruitment of phospholipid films at low bubble radius. Genetically decreased levels of SP-B combined with superimposed O(2)-induced injury reveals the distinct contribution of SP-C to pulmonary function in vivo.     

A radioimmunoprecipitation method in the serodiagnosis of HIV infection: the development of the optimal conditions for its performance and the evaluation of the possibilities for its use

The optimum conditions for using the method of radioimmunoprecipitation (RIP) for the detection of human immunodeficiency virus (HIV) in serum samples have been established. Out of several available cell lines persistently infected with HIV, specially selected line 17 has been chosen. The characteristic feature of this is the high and stable (under the conditions of prolonged cultivation) accumulation of virus-specific proteins in infected cells. The optimum conditions for making the test and its evaluation have also been established. The data of literature on the advantages of the method of RIP over such traditional methods as the enzyme immunoassay and immunoblotting have been confirmed. Thus, the presence of specific antibodies in several serum samples registered as false negative has been established. The intertypical reactivity of two serotypes of the virus, HIV-1 and HIV-2, has been studied. Cross reactivity of antibodies with respect to the HIV gene gag, but not with respect to viral glycoproteids, has been established. Ideas on the expediency and prospects of using RIP for the serological control of HIV infection are presented.     

Ricin-binding properties of acid hydrolases from isolated lysosomes implies prior processing by terminal transferases of the trans-Golgi apparatus

Acid hydrolases were isolated from the lysosome fraction of beta-galactosidase-deficient human fibroblasts and from the mannose 6-phosphate containing medium in which they were grown. Nearly half of the total beta-hexosaminidase and beta-glucuronidase from both sources bound to Ricin specifically. Lysosomal beta-hexosaminidase, metabolically labelled with [35S]-methionine, was also fractionated on Ricin-agarose. SDS-PAGE of immunoprecipitates from Ricin-binding and non-binding fractions revealed approximately equivalent amounts of cross-reacting material at the appropriate MW. We interpret these results to mean that acid hydrolases which are segregated to lysosomes are exposed to trans-Golgi processing enzymes to about the same extent as enzymes which are secreted, and that segregation by the Man 6-P receptor occurs after transit through the trans-Golgi compartment.     

Postnatal alveolar development of the rabbit.

Previous studies of alveolarization have used rats or lambs; however, neither closely reflects human alveolar development. We characterized alveolar development in rabbits (n = 3-7 /group) at 28 days gestation (dg) to 9 mo to determine whether they followed the human pattern more closely. The right lung was made up of 30% alveolar and 50% duct space at 28 dg to 3 days and of 50 and 30%, respectively, at 14 days to 9 mo. Tissue fraction and alveolar wall thickness decreased by 40% 28 dg to birth. At birth, approximately 4.5% of the number of alveoli seen at 9 mo were present, with alveolar number increasing progressively well into adulthood. The rate of alveolar formation was high around birth, decreasing progressively with age. Alveolar volume increased more than twofold (28 dg to birth) and continued to increase postnatally to 16 wk. Surface fraction decreased by 17% (28 dg to 3 days), after which it remained uniform. Our findings suggest that the timing of onset of alveolarization in humans and rabbits is similar and that rabbits may be used to model postnatal influences on alveolar development.     

Expression of human cation-independent mannose 6-phosphate receptor cDNA in receptor-negative mouse P388D1 cells following gene transfer

We recently reported the cDNA cloning, sequence, and expression of the human cation-independent mannose 6-phosphate receptor (hCI-MPR) (Oshima, A., Nolan, C. M., Kyle, J. W., Grubb, J. H., and Sly, W. S. (1988) J. Biol. Chem. 263, 2553-2562). The sequence of the hCI-MPR was virtually identical to that of the human insulin-like growth factor II receptor cDNA (Morgan, D. O., Edman, J. C., Standring, D. N., Fried, V. A., Smith, M. C., Roth, R. A., and Rutter, W. J. (1987) Nature 329, 301-307). To test the role of the putative bifunctional receptor in intracellular sorting of acid hydrolases, we studied its effect on lysosomal enzyme transport following gene transfer to receptor-negative cells. Receptor-negative mouse P388D1 cells were transfected with a cDNA construct containing the entire coding sequence of hCI-MPR under the control of the mouse metallothionine I promoter. Stable transformants were isolated and characterized. The expressed hCI-MPR was localized in membranes including the plasma membrane, bound mannose 6-phosphate containing ligands, and mediated endocytosis which could be specifically blocked by mannose 6-phosphate. We next measured the effect of the expressed hCI-MPR on intracellular and secreted acid hydrolases. The intracellular activity of the lysosomal marker enzymes beta-glucuronidase and beta-hexosaminidase increased up to 2-fold following transformation. In addition, expression of the receptor greatly reduced the fraction of acid hydrolases secreted. These phenotypic changes in the transformed cell lines support the proposed role of the cation-independent mannose 6-phosphate receptor in intracellular sorting and targeting of lysosomal enzymes.     

A comparison of lipid metabolism in hepatocytes isolated from fed and starved neonatal and adult rats.

1. The utilization of [1-14C]palmitate by hepatocytes prepared from fed and starved neonatal and adult rats has been examined by measuring isotopic incorporation into various products. 2. In cells from fed adult rats the principal products were esters (triglycerides and phospholipids) but ketone bodies were the main metabolic end products in cells from starved adult and fed and starved neonatal rats. Production of triglycerides exceeded that of phospholipids in fed adult cells whereas phospholipid formation always predominated in neonatal cells. 3. The high rate of fatty acid oxidation and hence NADH formation by neonatal cells is reflected by a lower acetoacetate--3-hydroxybutyrate ratio at the earlier stages of incubation of neonatal cells. 4. The addition of glycerol modified quantitatively the products of palmitate metabolism by adult hepatocytes but no such effects were observed with neonatal cells. 5. Compared with adult cells, neonatal hepatocytes showed very low rates of lipogenesis that were only enhanced a little by addition of lactate/pyruvate and did not show any effects of glucose concentration upon incorporation of tritium from 3H2O into lipids.     

Cleavage of human serum transferrin with N-bromosuccinimide.

Human serum transferrin was fragmented by N-bromosuccinimide and reduction-alkylation. It was observed that there were at least two each of tryptophanyl-serine and tryptophanyl-aspartic acid, and one each of tryptophanyl-alanine and tryptophanyl-glutamic acid bonds. The size of fragments detected by polyacrylamide gel electrophoresis ranged from 8,000 to 70,000 daltons. Several of the fragments were isolated in a homogeneous form with respect to molecular weight, but were shown to be mixtures of at least five molecular species each by end group analysis.     

Expression of the membrane-associated carbonic anhydrase isozyme XII in the human kidney and renal tumors.

Carbonic anhydrase isozyme XII (CA XII) is a novel membrane-associated protein with a potential role in von Hippel-Lindau carcinogenesis. Although Northern blotting has revealed positive signal for CA XII in normal human kidney, this is the first study to demonstrate its cellular and subcellular localization along the human nephron and collecting duct. Immunohistochemistry with a polyclonal antibody (PAb) raised against truncated CA XII revealed distinct staining in the basolateral plasma membrane of the epithelial cells in the thick ascending limb of Henle and distal convoluted tubules, and in the principal cells of the collecting ducts. A weak basolateral signal was also detected in the epithelium of the proximal convoluted tubules. In addition to the normal kidney specimens, this immunohistochemical study included 31 renal tumors. CA XII showed moderate or strong plasma membrane-associated expression in most oncocytomas and clear-cell carcinomas. The segmental, cellular, and subcellular distribution of CA XII along the human nephron and collecting duct suggests that it may be one of the key enzymes involved in normal renal physiology, particularly in the regulation of water homeostasis. High expression of CA XII in some renal carcinomas may contribute to its role in von Hippel-Lindau carcinogenesis.