A link between virulence and homeostatic responses to hypoxia during infection by the human fungal pathogen Cryptococcus neoformans

Fungal pathogens of humans require molecular oxygen for several essential biochemical reactions, yet virtually nothing is known about how they adapt to the relatively hypoxic environment of infected tissues. We isolated mutants defective in growth under hypoxic conditions, but normal for growth in normoxic conditions, in Cryptococcus neoformans, the most common cause of fungal meningitis. Two regulatory pathways were identified: one homologous to the mammalian sterol-response element binding protein (SREBP) cholesterol biosynthesis regulatory pathway, and the other a two-component-like pathway involving a fungal-specific hybrid histidine kinase family member, Tco1. We show that cleavage of the SREBP precursor homolog Sre1-which is predicted to release its DNA-binding domain from the membrane-occurs in response to hypoxia, and that Sre1 is required for hypoxic induction of genes encoding for oxygen-dependent enzymes involved in ergosterol synthesis. Importantly, mutants in either the SREBP pathway or the Tco1 pathway display defects in their ability to proliferate in host tissues and to cause disease in infected mice, linking for the first time to our knowledge hypoxic adaptation and pathogenesis by a eukaryotic aerobe. SREBP pathway mutants were found to be a hundred times more sensitive than wild-type to fluconazole, a widely used antifungal agent that inhibits ergosterol synthesis, suggesting that inhibitors of SREBP processing could substantially enhance the potency of current therapies.     

Disruption of methylarginine metabolism impairs vascular homeostasis

Asymmetric dimethylarginine (ADMA) and monomethyl arginine (L-NMMA) are endogenously produced amino acids that inhibit all three isoforms of nitric oxide synthase (NOS). ADMA accumulates in various disease states, including renal failure, diabetes and pulmonary hypertension, and its concentration in plasma is strongly predictive of premature cardiovascular disease and death. Both L-NMMA and ADMA are eliminated largely through active metabolism by dimethylarginine dimethylaminohydrolase (DDAH) and thus DDAH dysfunction may be a crucial unifying feature of increased cardiovascular risk. However, despite considerable interest in this pathway and in the role of ADMA as a cardiovascular risk factor, there is little evidence to support a causal role of ADMA in pathophysiology. Here we reveal the structure of human DDAH-1 and probe the function of DDAH-1 both by deleting the DDAH1 gene in mice and by using DDAH-specific inhibitors which, as we demonstrate by crystallography, bind to the active site of human DDAH-1. We show that loss of DDAH-1 activity leads to accumulation of ADMA and reduction in NO signaling. This in turn causes vascular pathophysiology, including endothelial dysfunction, increased systemic vascular resistance and elevated systemic and pulmonary blood pressure. Our results also suggest that DDAH inhibition could be harnessed therapeutically to reduce the vascular collapse associated with sepsis.     

Structural conservation and variation in the mitochondrial control region of fringilline finches (Fringilla spp.) and the greenfinch (Carduelis chloris)

We sequenced the entire control region and portions of flanking genes (tRNA(Phe), tRNA(Glu), and ND6) in the common chaffinch (Fringilla coelebs), blue chaffinch (F. teydea), brambling (F. montifringilla), and greenfinch (Carduelis chloris). In these finches the control region is similar in length (1,223-1,237 bp) and has the same flanking gene order as in other birds, and contains a putative TAS element and the highly conserved CSB-1 and F, D, and C boxes recognizable in most vertebrates. Cloverleaf-like structures associated with the TAS element at the 5' end and CSB-1 at the 3' end of the control region may be involved with the stop and start of D-loop synthesis, respectively. The pattern of nucleotide and substitution bias is similar to that in other vertebrates, and consequently the finch control region can be subdivided into a central, conserved G-rich domain (domain II) flanked by hypervariable 5'-C-rich (domain I) and 3'-AT-rich (domain III) segments. In pairwise comparisons among finch species, the central domain has unusually low transition/transversion ratios, which suggests that increased G + T content is a functional constraint, possibly for DNA primase efficiency. In finches the relative rates of evolution vary among domains according to a ratio of 4.2 (domain III) to 2.2 (domain I) to 1 (domain II), and extensively among sites within domains I and II. Domain I and III sequences are extremely useful in recovering intraspecific phylogeographic splits between populations in Africa and Europe, Madeira, and a basal lineage in Nefza, Tunisia. Domain II sequences are highly conserved, and are therefore only useful in conjunction with sequences from domains I and III in phylogenetic studies of closely related species.      

The divergent domains of the NEFA and nucleobindin proteins are derived from an EF-hand ancestor

The human protein NEFA (DNA binding, EF-hand, Acidic region) has previously been isolated from a KM3 cell line and immunolocalized on the plasma membrane, in the cytoplasma, and in the culture medium. Sequence analysis of a cDNA clone encoding NEFA identified a hydrophilic domain, two EF-hands, and a leucine zipper at the C- terminus. These characters are shared with nucleobindin (Nuc). In this paper we have further characterized NEFA and probed its evolutionary origins. Circular dichroism (CD) spectra of recombinant NEFA indicated a helical content of 51% and showed that the EF-hands are capable of binding Ca2+. Experiments with recombinant NEFA and synthesized peptides revealed that the leucine zipper cannot form a homodimer. The leucine zipper may allow heterodimer formation of NEFA and an unknown protein. Phylogenetic analyses suggest that this protein is derived from a four-domain EF-hand ancestor with subsequent duplications and fusions. The leucine zipper and putative DNA-binding domains of NEFA have evolved secondarily from existing EF-hand sequences. These analyses provide insights into how complex proteins may originate and trace the precursor of NEFA to the common ancestor of eukaryotes.      

Preferential hemolysis of postnatal calf red cells induced by internal alkanlinization

Red blood cells from neonatal calves, but not from adult cows, rapidly hemolyze in buffered 300 mM solutions of a variety of nonelectrolytes and amino acids. Of these compounds, sucrose is chosen to elucidate the mechanism by which this preferential hemolysis takes place. As in other mammalian red cells, both calf and cow cells are found to be impermeable to sucrose and, in an isosmolar sucrose solution, to undergo volume shrinkage caused by the net loss of chloride ions with concomitant increase in intracellular pH. To test the potential role of intracellular pH change associated with chloride loss in promoting hemolysis, intracellular pH was altered by: (a) a direct addition of fixed acid or base to sucrose solution; (b) the removal of dissolved CO(2) from sucrose solution; and (c) the addition of cells to isotonic NaHCO(3) solution in the absence of sucrose. In all cases, only calf and not cow cells underwent hemolysis. Moreover, 4-acetamido-4’-isothiocyano-2,2’-stilbene disulfonic acid, a potent anion transport inhibitor, completely protected calf cells from hemolysis and caused a nearly total inhibition of both chloride loss and intracellular alkalinization. Furthermore, the hemolytic process is closely related to the integrity of a membrane protein, the band 3 protein, which can be cleaved to varying degrees by the combined treatment of pronase and lipase. Hemolysis is progressively inhibited as the band 3 protein undergoes proteolysis, until a total inhibition of hemolysis takes place when almost all of the band 3 protein is digested into smaller protein components with a mol wt of 65,000 and 35,000 daltons. These results suggest that the intracellular alkalinization process leading to a structural instability of the membrane band 3 protein is responsible for this calf cell hemolysis.     

Pig Reticulocytes. III. Glucose permeability in naturally occurring reticulocytes and red cells from newborn piglets

The loss of facilitated glucose transport of red cells occurring in the newborn pig was monitored in 11 density-separated cells from birth to a 4 wk of age. At birth there was a threefold increase in glucose permeability from the lightest cells to the most dense, suggesting that cells having progressively less glucose permeability are released into the circulation as gestation proceeds. Because of extraordinary stimulation of erythropoietic activity, the uppermost top fraction constituting 2-3 percent of the total cells is composed purely of reticulocytes in the growing animal. The glucose permeability of these reticulocytes which at birth has a slow but significant rate of 3.7 μmol/ml cell x min at 25 degrees C is rapidly decreased within 3-4 days to the level of reticulocytes produced in the adult in response to phenylhydrazine assault. Moreover, reticulocytes themselves discard their membrane permeability to glucose in the course of maturation to red cells. Thus, even though reticulocytes at birth are permeable to glucose, they will become red cells practically impervious to glucose within a few days. These findings suggest that the transition from a glucose- permeable fetal state to a glucose-impermeable postnatal state is brought about by two mechanisms: (a) dilution of fetal cells by glucose-impervious cells produced coincidentally with or shortly after birth; and (b) elimination of fetal cells, which have a shorter half-life, from the circulation.