Nitrogen fixation in tomato (Lycopersicon esculentum Mill ‘Pusa Ruby’)

Nitrogen fixation (acetylene reduction) was found in intact tomato (Lycopersicon esculentum Mill ‘Pusa Ruby’) plants in the field, in pots and also in aseptic cultures. The unsterilized as well as sterilized rhizoplane and phylloplane of the plant when assayed separately also responded to the test. From root bits of tomato sterilized upto 20 minutes with 0.1% mercuric chloride, growth of the bacterium from the interior of the root into the medium was observed thereby indicating their presence within the endorhizosphere. Phase contrast and electron microscopic studies of the root system of tomato revealed the presence of bacterial colonies in the epidermis, cortex and vascular bundles. Bacterial numbers in the endosphere, of root and leaf were 30×10⁴ and 12×10⁴, respectively, per gram fresh weight of tissue. The bacteria were predominantly rod-shaped 1.4–4.8×0.9–1.95 μm in 24-h-old cultures, pleomorphic, polar or bipolary flagellated having β-hydroxy butyrate granules. The bacterium has been identified as a new species of Azospirillum.     

Biosynthesis of Cell Envelope-Associated Phenolic Glycolipids in Mycobacterium marinum

Phenolic glycolipids (PGLs) are polyketide synthase-derived glycolipids unique to pathogenic mycobacteria. PGLs are found in several clinically relevant species, including various Mycobacterium tuberculosis strains, Mycobacterium leprae, and several nontuberculous mycobacterial pathogens, such as M. marinum. Multiple lines of investigation implicate PGLs in virulence, thus underscoring the relevance of a deep understanding of PGL biosynthesis. We report mutational and biochemical studies that interrogate the mechanism by which PGL biosynthetic intermediates (p-hydroxyphenylalkanoates) synthesized by the iterative polyketide synthase Pks15/1 are transferred to the noniterative polyketide synthase PpsA for acyl chain extension in M. marinum. Our findings support a model in which the transfer of the intermediates is dependent on a p-hydroxyphenylalkanoyl-AMP ligase (FadD29) acting as an intermediary between the iterative and the noniterative synthase systems. Our results also establish the p-hydroxyphenylalkanoate extension ability of PpsA, the first-acting enzyme of a multisubunit noniterative polyketide synthase system. Notably, this noniterative system is also loaded with fatty acids by a specific fatty acyl-AMP ligase (FadD26) for biosynthesis of phthiocerol dimycocerosates (PDIMs), which are nonglycosylated lipids structurally related to PGLs. To our knowledge, the partially overlapping PGL and PDIM biosynthetic pathways provide the first example of two distinct, pathway-dedicated acyl-AMP ligases loading the same type I polyketide synthase system with two alternate starter units to produce two structurally different families of metabolites. The studies reported here advance our understanding of the biosynthesis of an important group of mycobacterial glycolipids.     

Chromosome mapping of the murine syndecan gene.

The chromosomal localization of the murine syndecan gene was determined by analysis of DNA from a panel of mouse-hamster cell hybrids containing various mouse chromosomes, detection of immunoreactive syndecan in culture medium of these cells, and linkage analysis of a mouse interspecific backcross. Southern analysis of the mouse-hamster cell hybrid DNA shows two distinct hybridizing sequences, one on mouse Chromosome 12 and the other on the X chromosome. Localization of the syndecan gene to mouse Chromosome 12 was determined by detection of immunoreactive syndecan in the culture medium of cell hybrids containing mouse Chromosome 12. Hybrids containing other mouse chromosomes were negative. Linkage analysis by Southern hybridization of DNA from a mouse interspecific backcross using a syndecan-specific probe localized the syndecan gene locus, Synd, to the proximal end of Chromosome 12, tightly linked to the Pomc-1 and Nmyc loci. The syndecan gene is likely on human Chromosome 2 because this region shows conservation of synteny between mouse and human chromosomes.     

Assignment of a serotonin 5HT-2 receptor gene (HTR2) to human chromosome 13q14-q21 and mouse chromosome 14

A gene for serotonin 5HT-2 receptor (HTR2) is assigned to human chromosome 13 by somatic cell hybrids and to region 13q14-q21 by in situ hybridization. It is assigned to mouse chromosome 14 by somatic cell hybrid analysis.     

Intrasplicing coordinates alternative first exons with alternative splicing in the protein 4.1R gene

In the protein 4.1R gene, alternative first exons splice differentially to alternative 3' splice sites far downstream in exon 2'/2 (E2'/2). We describe a novel intrasplicing mechanism by which exon 1A (E1A) splices exclusively to the distal E2'/2 acceptor via two nested splicing reactions regulated by novel properties of exon 1B (E1B). E1B behaves as an exon in the first step, using its consensus 5' donor to splice to the proximal E2'/2 acceptor. A long region of downstream intron is excised, juxtaposing E1B with E2'/2 to generate a new composite acceptor containing the E1B branchpoint/pyrimidine tract and E2 distal 3' AG-dinucleotide. Next, the upstream E1A splices over E1B to this distal acceptor, excising the remaining intron plus E1B and E2' to form mature E1A/E2 product. We mapped branchpoints for both intrasplicing reactions and demonstrated that mutation of the E1B 5' splice site or branchpoint abrogates intrasplicing. In the 4.1R gene, intrasplicing ultimately determines N-terminal protein structure and function. More generally, intrasplicing represents a new mechanism by which alternative promoters can be coordinated with downstream alternative splicing.     

The role of cholesterol and glycosylphosphatidylinositol-anchored proteins of erythrocyte rafts in regulating raft protein content and malarial infection

Human erythrocytes are terminally differentiated, nonendocytic cells that lack all intracellular organelles. Here we show that their plasma membranes contain detergent-resistant membrane rafts that constitute a small fraction (4%) of the total membrane protein, with a complex mixture of proteins that differentially associate with rafts. Depletion of raft-cholesterol abrogates association of all proteins with no significant effect on cholesterol:protein ratios in the rest of the membrane, lipid asymmetry, deformability, or transport properties of the bilayer, indicating that cholesterol is critical for protein assembly into rafts and suggesting that rafts have little influence on several erythrocyte functions. Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria, which lack glycosylphosphatidylinositol-anchored proteins, show significant elevation in raft-cholesterol but no increase in raft protein association, suggesting that raft assembly does not require glycosylphosphatidylinositol-anchored proteins, raft proteins do not bind directly to cholesterol, and only threshold levels of raft-cholesterol are critical for protein recruitment. Loss of glycosylphosphatidylinositol-anchored proteins had no effect on erythrocytic infection by malarial parasite or movement of raft markers into the parasite's vacuole. However, infection is blocked following raft-cholesterol disruption, suggesting that erythrocyte rafts can be functionally exploited and providing the first evidence for the involvement of host rafts in an apicomplexan infection.     

Characterization of human RhCG and mouse Rhcg as novel nonerythroid Rh glycoprotein homologues predominantly expressed in kidney and testis

In mammals, the Rh family includes the variable Rh polypeptides and invariant RhAG glycoprotein. These polytopic proteins are confined to the erythroid lineage and are assembled into a multisubunit complex essential for Rh antigen expression and plasma membrane integrity. Here, we report the characterization of RhCG and Rhcg, a pair of novel Rh homologues present in human and mouse nonerythroid tissues. Despite sharing a notable similarity to the erythroid forms, including the 12-transmembrane topological fold, the RHCG/Rhcg pair is distinct in chromosome location, genomic organization, promoter structure, and tissue-specific expression. RHCG and Rhcg map at 15q25 of human chromosome 15 and the long arm of mouse chromosome 7, respectively, each having 11 exons and a CpG-rich promoter. Northern blots detected kidney and testis as the major organs of RHCG or Rhcg expression. In situ hybridization revealed strong expression of Rhcg in the kidney collecting tubules and testis seminiferous tubules. Confocal imaging of transiently expressed green fluorescence protein fusion proteins localized RhCG exclusively to the plasma membrane, a distribution confirmed by cellular fractionation and Western blot analysis. In vitro translation and ex vivo expression showed that RhCG carries a complex N-glycan, probably at the (48)NLS(50) sequon of exoloop 1. These results pinpoint RhCG and Rhcg as novel polytopic membrane glycoproteins that may function as epithelial transporters maintaining normal homeostatic conditions in kidney and testis.     

Release of intracellular Zn<Superscript>2+</Superscript> in cultured neurons after brief exposure to low concentrations of exogenous nitric oxide

Several studies have shown intracellular Zn²⁺ release and concomitant cell death after prolonged exposure to exogenous NO. In the present study, we investigated whether cortical neurons briefly exposured to exogenous NO would demonstrate similar levels of intracellular Zn²⁺ release and subsequent cell death. Cortical neurons were loaded with the Zn²⁺ selective fluorophore FluoZin-3 and treated with various concentrations of the NO generator, spermine NONOate. Fluorescence microscopy was used to detect and quantify intracellular Zn²⁺ levels. Concomitant EDTA perfusion was used to eliminate potential effects of extracellular Zn²⁺. Neurons were perfused with the heavy metal chelator TPEN to selectively eliminate Zn²⁺ induced fluorescence changes. A significant increase of intracellular fluorescence was detected during a 5 min perfusion with spermine NONOate. The increase in intracellular Zn²⁺ release appeared to peak at 1 μM spermine NONOate (123.8 ± 28.5%, increase above control n = 20, P < 0.001). Further increases in spermine NONOate levels as high as 1 mM failed to further increase detectable intracellular Zn²⁺ levels. The NO scavenger hemoglobin blocked the effects of spermine NONOate and the inactive analog of the spermine NONOate, spermine, was without effect. No evidence of cell death induced by any of the brief treatments with exogenous NO was observed; only prolonged incubation with much larger amounts of exogenous NO resulted in significant cell death. These data suggest that in vivo release of NO may cause elevations of intracellular Zn²⁺ in cortical neurons. The possibility that release of intracellular Zn²⁺ in response to NO could play a role in intracellular signaling is discussed.     

Interactions of Plasmodium falciparum erythrocyte membrane protein 3 with the red blood cell membrane skeleton

Plasmodium falciparum parasites express and traffick numerous proteins into the red blood cell (RBC), where some associate specifically with the membrane skeleton. Importantly, these interactions underlie the major alterations to the modified structural and functional properties of the parasite-infected RBC. P. falciparum Erythrocyte Membrane Protein 3 (PfEMP3) is one such parasite protein that is found in association with the membrane skeleton. Using recombinant PfEMP3 proteins in vitro, we have identified the region of PfEMP3 that binds to the RBC membrane skeleton, specifically to spectrin and actin. Kinetic studies revealed that residues 38-97 of PfEMP3 bound to purified spectrin with moderately high affinity (K_D(kin) = 8.5 × 10^− 8 M). Subsequent deletion mapping analysis further defined the binding domain to a 14-residue sequence (IFEIRLKRSLAQVL; K_D(kin) = 3.8 × 10^− 7 M). Interestingly, this same domain also bound to F-actin in a specific and saturable manner. These interactions are of physiological relevance as evidenced by the binding of this region to the membrane skeleton of inside-out RBCs and when introduced into resealed RBCs. Identification of a 14-residue region of PfEMP3 that binds to both spectrin and actin provides insight into the potential function of PfEMP3 in P. falciparum-infected RBCs.