In vivo quantitative relationship between plastid division proteins FtsZ1 and FtsZ2 and identification of ARC6 and ARC3 in a native FtsZ complex

FtsZ1 and FtsZ2 are phylogenetically distinct homologues of the tubulin-like bacterial cell division protein FtsZ that play major roles in the initiation and progression of plastid division in plant cells. Both proteins are components of a mid-plastid ring, the Z-ring, which functions as a contractile ring on the stromal surface of the chloroplast IEM (inner envelope membrane). FtsZ1 and FtsZ2 have been shown to interact, but their in vivo biochemical properties are largely unknown. To gain insight into the in vivo biochemical relationship between FtsZ1 and FtsZ2, in the present study we investigated their molecular levels in wild-type Arabidopsis thaliana plants and endogenous interactions in Arabidopsis and pea. Quantitative immunoblotting and morphometric analysis showed that the average total FtsZ concentration in chloroplasts of 3-week-old Arabidopsis plants is comparable with that in Escherichia coli. FtsZ levels declined as plants matured, but the molar ratio between FtsZ1 and FtsZ2 remained constant at approx. 1:2, suggesting that this stoichiometry is regulated and functionally important. Density-gradient centrifugation, native gel electrophoresis, gel filtration and co-immunoprecipitation experiments showed that a portion of the FtsZ1 and FtsZ2 in Arabidopsis and pea chloroplasts is stably associated in a complex of approximately 200-245 kDa. This complex also contains the FtsZ2-interacting protein ARC6 (accumulation and replicatioin of chloroplasts 6), an IEM protein, and analysis of density-gradient fractions suggests the presence of the FtsZ1-interacting protein ARC3. Based on the mid-plastid localization of ARC6 and ARC3 and their postulated roles in promoting and inhibiting chloroplast FtsZ polymer formation respectively, we hypothesize that the FtsZ1-FtsZ2-ARC3-ARC6 complex represents an unpolymerized IEM-associated pool of FtsZ that contributes to the dynamic regulation of Z-ring assembly and remodelling at the plastid division site in vivo.     

Synthesis of a novel peptidic photoaffinity probe for the PTP-1B enzyme

The synthesis of a novel radioactive peptidic photoaffinity probe for the PTP-1B enzyme as well as some SAR leading to the choice of this compound as a photoaffinity probe are presented.     

Nitric oxide promotes differentiation of rat white preadipocytes in culture

The putative role of nitric oxide (NO) in modulating adipogenesis was investigated in cultured preadipocytes derived from rat white adipose tissue. The NO releasing reagent, hydroxylamine (HA), and nitric oxide synthase (NOS) substrate L-arginine (Arg) had no influence on cell replication. However, both HA and Arg exhibited significant induction on differentiation, as evidenced by increased lipoprotein lipase (LPL) and glycerol-3-phosphate dehydrogenase (GPDH) activities, as well as accelerated triacylglycerol (TG) accumulation. These observations suggested a positive role of NO in modulating adipogenesis. Preadipocytes were found to produce NO, and a approximately 50% increase over basal level was observed on the first 2 days of differentiation. Deprivation of endogenous NOS activity by a non-selective NOS inhibitor, N(G)-monomethyl-L-arginine (NMMA), partially abrogated the differentiation process, implicating a role for endogenous NO to stimulate preadipocyte differentiation. Both NOS isoforms, eNOS and iNOS, were detected in differentiating preadipocytes. Specific iNOS inhibitors (1400W and aminoguanidine) had little influence on NO production and differentiation, suggesting that eNOS rather than iNOS may be the major isoform involved in modulating adipogenesis.     

Structural basis of BLyS receptor recognition

B lymphocyte stimulator (BLyS), a member of the tumor necrosis factor (TNF) superfamily, is a cytokine that induces B-cell proliferation and immunoglobulin secretion. We have determined the three-dimensional structure of BLyS to 2.0 A resolution and identified receptor recognition segments using limited proteolysis coupled with mass spectrometry. Similar to other structurally determined TNF-like ligands, the BLyS monomer is a beta-sandwich and oligomerizes to form a homotrimer. The receptor-binding region in BLyS is a deeper, more pronounced groove than in other cytokines. The conserved elements on the 'floor' of this groove allow for cytokine recognition of several structurally related receptors, whereas variations on the 'walls' and outer rims of the groove confer receptor specificity.     

A novel spectrofluorimetric method for determination of lomefloxacin adopting on zinc(II) chelation strategy: Application in human plasma

A new sensitive and instantaneous spectrofluorimetric method for efficient determination of lomefloxacin (LMX) in its pure, dosage form and human plasma was designed. The developed method depends on formation of a metal-chelation compound of LMX as a ligand with zinc(II) in a buffer of acetate (pH 5.5). The following parameters; type of metal, concentration of metal, pH, type of buffer and diluting solvent were optimized. After carefully investigation; 0.2 mM zinc, 2.0 ml acetate buffer (pH 5.5) and water as diluting solvent were set as optimum reaction conditions. Under these conditions, a large increase in the intensity of the fluorescence of LMX was attained at 450 after excitation at 284 nm. The limits of detection and quantification were 5.8 and 1.9 ng ml⁻¹, respectively, with linearity range of 10.0 to 500.0 ng ml⁻¹. The binding mode of LMX and zinc(II) ion (Zn²⁺) was found to be 2:1, respectively, and confirmed by Job's plot method. Furthermore, it extended to the analysis of LMX in the spiked plasma of humans with percentage recovery (98.70 ± 0.97 to 100.30 ± 1.69%, n = 3).     

A vertebrate slow skeletal muscle actin isoform

Salmonids utilize a unique, class II isoactin in slow skeletal muscle. This actin contains 12 replacements when compared with those from salmonid fast skeletal muscle, salmonid cardiac muscle and rabbit skeletal muscle. Substitutions are confined to subdomains 1 and 3, and most occur after residue 100. Depending on the pairing, the 'fast', 'cardiac' and rabbit actins share four, or fewer, substitutions. The two salmonid skeletal actins differ nonconservatively at six positions, residues 103, 155, 278, 281, 310 and 360, the latter involving a change in charge. The heterogeneity has altered the biochemical properties of the molecule. Slow skeletal muscle actin can be distinguished on the basis of mass, hydroxylamine cleavage and electrophoretic mobility at alkaline pH in the presence of 8 m urea. Further, compared with its counterpart in fast muscle, slow muscle actin displays lower activation of myosin in the presence of regulatory proteins, and weakened affinity for nucleotide. It is also less resistant to urea- and heat-induced denaturation. The midpoints of the change in far-UV ellipticity of G-actin versus temperature are approximately 45 degrees C ('slow' actin) and approximately 56 degrees C ('fast' actin). Similar melting temperatures are observed when thermal unfolding is monitored in the aromatic region, and is suggestive of differential stability within subdomain 1. The changes in nucleotide affinity and stability correlate with substitutions at the nucleotide binding cleft (residue 155), and in the C-terminal region, two parts of actin which are allosterically coupled. Actin is concluded to be a source of skeletal muscle plasticity.     

Effects of mutations in Arabidopsis FtsZ1 on plastid division, FtsZ ring formation and positioning, and FtsZ filament morphology in vivo

In plants, chloroplast division FtsZ proteins have diverged into two families, FtsZ1 and FtsZ2. FtsZ1 is more divergent from its bacterial counterparts and lacks a C-terminal motif conserved in most other FtsZs. To begin investigating FtsZ1 structure-function relationships, we first identified a T-DNA insertion mutation in the single FtsZ1 gene in Arabidopsis thaliana, AtFtsZ1-1. Homozygotes null for FtsZ1, though impaired in chloroplast division, could be isolated and set seed normally, indicating that FtsZ1 is not essential for viability. We then mapped five additional atftsZ1-1 alleles onto an FtsZ1 structural model and characterized chloroplast morphologies, FtsZ protein levels and FtsZ filament morphologies in young and mature leaves of the corresponding mutants. atftsZ1-1(G267R), atftsZ1-1(R298Q) and atftsZ1-1(Delta404-433) exhibit reduced FtsZ1 accumulation but wild-type FtsZ2 levels. The semi-dominant atftsZ1-1(G267R) mutation caused the most severe phenotype, altering a conserved residue in the predicted T7 loop. atftsZ1-1(G267R) protein accumulates normally in young leaves but is not detected in rings or filaments. atftsZ1-1(R298Q) has midplastid FtsZ1-containing rings in young leaves, indicating that R298 is not critical for ring formation or positioning despite its conservation. atftsZ1-1(D159N) and atftsZ1-1(G366A) both have overly long, sometimes spiral-like FtsZ filaments, suggesting that FtsZ dynamics are altered in these mutants. However, atftsZ1-1(D159N) exhibits loss of proper midplastid FtsZ positioning while atftsZ1-1(G366A) does not. Finally, truncation of the FtsZ1 C-terminus in atftsZ1-1(Delta404-433) impairs chloroplast division somewhat but does not prevent midplastid Z ring formation. These alleles will facilitate understanding of how the in vitro biochemical properties of FtsZ1 are related to its in vivo function.     

Expression of Vesicular Glutamate Transporter 2 (vGluT2) on Large Dense-Core Vesicles within GnRH Neuroterminals of Aging Female Rats

The pulsatile release of GnRH is crucial for normal reproductive physiology across the life cycle, a process that is regulated by hypothalamic neurotransmitters. GnRH terminals co-express the vesicular glutamate transporter 2 (vGluT2) as a marker of a glutamatergic phenotype. The current study sought to elucidate the relationship between glutamate and GnRH nerve terminals in the median eminence—the site of GnRH release into the portal capillary vasculature. We also determined whether this co-expression may change during reproductive senescence, and if steroid hormones, which affect responsiveness of GnRH neurons to glutamate, may alter the co-expression pattern. Female Sprague-Dawley rats were ovariectomized at young adult, middle-aged and old ages (~4, 11, and 22 months, respectively) and treated four weeks later with sequential vehicle + vehicle (VEH + VEH), estradiol + vehicle (E₂ + VEH), or estradiol + progesterone (E₂+P₄). Rats were perfused 24 hours after the second hormone treatment. Confocal microscopy was used to determine colocalization of GnRH and vGluT2 immunofluorescence in the median eminence. Post-embedding immunogold labeling of GnRH and vGluT2, and a serial electron microscopy (EM) technique were used to determine the cellular interaction between GnRH terminals and glutamate signaling. Confocal analysis showed that GnRH and vGluT2 immunofluorescent puncta were extensively colocalized in the median eminence and that their density declined with age but was unaffected by short-term hormone treatment. EM results showed that vGluT2 immunoreactivity was extensively associated with large dense-core vesicles, suggesting a unique glutamatergic signaling pathway in GnRH terminals. Our results provide novel subcellular information about the intimate relationship between GnRH terminals and glutamate in the median eminence.