The hydrophobic recognition site formed by residues PsaA-Trp651 and PsaB-Trp627 of photosystem I in Chlamydomonas reinhardtii confers distinct selectivity for binding of plastocyanin and cytochrome c6

On the lumenal side of photosystem I (PSI), each of the two large core subunits, PsaA and PsaB, expose a conserved tryptophan residue to the surface. PsaB-Trp(627) is part of the hydrophobic recognition site that is essential for tight binding of the two electron donors plastocyanin and cytochrome c(6) to the donor side of PSI (Sommer, F., Drepper, F., and Hippler, M. (2002) J. Biol. Chem. 277, 6573-6581). To examine the function of PsaA-Trp(651) in binding and electron transfer of both donors to PSI, we generated the mutants PsaA-W651F and PsaA-W651S by site-directed mutagenesis and biolistic transformation of Chlamydomonas reinhardtii. The protein-protein interaction and the electron transfer between the donors and PSI isolated from the mutants were analyzed by flash absorption spectroscopy. The mutation PsaA-W651F completely abolished the formation of a first order electron transfer complex between plastocyanin (pc) and the altered PSI and increased the dissociation constant for binding of cytochrome (cyt) c(6) by more than a factor of 10 as compared with wild type. Mutation of PsaA-Trp(651) to Ser had an even larger impact on the dissociation constant. The K(D) value increased another 2-fold when the values obtained for the interaction and electron transfer between cyt c(6) and PSI from PsaA-W651S and PsaA-W651F are compared. In contrast, binding and electron transfer of pc to PSI from PsaA-W651S improved as compared with PSI from PsaA-W651F and admitted the formation of an inter-molecular electron transfer complex, resulting in a K(D) value of about 554 microm that is still five times higher than observed for wild type. These results demonstrate that PsaA-Trp(651) is, such as PsaB-Trp(627), crucial for high affinity binding of pc and cyt c(6) to PSI. Our results also indicate that the highly conserved structural recognition motif that is formed by PsaA-Trp(651) and PsaB-Trp(627) confers a differential selectivity in binding of both donors to PSI.     

Tec kinases mediate sustained calcium influx via site-specific tyrosine phosphorylation of the phospholipase Cgamma Src homology 2-Src homology 3 linker

Tyrosine phosphorylation of phospholipase Cgamma2 (PLCgamma2) is a crucial activation switch that initiates and maintains intracellular calcium mobilization in response to B cell antigen receptor (BCR) engagement. Although members from three distinct families of non-receptor tyrosine kinases can phosphorylate PLCgamma in vitro, the specific kinase(s) controlling BCR-dependent PLCgamma activation in vivo remains unknown. Bruton's tyrosine kinase (Btk)-deficient human B cells exhibit diminished inositol 1,4,5-trisphosphate production and calcium signaling despite a normal inducible level of total PLCgamma2 tyrosine phosphorylation. This suggested that Btk might modify a critical subset of residues essential for PLCgamma2 activity. To evaluate this hypothesis, we generated site-specific phosphotyrosine antibodies recognizing four putative regulatory residues within PLCgamma2. Whereas all four sites were rapidly modified in response to BCR engagement in normal B cells, Btk-deficient B cells exhibited a marked reduction in phosphorylation of the Src homology 2 (SH2)-SH3 linker region sites, Tyr(753) and Tyr(759). Phosphorylation of both sites was restored by expression of Tec, but not Syk, family kinases. In contrast, phosphorylation of the PLCgamma2 carboxyl-terminal sites, Tyr(1197) and Tyr(1217), was unaffected by the absence of functional Btk. Together, these data support a model whereby Btk/Tec kinases control sustained calcium signaling via site-specific phosphorylation of key residues within the PLCgamma2 SH2-SH3 linker.     

Functions of the C-terminal domain of varicella-zoster virus glycoprotein E in viral replication in vitro and skin and T-cell tropism in vivo

Varicella-zoster virus (VZV) glycoprotein E (gE) is essential for VZV replication. To further analyze the functions of gE in VZV replication, a full deletion and point mutations were made in the 62-amino-acid (aa) C-terminal domain. Targeted mutations were introduced in YAGL (aa 582 to 585), which mediates gE endocytosis, AYRV (aa 568 to 571), which targets gE to the trans-Golgi network (TGN), and SSTT, an "acid cluster" comprising a phosphorylation motif (aa 588 to 601). Substitutions Y582G in YAGL, Y569A in AYRV, and S593A, S595A, T596A, and T598A in SSTT were introduced into the viral genome by using VZV cosmids. These experiments demonstrated a hierarchy in the contributions of these C-terminal motifs to VZV replication and virulence. Deletion of the gE C terminus and mutation of YAGL were lethal for VZV replication in vitro. Mutations of AYRV and SSTT were compatible with recovery of VZV, but the AYRV mutation resulted in rapid virus spread in vitro and the SSTT mutation resulted in higher virus titers than were observed for the parental rOka strain. When the rOka-gE-AYRV and rOka-gE-SSTT mutants were evaluated in skin and T-cell xenografts in SCIDhu mice, interference with TGN targeting was associated with substantial attenuation, especially in skin, whereas the SSTT mutation did not alter VZV infectivity in vivo. These results provide the first information about how targeted mutations of this essential VZV glycoprotein affect viral replication in vitro and VZV virulence in dermal and epidermal cells and T cells within intact tissue microenvironments in vivo.     

Recombinant expression, purification, and functional analysis of two novel cystatins from sugarcane (Saccharum officinarum)

Phytocystatins are cysteine proteinase inhibitors from plants implicated in the endogenous regulation of protein turnover, programmed cell death, and in defense mechanisms against pathogens. To date, only few cystatin genes have been characterized in most plant species. We have previously characterized the protein Canecystatin, the first cystatin described in sugarcane. In an attempt to study novel Canecystatins, we identified two ORFs encoding cystatins (referred as CaneCPI-2 and CaneCPI-3) using the data from the Sugarcane EST genome project. These ORFs were then subcloned and expressed in Escherichia coli using pET28 expression vector. High amounts (approximately 20 mg/L) of pure recombinant proteins were obtained by affinity chromatography in a single step of purification. Polyclonal antibodies against the recombinant Canecystatins were raised, allowing the immunodetection of the endogenous proteins in the plant tissues. Moreover, the proteins were able to inhibit papain in a fluorometric assay with K(i) values of 0.2 and 0.25 microM for CaneCPI-2 and CaneCPI-3, respectively. These findings contribute to a better understanding of the activity of sugarcane cystatins and encourage future activity and structural studies of these proteins.     

Effect of microbial phytase on apparent ileal amino acid digestibility of phosphorus-adequate diets in growing pigs

Six ileally cannulated pigs (mean initial body weight 34.8 kg) were used to study the effect of microbial phytase on apparent ileal digestibility of P, total N and amino acids. Three P-adequate diets (digestible P concentration 2.3 g kg(- )l) containing barley (B), soyabean meal (S) or a mixture of the two (BS) with or without phytase supplement (1000 FTU x kg(-1)) were fed to pigs using a 6 x 6 Latin square design. The addition of phytase increased (p < 0.05) apparent ileal P digestibility of diets B, S and BS by 16.5, 19.2 and 19.2%, respectively. There was no effect of phytase on the ileal digestibility of total N. Apparent ileal digestibility of amino acids tended to increase in the BS diet supplemented with phytase (mean improvement of 2.2%); but no significant difference was found for any amino acid as compared with the unsupplemented diet. To asses the additivity of apparent amino acid digestibility, the determined values for the BS diet were compared to those calculated from digestibilities found in diets B and S. There were no significant differences between the determined and calculated values. It is concluded that the addition of microbial phytase to P-adequate diets does not affect ileal amino acid digestibility in growing pigs and that the apparent amino acid digestibility values determined in single ingredients may be additive when included into a complex diet.     

New ruthenium(II) mixed metallocene derived complexes: Synthesis, characterization by X-ray diffraction and evaluation on DNA interaction by atomic force microscopy

A new family of Ru(II) mixed metallocene complexes of the type [Ru(η⁵-C₅H₅)(η⁶-arene)][PF₆] has been synthesized and fully characterized by NMR and UV-Vis spectroscopy. X-ray analysis of single crystal was achieved for all the complexes and revealed the presence of two enantiomers expected for planar chirality originated by the η⁶ coordination of the arene prochiral ligands. Studies of interaction of the new complexes with pBR322 DNA by atomic force microscopy showed very strong and different types of interaction. Antiproliferative tests were examined on human leukemia cancer cells (HL-60) using the MTT assay, and the IC₅₀ values revealed low antiproliferative activity compared to cisplatin.     

Determinants governing the potency of STAT3 activation via the individual STAT3-recruiting motifs of gp130

In recent years, the elucidation of the structures of many signalling molecules has allowed new insights into the molecular mechanisms that govern signal transduction events. In the field of cytokine signalling, the solved structures of cytokine/receptor complexes and of key components involved in signal transduction such as STAT factors or the tyrosine phosphatase SHP2 have broadened our understanding of the molecular basis of the signalling events and provided key information for the rational design of therapeutic approaches to modulate or block cytokine signal transduction. Unfortunately, no structural data on the intracellular parts of cytokine receptors are available. The exact molecular mechanism underlying one of the first steps in signal transduction, namely the recruitment of signalling components to the cytoplasmic parts of cytokine receptors, remains elusive. Here we investigated possible mechanisms underlying the different potency of the STAT3-activating motifs of gp130 after IL-6 stimulation. Our data indicate that the extent of STAT3 activation by the different receptor motifs is not influenced by structural features such as contacts between the two gp130 chains. In addition, the proximity of the negatively regulating motif around tyrosine Y759 to the different STAT3-recruiting motifs does not seem to be responsible for their differential capacity to activate STAT3. However, the potency of a specific motif to activate STAT3 directly reflects the affinity for the binding of STAT3 to this motif.     

DNA polymerase X from Deinococcus radiodurans possesses a structure-modulated 3'-->5' exonuclease activity involved in radioresistance

Recently a family X DNA polymerase (PolXDr) was identified in the radioresistant bacterium Deinococcus radiodurans. Knockout cells show a delay in double-strand break repair (DSBR) and an increased sensitivity to gamma-irradiation. Here we show that PolXDr possesses 3'-->5' exonuclease activity that stops cutting close to a loop. PolXDr consists of a DNA polymerase X domain (PolXc) and a Polymerase and Histidinol Phosphatase (PHP) domain. Deletion of the PHP domain abolishes only the structure-modulated but not the canonical 3'-->5' exonuclease activity. Thus, the exonuclease resides in the PolXc domain, but the structure-specificity requires additionally the PHP domain. Mutation of two conserved glycines in the PolXc domain leads to a specific loss of the structure-modulated exonuclease activity but not the exonuclease activity in general. The PHP domain itself does not show any activity. PolXDr is the first family X DNA polymerase that harbours an exonuclease activity. The wild-type protein, the glycine mutant and the two domains were expressed separately in DeltapolXDr cells. The wild-type protein could restore the radiation resistance, whereas intriguingly the mutant proteins showed a significant negative effect on survival of gamma-irradiated cells. Taken together our in vivo results suggest that both PolXDr domains play important roles in DSBR in D. radiodurans.     

In vitro assessment of a motion-based optimization method for locating the talocrural and subtalar joint axes

The locations of the joint axes of the ankle complex vary considerably between subjects, yet no noninvasive method with demonstrated accuracy exists for locating these axes. The moments of muscle and ground reaction forces about the joint axes are dependent on axis locations, making knowledge of these locations critical to accurate musculoskeletal modeling of the foot and ankle. The accuracy of a computational optimization method that fits a two-revolute model to measured motion was assessed using computer-generated data, a two-revolute mechanical linkage, and three lower-leg cadaver specimens. Motions were applied to cadaver specimens under axial load while bone-mounted markers attached to the tibia, talus, and calcaneus were tracked using a video-based motion analysis system. Estimates of the talocrural and subtalar axis locations were computed from motions of the calcaneus relative to the tibia using the optimization method. These axes were compared to mean helical axes computed directly from tibia, talus, and calcaneus motions. The optimization method performed well when the motions were computer-generated or measured in the mechanical linkage, with angular differences between optimization and mean helical axes ranging from 1 deg to 5 deg. In the cadaver specimens, however, these differences exceeded 20 deg. Optimization methods that locate the anatomical joint axes of the ankle complex by fitting two revolute joints to measured tibia-calcaneus motions may be limited because of problems arising from non-revolute behavior.     

Cryopreservation of preantral ovarian follicles in situ from domestic cats (Felis catus) using different cryoprotective agents

The objective of this investigation was to verify the structural characteristics of preantral follicles (PAF) of cat ovarian tissue after cryopreservation in 1.5 M glycerol or ethylene glycol, using a slow-freezing procedure. Ovaries (n = 10) from domestic cats were divided into fragments. One fragment was immediately preserved for classical histology (fresh control), and additional fragments were immersed in minimum essential medium plus 10% bovine fetal serum (MEM+BFS), or MEM+BFS supplemented with 1.5 M glycerol or ethylene glycol. The samples were frozen and plunged into liquid nitrogen. After 1 wk, the samples were thawed. A total of 600 PAF were evaluated. In the fresh control, there were 71.3% normal PAF. After thawing, the rates of normal PAF were 26.0, 39.3 and 58.0% for samples without cryoprotectant or with glycerol or ethylene glycol, respectively. We concluded that ethylene glycol was useful for the cryopreservation of feline PAF in situ.