Beta 2-adrenergic receptor stimulated,G protein-coupled receptor kinase 2 mediated,phosphorylation of ribosomal protein P2

G protein-coupled receptor kinases are well characterized for their ability to phosphorylate and desensitize G protein-coupled receptors (GPCRs). In addition to phosphorylating the beta2-adrenergic receptor (beta2AR) and other receptors, G protein-coupled receptor kinase 2 (GRK2) can also phosphorylate tubulin, a nonreceptor substrate. To identify novel nonreceptor substrates of GRK2, we used two-dimensional gel electrophoresis to find cellular proteins that were phosphorylated upon agonist-stimulation of the beta2AR in a GRK2-dependent manner. The ribosomal protein P2 was identified as an endogenous HEK-293 cell protein whose phosphorylation was increased following agonist stimulation of the beta2AR under conditions where tyrosine kinases, PKC and PKA, were inhibited. P2 along with its other family members, P0 and P1, constitutes a part of the elongation factor-binding site connected to the GTPase center in the 60S ribosomal subunit. Phosphorylation of P2 is known to regulate protein synthesis in vitro. Further, P2 and P1 are shown to be good in vitro substrates for GRK2 with K(M) values approximating 1 microM. The phosphorylation sites in GRK2-phosphorylated P2 are identified (S102 and S105) and are identical to the sites known to regulate P2 activity. When the 60S subunit deprived of endogenous P1 and P2 is reconstituted with GRK2-phosphorylated P2 and unphosphorylated P1, translational activity is greatly enhanced. These findings suggest a previously unrecognized relationship between GPCR activation and the translational control of gene expression mediated by GRK2 activation and P2 phosphorylation and represent a potential novel signaling pathway responsible for P2 phosphorylation in mammals.     

Medroxyprogesterone priming and response to the ram effect in Corriedale ewes during the nonbreeding season

The "ram effect" (RE) is an inexpensive technique that allows farmers to obtain out-of-season lambs. Five hundred and ninety-six Corriedale ewes were used in three experiments to determine the effectiveness of different medroxyprogesterone (MAP) treatments associated with the ram effect during the nonbreeding season. The aim of the first experiment was to evaluate the effectiveness of short-term (6-day) MAP priming. We obtained similar results in estrus incidence and fertility after using MAP sponges for 6, 9, and 13 days. In the second experiment, we compared the effect of sponges containing 20, 40, or 60 mg of MAP used in 6-day priming. Estrous behavior and fertility were not affected by dosage. In the third experiment, 2.5mg of MAP was administered in single treatments 0, 1, 3, or 5 days before the introduction of the rams. Medroxyprogesterone administration 1, 3, or 5 days before the introduction of the rams concentrated estrus in ewes 17 to 20 days later.     

Additive polymorphisms and reticulation in an ITS phylogeny of thrifts (Armeria,Plumbaginaceae)

A parsimony analysis of 133 sequences of the nuclear ribosomal DNA ITS1+5.8S+ITS2 region from 71 taxa in Armeria was carried out. The presence of additive polymorphic sites (APS; occurring in 14 accessions) fits the reticulate scenario proposed in previous work for explaining the ITS pattern of variation on a much smaller scale and is based mainly on the geographical structure of the data, irrespective of taxonomic boundaries. Despite the relatively low bootstrap values and large polytomies, part of which are likely due to disruptive effects of reticulation and concerted evolution in these multicopy sequences, the ITS analysis has phylogenetic and biogeographic implications. APS detected in this study are consistent with hypothesized hybridization events, although biased concerted evolution, previously documented in the genus, needs to be invoked for specific cases and may be responsible for a possible "sink" effect in terminals from a large clade. The causes for sequences of the same species appearing in different clades (here termed transclade) are discussed.     

Death inducer-obliterator 1 triggers apoptosis after nuclear translocation and caspase upregulation

Death inducer-obliterator 1 (DIO-1) is a gene that is upregulated early in apoptosis. Here we report that in healthy cells, the DIO-1 gene product was located in the cytoplasm, where it formed oligomers. After interleukin-3 starvation or c-Myc-induced apoptosis in serum-free conditions, DIO-1 translocated to the nucleus, where it upregulated caspase levels and activity. A nuclear localization signal deletion mutant (DIO-1deltaNLS) was unable to translocate to the nuclear compartment in the absence of interleukin-3 and failed to upregulate procaspase levels or trigger cell death. In addition, cells stably expressing DIO-1deltaNLS were protected from apoptosis induced by interleukin-3 withdrawal. These results indicate that DIO-1 has a relevant role in regulating the early stages of cell death.     

A reversible abnormal form of myelin: an X-ray scattering study of human sural and rat sciatic nerves

A sural nerve dissected from a recently dead patient displayed an unusual X-ray diffraction pattern, suggesting that in situ and at the time of the patient's death the myelin sheaths were in a swollen state. Diffraction patterns of the swollen type were also recorded from: (1) a sural nerve from the corpse of a neurologically healthy person after soaking the nerve with Ringer solution at pH 5.5; (2) sciatic nerves dissected from rat cadavers at increasing time after death. In all the cases the swollen patterns reversed to the native type upon superfusion with Ringer solution at pH 7.3. The postmortem effect is to decrease the pH of the fluids surrounding the nerves in the cadavers. Our experiments show that the early postmortem processes have the effect of acidifying PNS nerves and that as a consequence of acidification the myelin sheaths swell.     

A muscle activation model of variable stimulation frequency response and stimulation history, based on positive feedback in calcium dynamics

 Muscle fiber response to a train of variable-frequency pulses includes the potentiation and catch-like effect. For better understanding of these phenomena, we built an activation model with emphasis on the calcium liberation from and re-sequestration into the sarcoplasmic reticulum, including calcium-induced calcium release. The model had two stable equilibrium points in the calcium concentration. Changes from the low to the high equilibrium point could be produced by high-frequency trains of pulses and would account for the potentiation. The model also showed a catch-like effect, as a long-lasting increment of muscle force after the application of a single extra pulse. The increase in force appeared in resting muscle, disappeared when the muscle was potentiated, and reappeared briefly if the stimulation was continued for long periods. Received: 31 January 2000 / Accepted in revised form: 2 August 2000     

Screening and characterization of Bacillus thuringiensis isolates from Brazil for the presence of coleoptera-specific cry genes

Forty-three Bacillus thuringiensis isolates from Brazil and 3 from Argentina were screened, using the polymerase chain reaction (PCR), for various coleoptera-specific cry genes. Seven isolates produced specific and/or nonspecific DNA fragments in a PCR reaction with primers specific for two coleopteran cry genes and 4 of these produced DNA fragments with primers specific for 7 known coleopteran cry genes. These isolates showed, by electron microscopy, the presence of spherical crystals. They also showed proteins of around 70 kDa which were immunologically similar to the Cry3Aa protein from B. thuringiensis subsp. tenebrionis. The 3 isolates from Argentina were toxic to T. molitor, and although no isolate from Brazil showed toxicity, they might show toxicity to another insect species.     

Adenosine deaminase prefers a distinct sugar ring conformation for binding and catalysis: kinetic and structural studies

Several recent X-ray crystal structures of adenosine deaminase (ADA) in complex with various adenosine surrogates have illustrated the preferred mode of substrate binding for this enzyme. To define more specific structural details of substrate preferences for binding and catalysis, we have studied the ADA binding efficiencies and deamination kinetics of several synthetic adenosine analogues in which the furanosyl ring is biased toward a particular conformation. NMR solution studies and pseudorotational analyses were used to ascertain the preferred furanose ring puckers (P, nu(MAX)) and rotamer distributions (chi and gamma) of the nucleoside analogues. It was shown that derivatives which are biased toward a "Northern" (3'-endo, N) sugar ring pucker were deaminated up to 65-fold faster and bound more tightly to the enzyme than those that preferred a "Southern" (2'-endo, S) conformation. This behavior, however, could be modulated by other structural factors. Similarly, purine riboside inhibitors of ADA that prefer the N hemisphere were more potent inhibitors than S analogues. These binding propensities were corroborated by detailed molecular modeling studies. Docking of both N- and S-type analogues into the ADA crystal structure coordinates showed that N-type substrates formed a stable complex with ADA, whereas for S-type substrates, it was necessary for the sugar pucker to adjust to a 3'-endo (N-type) conformation to remain in the ADA substrate binding site. These data outline the intricate structural details for optimum binding in the catalytic cleft of ADA.     

Potassium and Sodium Fluxes in the PhytopathogenicFungus Fusarium oxysporum var. lini

Rubidium uptake in potassium-starved cells followed biphasic kinetics in the micromolar and millimolar range and was independent of the temperature. In contrast, Rb⁺ uptake in normal-K⁺ cells followed a monophasic kinetics in the millimolar range and increased at temperatures higher than 30°C. Differences in the K _m values and in the Arrhenius plots of Rb⁺ uptake suggest different uptake systems in K⁺-starved and in normal-K⁺ cells. In addition, the substantial inhibition of Rb⁺ uptake caused by carbonyl cyanide-m-chlorophenyl hydrazone indicates that these systems are strongly dependent on membrane voltage. Lithium (sodium) tolerance, influx, and efflux were separately studied. F. oxysporum was shown to be very tolerant to sodium, while lithium caused a specific toxic effect. Li⁺ uptake in K⁺-starved cells exhibits a monophasic kinetics with low affinity. Li⁺ efflux was not affected by external pH or addition of potassium to the medium, suggesting that a Na⁺/cation antiporter is not involved in this process. Received: 14 March 2000 / Accepted: 5 June 2000     

The HPr kinase from Bacillus subtilis is a homo-oligomeric enzyme which exhibits strong positive cooperativity for nucleotide and fructose 1,6-bisphosphate binding

Carbon catabolite repression allows bacteria to rapidly alter the expression of catabolic genes in response to the availability of metabolizable carbon sources. In Bacillus subtilis, this phenomenon is controlled by the HPr kinase (HprK) that catalyzes ATP-dependent phosphorylation of either HPr (histidine containing protein) or Crh (catabolite repression HPr) on residue Ser-46. We report here that B. subtilis HprK forms homo-oligomers constituted most likely of eight subunits. Related to this complex structure, the enzyme displays strong positive cooperativity for the binding of its allosteric activator, fructose 1,6-bisphosphate, as evidenced by either kinetics of its phosphorylation activity or the intrinsic fluorescence properties of its unique tryptophan residue, Trp-235. It is further shown that activation of HPr phosphorylation by fructose 1,6-bisphosphate essentially occurs at low ATP and enzyme concentrations. A positive cooperativity was also detected for the binding of natural nucleotides or their 2'(3')-N-methylanthraniloyl derivatives, in either phosphorylation or fluorescence experiments. Most interestingly, quenching of the HprK tryptophan fluorescence by using either iodide or acrylamide revealed a heterogeneity of tryptophan residues within the population of oligomers, suggesting that the enzyme exists in two different conformations. This result suggests a concerted-symmetry model for the catalytic mechanism of positive cooperativity displayed by HprK.