Inheritance and expression of the cry1Ab gene in Bt ( Bacillus thuringiensis) transgenic rice

The inheritance and expression patterns of the cry1Ab gene were studied in the progenies derived from different Bt (Bacillus thuringiensis) transgenic japonica rice lines under field conditions. Both Mendelian and distorted segregation ratios were observed in some selfed and crossed F₂ populations. Crosses between japonica intra-subspecies had no significant effect on the segregation ratios of the cry1Ab gene, but crossing between japonica and indica inter-subspecies led to distorted segregation of the cry1Ab gene in the F₂ population. Field-release experiments indicated that the cry1Ab gene was stably transmitted in an intact manner via successive sexual generations, and the concentration of the Cry1Ab protein was kept quantitatively stable up to the R₆ generation. The cry1Ab gene, driven by the maize ubiquitin promoter, displayed certain kinds of spatial and temporal expression patterns under field conditions. The content of the Cry1Ab protein varied in different tissues of the main stems, the primary tillers and the secondary tillers. Higher levels of the Cry1Ab protein were found in the stems, leaves and leaf sheaths than in the roots, while the lowest level was detected in grains at the maturation stage. The content of the Cry1Ab protein in the leaves peaked at the booting stage and was lowest at the heading stage. Furthermore, the Cry1Ab content of cry1Ab expression in different tissues of transgenic rice varied individually with temperature. Received: 17 April 2001 / Accepted: 7 May 2001     

Effects of active immunization against GnRH on serum LH,inhibin A,sexual development and growth rate in Chinese female pigs

Surgical castration of young female pigs is common practice in Chinese pig farming today. The purpose of the present study is to investigate anti-GnRH immunization as a practical alternative to surgical castration for female pigs. Thirty-six Chinese female crossbred pigs (Chinese Yanan x Yorkshire) were selected from 12 litters, three pigs from each litter, at the age of 10-13 weeks. One pig from each litter was immunized with 62.5 microg D-Lys6-GnRH-tandem-dimer peptide conjugated to ovalbumin in Specol adjuvant at Week 0 (0 week post-vaccination, wpv), and a booster vaccination was given 8 weeks later (8 wpv). Its intact and castrate littermates (surgically castrated at the time of weaning, i.e. at 6 weeks of age) were administered the vehicle and served as controls. Antibody titers, serum LH and inhibin A were determined at the day of first vaccination, every 4 weeks thereafter and at the day of slaughter (18 wpv). At slaughter, ovaries were inspected for the presence of follicles and corpora lutea, and ovarian and uterine weights were recorded. Ten of twelve immunized pigs responded well to the immunization (immunocastrated animals), while the remaining two pigs responded poorly (nonresponders). Antibody titres in immunocastrated animals steadily increased after immunization, became maximal at 12 wpv and remained high until slaughter. Serum LH levels were reduced (P < 0.05) in immunocastrated pigs as compared to intact controls and surgical castrates. Serum inhibin A levels decreased after vaccination, and equaled surgical castrate levels from 8 wpv until the end of the experiment. Ovarian and uterine weights (1.3 +/- 0.2 and 43.9 +/- 11.4 g, respectively; mean +/- S.E.M.) were significantly lower (P < 0.05) in immunocastrates than in intact controls (9.4 +/- 1.1 and 390.9 +/- 67.2 g, respectively). Antibody titers were significantly lower (P < 0.05) in nonresponders than in immunocastrated pigs from 12 wpv to slaughter. Ovarian and uterine weights were similar in nonresponders and in intact controls. Macroscopically, no follicular structures were found in ovaries of immunocastrated pigs, while large follicles or corpora lutea were observed in the ovaries of both nonresponders and intact controls. Although not significant, immunocastrates had a numerically higher average daily gain than surgical castrates and intact controls (0.74 +/- 0.04 versus 0.66 +/- 0.04 versus 0.66 +/- 0.03 kg per day, respectively; mean +/- S.E.M., P = 0.09). Results obtained in the present study demonstrate that anti-GnRH immunization can be an attractive alternative to surgical castration for Chinese crossbred female pigs. Our results also question the beneficial effect of surgical castration on growth as compared to intact controls.     

YIL113w encodes a functional dual-specificity protein phosphatase which specifically interacts with and inactivates the Slt2/Mpk1p MAP kinase in S. cerevisiae

We show here that the YIL113w gene of Saccharomyces cerevisiae encodes a functional protein phosphatase. Yil113p shows no activity in vitro towards either phosphorylated casein or myelin basic protein. However, Yil113p dephosphorylates activated extracellular signal-regulated kinase 2 MAP kinase indicating that it is a dual-specificity MAP kinase phosphatase. In support of this we find that Yil113p specifically interacts with the stress-activated Slt2/Mpk1p MAP kinase of S. cerevisiae. Furthermore, expression of Yil113p causes the dephosphorylation of Slt2/Mpk1p in vivo, while expression of an inactive mutant of Yil113p causes the accumulation of phosphorylated Slt2/Mpk1p. We conclude that the physiological target of YIL113p is Slt2/Mpk1p.     

Substitution of the autophosphorylation site Thr516 with a negatively charged residue confers constitutive activity to mouse 3-phosphoinositide-dependent protein kinase-1 in cells

3-Phosphoinositide-dependent protein kinase-1 (PDK-1)is a serine/threonine kinase that has been found to phosphorylate and activate several members of the AGC protein kinase family including protein kinase B (Akt), p70 S6 kinase, and protein kinase Czeta. However, the mechanism(s) by which PDK-1 is regulated remains unclear. Here we show that mouse PDK-1 (mPDK-1) undergoes autophosphorylation in vitro on both serine and threonine residues. In addition, we have identified Ser(399) and Thr(516) as the major mPDK-1 autophosphorylation sites in vitro. Furthermore, we have found that these two residues, as well as Ser(244) in the activation loop, are phosphorylated in cells and demonstrated that Ser(244) is a major in vivo phosphorylation site. Abolishment of phosphorylation at Ser(244), but not at Ser(399) or Thr(516), led to a significant decrease of mPDK-1 autophosphorylation and kinase activity in vitro, indicating that autophosphorylation at Ser(399) or Thr(516) is not essential for mPDK-1 autokinase activity. However, overexpression of mPDK-1(T516E), but not of mPDK-1(S244E) or mPDK-1(S399D), in Chinese hamster ovary and HEK293 cells was sufficient to induce Akt phosphorylation at Thr(308) to a level similar to that of insulin stimulation. Furthermore, this increase in phosphorylation was independent of the Pleckstrin homology domain of Akt. Taken together, our results suggest that mPDK-1 undergoes autophosphorylation at multiple sites and that this phosphorylation may be essential for PDK-1 to interact with and phosphorylate its downstream substrates in vivo.     

Detection of viral DNA and immune responses to the human herpesvirus 6 101-kilodalton virion protein in patients with multiple sclerosis and in controls

Human herpesvirus 6 (HHV-6), a latent lymphotropic and neurotropic virus, has been suspected as an etiologic agent in multiple sclerosis (MS). The study was undertaken to correlate virologic evidence for HHV-6 activity with the state of host immunity to HHV-6 in MS patients and control subjects. The study revealed that cell-free DNA of HHV-6 was detected more frequently in both serum and cerebrospinal fluid of MS patients than in those of control subjects. T cells recognizing the recombinant 101-kDa protein (101K) corresponding to the major immunoreactive region unique to HHV-6 occurred at significantly lower precursor frequency in MS patients than in control subjects. The resulting HHV-6-specific T-cell lines obtained from MS patients exhibited skewed cytokine profiles characterized by the inability to produce interleukin-4 (IL-4) and IL-10. The decreased T-cell responses to HHV-6 and the altered cytokine profile were consistent with significantly declined serum immunoglobulin G (IgG) titers for HHV-6 of MS patients compared to those of control subjects. In contrast, elevated serum IgM titers for HHV-6 were detected in the majority of MS patients, which may reflect frequent exposure of B cells to HHV-6. The findings suggest that the decreased immune responses to HHV-6 may be responsible for ineffective clearance of HHV-6 in MS patients.     

Cloning and characterization of a testis and brain-specific isoform of mouse 3'-phosphoinositide-dependent protein kinase-1, mPDK-1 beta

3'-Phosphoinositide-dependent protein kinase-1 (PDK-1) phosphorylates and activates members of the protein kinase AGC family and plays a key role in receptor tyrosine kinase signaling. Here we report the cloning and characterization of a splice variant of mouse PDK-1, mPDK-1 beta. The cDNA encoding mPDK-1 beta contains two alternative start codons and translation from these start codons generates proteins that are, respectively, 27 or 51 amino acid residues shorter at the amino-terminus than the previously identified PDK-1 isolated from mouse liver (now renamed mPDK-1 alpha) [J. Biol. Chem. 274 (1999) 8117]. Analysis of mouse tissues shows that mPDK-1 beta is highly expressed in the testis and various functional regions of the brain. Expression of this isoform is increased in the brain of aged mice. Both mPDK-1 alpha and mPDK-1 beta are autophosphorylated at both serine and threonine residues in vitro and showed similar levels of tyrosine phosphorylation when co-expressed with either constitutively active Src or Fyn tyrosine kinases in cells. However, the mPDK-1 isoforms showed significant differences in their response to pervanadate- or insulin plus vanadate-stimulated tyrosine phosphorylation. Taken together, our findings suggest that the two PDK-1 isoforms may be differentially regulated in cells. The specific expression of mPDK-1 beta in mouse testis and brains of aged mice also suggests potential involvement of this kinase in regulating animal spermatogenesis and aging.     

The relationship between oxidative/nitrative stress and pathological inclusions in Alzheimer's and Parkinson's diseases

Alzheimer's (AD) and Parkinson's diseases (PD) are late-onset neurodegenerative diseases that have tremendous impact on the lives of affected individuals, their families, and society as a whole. Remarkable efforts are being made to elucidate the dominant factors that result in the pathogenesis of these disorders. Extensive postmortem studies suggest that oxidative/nitrative stresses are prominent features of these diseases, and several animal models support this notion. Furthermore, it is likely that protein modifications resulting from oxidative/nitrative damage contribute to the formation of intracytoplasmic inclusions characteristic of each disease. The frequent presentation of both AD and PD in individuals and the co-occurrence of inclusions characteristic of AD and PD in several other neurodegenerative diseases suggests the involvement of a common underlying aberrant process. It can be surmised that oxidative/nitrative stress, which is cooperatively influenced by environmental factors, genetic predisposition, and senescence, may be a link between these disorders.     

Use of online-dual-column extraction in conjunction with chiral liquid chromatography tandem mass spectrometry for determination of terbutaline enantiomers in human plasma

An online sample extraction chiral bioanalytical method was developed and validated for the quantification of terbutaline, a beta2-selective adrenoceptor agonist, spiked into human plasma by using two extraction columns and a chiral stationary phase (CSP) in conjunction with liquid chromatography tandem mass spectrometry (LC-MS/MS). In this method, two Oasis HLB extraction columns were used in parallel for plasma sample purification and a Chirobiotic T CSP was used for enantiomeric separation. Atmospheric pressure chemical ionization MS/MS was employed in multiple reaction monitoring mode for the detection and quantification. Subsequent to the addition of an internal standard solution, the plasma samples were directly injected onto the system for extraction and analysis. This method allowed the use of one of the extraction columns for purification while the other was being equilibrated. Hence, the time required for reconditioning the extraction columns did not contribute to the total analysis time per sample, which resulted in a shorter run time and higher throughput. A lower limit of quantification of 1.0 ng/mL was achieved using only 50 microliter of human plasma. The method was validated with a dynamic range of 1.0-200 ng/mL. The intra- and interday precision was no more than 11% CV and the assay accuracy was between 94-106%.