Abscisic acid biosynthesis and catabolism and their regulation roles in fruit ripening

Abscisic acid (ABA) plays a series of significant physiology roles in higher plants including but not limited to promote bud and seed dormancy, accelerate foliage fall, induce stomatal closure, inhibit growth and enhance resistance. Recently, it has been revealed that ABA also has an important regulator role in the growth, development and ripening of fruit. In higher plants ABA is produced from an indirect pathway from the cleavage products of carotenoids. The accumulation of endogenous ABA levels in plants is a dynamic balance controlled by the processes of biosynthesis and catabolism, through the regulation of key ABA biosynthetic gene and enzyme activities. It has been hypothesized that ABA levels could be part of the signal that trigger fruit ripening, and that ABA may play an important role in the regulation of ripening and senescence of both non-climacteric and climacteric fruit. The expensive costs of natural ABA and labile active ABA for its chemical synthesis limit its application in scientific research and agricultural production. These findings that ABA has various of important roles in the regulation of growth and development, quality formation, coloring and softening, ripening and senescence of fruit, are providing opportunities and challenges for Horticultural Science. This is to elucidate the specific mechanism of response and biosynthesis, signal transduction, and receptor recognition of ABA in fruit, employing comprehensive research methods, such as molecular biology, plant physiology and molecular genetics. Further and more in-depth research about ABA has a great, realistic significance for knowing the mechanisms behind the process of fruit ripening.     

18F-labeled sufentanil for PET-imaging of mu-opioid receptors

The synthesis of an (18)F-labeled sufentanil analogue with apparent high mu-opioid receptor selectivity is reported. Intravenous injection of N-[4-(methoxymethyl)-1-[2-(2-thienyl)ethyl]-4-piperidinyl]-N-phenyl-2-(+/-)-[(18)F]fluoropropan-amide in mice resulted in high brain uptake and a regional brain activity distribution corresponding to the mu-opioid receptor expression pattern. The developed ligand is a promising tracer for extended protocols in mu-opioid receptor mapping and quantitation with positron emission tomography.     

Long-term cytomegalovirus infection leads to significant changes in the composition of the CD8+ T-cell repertoire, which may be the basis for an imbalance in the cytokine production profile in elderly persons

In spite of the present belief that latent cytomegalovirus (CMV) infection drives CD8+ T-cell differentiation and induces premature immune senescence, no systematic studies have so far been performed to compare phenotypical and functional changes in the CD8+ T-cell repertoire in CMV-infected and noninfected persons of different age groups. In the present study, number, cytokine production, and growth potential of naive (CD45RA+ CD28+), memory (CD45RA- CD28+), and effector (CD45RA+ CD28- or CD45RA- CD28-) CD8+ T cells were analyzed in young, middle-aged, and elderly clinically healthy persons with a positive or negative CMV antibody serology. Numbers and functional properties of CMVpp65(495-503)-specific CD8+ T cells were also studied. We demonstrate that aging as well as CMV infection lead to a decrease in the size of the naive CD8+ T-cell pool but to an increase in the number of CD8+ effector T cells, which produce gamma interferon but lack substantial growth potential. The size of the CD8+ memory T-cell population, which grows well and produces interleukin-2 (IL-2) and IL-4, also increases with aging, but this increase is missing in CMV carriers. Life-long latent CMV infection seems thus to diminish the size of the naive and the early memory T-cell pool and to drive a Th1 polarization within the immune system. This can lead to a reduced diversity of CD8 responses and to chronic inflammatory processes which may be the basis of severe health problems in elderly persons.     

A mechanical unfolding intermediate in an actin-crosslinking protein

Many F-actin crosslinking proteins consist of two actin-binding domains separated by a rod domain that can vary considerably in length and structure. In this study, we used single-molecule force spectroscopy to investigate the mechanics of the immunoglobulin (Ig) rod domains of filamin from Dictyostelium discoideum (ddFLN). We find that one of the six Ig domains unfolds at lower forces than do those of all other domains and exhibits a stable unfolding intermediate on its mechanical unfolding pathway. Amino acid inserts into various loops of this domain lead to contour length changes in the single-molecule unfolding pattern. These changes allowed us to map the stable core of approximately 60 amino acids that constitutes the unfolding intermediate. Fast refolding in combination with low unfolding forces suggest a potential in vivo role for this domain as a mechanically extensible element within the ddFLN rod.     

Steroid analysis and xenosteroid potentials in two small streams in southwest Germany

The presence of estrogenic substances in thewater of the small streams Körsch (Kö)and Krähenbach (Kr), Southwest Germany, wasdetermined by chemical and biological analysis.Because a large proportion of the Kö waternear its mouth consists of sewage treatmentplant (STPs) effluents, the impact of STPs onlevels of estrogens in surface water is anenvironmental issue of concern. In July 1996,water samples were taken from Kr and Kö(four sites) and tested in the E-Screen assaywith human MCF-7 breast cancer cells. AllKö samples induced estrogen-dependent cellproliferation resulting in 17-estradiolequivalent concentrations (EEQ) between 3.3 and9.7 ng/L while the Kr water showed no effect.In 1998/99 eight samples from Kö (near itsmouth) and nine samples from Kr were collectedand tested in the E-Screen after solid phaseextraction. Some estrogenicity was detectablein three Kr samples but Kö samples had amedian EEQ of 3.1 ng/L (range: 1.2–42 ng/L).GC/MS analysis revealed differences in thelevels of 17-estradiol and estronebetween the two streams. 17-estradiolwas detectable in five Kö samples only (0.7–1.8 ng/L). Estrone was found in the Köfrom 2.5 to 38 ng/L (median: 7.6 ng/L) and inthe Kr between 0.8 and 22 ng/L (median: 1.7 ng/L). Analysis for nine phenolic xenoestrogensrevealed rather low levels for five compoundswhich occurred more frequently and in higherconcentrations in the Kö. After asemi-field exposure of adult male rainbow troutfor 4 weeks in Kö water, plasmavitellogenin levels were significantly highercompared to those levels detected in the sameanimals before exposure.     

Multimodality Multiparametric Imaging of Early Tumor Response to a Novel Antiangiogenic Therapy Based on Anticalins

Anticalins are a novel class of targeted protein therapeutics. The PEGylated Anticalin Angiocal (PRS-050-PEG40) is directed against VEGF-A. The purpose of our study was to compare the performance of diffusion weighted imaging (DWI), dynamic contrast enhanced magnetic resonance imaging (DCE)-MRI and positron emission tomography with the tracer [¹⁸F]fluorodeoxyglucose (FDG-PET) for monitoring early response to antiangiogenic therapy with PRS-050-PEG40. 31 mice were implanted subcutaneously with A673 rhabdomyosarcoma xenografts and underwent DWI, DCE-MRI and FDG-PET before and 2 days after i.p. injection of PRS-050-PEG40 (n = 13), Avastin (n = 6) or PBS (n = 12). Tumor size was measured manually with a caliper. Imaging results were correlated with histopathology. In the results, the tumor size was not significantly different in the treatment groups when compared to the control group on day 2 after therapy onset (P = 0.09). In contrast the imaging modalities DWI, DCE-MRI and FDG-PET showed significant differences between the therapeutic compared to the control group as early as 2 days after therapy onset (P<0.001). There was a strong correlation of the early changes in DWI, DCE-MRI and FDG-PET at day 2 after therapy onset and the change in tumor size at the end of therapy (r = −0.58, 0.71 and 0.67 respectively). The imaging results were confirmed by histopathology, showing early necrosis and necroptosis in the tumors. Thus multimodality multiparametric imaging was able to predict therapeutic success of PRS-050-PEG40 and Avastin as early as 2 days after onset of therapy and thus promising for monitoring early response of antiangiogenic therapy.     

Spatial and temporal variation in malaria transmission in a low endemicity area in northern Tanzania

Background

Current detection or screening for malaria infection necessitates drawing blood by fingerprick or venipuncture, which poses risks and limitations for repeated measurement. This study presents PCR detection of Plasmodium falciparum in human urine and saliva samples, and illustrates this potential application in genotyping malaria infections.

Methods

Urine and saliva were obtained from 47 thick film positive and 4 negative individuals one day after collection of blood slides and filter paper blood spots. P. falciparum DNA was extracted from blood, urine and saliva, in separate groups, using the Chelex method or Qiagen DNEasy^® kit (urine and saliva only). Blood, urine and saliva extracts were subjected to PCR in separate batches. Amplicons from the various sample types were examined for MSP2 polymorphisms and restriction fragment patterns on DHFR amino acid codon 59.

Results and discussion

Malaria infections exhibited primarily low-grade parasite densities, with a geometric mean of 775 asexual parasites/μl. Regularly matching polymorphic MSP2 genotypes were found between the corresponding urine, saliva and peripheral blood amplicons of each individual, with different inter-individual polymorphic genotypes. Amplicon yields were significantly dependent on DNA extraction method, parasite density and primer set (p < 0.001). A Qiagen^® kit extraction had more than 2× higher amplicon yield than the Chelex method, for both urine and saliva. Amplicon yields were 1.6 fold higher from saliva than urine. For each unit increase in log parasite density, the probability of amplicon enhanced 1.8 fold. Highest amplicon yields were obtained from the primer set with the shortest PCR product.

Conclusion

P. falciparum infection is detectable by PCR on human urine and saliva samples. Subject to further refinement of extraction technique and amplicon yields, large-scale malaria parasite screening and epidemiological surveys could be possible without the need to collect blood and use of needles or sharps.     

Subcellular Localization and Characterization of the ParAB System from Corynebacterium glutamicum

Faithful segregation of chromosomes and plasmids is a vital prerequisite to produce viable and genetically identical progeny. Bacteria use a specialized segregation system composed of the partitioning proteins ParA and ParB to segregate certain plasmids. Strikingly, homologues of ParA and ParB are found to be encoded in many chromosomes. Although mutations in the chromosomal Par system have effects on segregation efficiency, the exact mechanism by which the chromosomes are segregated into the daughter cells is not fully understood. We describe the polar localization of the ParB origin nucleoprotein complex in the actinomycete Corynebacterium glutamicum. ParB and the origin of replication were found to be stably localized to the cell poles. After replication, the origins move toward the opposite pole. Purified ParB was able to bind to the parS consensus sequence in vitro. C. glutamicum possesses two ParA-like partitioning ATPase proteins. Both proteins interact with ParB but show a slightly different subcellular localization and phenotype. While ParA might be part of a conventional partitioning system, PldP seems to play a role in division site selection.Bacterial cell division is a temporally and spatially tightly regulated process (1, 13, 16, 36, 37). Spatial regulation is achieved by division site selection and prevents fatal division across the nucleoids and aberrant division close to the cell poles (3, 40). Temporal control ensures that division does not precede chromosome replication and segregation. Replicated chromosomes are rapidly segregated into the daughter cells. However, the machinery that performs this active segregation is not fully elucidated. In contrast, plasmid segregation is somewhat better understood. Plasmids such as pB171 (8) encode a machinery composed of a tripartite system. Centromere-like DNA sequences, named parS sites, are composed of short inverted repeats. Centromere-binding proteins (ParB) are recruited to the parS sites, forming nucleoprotein complexes. Finally, a partitioning ATPase is recruited to the ParB-parS complex. The hydrolytic activity of ParA oligomers is believed to drive the active segregation process. Strikingly, many bacterial chromosomes encode orthologs of the plasmid partitioning genes parA and parB. A comparatively well-examined chromosomal partitioning system is that of Bacillus subtilis. B. subtilis encodes a ParA ATPase (called Soj) and a ParB protein (called Spo0J). B. subtilis contains eight parS sites that cluster around the oriC region and bind Spo0J. Subsequently Spo0J spreads across the DNA, thereby forming a huge nucleoprotein complex that could serve as a platform for anchoring the segregation machinery. The ParA protein Soj is a DNA-binding protein that dissociates from DNA upon ATP hydrolysis. A direct interaction of Soj and Spo0J has been described (35). Interestingly, analysis of knockout mutations revealed that only the loss of the ParB protein Spo0J increases the amount of anucleate cells slightly, while the loss of Soj has no significant effect on chromosome segregation (17, 18). However, knockout mutations in either parA or parB result only in subtle effects on chromosome segregation. Thus, although the two proteins might act together they have certainly multiple roles during chromosome segregation and cell division. Recently, it was shown that Spo0J (ParB) helps to recruit SMC proteins (for structural maintenance of chromosomes) to the oriC region, thereby ensuring correct chromosome organization, which seems essential for proper segregation (15, 39). The B. subtilis ParA homologue Soj was shown to play an role in the initiation of DNA replication by interacting with DnaA (32). Hence, the ParAB system is a central component connecting replication and segregation. Interestingly, Par proteins have been implicated with different developmental processes in other bacteria. In Caulobacter crescentus ParAB are involved in cell cycle progression and cell division. A ParA-like protein, MipZ, was shown to interact with ParB and directly inhibit FtsZ polymerization (42). Thus, chromosome segregation and cell division are directly coupled. Consequently, null mutations in ParA and ParB are lethal in C. crescentus. In Vibrio cholerae it was shown that ParA and ParB encoded on the large chromosome contribute to active chromosome segregation and anchor the oriC region of the chromosomes to the cell poles (10).Although these diverse properties of the Par system have been studied in some detail in the classical model organisms, the situation in other bacteria remains unknown. Corynebacteria are high GC Gram-positive bacteria and, depending on the growth medium, rod-shaped or club-shaped. A remarkable feature of corynebacteria and their close relatives is a special cell wall that has, in addition to the common peptidoglycan, an arabino-galactan and a mycolic acid layer. Notorious pathogens such as Mycobacterium tuberculosis, Mycobacterium leprae, and Corynebacterium diphtheriae are members of this family, and hence an understanding of fundamental cell biological mechanisms might reveal insights how to combat these organisms. We now report the subcellular localization of the chromosome partitioning system and the oriC in the actinomycete Corynebacterium glutamicum. We show localization and phenotypic consequences of the canonical ParAB proteins. Furthermore, we identified a ParA-like division protein (PldP) that plays a role in division site selection.