The effect of cross-links on the conformational dynamics of duplex DNA.

The base pair lifetimes and apparent dissociation constants of a 21 base DNA hairpin and an analog possessing a disulfide cross-link bridging the 3'- and 5'-terminal bases were determined by measuring imino proton exchange rates as a function of exchange catalyst concentration and temperature. A comparison of the lifetimes and apparent dissociation constants for corresponding base pairs of the two hairpins indicates that the cross-link neither increases the number of base pairs involved in fraying nor alters the lifetime, dissociation constant, or the opened structure from which exchange occurs for the base pairs that are not frayed. The cross-link does, however, stabilize the frayed penultimate base pair of the stem duplex. Significantly, it appears that the disulfide cross-link is more effective at preventing fraying of the penultimate base pair than is the 5 base hairpin loop. Because this disulfide cross-link can be incorporated site specifically, and does not adversely affect static or dynamic properties of DNA, it should prove very useful in studies of nucleic acid structure and function.     

Genetic manipulation of plant growth-promoting bacteria to enhance biocontrol of phytopathogens

Plant growth-promoting bacteria (PGPB) control the damage to plants from phytopathogens by a number of different mechanisms including: outcompeting the phytopathogen, physical displacement of the phytopathogen, secretion of siderophores to prevent pathogens in the immediate vicinity from proliferating, synthesis of antibiotics, synthesis of a variety of small molecules that can inhibit phytopathogen growth, production of enzymes that inhibit the phytopathogen and stimulation of the systemic resistance of the plant. Biocontrol PGPB may be improved by genetically engineering them to overexpress one or more of these traits so that strains with several different anti-phytopathogen traits which can act synergistically are created. In engineering these strains it is essential to ensure that the normal functioning of the bacterium is not impaired, i.e., that there is no problem with metabolic load.     

Lateral hypothalamic lesions and striatal dopamine levels

Rats with bilateral lateral hypothalamic lesions were killed on the third day after surgery and their brains were assayed for tel-diencephalic norepinephrine and striatal dopamine. Lesion-induced weight loss was highly correlated with depletion of striatal dopamine but not with tel-diencephalic norepinephrine. In rats with severe dopamine depletions, the degree of weight loss was related more to the striatum with the highest remaining level of dopamine suggesting that a critical level of dopamine in one striatum may be essential for lateral hypothalamic recovery.     

Integrated microfluidic approach for quantitative high-throughput measurements of transcription factor binding affinities

Protein binding to DNA is a fundamental process in gene regulation. Methodologies such as ChIP-Seq and mapping of DNase I hypersensitive sites provide global information on this regulation in vivo. In vitro methodologies provide valuable complementary information on protein–DNA specificities. However, current methods still do not measure absolute binding affinities. There is a real need for large-scale quantitative protein–DNA affinity measurements. We developed QPID, a microfluidic application for measuring protein–DNA affinities. A single run is equivalent to 4096 gel-shift experiments. Using QPID, we characterized the different affinities of ATF1, c-Jun, c-Fos and AP-1 to the CRE consensus motif and CRE half-site in two different genomic sequences on a single device. We discovered that binding of ATF1, but not of AP-1, to the CRE half-site is highly affected by its genomic context. This effect was highly correlated with ATF1 ChIP-seq and PBM experiments. Next, we characterized the affinities of ATF1 and ATF3 to 128 genomic CRE and CRE half-site sequences. Our affinity measurements explained that in vivo binding differences between ATF1 and ATF3 to CRE and CRE half-sites are partially mediated by differences in the minor groove width. We believe that QPID would become a central tool for quantitative characterization of biophysical aspects affecting protein–DNA binding.     

Expression of polypeptide GalNAc-transferases in stratified epithelia and squamous cell carcinomas: immunohistological evaluation using monoclonal antibodies to three members of the GalNAc-transferase family

Mucin-type O-glycosylation is initiated by a large family of UDP- GalNAc: polypeptide N -acetyl-galactosaminyltransferases (GalNAc- transferases). Individual GalNAc-transferases appear to have different functions and Northern analysis indicates that they are differently expressed in different organs. This suggests that O-glycosylation may vary with the repertoire of GalNAc-transferases expressed in a given cell. In order to study the repertoire of GalNAc-transferases in situ in tissues and changes in tumors, we have generated a panel of monoclonal antibodies (MAbs) with well defined specificity for human GalNAc-T1, -T2, and -T3. Application of this panel of novel antibodies revealed that GalNAc- transferases are differentially expressed in different cell lines, in spermatozoa, and in oral mucosa and carcinomas. For example, GalNAc-T1 and -T2 but not -T3 were highly expressed in WI38 cells, and GalNAc-T3 but not GalNAc-T1 or -T2 was expressed in spermatozoa. The expression patterns in normal oral mucosa were found to vary with cell differentiation, and for GalNAc-T2 and -T3 this was reflected in oral squamous cell carcinomas. The expression pattern of GalNAc-T1 was on the other hand changed in tumors to either total loss or expression in cytological poorly differentiated tumor cells, where the normal undifferentiated cells lacked expression. These results demonstrate that the repertoire of GalNAc-transferases is different in different cell types and vary with cellular differentiation, and malignant transformation. The implication of this is not yet fully understood, but it suggests that specific changes in sites of O-glycosylation of proteins may occur as a result of changes in the repertoire of GalNAc-transferases.      

Mechanisms of action of plant growth promoting bacteria

The idea of eliminating the use of fertilizers which are sometimes environmentally unsafe is slowly becoming a reality because of the emergence of microorganisms that can serve the same purpose or even do better. Depletion of soil nutrients through leaching into the waterways and causing contamination are some of the negative effects of these chemical fertilizers that prompted the need for suitable alternatives. This brings us to the idea of using microbes that can be developed for use as biological fertilizers (biofertilizers). They are environmentally friendly as they are natural living organisms. They increase crop yield and production and, in addition, in developing countries, they are less expensive compared to chemical fertilizers. These biofertilizers are typically called plant growth-promoting bacteria (PGPB). In addition to PGPB, some fungi have also been demonstrated to promote plant growth. Apart from improving crop yields, some biofertilizers also control various plant pathogens. The objective of worldwide sustainable agriculture is much more likely to be achieved through the widespread use of biofertilizers rather than chemically synthesized fertilizers. However, to realize this objective it is essential that the many mechanisms employed by PGPB first be thoroughly understood thereby allowing workers to fully harness the potentials of these microbes. The present state of our knowledge regarding the fundamental mechanisms employed by PGPB is discussed herein.