Escherichia coli integration host factor bends the DNA at the ends of IS1 and in an insertion hotspot with multiple IHF binding sites.

The integration host factor of Escherichia coli (IHF) is a small, histone-like protein which participates in the integration of bacteriophage lambda into the E. coli chromosome and in a number of regulatory processes. Our recent footprinting analysis has shown that IHF binds specifically to the ends of the transposable element IS1, as well as to several sites within a short segment of the plasmid pBR322. We have extended our studies of the binding of the IHF molecule to these sites in vitro using a gel retardation assay. We report here that IHF bends the DNA upon binding, as judged from the strong cyclic dependence of the protein-induced mobility shift on the position of the binding site. Using cloned, synthetic ends of IS1 as substrates, we have found that some mutations within the conserved bases of the IHF consensus binding sequence abolish binding, and that alterations of the flanking sequences can greatly reduce IHF binding. The presence of multiple IHF sites on a single DNA fragment increases binding very little, indicating that IHF does not bind cooperatively in this complex. We discuss the possibility that DNA bending is related to the role IHF plays in forming and stabilizing nucleoprotein complexes, and suggest that bending at the IHF sites may be important to its diverse effects in the cell.     

Optimizing restriction fragment fingerprinting methods for ordering large genomic libraries

We present a statistical analysis of the problem of ordering large genomic cloned libraries through overlap detection based on restriction fingerprinting. Such ordering projects involve a large investment of effort involving many repetitious experiments. Our primary purpose here is to provide methods of maximizing the efficiency of such efforts. To this end, we adopt a statistical approach that uses the likelihood ratio as a statistic to detect overlap. The main advantages of this approach are that (1) it allows the relatively straightforward incorporation of the observed statistical properties of the data; (2) it permits the efficiency of a particular experimental method for detecting overlap to be quantitatively defined so that alternative experimental designs may be compared and optimized; and (3) it yields a direct estimate of the probability that any two library members overlap. This estimate is a critical tool for the accurate, automatic assembly of overlapping sets of fragments into islands called "contigs." These contigs must subsequently be connected by other methods to provide an ordered set of overlapping fragments covering the entire genome.     

Replication of pSC101: effects of mutations in the E. coli DNA binding protein IHF

Summary We have shown that the plasmid pSC101 is unable to be maintained in strains of E. coli carrying deletions in the genes himA and hip which specify the pleitropic heterodimeric DNA binding protein, IHF. We show that this effect is not due to a modulation of the expression of the pSC101 RepA protein, required for replication of the plasmid. Inspection of the DNA sequence of the essential replication region of pSC101 reveals the presence of a site, located between the DnaA binding-site and that of RepA, which shows extensive homology with the consensus IHF binding site. The proximity of the sites suggests that these three proteins, IHF, DnaA, and RepA may interact in generating a specific DNA structure required for initiation of pSC101 replication.     

Microwave irradiation increases recovery of neuropeptides from brain tissues

The effect of focused high energy microwave treatment (MW) on brain concentrations and molecular forms of substance P, neurokinin A, neuropeptide Y, neurotensin, galanin and calcitonin gene-related peptide was investigated. Groups of rats were treated as follows: 1) MW, storage for 60 min at 22°C, 2) Decapitation, storage for 60 min at 22°C, 3) Decapitation, storage for 60 min at 22°C, MW treatment, 4) MW, decapitation, storage for 2 min at 22°C and 5) Decapitation, storage for 2 min at 22°C. Peptide concentrations were in all instances highest in the MW sacrificed groups. MW increased the concentration of intact peptides by rapid inhibition of peptidase activity and increase in peptide solubility/extractability.     

Acidification of small mountain lakes in the High Tatra Mountains,Poland

A hydrochemical investigation was carried out on eight small High Tatra mountain lakes (Poland). When comparing recent data with those from the period 1935–1965, a constant process of acidification of the lakes is found. The average pH of precipitation is 4.8 in the study area, but the lakes are in two stages of acidification: weak (pH 6.0–6.5) and intermediate (pH 5.5–5.8). The differences are due to differences in water sources.     

The MO15 gene encodes the catalytic subunit of a protein kinase that activates cdc2 and other cyclin-dependent kinases (CDKs) through phosphorylation of Thr161 and its homologues.

Phosphorylation of Thr161, a residue conserved in all members of the cdc2 family, has been reported to be absolutely required for the catalytic activity of cdc2, the major regulator of eukaryotic cell cycle. In the present work, we have purified from starfish oocytes a kinase that specifically activates cdc2 in a cyclin-dependent manner through phosphorylation of its Thr161 residue. Our most highly purified preparation contained only two major proteins of apparent M(r) 37 and 40 kDa (p37 and p40), which could not be separated from each other without loss of activity. The purified kinase was found to phosphorylate not only cdc2, but also cdk2 and a divergent cdc2-like protein from Caenorhabditis, in chimeric complexes including both mitotic and G1/S cyclins. Extensive microsequencing of p40 did not reveal any convincing homology with any known protein. In contrast, p37 is the starfish homologue of the M015 gene product, a kinase previously cloned by homology probing from a Xenopus cDNA library. As expected, immunodepletion of the MO15 protein depleted Xenopus egg extracts of CAK (cdk-activating kinase) activity, which was recovered in immunoprecipitates. Taken together, the above results demonstrate that MO15 is a gene conserved throughout evolution (at least from echinoderms to vertebrates) that encodes the catalytic subunit of a protein kinase that activates cdc2-cdks complexes through phosphorylation of Thr161 (or its homologues).     

αCT1 peptide sensitizes glioma cells to temozolomide in a glioblastoma organoid platform

Glioblastoma (GBM) is the most common form of brain cancer. Even with aggressive treatment, tumor recurrence is almost universal and patient prognosis is poor because many GBM cell subpopulations, especially the mesenchymal and glioma stem cell populations, are resistant to temozolomide (TMZ), the most commonly used chemotherapeutic in GBM. For this reason, there is an urgent need for the development of new therapies that can more effectively treat GBM. Several recent studies have indicated that high expression of connexin 43 (Cx43) in GBM is associated with poor patient outcomes. It has been hypothesized that inhibition of the Cx43 hemichannels could prevent TMZ efflux and sensitize otherwise resistance cells to the treatment. In this study, we use a three-dimensional organoid model of GBM to demonstrate that combinatorial treatment with TMZ and αCT1, a Cx43 mimetic peptide, significantly improves treatment efficacy in certain populations of GBM. Confocal imaging was used to visualize changes in Cx43 expression in response to combinatorial treatment. These results indicate that Cx43 inhibition should be pursued further as an improved treatment for GBM.     

Stereoselectivity in β-cyclodextrin complexation of 1,4-dihydropyridine derivatives

Structure–interaction relationships, stereoselectivity, and solubility enhancement in inclusion compexation of β-cyclodextrins (CDs) with some racemic and enantiomerically pure 1,4-dihydropyridine derivatives (DHPs) were investigated. 1:1 and 1:2 (mole ratio) complexes were prepared and characterized by X-ray powder diffraction, differential scanning calorimetry (DSC), MS-FAB spectrometry, ¹H-NMR spectroscopy, water and phase solubility. The solubility studies have revealed different complexation equilibria for optically pure DHP enantiomers, and corresponding racemic mixtures in water solutions. By means of ¹H-NMR chemical shift measurements, the inclusion of aromatic fragments of racemic and enantiomerically pure DHP molecules within the cavities of different CDs was elucidated. Considerable stereoselectivity in complexation interactions was observed. The results indicate the potential use of cyclodextrins as chiral selectors for enantiomeric resolution of 1,4-DHP calcium antagonists. © 1993 Wiley-Liss, Inc.