Mammalian Homolog of Drosophila Tumor Suppressor Lethal (2) Giant Larvae Interacts with Basolateral Exocytic Machinery in Madin-Darby Canine Kidney Cells

The Drosophila tumor suppressor protein lethal (2) giant larvae [l(2)gl] is involved in the establishment of epithelial cell polarity during development. Recently, a yeast homolog of the protein has been shown to interact with components of the post-Golgi exocytic machinery and to regulate a late step in protein secretion. Herein, we characterize a mammalian homolog of l(2)gl, called Mlgl, in the epithelial cell line Madin-Darby canine kidney (MDCK). Consistent with a role in cell polarity, Mlgl redistributes from a cytoplasmic localization to the lateral membrane after contact-naive MDCK cells make cell-cell contacts and establish a polarized phenotype. Phosphorylation within a highly conserved region of Mlgl is required to restrict the protein to the lateral domain, because a recombinant phospho-mutant is distributed in a nonpolar manner. Membrane-bound Mlgl from MDCK cell lysates was coimmunoprecipitated with syntaxin 4, a component of the exocytic machinery at the basolateral membrane, but not with other plasma membrane soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins that are either absent from or not restricted to the basolateral membrane domain. These data suggest that Mlgl contributes to apico-basolateral polarity by regulating basolateral exocytosis.     

The EF-hand domain: A globally cooperative structural unit

EF-hand Ca(2+)-binding proteins participate in both modulation of Ca(2+) signals and direct transduction of the ionic signal into downstream biochemical events. The range of biochemical functions of these proteins is correlated with differences in the way in which they respond to the binding of Ca(2+). The EF-hand domains of calbindin D(9k) and calmodulin are homologous, yet they respond to the binding of calcium ions in a drastically different manner. A series of comparative analyses of their structures enabled the development of hypotheses about which residues in these proteins control the calcium-induced changes in conformation. To test our understanding of the relationship between protein sequence and structure, we specifically designed the F36G mutation of the EF-hand protein calbindin D(9k) to alter the packing of helices I and II in the apoprotein. The three-dimensional structure of apo F36G was determined in solution by nuclear magnetic resonance spectroscopy and showed that the design was successful. Surprisingly, significant structural perturbations also were found to extend far from the site of mutation. The observation of such long-range effects provides clear evidence that four-helix EF-hand domains should be treated as a single globally cooperative unit. A hypothetical mechanism for how the long-range effects are transmitted is described. Our results support the concept of energetic and structural coupling of the key residues that are crucial for a protein's fold and function.     

Bioinspired polymers that control intracellular drug delivery

One of the important characteristics of biological systems is their ability to change important properties in response to small environmental signals. The molecular mechanisms that biological molecules utilize to sense and respond provide interesting models for the development of “smart” polymeric biomaterials with biomimetic properties. An important example of this is the protein coat of viruses, which contains peptide units that facilitate the trafficking of the virus into the cell via endocytosis, then out of the endosome into the cytoplasm, and from there into the nucleus. We have designed a family of synthetic polymers whose compositions have been designed to mimic specific peptides on viral coats that facilitate endosomal escape. Our biomimetic polymers are responsive to the lowered pH within endosomes, leading to disruption of the endosomal membrane and release of important biomolecular drugs such as DNA, RNA, peptides and proteins to the cytoplasm before they are trafficked to lysosomes and degraded by lysosomal enzymes. In this article, we review our work on the design, synthesis and action of such smart, pH-sensitive polymers.     

An integrative approach for studying the etiology of alcoholism and other addictions.

Studies of alcoholism etiology often focus on genetic or psychosocial approaches, but not both. Greater understanding of the etiology of alcohol, tobacco and other addictions will come from integration of these research traditions. A research approach is outlined to test three models for the etiology of addictions--behavioral undercontrol, pharmacologic vulnerability, negative affect regulation--addressing key questions including (i) mediators of genetic effects, (ii) genotype-environment correlation effects, (iii) genotype x environment interaction effects, (iv) the developmental unfolding of genetic and environmental effects, (v) subtyping including identification of distinct trajectories of substance involvement, (vi) identification of individual genes that contribute to risk, and (vii) the consequences of excessive use. By using coordinated research designs, including prospective assessment of adolescent twins and their siblings and parents; of adult substance dependent and control twins and their MZ and DZ cotwins, the spouses of these pairs, and their adolescent offspring; and of regular families; by selecting for gene-mapping approaches sibships screened for extreme concordance or discordance on quantitative indices of substance use; and by using experimental (drug challenge) as well as survey approaches, a number of key questions concerning addiction etiology can be addressed. We discuss complementary strengths and weaknesses of different sampling strategies, as well as methods to implement such an integrated approach illustrated for the study of alcoholism etiology. A coordinated program of twin and family studies will allow a comprehensive dissection of the interplay of genetic and environmental risk-factors in the etiology of alcoholism and other addictions.     

Violacein transformation by peroxidases and oxidases: implications on its biological properties

1,3-Dihydro-2H-indol-2-one derivative (violacein) is extracted from Chromobacterium violaceum and presents several biological activities as antibiotic, antitumoral, antichagasic and antioxidant. In order to increase its biological activities and decrease its toxicity, one strategy is to slightly transform the molecules through a specific group transformation. Peroxidases, which are hemoproteins, are known as excellent oxidants producing hydroxylation and ring cleavage. Laccases are phenol oxidases produced by fungi as plants and belong to the oxidase group which complexes copper. This enzyme generates phenolates, quinones and also ring cleavage even in the presence of a mediator as 1-hydroxybenzotriazol (HBT). Lignin peroxidase and manganese peroxidase (LiP/MnP) pool from Phanerochaete chrysosporium, Horseradish peroxidase (HRP-VI) from horseradish, Lactoperoxidase (LPO) from bovine milk and Laccase (Lac) from Trametes versicolor acting on violacein were studied. Kinetics parameters and products distribution indicated a fast and efficient violacein transformation with all of them. HRP-VI acting on violacein was studied in details and spectral evidence indicated that Horseradish peroxidase compound II was formed during the oxidation reaction. Similar behavior with LiP/MnP pool was observed. Laccase, in the presence and absence of mediator (HBT), also showed a rapid violacein transformation. In a more detailed study with the HRP-VI reaction, a hydroxilation in the indol unit and a ring contraction of the pyrrol moiety of violacein molecule occurred.     

General method of rapid Smith/Birnstiel mapping adds for gap closure in shotgun microbial genome sequencing projects: application to Pseudomonas putida KT2440

A physical mapping strategy has been developed to verify and accelerate the assembly and gap closure phase of a microbial genome shotgun-sequencing project. The protocol was worked out during the ongoing Pseudomonas putida KT2440 genome project. A macro-restriction map was constructed by linking probe hybridisation of SwaI- or I-CeuI-restricted chromosomes to serve as a backbone for the quick quality control of sequence and contig assemblies. The library of PCR-generated SwaI linking probes was derived from the sequence assembly after 3- and 6-fold genome coverage. In order to support gap closure in regions with ambiguous assemblies such as the repetitive sequence of the seven ribosomal operons, high-resolution Smith/Birnstiel maps were generated by Southern hybridisation of pulsed-field gel electrophoresis-separated rare-cutter complete/frequent-cutter partial digestions with rare-cutter fragment end probes. Overall 1.5 Mb of the 6.1 Mb P.putida KT2440 genome has been subjected to high-resolution physical mapping in order to align assemblies generated from shotgun sequencing.     

Criteria followed in the establishment of a filamentous fungal culture collection – Micoteca da Universidade do Minho (MUM)

In Portugal there are few culture collections of filamentous fungi and these are mostly in-house, and located mainly in the Lisbon area. Furthermore, open access to Portuguese databases on microorganisms is not well established. This knowledge and the continuing experimental activity in mycology, combined with an interest from the University in creating and financing reliable services to support research, led to the organization of a filamentous fungal culture collection, hosted at the Biological Engineering Centre of Minho University, in the North of Portugal. The Micoteca da Universidade do Minho (MUM), was established in 1996, with the aim of maintaining and providing strains for research on biotechnology and for use in teaching laboratories, and to act as a centre of expertise, information and training, complying with international quality standards. The use of a computerized system for the management of recorded data and the collection's holdings was envisaged from the start. The collection now holds 128 identified species, including recognized standard or test strains and isolates derived from research activities, mainly of the genera Penicillium and Aspergillus. The microbial strain database was built in-house and runs on Windows 2000. To ensure widespread availability of details of the collection's holdings, the fields intended for general searching and viewing are available on-line at http://www.micoteca.deb.uminho.pt/.     

The discovery of anthranilic acid-based MMP inhibitors. Part 3: incorporation of basic amines.

Anthranilic acid derivatives bearing basic amines were prepared and evaluated in vitro and in vivo as inhibitors of MMP-1, MMP-9, MMP-13, and TACE. Piperazine 4u has been identified as a potent, selective, orally active inhibitor of MMP-9 and MMP-13.