A nanotechnology-based delivery system: Nanobots. Novel vehicles for molecular medicine.

AIM: We previously demonstrated that adenovirus-mediated p53 gene transfer following balloon angioplasty, decreased neointimal hyperplasia. However, safety concerns arise because viral promoters can cause unrestricted transgene expression. The paucity of safe and efficient vehicles for gene transfer thus limits the potential for clinical utilization of gene therapy. Our objective was to design and clone a virus-free p53 construct, targeted to express specifically in vascular smooth muscle cells (SMCs), via a nanoparticle-based delivery system for therapeutic modulation in vascular wall. METHODS: Biodegradable poly(lactide-co-glycolide) (PLGA), an FDA approved polymer, was used to formulate the nanoparticles. Cloned constructs consisting of SMC promoter, SM22, and p53 cDNA sequences along with enhanced green fluorescent protein (EGFP) gene, were loaded into PLGA nanoparticles. The affect of these nanobots on cell growth was examined. RESULTS: The gene sequences carried by the nanobot are expressed in target cells. The p53/EGFP construct under the constitutive promoter was found to express in 293T human embryonic kidney cells, whereas the p53/EGFP with SMC promoter expressed only in human aortic SMCs. SMCs internalize these nanobots without compromising cell viability or growth kinetics. CONCLUSION: A novel genetic sequence that targets a specific cell population has been successfully designed, cloned and encapsulated in a nanoparticle. This experiment is a significant step towards the development of a nanoparticle-based delivery system for therapeutic delivery of targeted gene-therapy towards attenuation of restenosis. Further work is necessary to expand the repertoire of this delivery system and determine whether it could become a versatile vehicle in molecular medicine.

Drosophila development. Delimiting patterns by repression.

Patterning of the Drosophila embryo requires not only the proper activation of determinants at specific times, but also their restriction to specific places. Recent studies on transcriptional repressors show how they delimit the gene expression patterns to ensure normal development.

Human Slug is a repressor that localizes to sites of active transcription.

Snail/Slug family proteins have been identified in diverse species of both vertebrates and invertebrates. The proteins contain four to six zinc fingers and function as DNA-binding transcriptional regulators. Various members of the family have been demonstrated to regulate cell movement, neural cell fate, left-right asymmetry, cell cycle, and apoptosis. However, the molecular mechanisms of how these regulators function and the target genes involved are largely unknown. In this report, we demonstrate that human Slug (hSlug) is a repressor and modulates both activator-dependent and basal transcription. The repression depends on the C-terminal DNA-binding zinc fingers and on a separable repression domain located in the N terminus. This domain may recruit histone deacetylases to modify the chromatin and effect repression. Protein localization study demonstrates that hSlug is present in discrete foci in the nucleus. This subnuclear pattern does not colocalize with the PML foci or the coiled bodies. Instead, the hSlug foci overlap extensively with areas of the SC-35 staining, some of which have been suggested to be sites of active splicing or transcription. These results lead us to postulate that hSlug localizes to target promoters, where activation occurs, to repress basal and activator-mediated transcription.

Snail/slug family of repressors: slowly going into the fast lane of development and cancer.

The existence of homologous genes in diverse species is intriguing. A detailed comparison of the structure and function of gene families may provide important insights into gene regulation and evolution. An unproven assumption is that homologous genes have a common ancestor. During evolution, the original function of the ancestral gene might be retained in the different species which evolved along separate courses. In addition, new functions could have developed as the sequence began to diverge. This may also explain partly the presence of multipurpose genes, which have multiple functions at different stages of development and in different tissues. The Drosophila gene snail is a multipurpose gene; it has been demonstrated that snail is critical for mesoderm formation, for CNS development, and for wing cell fate determination. The related vertebrate Snail and Slug genes have also been proposed to participate in mesoderm formation, neural crest cell migration, carcinogenesis, and apoptosis. In this review, we will discuss the Snail/Slug family of regulators in species ranging from insect to human. We will present the protein structures, expression patterns, and functions based on molecular genetic analyses. We will also include the studies that helped to elucidate the molecular mechanisms of repression and the relationship between the conserved and divergent functions of these genes. Moreover, the studies may enable us to trace the evolution of this gene family.

Characterization of a MEN1 ortholog from Drosophila melanogaster.

Multiple endocrine neoplasia type 1 (MEN1) is a familial cancer syndrome characterized by tumors of the parathyroid, entero-pancreatic neuroendocrine and pituitary tissues and caused by inactivating mutations in the MEN1 gene. Menin, the 610-amino acid nuclear protein encoded by MEN1, binds to the transcription factor JunD and can repress JunD-induced transcription. We report here the identification of a MEN1 ortholog in Drosophila melanogaster, Menin1, that encodes a 763 amino acid protein sharing 46% identity with human menin. Additionally, 69% of the missense mutations and in-frame deletions reported in MEN1 patients appear in amino acid residues that are identical in the Drosophila and human protein, suggesting the importance of the conserved regions. Drosophila Menin1 gene transcripts use alternative polyadenylation sites resulting in 4.3 and 5-kb messages. The 4.3-kb transcript appears to be largely maternal, while the 5-kb transcript appears mainly zygotic. The binding of Drosophila menin to human JunD or Drosophila Jun could not be demonstrated by the yeast two-hybrid analysis. The identification of the MEN1 ortholog from Drosophila melanogaster will provide an opportunity to utilize Drosophila genetics to enhance our understanding of the function of human menin.

DNA methylation in mycobacteria: absence of methylation at GATC (Dam) and CCA/TGG (Dcm) sequences.

The presence of 6-methyladenine and 5-methylcytosine at Dam (GATC) and Dcm (CCA/TGG) sites in DNA of mycobacterial species was investigated using isoschizomer restriction enzymes. In all species examined, Dam and Dcm recognition sequences were not methylated indicating the absence of these methyltransferases. On the other hand, high performance liquid chromatographic analysis of genomic DNA from Mycobacterium smegmatis and Mycobacterium tuberculosis showed significant levels of 6-methyladenine and 5-methylcytosine suggesting the presence of DNA methyltransferases other than Dam and Dcm. Occurrence of methylation was also established by a sensitive genetic assay.

Genotoxicity studies with Cuman L in Drosophila melanogaster.

Cuman L, a dithiocarbamate fungicide, was tested for its ability to induce sex-linked recessive lethals (SLRLs) and II-III chromosome translocations in Drosophila melanogaster by the larval feeding method. The three concentrations of Cuman L, of 20, 40, and 60 microliters/100 ml, induced significant (P less than 0.01) increases in SLRLs but failed to induce any translocations.

Cytogenetic effects of Cuman L, a dithiocarbamate fungicide.

Cuman L, a dithiocarbamate fungicide, was assessed for its effects in the germ cells and the bone marrow erythrocytes of Swiss Albino male mice. The 3 sublethal doses of 350, 700 and 1050 mg/kg b.w. of Cuman L induced a significant (P less than 0.01) increase in the number of chromosomal aberrations in the germ cells. A significant increase (P less than 0.01) in the percentage of micronuclei in the erythrocytes was also induced by the three doses.

Mutagenicity studies in Drosophila melanogaster with Lannate 20.

Lannate 20 a carbamate pesticide was evaluated for its mutagenicity in Drosophila melanogaster by the sex-linked recessive lethals and chromosome II-III translocation tests by continuous larval feeding. The 3 sublethal doses of 0.2, 0.4 and 0.6 microliter of Lannate per 100 ml of the food medium induced a significant (P less than 0.01) increase in the number of sex-linked recessive lethals over the controls. However, no translocations were observed either in the treated or the control series.

The roles of histidine residues at the starch-binding site in streptococcal-binding activities of human salivary amylase.

Human salivary alpha-amylase participates in the initial digestion of starch and may be involved in the colonization of viridans streptococci in the mouth. To elucidate the role of histidine residues located near the starch-binding site on the streptococcal-binding activity, the wild type and three histidine mutants, H52A, H299A and H305A were constructed and expressed in a baculovirus system. While His52 is located near the non-reducing end of the starch-binding pocket (subsite S3/S4), the residues His299 and His305 are located near the subsites S1/S1'. For the wild type, the cDNA encoding the leader and secreted sequences of human salivary amylase was amplified by polymerase chain reaction from a human submandibular salivary-gland cDNA library, and subcloned into the baculovirus shuttle vector pVL1392 downstream of the polyhedrin promoter. Oligonucleotide-based, site-directed mutagenesis was used to generate the mutants expressed in the baculovirus system. Replacing His52 or His299 or His305 to Ala residue did not alter the bacterial-binding activity significantly, but these mutants did show differences in their catalytic activities. The mutant H52A showed negligible reduction in enzymatic activity compared to that of wild type for the hydrolysis of starch and oligosaccharides. In contrast, the H299A and H305A mutants showed a 12 to 13-fold reduction (90-92%) in starch-hydrolysing activity. In addition, the k(cat) for the hydrolysis of oligosaccharides by H299A decreased by as much as 11-fold for maltoheptaoside. This reduction was even higher (40-fold) for the hydrolysis of p-nitrophenyl maltoside, with a significant change in K(M). The mutant H305A, however, exhibited a reduction in k(cat) only, with no changes in the K(M) for the hydrolysis of oligosaccharides. The reduction in the k(cat) for the H305A mutant was almost 93% for maltoheptaoside hydrolysis. The pH activity profile for the H305A mutant was also significantly different from that of the wild type and the other two mutants. These results suggest that, although histidines at the starch-binding site of salivary amylase are involved in starch binding and catalysis, they may not participate in Streptococcus gordonii G9B binding.

Evaluation of lannate 20, a carbamate pesticide in the germ cells of male mice.

Lannate 20, a carbamate pesticide, was evaluated for its effects on the germ cells of Swiss albino male mice, by the sperm morphology assay and meiotic chromosome preparations. The three sublethal concentrations of 20, 40, and 60 mg/kg body wt administered by the oral intubation method produced significant results (P less than 0.01) with both the above protocols in the said test system under study, thus indicating that lannate 20 is mutagenic in mice.

Differential regulation of gastrulation and neuroectodermal gene expression by Snail in the Drosophila embryo.

The initiation of mesoderm differentiation in the Drosophila embryo requires the gene products of twist and snail. In either mutant, the ventral cell invagination during gastrulation is blocked and no mesoderm-derived tissue is formed. One of the functions of Snail is to repress neuroectodermal genes and restrict their expressions to the lateral regions. The derepression of the neuroectodermal genes into the ventral region in snail mutant is a possible cause of defects in gastrulation and in mesoderm differentiation. To investigate such possibility, we analysed a series of snail mutant alleles. We found that different neuroectodermal genes respond differently in various snail mutant background. Due to the differential response of target genes, one of the mutant alleles, V2, that has reduced Snail function showed an intermediate phenotype. In V2 embryos, neuroectodermal genes, such as single-minded and rhomboid, are derepressed while ventral invagination proceeds normally. However, the differentiation of these invaginated cells into mesodermal lineage is disrupted. The results suggest that the establishment of mesodermal cell fate requires the proper restriction of neuroectodermal genes, while the ventral cell movement is independent of the expression patterns of these genes. Together with the data showing that the expression of some ventral genes disappear in snail mutants, we propose that Snail may repress or activate another set of target genes that are required specifically for gastrulation.