Branched co-polymers of histidine and lysine are efficient carriers of plasmids

We previously determined that a linear co-polymer of histidine and lysine (HK) in combination with liposomes enhanced the transfection efficiency of cationic liposomes. In the current study, we designed a series of HK polymers with increased branching and/or histidine/lysine ratio to determine if either variable affects transfection efficiency. In the presence of liposomes, the branched polymer with the highest number of histidines, HHK4b, was the most effective at enhancing gene expression. Furthermore, when serum was added to the medium during transfection, the combination of HHK4b and liposomes as a gene-delivery vehicle increased luciferase expression 400-fold compared to liposomes alone. In contrast to linear HK polymers, the higher branched HHK polymers were effective carriers of plasmids in the absence of liposomes. Without liposomes, the HHK4b carrier enhanced luciferase expression 15-fold in comparison with the lesser branched HHK2b carrier and increased expression by 5-logs in comparison with the HHK or HK carrier. The interplay of several parameters including increased condensation of DNA, buffering of acidic endosomes and differential binding affinities of polymer with DNA have a role in the enhancement of transfection by the HK polymers. In addition to suggesting that branched HK polymers are promising gene-delivery vehicles, this study provides a framework for the development of more efficient peptide-bond-based polymers of histidine and lysine.

Co-Permeability of 3H-Labeled Water and 14C-Labeled Organic Acids across Isolated Plant Cuticles1 : Investigating Cuticular Paths of Diffusion and Predicting Cuticular Transpiration

Penetration of 3H-labeled water (3H2O) and the 14C-labeled organic acids benzoic acid ([14C]BA), salicylic acid ([14C]SA), and 2,4-dichlorophenoxyacetic acid ([14C]2,4-D) were measured simultaneously in isolated cuticular membranes of Prunus laurocerasus L., Ginkgo biloba L., and Juglans regia L. For each of the three pairs of compounds (3H2O/[14C]BA, 3H2O/[14C]SA, and 3H2O/[14C]2,4-D) rates of cuticular water penetration were highly correlated with the rates of penetration of the organic acids. Therefore, water and organic acids penetrated the cuticles by the same routes. With the combination 3H2O/[14C]BA, co-permeability was measured with isolated cuticles of nine other plant species. Permeances of 3H2O of all 12 investigated species were highly correlated with the permeances of [14C]BA (r2 = 0.95). Thus, cuticular transpiration can be predicted from BA permeance. The application of this experimental method, together with the established prediction equation, offers the opportunity to answer several important questions about cuticular transport physiology in future investigations.

Creation of a novel protein-coding region at the RNA level in black pine chloroplasts: the pattern of RNA editing in the gymnosperm chloroplast is different from that in angiosperms.

The phenomenon of RNA editing has been found to occur in chloroplasts of several angiosperm plants. Comparative analysis of the entire nucleotide sequence of a gymnosperm [Pinus thunbergii (black pine)] chloroplast genome allowed us to predict several potential editing sites in its transcripts. Forty-nine such sites from 14 genes/ORFs were analyzed by sequencing both cDNAs from the transcripts and the corresponding chloroplast DNA regions, and 26 RNA editing sites were identified in the transcripts from 12 genes/ORFs, indicating that chloroplast RNA editing is not restricted to angiosperms but occurs in the gymnosperm, too. All the RNA editing events are C-to-U conversions; however, many new codon substitutions and creation of stop codons that have not so far been reported in angiosperm chloroplasts were observed. The most striking is that two editing events result in the creation of an initiation and a stop codon within a single transcript, leading to the formation of a new reading frame of 33 codons. The predicted product is highly homologous to that deduced from the ycf7 gene (ORF31), which is conserved in the chloroplast genomes of many other plant species.

Creation of a reversible on/off system for site-specific in vivo control of exogenous gene activity in the renal glomerulus.

Using genetically engineered glomerular mesangial cells, an in vivo gene transfer approach was developed that specifically targets the renal glomerulus. By combining this system with a tetracycline (Tc)-responsive promoter, the present study aimed to create a reversible on/off system for site-specific in vivo control of exogenous gene activity within the glomerulus. In the Tc regulatory system, a Tc-controlled transactivator (tTA) encoded by a regulator plasmid induces target gene transcription by binding to a tTA-responsive promoter located in a response plasmid. Tc inhibits this tTA-dependent transactivation via its affinity for tTA. In double-transfected cells, therefore, the activity of a transgene can be controlled by Tc. Cultured rat mesangial cells were cotransfected with a regulator plasmid and a response plasmid that introduces a beta-galactosidase gene. In vitro, stable double-transfectant MtTAG cells exhibited no beta-galactosidase activity in the presence of Tc. However, following withdrawal of Tc from culture media, expression of beta-galactosidase was induced within 24 h. When Tc was again added, the expression was rapidly resuppressed. Low concentrations of Tc were sufficient to maintain the silent state of tTA-dependent promoter. MtTAG cells were then transferred into the rat glomeruli via renal artery injection. In the isolated chimeric glomeruli, expression of beta-galactosidase was induced ex vivo in the absence of Tc, whereas it was repressed in its presence. When Tc-pretreated MtTAG cells were transferred into the glomeruli of untreated rats, beta-galactosidase expression was induced in vivo within 3 days. Oral administration of Tc dramatically suppressed this induction. These data demonstrate the feasibility of using mesangial cell vectors combined with the Tc regulatory system for site-specific in vivo control of exogenous gene expression in the glomerulus.

Creation of drug-specific herpes simplex virus type 1 thymidine kinase mutants for gene therapy.

Herpes simplex virus type 1 (HSV-1) thymidine kinase is currently used as a suicide agent in the gene therapy of cancer. This therapy is based on the preferential phosphorylation of nucleoside analogs by tumor cells expressing HSV-1 thymidine kinase. However, the use of HSV-1 thymidine kinase is limited in part by the toxicity of the nucleoside analogs. We have used random sequence mutagenesis to create new HSV-1 thymidine kinases that, compared with wild-type thymidine kinase, render cells much more sensitive to specific nucleoside analogs. A segment of the HSV-1 thymidine kinase gene at the putative nucleoside binding site was substituted with random nucleotide sequences. Mutant enzymes that demonstrate preferential phosphorylation of the nucleoside analogs, ganciclovir or acyclovir, were selected from more than one million Escherichia coli transformants. Among the 426 active mutants we have isolated, 26 demonstrated enhanced sensitivity to ganciclovir, and 54 were more sensitive to acyclovir. Only 6 mutant enzymes displayed sensitivity to both ganciclovir and acyclovir when expressed in E. coli. Analysis of 3 drug-sensitive enzymes demonstrated that 1 produced stable mammalian cell transfectants that are 43-fold more sensitive to ganciclovir and 20-fold more sensitive to acyclovir.

Dated co-occurrence of Homo erectus and Gigantopithecus from Tham Khuyen Cave, Vietnam.

Tham Khuyen Cave (Lang Son Province, northern Vietnam) is one of the more significant sites to yield fossil vertebrates in east Asia. During the mid-1960s, excavation in a suite of deposits produced important hominoid dental remains of middle Pleistocene age. We undertake more rigorous analyses of these sediments to understand the fluvial dynamics of Pleistocene cave infilling as they determine how skeletal elements accumulate within Tham Khuyen and other east Asian sites. Uranium/thorium series analysis of speleothems brackets the Pleistocene chronology for breaching, infilling, and exhuming the regional paleokarst. Clast analysis indicates sedimentary constituents, including hominoid teeth and cranial fragments accumulated from very short distances and under low fluvial energy. Electron spin resonance analysis of vertebrate tooth enamel and sediments shows that the main fossil-bearing suite (S1-S3) was deposited about 475 thousand years ago. Among the hominoid teeth excavated from S1-S3, some represent Homo erectus and Gigantopithecus blacki. Criteria are defined to differentiate these teeth from more numerous Pongo pygmaeus elements. The dated co-occurrence of Homo erectus and Gigantopithecus blacki at Tham Khuyen helps to establish the long co-existence of these two species throughout east Asia during the Early and Middle Pleistocene.

In situ isolation of mRNA from individual plant cells: creation of cell-specific cDNA libraries.

A method for isolating and cloning mRNA populations from individual cells in living, intact plant tissues is described. The contents of individual cells were aspirated into micropipette tips filled with RNA extraction buffer. The mRNA from these cells was purified by binding to oligo(dT)-linked magnetic beads and amplified on the beads using reverse transcription and PCR. The cell-specific nature of the isolated mRNA was verified by creating cDNA libraries from individual tomato leaf epidermal and guard cell mRNA preparations. In testing the reproducibility of the method, we discovered an inherent limitation of PCR amplification from small amounts of any complex template. This phenomenon, which we have termed the "Monte Carlo" effect, is created by small and random differences in amplification efficiency between individual templates in an amplifying cDNA population. The Monte Carlo effect is dependent upon template concentration: the lower the abundance of any template, the less likely its true abundance will be reflected in the amplified library. Quantitative assessment of the Monte Carlo effect revealed that only rare mRNAs (< or = 0.04% of polyadenylylated mRNA) exhibited significant variation in amplification at the single-cell level. The cDNA cloning approach we describe should be useful for a broad range of cell-specific biological applications.