Mycobacterium tuberculosis remains one of the world's most important pathogens. Responsible for millions of new cases of tuberculosis annually (1), it is the leading cause of death from a single infectious agent. The emergence of multidrug-resistant M. tuberculosis underscores the need for new approaches to combat this pathogen (1).We recently have identified glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming); EC 6.3.1.2] as an important determinant of M. tuberculosis pathogenesis (2-4). An enzyme that plays a central role in nitrogen metabolism in every cell, glutamine synthetase is 1 of 10 proteins released in large quantity into the bacterium's extracellular milieu, whether the bacterium is growing axenically or intraphagosomally in human mononuclear phagocytes, the primary host cells (2). Of great interest, extracellular release of glutamine synthetase is unique to pathogenic mycobacteria and correlated with the presence of a poly-L-glutamate͞ glutamine (P-L-glx) component in the cell wall of these organisms, suggesting that not only is the enzyme involved in the synthesis of this heteropolymer but also that its presence is significant to virulence (4). Treatment of bacteria with L-methionine-Ssulfoximine, an irreversible inhibitor of glutamine synthetase, results in a decrease in extracellular glutamine synthetase activity, a marked reduction in the amount of cell wall P-L-glx, and inhibition of bacterial growth both in broth culture and in human macrophages (4).Antisense oligodeoxyribonucleotides (ODNs), which can base pair with a gene's transcript, constitute a new technology for the control of gene expression in prokaryotes and eukaryotes, including mammalian cells (5, 6). In addition, this technology shows promise as a means for developing new chemotherapeutic agents against human diseases, including antibiotics against such pathogens as Plasmodium falciparum, Toxoplasma gondii, and HIV (7-10). An antisense PS-ODN for treatment of cytomegalovirus retinitis in AIDS patients became the first antisense ODN-based drug approved for human use in the U.S. (11).In this report, we have utilized modified antisense ODNs, in which all internucleoside linkages are phosphorothioates (PSODNs), to study the expression and function of M. tuberculosis glutamine synthetase and the feasibility of using antisense PSODNs as antimicrobial agents against this pathogen. We find that glutamine synthetase-specific antisense PS-ODNs substantially inhibit the expression and activity of M. tuberculosis glutamine synthetase, the amount of the P-L-glx structure in the mycobacterial cell wall, and bacterial replication. These observations are consistent with entry of the antisense PS-ODNs into the bacterial cytoplasm. Materials and Methods PS-ODN Selection and Preparation.Three target sites for the binding of the antisense PS-ODNs were chosen (Table 1). One site, located near the 5Ј end of the glutamine synthetase mRNA, corresponds to codons 4-9 of the glutamine synthetase mRNA translation product, starting from the N-termi...

Background and Objectives: Fractional ablation offers the potential benefits of full-surface ablative skin resurfacing while minimizing adverse effects. The purpose of this study was to evaluate the safety, damage profile, and efficacy of erbium fractional lasers. Materials and Methods: Histology from animal and human skin as well as clinical evaluations were conducted with erbium YAG (2,940 nm) and erbium YSGG (2,790 nm) fractional lasers varying pulse width, microbeam (mb) energy, number of passes, and stacking of pulses. Results: Single-pulse treatment parameters from 1 to 12 mJ per 50-70 mm diameter microbeam and 0.25-5 milliseconds pulse widths produced microcolumns of ablation with border coagulation of up to 100 mm width and 450 mm depth. Stacking of pulses generated deeper microcolumns. Clinical observations and in vivo histology demonstrate rapid re-epithelization and limited adverse side effects. Facial treatments were performed in the periorbital and perioral areas using 1-8 passes of single and stacked pulses. Treatments were well-tolerated and subjects could resume their normal routine in 4 days. A statistically significant reduction in wrinkle scores at 3 months was observed for both periorbital and perioral wrinkles using blinded grading. For periorbital treatments of four passes or more, over 90% had !1 score wrinkle reduction (0-9 scale) and 42% had !2. For perioral wrinkles, over 50% had substantial improvements (!2). Conclusion: The clinical observations and histology findings demonstrate that micro-fractional ablative treatment with 2,790 and 2,940 nm erbium lasers resulted in safe and effective wrinkle reduction with minimal patient downtime. The depth and width of the ablated microcolumns and varying extent of surrounding coagulation can be controlled and used to design new treatment procedures targeted for specific indications and areas such as moderate to severe rhytides and photodamaged skin.

Optical imaging in the near-infrared (NIR) range enables detecting ligand-receptor interactions and enzymatic activity in vivo due to lower scattering and absorption of NIR photons in the tissue. We designed and tested prototype NIR fluorescent oligodeoxyribonucleotide (ODN) reporters that can sense transcription factor NF-B p50 protein binding. The reporter duplexes included donor NIR Cy5.5 indodicarbocyanine fluorochrome linked to the 3 end of the first ODN and NIR acceptor fluorochromes (indodicarbocyanine Cy7 or, alternatively, a heptamethine cyanine IRDye 800CW) that were linked at the positions ؉8 and ؉12 to the complementary ODN that encoded p50 binding sites. Both Cy7 and 800CW fluorochromes were linked by using hydrophilic internucleoside phosphate linkers that enable interaction between the donor and the acceptor with no base-pairing interference. We observed efficient fluorescence resonance energy transfer (FRET) both in the case of Cy5.5-Cy7 and Cy5.5-800CW pairs of fluorochromes, which was sensitive to the relative position of the dyes. Higher FRET efficiency observed in the case of Cy5.5-Cy7 pair was due to a larger overlap between the ODN-linked Cy5.5 emission and Cy7 excitation spectra. Fluorescent mobility shift assay showed that the addition of human recombinant p50 to ODN duplexes resulted in p50 binding and measurable increase of Cy5.5 emission. In addition, p50 binding provided a concomitant protection of FRET effect from exonuclease-mediated hydrolysis. We conclude that NIR FRET effect can be potentially used for detecting protein-DNA interactions and that the feasibility of detection depends on FRET efficacy and relative fluorochrome positions within ODN binding sites.carbocyanine ͉ transcription factor

The use of specially designed island mask combined with non-laser intensive pulse irradiation produces a lattice of islands of limited thermal damage in SC that substantially enhances the penetration rate of topically applied index-matching agents. The suggested technique gave comparable magnitudes of clearing dynamics enhancement for glucose solution, glycerol solution, and propylene glycol solution applied to mammalian skin.

We have investigated the effect of sequence-specific antisense phosphorothioate-modified oligodeoxyribonucleotides (PS-ODNs) targeting different regions of each of the 30͞32-kDa protein complex (antigen 85 complex) encoding genes on the multiplication of Mycobacterium tuberculosis. Single PS-ODNs to one of the three mycolyl transferase transcripts, added either once or weekly over the 6-wk observation period, inhibited bacterial growth by up to 1 log unit. A combination of three PS-ODNs specifically targeting all three transcripts inhibited bacterial growth by Ϸ2 logs; the addition of these PS-ODNs weekly for 6 wk was somewhat more effective than a one-time addition. Targeting the 5 end of the transcripts was more inhibitory than targeting internal sites; the most effective PS-ODNs and target sites had minimal or no secondary structure. The effect of the PS-ODNs was specific, as mismatched PS-ODNs had little or no inhibitory activity. The antisense PS-ODNs, which were highly stable in M. tuberculosis cultures, specifically blocked protein expression by their gene target. PS-ODNs targeting the transcript of a related 24-kDa protein (mpt51) had little inhibitory effect by themselves and did not increase the effect of PS-ODNs against the three members of the 30͞32-kDa protein complex. The addition of PS-ODNs against the transcripts of glutamine synthetase I (glnA1) and alanine racemase (alr) modestly increased the inhibitory efficacy of the 30͞32-kDa protein complex-specific PS-ODNs to Ϸ2.5 logs. This study shows that the three mycolyl transferases are highly promising targets for antituberculous therapy by using antisense or other antimicrobial technologies.phosphorothioate oligodeoxyribonucleotides ͉ mRNA secondary structure ͉ inhibition of translation initiation ͉ drug design T uberculosis, caused primarily by the facultative intracellular bacterium Mycobacterium tuberculosis, is the leading cause of death from a single infectious agent. The emergence of multidrug-resistant strains of M. tuberculosis has given urgency to the need for novel antimicrobial agents to combat this pathogen (1).M. tuberculosis expresses and secretes three closely related mycolyl transferases of 30-32 kDa mass, also known as the antigen 85 protein complex (antigen 85A, 85B, and 85C are referred to herein as the 32A, 30, and 32B proteins, respectively). All three enzymes catalyze the transfer of mycolic acid from one trehalose 6-monomycolate to another, resulting in trehalose 6,6Ј-dimycolate and free trehalose (2). The 3D structure of two of the enzymes, the 30 and 32A proteins, their catalytic sites, and their reaction mechanisms have been described in detail (3, 4). All three proteins are abundantly secreted. The 30-kDa protein is the most abundant extracellular protein of M. tuberculosis, responsible for nearly one quarter of the total extracellular protein in broth culture, and the 32A-kDa protein is the second most abundant extracellular protein (5). The three mycolyl transferases are also abundantly expressed in human macrophages i...