Activatable fluorescent probes in fluorescence-guided surgery: Practical considerations

Fluorescence-guided imaging during surgery is a promising technique that is increasingly used to aid surgeons in identifying sites of tumor and surgical margins. Of the two types of fluorescent probes, always-on and activatable, activatable probes are preferred because they produce higher target-to-background ratios, thus improving sensitivity compared with always-on probes that must contend with considerable background signal. There are two types of activatable probes: 1) enzyme-reactive probes that are normally quenched but can be activated after cleavage by cancer-specific enzymes (activity-based probes) and 2) molecular-binding probes which use cancer targeting moieties such as monoclonal antibodies to target receptors found in abundance on cancers and are activated after internalization and lysosomal processing (binding-based probes). For fluorescence-guided intraoperative surgery, enzyme-reactive probes are superior because they can react quickly, require smaller dosages especially for topical applications, have limited side effects, and have favorable pharmacokinetics. Enzyme-reactive probes are easier to use, fit better into existing work flows in the operating room and have minimal toxicity. Although difficult to prove, it is assumed that the guidance provided to surgeons by these probes results in more effective surgeries with better outcomes for patients. In this review, we compare these two types of activatable fluorescent probes for their ease of use and efficacy.     

Chemoenzymatic synthesis of four diastereomers of (6-fluoro-2-chromanyl) oxirane: An intermediate of a potent β-blocker

The synthesis of optically active 5-acetoxy-3-(p-fluorophenoxy)-1-pentanol 4, for the synthesis of the potent β-blocker R-67555, bis[2-(2-chromanyl-6-fluoro)-2-hydroxyethyl]amine 1, was investigated. The acetylation of 3-(p-fluorophenoxy)-1,5-pentanediol 5a using lipozyme and the hydrolysis of 1,5-diacetoxy-3-(p-fluorophenoxy)pentane 5b using lipase Amano P yielded (3S)- and (3R)-5-acetoxy-3-(p-fluorophenoxy)-1-pentanol 4, respectively, with high enantiomeric excess. Four diastereomers of (6-fluoro-2-chromanyl)oxirane 2, important intermediates for the synthesis of R-67555, were synthesized by chemical methods using (S)-4 and (R)-4.     

Analysis of Genetic Relationships and Antimicrobial Susceptibility of Verotoxin-Producing Escherichia coli Strains Isolated in Nagasaki Prefecture,Japan in 1996

A total of 19 Escherichia coli O157 isolates were obtained in Nagasaki Prefecture, in the southwestern part of Japan, between 1990 and 1996. Pulsed-field gel electrophoresis (PFGE) and computer-assisted analysis were applied to determine genetic relationships among these strains. Fragment patterns of the isolates in Nagasaki, as determined by PFGE, were compared with those of isolates in other areas where large outbreaks and sporadic cases of E. coli O157 infection occurred. Similarity values of all the strains isolated in Nagasaki Prefecture were over 0.65 except for E. coli O26. Some strains were identical to the strains isolated from the areas where large outbreaks occurred. All strains were susceptible to ampicillin, fosfomycin, minocycline, amikacin, ofloxacin and sulfamethoxazole-trimethoprim.     

A high molecular weight glycoprotein in seminal plasma is a sperm immobilizing factor in the teleost Nile tilapia, Oreochromis niloticus

Sperm that have acquired potential for motility are kept immotile in seminal plasma in the teleost, Nile tilapia. In order to investigate the mechanism of immobilization, several experiments were performed using a previously characterized monoclonal antibody (TAT-30) against a molecular weight (Mr) = 120,000 protein that is secreted by Sertoli cells and epithelial cells of the sperm duct, and is also bound to the head of the spermatozoon. First, we assessed sperm motility in the seminal plasma protein fraction (SPP), and demonstrated that the sperm motility is inhibited by SPP in a concentration-dependent manner. Furthermore, sperm motility was recovered if SPP was pretreated with TAT-30, suggesting that the TAT-30 antigen is one of the components of the sperm immobilizing factor. Calibration by gel filtration followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting with TAT-30 demonstrated that the sperm immobilizing factor was more than Mr = 1,000,000 in seminal plasma, suggesting that it is a homopolymer of the Mr = 120,000-TAT-30 positive protein. Additionally, lectin blot analysis showed that the TAT-30 antigen was reactive with Lens culinarin agglutinin (LCA) and Conavalia ensiformis agglutinin (ConA), indicating that it is a glycoprotein. Immunohistochemical studies showed that the TAT-30 antigen was localized specifically on the heads of spermatozoa and on the apical surface, lysosomes and rough endoplasmic reticulum of Sertoli cells.     

Mouse Lefty2 and zebrafish antivin are feedback inhibitors of nodal signaling during vertebrate gastrulation.

Mammalian lefty and zebrafish antivin form a subgroup of the TGF beta superfamily. We report that mouse mutants for lefty2 have an expanded primitive streak and form excess mesoderm, a phenotype opposite to that of mutants for the TGF beta gene nodal. Analogously, overexpression of Antivin or Lefty2 in zebrafish embryos blocks head and trunk mesoderm formation, a phenotype identical to that of mutants caused by loss of Nodal signaling. The lefty2 mutant phenotype is partially suppressed by heterozygosity for nodal. Similarly, the effects of Antivin and Lefty2 can be suppressed by overexpression of the nodal-related genes cyclops and squint or the extracellular domain of ActRIIB. Expression of antivin is dependent on Nodal signaling, revealing a feedback loop wherein Nodal signals induce their antagonists Lefty2 and Antivin to restrict Nodal signaling during gastrulation.     

Evaluation of Voltage-Sensitive Dyes for Long-Term Recording of Neural Activity in the Hippocampus

We searched for an optimal voltage-sensitive dye for optical measurements of neural activity in the hippocampal slice by evaluating several merocyanine-rhodanine and oxonol dyes. The wavelength dependence (action spectra), pharmacological effects of staining, signal size, signal-to-noise ratio, and the utility of the dyes for long-term continuous recording were examined for four merocyanine-rhodanine dyes (NK2761, NK2776, NK3224 and NK3225), which had been reported to be optimal in embryonic nervous systems, and for two oxonol dyes (NK3630 (RH482) and NK3041 (RH155)), which have been among the most popular potentiometric probes for the hippocampal slice preparation. NK2761, NK3224 and NK3225 provided large signal-to-noise ratios, and proved to be useful for optical recordings lasting several hours. NK3630 was most suitable for long-term recording, although the signal-to-noise ratio was slightly inferior to that of the merocyanine-rhodanines. Using NK3630 (RH482) on the hippocampal slice preparation, we demonstrate here that long-term potentiation can be monitored stably for more than 8 hr. Received: 16 June 1999/Revised: 4 August 1999     

Synergistic role of c-Myc and ERK1/2 in the mitogenic response to TGFβ-1 in cultured rat nucleus pulposus cells

Introduction  

Although transforming growth factor β1 (TGFβ1) is known to be a potent inhibitor of proliferation in most cell types, it accelerates proliferation in certain mesenchymal cells, such as articular chondrocytes and nucleus pulposus cells. The low ability for self-renewal of nucleus pulposus cells is one obstacle in developing new therapeutic options for intervertebral disc diseases, and utilizing cytokines is one of the strategies to regulate nucleus pulposus cell proliferation. However, the precise cell cycle progression and molecular mechanisms by which TGFβ1 stimulates cell growth remain unclear. The aim of this study was to elucidate a mechanism that enables cell proliferation with TGFβ1 stimulation.     

Inhibition of acetylcholinesterase by metabolites of copper pyrithione (CuPT) and its possible involvement in vertebral deformity of a CuPT-exposed marine teleostean fish

In a previous study, we demonstrated that exposure to an antifouling biocide, copper pyrithione (CuPT), early during life induced vertebral deformity in the larvae of a marine fish, the mummichog (Fundulus heteroclitus). Skeletal deformities may be caused by inhibition by of acetylcholiensterase (AChE) activity, and to elucidate the mechanism underlying the CuPT-associated vertebral deformity, we first examined whether CuPT, zinc pyrithione (ZnPT), and their degradation products could inhibit AChE activity in the fish. Two of the degradation products, 2,2′-dipyridyldisulfide [(PS)₂] and 2,2′-dithiobispyridine-N-oxide [(PT)₂], but neither CuPT nor ZnPT, exhibited prominent AChE-inhibiting activity. Secondly, thin-layer chromatography revealed that mummichog hepatic microsomes metabolized CuPT to produce (PS)₂ in a microsome-dependent manner. The AChE inhibition induced in CuPT-exposed fish is likely due to (PS)₂ that was produced through metabolism of acquired CuPT. (PS)₂ may cause therefore skeletal deformity in CuPT-exposed fish by means of its neuromuscular blocking properties, through a mechanism similar to that proposed for animals exposed to organophosphorous pesticides.     

A novel mechanism in maggot debridement therapy: protease in excretion/secretion promotes hepatocyte growth factor production

Maggot debridement therapy (MDT) is effective for treating intractable wounds, but its precise molecular mechanism, including the association between MDT and growth factors, remains unknown. We administered MDT to nine patients (66.3 ± 11.8 yr, 5 male and 4 female) with intractable wounds of lower extremities because they did not respond to conventional therapies. Significant increases of hepatocyte growth factor (HGF) levels were observed in femoral vein blood during 48 h of MDT (P < 0.05), but no significant change was found for vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor-β1 (TGF-β1), or tumor necrosis factor-α (TNF-α). We conducted NIH-3T3 cell stimulation assay to evaluate the relation between HGF and protease activity in excretion/secretion (ES) derived from maggots. Compared with the control group, HGF was significantly higher in the 0.05 μg/ml ES group (P < 0.01). Furthermore, protease inhibitors suppressed the increase of HGF (P < 0.05). The HGF expression was increased in proportion to the ES protein concentration of 0.025 to 0.5 μg/ml. In fact, ES showed stronger capability of promoting HGF production and less cytotoxicity than chymotrypsin or bromelain. HGF is an important factor involved in cutaneous wound healing. Therefore, these results suggest that formation of healthy granulation tissue observed during MDT results from the increased HGF. Further investigation to identify molecules enhancing HGF expression by MDT will contribute greatly to drug target discovery for intractable wound healing therapy.