Quantitation of the tumor-targeting properties of antibody fragments conjugated to cell-permeating HIV-1 TAT peptides

Human monoclonal antibodies are promising agents for the development of more selective anticancer therapeutics. However, the tumor-targeting efficiency of most anticancer antibodies is severely limited by their poor penetration into the tumor mass. Recent studies have shown that a peptide derived from the HIV TAT protein could improve the distribution of cytoplasmic reporter proteins when administered systemically as fusion proteins or cross-linked chimeras. In this article, we tested by quantitative biodistribtution analysis whether conjugation to TAT peptides could improve the tumor targeting properties of scFv(L19)-Cys: an engineered human antibody fragment specific for the ED-B domain of fibronectin, a marker located in the modified extracellular matrix surrounding tumor neovasculature. Our results show that TAT peptides, consisting either of L-amino acids or D-amino acids, can efficiently transduce target cells when conjugated to fluorophores and/or antibody fragments, suggesting a receptor-independent cell entry mechanism. However, conjugation of scFv(L19)-Cys to TAT peptides resulted in a severely reduced tumor targeting performance compared to the unconjugated antibody, as measured in murine F9 teratocarcinoma-bearing mice, after intravenous injection of the radiolabeled antibody preparations. Our results outline the usefulness of TAT peptides for the efficient in vitro transduction of cells with globular proteins. In particular, the use of TAT peptides composed of D-amino acids may significantly reduce proteolytic degradation. At the same time, the poor biodistribution properties of antibody-TAT conjugates cast doubts over the applicability of this methodology for the delivery of biopharmaceuticals in vivo.     

Human melanoma TrkC: its association with a purine-analog-sensitive kinase activity

The various members of the Trk tyrosine kinase family and p75 neurotrophin receptor (p75(NTR)) have been identified as signaling receptors for the structurally related members of the neurotrophins (NT) family. We have previously reported that NT treatment of murine and human brain-metastatic melanoma cells affects their invasive capacities and increases the production of extracellular-matrix degradative enzymes. These cells express aberrant levels of functional p75(NTR) and TrkC, the putative high-affinity receptor for the neurotrophin NT-3. Here we demonstrate that, by using sensitive immune-complex kinase assays in human brain-metastatic (70W) melanoma cells, TrkC receptors associate with a kinase activity exhibiting a dose-dependent susceptibility to inhibition by the purine-analogs 6-thioguanine and 2-aminopurine. The activity of this purine-analog-sensitive kinase (PASK) was induced by NT-3 in a time-dependent fashion, phosphorylating exogenous myelin basic protein (MBP) but not denatured enolase. It is similar to the one reported to relate with p75(NTR) and TrkA receptors and stimulated by the prototypic NT, nerve growth factor. Thus, PASKs may represent unique signaling components common to NT receptors that could engage joint downstream signaling effectors in brain-metastatic melanoma.     

Safety and efficacy of office-based surgery with monitored anesthesia care/sedation in 4778 consecutive plastic surgery procedures

Office-based surgery has several potential benefits over hospital-based surgery, including cost containment, ease of scheduling, and convenience to both patients and surgeons. Scrutiny of office-based surgery by regulators and state-licensing agencies has increased and must be addressed by improved documentation of safety and efficacy. To evaluate the safety and efficacy of the authors' office-based plastic surgery, a review was undertaken of 3615 consecutive patients undergoing 4778 outpatient plastic surgery procedures under monitored anesthesia care/sedation in a single office. The charts of 3615 consecutive patients who had undergone office-based surgery with monitored anesthesia care/sedation between May of 1995 and May of 2000 were reviewed. In all cases, the anesthesia protocol used included sedation with midazolam, propofol, and a narcotic administered by a board-certified registered nurse anesthetist with local anesthesia provided by the surgeon. Charts were reviewed for patient profile, types of procedures, multiple procedures, duration of anesthesia, American Society of Anesthesiologists class, and complications related to anesthesia. Outcomes measured included death, airway compromise, dyspnea, hypotension, venous thrombosis, pulmonary emboli, protracted nausea and vomiting lasting more than 24 hours, and unplanned hospital admissions. Statistical analyses were performed using the Microsoft Excel program and the SAS package. Results were as follows: 92.3 percent of the patients were female and 7.7 percent were male, with a mean age of 42.7 years (range, 3 to 83 years). Patients underwent aesthetic (95.6 percent) and reconstructive (4.4 percent) plastic surgery procedures. Same-session multiple procedures occurred in 24.8 percent of patients. The vast majority of patients were healthy: 84.3 percent of patients were American Society of Anesthesiologists class I, 15.6 percent were class II, and 0.1 percent were class III. The operations required a mean of 111 minutes. There were no deaths, ventilator requirements, deep venous thromboses, or pulmonary emboli. Complications were as follows: 0.05 percent (n = 2) of patients had dyspnea that resolved, 0.2 percent (n = 6) of patients had protracted nausea and vomiting, and 0.05 percent (n = 2) of patients had unplanned hospital admissions (<24 hours). One patient had an emergent intubation. No prolonged adverse effects were noted. There was a 30-day follow-up minimum. Outpatient surgery is an important aspect of plastic surgery. It was shown that office-based surgery with intravenous sedation, performed by board-certified plastic surgeons and nurse anesthetists, is safe. Appropriate accreditation, safe anesthesia protocols, and proper patient selection constitute the basis for safe and efficacious office-based outpatient plastic surgery.     

Proteomics application exercise of the Swiss Proteomics Society: report of the SPS'02 session

After the success of the mass spectrometry (MS) round table that was held at the first Swiss Proteomics Society congress (SPS'01) in Geneva, the SPS has organized a proteomics application exercise and allocated a full session at the SPS'02 congress. The main objective was to encourage the exchange of expertise in protein identification, with a focus on the use of mass spectrometry, and to create a bridge between the users' questions and the instrument providers' solutions. Two samples were sent to fifteen interested labs, including academic groups and MS hardware providers. Participants were asked to identify and partially characterize the samples. They consisted of a complex mixture of peptide/proteins (sample A) and an almost pure recombinant peptide carrying post-translational modifications (sample B). Sample A was an extract of snake venom from the species Bothrops jararaca. Sample B was a recombinant and modified peptide derived from the shrimp Penaeus vannamei penaeidin 3a. The eight labs that returned results reported the use of a wide range of MS instrumentation and techniques. They mentioned a variety of time and manpower allocations. The origin of sample A was generally identified together with a number of database protein entries. The difficulty of the sample identification lay in the incomplete knowledge of the Bothrops species genome sequence and is discussed. Sample B was generally and correctly identified as penaeidin. However, only one group reported the full primary structure. Interestingly, the approaches were again varied and are discussed in the text.     

Annual fishes of the genus Nothobranchius as a model system for aging research

Aging research in vertebrates is hampered by the lack of short-lived models. Annual fishes of the genus Nothobranchius live in East African seasonal ponds. Their life expectancy in the wild is limited by the duration of the wet season and their lifespan in captivity is also short. Nothobranchius are popular aquarium fishes and many different species are kept as captive strains, providing rich material for comparative studies. The present paper aims at reviving the interest in these fishes by reporting that: (1) Nothobranchius can be cultured, and their eggs stored dry at room temperature for months or years, offering inexpensive methods of embryo storage; (2) Nothobranchius show accelerated growth and expression of aging biomarkers at the level of histology and behaviour; (3) the species Nothobranchius furzeri has a maximum lifespan of only 3 months and offers the possibility to perform investigations thus far unthinkable in a vertebrate, such as drug screening with life-long pharmacological treatments and experimental evolution; (4) when the lifespan of different species is compared, a general correlation is found between wet season duration in their natural habitat and longevity in captivity; and (5) vertebrate aging-related genes, such as p66Shc and MTP, can be easily isolated in Nothobranchius by homology cloning. These fishes can become excellent models for aging studies. They can be employed to test the effects of experimental manipulation on aging at a pace comparable with that of Drosophila and to probe the effects of natural selection on the evolution of aging-related genes.     

Dendritic cell-associated lectin-1: a novel dendritic cell-associated,C-type lectin-like molecule enhances T cell secretion of IL-4

We have characterized dendritic cell (DC)-associated lectin-1 (DCAL-1), a novel, type II, transmembrane, C-type lectin-like protein. DCAL-1 has restricted expression in hemopoietic cells, in particular, DCs and B cells, but T cells and monocytes do not express it. The DCAL-1 locus is within a cluster of C-type lectin-like loci on human chromosome 12p12-13 just 3' to the CD69 locus. The consensus sequence of the DCAL-1 gene was confirmed by RACE-PCR; however, based on sequence alignment with genomic DNA and with various human expressed sequence tags, we predict that DCAL-1 has two splice variants. C-type lectins share a common sequence motif of 14 invariable and 18 highly conserved aa residues known as the carbohydrate recognition domain. DCAL-1, however, is missing three of the cysteine residues required to form the standard carbohydrate recognition domain. DCAL-1 mRNA and protein expression are increased upon the differentiation of monocytes to CD1a(+) DCs. B cells also express high levels of DCAL-1 on their cell surface. Using a DCAL-1 fusion protein we identified a population of CD4(+) CD45RA(+) T cells that express DCAL-1 ligand. Coincubation with soluble DCAL-1 enhanced the proliferation of CD4(+) T cells in response to CD3 ligation and significantly increased IL-4 secretion. In contrast, coincubation with soluble DC-specific ICAM-3-grabbing nonintegrin (CD209) fusion protein as a control had no effect on CD4(+) T cell proliferation or IL-4 and IFN-gamma secretion. Therefore, the function of DCAL-1 on DCs and B cells may act as a T cell costimulatory molecule, which skews CD4(+) T cells toward a Th2 response by enhancing their secretion of IL-4.     

Time-resolved methods in biophysics. 4. Broadband pump-probe spectroscopy system with sub-20 fs temporal resolution for the study of energy transfer processes in photosynthesis.

In this paper we discuss how to push the temporal resolution limits of transient absorption spectroscopy in order to detect very fast processes (energy relaxation, energy or charge transfer, vibrational coherence) taking place in molecules of biological relevance. After reviewing the main principles of femtosecond pump-probe spectroscopy, we describe an experimental setup based on two synchronized non-collinear optical parametric amplifiers (NOPAs). Each NOPA can be independently configured to generate ultra-broadband sub-10 fs visible pulses, tunable 10-15 fs visible pulses, tunable 15-40 fs near-infrared pulses (900-1500 nm). This system enables to perform pump-probe experiments over nearly two octaves of spectrum with sub-20 fs temporal resolution. We then present an application example highlighting the capability of this instrument to track excited state dynamics in biomolecules on the sub-100 fs timescale: the study of carotenoid-bacteriochlorophyll energy transfer processes in peripheral light-harvesting complexes (LH2) from purple bacteria. We show that, by comparing excited-state dynamics of the carotenoids in organic solvents and inside the LH2 complexes, it is possible to visualize in the time domain the primary events in photosynthesis.