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.     

Foodborne viruses

Foodborne and waterborne viral infections are increasingly recognized as causes of illness in humans. This increase is partly explained by changes in food processing and consumption patterns that lead to the worldwide availability of high-risk food. As a result, vast outbreaks may occur due to contamination of food by a single foodhandler or at a single source. Although there are numerous fecal-orally transmitted viruses, most reports of foodborne transmission describe infections with Norwalk-like caliciviruses (NLV) and hepatitis A virus (HAV), suggesting that these viruses are associated with the greatest risk of foodborne transmission. NLV and HAV can be transmitted from person to person, or indirectly via food, water, or fomites contaminated with virus-containing feces or vomit. People can be infected without showing symptoms. The high frequency of secondary cases of NLV illness and - to a lesser extent - of hepatitis A following a foodborne outbreak results in amplification of the problem. The burden of illness is highest in the elderly, and therefore is likely to increase due to the aging population. For HAV, the burden of illness may increase following hygienic control measures, due to a decreasing population of naturally immune individuals and a concurrent increase in the population at risk. Recent advances in the research of NLV and HAV have led to the development of molecular methods which can be used for molecular tracing of virus strains. These methods can be and have been used for the detection of common source outbreaks. While traditionally certain foods have been implicated in virus outbreaks, it is clear that almost any food item can be involved, provided it has been handled by an infected person. There are no established methods for detection of viruses in foods other than shellfish. Little information is available on disinfection and preventive measures specifically for these viruses. Studies addressing this issue are hampered by the lack of culture systems. As currently available routine monitoring systems exclusively focus on bacterial pathogens, efforts should be made to combine epidemiological and virological information for a combined laboratory-based rapid detection system for foodborne viruses. With better surveillance, including typing information, outbreaks of foodborne infections could be reported faster to prevent further spread.     

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.     

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.     

Dynamics of orientation selectivity in the primary visual cortex and the importance of cortical inhibition

To test theories of orientation selectivity in primary visual cortex (V1), we have done experiments to measure the dynamics of orientation tuning of single neurons in the V1 cortex of macaque monkeys. Based on our dynamics results, we propose that a V1 cell's orientation selectivity is generated mainly by both tuned enhancement and global suppression. Enhancement near the preferred orientation is probably caused by feed-forward input from LGN (plus amplification by cortical-cortical interaction). Global suppression could be supplied by cortical inhibition. Additionally, in about 1/3 of V1 neurons (usually the most sharply tuned) there is tuned suppression, centered near the cell's preferred orientation but broader than tuned enhancement. These mechanisms also can explain important features of steady-state selectivity in the V1 neuron population. Furthermore, similar neuronal mechanisms may be used generally throughout the cerebral cortex.