Liposomes functionalized with monoclonal antibodies or their antigen-binding fragments have attracted much attention as specific drug delivery devices for treatment of various diseases including cancer. The conjugation of antibodies to liposomes is usually achieved by covalent coupling using cross-linkers in a reaction that might adversely affect the characteristics of the final product. Here we present an alternative strategy for liposome functionalization: we created a recombinant Fab antibody fragment genetically fused on its C-terminus to the hydrophobic peptide derived from pulmonary surfactant protein D, which became inserted into the liposomal bilayer during liposomal preparation and anchored the Fab onto the liposome surface. The Fab-conjugated liposomes specifically recognized antigen-positive cells and efficiently delivered their cargo, the Alexa Fluor 647 dye, into target cells in vitro and in vivo. In conclusion, our approach offers the potential for straightforward development of nanomedicines functionalized with an antibody of choice without the need of harmful cross-linkers.

• Keywords
• Active targeting, Antibody engineering, Immunoliposome, Liposome functionalization, Recombinant Fab antibody fragment,
• MeSH
• CD48 Antigen metabolism   MeSH
• CD59 Antigens metabolism   MeSH
• Immunoglobulin Fab Fragments chemistry   immunology   metabolism   MeSH
• Jurkat Cells MeSH
• Humans MeSH
• Liposomes chemistry   MeSH
• Lymphoma immunology   metabolism   pathology   MeSH
• Antibodies, Monoclonal chemistry   immunology   metabolism   MeSH
• Mice MeSH
• Tumor Cells, Cultured MeSH
• Peptide Fragments immunology   metabolism   MeSH
• Pulmonary Surfactant-Associated Protein D immunology   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CD48 Antigen MeSH
• CD59 Antigens MeSH
• CD48 protein, human MeSH Browser
• CD59 protein, human MeSH Browser
• Immunoglobulin Fab Fragments MeSH
• Liposomes MeSH
• Antibodies, Monoclonal MeSH
• Peptide Fragments MeSH
• Pulmonary Surfactant-Associated Protein D MeSH

Several methods of molecular analysis of microbial diversity, including terminal restriction fragment length polymorphism (T-RFLP) analysis are based on measurement of the DNA fragment length. Significant variation between sequence-determined and measured length of restriction fragments (drift) has been observed, which can affect the efficiency of the identification of microorganisms in the analyzed communities. In the past, this variation has been attributed to varying fragment length and purine content. In this study, principal component analysis and multiple regression analysis were applied to find the contributions of those and several other fragment characteristics. We conclude that secondary structure melting point and G+C nucleotide content, besides the fragment length, contribute to the variation observed, whereas the contribution of purine content is less important. Incomplete denaturation of the sample at the start of electrophoretic separation of fragments has been excluded as a major cause of the variation observed. Our regression model explains the observed drift variation by approximately 56%, with standard deviation of the prediction equal to approximately 1.3 bp.

• MeSH
• Amplified Fragment Length Polymorphism Analysis methods   standards   MeSH
• DNA, Fungal chemistry   genetics   MeSH
• Fungi chemistry   genetics   isolation & purification   MeSH
• Nucleic Acid Conformation MeSH
• Phalaris microbiology   MeSH
• Polymorphism, Restriction Fragment Length * MeSH
• Transition Temperature MeSH
• Base Composition MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Fungal MeSH

Single-chain variable antibody fragments (scFvs) are molecules with immense therapeutic and diagnostic potential. Knowledge of their three-dimensional structure is important for understanding their antigen-binding mode as well as for protein-engineering approaches such as antibody humanization. A major obstacle to the crystallization of single-chain variable antibody fragments is their relatively poor homogeneity caused by spontaneous oligomerization. A new approach to optimization of the crystallizability of single-chain variable antibody fragments is demonstrated using a representative single-chain variable fragment derived from the anti-CD3 antibody MEM-57. A Thermofluor-based assay was utilized to screen for optimal conditions for antibody-fragment stability and homogeneity. Such an optimization of the protein storage buffer led to a significantly improved ability of the scFv MEM-57 to yield crystals.

• Keywords
• Thermofluor assay, crystallizability optimization, crystallization, differential scanning fluorimetry, oligomerization, single-chain antibody fragment,
• MeSH
• Chromatography, Gel MeSH
• Single-Chain Antibodies chemistry   MeSH
• Crystallization * MeSH
• Humans MeSH
• Molecular Sequence Data MeSH
• Amino Acid Sequence MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Single-Chain Antibodies MeSH

The Hageman factor fragment (HFf) concentrate was prepared from acetone and dextran sulphate activated human plasma by chromatography on a CM-Sephadex column. The preparation was free of the main kallikrein inhibitors--Cl-inactivator, alpha 2-macroglobulin, antithrombin III and alpha 1-antitrypsin. The HFf concentrate can serve as an activator of prekallikrein in patient plasma.

• MeSH
• Enzyme Activation MeSH
• Chromatography, Liquid MeSH
• Factor XII chemistry   isolation & purification   MeSH
• Blood MeSH
• Humans MeSH
• Peptide Fragments chemistry   isolation & purification   MeSH
• Prekallikrein metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Factor XII MeSH
• Peptide Fragments MeSH
• Prekallikrein MeSH

Complement consumption induced by Fab' fragments of rabbit IgG, irrespectively of their state of aggregation, requires the presence of homologous rabbit IgG. Up to a certain concentration of Fab' fragment, the amount of complement fixed is a linear function of the Fab' fragment dose, but further raising of the Fab'-fragment concentration does not result in complete complement consumption in the sample. The interaction between Fab' fragment and IgG is not strictly species specific.

• MeSH
• Species Specificity MeSH
• gamma-Globulins metabolism   MeSH
• Immunoglobulin Fab Fragments * MeSH
• Complement Fixation Tests * MeSH
• Rabbits MeSH
• Guinea Pigs MeSH
• Animals MeSH
• Check Tag
• Rabbits MeSH
• Guinea Pigs MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• gamma-Globulins MeSH
• Immunoglobulin Fab Fragments * MeSH

Reverse transcription-polymerase chain reaction (RT-PCR) technique and restriction fragment length polymorphism (RFLP) analysis were used to analyse six isolates of plum pox virus (PPV). Whole coat protein (CP) gene was amplified in four isolates using the unipoty-polyT primer pari. PPV-D was identified by RFLP analysis using SfuI and DraI enzymes in two of the isolates. Two isolates of PPV-M strain yielded RT-PCR products which could not be digested by the two enzymes. Other isolates were subjected to RT-PCR using P1-P2 primers. The specificity of the RT-PCR products was confirmed by AluI digestion, while RsaI digestion enabled strain differentiation. No mixed infection was found.

• MeSH
• Capsid genetics   MeSH
• Plant Diseases virology   MeSH
• Fruit MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Polymorphism, Restriction Fragment Length * MeSH
• RNA, Viral isolation & purification   MeSH
• Rosales virology   MeSH
• Trees virology   MeSH
• Plum Pox Virus classification   genetics   isolation & purification   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Viral MeSH

The article presents a study of the effect of a modified fragment of neuropeptide Y (H-L-Ile-L-Asn-L-Leu-L-Nle-L-Ser- L-Arg-L-Asn-L-Arg-L-Tyr-NH2) named nonapeptide NP9 on the memory phases and extrapolation escape of animals. The study was performed in the passive avoidance test with intact animals, scopolamine-treated animals, and the extrapolation escape task. NP9 was investigated in the dose range of 0.04-0.4 mg/kg with a single intranasal administration. The comparison drug used peptide nootropic medicine Semax® (Met-Glu-His-Phe-Pro-Gly-Pro) at a dose of 0.1 mg/kg. Efficiency was assessed by the retention latency, the percentage of animals that have reached the learning criterion, the number of incomplete attempts to enter, the antiamnestic activity index calculated by Butlers formula, and the number of animals that successfully performed the extrapolation escape task. Peptide NP9 was superior to Semax® in most indicators. It demonstrated the ability to improve memorization due to its effect on I phase of memory and facilitated extinction of negative experiences when administered after a stress stimulus. NP9 also increased the cognitive ability of animals in the conditions an aversive environment in the extrapolation escape test. Thus, peptide NP9 is promising for a further study as a potential drug for the treatment of cognitive impairment and therapy of post-traumatic stress disorder.

• Keywords
• extrapolation escape task, memory phases, modified fragment of neuropeptide Y, passive avoidance test,
• MeSH
• Neuropeptide Y * administration & dosage   pharmacology   MeSH
• Oligopeptides * administration & dosage   pharmacology   MeSH
• Memory drug effects   MeSH
• Peptide Fragments * administration & dosage   pharmacology   MeSH
• Amino Acid Sequence MeSH
• Escape Reaction drug effects   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Neuropeptide Y * MeSH
• Oligopeptides * MeSH
• Peptide Fragments * MeSH

Precipitating and non-precipitating anti-Dnp antibodies and S-sulpho non-specific IgG in gram quantities were subjected to limited cleavage by trypsin. Upon gel chromatography on Sephadex G-100 the fraction of Fab and Fc fragments was separated from incompletely split molecules and from tFc' fragments. The Fab and Fc fragments were separated from each other either by ion-exchange chromatography on QAE-Sephadex or by preparative electrophoresis in starch block. Both Fab and Fc fragments appeared to be heterogeneous as to electric charge. The Fc fragments were characterized by amino acid composition and N-terminal amino acids. The Fc fragment of non-specific IgG was cleaved by cyanogen bromide, and a C-terminal peptide containing 18 residues was isolated. Partial amino acid sequence of this peptide pointed to a high degree of homology with immunoglobulins of other animal species.

• MeSH
• Amino Acids analysis   MeSH
• Cyanogen Bromide MeSH
• Dinitrophenols immunology   MeSH
• Immunoglobulin G analysis   MeSH
• Immunoglobulin Fab Fragments isolation & purification   MeSH
• Immunoglobulin Fc Fragments isolation & purification   MeSH
• Immunoglobulin Fragments isolation & purification   MeSH
• Peptides analysis   MeSH
• Swine immunology   MeSH
• Precipitins analysis   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Amino Acids MeSH
• Cyanogen Bromide MeSH
• Dinitrophenols MeSH
• Immunoglobulin G MeSH
• Immunoglobulin Fab Fragments MeSH
• Immunoglobulin Fc Fragments MeSH
• Immunoglobulin Fragments MeSH
• Peptides MeSH
• Precipitins MeSH

The wavelength-dependent tryptophan (Trp) fluorescence decays of Ca-prothrombin fragment 1 (Ca-BF1), which contains three tryptophan residues, in the presence of pure phosphatidylcholine (PC) small unilamellar vesicles (SUV) and PC-SUV containing either 25% phosphatidyl-l-serine (PS), and 25% or 40% phosphatidylglycerol (PG) are characterized, using fluorescence lifetime distribution, conventional multiexponential, and global analysis. In analogy to previous investigations on apo- and Ca-BF1 (M. Hof, G.R. Fleming, V. Fidler, Proteins Struct. Func. Genet. 24 (1996) 485-494), the analysis resulted in a five exponential decay model in all investigated systems, where the five fluorescence lifetimes (e.g. 0. 04+/-0.02 ns (component A), 0.24+/-0.02 ns (B), 0.66+/-0.03 ns (C), 2.4+/-0.3 ns (D), and 5.4+/-0.4 ns (E) for Ca-BF1 in the presence of PC-SUV) are wavelength-independent. The fluorescence lifetimes and the corresponding amplitudes of the 'Gla-Trp' (components D and E) and of the two 'kringle-Trp' (components B, C, and D) remain unchanged when bound to the PS-containing vesicles. Saturation binding to PG-containing membranes leads to a prolongation of the Gla component E from 5.3 in solution to 7.5 ns, indicating a change in the Gla-domain conformation. The results represent the first experimental evidence of a lipid-specific conformational change in the N-terminal 'Gla domain' of a vitamin K-dependent protein.

• MeSH
• Spectrometry, Fluorescence MeSH
• Phosphatidylcholines MeSH
• Phosphatidylglycerols MeSH
• Phosphatidylserines MeSH
• Membranes, Artificial MeSH
• Peptide Fragments chemistry   MeSH
• Protein Precursors chemistry   MeSH
• Prothrombin chemistry   MeSH
• Cattle MeSH
• Tryptophan chemistry   MeSH
• Calcium MeSH
• Animals MeSH
• Check Tag
• Cattle MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Phosphatidylcholines MeSH
• Phosphatidylglycerols MeSH
• Phosphatidylserines MeSH
• Membranes, Artificial MeSH
• Peptide Fragments MeSH
• Protein Precursors MeSH
• prothrombin fragment 1 MeSH Browser
• Prothrombin MeSH
• Tryptophan MeSH
• Calcium MeSH

OBJECTIVES: To characterize by restriction fragment length polymorphism (RFLP) patterns, the distribution of different Mycobacterium tuberculosis strains isolated consecutively from 75 tuberculosis patients who resided in Prague and had culture-confirmed cases during a 4-month period in 1995. METHODS: The insertion sequence IS6110-based RFLP analysis of M. tuberculosis isolates was carried out. RESULTS: There were a total of 75 patients with various forms of tuberculosis (54 males; 21 females). The sources of M. tuberculosis isolates were sputum (n = 64), pleura or lymph node drainage (n = 8), and urine (n = 3). Fifty-three of the patients (70.7%) had isolates with unique RFLP patterns, while 22 (29.3%) had isolates that belonged to seven clusters of related RFLP patterns. The seven clusters consisted of four groups of two patients, two groups of four patients, and one group of six patients. Most of the patients whose isolates fell within a clustered RFLP pattern lived in different quarters of the city and had no identifiable contacts with other patients whose isolates had the same pattern. CONCLUSIONS: The finding that isolates from most patients (70.7%) had unique rather than clustered RFLP patterns suggests that endogenous reactivation rather than exogenous transmission is the major determinant of most of the tuberculosis cases in Prague. The occurrence of seven distinct clusters comprising 29.3% of the isolates suggests that approximately one third of cases developed active tuberculosis from recent exogenous transmission.

• MeSH
• DNA, Bacterial analysis   MeSH
• DNA Fingerprinting MeSH
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Lymph Nodes microbiology   MeSH
• Urine microbiology   MeSH
• Molecular Epidemiology MeSH
• Mycobacterium tuberculosis genetics   isolation & purification   MeSH
• Polymorphism, Restriction Fragment Length * MeSH
• Aged MeSH
• Sputum microbiology   MeSH
• Tuberculosis epidemiology   microbiology   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic epidemiology   MeSH
• Names of Substances
• DNA, Bacterial MeSH