The inhibitory effect of dinitrosyl iron complexes (NO donors) on myeloperoxidase activity

The effect of synthetic analogues of dinitrosyl mononuclear iron complexes (DNICs) with functional sulfur-containing ligands (NO donors) on the activity of myeloperoxidase (MPO) was studied, and their efficiency was evaluated. It was shown that the enzyme MPO is the molecular target of DNICs. It was found that six DNICs inhibited the activity of MPO and one compound potentiated it. The evaluation of their efficiency showed that two DNICs effectively inhibited the activity of MPO by 50% at IC₅₀ = 2 × 10^–4 M and IC₅₀ = 5 × 10^–7 M.     

Some features of the mutability of bacteria during nonlethal selection

We describe the mutability of the Trp(-) chromosomal +1 frameshift mutation trpE7999 during nonlethal selection, finding that the appearance of Trp(+) revertants behaves similarly to that of episomal Lac(+) revertants. In addition, we show that a feature of the Lac(+) and Trp(+) mutability is the accumulation of Trp(+) and Lac(+) revertants with additional unselected mutations, most of which are not due to heritable mutators. The cells undergoing nonlethal selection apparently experience an epigenetic change resulting in a subset of bacteria with elevated mutability that often remain hypermutable for the duration of selection. The epigenetic change provoked by nonlethal selection appears to be mediated by a unique function provided by the F'128 episome.     

Brain uptake of multivalent and multi-specific DVD-Ig proteins after systemic administration

Therapeutic monoclonal antibodies and endogenous IgG antibodies show limited uptake into the central nervous system (CNS) due to the blood-brain barrier (BBB), which regulates and controls the selective and specific transport of both exogenous and endogenous materials to the brain. The use of natural transport mechanisms, such as receptor-mediated transcytosis (RMT), to deliver antibody therapeutics into the brain have been studied in rodents and monkeys. Recent successful examples include monovalent bispecific antibodies and mono- or bivalent fusion proteins; however, these formats do not have the capability to bind to both the CNS target and the BBB transport receptor in a bivalent fashion as a canonical antibody would. Dual-variable-domain immunoglobulin (DVD-Ig) proteins offer a bispecific format where monoclonal antibody-like bivalency to both the BBB receptor and the therapeutic target is preserved, enabling independent engineering of binding affinity, potency, valency, epitope and conformation, essential for successful generation of clinical candidates for CNS applications with desired drug-like properties. Each of these parameters can affect the binding and transcytosis ability mediated by different receptors on the brain endothelium differentially, allowing exploration of diverse properties. Here, we describe generation and characterization of several different DVD-Ig proteins, specific for four different CNS targets, capable of crossing the BBB through transcytosis mediated by the transferrin receptor 1 (TfR1). After systemic administration of each DVD-Ig, we used two independent methods in parallel to observe specific uptake into the brain. An electrochemiluminescent-based sensitive quantitative assay and a semi-quantitative immunohistochemistry technique were used for brain concentration determination and biodistribution/localization in brain, respectively. Significantly enhanced brain uptake and retention was observed for all TfR1 DVD-Ig proteins regardless of the CNS target or the systemic administration route selected.     

A new approach to genome mapping and sequencing: slalom libraries

We describe here an efficient strategy for simultaneous genome mapping and sequencing. The approach is based on physically oriented, overlapping restriction fragment libraries called slalom libraries. Slalom libraries combine features of general genomic, jumping and linking libraries. Slalom libraries can be adapted to different applications and two main types of slalom libraries are described in detail. This approach was used to map and sequence (with ~46% coverage) two human P1-derived artificial chromosome (PAC) clones, each of ~100 kb. This model experiment demonstrates the feasibility of the approach and shows that the efficiency (cost-effectiveness and speed) of existing mapping/sequencing methods could be improved at least 5–10-fold. Furthermore, since the efficiency of contig assembly in the slalom approach is virtually independent of length of sequence reads, even short sequences produced by rapid, high throughput sequencing techniques would suffice to complete a physical map and a sequence scan of a small genome.     

The clinical picture of legionnaires' disease

The results of the clinico-laboratory study of 12 cases of acute pneumonia of Legionella etiology are presented. The laboratory diagnosis of Legionella infection was carried out by the study of paired sera in the passive hemagglutination test with the use of Legionella pneumophila (serotype 1) erythrocyte diagnosticum. The clinical picture of pneumonia was characterized by a severe and moderate course of the disease. Characteristic symptoms indicating the presence of indurations and infiltrations in the lung tissue were registered. Roentgenological examination revealed that the foci of pulmonary tissue infiltration appeared in the segments of the lower lobes of both lungs. In 6 patients neutrophil leukopenia, in 4 patients relative lymphocytopenia, in 5 patients monocytopenia, in 11 patients the increase of the erythrocyte sedimentation rate and in 4 patients normochromic anemia were registered. More seldom changes in the levels of residual nitrogen, urea, fibrinogen and transaminases were observed. In most cases the resolution of pneumonia was observed on weeks 2-3 of treatment. In this treatment erythromycin, rifampicin and oleandomycin, used in combination, used in combination with detoxication and infusion therapy, vitamins, vascular and other symptomatic remedies, proved to be most effective. The cases of Legionella infection under study were sporadic and epidemiologically unrelated. The severity of the course of the disease depended mainly on the general state of the patient prior to infection, age and concomitant diseases.     

NotI flanking sequences: a tool for gene discovery and verification of the human genome

A set of 22 551 unique human NotI flanking sequences (16.2 Mb) was generated. More than 40% of the set had regions with significant similarity to known proteins and expressed sequences. The data demonstrate that regions flanking NotI sites are less likely to form nucleosomes efficiently and resemble promoter regions. The draft human genome sequence contained 55.7% of the NotI flanking sequences, Celera’s database contained matches to 57.2% of the clones and all public databases (including non-human and previously sequenced NotI flanks) matched 89.2% of the NotI flanking sequences (identity ≥90% over at least 50 bp, data from December 2001). The data suggest that the shotgun sequencing approach used to generate the draft human genome sequence resulted in a bias against cloning and sequencing of NotI flanks. A rough estimation (based primarily on chromosomes 21 and 22) is that the human genome contains 15 000–20 000 NotI sites, of which 6000–9000 are unmethylated in any particular cell. The results of the study suggest that the existing tools for computational determination of CpG islands fail to identify a significant fraction of functional CpG islands, and unmethylated DNA stretches with a high frequency of CpG dinucleotides can be found even in regions with low CG content.     

Comparison of different delivery systems of DNA vaccination for the induction of protection against tuberculosis in mice and guinea pigs

The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs.