Analysis of the allelic diversity of the mycobacterial interspersed repetitive units in Mycobacterium tuberculosis strains of the Beijing family: practical implications and evolutionary considerations

A study set comprised 44 Mycobacterium tuberculosis strains of the Beijing family selected for their representativeness among those previously characterized by IS6110-RFLP and spoligotyping (Northwest Russia, 1997 to 2003). In the present study, these strains were subjected to mycobacterial interspersed repetitive units (MIRU) typing to assess a discriminatory power of the 12-MIRU-loci scheme (P. Supply et al., J. Clin. Microbiol. 39:3563-3571, 2001). The 44 Russian Beijing strains were subdivided into 12 MIRU types with identical profiles: 10 unique strains and two major types shared by 10 and 24 strains. Thus, basically, two distinct sublineages appear to shape the evolution of the Beijing strains in Russia. Most of the MIRU loci were found to be (almost) monomorphic in the Russian Beijing strains; the Hunter-Gaston discriminatory index (HGDI) for all 12 loci taken together was 0.65, whereas MIRU26 (the most variable in our study) showed a moderate level of discrimination (0.49). The results were compared against all available published MIRU profiles of Beijing strains from Russia (3 strains) and other geographic areas (51 strains in total), including South Africa (38 strains), East Asia (7 strains), and the United States (4 strains). A UPGMA (unweighted pair-group method with arithmetic averages)-based tree was constructed. Interestingly, no MIRU types were shared by Russian and South African strains (the two largest samples in this analysis), whereas both major Russian types included also isolates from other locations (United States and/or East Asia). This implies the evolution of the Beijing genotype to be generally strictly clonal, although a possibility of a convergent evolution of the MIRU loci cannot be excluded. We propose a dissemination of the prevailing local Beijing clones to have started earlier in South Africa rather than in Russia since more monomorphic loci were identified in Russian samples than in South African samples (mean HGDI scores, 0.08 versus 0.17). To conclude, we suggest to use a limited number of MIRUs for preliminary subdivision of Beijing strains in Russian (loci 26 + 31), South African (10 + 26 + 39), and global settings (10 + 26 + 39).     

Affinity fractionation of lymphocytes using a monolithic cryogel

A new type of continuous, supermacroporous, monolithic, cryogel affinity adsorbent was developed, allowing specific fractionation and separation of human peripheral blood lymphocytes in a chromatographic format. The affinity adsorbent was used to design a novel cell separation strategy, which was based on the interaction of protein A from Staphylococcus aureus with cells bearing IgG antibodies on the surface. After treating lymphocytes with goat anti-human IgG(H+L), the IgG-positive B-lymphocytes were efficiently separated from T-lymphocytes. Protein A covalently coupled to epoxy activated dimethylacrylamide (DMAA) cryogel matrix specifically bound IgG-bearing B-lymphocytes through the Fc region, while non-bound T-lymphocytes passed through the column. More than 90% of the B-lymphocytes were retained in the column while the cells in the breakthrough fraction were enriched in T-lymphocytes (81%). The viability of the T-lymphocytes isolated was greater than 90%. The bound lymphocytes released by human or dog IgG recovered 60-70% of the B-cells without significantly impairing the cell viability. The technique can be applied in general to cell separation systems where IgG antibodies against specific cell surface markers are available.     

Genetic Vaccination of Mice with Plasmids Encoding the NS1 Non-structural Protein from Tick-borne Encephalitis Virus and Dengue 2 Virus

Although there is a safe, inexpensive and efficacious vaccine against yellow fever, vaccination against other flavivirus diseases is less successful. There is no licensed vaccine against dengue fever and current vaccines against tick-borne encephalitis (TBE) and Japanese encephalitis are expensive and require several injections. Furthermore novel vaccines containing only virus envelope proteins may raise fears over antibody mediated enhancement (ADE) of disease. Here we report the successful use of genetic vaccination against TBE in an experimental animal model using a plasmid containing the coding sequence of a non-structural protein (NS1). Such vaccines would provide inexpensive protection against disease, without raising concerns over inducing ADE on subsequent exposure to heterotypic infectious virus. Attempts to generate chaemeric plasmids to protect against both TBE and dengue fever were less successful. Although these chaemeric plasmids directed the synthesis and secretion of the virus NS1 protein normally, no protection was observed against either TBE or dengue fever.     

Human temperature regulation during cycling with moderate leg ischaemia

The effect of graded ischaemia in the legs on the regulation of body temperature during steady-state exercise was investigated in seven healthy males. It was hypothesised that graded ischaemia in the working muscles increases heat storage within the muscles, which in turn potentiates sweat secretion during exercise. Blood perfusion in the working muscles was reduced by applying a supra-atmospheric pressure (+6.6 kPa) around the legs, which reduced maximal working capacity by 29%. Each subject conducted three separate test trials comprising 30 min of steady-state cycling in a supine position. Exercise with unrestricted blood flow (Control trial) was compared to ischaemic exercise conducted at an identical relative work rate (Relative trial), as well as at an identical absolute work rate (Absolute trial); the latter corresponding to a 20% increase in relative workload. The average (SD) increases in both the rectal and oesophageal temperatures during steady-state cycling was 0.3 (0.2)°C and did not significantly differ between the three trials. The increase in muscle temperature was similar in the Control (2.7 (0.3)°C) and Absolute (2.4 (0.7)°C) trials, but was substantially lower (P<0.01) in the Relative trial (1.4 (0.8)°C). Ischaemia potentiated (P<0.01) sweating on the forehead in the Absolute trial (24.2 (7.3) g m^–2 min^–1) compared to the Control trial (13.4 (6.2) g m^–2 min^–1), concomitant with an attenuated (P<0.05) vasodilatation in the skin during exercise. It is concluded that graded ischaemia in working muscles potentiates the exercise sweating response and attenuates vasodilatation in the skin initiated by increased core temperature, effects which may be attributed to an augmented muscle metaboreflex.     

Potassium model for slow (2-3 Hz) in vivo neocortical paroxysmal oscillations

In slow neocortical paroxysmal oscillations, the de- and hyperpolarizing envelopes in neocortical neurons are large compared with slow sleep oscillations. Increased local synchrony of membrane potential oscillations during seizure is reflected in larger electroencephalographic oscillations and the appearance of spike- or polyspike-wave complex recruitment at 2- to 3-Hz frequencies. The oscillatory mechanisms underlying this paroxysmal activity were investigated in computational models of cortical networks. The extracellular K(+) concentration ([K(+)](o)) was continuously computed based on neuronal K(+) currents and K(+) pumps as well as glial buffering. An increase of [K(+)](o) triggered a transition from normal awake-like oscillations to 2- to 3-Hz seizure-like activity. In this mode, the cells fired periodic bursts and nearby neurons oscillated highly synchronously; in some cells depolarization led to spike inactivation lasting 50-100 ms. A [K(+)](o) increase, sufficient to produce oscillations could result from excessive firing (e.g., induced by external stimulation) or inability of K(+) regulatory system (e.g., when glial buffering was blocked). A combination of currents including high-threshold Ca(2+), persistent Na(+) and hyperpolarization-activated depolarizing (I(h)) currents was sufficient to maintain 2- to 3-Hz activity. In a network model that included lateral K(+) diffusion between cells, increase of [K(+)](o) in a small region was generally sufficient to maintain paroxysmal oscillations in the whole network. Slow changes of [K(+)](o) modulated the frequency of bursting and, in some case, led to fast oscillations in the 10- to 15-Hz frequency range, similar to the fast runs observed during seizures in vivo. These results suggest that modifications of the intrinsic currents mediated by increase of [K(+)](o) can explain the range of neocortical paroxysmal oscillations in vivo.     

Automatic avoidance of obstacles is a dorsal stream function: evidence from optic ataxia

When we reach out to pick something up, our arm is directed to the target by visuomotor networks in the cortical dorsal stream. However, our reach trajectories are influenced also by nontarget objects, which might be construed as potential obstacles. We tested two patients with bilateral dorsal-stream (parietal lesions, both of whom were impaired at pointing to visual stimuli (optic ataxia). We asked them to reach between two cylinders, which varied in location from trial to trial. We found that the patients' reaches remained invariant with changes in obstacle location. In a control task when they were asked to point midway between the two objects, however, their responses shifted in an orderly fashion. We conclude that the dorsal stream provides the visual guidance we automatically build into our movements to avoid potential obstacles, as well as that required to ensure arrival at the target.     

Relationship between MUC5AC and altered expression of MLH1 protein in mucinous and non-mucinous colorectal carcinomas

The main purpose of this study was to examine the expression of mucins and mismatch repair proteins in colorectal carcinomas. The immunohistochemical distribution of apomucins MUC2, MUC5AC, and the expression of MLH1 and MSH2 proteins were examined in 76 mucinous and 60 non-mucinous colorectal carcinomas. MUC2 was noted in all mucinous carcinomas, whereas MUC5AC was present in 41 cases only (54%). In non-mucinous carcinomas, MUC2 was expressed in 61.7% of the tumors; by contrast, MUC5AC was present in 20% of the cases. The expression level of apomucins was significantly different in mucinous and non-mucinous lesions (p<0.001). Twenty-seven (35.5%) of the mucinous carcinomas showed no MLH1 expression, whereas 11 (18.3%) of the non-mucinous tumors did. This difference was statistically significant (p<0.005). Altered expression of MSH2 protein was never observed. The lack of MLH1 expression was considerably more frequent in carcinomas with secretion of MUC5AC (p<0.005). Our study has demonstrated this close relationship by immunohistochemical methods. In summary, our data show: (1) differences in the expression of mucins between mucinous and non-mucinous tumors; (2) a high frequency of altered MLH1 protein expression (35.5%) in mucinous carcinomas; (3) a significant relationship between the presence of MUC5AC and the altered expression of MLH1 protein in colorectal carcinomas.     

Cell-volume-dependent vascular smooth muscle contraction: role of Na+, K+, 2Cl− cotransport, intracellular Cl− and L-type Ca2+ channels

This study elucidates the role of cell volume in contractions of endothelium-denuded vascular smooth muscle rings (VSMR) from the rat aorta. We observed that hyposmotic swelling as well as hyper- and isosmotic shrinkage led to VSMR contractions. Swelling-induced contractions were accompanied by activation of Ca²⁺ influx and were abolished by nifedipine and verapamil. In contrast, contractions of shrunken cells were insensitive to the presence of L-type channel inhibitors and occurred in the absence of Ca²⁺_o. Thirty minutes preincubation with bumetanide, a potent Na⁺,K⁺,Cl^– cotransport (NKCC) inhibitor, decreased Cl^–_i content, nifedipine-sensitive ⁴⁵Ca uptake and contractions triggered by modest depolarization ([K⁺]_o=36 mM). Elevation of [K⁺]_o to 66 mM completely abolished the effect of bumetanide on these parameters. Bumetanide almost completely abrogated phenylephrine-induced contraction, partially suppressed contractions triggered by hyperosmotic shrinkage, but potentiated contractions of isosmotically shrunken VSMR. Our results suggest that bumetanide suppresses contraction of modestly depolarized cells via NKCC inhibition and Cl^–_i-mediated membrane hyperpolarization, whereas augmented contraction of isosmotically shrunken VSMR by bumetanide is a consequence of suppression of NKCC-mediated regulatory volume increase. The mechanism of bumetanide inhibition of contraction of phenylephrine-treated and hyperosmotically shrunken VSMR should be examined further.     

Surface tension of animal cartilage as it relates to friction in joints

Measurements of the surface tension of articular cartilage and friction experiments were carried out to provide further evidence in support of a new theory regarding the mechanism of friction in joints. To determine the surface tension of cartilage, contact angle measurements were used in conjunction with the equation of state for interfacial tensions. The advancing contact angle between saline drops and articular cartilage was found to be 100°±5°, indicating a highly hydrophobic surface. The corresponding surface tension value was calculated to be 22.5 ergs/cm². Friction of cartilage against hydrophobic surfaces is shown to be lower than the friction of cartilage against hydrophilic surfaces. All these results further support the theory that lubrication by nonwetting drops occurs in joints and may be responsible for the exceptional friction characteristics of the joints.