Third harmonic generation imaging for fast, label-free pathology of human brain tumors.

In brain tumor surgery, recognition of tumor boundaries is key. However, intraoperative assessment of tumor boundaries by the neurosurgeon is difficult. Therefore, there is an urgent need for tools that provide the neurosurgeon with pathological information during the operation. We show that third harmonic generation (THG) microscopy provides label-free, real-time images of histopathological quality; increased cellularity, nuclear pleomorphism, and rarefaction of neuropil in fresh, unstained human brain tissue could be clearly recognized. We further demonstrate THG images taken with a GRIN objective, as a step toward in situ THG microendoscopy of tumor boundaries. THG imaging is thus a promising tool for optical biopsies.

Enhancement of spatial coherence by surface plasmons.

We report on a method to generate a stationary interference pattern from two independent optical sources, each illuminating a single slit in Young's interference experiment. The pattern arises as a result of the action of surface plasmons traveling between subwavelength slits milled in a metal film. The visibility of the interference pattern can be manipulated by tuning the wavelength of one of the optical sources.

Plasmon-assisted two-slit transmission: Young's experiment revisited.

We present an experimental and theoretical study of the optical transmission of a thin metal screen perforated by two subwavelength slits, separated by many optical wavelengths. The total intensity of the far-field double-slit pattern is shown to be reduced or enhanced as a function of the wavelength of the incident light beam. This modulation is attributed to an interference phenomenon at each of the slits, instead of at the detector. The interference arises as a consequence of the excitation of surface plasmons propagating from one slit to the other.

Complete nucleotide sequence and molecular characterization of hemolysin II gene from Bacillus cereus.

Hemolysin II gene from Bacillus cereus VKM-B771 has been sequenced. The deduced primary translation product consists of 412 amino acid residues which corresponds to the protein with an M(r) of 45.6 kDa. The predicted mature Hly-II protein (residues 32 to 412) is of 42.3 kDa, which is in close agreement with the mini-cell electrophoresis analysis. Hly-II deletion variant lacking 96 C-terminal residues still has hemolytic activity. The protein primary structure analysis revealed no homology with any known Bacillus cytolysins. Significant general homology (31-28% identity) was found between the hemolysin II and Staphylococcus aureus alpha-toxin, gamma-hemolysin (HlgB), and leukocidins (LukF, LukF-R, LukF-PV). The data suggest that hemolysin II belongs to the group of beta-channel forming cytolysins.

Phylogenetic status of Anaerobacter polyendosporus, an anaerobic, polysporogenic bacterium.

The almost complete sequence of the 16S rRNA gene of the Gram-positive polysporogenic bacterium Anaerobacter polyendosporus was determined. This allowed phylogenetic analysis of A. polyendosporus by comparing sequences of the 16S rRNA gene of this bacterium to similar genes of other Gram-positive bacteria. It was shown that this polysporogenic bacterium belongs to the Clostridium cluster I, subcluster A. Phylogenetically, A. polyendosporus is distantly related to another polysporogenic, but non-cultivatable, bacterium, 'Metabacterium polyspora' and can be satisfactorily clustered within the saccharolytic clostridia with a low DNA G+C content grouped in subcluster A. A. polyendosporus was most closely related to Clostridium intestinale (94.8% identity of 16S rRNA genes) and Clostridium fallax (93.1%). Like other members of the Clostridium cluster I, subcluster A, A. polyendosporus possesses such common phenotypic features as a Gram-positive cell wall structure, anaerobiosis, derivation of energy from carbohydrate fermentation yielding butyric acid among other organic acids and the capacity for endogenous spore-formation. However, the scale of evolutionary change in the 16S rRNA gene between A. polyendosporus and phylogenetically related Clostridium species does not correspond to the profound changes in the phenotype of A. polyendosporus. Distinctive phenotypic features of the latter are large cell size, polysporogenesis (up to seven spores per cell), alternative modes of development and an unusual membrane ultrastructure.

Mechanism of action of hemolysin III from Bacillus cereus.

Bacillus cereus hemolysin III activity was tested in crude extracts, from Escherichia coli carrying the hly-III gene. It was concluded that hemolysin III is a pore-forming hemolysin with functional pore diameter of about 3-3.5 nm. Hemolysis occurs in at least three steps: (i) the temperature-dependent binding of the Hly-III monomers to the erythrocyte membrane; (ii) the temperature-dependent formation of the transmembrane oligomeric pore; (iii) the temperature-independent erythrocyte lysis.

Cloning and primary structure of a new hemolysin gene from Bacillus cereus.

A new hemolysin gene from Bacillus cereus VKM-B164 was cloned in Escherichia coli and sequenced. Deduced protein consists of 219 amino acids and has a molecular mass of 24.4 kDa. It has been concluded that the hemolysin, named 'hemolysin III', is distinct from the B. cereus hemolysins reported previously: cereolysin, sphingomyelinase, cereolysin AB, hemolysin II, and 'cereolysin-like' hemolysin (Honda, T., Shiba, A., Seo, S., Yamamoto, J., Matsuyama, J. and Miwatani, T. (1991) FEMS Microbiol. Lett. 79, 205-210).

Hemolysin II is more characteristic of Bacillus thuringiensis than Bacillus cereus.

To investigate the distribution of the hemolysin II determinant among strains of Bacillus cereus and Bacillus thuringiensis, thirteen strains of B. cereus and fourteen strains of B. thuringiensis strains were tested for hybridization of their chromosomal DNAs with a DNA probe containing the B. cereus hemolysin II gene. In addition, the production of hemolysin II, whose activity is not inhibited by cholesterol, was tested. The presence (absence) of the hydridization response in the microorganisms's genome correlated with the presence (absence) of cholesterol-unaffected hemolysin production. Only four out of thirteen B. cereus strains were found to give a positive response in hybridization experiments, whereas thirteen out of fourteen B. thuringiensis strains responded positively. DNAs from ten B. thuringiensis strains contained a 3.5 kb EcoRV fragment, which hybridized with the B. cereus hemolysin II gene probe. The 3.5 kb EcoRV DNA fragment from one of these strains (B. thuringiensis VKM-B1555) was cloned and expressed in Escherichia coli cells. The hemolysin encoded by the cloned DNA fragment was not inhibited by cholesterol and possessed all other properties of B. cereus hemolysin II. The obtained data clearly show limited distribution of hemolysin II among B. cereus strains and demonstrate that hemolysin II is more characteristic of B. thuringiensis than B. cereus.

Regions of homology in small colicinogenic plasmids.

Restriction maps have been constructed for the colicinogenic plasmids (ColA, ColD, and ColK. Their regions of homology with the ColE1 plasmid and its deletion derivative pAO3 carrying the region responsible for autonomous replication of ColE1 plasmid were determined by means of blotting hybridization and heteroduplex analysis. The plasmids ColA, ColD, and ColK were shown to contain DNA fragments homologous to the region of ColE1 involved in the regulation of replication.