Wide-field optical coherence tomography: imaging of biological tissues.

We describe a two-dimensional optical coherence tomography technique with which we were able to obtain multiple longitudinal slices of a biological sample directly in a single Z scan. The system is based on a femtosecond Cr4+:forsterite laser and an infrared camera for wide-field imaging of the sample with a depth resolution of 5 microm. With this imaging apparatus we were able to investigate human skin and mouse ear samples and to observe the different constitutive tissues.

Sequence and expression of an Eisenia-fetida-derived cDNA clone that encodes the 40-kDa fetidin antibacterial protein.

Fetidins are 40-kDa and 45-kDa hemolytic and antibacterial glycoproteins present in the coelomic fluid of the earthworm Eisenia fetida andrei. By screening a cDNA library with a polyclonal antifetidin serum, we have cloned a cDNA that encoded the 40-kDa fetidin. The clone contains an insert of 1.44 kb encoding a protein of 34 kDa, which corresponds to the size of deglycosylated fetidins. The recombinant protein inhibits Bacillus megaterium growth. Restriction fragment polymorphisms were observed on Southern blots and correspond to a known protein polymorphism. The sequence of the cDNA contains a peroxidase signature and fetidins from earthworm coelomic fluid have peroxidase activity. The 40-kDa and 45-kDa fetidins therefore represent two related polymorphic defence factors in invertebrates.

Purification, characterization and activities of two hemolytic and antibacterial proteins from coelomic fluid of the annelid Eisenia fetida andrei.

The coelomic fluid of the earthworm Eisenia fetida andrei exhibits antibacterial, hemolytic and hemagglutinating activities. These activities are mainly mediated by two proteins, named fetidins, of apparent molecular mass 40 kDa and 45 kDa, respectively. For the first time, the two proteins have been purified to homogeneity from dialysed coelomic fluid by means of anion-exchange chromatography. Three peaks had hemolytic activity. The first fraction was found to correspond to the 40 kDa fetidin, the second to mixed 40 and 45 kDa fetidins, the last one to the 45 kDa fetidin. Both purified proteins still exhibited their hemolytic and antibacterial activities as dialysed coelomic fluid did. In this study, the amino-acid sequence of purified proteins is compared to the amino-acid sequence predicted by cDNA. This cDNA was isolated by screening an expression cDNA library from earthworm total tissues (unpublished data).

In vitro generation of reactive oxygen species by free coelomic cells of the annelid Eisenia fetida andrei: an analysis by chemiluminescence and nitro blue tetrazolium reduction.

Coelomocytes of the earthworm Eisenia fetida andrei were activated in vitro with various stimulants in order to investigate their capacity to produce reactive oxygen species. Analysis by luminol-enhanced chemiluminescence and nitro blue tetrazolium reduction suggests the production in vitro of reactive oxygen species by both categories of free coelomocytes, leucocytes and chloragocytes, while affecting different modalities: a respiratory burst-like reaction is exhibited by leucocytes in the presence of zymosan but not bacteria; a moderate production of reactive oxygen species spontaneously occurs when chloragocytes aggregate together. A substantial reduction of reactive oxygen species production, rapidly occurring after addition of exogenous superoxide dismutase, was interpreted as indicating a prominent role of the superoxide anion, O2.-. The factors modulating the coelomocyte generation of reactive oxygen species, as well as the presence of lipofuscin in brown bodies, support the opinion that production of reactive oxygen species may be an immune defense process occurring under in vivo conditions.

Serological evidence and amino acid sequence of ubiquitin-like protein isolated from coelomic fluid and cells of the earthworm Eisenia fetida andrei.

1. A small protein of M(r) 10 kDa has been isolated by reverse-phase chromatography of the basic proteins contained in the coelomic fluid and cell lysate of the earthworm Eisenia fetida andrei. 2. The protein crossreacted in dot-blot with an anti-bovine ubiquitin antiserum. 3. Its N-terminal primary structure was determined by automatic Edman degradation on 26 consecutive amino acids and showed 69% (based on the 26 amino acids) or 82% (based on the first 19 consecutive amino acids) identity with many ubiquitins and similar charge and hydrophobicity profiles and secondary structure conformation.

Formation of brown bodies in the coelomic cavity of the earthworm Eisenia fetida andrei and attendant changes in shape and adhesive capacity of constitutive cells.

The formation of brown bodies in the coelomic cavity may result from an aggregation of coelomocytes around offending foreign cells such as bacteria, gregarines, incompatible graft fragments, and altered self structures such as setae or necrotic muscle cells. The initial nodule rapidly increases in volume by aggregation of new coelomocytes and various waste particles. When a brown body has reached a diameter of 1-2 mm, its external cells flatten and lose their adhesiveness toward free coelomocytes or waste particles and its pigment rapidly darkens. Brown bodies play an important role in homeostasis.

Antibacterial activity of Eisenia fetida andrei coelomic fluid: III--Relationship within the polymorphic hemolysins.

The antibacterial activity exhibited by 10 different hemolytic, genetic families was established by measuring the inhibition of spontaneous in vitro growth by cell-free coelomic fluid toward 2 bacteria which are pathogenic for the earthworm: Bacillus megaterium (Gram +) and Aeromonas hydrophila (Gram -). Only two families (B and K) displayed potent inhibitory activities. This finding is consistent with the fact that the B family occurs most frequently in both natural as well as in industrial breedings. Nevertheless, evidence of a poor antibacterial defense in some frequent families suggests the existence of alternative antibacterial mechanisms.

Antibacterial activity of Eisenia fetida andrei coelomic fluid: transcription and translation regulation of lysozyme and proteins evidenced after bacterial infestation.

1. After bacterial infestation lysozyme and antibacterial activities are enhanced, peaking at 4 hr and 3 days, respectively. 2. Both humoral defenses require RNA and protein de novo synthesis in response to pathogenic bacteria injection (actinomycin D and cycloheximide experiments). 3. Antibacterial activity exists naturally at some basic level, involving regular translation of stable RNAs. 4. When antibacterial activity reaches its maximum after bacterial injection, proteins responsible for it undergo a turn-over. 5. Lysozyme and antibacterial proteins cannot account for the whole response to bacterial infestation; some cellular defense mechanisms like phagocytosis are involved at the same time.

Altered sensitivity to cholchicine and PHA in human cultured cells.

PHA-stimulated lymphocytes cultivated from a pair of human monozygotic twins yielded mostly tetraploid cells when colchicine was not used to arrest the metaphases. The rate of tetraploidy was also enhanced by colchicine in fibroblasts cultured without PHA. In in situ condition, larger than usual cells were observed. Other defects found in parental lymphocyte cultures included C-anaphase cells and increased cell aggregation. These results suggest a membrane mutation resulting in hypersensitivity to PHA and variant response to colchicine.