Franz Brümmer

Characterization of gap junctions between osteoblast-like cells in culture

SummaryThe structure of gap junctions in osteoblast-like cells (OBs) and the connexins (cx) that build up these structures were characterized by ultrastructural, immunocytochemical, and molecular techniques. Ultrastructural studies revealed numerous gap junctions which were mostly located on processes of neighboring cells. Immunofluorescence labeling using two different antibodies (specific to mouse live cx26 and cx32 and to a peptide-specific rat heart gap junction protein cx43) gave evidence that in OBs, gap junctions consist mainly of cx43. The presence of cx43 in cultured OB was also confirmed by Western blot analysis. Dye-coupling with Lucifer yellow led to a staining of up to 30 neighboring cells. Parallel intracellular recordings showed that membrane potential amplitude changes (4–5 mV) are typically related to those in the coupled cells. Thus, there is morphological and functional evidence for intercellular communication between OB in culture. OBs in culture express the same connexins as observed in vivo and may serve as a model to investigate electrophysiological events in response to different stimulation signals.

Biological effects of shock waves

SummaryExtracorporeal shock wave lithotripsy has become established worldwide as the method of choice for the treatment of nephrolithiasis and ureterolithiasis over the last 10 years. Although initial studies showed no damaging effects of the shock waves on organs and tissues, numerous recent reports have presented evidence for severe acute effects and chronic complications after shock wave treatment. The pathophysiological effects on kidneys and the histopathological effects on organs or tissues in man and animal, and also the effects on cells in culture and tumors are sumarized. Suspended and immobilized cell cultures were used to characterize and quantify the efficacy of shock wave. Extended applications of shock waves and possible modifications to shock wave generators are discussed.

Cavitation-generated free radicals during shock wave exposure: investigations with cell-free solutions and suspended cells

Extracorporeally generated shock waves as used in lithotripsy of urinary and biliary stones exhibit side effects in vivo. Furthermore, these shock waves destroy eukaryotic cells during in vitro treatment in suspension. A possible cause of these damaging effects might be cavitation, the growth and collapse of bubbles in liquids exposed to tensile stresses. During the collapse, temperature inside these cavitation bubbles rises up to several thousand K, leading to the formation of free radicals. We demonstrated the occurrence of cavitation-generated free radicals by direct reaction with fluorescent dyes in solution after shock wave treatment and investigated the resulting cell killing by variation of the cellular antioxidative defense status. We present evidence for the generation of intracellular free radicals during shock wave treatment of suspended cells.

Cultivation of primmorphs from the marine sponge Suberites domuncula: morphogenetic potential of silicon and iron

Marine demosponges (phylum Porifera) are rich sources for potent bioactive compounds. With the establishment of the primmorph system from sponges, especially from Suberites domuncula, the technology to cultivate sponge cells in vitro improved considerably. This progress was possible after the elucidation that sponges are provided with characteristic metazoan cell adhesion receptors and extracellular matrix molecules which allow their cells a positioning in a complex organization pattern. This review summarizes recent data on the cultivation of sponges in aquaria and--with main emphasis--of primmorphs in vitro. It is outlined that silicon and Fe(+++) contribute substantially to the formation of larger primmorphs (size of 10 mm) as well as of a canal system in primmorphs; canals are probably required for an improved oxygen and food supply. We conclude that the primmorph system will facilitate a sustainable use of sponges in the production of bioactive compounds; it may furthermore allow new and hitherto not feasible insights into basic questions on the origin of Metazoa.

Effect of shock waves on suspended and immobilized L1210 cells

L1210 mouse leukemia cells have been exposed to different doses of shock waves generated by underwater spark discharge at 18 kV in an experimental lithotripter (XL1, Dornier). Histological and flow cytometric investigations revealed severe damage and a LD50 of about 420 shock waves when the cells were treated as suspensions. Cells immobilized in gelatine, however, were unaffected, indicating that secondary effects are responsible for the cellular damage. Possible mechanisms such as cavitation, jets, and shear forces are discussed.

Sponge-specific Bacterial Associations of the Mediterranean Sponge Chondrilla nucula (Demospongiae, Tetractinomorpha)

A stable and specific bacterial community was shown to be associated with the Mediterranean sponge Chondrilla nucula. The associated bacterial communities were demonstrated to be highly similar for all studied specimens regardless of sampling time and geographical region. In addition, analysis of 16S rDNA clone libraries revealed three constantly C. nucula-associated bacterial phylotypes belonging to the Acidobacteria, the Gamma- and Deltaproteobacteria present in sponge specimens from two Mediterranean regions with distinct water masses (Ligurian Sea and Adriatic Sea). For the first time, candidate division TM7 bacteria were found in marine sponges. A major part (79%) of the C. nucula-derived 16S rDNA sequences were closely related to other sponge-associated bacteria. Phylogenetic analysis identified 14 16S rRNA gene sequence clusters, seven of which consisted of exclusively sponge-derived sequences, whereas the other seven clusters contained additional environmental sequences. This study adds to a growing database on the stability and variability of microbial consortia associated with marine sponges.

Sustainable Production of Bioactive Compounds by Sponges—Cell Culture and Gene Cluster Approach: A Review

Sponges (phylum Porifera) are sessile marine filter feeders that have developed efficient defense mechanisms against foreign attackers such as viruses, bacteria, or eukaryotic organisms. Protected by a highly complex immune system, as well as by the capacity to produce efficient antiviral compounds (e.g., nucleoside analogues), antimicrobial compounds (e.g., polyketides), and cytostatic compounds (e.g., avarol), they have not become extinct during the last 600 million years. It can be assumed that during this long period of time, bacteria and microorganisms coevolved with sponges, and thus acquired a complex common metabolism. It is suggested that (at least) some of the bioactive secondary metabolites isolated from sponges are produced by functional enzyme clusters, which originated from the sponges and their associated microorganisms. As a consequence, both the host cells and the microorganisms lost the ability to grow independently from each other. Therefore, it was—until recently—impossible to culture sponge cells in vitro. Also the predominant number of “symbiotic bacteria” proved to be nonculturable. In order to exploit the bioactive potential of both the sponge and the “symbionts,” a 3D-aggregate primmorph culture system was established; also it was proved that one bioactive compound, avarol/avarone, is produced by the sponge Dysidea avara. Another promising way to utilize the bioactive potential of the microorganisms is the cloning and heterologous expression of enzymes involved in secondary metabolism, such as the polyketide synthases.

Histopathology of shock wave treated tumor cell suspensions and multicell tumor spheroids

L1210 mouse leukemia cell suspensions exposed to 500 shock waves (SW) in an experimental lithotripter (XL1, Dornier) revealed severe cellular damage. Apart from cell fragments and cellular debris, cells exhibited alterations of shape, vacuolization of the cytoplasm, perinuclear cisternae, swelling of mitochondria or rupture of the mitochondrial fine structure, and permeabilization of the cell membrane. Treatment of multicell tumor spheroids of both HeLa and EMT6/Ro cells in suspension with 500 SW resulted either in loss of peripheral cells and serious cellular damage in the outer regions or in a fragmentation of the spheroids. Many of the geometrically intact cells exhibited the same histopathological alterations as the suspended L1210 cells. Immobilization of the spheroids in agar or gelatine, however, prevented spheroids from being agitated and accelerated during SW-exposure. After treatment with 500 SW, spheroids immobilized in gelatine were not different from control cultures, as investigated with light- and electronmicroscopy. From our results we conclude that spheroids in suspension are subject to cavitation and liquid jet formation, causing not only acceleration and shearing forces but also collisions which account for the observed cell damage.

Bacteria of the genus Roseobacter associated with the toxic dinoflagellate Prorocentrum lima

The dinoflagellate Prorocentrum lima is known to produce diarrhetic shellfish poisons. However, it is yet unclear if the dinoflagellates themselves or the bacteria associated with them produce the toxins. Here we analyze the toxicity as well as the spectrum of bacteria in two cultures of P. lima, namely P. lima-SY and P. lima-ST, which initially derived from the same P. lima strain PL2V. Toxicity tests, applying the Artemia bioassay revealed in both cultures high levels of toxins. The bacteria, associated with the two cultures, were identified by PCR/nucleotide sequence analysis of the 16S rRNA gene. From cultures of P. lima-SY the dominant sequence was found to share a 93.7% similarity with the sequence of Roseobacter algocolus [R. algicola]; the relative abundance was determined to be 83%. In addition three further sequences of bacteria, grouped to the α-Protobacteria have been identified: Paracoccus denitrificans [90.8%], R. algocolus [94.4%] and Rhizobium huakuii [92.6%]. The identification of bacteria in P. lima-ST revealed that most share highest similarity with Bartonella taylorii but with a relatively low score of 87%. In addition to this sequence, two sequences with high similarity to the genus Roseobacter were obtained. The other sequences identified have not been detected in P. lima-SY. Studies with pure bacterial strains, previously isolated from a culture of P. lima-ST and subsequently cultured on agar plates, revealed that none of them was identical to those identified in the dinoflagellate culture itself. An explanation for the change of the spectrum of bacteria in the different cultures can only be expected when axenic cultures from P. lima are available.

Freeze tolerance, supercooling points and ice formation: comparative studies on the subzero temperature survival of limno-terrestrial tardigrades

SUMMARY Many limno-terrestrial tardigrades live in unstable habitats where they experience extreme environmental conditions such as drought, heat and subzero temperatures. Although their stress tolerance is often related only to the anhydrobiotic state, tardigrades can also be exposed to great daily temperature fluctuations without dehydration. Survival of subzero temperatures in an active state requires either the ability to tolerate the freezing of body water or mechanisms to decrease the freezing point. Considering freeze tolerance in tardigrades as a general feature, we studied the survival rate of nine tardigrade species originating from polar, temperate and tropical regions by cooling them at rates of 9, 7, 5, 3 and 1°C h–1 down to –30°C then returning them to room temperature at 10°C h–1. The resulting moderate survival after fast and slow cooling rates and low survival after intermediate cooling rates may indicate the influence of a physical effect during fast cooling and the possibility that they are able to synthesize cryoprotectants during slow cooling. Differential scanning calorimetry of starved, fed and cold acclimatized individuals showed no intraspecific significant differences in supercooling points and ice formation. Although this might suggest that metabolic and biochemical preparation are non-essential prior to subzero temperature exposure, the increased survival rate with slower cooling rates gives evidence that tardigrades still use some kind of mechanism to protect their cellular structure from freezing injury without influencing the freezing temperature. These results expand our current understanding of freeze tolerance in tardigrades and will lead to a better understanding of their ability to survive subzero temperature conditions.