Shaping of colony elements in Laomedea flexuosa Hinks (Hydrozoa, Thecaphora) includes a temporal and spatial control of skeleton hardening.

The colonies of thecate hydroids are covered with a chitinous tubelike outer skeleton, the perisarc. The perisarc shows a species-specific pattern of annuli, curvatures, and smooth parts. This pattern is exclusively formed at the growing tips at which the soft perisarc material is expelled by the underlying epithelium. Just behind the apex of the tip, this material hardens. We treated growing cultures of Laomedea flexuosa with substances we suspected would interfere with the hardening of the perisarc (L-cysteine, phenylthiourea) and those we expected would stimulate it (dopamine, N-acetyldopamine). We found that the former caused a widening of and the latter a reduction in the diameter of the perisarc tube. At the same time, the length of the structure elements changed so that the volume remained almost constant. We propose that normal development involves a spatial and temporal regulation of the hardening process. When the hardening occurs close to the apex, the diameter of the tube decreases. When it takes place farther from the apex, the innate tendency of the tip tissue to expand causes a widening of the skeleton tube. An oscillation of the position at which hardening takes place causes the formation of annuli.     

An H1-like protein from the macronucleus of Euplotes eurystomus

An H1-like protein has been purified from the macronucleus (MAC) of the hypotrichous ciliated protozoan, Euplotes eurystomus. It is present in amounts comparable to the inner histones and is extracted by treatment with 5% perchloric acid or 0.65 M NaCl, but not by 0.35 M NaCl. Treatment of soluble MAC chromatin with the ionic exchange resin AG 50W-X2 in 80 mM NaCl removes MAC H1 and yields H1-depleted chromatin. Mac H1 is lysine-rich and deficient in acidic amino acids. The stoichiometry of the H1 protein is reduced in mononucleosome preparations, consistent with its postulated interaction with linker DNA regions. Thermal denaturation and circular dichroism studies reveal that H1-depleted chromatin contains a larger portion of destabilized DNA than control chromatin. The molecular weight of Euplotes MAC H1 is significantly smaller than most reported H1 proteins. Comparisons are made with extracts of macronuclei from other hypotrichous ciliated protozoa and published reports of other lower eukaryotes.     

Correlation of adenosine receptor affinities and cardiovascular activity

Binding affinities of 28 adenosine analogs at A1 adenosine receptors (rat whole brain membranes, [3H]N6-cyclohexyladenosine, CHA), and at A2 adenosine receptors (rat striatal membranes, [3H]NECA) were compared to their EC25 values for decreasing heart rate and increasing coronary flow in the isolated rat heart. Heart rate (an A1 response) correlated with A1 binding affinity (r2 = 0.71, p less than 0.0001) but not with A2 binding affinity (r2 = 0.007, n.s.); conversely, coronary flow (an A2 response) correlated with A2 binding affinity (r2 = 0.83, p less than 0.0001) but not with A1 binding affinity (r2 = 0.05, n.s.). These results confirm that the brain A1 and A2 receptors, studied by binding methods, bear close similarities to their respective counterparts in the heart, studied by means of functional responses.     

Dsi-1, a region with frequent proviral insertions in Moloney murine leukemia virus-induced rat thymomas.

Dsi-1 is a region of chromosomal DNA that underwent proviral insertion in 3 of 24 Moloney murine leukemia virus-induced rat thymomas. In one of these tumors, a provirus is also integrated adjacent to the proto-oncogene c-myc. The proviruses in Dsi-1 have been characterized and appear to be complete. The proviruses were located within a 2-kilobase region that contained four prominent DNase I-hypersensitive sites. These hypersensitive sites were observed in Moloney murine leukemia virus-induced thymomas but not in NRK cells. The region of Dsi-1 immediately 3' to the insertions cross-hybridized with human and chicken DNA, indicating that it contains highly conserved sequences. No evidence could be found for the expression of this highly conserved region. Dsi-1 was mapped to mouse chromosome 4. This location demonstrates that Dsi-1 is different from 16 of the known proto-oncogenes (c-abl, c-erbA c-erbB, c-ets-1, c-ets-2, c-fes, c-fos, c-myb, c-myc, c-raf, A-raf, c-Ha-ras, c-Ki-ras, N-ras, c-sis, and c-src) and 12 cellular regions of tumor-associated integrations in retrovirus-induced tumors (c-erbB, Fis-1, int-1, int-2, Mis-1/pvt-1, Mlvi-1, Mlvi-2, c-mos, c-myb, c-myc, Pim-1, and c-Ha-ras). Hybridization experiments indicated that Dsi-1 is probably different from five additional proto-oncogenes (c-fgr, c-fms, c-mos, neu, and c-yes) and from two additional frequent integration regions (lck and Mlvi-3).     

Initiation of primary cell cultures from human intracranial tumors on extracellular matrix from bovine corneal endothelial cells

Tissue specimens from 105 human gliomas and 57 human meningiomas were obtained at surgery, dissociated into single cells and small cell aggregates and then plated onto plain plastic tissue culture dishes and dishes which had been precoated with an extracellular matrix (ECM) derived from bovine corneal endothelium. In 80% of the glioma cases we observed a marked improvement in initial plating efficiency, colony formation and speed of attachment when cells were plated on ECM. In 5 cases cells attached only to the ECM-coated dishes but remained afloat in the untreated dishes. In addition it could be noted that over the first 2 days, those cells which had been initiated on ECM showed more signs of morphological differentiation, i.e., extension of cytoplasmic processes or formation of fiber networks between cell groups. If adaptation occurred and proliferation began in vitro, either immediately or after a several days' lag phase, both the ECM-cultured cells as well as those which slowly had adapted to culture on plastic could be passed on to untreated culture ware and perpetuated thereon. In the case of well-differentiated low-grade gliomas where no growth in culture took place, the cultures on ECM could at least be used for initial experiments in the primary cultures (P0). Meningiomas usually attached well to both, plastic or ECM. In 50% of our cases the plating efficiency was higher on ECM but after successful initial culture, the delay until the cells on plastic reached confluence in comparison with those on ECM was 1 or 2 days. Again there were 2 cases in which the cells would not plate on plastic. Here the cells which after 1 day were still afloat plated to more than 80% within the first 2 h after transfer to ECM. In all cases the cells from plastic and ECM cultures were indistinguishable and could be passed onto untreated dishes henceforth. In later culture stages ECM offers several advantages: It is easier to shift cells to serum-free defined culture conditions, the cells will grow at a faster rate on ECM when in higher passages and the maximal number of passages possible is higher on ECM.     

High-affinity uptake of γ-[3H]aminobutyric acid by isolated mouse oligodendrocytes in culture

Oligodendrocytes were isolated from mixed glial cultures of neonatal mouse forebrain and further grown in serum-free hormone supplemented culture medium. Cell populations were identified by indirect immunofluorescence using a range of specific antibodies, revealing a predominantly immature population of oligodendrocytes, the majority expressing the myelin glycolipids galactocerebroside and sulfatide on their plasma membrane. Astroglial contamination was found to be minimal. Simultaneous autoradiography and immunofluorescence demonstrated the presence of a transport system for the major inhibitory neurotransmitter GABA in the oligodendrocytes. The transport system was found to be energy, sodium and temperature dependent. Kinetic analysis revealed a high affinity system, with aK _m of 6.27 M and aV _max of 0.714 nmol/min/mg protein, which is comparable to that found previously for CNS neurons and astrocytes.Special Issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

Relative sensitivity of photosynthesis and respiration to freeze-thaw stress in herbaceous species : importance of realistic freeze-thaw protocols

The relative effect of a freeze-thaw cycle on photosynthesis, respiration, and ion leakage of potato leaf tissue was examined in two potato species, Solanum acaule Bitt. and Solanum commersonii Dun. Photosynthesis was found to be much more sensitive to freezing stress than was respiration, and demonstrated more than a 60% inhibition before any impairment of respiratory function was observed. Photosynthesis showed a slight to moderate inhibition when only 5 to 10% of the total electrolytes had leaked from the tissue (reversible injury). This was in contrast to respiration which showed no impairment until temperatures at which about 50% ion leakage (irreversible injury) had occurred. The influence of freeze-thaw protocol was further examined in S. acaule and S. commersonii, in order to explore discrepancies in the literature as to the relative sensitivities of photosynthesis and respiration. As bath cooling rates increased from 1°C/hour to about 3 or 6°C/hour, there was a dramatic increase in the level of damage to all measured cellular functions. The initiation of ice formation in deeply supercooled tissue caused even greater damage. As the cooling rates used in stress treatments increased, the differential sensitivity between photosynthesis and respiration nearly disappeared. Examination of agriculturally relevant, climatological data from an 11 year period confirmed that air cooling rates in the freezing range do not exceed 2°C/hour. It was demonstrated, in the studies presented here, that simply increasing the actual cooling rate from 1.0 to 2.9°C/hour, in frozen tissue from paired leaflet halves, meant the difference between cell survival and cell death.