Cryopreservation of Digitalis lanata Ehrh. cell cultures: Preculture and freeze tolerance

Cryopreservation experiments were performed with Digitalis lanata cell cultures. The main stress was laid on the behaviour of the cells during the preculture period and the capacity of various preculture additives to induce freeze tolerance. The following compounds were used as preculture additives: trehalose, mannitol, sucrose, melibiose, proline, and sorbitol. They are listed in the order of their respective efficiency. Using trehalose, high post-thaw viability rates were achieved and the cells resumed growth after a short lag period. Melibiose was used as a preculture additive for the first time. Its suitability was in the range of that of sucrose. Proline and sorbitol were not able to induce freeze tolerance in Digitalis cells. Cell viability showed a considerable decrease at the beginning of the preculture period. This reduction was found to be transient in the presence of trehalose, mannitol, sucrose, and melibiose. The damaging effects of proline and sorbitol were too severe to be compensated for by the cells. The PAL activity increased markedly in the presence of proline, whereas the trehalose-treated and the control cells behaved nearly identical to one another.     

Application of good laboratory practice (GLP) to culture collections of microbial and cell cultures

Although the principles and the necessity for good laboratory practice (GLP) guidelines to confirm the credibility, integrity, and quality of non-clinical laboratory studies have been known for more than a decade, culture collection activities are not subject to them. Because of recent advances in biotechnology, culture collections face increased demands not only for quality cultures but also current information. When applied in culture collections, GLP guidelines prove to be an excellent management tool as well as a cost-effective system of providing authentic and reliable microbial and cell cultures and associated data.     

Competition between two primary parasitoids,Gyranusoidea tebygi Noyes and Anagyrus mangicola Noyes,attacking the mealybug Rastrococcus invadens Williams and the influence of a hyperparasitoid Chartocerus hyalipennis Hayat

Two primary parasitoids of the mealybug Rastrococcus invadens Williams, (Hemiptera: Pseudococcidae), Gyranusoidea tebygi Noyes and Anagyrus mangicola Noyes (Hymenoptera: Encyrtidae), were studied. Both primary parasitoids were capable of eliminating the mealybug host but on occasions the parasitoids went extinct before the mealybugs. Three of four parasitoids known to attack G. tebygi also attacked A. mangicola. The latter species was more heavily parasitized than the former, especially by the gregarious species Chartocerus hyalipennis Hayat. In competition experiments the presence of hyperparasitoids slightly slowed the speed of extinction of either the mealybug or the primary parasitoid. A. mangicola was heavily parasitized by C. hyalipennis and the primary went extinct while many suitable mealybug hosts were still available. There were two situations where the primary parasitoids were in direct competition; in the first G. tebygi was more successful than A. mangicola while the reverse was true for the second. It is likely that the superiority of either parasitoid would depend on particular conditions but the introduction of A. mangicola is unlikely to lessen the control being exerted in West Africa by G. tebygi.     

Isolation and partial sequence of goat spleen prothymosin α

Goat prothymosin , a highly acidic polypeptide of pl 3.5, 109 amino acid residues, has been isolated from lymphoid and non-lymphoid tissues of young female goats. Unlike rat, murine and porcine prothymosins , goat prothymosin appears at a higher concentration in the spleen compared with the thymus. The sequence of segments of the polypeptide involving known mutations has been determined, by automatic sequencing of its tryptic peptide fragments. The acidic amino acid-rich segment in the middle of the molecule, including residues 49–83, has not been sequenced. Goat prothymosin closely resembles bovine prothymosin , with only one substitution, proline for alanine at position 85. It also resembles human prothymosin , with only three substitutions. It differs more significantly from rat and murine prothymosins , by two deletions and three substitutions. The results show the highly conserved nature of the molecule, with substitutions at given positions only.Abbreviations ProT Prothymosin - T₁ Thymosin ₁ - MLR Mixed Lymphocyte Response - HPLC High Performance Liquid Chromatography - RIA Radioimmunoassay - B Aspartic acid or Asparagine - Z Glutamic acid or Glutamine     

Crustacean cardioactive peptide-immunoreactive neurons in the ventral nerve cord and the brain of the meal beetle Tenebrio molitor during postembryonic development

Summary By use of an antiserum against the crustacean cardioactive peptide (CCAP) several types of bilaterally symmetrical neurons have been mapped quantitatively in the ventral nerve cord and in the brain of the meal beetle, Tenebrio molitor. The general architecture of these neurons was reconstructed from peroxidase-antiperoxidase-labelled whole-mount preparations. From the subesophageal to the seventh abdominal ganglia two types of neurons show a repetitive organization of contralateral projection patterns in each neuromere. The first type has few branches in the central neuropil and a distinct peripheral projection. The second type is characterized by an elaborate central branching pattern, which includes ascending and descending processes. Some of its peripheral branches were found to supply peripheral neurohemal areas. In the protocerebrum, 10 CCAP-immunoreactive neurons occur with projections into the superior median protocerebrum and the tritocerebrum. Immunopositive neurons were mapped in larval and various pupal stages, as well as in the adult. All types of identified neurons were found to persist throughout metamorphosis maintaining their essential structural and topological characteristics. The CCAP-immunoreactive neurons of T. molitor are compared with those described for the locust. Putative structural homologies of subsets of neurons in both species are discussed.     

Selectivity of juxtaposition between cup-shaped lactotrophs and gonadotrophs from rat anterior pituitary in culture

Summary Semi-thin sections of three-dimensional reaggregates from adult female rat pituitary, cultured in serum-free defined medium, were stained for prolactin, gonadotropin, thyrotropin, growth hormone and S-100, using the double immunolabelling technique. The frequency of juxtaposition between lactotrophs and gonadotrophs was enumerated and compared with the expected frequency at random distribution of polygonal cell profiles in a hexagonal configuration. The proportions of lactotrophs and gonadotrophs in the aggregate sections were determined using stereometrical analysis. The observed frequency of juxtaposition did not differ significantly from the expected frequency. Hence, no reason was found to assume a selective adhesion between lactotrophs and gonadotrophs in adult female rat pituitary reaggregates. A constant proportion of lactotrophs was found to meet the criteria of a cup-shaped morphology, and 70%±9% (mean ±S.D.) of these so-called cupshaped lactotrophs were found to be juxtaposed at their concave side to gonadotrophs. Administration of 0.01 nM 17-oestradiol to the culture medium resulted in a significant reduction of the proportion of cup-shaped lactotrophs but did not affect the selectivity of juxtaposition to gonadotrophs. The selectivity of juxtaposition between cup-shaped lactotrophs and gonadotrophs may be the morphological correlate of the functional relationship between these cells, which are known to be involved in an intra-pituitary paracrine communication system.     

Neural coding of speech sound in the telencephalic auditory area of the mynah bird

Summary The responses of neurons in field L in the auditory neostriatum of the mynah bird, Gracula religiosa, were recorded during presentation of intact or manipulated mimic voices. A typical mimic voice konnichiwa elicited responses in most of the neurons. Neurons in the input layer (L2) of field L showed many peaks on peristimulus time histograms while those in other layers (L1 and L3) exhibited only one or two peaks. Several neurons in L1 and L3 responded only to the affricative consonant /t/ in the intact mimic voices. They did not respond to the affricative consonant in the isolated segment or to the one in the playbacked voice in reverse. Forty-five percent of the neurons (33/ 73) decreased in firing rates at the affricative consonant in the isolated segment compared with in the intact voice. Some of these neurons, in which neither the affricative consonant in the isolated segment nor bursts of noise alone elicited responses, exhibited clear phasic responses to /t/ in the case when bursts of noise with particular central frequencies preceded the affricative consonant. The responsiveness of these neurons appears to receive temporal facilitation. These results suggest that these neurons code the temporal relationship of speech sound.Abbreviations HVc hyperstriatum ventrale, pars caudale - TFN temporally facilitated neuron - TSN temporally suppressed neuron     

Exogenous gangliosides may affect methylation mechanisms in neuronal cell cultures

Primary neurons in culture from chick embryo cerebral hemispheres were treated with a mixture of gangliosides added to the growth medium (final concentration: 10^–5M and 10^–8M) from the 3rd to the 6th day in vitro. Under these conditions methylation processes measured with [³H] and [³⁵S] methionine and [³H]ethanolamine as precursors showed an increased methylation of [³H]ethanolamine containing phospholipids, a correspondent increased conversion of these compounds to [³H]choline containing phospholipids, and a general increased methylation of trichloroacetic acid precipitable macromolecules containing labeled methionine. A small increase in protein synthesis was observed after incubation of neurons with [³H]- and [³⁵S]methionine. This was confirmed after electrophoretic separation of a protein extract with increased³H-and³⁵S-labeling in protein bands with moecular weights between 50 and 60 KDaltons. A protein band of about 55 KDaltons appeared to be preferentially labelled when [³H] methionine was the precursor. The treatment with gangliosides increased the incorporation of [methyl-³H] label after incubation of neurons with [³H] methionine, into total DNA and decreased that of total RNA. The treatment of neurons in culture with exogenous gangliosides hence affects differently methylation processes, a finding which may confirm the involvement of gangliosides on the intracellular mediation of neuronal information mechanisms.     

Effect of ferric nitrilotriacetate on rostral mesencephalic cells

After murine fetal cells from the rostral mesencephalic tegmentum were isolated, prepared, and cultured; neuronal and glial cells in primary mixed cell cultures were exposed to ferric nitrilotriacetate (Fe-NTA) at varying concentrations. Studies were performed at 23 days in culture after 14 day exposure to Fe-NTA. In addition to morphologic studies, biochemical assays including specific [³H]flunitrazepam (FLU) binding, clonazepam (CLO)-displaceable [³H]-FLU binding, Ro5-4864-displaceable [³H]-FLU binding, [³H]dopamine (DA) uptake, [³H]haloperidol (HAL) binding, [³H]spiperone (SP) binding, glutamine synthetase activity (GS), and protein determinations were performed. The data demonstrate that chelated ferric iron has an adverse effect on these cells. The data also demonstrate that increasing concentrations of Fe-NTA resulted in massive neuronal dropout leaving the culture population virtually all glial; however, the specific binding of [³H]HAL and [³H]SP increased. There was a concomitant decrease in both glutamine synthetase activity and overall protein content. The mechanism of enhancement in the presence of Fe-NTA of [³H]HAL and [³H]SP binding is unknown and may be unique, but may be related to the known increase in D₂ receptor ligand affinity in the presence of other multivalent cations (Ca²⁺ and Mg²⁺).     

Bacteria and phosphorus regeneration in lakes. An experimental study

The aerobic decomposition of the green alga Chlamydomonas reinhardii by a mixed population of lake bacteria was studied in batch and chemostat cultures. Bacterial chemostats were supplied with continuously heatkilled algae. The dead algae rapidly released most of their phosphorus as SRP. In the batch experiments bacteria acted as consumers of the released algal phosphorus. This phosphorus uptake was dependent on the C:P ratio of the algae. During the death phase of the bacteria most of the bacterial phosphorus itself was released. The continuous supply of energy in form of dead algae in the chemostat experiments prevented the death phase of the bacteria and thus any net regeneration of phosphorus. The influence of the C:P stoichiometry of algae and bacteria on the regeneration of algal phosphorus is discussed.