Pharmacological evidence for cell surface control of oral regeneration in Stentor coeruleus

This study suggests that membrane perturbations can affect oral morphogenesis in Stentor, possibly by a mechanism involving calcium ions. Exposure of regenerating Stentor to micromolar concentrations of the membrane active local anesthetics dibucaine, tetracaine, or procaine greatly delayed the progress of oral regeneration. In the case of tetracaine and dibucaine the greatest delays were observed in the early stages of regeneration prior to stage 4, when the majority of essential synthetic activity is occurring. The effects of dibucaine were generally readily reversible upon removal of the cells from the drug, with some residual effects occurring at higher dibucaine concentrations. Regenerating cells in the presence of dibucaine and excess extracellular calcium were not delayed, suggesting that the effects of dibucaine were reversible by calcium ions. The effects of tetracaine were not reversible by calcium ions, however. Exposure of regenerating cells to medium either lacking in, or containing an excess of, extracellular calcium had no effect on the time required to complete oral regeneration. The plant lectin, phytohemagglutinin, can also delay oral regeneration. The possible implications of these findings on the control of oral regeneration are discussed.     

Biochemical and cytochemical evidence for ATPase activity in basal bodies isolated from oviduct

Biochemical and cytochemical techniques were used to determine whether oviduct basal bodies have ATPase activity. All studies were carried out on basal bodies isolated and purified from the chicken oviduct. These preparations contained structurally intact basal bodies with basal feet, rootlet, and alar sheet accessory structures. Whereas the specific activity of the basal body ATPase in 2 mM Ca++ or 2 mM Mg++, 1 mM ATP, pH 8.0, averaged 0.04 mumol Pi/min per mg protein, higher concentrations of either cation inhibited the enzyme activity. Furthermore, the pH optimum for this reaction was pH 8.5. In comparison, the ATPase activity in cilia purified and measured under conditions identical to those for determining the basal body ATPase activity averaged 0.07 mumol Pi/min per mg protein. However, the activity increased at higher concentrations of divalent cation, and the pH optimum was pH 10.0. By cytochemical procedures for localizing ATPase activity, ATP-dependent reaction product in isolated basal bodies was found to be confined to: (a) the cross-striations of the rootlet; (b) the outer portion of the basal foot; (c) the alar sheets; and (d) the triplet microtubules. It is concluded that basal bodiesve an intrinsic ATPase activity that, by a variety of criteria, can be distinguished from the ATPase activity found in cilia.     

Replicon size and rate of DNA replication in the macronucleus of Tetrahymena pyriformis]

The size of replication units (or replicons) measured in Tetrahymena pyriformis GL macronuclear DNA reaches 20--30 microns, according to the two independent methods: DNA fiber autoradiography, and alkaline isokinetic sucrose gradient centrifugation. The synthesis of new DNA fragments--replicons and their subsequent assembly are separated by time intervals (30 min). The rate of DNA synthesis for one fork averaged 0.6--0.7 microns/min. These data were obtained for cells of cultures being both in the expotential phase of growth, and those synchronized by starvation-refeeding. The generation time of T. pyriformis cells, calculated by the increase of the part of labeled nuclei, is almost 2 hours; the synthesis lasts 1 hour. Total amount of replication units in polyploid (polygenomic) Tetrahymena macronucleus is about 3000. Their initiation during S-period is presumably asynchronous.     

Further evidence for the usefulness of the salivary testosterone radioimmunoassay in the assessment of androgenicity in man in basal and stimulated conditions

Since correct assessment of testicular function and androgenic status in humans requires multiple sampling, a sensitive and accurate radioimmunoassay (RIA) of testosterone (T) was established for male and female saliva samples. This easily collected biological fluid, which contains nonprotein-bound T, may represent an attractive alternative or a complement to total plasma T assays. In saliva samples from 5 normal males, a clear circadian rhythm was observed, and morning concentrations (135 +/- 31 pg/ml) were significantly higher (p less than 0.02) than evening samples (85 +/- 23 pg/ml). In 11 normal females, morning saliva levels were 12.8 +/- 1.8 pg/ml. The levels of T in male saliva, in response to both exogenous T administration (100 mg i.m.) and HCG stimulation (2 X 2,000 IU i.m.), accurately reflected the changes observed in plasma T, and the magnitude of increase in T levels was clearly greater in saliva than in plasma samples during the intramuscular administration of the long-acting T preparation. In males, significant correlations were observed between salivary and plasma T concentrations in morning samples (r = 0.61, p less than 0.01), following HCG stimulation (r = 0.89, p less than 0.05) and during T administration (r = 0.87, p less than 0.05). In women, the correlation at 8 a.m. was also significant (r = 0.82, p less than 0.05).     

Genetic evidence that control of F replication is negative

Summary We have taken advantage of two situations in which the incompatibility barrier between F plasmids is overcome to show that wild-type genes controlling F copy number (cop ⁺) are dominant in trans over mutant genes. The simplest interpretation of our findings is that the cop mutations have inactivated a repressor gene that controls F replication. Since the cop. mutations all map in a region that others have shown by sequence analysis to theoretically encode four proteins, a strong possibility exists that one of these proteins is the repressor.     

The role of cortical pattern in timing of cell division and morphogenesis in Stentor.

This paper describes experiments involving reciprocal exchange of the oral apparatus between a large (L) and a small (S) strain of Stentor coeruleus. Mature L-strain stentors given and S-strain oral apparatus formed an oral primordium in response to the presence of abnormally small oral structures and mostly divided although a few reorganized instead. Conversely, when S-strain stentors in early division were given a large L-strain oral apparatus, they resorbed the developing primordium and returned to interphase. When combined with previous results, these findings provide evidence for the hypothesis that the time of cell division in Stentor is determined by a "critical" ratio between the size of the oral apparatus and the size of the cell body (somatic) cortex. This ratio develops because the somatic cortex grows during interphase and the oral apparatus does not. Cell division in Stentor therefore appears to be initiated by a cortical pattern change resulting from cell surface growth during interphase.     

Minichromosomal DNA replication in the macronucleus of the hypotrichous ciliate Stylonychia lemnae is independent of chromosome-internal sequences

The origins of DNA replication in prokaryotes and eukaryotes are typically defined by cis-acting sequences. However, in ciliates, evidence suggests that the replication of short macronuclear minichromosomes may not require such determinants. In hypotrichous ciliates, macronuclei contain millions of gene-sized minichromosomes, which generally have a single protein-coding region, two short noncoding flanks and, on each end, a short telomere consisting of a double-stranded repeat region and a single-stranded 3' overhang. Electron microscopic studies that showed that replication of minichromosomes initiates at or near telomeres and the discovery of a primase activity synthesizing RNA primers over the whole 3' telomeric overhang in vitro suggested that minichromosome replication starts directly at telomeres. Conversely, many minichromosomes contain an AT-rich, semi-conserved, palindromic sequence motif in their subtelomeric regions and it has been proposed that this motif is involved in regulating minichromosomal replication. To analyze what sequences or structures of the minichromosomes are essential for DNA replication, we stably transfected genetically modified alpha1-tubulin-encoding minichromosomes into the hypotrichous ciliate Stylonychia lemnae. Cotransfection of mutated and control minichromosomes revealed that noncoding regions can be deleted or replaced with unrelated sequences without affecting minichromosome replication efficiency in vegetatively growing cells. Similarly, replacement of the coding region resulted in a minichromosome that was stably maintained in transfected cells at the same high copy number for many months. In contrast, alpha1-tubulin-encoding minichromosomes without telomeres were rapidly lost after transfection. Hence, DNA replication of the alpha1-tubulin-encoding minichromosome does not depend on chromosome-internal sequences but may depend on telomeres.     

Replication of basal bodies and centrioles.

During the past year, studies on the centrioles and basal bodies of animal and algal cells, and the spindle pole bodies of yeast and other fungi, have added significantly to our knowledge of how these cell organelles form and how they function in initiating microtubule assembly throughout the cell cycle. Most of these studies have used antibodies to identify proteins within and around these organelles and, in some cases, to disrupt their ability to nucleate microtubules. Genetic methods have been used to identify specific proteins, including a new member of the tubulin superfamily, involved in the function and replication of spindle pole bodies and centrioles.     

Further evidence for the electron pool hypothesis

In continuation of experiments with photo-system II inhibitors [3-(3,4-dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6-isopropylbenzoquinone] the effect of photosystem I inhibitors was studied.

1. Neither the plastocyanin inhibitor, potassium cyanide, nor the ferredoxin antagonist, disalicyliden propandiamin, markedly affected those phobic reactions which are mediated by the electron transport via photosystem II into the electron pool.
2. On the other hand those phobic reactions, which are triggered by an increased flow of electrons out of the pool, are specifically inhibited by both substances.

These results are regarded as further evidence that there is only one electron pool, the level of which triggers photophobic reactions and is located in the linear electron transport chain near photosystem II.     

Rearrangement of the 5S ribosomal RNA gene clusters during the development and replication of the macronucleus in Tetrahymena thermophila

The organization of the 5S rRNA genes in the MACronuclear genome of Tetrahymena thermophila was examined during MAC development and replication. The 5S genes are arranged in several tandem arrays of alternating transcribed and spacer sequences in both MICronucleus and MAC. The number of EcoRI fragments bearing 5S gene clusters is similar in MIC and MAC. Most fragments occur in both the MIC and newly formed MAC genomes, a few being MIC-limited and a few MAC-limited. The same rearrangements are seen in the MACs of all four caryonides of a mating pair, and most rearrangements are seen in the newly formed MACs of different inbred strains. During replication of the MAC about half the fragments bearing 5S gene clusters disappear in different cell lines, and new fragments containing 5S genes appear. These fragments differ in size from those present in the MIC or newly formed MAC. These alterations occur in the MACs of all strains except strain B, which is more resistant to vegetative rearrangement. The losses and gains of fragments occur during clonal propagation of cell lines. The process begins by 35 fissions following conjugation, but once an alteration occurs, it is stably propagated. Clonal variation occurs with respect to which losses and gains occur, although a nonrandom distribution is seen among cell clones. We conclude that the alterations in MAC fragment size occur at two stages in the life cycle of Tetrahymena. The first stage occurs during conjugation, when the MAC develops from the MIC. The second stage becomes manifest during vegetative growth, when DNA replication occurs in the MAC and daughter molecules are distributed “amitotically” to daughter nuclei. The two-stage character to MAC alterations for the 5S genes is interpreted in terms of the two steps previously described for MAC differentiation: determination and phenotypic assortment. Possible molecular mechanisms are also discussed.     

Cortical Control over the Direction of Macronuclear Elongation in the Heterotrich Ciliate Stentor coeruleus1

Earlier experimental work involving macronuclear implants in Stentor coeruleus has shown that the cytoplasmic cortex of the nuclear site 1) attracts the macronucleus and 2) holds it in place during interphase. Now experiments indicate macronuclei transferred with overlying cortex elongate in the direction of the transferred cortical pigment stripes, whether or not the transferred stripes realign in the direction of the host stentor's stripes. Therefore the third function of the cortex is to determine the direction of elongation and thus assure that both daughter cells at division receive part of the macronucleus.     

Further evidence for the heterogeneity of serum albumin

Albumin samples from three species (avian, bovine and human) were electrophoresed on gradient polyacrylamide gels in the presence of sodium dodecyl sulphate (SDS-PAGE). The resulting electrophoregram from each sample of serum albumin investigated showed multiple protein bands of a wide range of molecular weights. All seven samples of human serum albumin were found, using gel immunodiffusion, to be contaminated with other proteins. All but one sample was contaminated with proteins such as haptoglobin, alpha 1-glycoprotein, alpha 1-trypsin inhibitor, and prealbumin. This contamination accounts for part of the heterogeneity of these samples. Immunoblots, where the proteins were transferred to nitrocellulose and incubated with antisera, gave a better demonstration of the heterogeneity than Coomassie Blue staining and the immunoblotting procedure appeared to be more sensitive than the gel immunodiffusion technique. The heterogeneity of serum albumin demonstrated by the former technique included that of the monomer which was shown to be contaminated with antithrombin III. The commercial samples of human serum albumin, claimed as pure, were found to vary greatly in their tryptophan content, which also indicated heterogeneity. Heat treatment of human serum albumin with 1% SDS, followed by chromatography on agarose, removed the protein contaminants and with it the tryptophan. The presence of tryptophan in human serum albumin, therefore, indicated the presence of impurities.     

Further evidence for the glycoside structure of cinerarin

The glycoside structure of cinerarin, a dicaffeoyltriglucoside of delphinidin isolated from cineraria, was further studied. Alkaline degradation products of methylated anthocyanin characterized the deacylcinerarin as 3,7,3′-triglucosidyldelphinidin.     

Further evidence for the presence of meiotic pairing control genes in Alopecurus L. (Gramineae)

Mathematical equations applied to data on the meiotic chromosome behaviour of diploid, triploid and tetraploid Alopecurus species, their hybrids and synthesised autopolyploids confirm that chromosome pairing among homologues does not occur at random. The genotypic control of preferential bivalent formation is demonstrated and its role in natural populations discussed.     

Further evidence for size-assortative schooling in sticklebacks

Using brook ( Culaea inconstans ) and 10-spined ( Pungitius pungitius ) sticklebacks we examined body-size related schooling behaviour. Small and large sticklebacks were allowed to choose between two test schools, of small and of large individuals, with and without a piscivorous fish ( Oncorhynchus mykiss ) visible. Sticklebacks of both species preferred the company of fish of matching body size: small fish associating with a school of small fish, large fish with a school of large fish. While no interspecific differences were found in responses to school selection, body size and predator presence did affect selection of school-type. In both species, small fish tended to show a stronger preference for matching schools. The preference was enhanced in small fish with presence of a predator, but not in large fish. These observations are further evidence for assortative schooling in sticklebacks.     

Arrangement of C-tubule protofilaments in mammalian basal bodies

The arrangement of C-tubule protofilaments was studied in basal bodies of respiratory epithelial cilia using tannic acid staining techniques. At the proximal end of the basal body, the C tubule consisted of 10 protofilaments arranged analogously to the 10 protofilaments of the B tubule. In the region of the basal foot, 5 of the C protofilaments sequentially terminated. First, the C10 and C9 filaments terminated. Then the C1 and C8 filaments terminated, and finally the C7 filament terminated leaving a curved sheet of 5 filaments C2-C6 coursing through the distal third of the basal body. This study validates previous suggestions that the C tubule is incomplete distally, and this study clearly demonstrates the extent and consistency of this incompleteness. This morphological study also raises again the question of the function of the C tubules of basal bodies in light of both the incomplete nature of the C tubule and the apparent minor role of the C tubule in providing attachment sites for the basal foot and the alar sheets.