Circadian Rhythms In the Epithelial Cells and the Pericryptal Fibroblast Sheath In Three Different Sites In the Murine Intestinal Tract

Variations in percentage labelling (LI) and mitotic activity (mitoses per crypt) have been studied over a 24-hr period in the epithelial cells and pericryptal fibroblast sheath (PCFS) of the small intestine, caecum, and colon of the mouse. All three tissues displayed clear, synchronized circadian rhythms in DNA-synthetic activity in both the epithelial cells and PCFS. Peak values were coincident within a tissue, but staggered between tissues. In the epithelial cells, peak mitotic values were found between 3 and 6 hr after the peak LI values. the low level of mitotic activity in the PCFS appeared to be synchronized with the rhythms in the adjacent epithelia. the epithelial cells of the lowest crypt third displayed the clearest circadian rhythms. However, the PCFS cells at all levels produced similar curves. A craniocaudal wave of proliferative activity is proposed.     

Localization and quantification of carbonic anhydrase activity in the symbiotic Scyphozoan Cassiopea xamachana

The relationship between density and location of zooxanthellae and levels of carbonic anhydrase (CA) activity was examined in Cassiopea xamachana. In freshly collected symbiotic animals, high densities of zooxanthellae corresponded with high levels of CA activity in host bell and oral arm tissues. Bleaching resulted in a significant loss of zooxanthellae and CA activity. Recolonization resulted in full restoration of zooxanthellar densities but only partial restoration of CA activity. High levels of CA activity were also seen in structures with inherently higher zooxanthellar densities, such as oral arm tissues. Similarly, the oral epidermal layer of bell tissue had significantly higher zooxanthellar densities and levels of CA activity than did aboral bell tissues. Fluorescent labeling, using 5-dimethylaminonapthalene-1-sulfonamide (DNSA) also reflected this tight-knit relationship between the presence and density of zooxanthellae, as DNSA-CA fluorescence intensity was greatest in host oral epithelial cells directly overlying zooxanthellae. However, the presence and density of zooxanthellae did not always correspond with enzyme activity levels. A transect of bell tissue from the margin to the manubrium revealed a gradient of CA activity, with the highest values at the bell margin and the lowest at the manubrium, despite an even distribution of zooxanthellae. Thus, abiotic factors may also influence the distribution of CA and the levels of CA activity.     

Song activity and variability in relation to male quality and female choice in Whitethroats Sylvia communis

To investigate whether song variability or activity could be used as a cue to male quality in the Whitethroat Sylvia communis and whether the song influenced female choice, songs from 18 males were recorded and analysed, and their perch song and flight song activity were registered. Song repertoire was measured as Rep1000 (the average number of different elements within sequences of 1000 elements), Max1000 (the maximum number of different elements in a 1000 element sequence) and number of elements per repertoire. Old males had larger Rep1000, Max1000 and number of different elements per song than 1-yr-old males. 1-yr-old males had higher perch song and flight song activity than old males. A principal component factor analysis on the song variables showed that (1) males with a large principal component factor score on the first component (PCFS1) typically were old males and (2) the PCFS1 was positively related to male wing length. Rep1000 and Max1000 and a low perch and flight song activity contributed to a positive factor score. Males that mated had larger PCFS1 than males that remained unmated, and there was a significant negative correlation between mating date and PCFS1.
The study suggests that song variability potentially could act as a cue to male quality. Females may prefer males with elaborate songs. However, arrival date could account for the mating pattern and it cannot be excluded that other factors, such as territory quality, also could have influenced the observed mating pattern.     

Purkinje cell maturation in the light of RNA synthesis

Wistar rats 1- to 90-day-old received an injection of 3H-uridine and were killed 20 min to 44 h later. Autoradiographic examination revealed the highest grain count densities in Purkinje cell nuclei around postnatal day (PD) 6 while the incidence of labelled nuclei stayed at the peak values till PD 15. Silver staining of Purkinje cell nuclei showed that the expression of nucleolar r-RNA coding genes is maximal at PD 15; in some cells it even slightly exceeds adult values. After PD 15, the percentage of labelled Purkinje cell nuclei declined; this was more pronounced in the nucleolar region than outside the nucleolus. The percentage of cells with cytoplasmic labelling culminated on PD 15. The highest grain counts were found in Purkinje cell cytoplasm on PD 6 at 44 h p.i. interval. Reversal in nuclear grain counts at 2 and 6 h p.i. intervals observed between PD 15 and PD 25 suggests faster degradation, or processing and export, of a newly synthesized nuclear RNA in these age groups. Frequency distribution analysis of grain count densities revealed a small group of Purkinje cells with higher incorporation of 3H-uridine both in the nucleolar region and the whole nucleus at PD 15. In situ hybridization of 3H-r-RNA revealed a slight binding excess to DNA of some Purkinje cell nuclei but not in granule cells of 1-month-old rats. These data, together with those published recently by Brodsky et al. (1985), indicate an uneven structural organization and partial overexpression of the genom coding r-RNA synthesis in the population of Purkinje cells.     

QUANTITATIVE TRITIUM AUTORADIOGRAPHY OF MAMMALIAN CHROMOSOMES : I. The Basic Method

A technique has been developed that allows repeated autoradiographs to be made of the isotope distribution in the chromosomes of a single cell. A series of 10 separate autoradiographs were made of a Chinese hamster diploid male metaphase cell which had been labeled with tritiated thymidine during the first 15 minutes of its DNA synthesis period in the previous interphase. Each autoradiograph had low grain densities above the chromosomes so that quantitation was feasible. The separate autoradiographs were photographically combined into a single composite in which grain images were converted to lines oriented at right angles to the chromosome axis. The line densities were then measured with a recording microdensitometer to yield graphs reflecting the isotope distribution along each chromosome. The area under each graph was directly proportional to the total number of grains counted above the corresponding chromosome in the 10 separate autoradiographs. The distribution of isotope along the chromosomes was different for each chromosome, and in some cases homologs also differed in their early labeling patterns.     

Blue fan palm distribution and seed removal patterns in three desert oases of northern Baja California, Mexico

Information on the current processes controlling the distribution patterns of relict palm populations at the limit of their northwestern distribution in America are poorly known. We explored the importance of post-dispersal seed removal by vertebrates, recruitment, and distribution patterns of the blue fan palm, Brahea armata, in the Northern Baja California peninsula, by evaluating (i) the levels of blue fan palm seed removal by vertebrates at two spatial scales and the initial fate of dispersed seeds, (ii) the spatial distribution and association of seedlings and adults at two spatial scales, (iii) seed removal levels and seedling densities based on density and distance to adult palm trees, and (iv) the population age structure. Overall, seed removal levels were low at all sites, varied at regional but not at local scales, with small rodents apparently being responsible of most removals. Adult density and distribution differed among oases, but seedling densities did differ locally. Whereas seed removal did not vary with distance from seed sources or parent trees, a weak positive association between seedlings and adults at the whole patch level indicated that establishment tended to occur in or near those grid cells where adults had established successfully. However, the analysis showed a negative association between seedling and adult densities within the patches, indicating that within the grid cells where growth was most successful, seedlings established preferentially in relatively open spaces. All life-history categories were adequately represented within each site and populations seemed to be in a fairly good conservation state. We did not find evidence to establish that post-dispersal seed removal by rodents is a main factor defining the recruitment patterns in these oases. In contrast, the effect of flood pulses seems to be significant and may have strong and conclusive effects on palm seedling distributions. We suggest that local biotic (nurse plants) and/or abiotic (canyon physiography, nurse objects) factors at each particular canyon have the potential to affect post-dispersal seed removal activity patterns by rodents, as well as to provide vital protection for palm seedling establishment from the extreme floods. This baseline information will help in further investigations on the possible mechanisms that sustain palm populations at their distribution limits and in stressful environments.     

Lipid signalling pathways in normal and ras-transfected NIH/3T3 cells

The role of ras oncogenes in cellular signalling pathways involving phospholipid breakdown was studied in untransfected and proto-H-ras and mutated H-, K- and N-ras transfected NIH/3T3 cells. When the cells were grown at low cell densities, all of the ras transfected cells had 2-4 fold higher diacylglycerol (DAG) levels compared to growing NIH/3T3 cells. At high cell densities, DAG levels decreased in the former and increased in contact inhibited NIH/3T3 cells. In this regard, only cells transformed by mutated cellular and viral H-ras oncogenes (but not by the H-ras proto-oncogene) had elevated DAG levels compared to contact inhibited NIH/3T3 cells. The basal levels of inositol phosphates in ras transfected cells were not significantly different from NIH/3T3 cells and did not vary with cell density. Thus, the elevated DAG levels are not a consequence of increased phosphoinositide hydrolysis. The latter was stimulated by serum and bombesin only in normal and proto-H-ras transfected cells. In contrast, stimulation by bradykinin was observed only in cells transformed by mutated cellular ras oncogenes. Furthermore, aluminum fluoride stimulated phosphoinositide breakdown in the latter cells indicating that there was no uncoupling of the G protein from phospholipase C. Treatment of ras transfected cells with dibutyryl cyclic AMP (DB-cAMP), which causes an inhibition of growth and a reversal of the transformed morphology, did not alter the basal levels of inositol phosphates, DB-cAMP, however, did lower DAG levels in some of the transformed cell lines, but elevated DAG levels in low density NIH/3T3 cells. These findings indicate that the ras gene product p21 is not involved in phosphoinositide hydrolysis and that DAG levels do not correlate with cell growth in either normal or ras transfected NIH/3T3 cells. Thus, p21 appears to alter cell growth through mechanism(s) independent of lipid signalling pathways.     

Examination and characterization of distribution system biofilms

Investigations concerning the role of distribution system biofilms on water quality were conducted at a drinking water utility in New Jersey. The utility experienced long-term bacteriological problems in the distribution system, while treatment plant effluents were uniformly negative for coliform bacteria. Results of a monitoring program showed increased coliform levels as the water moved from the treatment plant through the distribution system. Increased coliform densities could not be accounted for by growth of the cells in the water column alone. Identification of coliform bacteria showed that species diversity increased as water flowed through the study area. All materials in the distribution system had high densities of heterotrophic plate count bacteria, while high levels of coliforms were detected only in iron tubercles. Coliform bacteria with the same biochemical profile were found both in distribution system biofilms and in the water column. Assimilable organic carbon determinations showed that carbon levels declined as water flowed through the study area. Maintenance of a 1.0-mg/liter free chlorine residual was insufficient to control coliform occurrences. Flushing and pigging the study area was not an effective control for coliform occurrences in that section. Because coliform bacteria growing in distribution system biofilms may mask the presence of indicator organisms resulting from a true breakdown of treatment barriers, the report recommends that efforts continue to find methods to control growth of coliform bacteria in pipeline biofilms.     

Examination and characterization of distribution system biofilms.

Investigations concerning the role of distribution system biofilms on water quality were conducted at a drinking water utility in New Jersey. The utility experienced long-term bacteriological problems in the distribution system, while treatment plant effluents were uniformly negative for coliform bacteria. Results of a monitoring program showed increased coliform levels as the water moved from the treatment plant through the distribution system. Increased coliform densities could not be accounted for by growth of the cells in the water column alone. Identification of coliform bacteria showed that species diversity increased as water flowed through the study area. All materials in the distribution system had high densities of heterotrophic plate count bacteria, while high levels of coliforms were detected only in iron tubercles. Coliform bacteria with the same biochemical profile were found both in distribution system biofilms and in the water column. Assimilable organic carbon determinations showed that carbon levels declined as water flowed through the study area. Maintenance of a 1.0-mg/liter free chlorine residual was insufficient to control coliform occurrences. Flushing and pigging the study area was not an effective control for coliform occurrences in that section. Because coliform bacteria growing in distribution system biofilms may mask the presence of indicator organisms resulting from a true breakdown of treatment barriers, the report recommends that efforts continue to find methods to control growth of coliform bacteria in pipeline biofilms.     

Temperature-Sensitive Simian Virus 40-Transformed Cells: Phenomena Accompanying Transition from the Transformed to the “Normal” State

Temperature-sensitive simian virus (SV 40)-transformed 3T3 cells (tsSV3T3), which express the transformed phenotype when growing at 32 C but not at 39 C, were used to study changes in growth behavior during shift-up or shift-down experiments. In cultures of tsSV3T3 cells which had reached or were beyond monolayer density at 32 C, DNA synthesis reached very low levels within 24 to 48 h after shift-up. When cells which had been allowed to grow to high densities at 32 C were shifted to 39 C, not only cell growth stopped, but within two to three days the cultures shed a large number of cells into the medium. These cells were nonviable, and shedding stopped only when the number of cells attached had been reduced to that characteristic of the saturation density at 39 C. The remaining attached cells were viable and after the shift to 32 C were again able to grow from the monolayer to high cell densities. This behavior has been compared with that of normal 3T3 and wild-type SV3T3 cells under different conditions. We have also isolated new tsSV3T3 lines, using cells which had been infected with non-mutagenized wild-type SV40. This further demonstrates that the temperature sensitivity of these lines is due to a cellular rather than a viral mutation.     

Effects of feeding position of the aphids Sitobion avenae and Metopolophium dirhodum on wheat yield and quality

The effects of feeding site of Sitobion avenae and Metopolophium dirhodum on grain weight, number and nitrogen content were investigated in the laboratory and in the field. When the latter species was confined to particular leaves in a growth room, grain weight reductions occurred only when the aphid fed on the flag leaf; reductions in percentage grain protein, but not grain weight, followed lower leaf feeding. Leaves which had borne aphids had a higher proportion of nitrogen at death than control leaves, but this explained little of the shortfall in grain nitrogen, which was mainly due to direct removal during aphid feeding. Multiple regressions of grain weight on aphid species/feeding site in the field confirmed the lack of grain weight effects when lower leaves were colonised; because grain size was greatly reduced by aphid feeding, percentage grain nitrogen increased with increasing aphid density in one cultivar and in others was unchanged. Successive comparison of control grain weights with those from tillers bearing an increasing range of aphid densities resulted in a significant reduction in weight with tillers bearing 21 to 25 aphid units on their ear and flag leaf combined, one unit comprising one adult or fourth-instar aphid or three of earlier instars. These and other factors affecting the nature and extent of cereal aphid damage are discussed.     

Vertical Distribution of Adult Rice Weevils, <Emphasis Type="Italic">Sitophilus oryzae</Emphasis> L. (Coleoptera,Dryophthoridae), in a Stored Grain Bulk

The results of an experimental study of the vertical distribution of Sitophilus oryzae L. adults in a 10-m high wheat grain bulk during storage are described. At the beginning of the experiment, the model grain bulk was infested with beetles at a uniform density of 5 ind. per 1 kg of grain. After 151 and 224 days of storage, live beetles were found only in the surface grain layer down to 0.1 m deep where their densities were 693 and 3943 ind./kg, respectively. At the same time, the densities of dead beetles in the grain bulk decreased considerably with depth.     

Location of messenger specifying sequences in mammalian chromosomes

In situ hybridization of complementary DNA (cDNA) synthesized from total cytoplasmic polyadenylated RNA isolated from Chinese hamster cells was employed to investigate the distribution of messenger specifying sequences on mammalian chromosomes. The kinetics of cDNA-nuclear DNA annealing indicate that about 85% of the cDNA represents sequences which are transcribed from non-repetitive DNA sequences. When cDNA is hybridized back to its template RNA, the reaction kinetics show that more than 60% of the poly(A) RNA is at least 10⁴ times more complex than rabbit globin mRNA. In situ hybridization of cDNA to Chinese hamster cells fixed on slides shows no significant clustering of silver grains on interphase nuclei. On metaphase chromosomes the majority of silver grains are localized in euchromatic areas. It appears that all euchromatic segments have similar grain densities. Chromosomes 1 and 2, which have relatively little heterochromatin, do not have a higher grain density than the other chromosomes. However, the Y chromosome, which is entirely heterochromatic, contains only about 1/3 the grain density of the chromosomes 1 or 2. — When the cDNA, which anneals only to the high abundancy class of poly(A) RNA was fractionated and hybridized in situ to Chinese hamster chromosomes, the distribution of silver grains is localized in the euchromatic areas. The Y chromosome and the heterochromatic arm of the X chromosome contain less grains; telomeres of some autosomes have higher grain densities. The oligo-(dT) primer in cDNA did not affect the results of this study since no grains are found when ₃H-poly(dT) was used as probe for in situ hybridization. The majority (>90%) of the grains could be blocked by competition with excess repetitive DNA in the hybridization reaction, indicating that the in situ hybridization involved predominantly repetitive sequences.     

Pyridine nucleotide levels as a function of growth in normal and transformed 3T3 cells.

The levels of NAD (NAD⁺ + NADH) and NADP (NADP⁺ + NADPH) and their redox states were measured as a function of growth in 3T3 mouse fibroblasts which exhibit density-dependent inhibition of growth and SV40 (simian virus #40)-transformed 3T3 cells (SVT2) which have lost this property. The levels were related to cell numbers, protein content, and rates of DNA synthesis. At corresponding cell densities, 3T3 cells contain approximately twice as much total protein as SVT2 cells. The levels of NAD relative to total cellular protein are density dependent in both 3T3 and SVT2, increasing with increasing cell density. Over a 30-fold range of cell densities, the NAD levels in 3T3 increase 2.4-fold, while the levels in SVT2 increase 1.6-fold. The levels of NAD are very similar in dividing 3T3 and SVT2 cells at corresponding cell densities; however, a marked increase in the levels of NAD is observed in 3T3 cells, but not in SVT2 cells, at cell densities just prior to where 3T3 cells enter density-dependent inhibition of growth. This increase in NAD levels is correlated with the cessation of DNA synthesis. The NAD pools are 15–25% NADH for 3T3 and 5–15% NADH for SVT2. NADP levels relative to protein in 3T3 and SVT2 are less density dependent, with overall increases of 1.3- and 1.2-fold, respectively, observed over the range of cell densities examined. NADP levels relative to protein are nearly twice as high in SVT2 cells as in 3T3 cells of corresponding cell densities. The NADP pools are approximately 70–80% NADPH in both cell types.     

Association of tyrosine phosphatase SHP-2 with F-actin at low cell densities

SHP-2 is an intracellular SH2 domain-containing protein-tyrosine phosphatase with an essential role in cell signaling. Here we demonstrate that localization of SHP-2 is regulated by cell density in a cell adhesion-dependent manner. When cells were plated at low densities, SHP-2 was distributed in Triton X-100-insoluble fractions, whereas it was totally soluble when cells were plated at high densities or when low density cells approached confluency. In all cases, the total protein level of SHP-2 was not changed. Fluorescent cell staining revealed that SHP-2 was co-localized with actin stress fibers to the cell peripheral at low cell densities but was diffused in the entire cytoplasm at high cell densities. Transient transfection of cells with truncated forms of SHP-2 demonstrated that the catalytic domain of the enzyme was responsible for the density-regulated distribution of SHP-2, but the catalytic activity was not required. An in vitro co-sedimentation study demonstrated direct binding of full-length and SH2 domain-truncated forms of SHP-2 to F-actin. The data indicate that SHP-2 is regulated by cell density and that it may have a role in assembling and disassembling of the actin network.     

Population analysis of arrested human diploid fibroblasts by flow microfluorometry

Confluent cultures of human diploid fibroblasts were maintained for 28 days with medium containing 0.5% serum. Periodically during this time cells were exposed to ³H-thymidine for 72 h; harvested; and analysed by flow microfluorometric, ‘cell sorting’, and autoradiographic techniques. The results showed that cells cultured under these conditions maintain a stable population distribution similar to that occurring when a population reaches confluency in medium containing 10% serum. Low labeling indices, sparce grain densities, and the presence of some mitotic cells indicated that a limited amount of cell-cycle traverse did occur but that both the S and G2 phases were prolonged. This new state of reduced mitotic activity with prolonged cell-cycle times may mimic the long-term inhibition of cell-cycle traverse of expanding tissues in vivo.     

不同密度混播对玉米植株13C同化物分配和产量的影响

为了探讨不同密度混播对玉米植株¹³C同化物分配和产量的影响,选用‘郑单958’(ZD)和‘登海605’(DH)为试验材料,在不同密度下(LD,67500株·hm^-2;HD,97500株·hm^-2)设置单播(SZD、SDH)与混播(M、1∶1、2∶2)处理,研究玉米品种不同密度混播对植株光合特性、¹³C同化物分配、干物质积累量和产量的影响.结果表明: 随密度增加,籽粒产量、¹³C同化物在籽粒中的分配、干物质积累量和叶面积指数均提高;而叶绿素含量和净光合速率则降低.在67500株·hm^-2下,混播较单播处理无显著优势,但在97500株·hm^-2下,两品种混播提高了叶面积指数、叶绿素含量和穂位叶净光合速率,干物质积累量增加.混播促进茎等营养器官的干物质向籽粒的转运,提高了¹³C同化物在籽粒中的分配比例.混播处理较单播产量增加,主要因为千粒重显著增加.在高密度种植条件下,混播有助于扩大光合面积,维持较高的净光合速率,提高群体干物质积累量,改善干物质的分配状况,增加同化物向籽粒的分配,最终提高夏玉米产量.可见,混播栽培可显著增加黄淮海区密植夏玉米产量.