The effect of estradiol on the cell kinetics in the uterine and cervical epithelium of neonatal mice.

The effect of estradiol-17beta on the length of the various phases of the cell cycle was studied in the neonatal mouse uterine, and cervical epithelium. A double labelling method was used, and in addition labelled mitoses were counted. In the uterus proper, estradiol shortens the length of the total cell cycle. TC, from 17-9 hr to 15-7 hr, and the duration of S phase, Ts from 6-7 to 5-1 hr 6 hr after estradiol treatment. 12 hr after estradiol treatment, TC is shortened to 7-4 hr and Ts to 4-5 hr. The shortening of TC at 12 hr is manily due to an effect on TG1, which is shortened from 8-55 hr in untreated animals to 1-8 hr in estradiol treated animals. The TC of cervix epithelium cells in untreated animals was found to be 21-8 hr. After treating the mice for 6 hr with estradiol the tc was now increased to 47 hr and further to 61-2 hr following 12 hr treatment with the hormone. Ts increases from 8-3 hr to 15-2 hr following 6 hr estradiol treatment, and to 15-4 hr after 12 hr treatment. The effect is most pronounced in TG1, which is lengthened from 10-95 hr in untreated animals to 28-1 hr and 43 hr, respectively, in animals treated for either 6 or 12 hr with estradiol.     

Pigmentiphaga soli sp. nov., a bacterium isolated from soil

Strain BS12^T, a Gram-negative motile bacterium, was isolated from soil in South Korea and characterized to determine its taxonomic position. Phylogenetic analyses based on the 16S rRNA gene sequence revealed that the strain belonged to the family Alcaligenaceae in the class Betaproteobacteria. The highest degree of sequence similarities of strain BS12^T were found with Pigmentiphaga litoralis JSM 061001^T (98.3%), Pigmentiphaga daeguensis K110^T (98.2%), and Pigmentiphaga kullae K24^T (98.1%). Chemotaxonomic data revealed that strain BS12^T possessed ubiquinone-8, which is common in the family Alcaligenaceae, and the predominant fatty acids were C_16:0, C_17:0 cyclo, summed feature 3 (C_16:1 ω6c/ω7c), and summed feature 8 (C_18:1 ω6c/ω7c). The major polar lipids of strain BS12^T were phosphatidylethanolamine and phosphatidylglycerol. Based on these data, BS12^T (=KCTC 23577^T =JCM 17666^T =KEMB 9004-082^T) should be classified as a type strain of a novel species, for which the name Pigmentiphaga soli sp. nov. is proposed.     

Genome mining revealed polyhydroxybutyrate biosynthesis by Ramlibacter agri sp. nov., isolated from agriculture soil in Korea

A white-colony-forming, facultative anaerobic, motile and Gram-stain-negative bacterium, designated G-1-2-2 ^T was isolated from soil of agriculture field near Kyonggi University, Republic of Korea. Strain G-1-2-2 ^T synthesized the polyhydroxybutyrate and could grow at 10–35 °C. The phylogenetic analysis based on 16S rRNA gene sequence showed that, strain G-1-2-2 ^T formed a lineage within the family Comamonadaceae and clustered as a member of the genus Ramlibacter. The 16S rRNA gene sequence of strain G-1-2-2 ^T showed high sequence similarities with Ramlibacter ginsenosidimutans BXN5-27 ^T (97.9%), Ramlibacter monticola G-3-2 ^T (97.9%) and Ramlibacter alkalitolerans CJ661^T (97.5%). The sole respiratory quinone was ubiquinone-8 (Q-8). The major polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, and an unidentified phospholipid. The principal cellular fatty acids were C_16:0, cyclo-C_17:0, summed feature 3 (C_16:1ω7c and/or C_16:1ω6c) and summed feature 8 (C_18:1ω7c and/or C_18:1ω6c). The genome of strain G-1-2-2 ^T was 7,200,642 bp long with 13 contigs, 6,647 protein-coding genes, and DNA G?+?C content of 68.9%. The average nucleotide identity and in silico DNA–DNA hybridization values between strain G-1-2-2 ^T and close members were?≤?81.2 and 24.1%, respectively. The genome of strain G-1-2-2 ^T showed eight putative biosynthetic gene clusters responsible for various secondary metabolites. Genome mining revealed the presence of atoB, atoB2, phaS, phbB, phbC, and bhbD genes in the genome which are responsible for polyhydroxybutyrate biosynthesis. Based on these data, strain G-1-2-2 ^T represents a novel species in the genus Ramlibacter, for which the name Ramlibacter agri sp. nov. is proposed. The type strain is G-1-2-2 ^T (=?KACC 21616 ^T?=?NBRC 114389 ^T).

     

THE PROLIFERATION KINETICS OF NHIK 1922 CELLS IN VITRO AND IN SOLID TUMOURS IN ATHYMIC MICE

The proliferation kinetics of cells of the line NHIK 1922 grown in vitro and as solid tumours in the athymic mutant nude mouse has been studied. In vitro, growth curves were determined for exponentially growing populations and for populations synchronized by mitotic selection. The phase durations for these populations were determined by flow cytofluorometric measurements of DNA-histograms and pulsed incorporation of [³H]TdR respectively. The generation time and the phase durations for synchronized populations were found to be about equal to those for exponentially growing populations. The duration of the phases G₁, S and G₂+ M was found to be 8·5–9·5, 11·0–12·0 and 6·0–6·5 hr respectively, i.e. the generation time was 26·5–27·0 hr. The proliferation kinetics in vivo were studied by flow cytofluorometry and by the technique of percentage labelled mitoses. The median duration of S-phase and (G₂+ M)-phase in vivo was found to be approximately the same as that observed in vitro, while the median duration of G₁-phase was found to be approximately 5 hr longer in vivo than under the present in vitro growth conditions. The growth fraction in vivo was estimated to be approximately 50%. The non-proliferative compartment of the tumour cells was found to consist mainly of cells with the DNA-content of cells in G₁-phase. It is concluded that the reduced rate of proliferation of NHIK 1922 cells in vivo is correlated with alterations in the duration of G₁-phase and, hence, the proportion of cells in G₁-phase.     

Altererythrobacter flava sp. nov., a new member of the family Erythrobacteraceae,isolated from a surface seawater sample

A Gram-stain-negative, light yellow pigmented, non-motile and aerobic bacterial strain, designated HHU E2-1 ^T, was isolated from a surface seawater sample. The 16S rRNA gene sequence analysis indicated that HHU E2-1 ^T shared the highest sequence similarity to the type strain Qipengyuania gaetbuli DSM 16225 ^T (96.90%), which belongs to the family Erythrobacteraceae. Combined phylogeny of 288 single-copy orthologous gene clusters, analysis of average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH), average amino acid identity (AAI) and evolutionary distances suggested that HHU E2-1 ^T can be considered as a member of the genus Altererythrobacter based on the recently proposed standard for defining genera of Erythrobacteraceae. Strain HHU E2-1 ^T grew at 15–35 °C and pH 5.0–8.0, with optimum growth at 28 °C and pH 7.0. Tolerance to NaCl was up to 4% (w/v) with optimum growth in 2–3% NaCl. The major fatty acids (>?10%) were C_18:1ω7c11-methyl, summed feature 3 (C_16:1ω7c and/or C_16:1ω6c), and summed feature 8 (C_18:1ω7c and/or C_18:1ω6c). The predominant isoprenoid quinone was ubiquinone-10. The genomic G?+?C content was 57.40%. On the basis of the phenotypic, phylogenetic and chemotaxonomic characterizations, HHU E2-1 ^T represents a novel species of the genus Altererythrobacter, for which the name Altererythrobacter flava sp. nov. is proposed. The type strain is HHU E2-1 ^T (=?CGMCC 1.17394 ^T?=?KCTC 72835 ^T?=?MCCC 1K04226^T).

     

STUDIES ON THE POPULATION KINETICS OF THE WALKER CARCINOMA BY AUTORADIOGRAPHY AND PULSE CYTOPHOTOMETRY

The proliferation parameters of the Walker carcinoma were estimated from both in vivo and in vitro measurements. The transplantable Walker carcinoma 256 was grown in male inbred BD1 rats. During exponential growth, 5-6 days after transplantation, a PLM curve was performed, yielding estimates of Tc ? 18.0 hr, Ts ? 6.4 hr, T_G2+M? 4.1 hr. With the double labelling technique in vitro under 2.2 atm oxygen we obtained: Tc ? 18.2hr, Ts ? 8.2 hr, T_G2+M? 2.0hr. From pulse cyto-photometry DNA content histograms the fractions of cells in the cell cycle phases were calculated using a computer program: f_G1? (47.6 ± 1.1)%, f_s? (34.1 ± 1.0)%, f_G2+M? (18.3 ± 1.5)%. These fractions remained constant between the fifth and the twelfth day after transplantation. At that time the tumour growth had already slowed down appreciably. The growth fraction determined by repetitive labelling was 0.96 on the fifth and 0.93 on the seventh and eleventh day. The cell loss factor was φ? 17% during exponential tumor growth and increased to about 100% between the tenth and twelfth day. The agreement of the cell kinetic data determined by autoradiography from solid tumours in vivo (PLM, continuous labelling) and autoradiography as well as pulse cytophotometry from in vitro experiments (excised material) was satisfactory.     

CELL CYCLE STUDY UP TO THE TIME OF HATCHING IN THE EMBRYOS OF THE SEA URCHIN,HEMICENTROTUS PULCHERRIMUS*

Cell cycles have been analyzed in 10 divisions up to the time of hatching in the embryos of the sea urchin, Hemicentrotus pulcherrimus. In the first 5 cleavages, division synchrony is very high. On the average, T_GC= 55.4 min, T_G1= 0 min, T_s= 12 min, T_G2=±0 min, T_M= 42 min. In the remaining 5 cleavages, T_GC becomes longer: 70 min for the 7th to 246 min for the 10th cleavage. G₁ and G₂ become definitely recognizable and become longer along with T_s. T_M stays more or less constant. Plots of the changing lengths of the four compartments (G₁, S, G₂, M) on the Y-axis against T_GC (X-axis) can be fitted to the following 4 regression equations; T_G1= 0.28T_GC - 19.7, T_s= 0.609T_GC - 15.2, T_G2= 0.104T_GC - 4.72 and T_M= 0.007T_GC+ 39.6.     

Neiella holothuriorum sp. nov., isolated from the gut of a sea cucumber Apostichopus japonicus

A Gram-stain negative, aerobic, rod-shaped bacterium, designated 126^T, was isolated from the intestinal content of a sea cucumber, Apostichopus japonicus, in China. Strain 126^T was found to grow optimally at 25–28 °C and pH 7.5–8.0 in marine 2216 E medium, with tolerance of 1–7% (w/v) NaCl. Strain 126^T is motile by means of one to several polar flagella. The dominant fatty acids of strain 126^T were identified as C_16:1 ω7c/C_16:1 ω6c (29.5%), C_18:1 ω7c/C_18:1 ω6c (19.8%) and C_16:0 (16.7%). The respiratory quinone was found to be Q-8. The polar lipid profile was found to be mainly composed of phosphatidylglycerol and phosphatidylethanolamine. The total length of the draft genome is approximately 4.2?×?10⁶ bp, encoding 3655 genes and 3576 coding sequences. The G?+?C content of the genomic DNA is 48.0%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain 126^T belongs to the genus Neiella and is closely related to Neiella marina J221^T (96.5%). Genomic comparisons of 126^T to N. marina J221^T revealed that they had similar genome size, G?+?C content and complement of clusters of orthologous groups. However, average nucleotide identity and digital DNA–DNA hybridization values between strains126^T and N. marina J221^T was 75.5% and 19.7%, which could distinguish the strains. On the basis of these phenotypic and genotypic data, strain 126^T is concluded to represent a novel species, for which the name Neiella holothuriorum sp. nov. is proposed. The type strain is 126^T (=?GDMCC 1.2530^T?=?KCTC 82829^T).

     

Rat C-6 Glioma Cells Maintained In an Organ Culture System: A Study of Kinetic Parameters

Rat C-6 glioma cells were grown on a sponge foam matrix in an organ culture system and the cell cycle parameters, including the growth fraction (GF), were assessed after autoradiography. the zones of growth consisted of a compact upper layer (UL) at the gaseous interface, a central necrotic layer and a deeper lower layer (LL) which invaded the matrix. the fraction of continuously labeled mitoses (FCLM) was similar in both the UL and LL cells. the derivatives of the FCLM curves obtained in three experiments gave an average modal T_G2 of 5 hr. A mathematical model relating GF, T_G2, T_C and labeling index as a function of time, LI(t), was devised for cells in a steady state exposed continuously to tritiated thymidine and was applied to data obtained from UL cells. A mean GF of 9% (range: 8–10%) and a mean cell cycle time (T_C') of 27 hr (range: 13–47 hr) were obtained. the mean T_S was calculated to be 11 hr (range: 8–16 hr) by the method of grain counts per mitotic figure or grain index (GI). Knowledge of T_S permitted alternative calculation of the cell cycle time from the equation T_S/T_C= LI(0)/GF: this gave a mean cell cycle time (T_C) of 29 hr (range: 20–45 hr). Except for the GF, the cell kinetics were comparable to those of the same cell line grown in monolayer culture. the GF in the in vitro system described is in the lower range reported in some human malignant gliomas in vivo.     

ON THE EXISTENCE OF A Go-PHASE IN THE CELL CYCLE

The model is based on the assumption that the cell cycle contains a G_o-phase which cells leave randomly with a constant probability per unit time, γ. After leaving the G_o-phase, the cells enter the C-phase which ends with cell division. The C-phase and its constituent phases, the‘true’G₁-phase, the S-phase, the G₂-phase and mitosis are assumed to have constant durations of T, T₁T_s, T₂ and T_m, respectively. For renewal tissue it is assumed that the probability per unit time of being lost from the population is a constant for all cells irrespective of their position in the cycle. The labelled mitosis curve and labelling index for continuous labelling are derived in terms of γ, T, and T_s. The model generates labelled mitosis curves which damp quickly and reach a constant value of twice the initial labelling index, if the mean duration of the G_o-phase is sufficiently long. It is shown that the predicted labelled mitosis and continuous labelling curves agree reasonably well with the experimental curves for the hamster cheek pouch if T has a value of about 60 hr. Data are presented for the rat dorsal epidermis which support the assumption that there is a constant probability per unit time of a cell being released from the Go-phase.     

The Genes Encoding the Somatic and Testis-Specific Isotypes of the Mouse Cytochrome c Genes Map to Paralogous Regions of Chromosomes 6 and 2

Using mouse probes specific to cytochrome c_S and to cytochrome c_T, the single-copy genes encoding these two proteins have been mapped to paralogous chromosomal regions by analysis of restriction fragment length variants in interspecific crosses. The gene for cytochrome c_S, Cycs, maps to a position between Tcrb and Cbl-1 on proximal mouse Chromosome 6, and the gene for cytochrome c_T, Cyct, maps between Gad-1 and Sfpi-1 on mouse Chromosome 2.     

<Emphasis Type="Italic">Roseomonas aeriglobus</Emphasis> sp. nov., isolated from an air-conditioning system

A novel pale pink-coloured, strictly aerobic, Gram-stain negative bacterial strain, designated strain KER25-12^T, was isolated from a laboratory air-conditioning system in South Korea. Cells were observed to be non-motile cocci showing positive catalase and oxidase reactions. Strain KER25-12^T was found to grow at 10–30 °C (optimum, 25–30 °C), at pH 4.0–9.0 (optimum, pH 6.0–7.0) and in the presence of 0–2% (w/v) NaCl (optimum, 0%). Ubiquinone-10 and spermidine were detected as the sole respiratory quinone and the predominant polyamine, respectively. The major fatty acids were identified as summed feature 8 (comprising C_18:1 ω7c and/or C_18:1 ω6c), summed feature 3 (comprising C_16:1 ω7c and/or C_16:1 ω6c), C_16:0 and C_18:0. The genomic DNA G+C content of strain KER25-12^T was determined to be 70.0 mol%. The major polar lipids were identified as diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and an unidentified aminolipid. Phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that strain KER25-12^T belongs to the genus Roseomonas and shows high sequence similarity to Roseomonas aerilata 5420S-30^T (98.57%), Roseomonas pecuniae N75^T (97.44%) and Roseomonas vinacea CPCC 100056^T (97.40%). Based on the morphological, physiological, chemotaxonomic and phylogenetic features, strain KER25-12^T is concluded to represent a novel species of the genus Roseomonas, for which the name Roseomonas aeriglobus sp. nov. is proposed. The type strain is KER25-12^T (= KACC 19282^T = JCM 32049^T).     

Spongiibacter pelagi sp. nov., a marine gammaproteobacterium isolated from coastal seawater

A novel gammaproteobacterium, designated as KMU-158^T, was isolated from seawater collected on the coastline of Dadaepo in the Republic of Korea, and characterized using a polyphasic taxonomic approach. Strain KMU-158^T was Gram-staining-negative, pale beige-colored, strictly aerobic, rod-shaped, motile, and chemoorganoheterotrophic. The novel isolate was able to grow at 0–3% NaCl concentrations (w/v), pH 6.5–9.5, and 15–40 °C. The analysis based on 16S rRNA gene sequences indicated that strain KMU-158^T belongs to the family Spongiibacteraceae and shared the highest similarity with Spongiibacter tropicus CL-CB221^T (96.1%). The major cellular fatty acids were summed feature 3 (C_16:1 ω7c and/or C_16:1 ω6c) and C_17:1 ω8c. The only respiratory quinone was identified as ubiquinone-8. The polar lipids of strain KMU-158^T contained phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, and two unidentified lipids. The assembled draft genome size of strain KMU-158^T was 3.29 Mbp with a DNA G?+?C content of 51.3%. The average nucleotide identity, digital DNA–DNA hybridization, and average amino acid identity values of KMU-158^T and the representatives of the genus Spongiibacter were found to be 78.5–79.1%, 13.8–14.1%, and 66.6–66.8%, respectively. From the distinct phenotypic, phylogenetic, genomic, and chemotaxonomic properties, the strain KMU-158^T is considered to represent a novel species of the genus Spongiibacter, for which the name Spongiibacter pelagi sp. nov. is proposed. The type strain of the species S. pelagi sp. nov. is KMU-158^T (=?KCCM 90448^T?=?NBRC 114307^T).

     

Pig Epidermis: A Cell Kinetic Study

The basal cell density (BCD), labelling index (LI), duration of DNA synthesis (T_s) and cell cycle time (T_c) have been calculated for the epidermis of pigs in the age range 4–27 months. the BCD declined progressively from 143.4 ± 6.5 cells/mm at 4 months to 128.8 ± 8.3 cells/mm at 15 months, whereafter the values showed little change. There was a small decrease in LI with increasing age, from 7.9 ± 1.5% at 4 months to 5.9 ± 1.0% at 27 months. However, the change to housing animals outdoors as compared with indoors had a greater effect on the LI (～10%). Severe weathering in the skin of animals housed outdoors resulted in a very high LI (～20%). Neither T_s or T_c varied significantly with age. T_s was within the range 8.8–9.2 hr and T_c 127–161 hr. In animals housed outdoors T_c was reduced relative to animals housed indoors. the BCD and T_s were not affected by housing conditions. the kinetic parameters investigated in the pig were similar to those reported for man.     

Hyphomonas sediminis sp. nov., isolated from marine sediment

A Gram-staining-negative, aerobic and pear-shaped bacterial strain, designated WL0036^T, was isolated from coastal sediment sample collected in Nantong city, Jiangsu province of China (120° 51′ 13″ E, 32° 6′ 26″ N) in October 2020. Strain WL0036^T was found to grow at 20–37 °C (optimum, 28 °C) with 0–9.0% NaCl (optimum, 2.5–4.0%) and displayed alkaliphilic growth with the pH range of pH 6.0–10.0 (optimum, pH 7.0–8.0). The polar lipids profile of strain WL0036^T included phosphatidylcholine, phosphatidylethanolamine, glycolipid and an unidentified lipid. The major isoprenoid quinone was determined to be Q-11 and the major fatty acids were C_16:0, 11-methyl-C_18:1ω7c, and summed features 8 (C_18:1ω6c and/or C_18:1ω7c). The G?+?C content of genomic DNA was 61.8%. Phylogenetic trees constructed based on 16S rRNA gene sequence and bac120 gene set (a collection of 120 single-copy protein sequences prevalent in bacteria) indicted that strain WL0036^T clustered with strains Hyphomonas neptunium ATCC 15444^T and H. polymorpha PS728^T. The average nucleotide identities between strain WL0036^T and strains H. neptunium ATCC 15444^T and H. polymorpha PS728^T were 80.7% and 81.2%, respectively. Strain WL0036^T showed 22.8% and 23.2% of digital DNA-DNA hybridization identities with H. neptunium ATCC 15444^T and H. polymorpha PS728^T, respectively. As inferred from the phenotypic and genotypic characteristics and the phylogenetic trees, strain WL0036^T ought to be recognized as a novel species in genus Hyphomonas, for which the name Hyphomonas sediminis sp. nov. is proposed. The type strain is WL0036^T (=?MCCC 1K05843^T?=?JCM 34658^T?=?GDMCC 1.2413^T).

     

Effect of temperature on the duration of egg development, and moulting and growth in juveniles of Crangonyx pseudogracilis (Crustacea: Amphipoda) in the laboratory

SUMMARY. The interval between moults is an extension of egg development time, increasing from birth to sexual maturity which is probably reached at instar 6 or 7. The duration of each instar increased with the animal's age. Incubation time for eggs and the intermoult interval have the same curvilinear inverse relationship with water temperature in the range 3.5–25°C. Results are expressed as degree-days above predicted threshold temperatures of 3.8°C for eggs and 3.2°C for instar 1 after birth, but inverse power-law relationships were a better fit to the results, with exponents of - 1.355 for eggs, - 1.263 for instar 1 and - 1.37 to - 1.92 for instars 2–4. Temperature — dependence apparently altered in instars 5 and 6 at 15–25°C. From a multiple regression of geometric mean moult interval (M_i, days) against mean age (A) and temperature (T, °C), M_i= 56.4 T^?0.7 e^0.016A, with mean ages of 106 days at 15°C and 85 days at 25°C after six moults. The mean number of primary flagellar segments on the antennules increased from 4.0 in instar 1 to 6.0 in instar 2 and 8.0 in instar 3. Thereafter, segments were added less regularly to give a mean of 13.2 in instar 7. In a natural population, when the sexes became distinctive they had 11–13 flagellar segments. From birth at c. 0.05 mg wet wt, individual growth rates were highly variable; mean growth rates (G_s, % wet wt day^?1) were similar in animals fed on dried, leached elm leaves and living, green leaves of Callitriche; there was a power-law relationship with temperature in the range 3.5–25°C, (G_s= 0.27 T^0.59). Faster growth rates were obtained on living leaves of Elodea. Sexual maturity is reached at c. 0.4–0.5 mg wet wt. A brief comparison is made with Gammarus pulex; C. pseudogracilis may be better adapted to warm-water habitats.     

COMPARISON OF GROWTH CHARACTERISTICS OF EXPERIMENTAL TUMOURS AND DERIVED CELL CULTURES

Cells from seven different rat tumours and a mouse sarcoma have been transplanted in syngeneic animals and were cultured in vitro. Tumours produced by inoculation of cultured cells in animals have been compared with the primary tumours. For the transplanted tumours, volume doubling times, T_d, have been compared with doubling times, T_d(cult), of cell numbers in cultures. Volume doubling times of the transplanted tumours generally decrease with increasing volume. At volumes of about 0.5 cm³, T_d values range from 2.2 days to 10 days, while T_d(cult) values ranged from 11 to 24 hr. A systematic correlation between T_d and T_d(cult) could not be established. During sequential transplantation of the tumours for many generations, as well as during continuous propagation of derived cell cultures, significant changes occurred which resulted in a decrease in the expression of differentiation characteristics in tumours.     

Hydrocortisone increases the number of receptors for thyrotropin-releasing hormone on pituitary cells in culture.

Hydrocortisone (cortisol) increased the binding of thyrotropin-releasing hormone (TRH) to specific membrane receptors in 4 clonal strains of rat pituitary cells. At the highest effective cortisol concentration (3–5 × 10^?6 M), the increase was observed within 6–8 hr and became maximal (140 to 160% of control binding) by 18–24 hr. Half-maximum stimulation occurred in serum-containing medium at 9 × 10^?8 M cortisol, and a significant increase in TRH binding was seen at 3 × 10^?8 M. Equilibrium binding studies showed that enhanced TRH binding was explained by an increase in receptor number with no change in affinity. Similar effects were seen with Dexamethasone, but no increase in TRH binding was noted when testosterone, methyltestosterone, progesterone, estradiol or the antiestrogen Lilly 88571 were added to the culture medium. Cortisol treatment did not cause the appearance of specific TRH binding sites in cell strains previously shown to lack receptors for the tripeptide (F₄C₁, GH₁2C₁ and R₅ cells). When added cortisol was removed from medium, receptor number decayed to control values with a $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of about 30 hr. Previous studies have shown that TRH receptors in GH-cells can be down-modulated by TRH and thyroid hormones; the present findings demonstrate that glucocorticoid hormones can increase the number of TRH receptors in GH-cells.     

RECYCLING OF RESTING CELLS IN THE JB-1 ASCITES TUMOUR AFTER TREATMENT WITH 1-β-D-ARABINOFURANOSYLCYTOSINE (Ara-C)

Resting cells in tumours present a major problem in cancer chemotherapy. In the plateau phase of growth of the murine JB-1 ascites tumour (i.e. 10 days after 2–5 × 10⁶ cells i.p.) large fractions of non-cycling cells with G₁ and G₂ DNA content (Q₁ and Q₂ cells) are present, and the fate of these resting cells was investigated after treatment with l-β-d-arabinofuranosylcytosine (Ara-C). The experimental work consisted of growth curves, percentage of labelled mitoses curves after continuous labelling with ³H-TdR, and cytophotometric determination of single-cell DNA content in unlabelled tumour cells. Treatment with an i.p. single injection of Ara-C 200 mg/kg in the plateau JB-1 tumour resulted in a significant reduction in the number of tumour cells 1 and 2 days later as compared with untreated controls, while no difference in the number of tumour cells was observed after 3 days. In tumours prelabelled with ³H-TdR 24 hr before Ara-C treatment, a significant decrease in the percentage of labelled mitoses was observed 6–8 hr later followed by a return to the initial value after 12 hr, and a new pronounced fall from 20 hr after Ara-C. The second fall in the percentage of labelled mitoses disappeared when the labelling with ³H-TdR was continued also after Ara-C treatment. Cytophotometry of unlabelled tumour cells prelabelled for 24 hr with ³H-TdR before Ara-C treatment showed 20 hr after Ara-C a pronounced decrease in the fraction of Q_t cells paralleled by an increase in the fraction of unlabelled cells with S DNA content. These results indicate recycling of resting cells first with G₂ and later with G_x DNA content, which contribute to the regrowth of the tumours.