Effects of different states of sheep fetal fibroblasts as donor cells on the early development in vitro of reconstructed sheep embryos

To investigate the effects of different states of donor cells on the development of reconstructed sheep embryos, we designed five treatments of donor cells, including cell passage, cell size, serum starvation, colchicine treatment and gene transfection. Results are as follows: (Ⅰ) Compared with 16-18 passage cells, the morula/blastocyst rate of 5-7 passage cells as donor nuclei was significantly higher (17.3% vs. 4.9%, P<0.05), suggesting the advantage of short-time cultured cells in supporting the development of reconstructed embryos. (Ⅱ) The morula/blastocyst rate of reconstructed embryos derived from medium cells (15-25μm) as donor nuclei was higher than that from large cells (25-33μm) and small cells (8-15μm)( 20.0% vs. 8.0%, 9.7%), indicating that reconstructed embryos from medium cells had a greater potentiality to develop into morula/blastocysts than those from small or large ones. (Ⅲ) The morula/blastocyst rate of reconstructed embryos from donor cells of SS (serum starvation) was lower than that from donor cells of NSS (non-serum starvation), but no significant difference was detected between SS and NSS(11.8% vs. 18.6%, P>0.05). (Ⅳ) Fetal fibroblasts treated with 0.05μmol/L colchicine exhibited a higher morula/blastocyst rate of reconstructed embryos than those treated with 0.10 μmol/L colchicine and untreated ones (27.5% vs. 12.1%, 17.1%), however, no significant difference among the three treatments was detected (P>0.05). (Ⅴ) The morula/blastocyst rate of reconstructed embryos from fetal fibroblasts transfected with GFP gene only was 3.1%, significantly lower than that from non-transgenic cells (3.1% vs. 20.4%, P<0.05). In conclusion, our results demonstrated that fetal fibroblasts of fewer passages, medium size could ensure a higher morula/blastocyst rate of reconstructed embryos. Serum starvation of donor cells might be unnecessary to the development of reconstructed embryos. Donor cells treated with 0.05μmol/L colchicine could facilitate the development of reconstructed embryos. Additionally, as cells transfected with GFP gene were used as donor nuclei, adverse effect on the development of reconstructed embryos was observed. Therefore, the developmental efficiency of reconstructed embryos could be improved if proper treatments to donor cells were used.     

Cloned calves produced by nuclear transfer from cultured cumulus cells

Short-term cultured cumulus cell lines (1-5BCC) derived from 5 individual cows were used in nuclear transfer (NT) and 1188 enucleated bovine oocytes matured in vitro were used as nuclear recipients. A total of 931 (78.4%) cloned embryos were reconstructed, of which 763 (82%) cleaved, 627 (67.3%) developed to 8-cell stage, and 275 (29.5%) reached blastocyst stage. The average cell number of blastocysts was 124±24.5 (n=20). In this study, the effects of donor cell sources, serum starvation of donor cells, time interval from fusion to activation (IFA) were also tested on cloning efficiency. These results showed that blastocyst rates of embryos reconstructed from 5 different individuals cells were significantly different among them (14.1%, 45.2%, 27.3%, 34.3%, vs 1.5%, P<0.05); that serum starvation of donor cells had no effect on blastocyst development rate of NT embryos (47.1% vs 44.4%, P>0.05); and that blastocyst rate (20.3%) of the group with fusion/activation interval of 2–3 h, was significantly lower than that of the 3–6 h groups (31.0%), while not significantly different among 3–4 h (P < 0.05), 4–5 h, and 5–6 h groups (P≥0.05). Sixty-three thawed NT blastocysts were transferred to 31 recipient cows, of which 4 pregnancies were established and two cloned calves were given birth. These results indicate that serum starvation of cumulus cells is not a key factor for successful bovine cloning, while IFA treatment and sources of donor cells have effects on cloning efficiency.     

High-frequency plant regeneration from coleoptile tissue of indica rice (Oryza sativa L.)

Summary An efficient method was established for high-frequency embryogenic callus induction and plant regeneration from 3-,4-, 5- and 7-d-old coleoptile segments of Indica rice (Oryza sativa L. cv. Kasturi), Compact and friable callus developed from the cut ends and also on the entire length of the coleoptile segments cultured on Murashige and Skoog (MS) basal medium (1962) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 4.50–18.0 μM), kinetin (2.32 μM) and sucrose (3%, w/v). High frequency embryogenic callus induction and somatic embryo development was achieved when embryogenic calluses were transferred to MS medium supplemented with 2.25 μM 2,4-D, 2.32 μM kinetin, 490 μM L-tryptophan and 3% (w/v) sucrose. Plant regeneration was achieved by transferring clumps of embryogenic callus onto MS medium containing 2.85 μM indole-3-acetic acid (IAA), 17.77 μM 6-benzylaminopurine (BA) and 3% (w/v) sucrose. Histological observations of embryogenic calluses revealed the presence of somatic embryos and also plant regeneration via multiple shoot bud formation. Three, 4- and 5-d-old coleoptile segments showed a significantly (P<0.05) higher frequency of plant regeneration and mean number of plantlets per explant in comparison to 7-d-old coleoptile segments. The highest frequency (73.5%) of plant regeneration and mean number of plantlets (11.9±1.0) was obtained from 4-d-old coleoptile segments. Regenerated shoots were rooted on MS basal medium containing 4.92 μM indole-3-butyric acid (IBA) and plants were successfully transferred to soil and grown to maturity.     

Inhibition of fibroblast proliferation in cardiac myocyte cultures by surface microtopography

Cardiac myocyte cultures usually require pharmacological intervention to prevent overproliferation of contaminating nonmyocytes. Our aim is to prevent excessive fibroblast cell proliferation without the use of cytostatins. We have produced a silicone surface with 10-µm vertical projections that we term "pegs," to which over 80% of rat neonatal cardiac fibroblasts attach within 48 h after plating. There was a 50% decrease in cell proliferation by 5 days of culture compared with flat membranes (P < 0.001) and a concomitant 60% decrease (P < 0.01) in cyclin D1 protein levels, suggesting a G₁/S₁ cell cycle arrest due to microtopography. Inhibition of Rho kinase with 5 or 20 µM Y-27632 reduced attachment of fibroblasts to the pegs by over 50% (P < 0.001), suggesting that this signaling pathway plays an important role in the process. Using mobile and immobile 10-µm polystyrene spheres, we show that reactive forces are important for inhibiting fibroblast cell proliferation, because mobile spheres failed to reduce cell proliferation. In primary myocyte cultures, pegs also inhibit fibroblast proliferation in the absence of cytostatins. The ratio of aminopropeptide of collagen protein from fibroblasts to myosin from myocytes was significantly reduced in cultures from pegged surfaces (P < 0.01), suggesting an increase in the proportion of myocytes on the pegged surfaces. Connexin43 protein expression was also increased, suggesting improved myocyte-myocyte interaction in the presence of pegs. We conclude that this microtextured culture system is useful for preventing proliferation of fibroblasts in myocyte cultures and may ultimately be useful for tissue engineering applications in vivo. tissue engineering; cell culture; cell cycle     

The UvrY response regulator of the BarA–UvrY two-component system contributes to Yersinia ruckeri infection of rainbow trout (Oncorhynchus mykiss)

To identify virulence-associated genes of a fish pathogen Yersinia ruckeri, we screened a total of 1056 mini-Tn5-Km2 signature-tagged mutants in rainbow trout by immersion challenge. Of 1056, 25 mutants were found survival-defective as they could not be re-isolated from fish kidney 7 days after infection. Mutated gene in F2-4 mutant, one of the 25 mutants, was homologous to uvrY that encodes UvrY response regulator of BarA–UvrY two-component system (TCS). Mutant F2-4 was significantly more sensitive (P < 0.05) to H₂O₂-mediated killing and was less able to infect Epithelioma papulosum cyprini cells. However, UvrY mutation did not affect survival of F2-4 mutant in the presence of non-immune fish serum and its ability to grow under iron starvation. In a time-course co-infection, mutant F2-4 had lower bacterial loads on day 1 itself, and by day 5 there was nearly a 1,000-fold difference in infection levels of the parent and mutant strains. The barA homolog of Y. ruckeri was PCR-amplified and sequence analyses identified four domains that were characteristic of hybrid histidine kinases. To conclude, the BarA–UvrY TCS contributes to the pathogenesis of Y. ruckeri in its natural host rainbow trout, possibly by regulating invasion of epithelial cells and sensitivity to oxidative stress induced by immune cells.     

Genetic variation in catechol-<Emphasis Type="Italic">O</Emphasis>-methyltransferase (<Emphasis Type="Italic">COMT</Emphasis>) and obesity in the prostate,lung, colorectal,and ovarian (PLCO) cancer screening trial

Catechol-O-methyltransferase (COMT) is an important modulator in the catabolism of extraneural dopamine, which plays an important role in drug reward mechanisms. It is hypothesized that genetic variations in the COMT gene, which can result in a three to fourfold difference in COMT enzyme activity, may be associated with several reward-motivated behaviors. The aim of our study was to examine the relationship between COMT polymorphisms with smoking, obesity and alcohol. Three single nucleotide polymorphisms (SNPs) in COMT were genotyped in 2,371 participants selected randomly from the screening arm of the PLCO Cancer Screening Trial after stratifying by sex, age, and smoking status. Smoking, obesity, and alcohol consumption were assessed by questionnaire. SNP and haplotype associations were estimated using odds ratios (ORs) and 95% confidence intervals (CIs) derived from conditional logistic regression models, adjusted for race/ethnicity. The COMT Ex4-76C > G (Leu136Leu) polymorphism was statistically significantly associated with individuals who had >30% increases in BMI from ages 20 to 50 years, compared to those with 0–5% increase in BMI (0–5%) over the same age period: (CC is referent; OR_CG = 1.42, OR_GG = 1.46, P _trend = 0.06). By sex, the increased risk was further pronounced among females (OR_CG = 1.50, OR_GG = 2.10, P _trend = 0.03). Consistent with our analyses of single polymorphisms, individuals whose BMI increased >30% from ages 20 to 50 years were more likely than individuals with 0–5% increases in BMI to possess COMT haplotypes [COMT Ex3-104C > T–COMT Ex4-76 C > G–COMT Ex4-12 A > G] that included the variant allele for COMT Ex4-76 C > G: C-G-G (T-C-A is referent: OR_C-G-G = 1.33, 95% CI 1.01–1.77) and C-G-A (OR_C-G-A = 1.79, 95% CI 0.72–4.49). We observed no association between any of the COMT polymorphisms with smoking behavior or alcohol intake. The COMT Ex4-76C > G (Leu136Leu) polymorphism appears to play a role in large increases in BMI. The null association with smoking and alcohol and the pronounced association with increasing BMI among women further implicates COMT’s role in estrogen metabolism as a potentially culpable pathway. Our results support a need for comprehensive evaluation of COMT variations and their functional relevance as COMT may be an important molecular target to evaluate for new treatments regarding obesity.     

Cell cycle propagation is driven by light–dark stimulation in a cultured symbiotic dinoflagellate isolated from corals

Endosymbiosis is an intriguing plant–animal interaction in the dinoflagellate–Cnidaria association. Throughout the life span of the majority of corals, the dinoflagellate Symbiodinium sp. is a common symbiont residing inside host gastrodermal cells. The mechanism of regulating the cell proliferation of host cells and their intracellular symbionts is critical for a stable endosymbiotic association. In the present study, the cell cycle of a cultured Symbiodinium sp. (clade B) isolated from the hermatypic coral Euphyllia glabrescens was investigated using flow cytometry. The results showed that the external light–dark (L:D) stimulation played a pivotal role in regulating the cell cycle process. The sequential light (40–100 μmol m⁻² s⁻¹ ~ 12 h) followed by dark (0 μmol m⁻² s⁻¹ ~ 12 h) treatment entrained a single cell cycle from the G₁ to the S phase, and then to the G₂/M phase, within 24 h. Blue light (~450 nm) alone mimicked regular white light, while lights of wavelengths in the red and infrared area of the spectrum had little or no effect in entraining the cell cycle. This diel pattern of the cell cycle was consistent with changes in cell motility, morphology, and photosynthetic efficiency (F _v /F _m ). Light treatment drove cells to enter the growing/DNA synthesis stage (i.e., G₁ to S to G₂/M), accompanied by increasing motility and photosynthetic efficiency. Inhibition of photosynthesis by 3-(3, 4-dichlorophenyl)-1, 1-dimethyl-urea (DCMU) treatment blocked the cell proliferation process. Dark treatment was required for the mitotic division stage, where cells return from G₂/M to G₁. Two different pools of adenylyl cyclase (AC) activities were shown to be involved in the growing/DNA synthesis and mitotic division states, respectively. Communicated by Biology Editor Dr Michael Lesser     

Structural and immunochemical characterization of β-1,2-linked mannobiosyl phosphate residue in the cell wall mannan of Candida glabrata

A mannan of Candida glabrata IFO 0622 digested by Arthrobacter exo-α-mannosidase and a β-1,2-linked mannobiose obtained from the parent mannan by acid treatment was analyzed using ¹³C nuclear magnetic resonance spectroscopy. The results show that the β-1,2-linked mannobiosyl residue is esterified to a phosphate group through position C-1 in the α-configuration, Manβ1– 2Manα1–HPO₃–. The results of immunochemical assays of these mannans using the commercial antigenic factor sera of the genus Candida (Candida Check, Iatron) indicate that the main recognition site of serum no. 6 in this kit is the mannotetraosyl side-chain Manβ1–2Manα1– 2Manα1–2Man in C. glabrata mannan and also suggest that the phosphate-containing unit (such as Manβ1– 2Manα1–HPO₃– in this mannan) behaves as one of the antigenic determinants of serum no. 6, but not of serum no. 5. Therefore, the present and previous findings indicate that serum no. 5 recognizes relatively longer β-1,2-linked oligomannosyl side-chains, Manβ1–[2Manβ1–]_n 2Man (n = 1–6), attached to the phosphate groups previously observed in the cell wall mannans of Candida albicans, Candida stellatoidea, and Candida tropicalis. Received: 18 March 1997 / Accepted: 16 September 1997     

Influence of Acute and Chronic Treadmill Exercise on Rat Plasma Lactate and Brain NPY,L-ENK,DYN A<Subscript>1–13</Subscript>

SUMMARY This study was designed to investigate the effect of acute and chronic high-intensity treadmill exercise on changes in plasma lactate and brain neuropeptide (NPY), leucine-enkephalin (L-ENK), and dynorpin A_1–13 (DYN A_1–13). Avidin–biotin complex (ABC) immunohistochemistry and image pattern analysis were used to observe the effect of chronic (total 7 weeks) and acute treadmill exercise (an initial speed of 15 m min⁻¹ gradually increased to 35 m min⁻¹ with 0°, 20–25 min per day duration) on the changes of NPY, L-ENK, and DYN A_1–13 in different areas of rat brain. Plasma lactate was also measured in response to such exercise. Compared with preexercise control (P < 0.01), plasma lactate concentration significantly increased in the immediate postexercise; but it returned to the normal level soon after the 30 min postexercise. The content of NPY in paraventricular (PVN), dorsomedial (DMN), and ventromedial (VMN) hypothalamic nuclei continued to increase in 0, 30, and 180 min postexercise compared with preexercise control (P < 0.01). The content of L-ENK in caudate-putamen (CPu) significantly increased in the immediate postexercise compared with preexercise control (P < 0.01), but it gradually returned to the normal level after the 180 min postexercise. However, the content of DYN A_1–13 in PVN rose substantially only in 30 min postexercise in comparison with the preexercise control (P < 0.01). Thus, different changes of NPY, L-ENK, and DYN A_1–13 in response to such high-intensity exercise depend on the brain region and the time examined, especially, the contents of NPY in different brain regions continuously remain at a high level after such high-intensity exercise. And this high level might reduce energy expenditure and thus contribute to the stimulation of brain NPY neurons.     

Seasonal changes in the photosynthetic capacity of cones on a larch (Larix kaempferi) canopy

The seasonal changes of photosynthesis of cones of Japanese larch (Larix kaempferi Carr.) trees showed that gross photosynthetic rate of young cones (P _G) was 2–3 μmol m⁻² s⁻¹ at surface area unit and P _G/R _D (dark respiration of cones) peaked about 0.7 in the same period, indicating that 70 % of respiratory CO₂ was re-fixed. With maturation, P _G and P _G/R _D sharply decreased. Chlorophyll content in cones was 3–20 % of that in leaves, which made it a limiting factor for photosynthesis and its content was closely correlated with photosynthetic capacity. Although sunken and linearly arranged stomatal organs were found on the scale of young cones, differently from the significant regulation of leaf photosynthesis, these stomata tended to be non-functional since CO₂ is not limiting factor for cone photosynthesis. Thus photosynthesis of larch cones is an additional contribution to their development.     

Lack of endothelial nitric oxide synthase decreases cardiomyocyte proliferation and delays cardiac maturation

We recently demonstrated that deficiency in endothelial nitric oxide synthase (eNOS) results in congenital septal defects and postnatal heart failure. The aim of this study was to investigate the role of eNOS in cardiomyocyte proliferation and maturation during postnatal development. Cultured eNOS knockout (eNOS^–/–) cardiomyocytes displayed fewer cells and lower bromodeoxyuridine (BrdU) incorporation in vitro compared with wild-type (WT) cardiomyocytes (P < 0.05). Treatment with the nitric oxide (NO) donor diethylenetriamine NONOate increased BrdU incorporation and cell counts in eNOS^–/– cardiomyocytes (P < 0.05). Inhibition of nitric oxide synthase activity using N^G-nitro-L-arginine methyl ester decreased the level of BrdU incorporation and cell counts in WT cardiomyocytes (P < 0.05). Vascular endothelial growth factor (VEGF) increased the level of BrdU incorporation in cultured WT cardiomyocytes in a dose- and time-dependent manner (P < 0.05). Conversely, VEGF did not alter BrdU incorporation in eNOS^–/– cardiomyocytes (P = not significant). Furthermore, deficiency in eNOS significantly decreased BrdU labeling indexes in neonatal hearts in vivo. Although WT hearts displayed a rapid decrease in atrial natriuretic peptide (ANP) expression in the first week of neonatal life, ANP expression in eNOS^–/– hearts remain elevated. Our study demonstrated that NO production from eNOS is necessary for postnatal cardiomyocyte proliferation and maturation, suggesting that eNOS plays an important role during postnatal heart development. proliferation; heart development     

Chloride Conductance and P i Transport are Separate Functions Induced by the Expression of NaPi-1 in Xenopus Oocytes

Expression of the protein NaPi-1 in Xenopus oocytes has previously been shown to induce an outwardly rectifying Cl⁻ conductance (G_Cl), organic anion transport and Na⁺-dependent P _i -uptake. In the present study we investigated the relation between the NaPi-1 induced G_Cl and P _i -induced currents and transport. NaPi-1 expression induced P _i -transport, which was not different at 1–20 ng/oocyte NaPi-1 cRNA injection and was already maximal at 1–2 days after cRNA injection. In contrast, G_Cl was augmented at increased amounts of cRNA injection (1–20 ng/oocyte) and over a five day expression period. Subsequently all experiments were performed on oocytes injected with 20 ng/oocytes cRNA. P _i -induced currents (Ip) could be observed in NaPi-1 expressing oocytes at high concentrations of P _i (≥ 1 mm P _i ). The amplitudes of Ip correlated well with G_Cl. Ip was blocked by the Cl⁻ channel blocker NPPB, partially Na⁺-dependent and completely abolished in Cl⁻ free solution. In contrast, P _i -transport in NaPi-1 expressing oocytes was not NPPB sensitive, stronger depending on extracellular Na⁺ and weakly affected by Cl⁻ substitution. Endogenous P _i -uptake in water-injected oocytes amounted in all experiments to 30–50% of the Na⁺-dependent P _i -transport observed in NaPi-1 expressing oocytes. The properties of the endogenous P _i -uptake system (K _m for P _i > 1 mm; partial Na⁺- and Cl⁻-dependence; lack of NPPB block) were similar to the NaPi-1 induced P _i -uptake, but no Ip could be recorded at P _i -concentrations ≤3 mm. In summary, the present data suggest that Ip does not reflect charge transfer related to P _i -uptake, but a P _i -mediated modulation of G_Cl. Received: 22 October 1997/Revised: 24 March 1998     

The effect of transforming growth factor-β on fibroblast cell proliferation in intact connective tissue in vitro

Summary Transforming growth factor-β (TGF-β) has varying effects on cell proliferation, stimulating some cell types while inhibiting others. Its effect on proliferation has mostly been assessed in cell cultures without consideration for the influence of a tissue matrix. In the present investigation we studied the effect of TGF-β on fibroblast cell proliferation in intact connective tissue in vitro using the membranous part of the rat mesentery. Mesenteric membranes were spread over the hole of a cytocentrifuge paper, incubated in vitro, and exposed to various concentrations of TGF-β with or without serum added. At designated times after incubation, the specimens were fixed, spread out on microscope slides, and stained by the Feulgen reaction. Cell proliferation was estimated by counting mitoses in fibroblasts and mesothelial cells and by DNA cytometry of fibroblast nuclei using computer assisted image analyses. Higher concentrations of TGF-β significantly increased proliferation estimated as either the percentage of cells in the S+G₂ phase of the cell cycle or the mitotic index when serum was added. In medium without serum, TGF-β did slightly, but not significantly, increase proliferation. The results show that TGF-β stimulates connective tissue cell proliferation dose-dependently in intact connective tissue in vitro and that addition of serum to the medium is a prerequisite for optimal stimulation.     

Effects of starvation on morphometric characteristics of olive flounder, <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

An experiment was conducted for 12 weeks to determine the effect of feeding and starvation on truss and classical parameters in the external body of olive flounder, Paralichthys olivaceus. There was an increase in the truss dimension of body depth in the trunk region of the fed group at the end of the experiment (P < 0.05). In the olive flounder, the trunk region dimensions, including body depth measurements, are likely to be compromised by variability related to differences in the feeding of fish from different habitats. Classical dimensions in relation to the anterior–posterior body axis decreased and classical dimensions of head characteristics increased upon starvation but decreased upon feeding (P < 0.05). These results suggest that these morphometric parameters may be a useful index of nutritional status in olive flounder.     

Genetic control of variation in human gingival fibroblast proliferation rate

Summary Logarithmic proliferation rate (Days 1 to 6) of gingival fibroblasts derived from 15 pairs of monozygotic (MZ) and 9 pairs of dizygotic (DZ) human twins was compared under optimal and suboptimal growth conditions. Cell proliferation rates exhibited considerable variability among strains. For Caucasian donors (13 MZ, 6 DZ pairs) DZ twins demonstrated significantly greater (P<0.01) within-pair variance in cell proliferation rate compared to MZ twins when evaluated under optimal growth conditions. Heritability analysis indicated strong genetic control of proliferation rate of human gingival fibroblasts (HGF) under optimal growth conditions (1.0±0.67), whereas proliferation rate of HGF under suboptimal growth conditions revealed less genetic control (0.42±0.61). These findings emphasize the importance of carefully matching control and test HGF in assays dependent on cellular proliferation. This work was supported by grants DE-06671 and DE-07841 from the National Institutes of Health.     

A Th1 cytokine–enriched microenvironment enhances tumor killing by activated T cells armed with bispecific antibodies and inhibits the development of myeloid-derived suppressor cells

In this study, we investigated whether activated T cells (ATC) armed with bispecific antibodies (aATC) can inhibits tumor growth and MDSC development in a Th₁ cytokine–enriched (IL-2 and IFN-γ) microenvironment. Cytotoxicity mediated by aATC was significantly higher (P < 0.001) against breast cancer cell lines in the presence of Th₁ cytokines as compared with control co-cultures. In the presence of aATC, CD33⁺/CD11b⁺/CD14⁻/HLA-DR⁻ MDSC population was reduced significantly under both control (P < 0.03) and Th₁-enriched (P < 0.036) culture conditions. Cytokine analysis in the culture supernatants showed high levels of MDSC suppressive chemokines CXCL9 and CXCL10 in Th₁-enriched culture supernatants with highly significant increase (P < 0.001) in the presence of aATC. Interestingly, MDSC recovered from co-cultures without aATC showed potent ability to suppress activated T-cell-mediated cytotoxicity (P < 0.001), IFN-γ production (P < 0.01) and T-cell proliferation (P < 0.05) compared to those recovered from aATC-containing co-cultures. These data suggest that aATC can mediate enhanced killing of tumor cells and may suppress MDSC and T_reg differentiation, and presence of Th₁ cytokines potentiates aATC-induced suppression of MDSC, suggesting that Th₁-enriching immunotherapy may be beneficial in cancer treatment.     

Inhibition of cellular transition from G1-resting to G1-prereplicative phase by aminonucleoside or puromycin

Summary Human embryonic lung fibroblasts (IMR-90 and WI-38) were arrested in the G₁ phase of the cell cycle by serum deprivation and high population density. Within 1 hr after the addition of medium containing fresh serum, these cells showed an increase in rRNA synthesis. The inclusion of 100 μg per ml aminonucleoside of puromycin (AMS) in the fresh medium eliminated the serum stimulation of rRNA synthesis and prevented the cells from making the G₁-resting phase to G₁-prereplicative phase transition. AMS also prevented the synthesis of HnRNA normally found within 10 hr after serum stimulation. Serum-stimulated RNA synthesis in starved, SV-40 transformed fibroblasts (WI-38-VA-13 cells) was inhibited, but not completely prevented, by AMS indicating that transformed cells may produce specific RNA's that are not AMS-sensitive and that may be responsible for the failure of transformed cells to be arrested in G₁. This work was supported by PHS Research Grant CA19750-02 from the National Cancer Institute. These results were reported previously in a preliminary form (7).     

Culture,characteristics and chromosome complement of Siberian tiger fibroblasts for nuclear transfer

Tiger (Panthera tigris Linnaeus, 1758) is a characteristic species of Asia, which is in severe danger. Siberian tiger (Panthera tigris altaica) is the largest one of the five existent tiger subspecies. It is extremely endangered. One new way for tiger protection and rescue is to study interspecies cloning. But there is few research data about Siberian tiger. In this study, we cultured Siberian tiger fibroblasts in vitro, analyzed their biological characteristics, chromosomes, and cell cycles, to provide not only nuclear donors with good morphology, normal biological characteristics, and chromosome quantity for tiger interspecies cloning, but also reliable data for further studying Siberian tiger. The results indicated that Siberian tiger ear fibroblasts can be successfully obtained by tissue culture either with or without overnight cold digestion, the cultured cells were typical fibroblasts with normal morphology, growth curve, and chromosome quantity; G0/G1 percentage increased and S percentage decreased with the confluence of cells. G0/G1 and S stage rate was significantly different between 40–50% and 80–90%, 95–100% confluence; there is no distinct difference between 80–90% and 95–100% confluence. The cells at the same density (80–90% confluence) were treated with or without 0.5% serum starving, GO/G1 rate of the former was higher than the latter, but the difference was not significant. GO/G1 proportion of 95–100% confluence was slightly higher than serum starving (80–90% confluence), but no significant difference. Therefore, the Siberian tiger fibroblasts we cultured in vitro can be used as donor cells, and the donor cells do not need to be treated with normal serum starvation during nuclear transfer; if we will just consider the rate of the G0/G1 stage cells, serum starvation can be replaced by confluence inhibition when cultured cells were more than 80–90% confluence.     

Induction of accessory cell function of human alveolar macrophages by inhalation of human natural interleukin-2

 Accessory function allows antigen-presenting cells to produce sufficient secondary signals for optimum T cell proliferation and interleukin-2 (IL-2) production. Alveolar macrophages are inferior accessory cells compared to monocytes (PBM). We report here that the accessory index (AI) of alveolar macrophages and PBM of patients with lung metastases of solid tumors treated with inhalations of human natural IL-2 (hnIL-2) increased following its administration (P<0.005). The accessory index was significantly elevated from baseline values after 2 weeks of inhalation of 300 000 IU hnIL-2/day (8.2±10.2 compared to 1.1±1; P<0.001). The inhalation of 150 000 IU also induced increases in the index (AI = 2.3±1.9), however, without reaching statistical significance. In addition at 300 000 IU IL-2/day a significant increase in the accessory index was observed for PBM (4±2.5; P<0.05). The indices of PBM and alveolar macrophages prior to inhalation showed a significant negative correlation with the age of the patients (r _s =  – 0.5; r _s =  – 0.8, respectively; P<0.03 for all comparisons). Our data demonstrate that the inhalational application of hnIL-2 enhances the accessory function of alveolar macrophages and, to lesser extent, the accessory index of PBM, indicating the occurrence of pharmacological immunostimulation. Received: 16 August 1995 / Accepted: 4 January 1996     

Down syndrome: increased frequency of maternal meiosis I nondisjunction during the transitional stages of the ovulatory seasons

Summary In a month-of-birth study of Down syndrome (DS) individuals, we found—in agreement with a previous collaborative European study (Jongbloet et al. 1982)—a distribution of maternal meiosis I (Mat MI)-DS births, both graphically and statistically differing from that of both total births (P<0.05) and from that of the three other (Mat MII+Pat MI+Pat MII)-DS subcategories (P<0.001). In four subgroups, namely the DS individuals born in the years 1960–1979 and 1980–1983 and those from mothers ≤30 years, and ≥31 years, analogous results were found. In addition, the Mat MI-DS individuals were shown to have been conceived more frequently during the “strong” change rate periods. i.e. the transitional stages of ovulatory breakthrough and breakdown, and less during the “weak” change rate periods, i.e. the periods of a rather stable ovulatory rate (P<0.05). The same results were found in two subgroups of individuals born 1960–1970 (P<0.055) and from mothers≥31 years (P<0.02). This interdependence estimated by linear regression in the total group (r _s =0.503; P<0.055) and in the subgroup ≥31 years (r _s =0.556; P<0.05), supports the hypothesis that Mat MI nondisjunctions occur more frequently during the transitional stages of the ovulatory seasons, i.e. they are caused by seasonal pre-ovulatory overripeness ovopathy (SPOO).