Spliced exons of adenovirus late RNAs colocalize with snRNP in a specific nuclear domain.

Posttranscriptional steps in the production of mRNA include well characterized polyadenylation and splicing reactions, but it is also necessary to understand how RNA is transported within the nucleus from the site of its transcription to the nuclear pore, where it is translocated to the cytoplasmic compartment. Determining the localization of RNA within the nucleus is an important aspect of understanding RNA production and may provide clues for investigating the trafficking of RNA within the nucleus and the mechanism for its export to the cytoplasm. We have previously shown that late phase adenovirus-infected cells contain large clusters of snRNP and non-snRNP splicing factors; the presence of these structures is correlated with high levels of viral late gene transcription. The snRNP clusters correspond to enlarged interchromatin granules present in late phase infected cells. Here we show that polyadenylated RNA and spliced tripartite leader exons from the viral major late transcription unit are present in these same late phase snRNP-containing structures. We find that the majority of the steady state viral RNA present in the nucleus is spliced at the tripartite leader exons. Tripartite leader exons are efficiently exported from the nucleus at a time when we detect their accumulation in interchromatin granule clusters. Since the enlarged interchromatin granules contain spliced and polyadenylated RNA, we suggest that viral RNA may accumulate in this late phase structure during an intranuclear step in RNA transport.

Evidence for involvement of activin A and bone morphogenetic protein 4 in mammalian mesoderm and hematopoietic development.

Xenopus in vitro studies have implicated both transforming growth factor beta (TGF-beta) and fibroblast growth factor (FGF) families in mesoderm induction. Although members of both families are present during mouse mesoderm formation, there is little evidence for their functional role in mesoderm induction. We show that mouse embryonic stem cells, which resemble primitive ectoderm, can differentiate to mesoderm in vitro in a chemically defined medium (CDM) in the absence of fetal bovine serum. In CDM, this differentiation is responsive to TGF-beta family members in a concentration-dependent manner, with activin A mediating the formation of dorsoanterior-like mesoderm and bone morphogenetic protein 4 mediating the formation of ventral mesoderm, including hematopoietic precursors. These effects are not observed in CDM alone or when TGF-beta 1, -beta 2, or -beta 3, acid FGF, or basic FGF is added individually to CDM. In vivo, at day 6.5 of mouse development, activin beta A RNA is detectable in the decidua and bone morphogenetic protein 4 RNA is detectable in the egg cylinder. Together, our data strongly implicate the TGF-beta family in mammalian mesoderm development and hematopoietic cell formation.

Mevalonic acid products as mediators of cell proliferation in simian virus 40-transformed 3T3 cells.

Effects of treatment with serum-free medium and 25-hydroxycholesterol (25-OH) on the cell cycle of simian virus 40-transformed 3T3 fibroblasts, designated SV-3T3 cells, were studied and compared with simultaneous effects on the activity of 3-hydroxy-3-methylglutaryl (HMG) CoA reductase and incorporation of [3H]mevalonic acid into cholesterol, Coenzyme Q, and dolichol. The data confirm our previous finding (O. Larsson and A. Zetterberg, Cancer Res., 46: 1233-1239, 1986) that 25-OH inhibits the cell cycle traverse of SV-3T3 cells specifically in early G1. In contrast, treatment with serum-free medium had no effect on cell cycle progression. The effect of 25-OH on the cell cycle traverse was correlated to a substantial decrease in the activity of HMG CoA reductase, whereas there was no change in the rate of [3H]mevalonic acid incorporated into cholesterol, Coenzyme Q, and dolichol. When the cells were exposed to serum-free medium, there was no depression of activity of HMG CoA reductase, and the rate of [3H]mevalonic acid incorporated into dolichol and cholesterol was not affected in any appreciable degree. In contrast the rate of Coenzyme Q synthesis was substantially decreased as a result of serum depletion. A similar decrease in Coenzyme Q synthesis was also achieved by treating the cells with cholesterol-poor serum. This indicates that the rate of Coenzyme Q synthesis is dependent on the concentration of cholesterol in the culture medium. In order to analyze whether some of the products in the mevalonic acid biosynthetic pathway may be of importance in the control of G1 traverse and cell proliferation of SV-3T3 cells, cholesterol, Coenzyme Q, and dolichol were added as supplements to cells treated with 25-OH. It was shown that dolichol was capable of overcoming the 25-OH-induced inhibition of G1 traverse efficiently, whereas cholesterol and Coenzyme Q were considerably less effective. Considered together with the fact that the activity of HMG CoA reductase and incorporation of mevalonic acid into dolichol were unaffected following serum-free treatment, the results suggest that maintenance of a certain level of de novo synthesis of dolichol may contribute to the capability of SV-3T3 cells to proliferate in serum-free medium.

Analysis of factors controlling primary germ layer formation and early hematopoiesis using embryonic stem cell in vitro differentiation.

Differentiation and subsequent development are intricately interwoven processes operating as an integrated whole to form the organism. As an approach to examine these interactions in early mammalian development, we used embryonic stem (ES) cell in vitro differentiation. ES cells can, depending upon the environment differentiated to neuroectoderm, mesoderm and hematopoietic cells. We developed a serum-free, chemically defined medium (CDM) in which ES cells survive and differentiate. In CDM, in the absence of exogenous factors, ES cells form neuroectoderm, upregulating the early neural marker Pax-6. This is consistent with the view that neuroectoderm development can represent a default state, where the absence or sequestration of mesoderm inducing factors permits neuroectoderm formation. In contrast, if CDM is supplemented with bone morphogenetic protein (BMP) 2 or 4 a process resembling primitive streak formation, least at the molecular level occurs, with the formation of mesoderm and subsequently endothelial and hematopoietic cells. If used with care, ES cell in vitro differentiation can act as a guide in understanding the environment which controls early differentiation events in mammals.

Content of trans-octadecenoic acid in vegetarian and normal diets in Sweden, analyzed by the duplicate portion technique.

The intake of trans fatty acids by subjects adhering to the normal Swedish diet or to different vegetarian regimes was studied, using chemical analysis of duplicate portions. Trans-octadecenoic acid was 5.0, 3.9, and 1.8% of dietary fatty acid in the normal, lactovegetarian, and vegan diets, respectively, corresponding to 2.0, 1.3, and 0.5% of energy intake. The results are related to the content of trans-octadecenoic acid in some edible fats.

[New documentation routines in psychiatry in Västerbotten: unified structure for better quality of care]. / Nya dokumentationsrutiner i psykiatrin i Västerbotten: Gemensam journalstruktur för bättre vårdkvalitet.

During recent decades psychiatric health care has become increasingly complex due to substantial clinical improvements and to the growing need of integrating psychiatric services with other health and welfare services in the community. The traditional psychiatric record format is incompatible both with these requirements and with the practical advantages and difficulties of modern computer technology. In a collaborative effort involving most professional categories at three psychiatric units in the county of Västerbotten in northern Sweden, a new structured format for medical records was developed. The basic feature is a structured summary of background factors, social situation, drug habits, and general health, which is reviewed and updated as necessary. The psychiatric condition is described in some detail, including onset and course, symptomatology, personality factors, diagnosis, treatment results, suicidality, etc. Day to day treatment is outlined in in- and out-patient treatment plans, which are evaluated and revised at regular intervals. The new record format, which is used by all categories of health care professionals, is intended to promote goal-directed treatment and professional collaboration, and is easily adapted to computer technology.

Autologous hematopoietic stem cell transplantation in multiple myeloma and lymphoma: an analysis of factors influencing stem cell collection and hematological recovery.

Autologous stem cell transplantation is standard treatment for newly diagnosed younger patients with multiple myeloma and for relapsed or refractory Hodgkin or non-Hodgkin lymphoma. Patient characteristics influencing the yield from stem cell collection and time from transplant to platelet recovery were retrospectively analyzed in 630 consecutive patients, attempting to define adequate amounts of CD34+ cells to collect and reinfuse; 509/630 patients (81%) mobilized the requested CD34+ cell number. Factors influencing the harvest yield were age (P < 0.001) and gender, where 85% of men and 78% of women (P < 0.02) attained the requested stem cell amount. Time to platelet recovery was significantly faster for multiple myeloma patients compared to all other diagnoses (14.6 days compared to 19.8, P < 0.0001). Multiple myeloma patients were older than lymphoma patients but received stem cell transplant up-front as opposed to second line therapy for other patient groups. Multivariate analysis revealed that the most important factor influencing platelet recovery was diagnosis, followed by the amount of reinfused CD34+ cells (P < 0.001, P < 0.05). Blood group O+ had the fastest platelet recovery, whereas blood group A harvested the highest cell amounts. In conclusion, we demonstrate a significant importance of the number of reinfused CD34+ cells on the time to platelet recovery.

Embryonic stem cell development in a chemically defined medium.

Vertebrate germ layer development is an intricately interwoven process with the organism operating as an integrated whole. To examine these processes we have used embryonic stem (ES) cell in vitro differentiation in a serum-free, chemically defined medium (CDM). In CDM, ES cells differentiate as embryoid bodies to neuroectoderm with upregulation of pax-6, without commensurate expression of Brachyury. In the presence of Activin A, pax-6 and Brachyury mRNAs are readily detectable, suggestive of both neuroectoderm and mesoderm formation, while in the presence of BMP-4 a process resembling primitive streak formation at the molecular level occurs. Neuroectoderm development in CDM alone is consistent with the view that this process can occur by default, as reported in Xenopus, due to the absence or sequestration of mesoderm-inducing factors. Additionally, these data show that BMP-4 alone is capable of instigating a process resembling primitive streak formation in ES cells and possibly in vivo.

In vivo effect of noradrenaline on the cyclic AMP level in rat corpora lutea.

Under in vitro conditions adrenaline and noradrenaline can stimulate the production of cyclic AMP in rat corpora lutea to a similar extent as a maximal dose of LH. The present study was undertaken to compare the effects of noradrenaline and LH under in vivo conditions. Anaesthetized rats bearing 2 days old corpora lutea were either infused intraarterially with noradrenaline (0.4 microgram/min) or given a single intraarterial injection of 5 micrograms LH. The cAMP levels in whole ovaries had increased already after 30 sec of noradrenaline infusion. These levels remained high for a few minutes and, thereafter the effect decreased with time. No effect was seen after 20 min of infusion. Infusion of noradrenaline for 20 min increased cAMP in corpora lutea, but not in non-luteal tissue. LH was effective both in luteal and non-luteal tissue. The results show that noradrenaline in vivo can increase the cAMP content only in corpus luteum and within a rather short time period (5 min). This effect is different from that seen with LH which, instead, acts on both corpus luteum and stromal tissue.

Control of adenovirus gene expression: cellular gene products restrict expression of adenovirus host range mutants in nonpermissive cells.

Adenovirus type 5 (Ad5) host range mutants dl312 and hr-1, with lesions in region E1A (0 to 4.5 map units) of the viral genome, fail to accumulate virus-specific early RNA during infection in HeLa cells. In a recent report, we showed that the addition of anisomycin, a stringent inhibitor of protein synthesis, at 1 h after infection of HeLa cells with hr-1 virus resulted in the accumulation of properly spliced and translatable mRNA from all early regions (M. G. Katze, H. Persson, and L. Philipson, Mol. Cell. Biol. 1:807-813, 1981). Based on these results we proposed a model in which expression of early mutant RNA was achieved through inactivation of a cellular protein normally causing a reduction in the amount of viral RNA. These studies have been extended in the present report, which shows that early viral proteins can be detected in Ad5 dl312- and Ad5 hr-1-infected HeLa cells which have been treated for several hours with anisomycin either shortly after infection or before infection. A pulse of drug treatment also resulted in expression of substantial amounts of adenovirus structural proteins after infection with both Ad5 hr-1 and Ad5 dl312, whereas in drug-free controls no late proteins were detected. The Ad5 hr-1 virus previously reported to be DNA replication negative in nonpermissive HeLa cells was found to replicate its DNA, albeit at low levels, when anisomycin was present either from 1 to 5 h postinfection or for 5 h before infection. When infectious virus production was examined in mutant-infected cells the titer of Ad5 dl312 virus was found to increase at least 500-fold in anisomycin-treated HeLa cells. Taken together, these and our previous results suggest that the block in gene expression characteristic for complementation group I Ad5 host range mutants in HeLa cells can be overcome by inactivating cellular gene products serving as negative regulators of viral gene expression.

Characterization of an in vitro perfused rat ovary model: ovulation rate, oocyte maturation, steroidogenesis and influence of PMSG priming.

An in vitro perfused rat ovary model was characterized with respect to ovulation rate, oocyte maturation and steroidogenesis after priming with various doses of pregnant mare's serum gonadotrophin (PMSG) (10, 20 or 30 IU PMSG s.c. in the morning of day 28). In ovaries stimulated with LH 0.1 microgram ml-1 after 1 h of perfusion the number of ovulations was significantly higher in the 20 IU PMSG group than in the to IU PMSG group (6.8 +/- 1.0 ovulations per treated ovary versus 2.4 +/- 1.2, P less than 0.01). Priming with 30 IU PMSG did not result in significantly more or fewer ovulations than did 10 or 20 IU PMSG. No ovaries in the non-stimulated control groups ovulated. Oocytes retrieved directly at ovulation were mature (GVB or PB stage). Among oocytes retrieved from pre-ovulatory follicles punctured at 0, 2, 4, 6 and 8 h after stimulation with LH 0.1 microgram ml-1 there was an increasing number of oocytes with GVB (0/10 at 0 h and 10/10 at 8 h). Stimulation with LH resulted in a rapid release of progesterone, testosterone and oestradiol into the medium, whereas non-stimulated control ovaries exhibited a slow rise of all steroids throughout the perfusion period. It is concluded that priming with 20 IU PMSG in this model results in an optimal number of ovulations and that this model provides a useful tool in studies of various kinds of modifying influences on the ovulatory process.