Investigation of factors influencing the immunogenicity of hCG as a potential cancer vaccine.

Human chorionic gonadotrophin (hCG) and its ß-subunit (hCGß) are tumour autocrine growth factors whose presence in the serum of cancer patients has been linked to poorer prognosis. Previous studies have shown that vaccines which target these molecules and/or the 37 amino acid C-terminal hCGß peptide (hCGßCTP) induce antibody responses in a majority of human recipients. Here we explored whether the immunogenicity of vaccines containing an hCGß mutant (hCGßR68E, designed to eliminate cross-reactivity with luteinizing hormone) or hCGßCTP could be enhanced by coupling the immunogen to different carriers [keyhole limpet haemocyanin (KLH) or heat shock protein 70 (Hsp70)] using different cross-linkers [1-ethyl-3(3-dimethylaminopropyl)carboiimide (EDC) or glutaraldehyde (GAD)] and formulated with different adjuvants (RIBI or Montanide ISA720). While there was little to choose between KLH and Hsp70 as carriers, their influence on the effectiveness of a vaccine containing the BAChCGßR68E mutant was less marked, presumably because, being a foreign species, this mutant protein itself might provide T helper epitopes. The mutant provided a significantly better vaccine than the hCGßCTP peptide irrespective of the carrier used, how it was cross-linked to the carrier or which adjuvant was used when hCG was the target. Nonetheless, for use in humans where hCG is a tolerated self-protein, the need for a carrier is of fundamental importance. Highest antibody titres were obtained by linking the BAChCGßR68E to Hsp70 as a carrier by GAD and using RIBI as the adjuvant, which also resulted in antibodies with significantly higher affinity than those elicited by hCGßCTP peptide vaccine. This makes this mutant vaccine a promising candidate for therapeutic studies in hCGß-positive cancer patients.

Exome sequencing-driven discovery of coding polymorphisms associated with common metabolic phenotypes.

AIMS/HYPOTHESIS: Human complex metabolic traits are in part regulated by genetic determinants. Here we applied exome sequencing to identify novel associations of coding polymorphisms at minor allele frequencies (MAFs) >1% with common metabolic phenotypes. METHODS: The study comprised three stages. We performed medium-depth (8×) whole exome sequencing in 1,000 cases with type 2 diabetes, BMI >27.5 kg/m(2) and hypertension and in 1,000 controls (stage 1). We selected 16,192 polymorphisms nominally associated (p < 0.05) with case-control status, from four selected annotation categories or from loci reported to associate with metabolic traits. These variants were genotyped in 15,989 Danes to search for association with 12 metabolic phenotypes (stage 2). In stage 3, polymorphisms showing potential associations were genotyped in a further 63,896 Europeans. RESULTS: Exome sequencing identified 70,182 polymorphisms with MAF >1%. In stage 2 we identified 51 potential associations with one or more of eight metabolic phenotypes covered by 45 unique polymorphisms. In meta-analyses of stage 2 and stage 3 results, we demonstrated robust associations for coding polymorphisms in CD300LG (fasting HDL-cholesterol: MAF 3.5%, p = 8.5 × 10(-14)), COBLL1 (type 2 diabetes: MAF 12.5%, OR 0.88, p = 1.2 × 10(-11)) and MACF1 (type 2 diabetes: MAF 23.4%, OR 1.10, p = 8.2 × 10(-10)). CONCLUSIONS/INTERPRETATION: We applied exome sequencing as a basis for finding genetic determinants of metabolic traits and show the existence of low-frequency and common coding polymorphisms with impact on common metabolic traits. Based on our study, coding polymorphisms with MAF above 1% do not seem to have particularly high effect sizes on the measured metabolic traits.

The frequent UCP2 -866G>A polymorphism protects against insulin resistance and is associated with obesity: a study of obesity and related metabolic traits among 17 636 Danes.

CONTEXT: Uncoupling protein 2 (UCP2) is involved in regulating ATP synthesis, generation of reactive oxygen species and glucose-stimulated insulin secretion in ß-cells. Polymorphisms in UCP2 may be associated with obesity and type 2 diabetes mellitus. OBJECTIVE: To determine the influence of a functional UCP2 promoter polymorphism (-866G>A, rs659366) on obesity, type 2 diabetes and intermediary metabolic traits. Furthermore, to include these and previously published data in a meta-analysis of this variant with respect to its impact on obesity and type 2 diabetes. DESIGN: We genotyped UCP2 rs659366 in a total of 17 636 Danish individuals and established case-control studies of obese and non-obese subjects and of type 2 diabetic and glucose-tolerant subjects. Meta-analyses were made in own data set and in publicly available data sets. Quantitative traits relevant for obesity and type 2 diabetes were analysed within separate study populations. RESULTS: We found no consistent associations between the UCP2 -866G-allele and obesity or type 2 diabetes. Yet, a meta-analysis of data from 12 984 subjects showed an association with obesity (GA vs GG odds ratio (OR) (95% confidence interval (CI)): 0.894(0.826-0.968) P=0.00562, and AA vs GG OR(95% CI): 0.892(0.800-0.996), P=0.0415. Moreover, a meta-analysis for type 2 diabetes of 15 107 individuals showed no association. The -866G-allele was associated with elevated fasting serum insulin levels (P=0.002) and HOMA insulin resistance index (P=0.0007). Insulin sensitivity measured during intravenous glucose tolerance test in young Caucasian subjects (n=377) was decreased in carriers of the GG genotype (P=0.05). CONCLUSIONS: The UCP2 -866G-allele is associated with decreased insulin sensitivity in Danish subjects and is associated with obesity in a combined meta-analysis.

Association studies of novel obesity-related gene variants with quantitative metabolic phenotypes in a population-based sample of 6,039 Danish individuals.

AIMS/HYPOTHESIS: Genome-wide association studies have identified novel WHR and BMI susceptibility loci. The aim of this study was to elucidate if any of these loci had an effect on quantitative measures of glucose homeostasis, including estimates of insulin release and insulin sensitivity in an epidemiological setting. METHODS: By applying an additive genetic model, 14 WHR-associated gene variants and 18 BMI-associated variants were investigated for their relationships with glucose-related metabolic traits in treatment-naive individuals from the population-based Inter99 study sample (n = 6,039). RESULTS: Of the variants associated with BMI, the QPCTL rs2287019 C allele was associated with an increased insulinogenic index of 7.4% per risk allele (p = 4.0 × 10⁻7) and increased disposition index of 5.6% (p = 6.4 × 10⁻5). The LRP1B rs2890652 C allele was associated with insulin resistance, showing a 3.3% increase (p = 0.0011) using the HOMA-insulin resistance (HOMA-IR) index and a 2.2% reduction (p = 0.0014) with the Matsuda index. Of the variants associated with WHR, LYPLAL1/SLC30A10 rs4846567 G allele carriers showed a 5.2% lower HOMA-IR (p = 0.00086) in women, indicating improved insulin sensitivity. Female carriers of the VEGFA rs6905288 A allele were insulin resistant, with a 3.7% increase in HOMA-IR (p = 0.00036) and 4.0% decrease in Matsuda index (p = 2 × 10⁻4). CONCLUSIONS: Our correlative findings from analysing single-locus data suggest that some variation in validated BMI and WHR loci are associated with either increased or decreased insulin sensitivity and thereby potentially with metabolically healthy or metabolically unhealthy subsets of obesity. The results call for testing in larger study samples and for further physiological exploration of the possible metabolic implications of these loci.

Class-1 translation termination factors: invariant GGQ minidomain is essential for release activity and ribosome binding but not for stop codon recognition.

Previously, we have shown that all class-1 polypeptide release factors (RFs) share a common glycine-glycine-glutamine (GGQ) motif, which is critical for RF activity. Here, we subjected to site-directed mutagenesis two invariant amino acids, Gln185 and Arg189, situated in the GGQ minidomain of human eRF1, followed by determination of RF activity and the ribosome binding capacity for mutant eRF1. We show that replacement of Gln185 with polar amino acid residues causes partial inactivation of RF activity; Gln185Ile, Arg189Ala and Arg189Gln mutants are completely inactive; all mutants that retain partial RF activity respond similarly to three stop codons. We suggest that loss of RF activity for Gln185 and Arg189 mutants is caused by distortion of the conformation of the GGQ minidomain but not by damage of the stop codon recognition site of eRF1. Our data are inconsistent with the model postulating direct involvement of Gln185 side chain in orientation of water molecule toward peptidyl-tRNA ester bond at the ribosomal peptidyl transferase centre. Most of the Gln185 mutants exhibit reduced ability to bind to the ribosome, probably, to rRNA and/or (peptidyl)-tRNA(s). The data suggest that the GGQ motif is implicated both in promoting peptidyl-tRNA hydrolysis and binding to the ribosome.

Cell volume increase in murine MC3T3-E1 pre-osteoblasts attaching onto biocompatible tantalum observed by magnetic AC mode atomic force microscopy.

Magnetic AC mode (MACmode) atomic force microscopy (AFM) was used to study murine (mouse) MC3T3-E1 preosteoblastic cells attached to biocompatible tantalum substrates. Cell volumes of attached cells derived from AFM images were compared to volumes of detached cells in suspension measured by the Coulter sizing technique. An increase of approximately 50% in cell volume was observed when the cells attached to planar tantalum substrates and developed a flattened structure including lamellipodia. We address thoroughly the issues general to the AFM determination of absolute cell volumes, and compare our magnetic AC mode AFM measurements to hitherto reported cell volume determinations by contact mode AFM.

Major proteins induced and down-regulated by interferons in human cultured cells: identification of a unique set of proteins induced by interferon-alpha in epithelial, fibroblast, and lymphoid cells.

In all, 40 major polypeptides ranging in molecular weights from 14.5 to 83 kDa were shown to be induced by IFNs alpha (also by IFN-alpha 2b and beta in a few cases) and gamma in human cultured cells of epithelial (transformed amnion cells (AMA)), fibroblast (proliferating and quiescent MRC-5 fibroblasts), and lymphoid origin (Molt-4). With the exception of a heat shock protein (IEF14 or hs x 70) and two tropomyosins (IEFs 52x and 55), none of these proteins corresponded to polypeptides (proliferation-sensitive or others) previously identified and catalogued by us. IFN-alpha induced the highest number of polypeptides in lymphoid cells, while the response to IFN-gamma was more pronounced in cultured epithelial and fibroblast cells. Several of the polypeptides induced by IFNs alpha and gamma were synthesized (albeit at different rates) by the control untreated cells, and in some cell types such as normal human peripheral blood mononuclear cells many were expressed at high levels. Only IFN-alpha-induced a unique set of proteins (alpha 1, 51 kDa; alpha 2, 15 kDa; alpha 19, 78 kDa; and gamma 10, 83 kDa) in all cultured cell types studied, implying that response to this IFN involves a shared biochemical pathway(s). Both IFN-alpha (also IFN-alpha 2b) and beta induced an identical group of proteins in AMA cells in agreement with the fact that type I IFNs share common receptors. IFNs alpha and gamma induced a few common polypeptides, but only gamma 10 (83 kDa) showed increased synthesis in all cell types exposed to either of these IFNs. A total of 28 major cellular polypeptides were down-regulated by IFNs in the various cell type studied. Different sets of proteins were affected, however, in each system, emphasizing the complexity of the mechanisms underlying the action of these factors. Treatment of synchronized G1 AMA cells with IFNs alpha, beta, or gamma (500 IU/ml, final concentration) did not inhibit their progression from G1 to S-phase as determined by indirect immunofluorescence using PCNA autoantibodies specific for cyclin. These observations were in line with the fact that IFNs did not affect dividin or cyclin(PCNA) synthesis (S-phase specific proteins) at least within the first 17 hr after their addition.

A genetic risk score of 45 coronary artery disease risk variants associates with increased risk of myocardial infarction in 6041 Danish individuals.

BACKGROUND: In Europeans, 45 genetic risk variants for coronary artery disease (CAD) have been identified in genome-wide association studies. We constructed a genetic risk score (GRS) of these variants to estimate the effect on incidence and clinical predictability of myocardial infarction (MI) and CAD. METHODS: Genotype was available from 6041 Danes. An unweighted GRS was constructed by making a summated score of the 45 known genetic CAD risk variants. Registries provided information (mean follow-up = 11.6 years) on CAD (n = 374) and MI (n = 124) events. Cox proportional hazard estimates with age as time scale was adjusted for sex, BMI, type 2 diabetes mellitus and smoking status. Analyses were also stratified either by sex or median age (below or above 45 years of age). We estimated GRS contribution to MI prediction by assessing net reclassification index (NRI) and integrated discrimination improvement (IDI) added to the European SCORE for 10-year MI risk prediction. RESULTS: The GRS associated significantly with risk of incident MI (allele-dependent hazard ratio (95%CI): 1.06 (1.02-1.11), p = 0.01) but not with CAD (p = 0.39). Stratification revealed association of GRS with MI in men (1.06 (1.01-1.12), p = 0.02) and in individuals above the median of 45.11 years of age (1.06 (1.00-1.12), p = 0.03). There was no interaction between GRS and gender (p = 0.90) or age (p = 0.83). The GRS improved neither NRI nor IDI. CONCLUSION: The GRS of 45 GWAS identified risk variants increase the risk of MI in a Danish cohort. The GRS did not improve NRI or IDI beyond the performance of conventional European SCORE risk factors.

Number and proliferative capacity of osteogenic stem cells are maintained during aging and in patients with osteoporosis.

Decreased bone formation is an important pathophysiological mechanism responsible for bone loss associated with aging and osteoporosis. Osteoblasts (OBs), originate from mesenchymal stem cells (MSCs) that are present in the bone marrow and form colonies (termed colony-forming units-fibroblastic [CFU-Fs]) when cultured in vitro. To examine the effect of aging and osteoporosis on the MSC population, we quantified the number of MSCs and their proliferative capacity in vitro. Fifty-one individuals were studied: 38 normal volunteers (23 young individuals [age, 22-44 years] and 15 old individuals [age, 66-74 years]) and 13 patients with osteoporosis (age, 58-83 years). Bone marrow was aspirated from iliac crest; mononuclear cells were enriched in MSCs by magnetic activated cell sorting (MACS) using STRO-1 antibody. Total CFU-F number, size distribution, cell density per CFU-F, number of alkaline phosphatase positive (ALP+) CFU-Fs, and the total ALP+ cells were determined. In addition, matrix mineralization as estimated by alizarin red S (AR-S) staining was quantified. No significant difference in colony-forming efficiency between young individuals (mean +/- SEM; 87 +/- 12 CFU-Fs/culture), old individuals (99 +/- 19 CFU-Fs/culture), and patients with osteoporosis (129 +/- 13 CFU-Fs/culture; p = 0.20) was found. Average CFU-F size and cell density per colony were similar in the three groups. Neither the percentage of ALP+ CFU-Fs (66 +/- 6%, 65 +/- 7%, and 72 +/- 4% for young individuals, old individuals, and patients with osteoporosis, respectively) nor the percentage of ALP+ cells per culture (34 +/- 5%, 40 +/- 6%, and 41 +/- 4%) differed between groups. Finally, mineralized matrix formation was similar in young individuals, old individuals, and patients with osteoporosis. Our study shows that the number and proliferative capacity of osteoprogenitor cells are maintained during aging and in patients with osteoporosis and that other mechanisms must be responsible for the defective osteoblast (OB) functions observed in these conditions.

2'-adenylated derivatives of Ap3A activate RNase L.

The exact physiological function of Ap3A (A5'ppp5"A, 5'5" diadenosine triphosphate) remains unclear. Previously we have demonstrated that the human p46 2-5A synthetase (OAS1) efficiently utilises Ap3A as an acceptor substrate for oligoadenylate synthesis. Here we show that Ap3A(2'p5'A)n oligonucleotides can activate the 2-5A-dependent RNase (RNase L), when the number of 2',5'-linked adenyl residues is two or more. Under the experimental conditions applied the half-maximal activation (AC50) of RNase L for 2'-adenylated Ap3A derivatives was determined to be in nanomolar range while the AC50 for 2-5A3 was 0.4 nM. The Ap3A(2'p5'A)n oligonucleotides are thus less effective in activating RNase L than 2-5A. We also investigated the occurrence of 2'-adenylated Ap3A in interferon and poly(I).poly(C)-treated HeLa cells. In purified trichloroacetic acid-soluble extracts about 40% of RNase L-activating material is resistant to phosphatase treatment, whereas the removal of 5'-terminal phosphates greatly reduces the activating properties of 2-5A. We assume that this activity at least partly may be associated with the presence of 2'-adenylated ApnA derivatives with blocked 5'-terminal phosphates.

Inhibition of 2'-5' oligoadenylate synthetase by divalent metal ions.

OAS1 is the small form and OAS2 is the medium form of the human interferon-induced 2'-5' oligoadenylate synthetases. The p42 isoform of OAS1 and the p69 isoform of OAS2 have been expressed in insect cells and purified to give pure, highly active 2'-5' oligoadenylate synthetase. The catalysis of 2'-5' oligoadenylate synthesis is strictly dependent on double-stranded RNA and magnesium ions. We have examined the effect of a series of divalent metal ions: copper, iron and zinc ions strongly inhibited the enzymatic activity, cobalt and nickel ions were partly inhibitory whereas calcium and manganese ions were without effect. However, manganese ions can replace magnesium ions as activator. The inhibitory effect of zinc ions was characterised in detail. The inhibitory constants of Zn(2+) were estimated to be 0.10 mM for OAS1p42 and to 0.02 mM for OAS2p69. Cross-linking experiments showed that zinc ions can control the oligomerisation by enhancing the formation of tetrameric forms of OAS1p42

Ap3A and Ap4A are primers for oligoadenylate synthesis catalyzed by interferon-inducible 2-5A synthetase.

The biological role of Ap3A synthesized in cells by tryptophanyl-tRNA synthetase (WRS) is unknown. Previously we have demonstrated that the cellular level of Ap3A significantly increases after interferon treatment. Here we show that the human 46 kDa 2-5A synthetase efficiently utilizes Ap3A as a primer for oligoadenylate synthesis. The Km for Ap3A is several-fold lower than for Ap4A and 100-fold lower than for ATP. This implies that Ap3A might be a natural primer for the 2'-adenylation reaction catalysed by 2-5A synthetase. Since WRS and 2-5A synthetase are both interferon-inducible proteins, a new link between two interferon-dependent enzymes is established.

Diadenosine oligophosphates (Ap(n)A), a novel class of signalling molecules?

The diadenosine oligophosphates (Ap(n)A) were discovered in the mid-sixties in the course of studies on aminoacyl-tRNA synthetases (aaRS). Now, more than 30 years later, about 300 papers have been published around these substances in attempt to decipher their role in cells. Recently, Ap(n)A have emerged as intracellular and extracellular signalling molecules implicated in the maintenance and regulation of vital cellular functions and become considered as second messengers. Great variety of physiological and pathological effects in mammalian cells was found to be associated with alterations of Ap(n)A levels (n from 2 to 6) and Ap3A/Ap4A ratio. Cell differentiation and apoptosis have substantial and opposite effects on Ap3A/Ap4A ratio in cultured cells. A human Ap3A hydrolase, Fhit, appeared to be involved in protection of cells against tumourigenesis. Ap3A is synthesised by mammalian u synthetase (TrpRS) which in contrast to most other aaRS is unable to synthesise Ap4A and is an interferon-inducible protein. Moreover, Ap3A appeared to be a preferred substrate for 2-5A synthetase, also interferon-inducible, priming the synthesis of 2' adenylated derivatives of Ap3A, which in turn may serve as substrates of Fhit. Tumour suppressor activity of Fhit is assumed to be associated with involvement of the Fhit.Ap3A complex in cytokine signalling pathway(s) controlling cell proliferation. The Ap(n)A family is potentially a novel class of signal-transducing molecules whose functions are yet to be determined.

The physiology of stringent factor (ATP:GTP 3'-diphosphotransferase) in Escherichia coli.

The enzyme ATP:GTP 3'-diphosphotransferase catalyzes the transfer of the beta, gamma-pyrophosphate of ATP to the 3' position of GTP or GDP. The amounts of enzyme were measured in cell extracts of a relA+ strain of E. coli grown at different growth rates between 0.4 and 1.9 generations per hour, using precipitation with specific antibodies to purify the enzyme. The amount of enzyme was found to be a constant fraction of total protein at all growth rates corresponding to about 45 molecules of enzyme per genome equivalent of DNA. The purified enzyme has little catalytic activity by itself but has to be activated either by a complex of 70S ribosomes, mRNA and uncharged tRNA or by a solvent like ethanol at a concentration of about 20%. The kinetic constants of the enzyme for the transfer pyrophosphate from ATP to GTP in the ribosome-activated state were determined. The Vmax was estimated to be 140 mumol/min X mg at 37 degrees C and the S0.5 values for GTP and ATP were 0.35 and 0.53 mM, respectively. The reaction was estimated to have an equilibrium constant of about 300. In the pyrophosphate transfer from ATP to GDP the Vmax was estimated to be 90 mumol/min X mg at 37 degrees C and the S0.5 for GDP as 0.3 mM. During amino acid starvation of a relA+ strain of E. coli the amounts of enzyme and the catalytic capacity of the enzyme are sufficient to maintain the observed ppGpp levels in the cells at all growth rates.

The interferon-induced enzyme 2-5A synthetase adenylates tRNA.

The interferon-induced enzyme 2-5A synthetase is shown to adenylate tRNA. Yeast tRNAPhe was incubated with the enzyme in the presence of double stranded RNA (in this case polyI-polyC) and ATP or deoxyATP. The reaction products were analyzed by ribonuclease T1 digestion of the tRNA, polyacrylamide gel electrophoresis and autoradiography. Using ATP, the 2-5A synthetase adds one, two or three AMP residues to the 3'-end of the tRNA whereas when dATP is replacing ATP, only one nucleotide unit is added. It is concluded that one of the mechanisms of the interferon-induced antiviral effect may be an inhibition of the translation process caused by an inactivation of tRNA molecules by a 2-5A synthetase catalyzed 2'-adenylation of the 3'-end.

Specific inhibitors prevent proteolytic degradation of recombinant proteins expressed in High Five cells.

The insect cell line BTI-TN-5B1-4 (High Five) is frequently used to express recombinant proteins in large amounts using the baculovirus expression system. However, extensive proteolytic degradation of recombinant proteins is often encountered. Furthermore, we have observed that recombinant proteins migrate in SDS-PAGE in agreement with poly-ubiquitinated forms of the protein, suggesting a ubiquitin/proteasome degradation pathway. Here, we describe a systematic study unraveling the effect of adding proteasome inhibitors or specific protease inhibitors to the growth medium of High Five insect cells infected with recombinant baculovirus. Furthermore, protease inhibitors were added to the lysis buffer to establish the most efficient way to inhibit proteolytic activity after lysis of baculovirus-infected cells expressing recombinant proteins. We conclude that a combination of adding protease inhibitors to the growth medium and to the lysis buffer minimizes the proteolytic activity in High Five cells. The most efficient protease inhibitors were E-64 in the growth medium together with Leupeptin in the lysis buffer at concentrations higher than with available cocktails of inhibitors. The optimal treatment of High Five cells is different from the optimal treatment of Sf9 cells. For proteins susceptible to ubiquitinylation, a treatment of insect cell cultures with the proteasome inhibitor MG132 (LLL) leads to a considerable reduction of the yield of production of recombinant protein.

Basal and interferon-induced 2',5'-oligoadenylate synthetase activity in human placental trophoblast and trophoblast-derived malignant cell lines.

Human placental trophoblasts produce interferon (tro-IFNs) when stimulated with viral inducers. Since the antiviral and cellular functions of IFNs are partly mediated by the 2',5'-oligoadenylate synthetase (2-5A synthetase) pathway, the aim of the present study was to determine the basal and IFN-induced levels of 2-5A synthetase in villous trophoblast cultures. A considerable basal level of 2-5A synthetase was observed in syncytiotrophoblast cultures from both first and third trimester. In contrast no basal activity was detectable in placental fibroblast- and trophoblast-derived malignant cell lines (Far, FEG-3, and BeWo). Stimulation with tro-IFN-beta, -alpha and leucocyte-IFN (leu-IFN)-alpha increased the enzyme activity in first and third trimester human syncytiotrophoblast cultures. Treatment with recombinant-IFN (rec-IFN)-gamma significantly enhanced 2-5A synthetase activity in first trimester syncytiotrophoblast, but had no effect on third trimester syncytiotrophoblast. Tro-IFN-beta, -alpha and leu-IFN-alpha induced high levels of 2-5A synthetase activity in placental fibroblast, BeWo and FEG-3 cell-lines, whereas rec-IFN-gamma treatment did not induce 2-5A synthetase activity in any of these cells. No detectable 2-5A synthetase activity was found in the Far cell line. The capability of cells deriving from the fetoplacental unit to raise an antiviral response by the induction of 2-5A synthetase may be important in defending the fetus against viral infection from the mother. Furthermore 2-5A synthetase in cells of the fetoplacental unit may play a role in their normal growth and development.

Variation in the expression of Hsp70, the major heat-shock protein, and thermotolerance in larval and adult selection lines of Drosophila melanogaster.

(1) Lines of Drosophila melanogaster were "laboratory naturally" or artificially selected under five thermal regimes. (2) Hsp70 expression per unit protein after heat hardening and heat-shock resistance with and without prior hardening were measured. (3) Differences between the selection regimes in the responses of these traits suggest that thermal resistance can be changed independently of inducible Hsp70 expression. (4) Adult males had higher survival than females but did not differ in inducible Hsp70 expression per unit protein after heat hardening. (5) Larvae expressed less Hsp70 per unit protein than adults after heat hardening.

[Identification of a novel termination release factor eRF3b expressing the eRF3 activity in vitro and in vivo]. / Identifikatsiia novogo faktora terminatsii transliatsii eRF3b, obladaiushchego in vitro i in vivo aktivnost'iu eRF3.

Termination of translation in eukaryotes is governed by the ribosome, a termination codon in the mRNA, and two polypeptide chain release factors (eRF1 and eRF3). We have identified a human protein of 628 amino acids, named eRF3b, which is highly homologous to the known human eRF3 henceforth named eRF3a. At the nucleotide and at the amino acid levels the human eRF3a and eRF3b are about 87% identical. The differences in amino acid sequence are concentrated near the amino terminus. The most important difference in the nucleotide sequence is that eRF3b lacks a GGC repeat close to the initiation codon in eRF3a. We have cloned the cDNA encoding the human eRF3b, purified the eRF3b expressed in Escherichia coli, and found that the protein is active in vitro as a potent stimulator of the release factor activity of human eRFl. Like eRF3a, eRF3b exhibits GTPase activity, which is ribosome- and eRFl-dependent. In vivo assays (based on suppression of readthrough induced by three species of suppressor tRNAs: amber, ochre, and opal) show that the human eRF3b is able to enhance the release factor activity of endogenous and overexpressed eRFl with all three stop codons.

[Mesenchymal stem cells. Potential use in cell and gene therapy of bone loss caused by aging and osteoporosis]. / Mesenkymale stamceller. Potentielle anvendelser i celle- og genterapi ved knogletab forårsaget af aldring og osteoporose.

Mesenchymal stem cells (MSC) originate from bone marrow and give rise to various cell types, including osteoblasts, chondrocytes, adipocytes, and myocytes. Lineage-specific differentiation is dependent on activation of specific transcription factors. MSCs play an important role in bone modelling and remodelling where they give rise to the osteoblasts necessary for bone formation. Human studies have shown that the number and differentiation potential of MSC are unchanged with age and osteoporosis. In recent years, there has been an increasing interest in the clinical use of MSCs. These have been used to augment healing of bone fractures. Some animal studies have shown that MSCs infused intravenously target bone and possibly participate in bone formation. These studies represent the beginning of a new era, where transplantation with autologous MSCs or genetically-modified MSCs expanded in vitro is a potential treatment strategy to augment bone formation in patients with diverse metabolic and genetic bone diseases, including osteoporosis.