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PubMed1971

Role of exonuclease and beta protein of phage lambda in genetic recombination. V. Recombination of lambda DNA in vitro.

The sequential action of lambda exonuclease and polynucleotide ligase upon redundant joint molecules is sufficient to produce intact polynucleotide chains and heat-stable, biologically active molecules of lambda DNA, whereas the action of ligase alone is insufficient. These results (a) confirm the previously described mechanism of single-strand assimilation, including a subsidiary mechanism by which the further action of lambda exonuclease is arrested when a redundant strand is completely assimilated, and (b) represent a simulation of the steps in genetic recombination that follow the formation of biparental complexes (synapsis). lambda exonuclease is postulated to catalyze a concerted reaction that includes exposure of complementary sequences, formation of heteroduplex regions, and elimination of redundant branches.

Biological Assay↗
PubMed1989

Phase I study of a combination of recombinant tumor necrosis factor-alpha and recombinant interferon-gamma in cancer patients.

Combinations of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) demonstrate synergistic antiproliferative activity in vitro. Therefore, we initiated a clinical study of recombinant TNF-alpha (rTNF-alpha) and rIFN-gamma combination therapy in humans. Twenty-five patients with metastatic cancer received both rTNF-alpha and rIFN-gamma by intramuscular injection for 5 consecutive days every 2 weeks for a total of 4 weeks. The dose levels were 5/5, 10/5, 10/10, 25/10, 25/25, 50/25, 50/50, 75/50, and 75/75 micrograms/m2 per day of rTNF-alpha/rIFN-gamma. A minimum of 2 patients were entered sequentially at each dose level. If the first 2-week cycle of therapy was well tolerated, the dose was increased to the next highest dose level during the second 2-week cycle. Fever, chills, and fatigue were observed at all doses. Severity of symptoms corresponded to increasing dose levels. Although TNF is identical to a molecule known as "cachectin," the vast majority of our patients did not lose weight while on study. However, alterations in lipid metabolism occurred and were manifested by a median change in triglyceride values of +40 mg/dl (range, -130 to +318 mg/dl), and in cholesterol values of -30 mg/dl (range, -103 to +2 mg/dl). The maximum tolerated dose was 75 micrograms/m2 of rTNF-alpha combined with 50 micrograms/m2 of rIFN-gamma, with dose-limiting side effects being mainly constitutional symptoms. A dose-related suppression in granulocyte and platelet counts was observed. Hematologic parameters returned to baseline within 72 h after therapy was discontinued, and neither infection nor bleeding occurred. Ten of 22 evaluable patients had stable disease for a median of 8 weeks (range, 4-21 weeks); 12 patients showed progressive disease. This study will form the framework for phase II trials of rTNF-alpha and rIFN-gamma combination therapy.

Adolescent↗
PubMed2005

Recombinant human thyrotropin is helpful in the follow-up and 131I therapy of patients with thyroid cancer: a report of the results and benefits using recombinant human thyrotropin in clinical routine.

There is no doubt that the availability of recombinant human thyrotropin (rhTSH) is one of the milestones in the management of patients with differentiated thyroid cancer (DTC). It offers the opportunity to obtain representative serum thyroglobulin (Tg) levels and diagnostic whole-body scanning (Dx WBS) with 131I under adequate TSH elevation, while the patient continues to receive thyroid hormone. But rhTSH is also used with success in the treatment of local recurrences and distant metastases. In this retrospective analysis we were able to show that our excellent clinical experiences with the use of rhTSH (rare side effects and high compliance) could also be demonstrated by sufficiently elevated TSH levels and representative stimulated Tg measurements. Since April 2001 most of the patients with thyroid cancer in our hospital have undergone diagnostic examination (205 patients underwent 319 examinations) and 131I therapy (a total of 68 treatments) with rhTSH stimulation excluding the first radioiodine ablation of remnants after initial thyroidectomy. Our results show that under rhTSH stimulation 83.5% (diagnostic group) and 88% (therapy group) of our patients with DTC obtained a TSH level of greater than 80 mU/L after two injections of rhTSH (Thyrogen, Genzyme Corp., Cambridge, MA) 0.9 mg intramuscularly 24 hours and 48 hours before the administration of 131I. Only 2.3% (diagnostic group) and 0% (therapy group) demonstrated TSH levels less than 50 mU/L. Serum Tg levels under rhTSH-stimulated conditions showed that in 81.2% the serum Tg maximum was obtained on day 5. Because of the costs associated with periodically rhTSH-assisted Tg testing and based on the data of other studies we are now testing mainly on day 5 to identify residual tumor mass and to compare these Tg levels in the follow-up. Our experience demonstrates that the administration of rhTSH is a safe, effective, and-from an economic point of view- valuable tool in the management of patients with DTC.

Follow-Up Studies↗
PubMed1989

Meiosis in Mesostoma ehrenbergii ehrenbergii. IV. Recombination nodules in spermatocytes and a test of the correspondence of late recombination nodules and chiasmata.

Male meiosis in Mesostoma ehrenbergii ehrenbergii (2x = 10) is characterized by extreme restriction of chiasma formation; 3 pairs of chromosomes form bivalents at metaphase I which are associated by single very distally localized chiasma, while two pairs of chromosomes remain as unpaired univalents. Electron microscopical three-dimensional reconstruction analysis of serial sections has been applied to 20 pachytene spermatocyte nuclei. In each nucleus three short stretches of synaptonemal complex (SC) were found, confined to a localized branched lobe of the nucleus, confirming the findings of an earlier study. The majority of reconstructed nuclei show that each of the three SC segments has a single prominent recombination nodule ("late" RN) associated with it. Late RNs in this system therefore show an excellent correspondence with metaphase I chiasmata, in contrast to a previous report. M.e. ehrenbergii is therefore not an exception to the hypothesis that meiotic exchange requires a functional late RN. A few nuclei had two, one or no RNs; these presumably represent nuclei that are not at the stage of maximum RN presence. Although M. e. ehrenbergii shows pronounced chiasma localization at the light microscope level, at the ultrastructural level RNs are widely distributed along the 5-10 microns of SC formed in each bivalent, indicating that genetic exchange are not restricted to particular localized sites but occur at a large number of DNA sequence.

Animals↗
PubMed1999

Activation of the recombination activating gene 1 (RAG-1) transcript in bone marrow of senescent C57BL/6 mice by recombinant interleukin-7.

The level of the recombination activating gene 1 (RAG-1) mRNA in bone marrow cells decreases to a minimal level by the age of 10 months. Recominbant interleukin-7 (rIL-7) is a potent proliferative stimulus for B cell progenitors and upregulates RAG-1 expression in lymphocyte precursors. To investigate the stimulatory effect of rIL-7 on the expression of RAG-1 in old mice, we compared the level of RAG-1 message in short-term bone marrow cultures of cells from mice aged 1 month and 18 months. We found similar levels of RAG-1 mRNA in bone marrow cells of young mice before and after 24 hours of incubation. No RAG-1 mRNA was detected in bone marrow cell cultures prepared from old mice after 24 hours of incubation. However, when rIL-7 was added to the culture medium, RAG-1 mRNA was detected after 24 hours of incubation and its level was similar to that measured in cells from young mice. The expression of RAG-1 was dose-dependent, with 20 ng of rIL-7 per 10(6) old nucleated cells yielding the maximal response. Our results indicate that despite the low or no RAG-1 expression in bone marrow cultures of old mice, the potential to activate RAG-1 in B-cell precursors is still present, and immunoglobulin heavy chain (V(H)D(H)J(H)) rearrangement may be enhanced by rIL7.

Aging↗
PubMed2003

A prospective study of predictive factors of ovarian response in 'standard' IVF/ICSI patients treated with recombinant FSH. A suggestion for a recombinant FSH dosage normogram.

BACKGROUND: The aim was to identify independent predictors of ovarian response to recombinant (r)FSH through a multiple regression analysis. METHODS: Prospective study including 145 'standard' patients treated with 150 IU/day of rFSH during their first IVF/ICSI cycle. Down-regulation was achieved with long agonist protocol. The following were examined as possible predictive factors: age, body mass index, cycle length, smoking status and on day 2-5: total ovarian volume, total number of antral follicles (<10 mm), total Doppler score of the ovarian stromal blood flow, serum FSH, LH, estradiol, inhibin B, and testosterone. RESULTS: Total number of antral follicles, total Doppler score, serum FSH, LH, estradiol, inhibin B, smoking status and cycle length were independent predictors of the number of aspirated follicles. The number of oocytes was predicted by the total number of antral follicles, total Doppler score, serum testosterone and smoking status. In bivariate linear regression analyses ovarian volume was a highly significant predictor of both the number of follicles (P < 0.001) and the number of oocytes (P < 0.001). CONCLUSIONS: Among 12 investigated possible predictive factors in 'standard' patients, the total number of antral follicles and ovarian stromal blood flow assessed by total Power Doppler score are the two most significant predictors of ovarian response. Suggestion for an rFSH dosage normogram is presented.

Adult↗
PubMedDate not supplied

Recombinant follicle stimulating hormone: development of the first biotechnology product for the treatment of infertility. Recombinant Human FSH Product Development Group.

Genes encoding the common gonadotrophin alpha subunit and follicle stimulating hormone (FSH)-specific beta subunit were isolated from a DNA library derived from human fetal liver cells, and inserted into separate expression vectors containing a selectable/amplifiable gene. These vectors were inserted into the genome of the Chinese hamster ovary cell line, resulting in expression of large amounts of biologically active human (h)FSH. This cell line was cultured on microcarrier beads in a large-scale bioreactor. hFSH in the cell culture supernatant was purified to homogeneity by a multistep process. The mature beta subunit had seven fewer amino acid residues than reported in the literature and three other differences were found in the sequence. Similar oligosaccharide structures were present on recombinant (r)-hFSH and a purified urinary (u)-hFSH preparation. In-vitro and in-vivo, the biological activities of u- and r-hFSH were indistinguishable. r-hFSH was formulated in ampoules containing 75 IU FSH activity (approximately 7.5 microg FSH), which accounts for >99% of the protein content of the preparation. Studies in non-human primates and human volunteers showed the pharmacokinetics of u- and r-hFSH to be similar. In healthy volunteers, r-hFSH stimulated follicular development and induced significant increases in serum oestradiol and inhibin. Clinical experience with r-hFSH has shown it is more effective at stimulating ovarian follicle growth than urinary gonadotrophins. It is also effective at initiating spermatogenesis when given together with human chorionic gonadotrophin.

Animals↗
PubMed1997

The interaction of recombinant tissue type plasminogen activator and recombinant plasminogen activator (r-PA/BM 06.022) with human endothelial cells.

The Escherichia coli-expressed recombinant plasminogen activator (r-PA) comprising the kringle 2 and protease domains of human tissue-type plasminogen activator (t-PA) has a four-fold longer half-life time in the circulation than t-PA, possibly resulting in an increased opportunity for r-PA to interact with the endothelial lining. In the present study we investigated the interaction of r-PA and t-PA with human umbilical vein endothelial cells (HUVEC). Specific binding of 125I-t-PA and 125I-r-PA were similar at 4 degrees C (Kd 6 nmol/l; Bmax about 120 fmol/mg cell protein). About half of the specific binding sites were shared by t-PA and r-PA, because unlabeled t-PA and r-PA competed equally with 125I-labeled t-PA and r-PA for binding to HUVEC. The low affinity interaction of 125I-t-PA was several-fold higher than that of 125I-r-PA. When PA binding was studied at 37 degrees C, HUVEC bound more t-PA than r-PA to both specific and non-specific binding sites. Both t-PA and r-PA were internalized and degraded, but t-PA internalization proceeded more efficiently than that of r-PA. In the presence of 100 microM chloroquine, the degradation of t-PA and r-PA was inhibited by 75% and 40%, respectively, indicating lysosomal degradation. When the active sites of t-PA and r-PA were blocked by PPACK, part of the cell association and most of the degradation of both t-PA and r-PA were inhibited. This points to plasminogen activator inhibitor-1 (PAI-1) as one of the specific binding sites. A possible role of LDL-receptor related protein (LRP) or related members of this receptor family was investigated by using the 39 kD receptor associated protein (RAP) which prevents interaction of ligands with these receptors. RAP reduced the association of 125I-t-PA by 25% and the degradation of 125I-t-PA and 125I-r-PA by 65% and 50%, respectively. Our data show that both t-PA and r-PA bind to HUVEC and can subsequently be internalized and degraded. However, r-PA interacts less effectively with HUVEC than t-PA. This indicates that binding to the endothelium does not prevent the clearance of r-PA and is not the cause of its long half-life.

Binding Sites↗
PubMed1991

Thyroid function abnormalities associated with the chronic outpatient administration of recombinant interleukin-2 and recombinant interferon-alpha.

We prospectively examined thyroid function during and following chronic, outpatient therapy with recombinant interleukin-2 (rIL-2) and Roferon-A (rIFN-alpha 2a). Twenty-two of 30 patients with advanced renal cell carcinoma treated on a phase II open pilot study of concomitant rIL-2 and rIFN-alpha 2a were included. Serum levels of thyroxine, triiodothyronine, free thyroxine index, thyrotropin, antithyroid antibodies, and thyrotropin (TSH) receptor binding antibodies were measured before therapy and after every other cycle. Selected patients underwent studies after every cycle and following completion of therapy. Twenty patients (91%) developed laboratory evidence of thyroid dysfunction, 11 (50%) developed hypothyroidism, five (23%) had a biphasic pattern, and four (18%) had hyperthyroidism. The incidence of thyroid dysfunction increased with increased number of treatment cycles. Transient hyperthyroidism was noted in six of the 11 patients studied after the first cycle and persisted after cycle three in only two patients. Hypothyroidism was not observed after cycle 1, but became increasingly frequent between cycles 2 (56%) and 6 (90%). Thyroid function normalized following therapy in nine of 12 patients tested. Antithyroid antibodies were identified pretherapy in five patients (23%) and de novo in none; TSH receptor binding antibodies were not detected. This study demonstrates a remarkably high frequency of reversible thyroid dysfunction in patients with advanced renal cell carcinoma treated with repeated cycles of rIL-2 plus rIFN-alpha 2a. We conclude that chronic therapy with rIL-2 and rIFN-alpha 2a produces thyroid dysfunction in virtually all patients most likely secondary to a nonspecific, nonautoimmune, toxic manifestation of prolonged treatment. IL-2 therapy may, therefore, produce thyroid dysfunction by more than one mechanism.

Adult↗
PubMed2006

Inhibition of homologous recombination by a cohesin-associated clamp complex recruited to the rDNA recombination enhancer.

Silencing within the yeast ribosomal DNA (rDNA) repeats protects the integrity of this highly repetitive array by inhibiting hyperrecombination and repressing transcription from foreign promoters. Using affinity purification combined with highly sensitive mixture mass spectrometry, we have analyzed the protein interaction network involved in suppressing homologous recombination within the rDNA locus. We show that the Net1 and Sir2 subunits of the RENT (regulator of nucleolar silencing and telophase exit) silencing complex, and Fob1, which recruits RENT to the nontranscribed spacer I (NTS1) region of rDNA, are physically associated with Tof2. In addition to RENT components and Fob1, Tof2 copurified with a two-subunit complex composed of Lrs4 and Csm1. Tof2, Lrs4, and Csm1 are recruited to the NTS1 region by Fob1 and are specifically required for silencing at this rDNA region. Moreover, Lrs4 and Csm1 act synergistically with Sir2 to suppress unequal crossover at the rDNA and are released from the nucleolus during anaphase. Together with previous observations showing that Csm1 physically associates with cohesin, these findings suggest a possible model in which RENT, Tof2, and Lrs4/Csm1 physically clamp rDNA to the cohesin ring, thereby restricting the movement of rDNA sister chromatids relative to each other to inhibit unequal exchange.

Cell Cycle Proteins↗
PubMed1990

Phase I trial with recombinant interleukin-2 (rIL-2): immune activation by rIL-2 alone or following pretreatment with recombinant interferon-gamma.

Alterations of immunological parameters were analysed in patients with advanced malignancies during a phase I trial with rIL-2. Five-day infusions of rIL-2 at doses from 1 x 10(6) to 24 x 10(6) biological response modifiers program (BRMP) U/m2 per day were given to 29 patients, with a minimum of three patients per dose. The dose of 24 x 10(6) U/m2 per day was the maximal tolerated dose (MTD). Immunological parameters were analyzed at days 0, 8 and 11 of the rIL-2 courses. Following a leucopenia during rIL-2 infusion, a lymphocytosis was found in all patients except one. The lymphocytosis peaked at day 8 and was detected at doses of rIL-2 as low as 1 x 10(6) U/m2 per day, reaching a plateau at a dose of 16 x 10(6) U/m2 per day. Although all lymphocyte subsets were increased in patients receiving rIL-2, some patients had predominant T cells (CD3+, NKH1(CD56)-), others had predominant natural killer (NK) cells (CD3-, NKH1 (CD56)+), and yet others showed a mixed profile. A strong induction of cells cytotoxic for K562 targets was found in all patients at days 8 and 11. Eighteen patients received, 1 month later, a second treatment in which infusion of rIL-2 was preceded by a course of 5 days infusion of 2 x 10(6) U/m2 per day recombinant interferon-gamma (rIFN-gamma). The infusion of rIFN-gamma prior to rIL-2 had no effect on the rIL-2-induced alterations of immunological parameters. Taken together, our results suggest that immune stimulation by rIL-2 occurs even at low doses and is maximal at a dose below the MTD; and that pretreatment with low-dose rIFN-gamma does not modify the immune stimulation by rIL-2.

Cytotoxicity, Immunologic↗
PubMed1993

Effects of recombinant tumour necrosis factor (rTNF-alpha) in cancer. Observations on the acute phase protein reaction and immunoglobulin synthesis after high dose recombinant TNF-alpha administration in isolated limb perfusions in cancer patients.

To obtain insight in the effect of TNF on the synthesis of acute phase proteins like CRP, alpha 1-antitrypsine, alpha 1-acidglycoprotein, C3 and C4 and the immunoglobulins (IgG-M-A), nine cancer patients who were treated with an isolated limb perfusion (ILP) with high dose recombinant TNF-alpha (rTNF-alpha) were investigated during a 7-day period after the end of the perfusion. Resorption of rTNF-alpha from out of these limbs into the circulation after the ILP induced within 30 min to 6 h in all patients elevated serum levels of IL-6. At the same time C-reactive protein became detectable in serum. The highest serum levels were obtained at 48 h after ILP. The serum levels of the other acute phase proteins (alpha 1-acidglycoprotein, alpha 1-antirypsine, C3, C4), rose more slowly and the highest serum levels were found at the third day. All investigated proteins declined after they had reached their peak levels. Levels of alpha 1-acidglycoprotein and alpha 1-anti-trypsin alpha 1-acid declined slower than both complement component. In regard to the immunoglobulin levels a nearly continuous increase in the serum level of specifically IgM was observed. This study clearly shows the interrelationship between TNF-alpha and IL-6 in regard to the synthesis of the different acute phase proteins; and moreover also a striking effect on IgM synthesis.

Acute-Phase Proteins↗
PubMed1993

In vivo biological activity of retinoids partially correlates to their affinity to recombinant retinoic-acid receptor alpha and recombinant-cellular retinoic-acid-binding protein I.

Several known and some new retinoids were synthesized and their in vivo activity was investigated by an assay, based on induction of alkaline phosphatase in P19 teratocarcinoma cells, human prostate carcinoma cells and primary cultures of neonatal rat heart cells. The assay used in this study was found to be reproducible and useful for rapid screening of retinoids for biological activity. Two newly synthesized compounds exhibit high biological activity. The biological potency of the compounds was compared to their ability to bind to recombinant retinoic-acid receptor alpha and to cellular retinoic-acid-binding protein I determined by Charsorb-binding assay. mRNA of both retinoic-acid-binding proteins could be detected in the three cell lines investigated. As expected from the number of different retinoic-acid receptors, the results suggest that retinoids do not need to bind retinoic-acid receptor alpha nor cellular retinoic-acid-binding protein I in order to exhibit biological activity, but most retinoids investigated show a clear correlation between binding to these proteins and their biological activity.

Alkaline Phosphatase↗
PubMed1996

An alternative pathway of recombination of chromosomal fragments precedes recA-dependent recombination in the radioresistant bacterium Deinococcus radiodurans.

Deinococcus radiodurans R1 and other members of this genus are able to repair and survive extreme DNA damage induced by ionizing radiation and many other DNA-damaging agents. The ability of R1 to repair completely > 100 double-strand breaks in its chromosome without lethality or mutagenesis is recA dependent. However, during the first 1.5 h after irradiation, recA+ and recA cells show similar increases in the average size of chromosomal fragments. In recA+ cells, DNA continues to enlarge to wild-type size within 29 h. However, in recA cells, no DNA repair is observed following the first 1.5 h postirradiation. This recA-independent effect was studied further, using two slightly different Escherichia coli plasmids forming adjacent duplication insertions in the chromosome, providing repetitive sequences suitable for circularization by non-recA-dependent pathways following irradiation. After exposure to 1.75 Mrad (17,500 Gy), circular derivatives of the integration units were detected in both recA+ and recA cells. These DNA circles were formed in the first 1.5 h postirradiation, several hours before the onset of detectable recA-dependent homologous recombination. By comparison, D. radiodurans strains containing the same E. coli plasmids as nonrepetitive direct insertions did not form circular derivatives of the integration units before or after irradiation in recA+ or recA cells. The circular derivatives of the tandemly integrated plasmids were formed before the onset of recA-dependent repair and have structures consistent with the hypothesis that DNA repair occurring immediately postirradiation is by a recA-independent single-strand annealing reaction and may be a preparatory step for further DNA repair in wild-type D. radiodurans.

Alleles↗
PubMed2000

Recruitment of single-stranded recombinant adeno-associated virus vector genomes and intermolecular recombination are responsible for stable transduction of liver in vivo.

Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Following portal-vein administration of rAAV vectors in vivo, single-stranded (ss) rAAV genomes become double stranded (ds), circularized, and/or concatemerized concomitant with a slow rise and, eventually, steady-state levels of transgene expression. Over time, at least some of the stabilized genomes become integrated into mouse chromosomal DNA. The mechanism(s) of formation of stable ds rAAV genomes from input ss DNA molecules has not been delineated, although second-strand synthesis and genome amplification by a rolling-circle model has been proposed. To begin to delineate a mechanism, we produced rAAV vectors in the presence of bacterial PaeR7 or Dam methyltransferase or constructed rAAV vectors labeled with different restriction enzyme recognition sites and introduced them into mouse hepatocytes in vivo. A series of molecular analyses demonstrated that second-strand synthesis and rolling-circle replication did not appear to be the major processes involved in the formation of stable ds rAAV genomes. Rather, recruitment of complementary plus and minus ss genomes and subsequent random head-to-head, head-to-tail, and tail-to-tail intermolecular joining were primarily responsible for the formation of ds vector genomes. These findings contrast with the previously described mechanism(s) of transduction based on in vitro studies. Understanding the mechanistic process responsible for vector transduction may allow the development of new strategies for improving rAAV-mediated gene transfer in vivo.

Animals↗
PubMed2006

Recombinant factor VII (activated) for haemorrhagic complications of severe sepsis treated with recombinant protein C (activated).

We report on 2 cases of severe sepsis treated with drotrecogin-alpha (Xigris, Eli Lilly), where massive perioperative haemorrhage required administration of recombinant factor VIIa. The first patient developed severe sepsis after surgery (laparoscopic cholecystectomy, laparotomy due to peritonitis). After 18 h of treatment with Xigris, the patient developed massive, refractory gastrointestinal and abdominal bleeding. Effective haemostasis was achieved after 2 doses of NovoSeven (Novo Nordisk, Denmark). The patient died due to a cerebral bleed. The second patient developed septic shock in the course of pyelonephritis and right hydronephrosis. She was treated with Xigris and nephrectomy. Uncontrollable perioperative bleeding was effectively treated with 2 doses of NovoSeven. The patient survived.

Adult↗