Stromatolite-like Structures Within Microbially Laminated Sandstones of the Paleoarchean Moodies Group, Barberton Greenstone Belt, South Africa.

We report abundant small calcareous mounds associated with fossilized kerogenous microbial mats in tidal-facies sandstones of the predominantly siliciclastic Moodies Group (ca. 3.22 Ga) of the Barberton Greenstone Belt (BGB), South Africa and Eswatini. Most of the bulbous, internally microlaminated mounds are several centimeters in diameter and formed at the sediment-water interface contemporaneously with sedimentation. They originally consisted of Fe-Mg-Mn carbonate, which is now largely silicified; subtle internal compositional laminations are composed of organic matter and sericite. Their presence for >6 km along strike, their restriction to the inferred photic zone, and the internal structure suggest that mineral precipitation was induced by photosynthetic microorganisms. Similar calcareous mounds in this unit also occur within and on top of fluid-escape conduits, suggesting that carbonate precipitation may either have occurred abiogenically or involved chemotrophic metabolism(s) utilizing the oxidation of organic matter, methane, or hydrogen, the latter possibly generated by serpentinization of underlying ultramafic rocks. Alternatively or additionally, carbonate may have precipitated abiotically where heated subsurface fluids, sourced by the intrusion of a major Moodies-age sill, reached the tidal flats. In summary, precipitation mechanisms may have been variable; the calcareous mounds may represent "hybrid carbonates" that may have originated from the small-scale overlap of bioinduced and abiotic processes in space and time. Significantly, the widespread occurrence of these stromatolite-like structures in a fully siliciclastic, high-energy tidal setting broadens search criteria in the search for life on Mars while their possible hybrid origin challenges our ability to unambiguously identify a biogenic component.

Tuning excitability by alloying: the Rh(111)/Ni/H2 + O2 system.

The dynamic behavior of the O2 + H2 reaction on a Rh(111) surface alloyed with Ni has been studied in the 10(-5) mbar range using photoemission electron microscopy (PEEM) as a spatial resolving method. For T = 773 K and p(O2) = 5 × 10(-5) mbar the bifurcation diagram has been mapped out as a function of the Ni coverage in a range of 0 ML ≤ Θ(Ni) ≥ 1.3 ML. A critical Ni coverage of Θ(Ni,crit) = 0.13 monolayers (ML) is required for excitability. In the excitable parameter range pulse trains and irregular chemical wave patterns are found. Whereas the propagation speed of the pulses exhibits no clear-cut dependence on the Ni coverage, the frequency of the local PEEM intensity oscillations increases linearly with Ni coverage in the range from Θ(Ni) = 0.13 ML to Θ(Ni) = 1.3 ML.

[Interhospital transport of intensive care patients in Lower Saxony : statewide need-based and effective management]. / Intensivverlegung in Niedersachsen : Landesweite bedarfsgerechte und effektive Sicherstellung.

BACKGROUND: Since 2007 interhospital transport of intensive care patients in Lower Saxony appertains to the performance requirements of emergency medical services. Against this background the Working Group for Evaluation of Intensive Care Transport (Arbeitsgemeinschaft Evaluation Intensivverlegung) was established. This group formulated standardized definitions for the requirements of intensive care transport vehicles and a federal statewide monitoring of intensive care transport was implemented to analyze if simultaneously on-call intensive care transport systems (intensive care helicopter and ground based mobile intensive care units) can be deployed need-based and efficiently. METHODS: A prospective follow-up study and evaluation of intensive care transport in Lower Saxony between April 1(st) 2008 and July 31(st) 2010 was carried out. RESULTS: A total of 6,779 data records were evaluated in this study of which 4,941 (72.9%) missions were located in Lower Saxony, 2,928 (43.2%) missions were carried out by helicopters and 3,851 (56.8%) by ground based mobile intensive care units. The mean duration of a mission was 3 h 59min±2 h 25 min, 4 h 39 min±2 h 23 min by ground based mobile intensive care units and 2 h 21 in±30 min by helicopter units. All systems proved to be feasible for intensive care transport. The degree of urgency was estimated correctly in 94.8% of the evaluated missions and 58.0% of the transfers could not be deployed. In 76.8% patients were transferred to hospitals with a higher level of medical care, 51.7% of patients were transferred for intensive care therapy and 40.4% for an operation/intervention. Of the patients 38.2% required mechanical ventilation and in 48.3% invasive monitoring was carried out. CONCLUSION: Interhospital transfer of intensive care patients can be carried out need-based with a limited number of intensive care transport vehicles if the missions are deployed effectively by standardized disposition in accordance with performance requirements.

Isoflurane inhalation after induction of endotoxemia in rats attenuates the systemic cytokine response.

BACKGROUND/AIMS: Volatile anesthetics are frequently utilized in clinical routine. Isoflurane has been shown to attenuate the response to inflammatory stimuli such as lipopolysaccharide (LPS) when administered before induction of endotoxemia. We aimed therefore to evaluate the effect of isoflurane after administration of LPS on the cytokine release as a therapeutic option. MATERIALS AND METHODS: 21 male Sprague-Dawley rats were randomly assigned to the following groups: animals that received LPS (5 mg/kg, i.v.) without further intervention (LPS group), animals that received continuous inhalation of 1 minimum alveolar concentration (MAC) isoflurane 15 min after administration of LPS (Iso group) and no specific intervention (sham group). Four hours following LPS injection, plasma levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), IL-6 and IL-10 were determined. Furthermore, nitrite release from cultured alveolar macrophages was analyzed. RESULTS: Inhalation of isoflurane after induction of endotoxemia attenuated the release of TNF-alpha (-52%, p < 0.05) and IL-1 beta (-39%, p < 0.05) as compared to the LPS group, while IL-6 and IL-10 levels were not significantly altered. Nitrite release was significantly increased in the Iso group as compared to the LPS group (+115%, p < 0.05). CONCLUSION: Inhalation of 1 MAC isoflurane after induction of endotoxemia in rats attenuates the systemic release of proinflammatory cytokines and concurrently enhances the production of nitrite in cultured alveolar macrophages.

[Epidemiology and management in emergency room patients with maxillofacial fractures]. / Epidemiologie und Behandlungsmanagement bei Schockraumpatienten mit Gesichtsschädelverletzungen.

OBJECTIVE: To minimize overall mortality and optimise reconstructive and cosmetic outcome in severely injured patients with maxillofacial injuries the interdisciplinary coordination of several surgical disciplines is required. It is still discussed controversy whether patients with maxillofacial fractures benefit from early fracture repair or if delayed operative management also yields in good results. METHODS: Herein we analysed the data of 1252 severely injured patients between May 1998 through June 2002 in our trauma department regarding fractures of the maxillofacial region, injury severity, length of ICU stay and postoperative complications in patients with either early (within 72 hours) or delayed ( > 3 days) facial fracture repair. RESULTS: 147 patients had severe facial fractures. Average age was 39.8 years (3-87 years), mean ICU was 25 (+/- 16) and the overall mortality 12% (n = 18). The most common cause for the injuries were traffic accidents in 45%. 78 patients (53%) underwent surgical repair of the maxillofacial fractures; 18 patients had early fracture repair and 60 patients had delayed operative repair. We found 4 complications (22%) in the early repair group and 13 local complications (21%) in the group with delayed surgical repair. CONCLUSION: Delayed repair of maxillofacial injuries in severely injured patients is feasible and yields in good results compared to early fracture repair.

Anti-inflammatory effects of sevoflurane and mild hypothermia in endotoxemic rats.

BACKGROUND: Volatile anesthetics and hypothermia attenuate the inflammatory response. We aimed to compare the anti-inflammatory effects of sevoflurane and mild hypothermia during experimental endotoxemia in the rat. METHODS: Anesthetized, ventilated Sprague-Dawley (SD) rats were randomly treated as follows (n = 6 per group): lipopolysaccharide (LPS) only, animals received LPS [LPS 5 mg/kg, intravenously (i.v.)] with no further treatment. In the LPS-hypothermia group, rats were cooled down to a temperature of 33 degrees C 15 min after LPS-injection (LPS 5 mg/kg i.v.). In animals of the LPS-sevoflurane group, sevoflurane inhalation (1 MAC) was initiated 15 min after induction of endotoxemia. The LPS-sevoflurane-hypothermia group received combined sevoflurane and hypothermia 15 min after induction of endotoxemia. A Sham group served as control without endotoxemia or treatment. After 4 h of endotoxemia, plasma levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and IL-10 were measured. Alveolar macrophages (AM) were ex vivo cultured for nitrite assay. RESULTS: Inhalation of sevoflurane significantly attenuated plasma levels of TNF-alpha (-60%, P < 0.05) and IL-1beta (-68%, P < 0.05) as compared with the LPS-only group. Hypothermia and its combination with sevoflurane significantly reduced TNF-alpha levels (-46% and -58%, each P < 0.05), but not IL-1beta. Application of mild hypothermia and also its combination with sevoflurane resulted in a significant increase in plasma IL-10 as compared with endotoxemic controls. Nitrite release from AM was found to be significantly suppressed by sevoflurane (-83%), hypothermia (-73%) and by the combination of both (-67%) (P < 0.05, each). CONCLUSION: Our data suggest that sevoflurane and mild hypothermia attenuate the inflammatory response during endotoxemia in vivo thus contributing to their beneficial role in clinical organ protection.

RNA aptamers as potential pharmaceuticals against infections with African trypanosomes.

Protozoal pathogens cause symptomatic as well as asymptomatic infections. They have a worldwide impact, which in part is reflected in the long-standing search for antiprotozoal chemotherapy. Unfortunately, effective treatments for the different diseases are by and large not available. This is especially true for African trypanosomiasis, also known as sleeping sickness. The disease is an increasing problem in many parts of sub-Saharan Africa, which is due to the lack of new therapeutics and the increasing resistance against traditional drugs such as melarsoprol, berenil and isometamidium. Considerable progress has been made over the past 10 years in the development of nucleic acid-based drug molecules using a variety of different technologies. One approach is a combinatorial technology that involves an iterative Darwinian-type in vitro evolution process, which has been termed SELEX for "systematic evolution of ligands by exponential enrichment". The procedure is a highly efficient method of identifying rare ligands from combinatorial nucleic acid libraries of very high complexity. It allows the selection of nucleic acid molecules with desired functions, and it has been instrumental in the identification of a number of synthetic DNA and RNA molecules, so-called aptamers that recognize ligands of different chemical origin. Aptamers typically bind their target with high affinity and high specificity and have successfully been converted into pharmaceutically active compounds. Here we summarize the recent examples of the SELEX technique within the context of identifying high-affinity RNA ligands against the surface of the protozoan parasite Trypanosoma brucei, which is the causative agent of sleeping sickness.

[Emergency room patients]. / Der Schockraumpatient. Einschätzung ernster Schädelverletzungen anhand der GCS bei vermeintlich leichtem und mittelschwerem Schädel-Hirn-Trauma.

UNLABELLED: The aim of the study was to examine the predictive value of CT for severe intracranial lesions of mild (GCS 15-13) and moderate (12-9) head injuries. Further,we examined the possibility of predicting these lesions by various variables/factors. Data were collected prospectively from the trauma registry of the DGU (Deutsche Gesellschaft für Unfallchirurgie). Patients with a GCS score from 15-13 and from 12-9 were included in this study and examined for intracranial lesions (AIS(head) 3-6). Over a time period from 1993 to 1999, 1778 patients with mild head injury and 235 patients with moderate head injury were analyzed. Severe intracranial lesions were suffered by 18.6% of the patients with mild head injury and 50.4% of the patients with moderate head injuries. Of the predictive variables, heart rate,patients' age,and primary assessment by the emergency physician showed a strong correlation with the later observed intracranial lesions. IN CONCLUSION: (1) independently of the initially good GCS, a high percentage of patients suffered from severe intracranial lesions and (2) besides the GCS only the patients'age and primary assessment by the emergency physician were useful for identifying patients at risk for an intracranial lesion.

Fibromyxoma of the iliac wing.

Myxoma/fibromyxoma of extragnathic bone is a rare, benign tumor. We report the case of a 19-year-old man with a myxoma/fibromyxoma of the pelvic bone, who presented with abdominal pain in the right lower quadrant. Radiographs demonstrated a tumor with central scattered calcifications and ossification in the left ilium. CT scans disclosed a solitary ellipsoid tumor with sclerotic borders and no evidence of cortical disruption. MRI showed homogeneous signal enhancement of the bone marrow with slight thinning of the medial iliac cortex. Final diagnosis by radiological characteristics was not conclusive and histopathological examination suggested the diagnosis of a myxoma/fibromyxoma.

Uptake and intracellular transport of RNA aptamers in African trypanosomes suggest therapeutic "piggy-back" approach.

African trypanosomes are protozoan organisms that multiply as extracellular parasites in the blood of humans and other mammals. The parasites escape destruction by the host immune system by periodically changing their glycoprotein surface coat. This phenomenon is known as antigenic variation and is responsible for the inability of the infected host to clear the infection. Previously we reported the selection of RNA aptamers that bind to a 42 kDa surface protein of Trypanosoma brucei. The polypeptide is localised within a specific substructure on the parasite surface, the so-called flagellar pocket. Here we analyse the fate of the aptamers upon binding to the flagellar pocket. At elevated temperatures, both terminal ends of the RNAs are degraded to form a stable core structure of approximately 50 nucleotides. The RNAs become rapidly internalised by endocytosis and are transported to the lysosome by vesicular transport. The endocytotic process is sequence specific and does not occur with randomised RNA sequences or significantly shortened aptamer fragments. Co-localisation experiments with transferrin suggest a receptor-mediated uptake. The identified internalisation and transport pathway was used to target aptamer-coupled biotin molecules to the lysosome. This demonstrates that the RNAs can be used as 'piggy-back' molecules to target aptamer-coupled compounds/toxins to the lysosomal compartment of the parasite.

Performance of allografts and xenografts for right ventricular outflow tract reconstruction.

BACKGROUND: We compared the long-term durability of allografts and xenografts implanted for reconstruction of the right ventricular outflow tract. METHODS: A total of 401 patients were studied from January 1974 to June 2000 (145 xeno- and 256 allografts), follow-up being 98% complete. We analyzed freedom from reoperation and allograft specific factors that may indicate degeneration. RESULTS: The age at implantation was 2 days to 31 years (median 4.0 years). Conduit exchange rate was similar (p = 0.2) for conduit diameters less than 15 mm (41%+/-9% for allografts, 30%+/-6% for xenografts), but significantly different (p = 0.02) for diameters of 15 mm or larger (60%+/-8% for allografts, 30%+/-10% for xenografts). Diagnosis-related 20-year survival analysis showed a significantly (p = 0.01) better survival of patients with tetralogy of Fallot/pulmonary atresia (83%+/-5%) and Rastelli-type surgery (81%+/-8%) compared with patients with truncus arteriosus communis (69%+/-8%). ABO-compatibility, preservation method, and aortic or pulmonary allograft could not be identified as risk factors for allograft longevity. CONCLUSIONS: For smaller diameters (less than 15 mm), allografts exhibit no advantage over xenografts, whereas in larger diameters (15 mm or larger) allografts are the conduit of choice for the right ventricular outflow tract.

Reconstruction of the RVOT with valved biological conduits: 25 years experience with allografts and xenografts.

OBJECTIVE: The reconstruction of the RVOT in congenital heart disease often requires the implantation of a valved conduit. Although allografts are considered the conduit of choice their availability is limited and therefore xenografts are implanted as well. We compared the long-term durability of both grafts in the RVOT over a 25-year period. METHODS: Between January 1974 and August 1999, 505 patients (median age 4.0 years, range 2 days-31 years; median weight 14.5 kg, range 2.2-76.6 kg; median body length 103 cm, range 48-183 cm) with congenital malformations (PA 25.3%, TOF 14.5%, TOF+PA 2.4%, DORV 4.2%, TGA+PS 8.7%, TAC 24.8%, and other 20.2%) received their first valved conduit (174 xenografts: median diameter 14 mm, range 8-27 mm; 331 allografts: median diameter 19 mm, range 8-30 mm). RESULTS: Follow-up is 3017 patient-years. The 10-year survival-probability for all patients. was 66% with a mean reoperation-free interval for conduit-exchange of 13.3 years (mean reoperation-free interval for allografts, 16.0 years; mean reoperation-free interval for xenograft, 10.3 years). One hundred and thirteen patients underwent a conduit-exchange, mostly due to conduit stenosis. Fourteen patients had a second exchange and three patients a third exchange. For patients with conduit diameters <18 mm (n=235: allograft n=116, xenograft n=119; median age 9 months, range 0-27.3 years), the mean reoperation-free interval was 11.2 years (mean interval allograft, 13.1 years; mean interval xenograft, 8.6 years, P=0.03). For conduit diameters >/=18 mm (n=270: allograft n=215, xenograft n=55, median age 7.4 years, range 0-34.3 years) the mean interval from freedom of conduit exchange was 15.1 years (for allografts 14.1 years, for xenografts 12.5 years, P<0.01). Comparing xenografts to allografts, we found no difference in patient survival probability (P=0.62). There was no significant difference between antibiotic (n=198) preserved vs. cryopreserved (n=133) allografts (P=0.06). Blood group compatibility of allografts to recipients had no significant influence on allograft function (P=0.42). The donors allograft origin, whether aortic or pulmonary valve, had also no significant influence on allograft long-term function (P=0.15). CONCLUSION: For the reconstruction of the right ventricular outflow tract (RVOT) allografts show significantly better long-term durability than xenografts regardless of the age at implantation and the diameter.

Combinatorial selection of high affinity RNA ligands to live African trypanosomes.

African trypanosomiasis is a parasitic disease caused by a specific class of protozoan organisms. The best-studied representative of that group is Trypanosoma brucei which is transmitted by tsetse flies and multiplies in the blood of many mammals. Trypanosomes evade the immune system by altering their surface structure which is dominated by a layer of a variant surface glycoprotein (VSG). Although invariant surface proteins exist, they are inaccessible to the humoral immune response. Using a combinatorial selection method in conjunction with live trypanosomes as the binding target, we show that short RNA ligands (aptamers) for constant surface components can be isolated. We describe the selection of three classes of RNA aptamers that crosslink to a single 42 kDa protein located within the flagellar pocket of the parasite. The RNAs associate rapidly and with high affinity. They do not discriminate between two different trypanosome VSG variant strains and, furthermore, are able to bind to other trypanosome strains not used in the selection protocol. Thus, the aptamers have the potential to function as markers on the surface of the extracellular parasite and as such they might be modified to function as novel drugs against African trypanosomiasis.

Kinetic selection of HPV 16 E6/E7-directed antisense nucleic acids: anti-proliferative effects on HPV 16-transformed cells.

The E6/E7-coding sequences of the human papillomavirus type 16 (HPV 16) were probed for kinetic accessibility in vitro by pools of catalytic antisense RNA. Only long-chain complementary RNA and very few antisense sequences with a 3' portion complementary to a 10 nt window within unspliced and spliced E6-coding target sequences showed fast annealing with k(ass) values of up to 10(4) M-1s-1 indicating that the majority of E6/E7 RNA sequences are unfavourable targets for antisense inhibitors and ribozymes. Fast-annealing antisense oligodeoxyribonucleotides directed against the window of 10 nt inhibited cell proliferation of HPV 16-transformed SiHa cells but not slow-annealing antisense species. Antisense RNA of several hundred nucleotides in length also showed significant anti-proliferative activity. Biological effects of antisense oligodeoxyribonucleotides were specific for the antisense sequence, could only be found in HPV-positive but not in HPV-negative cell lines, and were related to decreased levels of E7 protein and E6/E7-specific transcripts. This work suggests that HPV 16 E7/E6 sequences exhibit a low accessibility for antisense oligonucleotides. This can be overcome, however, by exploiting the relationship between fast annealing of antisense species and their increased efficacy in human cells.

The association of complementary ribonucleic acids can be strongly increased without lowering Arrhenius activation energies or significantly altering structures.

The association rates of complementary nucleic acids can be increased by 2-3 orders of magnitude in vitro by cellular proteins and low molecular weight compounds including cetyltrimethylammonium bromide (CTAB). In this work, we provide experimental evidence that the CTAB-mediated enhancement of RNA-RNA annealing by approximately 3 orders of magnitude is due to a favorable activation entropy (DeltaS) and not due to a decrease of the Arrhenius activation energy (Ea) nor to major structural changes of the RNA. Two alternative models for the CTAB-facilitated RNA-RNA annealing will be discussed. First, CTAB could form a positively charged liquid matrix which could steer complementary RNA molecules and thereby increase their collision frequency and annealing rate. Second, increased annealing rates could be explained by stabilization of a non-base-specific precomplex of both complementary RNA molecules in solution.

The subcellular localization and length of hammerhead ribozymes determine efficacy in human cells.

The length requirements of the antisense portion of hammerhead ribozymes for efficacy in living cells was investigated. The HIV-1tat-directed asymmetric hammerhead ribozyme alphaYRz195 was used with a 195 nt 3'-antisense arm and a 3 nt 5'-antisense portion as well as a set of successively 3'-shortened derivatives thereof. In the 3'-antisense arm a minimum length of 20 complementary nucleotides was required for efficient association with a 645 nt target RNA transcript in vitro(for all constructs kass ranged between 0.3 and 1.8x104/M/s). The cleavage rate constants (kcleav) were independent of the length of the antisense flank and ranged between 0.8 and 1.2x10-4/s. However, the length of the antisense arms, as well as the mode of delivery and the subcellular location of the ribozymes, had a dramatic effect on efficacy in HIV-1-producing human cells. When proviral HIV-1 DNA and ribozymes were co-microinjected into the nucleus of human cells, a minimum length of 51 nt in the antisense arm was necessary for antisense- and ribozyme-mediated inhibition of HIV-1 replication. Ribozymes with shorter antisense arms were almost ineffective. Conversely, short chain ribozymes, including those with chemical modifications, were superior to long chain ribozymes when co-microinjected into the cytoplasm. When transfected, all ribozymes showed an antisense effect as well as an additional ribozyme-mediated increase in inhibition. Consequences for the design and application of ribozymes are discussed.

Dissociation of long-chain duplex RNA can occur via strand displacement in vitro: biological implications.

Hammerhead ribozymes with long antisense flanks (>50 bases) have been used successfully to inhibit replication of human immunodeficiency virus type 1 (HIV-1) in living cells. To explain their increased efficacy versus antisense controls or catalytically inactive derivatives, one can consider dissociation of the ribozyme-product complex to allow a complete catalytic cycle. In this work we investigated the dissociation of a double-stranded RNA with 56 bp in vitro. Dissociation was observed in the presence of single-stranded RNA with sequence complementarity to one of the duplex strands. A displacement reaction between RNA single strands and the duplex, but not simple dissociation, was strongly suggested by the concentration dependence of this process, the influence of additional non-complementary sequences on the single strand and by the unusually low Arrhenius activation energy. The strand displacement reaction was slow in vitro at 37 degrees C and physiological ionic strength, but was increased to k approximately 10(3)-10(4)/M/s (approximately 10(4)-fold) at higher temperatures by cetyltrimethylammonium bromide. This compound is thought to enhance non-sequence-specific association of nucleic acids in a mechanistically similar way to that in which cellular hnRNP proteins are thought to act, indicating that strand displacement can be fast and, more importantly, could be tightly regulated in vivo.

Allograft implantation in pediatric cardiac surgery: surgical experience from 1982 to 1994.

Between July 1982 and April 1994, a total of 290 patients (median age 6.5 years, range 1 month to 32.1 years, 69 patients younger than 1 year) underwent repair of their cardiac malformation by insertion of an allograft. The diagnoses were truncus arteriosus communis (n = 78, 27.0%), tetralogy of Fallot (n = 59, 20.0%), pulmonary atresia (n = 72, 25.0%), double outlet right ventricle (n = 15, 5.0%), complex transposition of the great arteries plus pulmonary stenosis (n = 37, 13.0%), and others (n = 29, 10.0%). Either pulmonary (n = 69) or aortic (n = 221) cadaver allografts were implanted. Two hundred twenty-nine of the allografts were antibiotic preserved. Since January 1991 (n = 61), a new cryopreservation procedure was employed for standardized uniform cooling using heat sinks and defined package geometry. Follow-up was complete for 95.2% (n = 276, 1,320 patient-years). Thirty-day mortality was 9.0% (n = 26) and late mortality was 12.1% (n = 35). Kaplan-Meier analysis revealed that patient survival was determined mainly by their underlying cardiac disease. All allografts with valve sizes less than 15.0 mm had to be exchanged within 7 years as these patients had outgrown their conduits. When the allograft was larger than 15.0 mm, exchange was necessary in 20% at 10 years. ABO compatibility and aortic or pulmonary origin of the allograft were not significant influences on allograft survival.(ABSTRACT TRUNCATED AT 250 WORDS)

Extension of helix II of an HIV-1-directed hammerhead ribozyme with long antisense flanks does not alter kinetic parameters in vitro but causes loss of the inhibitory potential in living cells.

When designed to cleave a target RNA in trans, the hammerhead ribozyme contains two antisense flanks which form helix I and helix III by pairing with the complementary target RNA. The sequences forming helix II are contained on the ribozyme strand and represent a major structural component of the hammerhead structure. In the case of an inhibitory 429 nucleotides long trans-ribozyme (2as-Rz12) which was directed against the 5'-leader/gag region of the human immunodeficiency virus type 1 (HIV-1), helix II was not pre-formed in the single-stranded molecule. Thus, major structural changes are necessary before cleavage can occur. To study whether pre-formation of helix II in the non-paired 2as-Rz12 RNA could influence the observed cleavage rate in vitro and its inhibitory activity on HIV-1 replication, we extended the 4 base pair helix II of 2as-Rz12 to 6, 10, 21, and 22 base pairs respectively. Limited RNase cleavage reactions performed in vitro at 37 degrees C and at physiological ion strength indicated that a helix II of the hammerhead domain was pre-formed when its length was at least six base pairs. This modification neither affected the association rate with target RNA nor the cleavage rate in vitro. In contrast to this, extension of helix II led to a significantly decreased inhibition of HIV-1 replication in human cells. Together with the finding of others that shortening of helix II to less than two base pairs reduces the catalytic activity in vitro, this observation indicates that the length of helix II in the naturally occurring RNAs with a hammerhead domain is already close or identical to the optimal length for catalytic activity in vitro and in vivo.

A three-nucleotide helix I is sufficient for full activity of a hammerhead ribozyme: advantages of an asymmetric design.

Trans-cleaving hammerhead ribozymes with long target-specific antisense sequences flanking the catalytic domain share some features with conventional antisense RNA and are therefore termed 'catalytic antisense RNAs'. Sequences 5' to the catalytic domain form helix I and sequences 3' to it form helix III when complexed with the target RNA. A catalytic antisense RNA of more than 400 nucleotides, and specific for the human immunodeficiency virus type 1 (HIV-1), was systematically truncated within the arm that constituted originally a helix I of 128 base pairs. The resulting ribozymes formed helices I of 13, 8, 5, 3, 2, 1 and 0 nucleotides, respectively, and a helix III of about 280 nucleotides. When their in vitro cleavage activity was compared with the original catalytic antisense RNA, it was found that a helix I of as little as three nucleotides was sufficient for full endonucleolytic activity. The catalytically active constructs inhibited HIV-1 replication about four-fold more effectively than the inactive ones when tested in human cells. A conventional hammerhead ribozyme having helices of just 8 nucleotides on either side failed to cleave the target RNA in vitro when tested under the conditions for catalytic antisense RNA. Cleavage activity could only be detected after heat-treatment of the ribozyme substrate mixture which indicates that hammerhead ribozymes with short arms do not associate as efficiently to the target RNA as catalytic antisense RNA. The requirement of just a three-nucleotide helix I allows simple PCR-based generation strategies for asymmetric hammerhead ribozymes. Advantages of an asymmetric design will be discussed.