Cytokinin activity: localization in transfer RNA preparations.

Transfer RNA from yeast, liver, and Escherichia coli has cytokinin activity in the tobacco callus bioassay, whereas ribosomal RNA from yeast is inactive. In contrast to fractions of yeast transfer RNA rich in serine acceptor and cytokinin activity, preparations (70 to 90 percent pure) of arginine transfer RNA(2), glycine transfer RNA, phenylalanine transfer RNA, and valine transfer RNA(1) and of highly purified alanine transfer RNA from yeast were inactive at concentrations of 20 to 2500 micrograms per liter. One molecule of 6-(gamma,gamma-dimethylallylamino) purine per 20 molecules of yeast tRNA would account for the observed cytokinin activity. The number of major molecular species contributing to cytokinin activity of transfer RNA, therefore, must be small.

Single crystals of transfer RNA from formylmethionine and phenylalanine transfer RNA's.

Reproducible conditions have been developed for crystallization of transfer RNA. The conditions may be applicable to many pure transfer RNA species since identical procedures (except for initial transfer-RNA concentration) yielded good crystals from both yeast and Escherichia coli transfer RNA. These crystals, which must be kept at temperatures below about 10 degrees C and handled in vapor of controlled alcohol concentration, have been studied by x-ray crystallography. The availability of crystals of a nucleic acid opens a route for extending knowledge of the tertiary structure of transfer RNA and its relation to important biological functions.

[Hepatitis A immunity in refugees in Germany during the current exodus]. / Hohe Hepatitis-A-Immunitätsrate bei Flüchtlingen in Deutschland.

BACKGROUND: Germany is facing a huge humanitarian challenge with rapidly rising numbers of refugees entering the country. Data on hepatitis A seroprevalence and infection in refugees and asylum seekers in Europe during the current refugee exodus is scarce. OBJECTIVES: To assess hepatitis A (HAV) seroprevalence and immunity in refugees arriving in northern Germany in 2015. MATERIALS AND METHODS: A cross-sectional study of 235 refugees seeking shelter in reception centers in Northern Germany in August 2015 was performed, as acute Hepatitis A had been detected in one refugee in this camp. In order to analyze acute HAV infection and overall immunity, serological screening for HAV antibodies (combined IgG and IgM) was performed. The immunity threshold was defined as <20 IU/l. In all positive screening results, separate IgM testing was performed to detect acute infections. RESULTS: Males accounted for 84.3 % of HAV screened refugees and the mean age of refugees was 29.1 ± 11.2 years. Children and adolescents below the age of 18 years made up 8.8 % of the migrants. Overall HAV immunity within the cohort was 90 %, and a mild age-dependent increase in HAV immunity was observed, with 81.1 % immunity in children <18 years and a 100 % seropositivity in subjects >50 years. One 20-year-old female refugee had positive IgM results with high HAV antibodies, most likely due to subacute HAV infection. CONCLUSIONS: This comparably high rate of HAV protected refugees in our cohort supports the notion that the probability of large HAV outbreaks in current German refugee centers is low. However, depending on their current living situation, HAV vaccination should be considered for each refugee child, and healthcare providers and personnel working in refugee centers should be vaccinated against HAV.

The hairpin ribozyme: discovery, two-dimensional model, and development for gene therapy.

This review chronicles the discovery of the hairpin ribozyme, its characterization, and determination of the two-dimensional structure, culminating with its use for human gene therapy as an AIDS therapeutic. The minimal sequence constituting the hairpin ribozyme catalytic domain was identified from a small plant viral satellite RNA. Biochemical characterization showed it to be among the most efficient of all known ribozymes. Mutagenesis determined that the two-dimensional structure had four helices, consisting of 17 Watson-Crick base pairs and one A:G pair for a total of 18 bp. The helices were interspersed with five single-stranded loops. Helices 1 and 2 were located between the ribozyme and substrate, allowing the ribozyme to recognize the substrate. The substrate had a sequence preference of BN*GUC where * is the site of cleavage and N*GUC the substrate loop between these two helices. By using sequences of this type, it was possible to design the ribozyme to base pair with the substrate and cleave heterologous RNA substrates-leading to design of the hairpin ribozyme for gene therapy. The HIV-1 sequence was searched for suitable target sites, and ribozymes were designed, optimized, catalytically characterized, and tested in vivo against HIV-1 targets. Two ribozymes had excellent in vitro catalytic parameters and inhibited in vivo expression of viral proteins by 3-4 logs in tissue culture cells. Viral replication was inhibited as well. They have been developed as human AIDS therapeutics, and will likely be the first ribozymes to be tested as human drugs in clinical trials.

[Low seroprevalence of syphilis and HIV in refugees and asylum seekers in Germany in 2015]. / Niedrige Seroprävalenz von Syphilis und HIV bei Flüchtlingen in Deutschland im Jahr 2015.

Background | Currently only estimates exist of seroprevalence of syphilis and human immunodeficiency virus (HIV) in refugees arriving in Germany during the current refugee crisis. Objectives | To assess the prevalence of syphilis and human immunodeficiency virus (HIV) in refugees arriving in northern Germany in 2015. Materials and methods | In a cross-sectional study in 790 patients from all age groups tests for serological markers of treponema pallidum and in 789 patients for human immunodeficiency virus (HIV) were performed in August 2015 in reception centers in northern Germany. Results | The overall prevalence of treponema pallidum antibodies was 0.13 % (1/790; [95 % CI: 0 - 0.4]). HIV antibodies were positive in two refugees from sub-Saharan Africa (2/789; 0.25 %, [95 % CI: 0 - 0.6]). Conclusions | This study showed a low prevalence of treponema pallidum antibodies and human immunodeficiency virus infection (HIV) in a German refugee cohort, not significantly different from German controls.

Altered leucyl-transfer RNA synthetase from a mammalian cell culture mutant.

Altered leucyl-tRNA synthetase from a mammalian cell culture temperature-sensitive mutant, tsHl, was compared with enzyme from normal wild type Chinese hamster ovary cells. The mutant enzyme had a Km for leucine four times larger than that of wild type and enzyme levels 3-10% that of wild type. The presence of tRNA was necessary during in vitro heating of the mutant enzyme to allow expression of thermolability while the presence of tRNA protected wild type enzyme against thermal inactivation. The tsHl enzyme was stable when heated alone or in the presence of tRNA, leucine, and ATP simultaneously. The mutant's enzymes aminoacylated tRNALeu, tRNAVal, and tRNAIle with fidelity in vitro as determined by cochromatography of the amino-acyl-tRNA isoacceptors on RPC-5 reversed phase chromatography. The mutant failed to show any defect other than the direct formation of leucyl tRNALeu by leucyl-tRNA synthetase.

A unique mechanism for RNA catalysis: the role of metal cofactors in hairpin ribozyme cleavage.

BACKGROUND: Ribozymes are biological catalysts that promote the hydrolysis and transesterification of phosphate diesters of RNA. They typically require divalent magnesium ions for activation, although it has proven difficult to differentiate structural from catalytic roles for the magnesium ions and to identify the molecular mechanism of catalysis. Direct inner-sphere coordination is usually invoked in the catalytic step, although there is no evidence to support the generality of such a pathway for all ribozymes. RESULTS: We studied the catalytic pathway for the hairpin class of ribozyme. The substitutionally inert transition metal complex cobalt hexaammine [Co(NH3)6(3+)] was shown to be as active as Mg2+(aq) in promoting hairpin ribozyme activity, demonstrating that inner-sphere pathways are not used by this class of ribozyme. These results were confirmed by studies with Rp- and Sp-phosphorothioate substrate analogs which show a similar reactivity to that of the native substrate towards the magnesium-activated ribozyme. Monovalent cations enhance the activity of Co(NH3)6(3+)-promoted reactions, but inhibit Mg(2+)-activated catalysis, demonstrating a requirement for hydrated cations at several key sites in the ribozyme. CONCLUSIONS: These results provide clear support for a model of RNA catalysis that does not involve direct coordination of magnesium to the phosphate ester, nor activation of a bound water molecule. A mechanism in which catalysis is carried out by functional groups on the RNA ribozyme itself is possible; such functional groups are likely to have pKa values that are appropriate for carrying out this catalysis. The metal cofactor would then serve to define the architecture of the catalytic pocket and contribute to the stabilization of transient species, as has been described earlier. Hydrolytic pathways in nucleic acid reactions are apparently more diverse than was previously thought, and the hairpin ribozyme falls into a mechanistically distinct class from the Tetrahymena and the hammerhead ribozymes.

A method for generating transcripts with defined 5' and 3' termini by autolytic processing.

Plasmids containing both the hammerhead and hairpin ribozyme autocatalytic cassettes were constructed for the purpose of generating RNA transcripts with specific termini at both the 5' and 3' ends. Following transcription, the RNA encoded by these cassettes was capable of intramolecular cleavage. This resulted in the generation of a processed RNA, which was located between the two cassettes, with specifically engineered 5' and 3' ends. The two different ribozymes were selected for their efficient intramolecular cleavage ability and to reduce the possibility of DNA recombination that could occur if identical cassettes were used. An application of this technique was the generation of a processed RNA which was itself a ribozyme, with specific 5' and 3' termini. The ribozyme generated was a hairpin ribozyme specific for a sequence in the gene encoding hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCoA reductase). The processed ribozyme was fully catalytically active against an RNA substrate sequence of HMGCoA reductase.

Detection of cleavage products from an in vivo transcribed cis hairpin ribozyme in turnips using the CaMV plant virus.

In order to examine ribozyme (Rz) activity in vivo, we have adapted a virus to deliver Rz to plants. DNA fragments that code for both active and mutant cis-hairpin Rz were cloned into the double-stranded DNA plant virus, cauliflower mosaic virus (CaMV). These Rz constructs successfully infected Brassica campestris rapa (turnip). The plants that were infected with the active-Rz construct showed, on average, a one-week delay in the appearance of viral symptoms, when compared to the mutant-Rz control. Since CaMV replicates through reverse transcription of a full-length RNA intermediate, Rz cloned into the CaMV DNA should be transcribed within this viral RNA. If these Rz constructs cleave, the amount of intact virus RNA should be reduced, resulting in attenuated viral symptoms. In addition, lysate RNase protection assays showed fragments corresponding to the sizes of both the 5' and 3' cis cleavage products in the active Rz tissue. No cleavage products were observed from plant tissue infected with the mutant Rz. Both the attenuated systemic viral symptoms and the cleavage products from the protection assay strongly support in vivo transcription and cleavage of this hairpin Rz. This is the first report of an in vivo transcribed Rz showing cleaved products by direct RNA analysis (non-PCR) in plants or animals.

Leucine-tRNA ligase complexes.

The methodologies described in this chapter allow the reproducible preparation of native high-molecular-weight synthetase complexes of leuRL. These complexes have the ability to preferentially utilize extracellular leucine immediately upon transport and are likely the forms of the enzyme most important in the utilization of leucine for protein synthesis.

Screening for important base identities in the hairpin ribozyme by in vitro selection for cleavage.

Random mutagenesis followed by an in vitro selection procedure was shown to be capable of identifying important bases of the hairpin ribozyme for cleavage of an RNA target sequence. The selection scheme enriched the RNA population for those molecules capable of efficient site-specific self-cleavage in the absence of ligation. Cleavable mutants were selected for all positions in loop 4 except for position A38, supporting the notion that A38 is an important base in the hairpin ribozyme. This has been confirmed by direct mutagenesis, validating the utility of this procedure. Thus, the method developed and reported here has utility for the selection of efficient hairpin ribozymes capable of highly efficient cleavage of a substrate RNA without a requirement for ribozyme-catalyzed ligation, conditions desired for many applications of catalytic RNA such as gene therapy.

The hairpin ribozyme. Discovery, mechanism, and development for gene therapy.

The hairpin ribozyme is a member of a family of small RNA endonucleases, which includes hammer-head, human hepatitis delta virus, Neurospora VS, and the lead-dependent catalytic RNAs. All these catalytic RNAs reversibly cleave the phosphodiester bond of substrate RNA to generate 5'-hydroxyl and 2',3'-cyclic phosphate termini. Whereas the reaction products from family members are similar, large structural and mechanistic differences exist. Structurally the hairpin ribozyme has two principal domains that interact to facilitate catalysis. The hairpin ribozyme uses a catalytic mechanism that does not require metals for cleavage or ligation of substrate RNA. In this regard it is presently unique among RNA catalysts. Targeting rules for cleavage of substrate have been determined and required bases for catalysis have been identified. The hairpin ribozyme has been developed and used for gene therapy and was the first ribozyme to be approved for human clinical trials.

Design of the hairpin ribozyme for targeting specific RNA sequences.

The following steps should be taken when designing the hairpin ribozyme to cleave a specific target sequence: 1. Select a target sequence containing BN*GUC where B is C, G, or U. 2. Select the target sequence in areas least likely to have extensive interfering structure. 3. Design the conventional hairpin ribozyme as shown in Fig. 1, such that it can form a 4 bp helix 2 and helix 1 lengths up to 10 bp. 4. Synthesize this ribozyme from single-stranded DNA templates with a double-stranded T7 promoter. 5. Prepare a series of short substrates capable of forming a range of helix 1 lengths of 5-10 bp. 6. Identify these by direct RNA sequencing. 7. Assay the extent of cleavage of each substrate to identify the optimal length of helix 1. 8. Prepare the hairpin tetraloop ribozyme to determine if catalytic efficiency can be improved.

External auditory canal dysplasia: CT evaluation.

Dysplasias of the external auditory canal can be evaluated with high resolution CT using proper technique. Images are studied to determine the type of atresia (membranous or bony) and the thickness of the associated atretic plate (if present). Other findings sought include the degree of pneumatization, the status of the fenestrae, the size of the tympanic cavity, and the location of both the mandibular condyle and the facial nerve canal.

Flexible neonatalscope.

Since the introduction of small flexible fiberoptic scopes for the examination of airways, efforts have been made to reduce their size to fit the smallest patients. The first practical scope of this type was the 3.9-mm nasopharyngolaryngoscope introduced in 1975. Modifications over the years have led to the development of the 1.7-mm neonatalscope, primarily designed for the examination of intubated neonates. This neonatalscope has been of great help in determining placement of endotracheal and tracheotomy tubes, as well as in localizing areas of obstruction within the tubes. By means of a special adapter the scope may be used while the patient is on a respirator. Introduction of this new generation of flexible scopes will undoubtedly lead to other valuable and interesting applications of this instrument.

New technique for the sterile introduction of flexible nasopharyngolaryngoscopes.

Since the inception of flexible fiberoptic endoscopes, disinfection of these instruments has been a problem. Soaking in glutaraldehyde does not always achieve sterilization, and often damages the scopes. Ethylene oxide can sterilize endoscopes; however, it is economically impractical because of a required downtime of 24 hours. Thus, it is obvious, especially with respect to human immunodeficiency virus, hepatitis B virus, and Mycobacterium, that a new technique to attain sterility is necessary. This paper discusses a new method of sterile introduction of the flexible nasopharyngolaryngoscope. The technique employs disposable sterile sheaths that are prepackaged and made from a thermoplastic elastomer with a clear optical end. The sheaths can be applied in seconds and tightly adhere to the flexible insertion portion of the scope. Results to date indicate that the performance of the endoscope is unhindered by using the sheaths. Furthermore, there has been no break in the integrity of the sheaths or damage to instruments. It is our opinion that these devices will greatly improve the level of sterility while at the same time reducing costs and downtime.

Computerized tomographic evaluation of the middle ear and mastoid for posttraumatic hearing loss.

High resolution computerized tomography has been proven valuable in all aspects of temporal bone study. The importance of the procedure in evaluation of traumatic lesions, particularly of the middle ear, has been underemphasized. Damage to the middle ear may occur with blunt trauma, penetrating injury, or barotrauma. Conductive hearing loss and/or CSF otorrhea may result. Detailed evaluation of the ossicles is the hallmark of properly performed computerized tomographic evaluation.

A typodont used exclusively for practical examinations.

The use of a testing typodont has eliminated the problems associated with the use of the students' typodont for practical examinations and provided increased objectivity, conformity, security, efficiency, and flexibility in the testing and evaluation process. The cost benefit comparison of purchasing separate testing typodonts for use in preclinical practical examinations can be measured only in such intangibles as student and faculty acceptance and confidence in the examination and evaluation procedures.