Safe biotechnology. 8. Transport of infectious and biological materials. Working Party "Safety in Biotechnology" of the European Federation of Biotechnology.

The transport of infectious and biological material is regulated by a number of international organizations. This mini-review has been compiled to increase awareness within the scientific community of problems caused by differences in terminology (such as infectious materials/substances, biological products, diagnostic specimens, genetically modified microorganisms) and certain technical aspects of the main international guidelines, and to assist policy makers in the creation of harmonized guidelines. A list of relevant Internet resources has been compiled.

Safe biotechnology. 7. Classification of microorganisms on the basis of hazard. Working Party "Safety in Biotechnology" of the European Federation Biotechnology.

The current systems for classifying human pathogens on the basis of hazard are well developed and their basic criteria are in general agreement one with another. Of more importance, the safety practices based on these classifications have generally been successful. They have enabled extensive research activities, medical practice and industrial production to be conducted on an ever-increasing scale, involving dangerous microorganisms (e.g. in vaccine production and treatment of infected patients) with a very low incidence of adverse effects on the workers involved and the general public. Although the EU has adopted a harmonised list of agents in groups 1-4 there is as yet no complete agreement among member states and individual microbiologists. The purpose of this paper is to present a historical survey and to discuss the current processes for identifying and classifying the hazards posed by the use of microorganisms in research and technology. This is essential in the design of appropriate methods of counteracting potential risks.

Safe biotechnology. Part 6. Safety assessment, in respect of human health, of microorganisms used in biotechnology.

The assessment of microorganisms in respect to human health is an important step for the introduction of new natural and genetically modified production strains to biotechnology. This report outlines the potential hazards posed by industrial microorganisms, important considerations related to pathogenicity, such as routes and portals of entry into the human body, mechanisms of spread of biological material and a definition of pathogenicity. Furthermore the most important steps in the assessment of pathogenicity of unknown strains are described. A short overview on characterization and in vitro and in vivo tests is presented. The hazard related to allergens and toxic metabolites is reviewed and the choice of methods and the handling of strains with unknown potential are discussed.

Safe biotechnology (4). Recommendations for safety levels for biotechnological operations with microorganisms that cause diseases in plants.

The Working Party on Safety in Biotechnology of the European Federation of Biotechnology has proposed a classification of microorganisms that cause diseases in plants. In this paper appropriate safety levels are proposed for these classes of microorganisms in order to ensure that research, development and industrial fermentation work with plant pathogens will limit the risk of outbreaks of diseases in crops that could result from work with such microorganisms when they are cultivated in laboratories, glasshouses and biotechnology installations.

An overview of the biotechnology research activities in the European Community.

The research in Biotechnology is an important element for the competitive environment of European bioindustrial activities. In this article the evolution of the Biotechnology programmes in the European Community is presented.

[Argon-green laser photocoagulation in the treatment of cystoid macular edema secondary to branch vein occlusion]. / Traitement de l'oedème maculaire cystoïde secondaire à une occlusion de branche veineuse par photocoagulation au laser monochromatique vert.

The present study evaluates the use of argon-green photocoagulation in the absorption rate of macular oedema and the visual recovery of patients suffering of branch venous occlusion of non-diabetic aetiology. Twenty patients suffering from branch venous occlusion and angiographically proven cystoid macular oedema were entered in this prospective study; they were randomly divided in two equal groups and were treated with photocoagulation 1-4 months after the onset of the disease. Patients in the first group were treated according to the clinical regime with focal and scattered photocoagulation which was applied over the affected area. Patients in the second group were photocoagulated so as to reduce the blood flow of the afferent arteriole to a volume that can be drained away by the diseased efferent venule. All patients were carefully followed-up for 6-24 months, particularly for the degree of macular oedema and their visual status. We discuss the clinical observations and present our results.

RNA binding to the untransformed glucocorticoid receptor. Sensitivity to substrate-specific ribonucleases and characterization of a ribonucleic acid associated with the purified receptor.

The cytosolic untransformed molybdate-stabilized glucocorticoid-receptor complex from rat liver was eluted as a heterogenous peak containing two components with Stokes radii (Rs) of 8.3 nm and 7.1 nm when analyzed by size-exclusion HPLC even in the absence of molybdate. In contrast, the highly purified glucocorticoid receptor yielded a sharp symmetrical peak of Rs = 7.1 nm. We demonstrate that the 7.1-nm component could not result from a proteolytic degradation of the 8.3-nm receptor form. The same receptor heterogeneity was observed in thymus cytosol which contains less proteases than liver. After labeling with [3H]dexamethasone 21-mesylate and SDS/PAGE the same 94-kDa receptor band was revealed in both the 8.3-nm and 7.1-nm forms. Immunoblotting experiments showed that both the 94-kDa hormone-binding subunit and the 90-kDa heat-shock protein were present in the two different receptor forms. The 8.3-nm receptor form was converted to the 7.1-nm receptor form after treatment by ribonuclease A in the presence of molybdate and this effect was dose-dependent, being completely prevented by placental ribonuclease inhibitor (RNasin). In contrast, in the presence of molybdate, the 7.1-nm receptor form was ribonuclease-insensitive. Treatment of cytosol with RNase A in the absence of molybdate, partially shifted the untransformed receptor towards the 5.2-nm transformed receptor form. This effect was abolished by placental ribonuclease inhibitor. RNase S protein, an enzymatically inactive proteolytic fragment of RNase A, or S1 nuclease, which is specific for single-stranded nucleic acids, were ineffective when used instead of RNase A. In contrast, cobra venom endonuclease, which preferentially attacks double-stranded regions of small RNAs, caused a complete conversion of the 7-8-nm untransformed receptor to the 5.2-nm transformed receptor form. These results were not observed in the presence of molybdate. Addition of RNasin prior to heating cytosol in the absence of molybdate did not prevent the receptor from dissociating to the 5.2-nm form, suggesting that an endogenous RNase is not involved in the transformation process. The 7.1-nm receptor form was shifted to a 9.2-nm complex when incubated with an excess of GR 49 antireceptor antibody, whereas the 8.3-nm receptor form did not bind to the antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

Glucocorticoid receptors bound to the antagonist RU486 are not downregulated despite their capacity to interact in vitro with defined gene regions.

Modulation of gene expression by glucocorticoids involves interaction of these hormones with an intracellular receptor followed by 'transformation' of the hormone-receptor complex into a nuclear binding form. The molecular basis for the antiglucocorticoid action of high-affinity steroid analogues such as RU486 remains controversial. The effects of dexamethasone and RU486 on in vitro and in vivo properties of the receptor were compared using human lymphoblastoid IM-9 cells. In these cells, RU486 fully antagonized the glucocorticoid-specific induction of 5'-nucleotidase activity by dexamethasone. In vitro, however, RU486-bound receptor could be transformed and shown to interact specifically with cloned DNA fragments containing glucocorticoid response elements. These fragments included one from the mouse mammary tumour virus and two from the human growth hormone gene. In vivo, RU486-bound receptor did not behave like dexamethasone-bound receptor. While receptor downregulation, a property of the transformed receptor, was achieved by dexamethasone, this did not occur with RU486. Likewise, RU486 did not affect receptor half-life under conditions when this was shortened by dexamethasone. These seemingly contradictory results can be reconciled by proposing that receptor transformation by agonists involves dissociation of the receptor oligomer to reveal a DNA-binding site that pre-exists on this protein. Although cell-free receptor dissociation and therefore DNA binding can occur even when the receptor is bound to RU486, this steroid maintains receptors in the untransformed state in the intact cell and therefore behaves a glucocorticoid antagonist in vivo.

Approach to the molecular mechanisms of the modulation of growth hormone gene expression by glucocorticoid and thyroid hormones.

Glucocorticoid and thyroid hormones modulate the expression of the growth hormone gene. To investigate this control mechanism, we have determined whether this gene contains sites that bind the human glucocorticoid and thyroid hormone receptors in vitro. To do so, we have designed a novel assay for studying binding of the purified glucocorticoid receptor to cloned fragments of the human growth hormone (hGH) gene, and have adapted a DNA-competition assay for the thyroid receptor in nuclear cell extracts. Two glucocorticoid receptor binding regions were found in the hGH gene, one of high affinity in a fragment of the gene containing the first intron, and one of low affinity located within a 290 bp-fragment of 5'-flanking DNA. In contrast, the thyroid receptor bound with high affinity to the 5'-flanking fragment. Homologous binding regions for the two types of receptor were found in the human placental lactogen (chronic somatomammotropin) gene. DNA binding of the two receptor types appeared to depend on the presence of the hormone, yet antagonist-bound glucocorticoid receptor was still capable of interacting specifically with DNA. There was no evidence for synergism or antagonism of the two receptor types in binding to their respective sites on the hGH gene. The data also make it unlikely that the thyroid receptor negatively controls gene transcription and that the stimulatory effect of thyroid hormone results from a derepression mechanism.

Thyroid hormone receptors bind to defined regions of the growth hormone and placental lactogen genes.

The intracellular receptor for thyroid hormone is a protein found in chromatin. Since thyroid hormone stimulates transcription of the growth hormone gene through an unknown mechanism, the hypothesis that the thyroid hormone-receptor complex interacts with defined regions of this gene has been investigated in a cell-free system. Nuclear extracts from human lymphoblastoid IM-9 cells containing thyroid hormone receptors were incubated with L-3,5,3'-tri[125I]iodothyronine and calf thymus DNA-cellulose. Restriction fragments of the human growth hormone gene were added to determine their ability to inhibit labeled receptor binding to DNA-cellulose. These fragments encompassed nucleotide sequences from about three kilobase pairs upstream to about four kilobase pairs downstream from the transcription initiation site. The thyroid hormone-receptor complex bound preferentially to the 5'-flanking sequences of the growth hormone gene in a region between nucleotide coordinates -290 and -129. The receptor also bound to an analogous promoter region in the human placental lactogen gene, which has 92% nucleotide sequence homology with the growth hormone gene. These binding regions appear to be distinct from those that are recognized by the receptor for glucocorticoids, which stimulate growth hormone gene expression synergistically with thyroid hormone. The presence of thyroid hormone was required for binding of its receptor to the growth hormone gene promoter, suggesting that thyroid hormone renders the receptor capable of recognizing specific gene regions.

Association of the glucocorticoid hormone receptor with ribonucleic acid.

The hypothesis that the glucocorticoid hormone receptor interacts with RNA has been tested in cultured rat hepatoma cells. The receptor was covalently labeled with radioactive dexamethasone mesylate, and putative RNA-receptor complexes were stabilized by either cell-free crosslinking using formaldehyde or irradiation of intact cells. After chemical cross-linking in vitro, the receptor displayed the buoyant density of a ribonucleoprotein in CsCl gradients. After photochemical crosslinking in cells labeled with radioactive uridine, the receptor analysed by polyacrylamide gel electrophoresis was carrying labeled ribonucleotides.

In vitro assembly of a pre-messenger ribonucleoprotein.

Transcription of the Bal I E restriction fragment of adenovirus DNA by RNA polymerase II in a HeLa cell extract produces a RNA transcript 1,712 nucleotides in length. This transcript contains the first two elements of the tripartite leader that, in vivo, is spliced onto the late mRNAs. We have found that this adenovirus 2 transcript forms a specific ribonucleoprotein complex (RNP) in this in vitro system. The RNP particle sediments in sucrose gradients as a monodisperse peak at 50 S and has a buoyant density of 1.34 g/cm3 in Cs2SO4, indicating the same 4:1 protein/RNA composition as native nuclear RNPs that contain pre-mRNA sequences (hnRNP). Moreover, the in vitro-assembled RNP is resistant to concentrations of NaCl that are known to dissociate nonspecific RNA-protein complexes. The adenovirus 2 transcript is precipitated by a monoclonal antibody for hnRNP core proteins. In addition, RNA-protein crosslinking of [alpha-32P]UTP-labeled transcript/RNP complexes reveals that the major proteins in contact with the RNA are the Mr 32,500-41,500 species known to be associated with hnRNA in vivo. These results demonstrate the in vitro assembly of a specific RNA polymerase II transcript into RNP. Moreover, because the 1,712-nucleotide adenovirus 2 transcript lacks poly(A) addition sites and because the leader sequences are not spliced appreciably in this in vitro system, it follows that RNP formation requires neither polyadenylylation nor splicing, nor is it sufficient to cause the latter.

Structure of nuclear ribonucleoprotein: heterogeneous nuclear RNA is complexed with a major sextet of proteins in vivo.

Mouse erythroleukemia cells were pulse-labeled with [3H]uridine and irradiated with 254-nm light to produce covalent crosslinks between RNA and proteins in close proximity to one another in vivo. Nuclear ribonucleoprotein particles containing heterogeneous nuclear RNA were isolated and digested with nucleases, and the resulting proteins were subjected to gel electrophoresis. Proteins carrying covalently crosslinked [3H]uridine nucleotides were identified by fluorography. The results demonstrate that heterogeneous nuclear RNA is complexed in vivo with a set of six major proteins having molecular weights between 32,500 and 41,500. Analysis of chromatin fractions indicates that nascent heterogeneous nuclear RNA chains assemble with these six proteins as a very early post-transcriptional event. These data, and other results [Nevins, J. R. & Darnell, J. E. (1981) Cell 15, 1477-1493], lead us to propose the usual order of post-transcriptional events to be: heterogeneous nuclear RNA-ribonucleoprotein particle assembly leads to poly(A) addition leads to splicing.

Assembly of nuclear ribonucleoprotein particles during in vitro transcription.

The assembly of heterogeneous nuclear RNA (hnRNA) into ribonucleoprotein (RNP) particles has been investigated during in vitro transcription in isolated nuclei. Approximately 80% of the in vitro transcription observed in mouse Friend erythroleukemia cell nuclei is attributable to the activity of RNA polymerase II. In vitro hnRNA transcripts are assembled into particles having the same properties as the nuclear ribonucleoprotein (hnRNP) particles in which hnRNA is found in vivo. Direct contact of hnRNP proteins with newly transcribed hnRNA was demonstrated by nuclease protection experiments and by the covalent transfer of 32P-labeled nucleotides from [alpha-32P]UTP-labeled hnRNA transcripts to specific proteins by RNA--protein crosslinking followed by nuclease digestion and electrophoresis of the nucleotide-bearing proteins. The availability of an in vitro system for hnRNP assembly opens a new route for investigating the functional relationship between nuclear structure and mRNA processing.

Studies on the regulation of the branched chain amino acyl-tRNA synthetases of the fungusNeurospora crassa.

The specific activities of the branched chain amino acyl-tRNA synthetases from the cytosolic and mitochondrial fractions ofN. crassa were low in dormant conidia and increased during germination, reaching a maximum 8 h after inoculation. This stage of development is characterised by high rates of many other cellular activities.The increases in activity of synthetases of both cytosol and mitochondria are inhibited by cycloheximide indicating that they are synthesized on cytoplasmic ribosomes. The mitochondrial synthetases show a stimulation of their specific activity when mitochondrial RNA and protein synthesis are inhibited by either ethidium bromide or chloramphenicol suggesting that a mitochondrial translation product regulates the synthesis of the mitochondrial synthetases.The activities of amino acyl-tRNA synthetases are dependent on energy production. When respiration is uncoupled from oxidative phosphorylation, synthetase specific activities decrease although the activities of other mitochondrial enzymes like NADH-dehydrogenase increase. This phenomenon suggests that more than one mechanism regulates the synthesis of mitochondrial proteins which are formed on cytoplasmic ribosomes.The synthesis of branched chain amino acyl-tRNA synthetases ofNeurospora is neither repressed by their cognate amino acids, nor is there inhibition by the precursors of these amino acids, as has been observed in other amino acyl-tRNA synthetases of various organism includingNeurospora.

Keratoconjunctivitis sicca following excision of a dermolipoma of the lacrimal gland.

A 20-year-old woman with subconjunctival mass of the superotemporal region of the globe was operated upon. The mass was found to be a dermolipoma of the lacrimal gland. The removal of the mass caused severe keratoconjunctivitis sicca, which could only be helped with frequent applications of artificial tears.

Corneal thickness after cataract surgery.

An increase in corneal thickness occurs so regularly after cataract surgery as to be considered to be the rule. It reflects the state of endothelial activity and is therefore affected both by the preoperative condition of the endothelium and the degree of surgical manipulation. The question how quickly the endothelium can be expected to recover with consequent disappearance of corneal swelling is still open to discussion. The central corneal thickness of 55 eyes with normal slit-lamp endothelial appearance undergoing cataract surgery was measured preoperatively and on the 3rd, 7th, 14th, 21st, 28th, and 42nd postoperative days. The initial corneal thickness was 0.56 mm. The greatest corneal swelling, found on the third postoperative day, was 16%. Corneal thickness returned to preoperative values in four eyes within 7 days, in 19 eyes within 14 days, in 39 eyes within 21 days, in 50 eyes within 28 days, and in all 55 eyes within 42 days. Corneal swelling, as measured on the third postoperative day, was greatest (19%) when chromic catgut was used, less (13-16%) when black silk 7/0 was used, and least (9%) when virgin silk 8/0 was used.