Extraordinary stability of enzymes dried in trehalose: simplified molecular biology.

We show that extremely fragile biomolecules such as DNA restriction and modifying enzymes can be dried in vitro in the presence of trehalose with no loss of activity, even after prolonged storage. A remarkable and unexpected property of the dried enzyme preparations is their ability to withstand prolonged exposure to temperatures as high as +70 degrees C. This stability is unique to trehalose and is not found with other sugars irrespective of their physical or chemical properties. The immediate significance of these observations is the ability to convert enzymes used in molecular biology into stable reagents. The indefinite stability and high temperature tolerance of these dried enzymes should permit the design of convenient formats that may be of particular significance in the automation of genome mapping and sequencing projects. The stabilization of a wide range of biomolecules by trehalose also has practical implications for a number of areas ranging from basic science, through healthcare and agriculture, to bio-electronics.


The effect of signal sequences on the efficiency of secretion of a heterologous phosphotriesterase by Streptomyces lividans.

A heterologous phosphotriesterase (parathion hydrolase) containing the native Flavobacterium species signal sequence was previously shown to be secreted by Streptomyces lividans. Western blot analysis of the recombinant phosphotriesterase produced by S. lividans demonstrated only the mature form extracellularly but both processed and unprocessed forms in cell-associated samples. To investigate the efficiency of secretion in Streptomyces, a construction was made that substituted a native Streptomyces beta-galactosidase signal sequence for the Flavobacterium signal sequence. This resulted in a higher proportion of hydrolase in the extracellular fluid and a lower proportion of parathion hydrolase remaining cell-associated. These results suggest that use of a native Streptomyces signal sequence may result in more efficient secretion of heterologous proteins.


Inhibitory effect of acylphloroglucinol derivatives on the replication of vesicular stomatitis virus.

The antiviral activity of natural phloroglucinols and of synthesized mono- and diacylphloroglucinols, and 2,6-diacyl-4,4-dialkylcyclohexa-1,3,5-triones was investigated. A correlation between the acyl chain length and inhibitory activity against vesicular stomatitis virus (VSV) was observed. Potent antiviral activity was found in di-isovalerylphoroglucinol. 2,6-Diacyl-4,4-dialkylcyclohexa-1,3,5-triones inhibited replication of the virus with low cytotoxicity.


Epiderstatin induces the flat reversion of NRK cells transformed by temperature-sensitive Rous sarcoma virus.

Epiderstatin is a unique glutarimide antibiotic which was found by screening for inhibitors of the signal transduction of epidermal growth factor (EGF). The antibiotic (0.01 microM) was found to reverse the morphology of NRK cells that were infected with a temperature-sensitive mutant of Rous sarcoma virus (srcts-NRK) from the transformed phenotype to the normal phenotype at the permissive temperature (32 degrees C). Epiderstatin did not inhibit the protein kinase activity of p60v-src. The cell cycle progression of src(ts)-NRK cells was blocked at G0/G1 phase, which was caused by the inhibition of biosynthesis of p60v-src but not the transcription of v-src mRNA.


Control of in vivo proteolysis in the production of recombinant proteins.

Proteolytic degradation of protein products causes many problems in the bioprocess industry. The increasing production of proteins in heterologous hosts through the use of recombinant DNA technology, has recently brought the problem into focus, since it seems that heterologous proteins are more prone to proteolysis. The result of proteolytic attack may vary from complete hydrolysis of the product to minor truncation of the protein without impairment of its biological function. The degree to which such partial proteolysis is a problem depends on the requirements of the ultimate use of the protein product.


Multiple drug-resistance in variant of a human non-small cell lung carcinoma cell line, DLKP-A.

A 300-fold adriamycin resistant variant (DLKP-A) of the human lung squamous cell carcinoma line DLKP was established by stepwise selection in increasing concentrations of adriamycin. Different levels of cross-resistance were observed towards VP-16, VM-26, colchicine, vincristine and, somewhat unexpectedly, cis-platin. Resistance was stable for at least 3 months in culture in the absence of drug. P-glycoprotein overexpression was detected by immunofluorescence and Western Blotting, and a direct causal role for P-glycoprotein overexpression in the resistant phenotype was established by transfection with an mdr1 specific antisense oligonucleotide. A modified cryopreservation procedure was necessary for the resistant variant line. The resistant population displays clonal heterogeneity with respect to resistance level. A higher frequency of double minute chromosomes was observed in DLKP-A when compared with the parental cell line.


Tests to combat lentiviruses in domestic animals. Biotechnology Research Group.

Since the discovery of human immunodeficiency virus (HIV) as the cause of AIDS the study of other members of the virus group lentivirinal has intensified. A major outcome of this research have been the development of "state of the art" diagnostic tests. While it has long been realised that these viruses were important causes of slowly developing diseases in sheep and horses other lentiviruses have recently been implicated in similar disease situations in goats, cats and cattle. Membership of the lentivirus group of viruses has increased dramatically over the past fifteen years and it is likely that most if not all mammalian species will be found to be infected with their own lentivirus. This paper will describe the problems caused by these viruses in domestic animals and the steps being taken to prevent their spread. Biotechnology companies have a major role in devising simple and economical tests to detect the viruses and antibodies to these viruses. In addition if a vaccine can be produced against any of the animal viruses this will perhaps enable a similar vaccine to be prepared against HIV.


Transcriptional regulation by the nuclear receptor superfamily.

In the past year, additional experimental data have expanded our understanding of the molecular mechanisms that underlie nuclear receptor control of regulatory programs. It is increasingly clear that steroid members (e.g. glucocorticoid and estrogen) and non-steroid members (e.g. retinoic acid, thyroid hormone, and vitamin D) of the nuclear receptor superfamily may utilize distinct strategies in achieving their complex control of gene regulation.


Viral proteases as targets for chemotherapeutic intervention.

Many viruses encode proteinases that are essential for infectivity, and are consequently attractive chemotherapeutic targets. The biochemistry and structure of the human immunodeficiency virus proteinase have been characterized extensively, and potent peptide-mimetic inhibitors have been developed. Techniques and strategies used to improve the efficiency of these compounds are likely to be applicable to other viral proteinases.


Neutrophil adhesion: a point for therapeutic intervention?

It has become increasingly clear over the last 20 years that the potential exists to modulate inflammatory responses with compounds that interfere with intercellular adhesion. This review highlights the adhesion interactions that occur during neutrophil extravasation and indicates some of the possible ways of disrupting these interactions.


Transformation of vitamin D3 to 1 alpha,25-dihydroxyvitamin D3 via 25-hydroxyvitamin D3 using Amycolata sp. strains.

To enzymatically synthesize active metabolites of vitamin D3, we screened about 500 bacterial strains and 450 fungal strains, of which 12 strains were able to convert vitamin D3 to 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] via 25-hydroxyvitamin D3 [25(OH)D3]. The conversion activity was only detected in strains belonging to the genus Amycolata among all the organisms tested. A preparative-scale conversion of vitamin D3 to 25(OH)D3 and 1 alpha,25(OH)2D3 in a 200-1 tank fermentor using A. autotrophica FERM BP-1573 was accomplished, yielding 8.3 mg 25(OH)D3/l culture and 0.17 mg 1 alpha,25(OH)2D3/l culture. A related compound, vitamin D2, could be also converted to 25-hydroxyvitamin D2 and 1 alpha,25-dihydroxyvitamin D2 using the same strain. The cytochrome P-450 of FERM BP-1573 was detected by reduced CO difference spectra in whole-cell suspensions. Vitamin D3 in the culture induced cytochrome P-450 and the conversion activity simultaneously, suggesting that the hydroxylation at C-25 of vitamin D3 and at C-1 of 25(OH)D3 originates from cytochrome P-450.


An integrated process for the production and biotransformation of penicillin.

The extraction of Penicillin G (PG) from the filtered cultivation medium of Penicillium chrysogenum and its conversion into 6-amino penicillanic acid (6-APA) and phenyl acetic acid (PhA) at pH 8 was performed in a 10 l kühni extractor during the production by means of penicillin-G-amidase immobilized in a liquid membrane carrier system (LM). 6-APA was enriched in LM, and the PhA returned to the cultivation medium. After electrocoalescence of LM, the 6-APA was converted into ampicillin with the same enzyme at pH 6, while the liquid membrane phase and enzyme were recycled and reused.


Transformation of Bowes melanoma cells with SV40 T antigen.

A serum-dependent and two serum-independent variants of the Bowes melanoma cell line, RPMI7272, were transfected with plasmids containing a geneticin-resistance (neo) gene transcribed by the HSV thymidine kinase promoter and an SV40 T antigen gene under control of the mouse metallothionein I promoter. T-antigen increased the cloning efficiency of the serum-dependent cell line in soft-agar more than 50-fold, but cloning efficiency of serum-independent lines was not increased. Trypsinization of serum-independent lines required 100 times lower concentrations of trypsin than serum-dependent cells. Human metal-inducible T-antigen-producing (HMT) melanoma cells supported replication of transfected plasmids containing an SV40 origin of replication. Transient expression of interferon or plasminogen activator from such plasmids was 40-fold higher than in untransformed melanoma cells and could be enhanced 30-fold more by stimulation of transcription of the T antigen gene with cadmium chloride. HMT cells can be grown in suspension and thus may represent an attractive alternative to monkey kidney COS cells.


Solubilization and activity of proteins in compressible-fluid based microemulsions.

We have used the pressure dependency of density in near critical propane to design a novel protein extraction system. Active, structurally intact, proteins have been extracted from an aqueous phase into subcritical propane containing the non-ionic detergent polyoxyethylene sorbitan trioleate (Tween-85). The pressure dependence of protein transfer from the aqueous to organic phase is described for cytochrome C and subtilisin. The effect of the microemulsion and compressible propane on protein structure and function is also discussed. The first determinations of kinetic constants for an enzyme-catalyzed reaction in such a system are also presented.


Separation of X- and Y-chromosome-bearing murine sperm by free-flow electrophoresis: evaluation of separation using PCR.

The effectiveness of separation of murine X- and Y-bearing sperm by free-flow electrophoresis was evaluated by the polymerase chain reaction (PCR). The ratio of X- and Y-bearing sperm from cauda epididymis was analyzed before and after free-flow electrophoresis. A Y-chromosome-specific sequence (pY353/B) and an autosomal sequence (myogenin) were used to estimate the ratio between X- and Y-sperm in the separated fractions. Cauda epididymal mice sperm were separated into two peak fractions under the electric field. Each peak fraction contained sperm of normal shape, however, the motility of the sperm was extremely diminished after separation by electrophoresis. DNA was extracted from 10(4) sperm from each fraction and from the unseparated sperm, and Y-chromosome specific PCR was performed. The PCR experiment revealed that fraction No. 16 (the peak near the cathode) was a Y-sperm rich fraction, whereas fraction No. 22 (the peak near the anode) was a Y-sperm poor one. These results suggested that murine X- and Y-sperm could be successfully separated by free-flow electrophoresis. Analysis of the chromosome-specific sequence by PCR was demonstrated to be a direct and adequate method to evaluate the separation of X- and Y-sperm.


Beneficial effect of hydrotropes on partitioning behaviour of proteins in aqueous two phase systems.

Proteins have been partitioned in poly(ethylene glycol) (PEG)-salt systems containing hydrotropes. Hydrotropes are surface active compounds with strong ionic nature with a smaller hydrophobic part as compared to surfactants. The effect of pH, hydrotrope concentration, polymer molecular weight and protein molecular weight on partitioning of proteins including enzymes and their separation has been investigated. The effects of hydrotropes may be explained on the basis of interaction of PEG and the hydrotropes. This is substantiated by measuring the concentration of hydrotropes in both the phases. The practical utilization of the described effect is to enhance the separation of a mixture of proteins by many folds. Preliminary experiments have shown that hydrotropes at low concentrations do not affect enzyme activity and do not have any bactericidal activity.


Hydrophobic interaction chromatography of recombinant human growth hormone, Genotropin.

A recombinant human growth hormone preparation (rhGH) has been used to study the effects of temperature, pH and detergent (Brij 35) concentration on the selectivity, recovery and retention time, during hydrophobic interaction chromatography (HI-HPLC) on a TSK-phenyl-5PW column. The rhGH preparation used in the study contained two rhGH variants, e.g. LMWGH and Clip I. These variants are structurally very similar to rhGH and exhibited very similar chromatographic behaviour to rhGH, important in evaluation of selectivity. Structural studies revealed that LMWGH had lost the first N-terminal amino acid residue, phenylalanine. Clip I exhibited an increased molecular mass of 32.7 Da for the C-terminal tryptic fragment T18-T19. Temperature and pH were found to influence retention time, sharpness of peaks and selectivity. Furthermore, recoveries were improved from 50% to 99% for rhGH upon the introduction of 0.075% Brij 35, a non-ionic detergent, in the presence of 5% acetonitrile. The optimized HI-HPLC system was found to exhibit good recoveries and excellent selectivity. The system is suitable both as an in-process chromatographic purification step for rhGH, as well as an analytical test method for purity and potency.


Shear thickening of DNA in SDS lysates.

Cells of Escherichia coli were subjected to lysis by an alkali detergent treatment (sodium dodecyl sulphate). The rate of detergent lysis was not first order. The rheological properties of the lysate were measured using a controlled stress rheometer. Detergent-lysed cells produced a lysate which showed marked non-Newtonian properties. The material showed a shear thickening at specific low shear stress conditions. The acceleration of the cone during the ascent stage of a flow curve, influenced the shape of the flow curves. These findings are discussed in relation to the form of bacterial DNA in solution.


Metal-based affinity separation of alpha- and gamma-chymotrypsin and thermal stability analysis of isolates.

Chymotrypsin preparations are contaminated by autolysis products and other post-translational products derived from the zymogen. Some prevalent contaminants are difficult to detect with activity assays and molecular weight separation. However, our differential scanning calorimetry (DSC) studies of alpha-chymotrypsin have revealed that gamma-chymotrypsin is a common contaminant in commercial preparations, and the alpha- and gamma-species differ significantly in thermal stability. Thus, DSC analysis provides a sensitive and rapid means to assess the homogeneity of preparations. Moreover, free metal ion binding studies conducted indicate that the alpha- and gamma-species differ substantially in the number of metal binding sites and metal affinity. Therefore, to attempt to repurify a commercial preparation of alpha-chymotrypsin, a resolubilized sample of alpha-chymotrypsin was subjected to immobilized metal (Cu+2) affinity chromatography with pH elution and the fractions were subjected to DSC analysis. The process successfully removed the majority of the contaminating gamma-chymotrypsin.


DNA fingerprinting of the mosquito pathogen Bacillus sphaericus with M13 DNA as a probe.

Hypervariable nucleotide sequences were detected in Bacillus sphaericus by hybridization with radioactively labelled M13 DNA. Different serotypes could be distinguished by their hybridization profiles. The appearance of bands common for mosquito-pathogenic strains and their absence in an apathogenic strain opens the probability that M13 could hybridize to specific alleles, related to insect toxicity.


Purification of bacterial exotoxins. The case of botulinum, tetanus, anthrax, pertussis and cholera toxins.

Bacterial protein toxins and their fragments have been isolated and purified for various reasons, including the development of efficient vaccines and for methods of identification of bacterial agents causing disease. This activity continues today but a new area of bacterial protein toxin research has recently emerged. Since it was shown that toxin molecules comprise several types of biological activity within their structural domains, it was suggested to use these domains (and their combinations) as biochemical tools for developing novel agents for disease imaging and and/or relieving. In this way eukaryotic cell-receptor specific fusion toxins have been developed to prevent malignancy in human. While human clinical trials of these preparations have only recently begun, the preliminary clinical findings are promising. Also fusion proteins which combine independent immunodominant epitopes from different antigens have also been developed thus opening a way for the generation of new vaccines for both human and veterinary use. Receptor binding fragments of microbial toxins when combined with other molecules may be useful in delivering these molecules into the cell. In this way novel agents may be developed with a potential for inducing specific changes at the molecular level for the correction of metabolic disorders causing human and animal diseases. Bacterial protein toxins such as anthrax, botulinum, cholera, pertussis and tetanus for which considerable progress has been achieved in structure-function analysis are promising candidates for such research. Particularly exciting appears the idea of extending this research to the cells of the nervous system, exploiting the unique specificity of the botulinum or tetanus toxin fragments which may bring long desired methods for treatment of various disorders of the nervous system. Data on functional domains of these toxins as well as methods of purification of the whole toxins and their fragments are considered in this review as they form a base for their further structure-function analysis and engineering applications.


Protein-catalysed protein folding.

A number of proteins, termed chaperonins, have been identified as part of the mechanism of folding other proteins into their biologically active forms. The role of chaperonins appears to be twofold--to prevent illegitimate interactions with other proteins and to facilitate folding, possibly through an energy-dependent, catalytic function. Controlled overexpression of chaperonins may be of therapeutic value in manipulating human immune response and rescuing certain inherited human mutations.


Mathematical modelling and simulation of aqueous two-phase continuous protein extraction.

A mathematical model has been developed to describe the continuous, steady-state operation of an aqueous two-phase system for protein extraction. The model is based on steady-state mass balances of the main components and phase equilibrium data. Experimental data on the separation of thaumatin from contaminant proteins of an homogenate of E. coli in a PEG4000/Phosphate system was used. The data shows the effect of the presence and absence of NaCl which was used to carry out the extraction of thaumatin into the PEG phase and back into the PO4(-3) phase. Simulation results showing the sensitivity to key process parameters, and the effect of process variables on performance are presented and discussed. The model can be used to predict performance and thus 'robustness' of process conditions as well as predict protein recovery yield and purity. This model can also be used to implement a suitable control strategy to maintain process stability.


Ambruticin S production in amino acid rich media.

The antimycoticum ambruticin was produced by cultivating the myxobacterium Sorangium cellulosum in shake flasks and stirred tank reactors with 10- and 30-l working volumes. During growth and product formation amino acid concentrations in the complex media were measured by HPLC. In the logarithmic growth phase free aspartate and glutamate were assimilated without product formation. After consumption of aspartate and glutamate, growth was limited and ambruticin production set in. The effects of biological parameters such as age, amount of inoculum, pretreatment of cells before inoculation and time of precultivation were also investigated.


Effects of nutritional conditions on plasmid stability and production of tryptophan synthase by a recombinant Escherichia coli.

Effects of nutritional conditions and insertion direction of the tryptophan synthase (TSase) gene into a plasmid vector on the plasmid stability and the production of TSase in high cell concentration cultures were examined using recombinant Escherichia coli (E. coli K12 IFO 3301) harboring pBR322trpAB(1) (the TSase gene was inserted at the EcoRI site of pBR322 in the clockwise direction) and pBR322trpAB(2) (counterclockwise direction). As to the effects of the insertion direction, the cells harboring pBR322trpAB(2) were slightly lower in the growth rate and the plasmid stability than those harboring pBR322trpAB(1). However, the former was higher in the productivity of TSase and the final cell concentration attained than the latter. On the other hand, the addition of organic nutrients, especially yeast extract, to TK-25 medium was very effective to improve the plasmid stability. Among the components of yeast extract, L-glutamic acid was found to be effective to improve both the plasmid stability and the production of TSase. When 1 g/l of L-glutamic acid was added to TK-25 medium, a mineral synthetic medium developed for a high concentration culture, 115g (dry basis)/l of recombinant cells were obtained in 14 hr and the expression of TSase was maintained at 240-300 U/mg-protein during the cultivation.


Genetic breeding of L-tyrosine producer from Brevibacterium lactofermentum.

A wild-type parent of Brevibacterium lactofermentum was converted into an L-Tyr producer by three steps of genetic breeding. First, acquirement of m-fluoro-D, L-phenylalanine resistance (1,000 microgram/ml) brought about MF1317 which produced 3.5 g/l of L-Tyr and a byproduct of 2.8 g/l of L-Phe. Second, increase in the drug resistance (5,000 microgram/ml) gave MF358 that produced 6.4 g/l of L-Tyr and a byproduct of 6.0 g/l of L-Phe. Third, an L-Phe auxotrophic mutant (FT-1) derived from MF358 accumulated 16 g/l of L-Tyr. In FT-1, L-Phe was not accumulated at all, but a small amount of anthranilate (0.4 g/l) was. A key enzyme in the biosynthesis of L-Tyr, 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, was free from synergistic feedback inhibition by L-Tyr and L-Phe in the producers, and so L-Tyr accumulation occurred independently of L-Phe concentration in the production medium.


Interfacial protein adsorption and inactivation.

Protein inactivations at liquid-liquid, gas-liquid, and liquid-solid interfaces are presented. Wherever possible the mechanisms of protein inactivation, the extent of inactivation, and means by which this inactivation may be minimized are presented. Emphasis is placed on the 'quality' or the heterogeneity of the protein absorbed at the different types of interfaces. The analysis of the adsorption of proteins at different types of interfaces presented together provides novel physical insights into protein interactions at interfaces. The influence of protein adsorption at interfaces on bioseparations is analyzed by discussing examples on two-phase separations, fermentation systems, membrane separation systems, and chromatographic separations. Valuable knowledge gained during protein adsorption for biomedical applications may be applied with caution to bioseparation systems wherever appropriate. Future theoretical and experimental analysis on protein adsorption in bioseparation systems should pay more attention to the 'quality' of the protein adsorbed at the interface.


Cloning and characterization of genes responsible for m-fluoro-D,L-phenylalanine resistance in Brevibacterium lactofermentum.

Two kinds of 3-deoxy-D-arabino-hepturosonate-7-phosphate (DAHP) synthase genes were cloned from an L-Phe-producing mutant of Brevibacterium lactofermentum, AJ11957, which was resistant to m-fluoro-D,L-phenylalanine (mFP) and p-fluoro-D,L-phenylalanine (pFP) and which had DAHP synthase free from feedback inhibition. Both genes were cloned using a vector plasmid, pAJ1844, and the resulting recombinant plasmids were named pAR1 and pAR2. They had different structures on the restriction maps. Both plasmids, pAR1 and pAR2, conferred mFP and pFP resistance and L-Phe and L-Tyr productivity on a wild-type strain of B. lactofermentum, AJ12036. The degrees of L-Phe-analogue resistance and aromatic amino acid productivity of the pAR1-transformant were larger than those of the pAR2-transformant. The introduction of pAR1 to AJ12036 resulted in the alteration of DAHP synthase activity from a feedback-sensitive mechanism to a feedback-insensitive one accompanied by an elevated level of specific activity. A DAHP synthase deficient mutant of Escherichia coli was complemented by pAR2, but not by pAR1. Characteristics of the two kinds of DAHP synthase genes are discussed.


Characterization of recombinant factor XIIIa produced in Saccharomyces cerevisiae.

Recombinant factor XIIIa (FXIIIa), produced in Saccharomyces cerevisiae, was recovered as a fully active cytosolic component and rigorously compared to natural F XIIIa from human placenta with respect to physicochemical and functional properties. Identical parameters were found in SDS polyacrylamide gel electrophoresis, analytical ultracentrifugation and HPLC gel filtration, and all spectral characteristics including derivative UV absorbance, fluorescence and circular dichroism were identical. Similarly, the interaction of both proteins with polyclonal antibodies directed against the entire FXIIIa or its N-terminal 4 kD activation peptide were identical. Furthermore, thrombin cleavage and fibrin cross-linking showed indistinguishable patterns. The only difference we observed was with respect to endgroup analysis. The recombinant protein is homogeneous, whereas placental FXIIIa shows multiple electrophoretic bands caused by microheterogeneity in the C-terminal part of the protein.


Genotyping of bovine kappa-casein loci following DNA sequence amplification.

Caseins are a family of milk proteins that exist in several molecular forms (alpha s1, alpha s2, beta and kappa) with variant alleles of each. Kappa-caseins (kappa-CN) exist as A or B variants and are of particular importance to the quality of the milk. We report a rapid non-radioactive method for differentiating bovine k-CN alleles. Unlike direct milk protein analysis, this method can be utilized for genotyping sperm, or animals of either sex at an early age. A 350bp fragment of the bovine kappa-CN gene was amplified by the polymerase chain reaction (PCR) technique. The amplified product digested with HinfI endonuclease generated restriction fragment length polymorphisms (RFLP's) that allowed precise identification of kappa-CN AA, AB or BB genotypes. Hybridization with allele specific oligo-probes confirmed the RFLP identification of genotypes. The accurate and early identification of milk protein genotypes can have a direct impact on dairy cattle breeding strategies.


Commercialization of biotechnology in the Asian region. Report of meeting of Asian Productivity Organization (APO) in Osaka, Japan.

Detailed studies on the commercial development of biotechnology in five countries in the Asian Region (viz. Japan, Korea, Taiwan, Thailand, India) indicate a very significant role played by government in strategic planning and R & D financing. Strong growth is projected in some traditional biotechnology products e.g. amino acids such as monosodium glutamate and L-lysine, animal and human vaccines, and in environmental applications of biotechnology. The cash flow from these products will underpin future development and marketing of diagnostics, anticancer and antiviral drugs, particularly in Japan and Korea.


Molecular and developmental biology of the Cnidaria--basic aspects and phylogenetic implications.

Cnidarians are generally considered to be the most primitive group of eumetazoans. They are therefore of considerable interest for comparative studies of metazoan development and evolution. The application of molecular techniques to cnidarians can provide important insights into developmental processes and phylogenetic relationships both within the phylum Cnidaria and among the eumetazoa. This paper reviews current knowledge of molecular and developmental biology of cinidarians with particular emphasis on tropical scleractinian corals and soft corals from the Great Barrier Reef region. Nucleotide sequencing of the histone gene cluster has unequivocally established that cnidarians are typical eumetazoans. Repeated sequence analysis has been applied to the staghorn corals, and implies a major divergence within this group, which corresponds with differences in sperm ultrastructure. Recent developmental studies of corals have indicated that some species have an unusual method of germ layer formation. Molecular events associated with this process, and with other important developmental stages, are currently under investigation. Other areas of current research interest, including targets for PCR-based investigations into cnidarian phylogeny, are highlighted.


Typing of Legionella pneumophila serogroups 2-14 strains by analysis of restriction fragment length polymorphisms.

A typing method based on analysis of restriction fragment length polymorphisms has previously been developed for Legionella pneumophila serogroup 1. Here data are presented demonstrating the utility of this method for typing strains of all other L. pneumophila serogroups described to date. The method, which is highly discriminatory, should be of considerable value in epidemiological investigations of legionella infections.


Chaperonin assisted polypeptide folding and assembly: implications for the production of functional proteins in bacteria.

Production of biologically active foreign proteins with correct three-dimensional structures is often difficult in bacteria. Recent advances demonstrate that, for some proteins at least, their correct folding and assembly is facilitated by a class of proteins known as molecular chaperones. An understanding of the function of molecular chaperones may assist in the synthesis in bacteria of functional foreign proteins produced by recombinant techniques.


Amplification of the ZFY and ZFX genes for sex identification in humans, cattle, sheep and goats.

We describe a quick and efficient method of determining the sex of DNA samples from humans, cattle, sheep, and goats. Using universal primers we have amplified 447 or 445 bp fragments from male or female genomic DNA corresponding to the ZFY or ZFX genes. Restriction fragment length polymorphism (RFLP) analysis of the fragments yielded specific banding patterns between the two sexes in these species. Horse, pig and rainbow trout fragments did not show RFLPs with the group of enzymes chosen for the other species. Use of this method would allow animal breeders to determine the sex of preimplantation embryos before transfer to recipient females. Human embryos at risk for sex-linked disorders could be sexed soon after in vitro fertilization (IVF) and options considered before implantation.


An inducible gene expression system for Neurospora crassa.

Neurospora crassa acetyl CoA synthetase is highly induced when the growing mycelium is transferred from sucrose- to acetate-based medium. The inducible promoter of this gene has been isolated and used to control the expression of glutamate dehydrogenase. Transformants containing this expression cassette show gdh levels up to 25 times higher than the nontransformed host strain. This expression cassette will form the basis of a system of heterologous gene expression.


Regulated expression allows high level production and secretion of HIV-1 gp120 envelope glycoprotein in Drosophila Schneider cells.

We have established a stable, continuous culture Drosophila Schneider 2 cell line that efficiently expresses a secreted, truncated form of the HIV envelope gp120 protein in a regulated manner. The Drosophila produced recombinant gp120 protein is highly glycosylated, is recognized by gp120-specific monoclonal antibodies, binds to the CD4 receptor and has the ability to inhibit syncytia formation between uninfected CD4+ cells and HIV infected cells. We conclude that this recombinant Drosophila envelope protein is an appropriate mimic of the authentic viral envelope protein. Thus, the Drosophila cell provides a continuous, stable culture system for the efficient expression of secreted forms of complex surface glycoproteins in quantities sufficient for detailed analyses.


Construction of a 7-aminocephalosporanic acid (7ACA) biosynthetic operon and direct production of 7ACA in Acremonium chrysogenum.

We have used cDNA encoding D-amino acid oxidase, and genomic DNA encoding cephalosporin acylase from Fusarium solani and Pseudomonas diminuta, respectively, to construct a novel hybrid 7-aminocephalosporanic acid (7ACA) biosynthetic operon under the control of regulatory elements from the alkaline protease gene of Acremonium chrysogenum. Transformants of A. chrysogenum BC2116, a high cephalosporin-producing strain, containing this operon, synthesized and secreted low levels of 7ACA. Although the amounts are not yet commercially significant, this represents the first microbial production of 7ACA and demonstrates the feasibility of introducing new biosynthetic capabilities into industrial microorganisms by combining fungal and bacterial genes.


Production of a site specifically cleavable P-glycoprotein-beta-galactosidase fusion protein.

We have fused full length and the carboxyl-half of human MDR1 cDNA with the E. coli lacZ gene via a collagen linker and allowed their expression in yeast Saccharomyces cerevisiae. Using antibodies against beta-galactosidase we partially purified the fusion proteins by immunoprecipitation and show here that the full length fusion protein has ATPase activity. By contrast, the fusion protein containing the carboxyl-half of P-glycoprotein did not show ATPase activity, indicating that both domains of P-glycoprotein are necessary. By treatment of the immunoprecipitated fusion protein with collagenase, P-glycoprotein was released from the beta-galactosidase moiety. The results shown here open the possibility for a large scale purification of P-glycoprotein using this site specifically cleavable fusion protein.


Expression of the human blood coagulation protein factor XIIIa in Saccharomyces cerevisiae: dependence of the expression levels from host-vector systems and medium conditions.

The human blood coagulation protein Factor XIIIa (FXIIIa) was expressed in Saccharomyces cerevisiae employing Escherichia coli-yeast shuttle vectors based on a 2-mu plasmid. Several factors affecting high production yield of recombinant FXIIIa were analysed. The use of the regulatable GAL-CYC1 hybrid promoter resulted in higher FXIIIa expression when compared with the constitutive ADCI promoter. Screening for suitable yeast strains for expression of FXIIIa under the transcriptional control of the GAL-CYC1 hybrid promoter revealed a broad spectrum of productivity. No obvious correlation between the expression rate and the genetic markers of the strains could be identified. The medium composition markedly influenced the FXIIIa expression rates. The expression of FXIIIa was strictly regulated by the carbon source. Glucose as the only sugar and energy source repressed the synthesis of FXIIIa, whereas addition of galactose induced FXIIIa expression. Special feeding schemes resulted in a productivity of up to 100 mg FXIIIa/l in shake flasks.


Regulatory sequences controlling short chain fatty acid metabolism in Escherichia coli.

Acetoacetate in Escherichia coli is metabolized via the combined enzymatic action of a CoA-transferase and a thiolase. Growth of E. coli on short chain fatty acids such as butyrate and valerate is also predicated upon the expression of these enzymes. The genes for these enzymes (atoDAB) are arranged in an operon and are coordinately transcribed in response to the inducer acetoacetate. A positive regulatory element, the product of the atoC gene, regulates expression of the operon. The atoC gene lies adjacent to the atoDAB operon and all the ato genes have been cloned as a single 6.2 kbp restriction fragment (kindly provided by Dr. Lauren Sallus Jenkins). We have isolated a series of mutant E. coli strains with altered regulatory properties that are either inducible by an alternate substrate, or that show constitutive expression of the atoDAB genes. The -10 and -35 regions upstream of the atoDAB operon poorly match consensus sequences. In addition, the transcriptional start is preceded by a catabolite activator protein binding site (CAP site), as well as a putative binding site for the atoC gene product as represented by a region of dyad symmetry.


Genetic control of flocculation in Escherichia coli.

Escherichia coli cells form flocs or aggregates by overproducing type 1 pili. When the pil operon is placed under the control of a tac or lac promoter-operator sequence, the bacterial cells can be induced to form flocs by adding isopropyl-beta-D-thiogalactopyranoside to the culture medium. This phenomenon of genetically induced flocculation can aid in the downstream of biological products. This paper describes the construction of two artificially controlled plasmids which cause cell flocculation. Cell aggregates 50 microns in mean diameter were obtained 1 h after the cells were induced.


Protein secretion in bacteria.

Most secretory proteins are synthesized as precursors with an amino-terminal signal peptide. Genetic identification of proteins essential for signal peptide dependent translocation to the Escherichia coli periplasm has led to the biochemical dissection of the secretion pathway. Additional mechanisms exist in Gram-negative bacteria for protein secretion to the extracellular environment.


Fab assembly and enrichment in a monovalent phage display system.

We have developed a system that allows the expression of a single copy of an antibody Fab molecule on the surface of the filamentous bacteriophage M13 and demonstrate the utility of this system for enrichment of specific "Fab phage". A "humanized" version of antibody 4D5 (h4D5) directed against the extracellular domain of the HER2 (neu) receptor, was used as prototype to assess the assembly of Fab molecules on the phage and to determine the power of the enrichment system. The h4D5 Fab phage showed specific binding to the extracellular domain of the receptor and exhibited an affinity for its antigen virtually identical to the Fab itself. By virtue of its antigen binding, the h4D5 Fab phage could be sorted from a million-fold excess of non-cognate Fab phage in only two rounds of sorting. Further experiments demonstrated that the h4D5 Fab phage could be preferentially enriched from mixtures of variant Fab phages that had lower affinities for the HER2 receptor.


Expression of human P-glycoprotein in yeast cells--effects of membrane component sterols on the activity of P-glycoprotein.

A human MDR1 cDNA was introduced into yeast cells. Immunoblot analysis and indirect immunostaining showed that some of the P-glycoprotein produced was situated in its native orientation in the yeast plasma membrane. Drug-binding activities of the recombinant P-glycoproteins were markedly decreased compared to that of the authentic P-glycoprotein. To identify the bases of decreased binding we studied the effects of membrane component sterols on the azidopine binding and found that ergosterol, which is the main sterol in the yeast membrane, and calciferol, which is produced from ergosterol by UV irradiation, inhibited azidopine binding. These sterols in yeast membrane probably inhibit the function of human P-glycoprotein as a multidrug transporter in yeast cells, because expression of P-glycoprotein in yeast cells did not confer resistance to doxorubicin.


Molecular basis of multidrug resistance mediated by P-glycoprotein.

In the past year, our understanding of the biology and molecular basis of multidrug resistance of tumours has advanced on several fronts. Intriguing clues to some of the key questions in the area provide optimism for future understanding and, with luck, eventual prevention and/or treatment.


The Hox gene family in transgenic mice.

Evidence is accumulating which suggests that the vertebrate Hox homeobox gene family plays an important role in pattern formation, particularly in the specification of regional diversity. In the last year important advances in the understanding of their regulation and function have been provided using transgenic mice.


Use of colony-stimulating factors in combination with high-dose chemotherapy.

Recent clinical trials of two hematopoietic cytokines, granulocyte- and granulocyte-macrophage colony stimulating factors, in combination with chemotherapy, have shown that they allow substantially higher doses of chemotherapeutic agents to be used. Administering cytokines decreased the length of time for granulocyte recovery, the incidence of infection, the need for antibiotics, as well as shortening the duration of hospitalizations. Current work is focusing on the use of cytokines to stimulate circulating progenitor cells for pheresis and replacement. There are still striking gaps, however, in our knowledge of the interactions of the various cytokines with each other and with chemotherapeutic agents.


Forensic applications.

The introduction of DNA based identity testing has revolutionized the field of forensic analysis of biological materials. Most of the publications during the past year expand earlier studies that began more than seven years ago, on the properties of DNA recovered from different types of evidential materials; these studies examine the suitability of these samples for identity testing, the effect of different environmental insults the population genetics of loci used for identity testing, and the methods used for statistical analysis of the DNA patterns.


DnaK-mediated alterations in human growth hormone protein inclusion bodies.

Protein overproduction in microbes frequently results in protein misfolding and aggregation though the molecular basis for this process is unclear. The HSP70 chaperonin, DnaK, was identified as an important factor controlling heterologous protein aggregation in Escherichia coli. Co-overproduction of DnaK significantly reduced human growth hormone (HGH) protein inclusion body formation and the extent of HGH aggregation.


High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker.

We report a method for introducing a glutamine synthetase (GS) selectable marker into myeloma cells in which transfectants are selected by growth in a glutamine-free medium. Vector amplification can subsequently be selected using the specific inhibitor of GS, methionine sulphoximine (MSX). Using this system, DNA sequences encoding a chimeric B72.3 IgG4 antibody were expressed from hCMV-MIE promoters in NSO myeloma cells. A cell line was isolated after a single round of selection for vector amplification which contains approximately 4 copies of the vector, secretes 10-15 pg/cell/day cB72.3 antibody during exponential growth and can accumulate 560 mg/l antibody in a fed-batch air-lift fermentation system. Productivity is stable in the absence of MSX selection.


The DNA sequence of gamma-glutamyltranspeptidase gene of Bacillus subtilis (natto) plasmid pUH1.

The gamma-glutamyltranspeptidase (gamma-GTP) gene of Bacillus subtilis (natto) plasmid designated pUH1, which is responsible for polyglutamate production, has been cloned and the nucleotide sequence determined. The sequence contains a single open-reading frame stretching for 1260 bp with a relative molecular mass of 49,356. Putative -35 and -10 sequences, TTCAAA and TATTAT, were observed as the consensus sequence for the promoter recognized by the sigma 43 RNA polymerase of B. subtilis, and the ribosome binding site, the sequence of which was AACGAG, was complementary to the binding sequence of B. subtilis 16S rRNA except for one base. The amino acid sequence of the gene with the segment of putative protein C403 of staphylococcal plasmid pE194 indicates homology, whereas that with Escherichia coli and mammalian gamma-GTPs does not show any similarity at all.


Molecular design and catalytic function of a B12-enzyme mimetic.

Developing artificial holoenzymes requires attention to the molecular design, not only of the active site, but also of its microenvironment, which in the naturally occurring enzyme is provided by the apoprotein. Microenvironmental effects play a major role in determining the catalytic performance of an enzyme. Using the development of a B12-dependent holoenzyme mimetic as an example, the important factors and concepts underlying the approaches to developing an artificial holoenzyme are discussed.


Overexpression and purification of asparagine synthetase from Escherichia coli.

Overexpression of the asnA gene from Escherichia coli K-12 coding for asparagine synthetase (EC 6.3.1.1) was achieved with a plasmid, pUNAd37, a derivative of pUC18, in E. coli. The plasmid was constructed by optimizing a DNA sequence between the promoter and the ribosome binding region. The enzyme, comprising ca. 15% of the total soluble protein in the E. coli cell, was readily purified to apparent homogeneity by DEAE-Cellulofine and Blue-Cellulofine column chromatographies. The amino-terminal sequence, amino acid composition, and molecular weight of the purified protein agreed with the predicted values based on the DNA sequence of the gene. Furthermore the native molecular weight measured by gel filtration confirmed that asparagine synthetase exists as a dimer of identical subunits.


Catalytic properties of X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis subsp. cremoris nTR.

An X-prolyl dipeptidyl aminopeptidase (X-PDAP; EC 3.4.14.5) was identified to be loosely bound on the inner cell membrane fraction of Lactococcus lactis subsp. cremoris nTR. The biosynthesis of X-PDAP was continuously increased before the late-log growth phase of the bacteria. Both Gly-Pro-pNA and Ala-Ala-pNA were hydrolyzed by X-PDAP; the kcat/Km value of the former was about 10-fold that of the latter. The Ki of X-Pro and Pro-X were more specific to X-PDAP than those of X-Ala. The enzyme splitting a dipeptide sequentially from beta-casomorphin as a model catalytic pattern was identified and some properties of the enzyme were further characterized.


Construction, bacterial expression and characterization of a bifunctional single-chain antibody-phosphatase fusion protein targeted to the human erbB-2 receptor.

We have constructed genes expressing single-chain antigen binding proteins (scFv) which recognize the human erbB-2 receptor. These genes encode the heavy and light chain variable domains of an erbB-2 receptor specific monoclonal antibody, MAb FRP5, connected by a peptide linker. In order to express a bifunctional molecule, a bacterial alkaline phosphatase gene was fused 3' to the scFv gene. The scFv(FRP5) and scFv(FRP5)-alkaline phosphatase fusion protein (scFv(FRP5)-PhoA) expressed in E. coli specifically recognize the human erbB-2 protein and compete with MAb FRP5 for binding to the receptor. The bound scFv(FRP5)-PhoA protein can be detected directly on tumor cells using a substrate for alkaline phosphatase, showing that the chimeric protein retains both binding and enzymatic activity.


Fatty acid alteration by a delta 9 desaturase in transgenic tobacco tissue.

Nicotiana tabacum tissue was transformed with a rat stearyl-CoA desaturase gene. Gas chromatographic analysis showed an increase in monounsaturated 16 and 18 carbon fatty acids in selected transformed calli and leaves. Fractionation of lipid classes indicated that palmitoleic acid was found in the phosphatidylcholine fraction of desaturase-transformed leaves, but not in leaves transformed with vector sequences. Plant transformation was verified by polymerase chain reaction (PCR) amplification of total leaf DNA.


Renaturation of a single-chain immunotoxin facilitated by chaperones and protein disulfide isomerase.

B3(Fv)-PE38KDEL, a recombinant immunotoxin, forms inclusion bodies when produced in Escherichia coli. In renaturation experiments, nonspecific aggregation of non-native polypeptide chains, and the formation of incorrect disulfide linkages lead to inactive molecules. To prevent these side reactions, we added molecular chaperones and protein disulfide isomerase (PDI) to the refolding buffer. Both DnaK and GroEL/S influenced the reactivation process. GroEL alone inhibited reactivation, but in the presence of ATP, GroEL and GroES significantly increased the yield of active protein. DnaK also increased the yield of properly folded protein and the stimulating effect of DnaK was also observed using immobilized DnaK, which can be used repeatedly without significant loss of activity. PDI, which catalyzes disulfide bridging of proteins, also stimulated reactivation of the immunotoxin. Under optimum conditions, reactivation yields in the presence of PDI were about twice that obtained with nonenzymatic disulfide bond formation. Furthermore, DnaK and PDI were additive when renaturation was performed in the presence of both proteins.


Enzymatic preparation of 1,6-anhydro-muropeptides by immobilized murein hydrolases from Escherichia coli fused to staphylococcal protein A.

In order to produce biologically active 1,6-anhydro-muropeptides in large amounts by enzymatic degradation of isolated bacterial murein polymer highly specific periplasmic murein-metabolizing enzymes from Escherichia coli are made available. The genes slt, dacB, and mepA, encoding the soluble lytic transglycosylase (Slt), the penicillin-sensitive DD-endopeptidase (PBP4), and the penicillin-insensitive murein endopeptidase A (MepA), were independently fused to the N-terminal encoding sequence of staphylococcal protein A (SpA) under control of the temperature-inducible phage lambda pR promoter. The SpA fusion proteins were stably over-produced at high levels in E. coli upon temperature induction at 42 degrees C and account for 3% (5 mg SpASlt/l culture), 3% (5 mg SpAPBP4/l culture), and 0.3% (0.5 mg SpAMepA/l culture) of total protein. The SpA fusion proteins, immobilized on IgG Sepharose, are proteolytically sensitive, in vitro, resulting in complete degradation of the SpA portion of the fusion proteins and release of the murein hydrolases in intact and enzymatically active form into the supernatant. Proteolytic degradation could be prevented by p-hydroxymercuribenzoic acid (PHMB) or ethylenediaminetetraacetate (EDTA) suggesting the involvement of the periplasmic protease Pi from E. coli. The immobilized fusion proteins were enzymatically active and could be used for the batch production of biologically active 1,6-anhydro-muropeptides, which were successively separated on HPLC. Isolated murein polymer was degraded quantitatively to monomeric 1,6-anhydro-muropeptides when immunoglobulin G (IgG)-SpASlt was used in combination with IgG-SpAMepA. A combination of IgG-SpASlt with IgG-SpAPBP4 left the 1,6-anhydro-dimers and oligomers being cross-linked via an LD-peptide bond (m-DAP-m-DAP) uncleaved.


Purification and characterization of benzoyl-CoA ligase from a syntrophic, benzoate-degrading, anaerobic mixed culture.

The benzoyl-CoA ligase from an anaerobic syntrophic culture was purified to homogeneity. It had a molecular mass of around 420 kDa and consisted of seven or eight subunits of 58 kDa. The temperature optimum was 37-40 degrees C, the optimum pH around 8.0 and optimal activity required 50-100 mM TRIS-HCl buffer, pH 8.0 and 3-7 mM MgCl2; MgCl2 in excess of 10 mM was inhibitory. The activation energy for benzoate was 11.3 kcal/mol. Although growth occurred only with benzoate as a carbon source, the benzoyl-coenzyme A (CoA) ligase formed benzoyl-CoA esters with benzoate, 2-, 3- and 4-fluorobenzoate, picolinate, nicotinate and isonicotinate. Acetate was activated to acetyl-CoA by an acetyl-CoA synthetase. The Km values for benzoate, 2-, 3- and 4-fluorobenzoate were 0.04, 0.28, 1.48 and 0.32 mM, the Vmax values 1.05, 1.0, 0.7 and 0.98 units (U)/mg, respectively. For reduced CoA (CoA-SH) a Km of 0.07 mM and a Vmax of 1.05 U/mg and for ATP a Km of 0.16 mM and a Vmax of 1.08 U/mg was determined. Benzoate activation was inhibited by more than 6 mM ATP, presumably by pyrophosphate generation from ATP. The inhibition constant (Ki) for pyrophosphate was 5.7 mM. No homology of the N-terminal amino acid sequence with that of a 2-aminobenzoyl-CoA ligase of a denitrifying Pseudomonas sp. was found.


Regulating gene expression in transgenic animals.

Regardless of the field of application, the raison d'etre of transgenic animals is to study gene regulation and function. With increasing frequency, mammalian genes are being isolated with no concomitant knowledge of their function. The human genome mapping initiative will undoubtedly produce a cornucopia of such genes. While the merit of taking a transgenic route to study genes of unknown function is axiomatic, the choices of strategies for gene regulation in vivo may not be fully appreciated. This review will address two main points: first, the targeted and regulated expression of genes, and second, the structural and functional ablation of genes.


Isolation and characterization of the groES and groEL genes of Bacillus subtilis Marburg.

The complete set of groES and groEL gene homologues from Bacillus subtilis Marburg 168 was identified, cloned, and characterized. The nucleotide sequence indicated the presence of two open reading frames corresponding to the groES and groEL genes. The presumptive GroES and GroEL proteins were calculated to be polypeptides of 10,175 and 57,175 Da, respectively, and showed extensive sequence similarities with the known GroES and GroEL proteins of Escherichia coli and Mycobacterium tuberculosis. A heat-inducible transcript initiated upstream of the groES coding region was identified by primer-extension analysis of in vivo transcripts, indicating that the two genes consist of an operon. At least six heat-shock inducible proteins were identified in the cell extract of heat treated B. subtilis. Two proteins of 10 and 60 kDa overproduced in B. subtilis cells carrying a multi-copy groES and groEL plasmid were demonstrated to correspond to two out of the six heat-shock inducible proteins. The groES and groEL genes of B. subtilis were physically mapped on the 60 degrees region of a 360 degrees map and genetically mapped at the position of 40% linkage with the purB locus using PBS1 transduction of the groEL genes tagged with a chloramphenicol resistance (chlr) marker.


Stimulative effect of non-parenchymal liver cells on ability of tyrosine aminotransferase induction in hepatocytes.

Hepatocytes and non-parenchymal liver cells were isolated from adult rat liver and co-cultured for 48 hours as a monolayer on polystyrene culture dishes. The ability of tyrosine aminotransferase (TAT) induction in hepatocytes was examined in the presence of dexamethasone and dibutyryl cAMP. Non-parenchymal cells greatly enhance the ability of TAT induction of hepatocytes. A soluble factor with molecular weight of more than 10,000 is responsible for this enhancement, because conditioned medium prepared from non-parenchymal cells is also stimulatory. Non-parenchymal cells restored the ability in hepatocytes damaged with the addition of D-galactosamine. Conditioned medium prepared from non-parenchymal cells treated with D-galactosamine had higher activity of enhancement than the medium from normal cells. The soluble factor might be released in response to some signal of injury. Hepatocytes and non-parenchymal cells were immobilized within Ca-alginate, and although immobilized hepatocytes rapidly lost the ability to induce TAT, hepatocytes co-immobilized with non-parenchymal cells maintained the ability during 4 days of culture. These results indicated that non-parenchymal liver cells, as well as hepatocytes, could be used to construct a bioartificial liver support system.


Demonstration of exopolysaccharide production by enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli O157:H7 produces visibly slimy colonies when grown on Sorbitol/MacConkey or Maloney's agar plates at room temperature, indicative of exopolysaccharide (EPS) production. Eighteen of 27 (67%) wild-type E. coli O157:H7 isolates produced enough EPS to be visually distinguishable. Of five strains that showed no visible EPS production on these media, four (80%) did produce slimy colonies on media containing higher salt concentrations. Measurements of EPS production by colorimetric determination of uronic acid indicated that EPS production was affected by growth temperature, atmosphere, and medium. Wild-type E. coli O157:H7 strain 932 produced the greatest amounts of EPS when grown anaerobically at 37 degrees C, whereas its plasmid-cured derivative 932P produced large quantities of EPS when grown aerobically at room temperature. Electron micrographs revealed thin, flexible fibers extending from the bacterial cell surface. Cells of strain 932P grown aerobically at room temperature were completely encased in a thick EPS matrix. Chemical analysis of purified EPS revealed that it is very similar or identical to colanic acid. E. coli O157:H7 adheres better to INT 407 cells when grown under conditions that favor high EPS production than when grown under conditions that repress EPS production.


Hemagglutination (fimbriae) and hydrophobicity in adherence of Serratia marcescens to urinary tract epithelium and contact lenses.

The capacity of 59 isolates of Serratia marcescens, obtained from urinary tract infections, wounds, and contact lenses or their paraphernalia, to agglutinate erythrocytes from different animal species was tested. Three main patterns were found: mannose-sensitive agglutination of guinea-pig, fowl or horse erythrocyte; mannose-resistant agglutination of chicken or pigeon erythrocytes alone or in combination with mannose-sensitive agglutination; and no agglutination. Hemagglutination capacity was associated with isolates from urinary tract infection, but not with isolates associated with contact lenses. Adherence to human urinary tract epithelium did not correlate with the hemagglutination patterns nor with the origin of the isolates. Some strains of different hemagglutination pattern were selected for the study of hydrophobicity and adherence to contact lens polymers. Hydrophobicity, as determined by degree of partition in hexadecane and water (BATH-values), correlated neither with degree of adherence to contact lens polymers nor with the hemagglutination pattern. For a representative strain there was an excellent correlation (r2 = 0.98) between adherence and the water content (hydrophobicity) of the lens polymers. These results suggest that, as with tissues, other factors interact with hydrophobicity in causing adherence to plastics.


Restriction polymorphisms in the internal transcribed spacers and 5.8S rDNA of Saccharomyces.

Polymorphisms in enzymatically amplified ribosomal DNA (rDNA) were examined in 18 strains of Saccharomyces. Restriction patterns generated from the region spanning the internal transcribed spacers (ITS) and the 5.8S rDNA produced two clusters corresponding to S. bayanus and S. cerevisiae. The type culture of S. carlsbergensis (ATCC 76529), which could not be separated from the S. cerevisiae group by small subunit (SSU) rDNA patterns, showed a ScrfI profile that was distinct from all the other strains. The type culture of S. intermedius var. turicensis (ATCC 76519) is assigned to S. bayanus on the basis of the combined results of SSU and ITS restriction analyses. S. kluyveri occurred at a separate branch of the distance tree and is unrelated to any of the strains. Results were in general agreement with reported DNA homologies and are discussed in relation to other molecular and genetic data.


Cell culture of Taxus as a source of the antineoplastic drug taxol and related taxanes.

Callus cultures of Taxus cuspidata and Taxus canadensis were induced using different tissue explants including green and red arils, seed contents, young stems and needles. Callus derived from stem segments displayed the best growth in defined media. The culture medium was supplemented with reducing agents and phenolic-binding compounds to inhibit callus darkening and subsequent growth reduction. T. cuspidata explant growth was affected by different concentrations and ratios of 2,4-D and kinetin. Callus tissues of T. cuspidata were extracted for taxol after 2 months in culture and analysed by HPLC. The presence of taxol (0.020 +/- 0.005% of the extracted dry weight) was indicated based on retention time, U.V. spectra, peak purity as assessed by photo-diode array spectroscopy and compared with an authentic taxol standard, as well as by 1H-NMR analysis. Suspension cultures of T. cuspidata were established from the callus cultures, subsequently immobilized onto glass fiber mats, and maintained as immobilized cultures for 6 months. The immobilized cell cultures also produced taxol at levels up to 0.012 +/- 0.007% of the extracted dry weight.


[Pulmonary small cell carcinoma: late sequelae in long-term survivors].

To assess the long term survivors rate and its complications in a group of patients diagnosed of small cell lung cancer. Clinical records of 227 patients diagnosed of small cell lung cancer over a period of ten years have been retrospectively reviewed. Global survival, survival free of disease, type of therapy, and complications developed after the initial diagnosis have been analyzed in patients surviving two or more years after the initial diagnosis of small cell lung cancer. Global survival has been of 7% at two years and 2% at five years. Three patients (19%) reached the two years survival with active disease. Other three patients developed late relapses after a minimum follow up of two years free of disease. 19% of survivors suffered a second neoplasm. 31% developed signs and symptoms of lung fibrosis. A patient (6%) developed ischemic cardiopathy and another one presented a neurologic picture consisting in progressive neurologic changes and dementia that could not be attributed to his initial disease. Long term survival in small cell lung cancer is still a relatively rare event however the potential complications for these patients are significant. Balance between benefit of therapy and its toxicity must be optimized.


Effect of digoxin on experimental adrenaline-induced hyperglycemia and insulin-induced hypoglycemia.

The effect of digoxin (0.035 mg/kg b.w., i.v.) on adrenaline-induced hyperglycemia (adrenaline: 50 micrograms/kg b.w., s.c.) and on insulin-induced hypoglycemia (insulin: 0.4 mU/kg b.w., s.c.) was studied in experiments on rabbits. Digoxin intensified the adrenaline-induced hyperglycemia at the 30th and 60th minutes of application. The hyperglycemia in this case subsided more rapidly. Digoxin alone caused on elevation of the blood sugar levels that was most pronounced at the 30th minute of introduction. These elevated levels fell to the initial value by the 180th minute. The blood sugar levels in the rabbits treated with physiological solution rose slightly. This was most noticeable at the 120th minute. Digoxin attenuated the insulin-induced hypoglycemia significantly at the 120th, 150th, and 180th minutes (p < 0.05). We suggest that the increase of the adrenaline-induced hyperglycemia and the attenuation of the insulin-induced hypoglycemia could be linked to the release of catecholamines in the acute stage of the action of Digitalis glycosides.


Elastoid transformation of the collagen fibrils in scleroderma skin.

Electron microscope study was conducted on bioptic material taken from the skin of 20 scleroderma patients (15 with progressive scleroderma and five with circumscribed scleroderma). In all cases, we observed elastoid transformation of the collagen fibrils. This phenomenon has a focal character and can be formed either directly or indirectly. The direct elastoid transformation is a result of aggregation of superficially damaged collagen fibrils. In such cases the elastoid acquires a fibrous structure. The indirect elastoid transformation is preceded by the complete destruction of the collagen fibrils. The fibrous elastoid is considered to be a pathologic form of fibrogenesis in scleroderma.


Physical and intellectual development in children with congenital hypothyroidism.

Twenty four patients (14 girls and 10 boys aged six months to 23 years) with congenital hypothyroidism were studied. The diagnoses of all subjects were made 15 days to one year after birth. After being diagnosed, each child was started immediately on replacement hormonotherapy. Parameters of their physical and mental development were analyzed depending on the type of thyroid disorder, patient age at the beginning of treatment and the adequacy of treatment. These parameters were within the normal limits for 71 percent of the patients. However, the remaining 29 percent of the patients were mentally retarded. The authors' assumption is that the lower intellectual parameters for these patients were primarily a result of delayed diagnoses and irregularly conducted treatments in household surroundings.


Therapeutic effect of the laser system Prometeus in the treatment of vertebrogenic lumboradiculalgia.

The authors report their experience in the application of laser therapy using 904 nm laser irradiation and impulse power of 10 W on patients with vertebrogenic lumboradiculalgia (osteochondrosis and herniation of the intervertebral disk). The therapeutic effect of the PROMETEUS type laser system is compared to that of similar equipment reported in literature. Pain was usually relieved between days five and 10 of laser therapy, or, in cases of slower convalescence, by day 15. Clinical symptoms of the disorders also disappeared simultaneously with the alleviation of pain. Our results are similar to those obtained by A.S. Krjuk (1986) and S. Gatev (1985), better than those of Van Lyancin (1988) and worse than those of D. Milani (1987). Laser therapy produces no side effects. It is an important contribution to the armamentarium of modern physiotherapeutic methods. When it is administered properly, it is painless, aseptic and does not cause trauma or allergic reactions. Moreover, it reduces the usage of drugs and consumable materials.


Suppression of experimental barbiturate and phenytoin withdrawal syndrome using valproate sodium.

A neuropharmacological study was performed to investigate the effect of valproate sodium (VS) used to suppress the barbiturate and phenytoin withdrawal syndrome induced experimentally in 1152 male Wistar rats. Valproate sodium was administered by mouth according to the following protocol: initial bolus dose of 400 mg/kg b.w. followed eight hours later by the first maintaining dose of 50 mg/kg b.w. This was given every 12 hours for five days. VS plasma levels were determined by gas chromatography. The therapeutic effect of valproate sodium was assessed by the seizure reactivity of the experimental and the control animals to an electroshock (50 mA/0.1 sec.). The results strongly indicate that valproate sodium inhibits the experimentally induced barbiturate and phenytoin withdrawal syndrome without causing drug dependance. It is suggested that some of the valproate sodium metabolites possess antiwithdrawal effect.


DNA-analysis of patients with autosomal dominant polycystic kidney disease.

Indirect DNA analysis was performed on 12 families totalling 80 people. The analysis used five genetic markers flanking the gene: 3'HVR, pGGG, 218 EP6, 24-1, 26-6. In 11 of the families (92%), a linkage with the PKD1 gene in chromosome 16 was established. In one family, the disease did not segregate with the polymorphic markers of PKD1-locus, thus excluding any possibility that a mutation in this locus was the cause of the autosomal dominant polycystic kidney disease (ADPKD). A correlation was discovered between the positive echographic diagnosis and the genotype in the PKD1-dependent patients with ADPKD. In 28.6 percent of the children studied, and in 12.5 percent of subjects under the age of 30, the echographic diagnosis was corrected through DNA analysis.


Computer tomographic densohistogramic characteristic of the renal cortex in children with suspected autosomal dominant polycystic kidney disease.

Computertomographic densohistogramic analysis of the renal cortex was performed on 36 children aged seven to 19. The clinicogenealogical and echographic findings suggest the presence of autosomal dominant polycystic kidney disease (ADPKD). Diagnoses made on the basis of two methods (echography and computer tomography) coincided in only 46.15 percent of examined cases. Histogramic analysis confirmed the echographic diagnosis of ADPKD in 38.46 percent of the remaining cases while it rejected the ADPKD diagnosis in 15.39 percent. Computer tomographic densohistogramic analysis is recommended for children when echographic findings suggest the presence of ADPKD. It allows early detection of the disease, effective medical surveillance and prophylaxis of complications.


Digital subtraction angiography in renal tumours.

By their nature, more than 98 percent of renal tumours are malignant. Clinically, early symptoms of malignancy are difficult to detect. Because of this, treatment of tumorous formations in the kidneys still is unsatisfactory. In general diagnostic terms, angiographic study can play an important role as a preoperative method that more accurately defines the degree of the spread of cancer as well as the extent of renal vessel involvement in this process. The authors report their experience in performing digital subtraction angiography (DSA) in 39 patients with renal tumours. The advantages of the method are emphasized by a comparison of 38 intra-arterial digital subtraction renal vasographic studies and 11 cavographic studies with respect to reduced irradiation doses and contrast material. In addition, the method allows qualitative image processing and the performance of therapeutic procedures such as embolization, which was done in 17 patients.


Ionized calcium in patients with chronic renal failure.

Parameters of various renal disorders were studied in 31 patients (17 men and 14 women) who all were in the stage of chronic renal failure (CRF). These parameters included total serum creatinine, ionized calcium, serum creatinine, creatinine clearance and acid-base balance. The results were compared with a control group of 30 healthy subjects. In the patients with first stage CRF, the total serum calcium did not significantly decrease (mean = 2.19 +/- 0.12 mmol/l) and ionized calcium was within reference value limits (mean = 1.28 +/- 0.02 mmol/l). The percentage of ionized calcium in total serum calcium was higher for these patients (mean = 58.43%) than for the control group (mean = 52.25%). For patients with first and second stage CRF, the total serum was significantly lower (mean = 1.92 +/- 0.03 mmol/l) than for the healthy controls (p < 0.001) as well as for patients with first stage CRF (p < 0.05). The ionized calcium in these patients was not significantly lower (mean = 1.14 +/- 0.01 mmol/l); however, its percentage was higher than that of serum calcium (mean = 59.38%). There was a slight negative correlation between the levels of ionized calcium and serum creatinine in patients with first stage CRF (r = -0.30) and a significant correlation in patients with second and third stage CRF.


Arteriovenous aneurysms in patients on hemodialysis.

Realization of vascular access for patients with chronic renal failure on chronic hemodialysis sometimes proves to be a difficult task. Problems arise when these routes start creating complications and technical difficulties after longterm hemodialysis. A similar problem arises in dealing with arteriovenous aneurysms in patients on chronic hemodialysis. Along with technical difficulties, there also are a number of problems related to hemodynamics and hemostasis. In the present study, 32 patients were analyzed. These patients had advanced arteriovenous aneurysmal dilatations which necessitated surgical intervention. The state of advancement, the causes and the possible types of aneurysms were taken into account. Serious surgical aneurysmal complications were observed in more than 40 percent of the cases. After surgical removal of the arteriovenous aneurysms, the problem of creating new routes for continuing hemodialysis always arose and frequently led to atypical and nonconventional solutions. Suggestions are made for prolongation of life and maximal avoidance of aneurysmal dilatation development in the vascular accesses of patients on chronic hemodialysis.


Ultrastructural changes of cutaneous nerves in scleroderma.

Ultrastructural changes in cutaneous nerves of 38 patients with progressive scleroderma and eight patients with circumscribed scleroderma are described in the present study. Changes in the myelinated as well as in the unmyelinated fibers were observed in all cases. Myelinated fibers exhibited progressive disintegration of their myelin sheaths. Edema of the cytoplasm and various lesions of the organelles were observed in the Schwann cells. The basal membranes of both myelinated and unmyelinated fibers were irregularly thickened and flimsy in appearance. The axis cylinders were affected to a lesser extent. Edema of the axoplasm, and, less often, reduction of the number of neurofibrils and microtubules were observed there. Sometimes, the nerve fibers were enwrapped compactly by a considerable amount of collagen fibrils. The observed changes of the peripheral skin nerves are very often secondary. They are the morphologic basis of a limited or generalized neuropathy which sometimes develops.


Epidemiological study of the frequency of prescribing antiepileptic drugs.

This epidemiological study reviewed the frequency of prescribing antiepileptic drugs for the period 1982 to 1991 in four regions of Bulgaria. The study included 8,340 outpatients with epilepsy. The therapeutic approaches to primary generalized, focal, and secondary types of epilepsy were evaluated. The drug of preference in all types of epilepsy was found to be phenobarbital (26.3 to 37.8%), followed by carbamazepine (14.0 to 29.8%), bellonal, sacerno, phenytoin, etc. Valproate was prescribed only in a few cases (0 to 2.1%). Combinations of phenobarbital anc carbamazepine prevailed in the therapeutic approach to epilepsy. The share of polytherapy in the treatment of epilepsy tended to decrease in favour of monotherapy. The data on the antiepileptic drug utilization given in the present study are calculated in the internationally accepted unit, a daily defined dose.


Abdominal wall reconstruction with a vascular external abdominal oblique myofascial flap.

A myofascial island flap for abdominal wall reconstruction was based on the lumbar component of the external abdominal oblique muscle and supplied by a major neurovascular pedicle consisting of branches of the cranial abdominal artery, cranial hypogastric nerve, and a satellite vein. The flap was elevated and sutured into a 10 cm x 10 cm body wall defect in five dogs. The dogs were observed for 26 to 28 days. Abdominal wall contour and function were preserved. All dogs developed seromas, two of which became infected. One dog developed a hernia at the dorsal margin of the flap, which was repaired. At necropsy, there was no evidence of dehiscence in any of the dogs. Loose adhesions of omentum to the inner surface of the flap occurred in four dogs. Results of histologic examination confirmed the clinical impression of flap viability. The myofascial island flap has a wide range of mobility over the ventral and caudal areas of the abdomen and lateral thoracic wall. It has potential clinical use for reconstruction of defects within its arc of rotation.


Open intestinal anastomosis with surgical stapling equipment in 24 dogs and cats.

Surgical stapling equipment was used to perform open antiperistaltic side-to-side ("functional end-to-end") entero-anastomoses in 20 dogs and 4 cats. Twenty-one anastomoses healed uneventfully. Seven animals with severe bacterial peritonitis required open peritoneal drainage and delayed abdominal closure. There was postoperative leakage at the anastomotic site in two dogs and a localized abscess at the staple line in one cat. No long-term complications occurred in follow-up periods of 3 to 29 months.


A comparison of surgical training with live anesthetized dogs and cadavers.

Cadavers were compared with live anesthetized dogs for their effectiveness as models for surgical training of veterinary medical students. One group of students was trained using cadavers, and a peer group was trained using live anesthetized dogs. Both groups then performed an intestinal anastomosis using a live subject. The time to completion of the procedure was recorded. The anastomoses and celiotomy closures were evaluated. Each anastomosis was isolated and pressure tested. Reviewers blindly scored each surgical team's performance based on actual inspection of the surgical site and on viewing videotapes of the procedure. The participants' attitudes toward the use of live animals in teaching and research were documented before and after training. No statistically significant differences could be detected between the two groups. The results suggest that some substitution of cadavers for live dogs in surgical training might be feasible.


Results of partial mandibulectomy for the treatment of oral tumors in 142 dogs.

Partial mandibulectomy was performed for the treatment of benign or malignant oral tumors in 142 dogs. Forty-two dogs with a benign tumor (ameloblastoma) had a 22.5 month (range, 6 to 74 months) median disease-free interval, with a 97% 1-year survival rate; there was local recurrence in one dog. Twenty-four dogs with squamous cell carcinoma had a disease-free interval of 26 months (range, 6 to 84 months), with a 91% 1-year survival rate; recurrence and metastasis developed in two dogs and metastatic disease in one dog. Based on survival curves, 37 dogs with a melanoma had a median survival time of 9.9 months (range, 1 to 36 months), with a 21% 1-year survival rate; 20 dogs died or were euthanatized for recurrent or metastatic disease. Twenty dogs with osteosarcoma had a median survival time of 13.6 months (range, 3 to 28 months), with a 35% 1-year survival rate; nine dogs died or were euthanatized for recurrent or metastatic disease. Nineteen dogs with fibrosarcoma had median survival time of 10.6 months (range, 3 to 32 months), with a 50% 1-year survival rate; 12 dogs died or were euthanatized for recurrent or metastatic disease. Results of this and previous studies demonstrated that partial mandibulectomy was effective in prolonging survival and decreasing recurrence for squamous cell carcinoma and ameloblastoma. Progressive disease and corresponding low survival times were common in dogs with melanoma, osteosarcoma, and fibrosarcoma. There were no differences in survival times or the progression of disease among five partial hemimandibulectomy procedures. The high rates of recurrence and metastasis in dogs with these tumors suggest a need for evaluation of ancillary chemotherapy and local radiation therapy to decrease the prevalence of progressive disease.


Treatment of forelimb growth plate deformity in 11 dogs by radial dome osteotomy and external coaptation.

Radial dome osteotomy and external coaptation were used to correct forelimb growth deformities with carpal valgus angles of 15 to 43 degrees in 11 dogs (12 forelimbs). Osteotomy sites were clinically healed by week 4. Appearance and function in 10 limbs was good to excellent. The angular deformity recurred in one dog by week 4 due to partial closure of the distal radial growth plate. Carpal valgus recurred in one dog by week 2 because the ulnar osteotomy healed before radial growth ceased.


Effect of tamoxifen citrate on canine immobilization (disuse) osteoporosis.

The effect of tamoxifen citrate on bone mass in immobilization osteoporosis was studied in 11 growing dogs. Immobilization osteoporosis was induced by fiberglass cast immobilization of the right hindlimb for 28 days, while the left hindlimb served as a nonimmobilized control. Six dogs received tamoxifen citrate (1.5 mg/kg per os) once daily for 28 days; five dogs received no treatment. All dogs were euthanatized on day 28 and bone samples were collected. Bone mineral content of the distal tibial metaphysis of casted and uncasted limbs was measured by single photon absorptiometry. Immobilization resulted in a significant reduction in bone mass in the casted limb of untreated and tamoxifen-treated dogs. However, tamoxifen-treated dogs had less severe immobilization osteoporosis than untreated dogs. The calculated bone mass sparing effect of tamoxifen was 24.4%. Because of the complexity of pathologic bone remodeling, use of a single therapeutic agent may not be the optimal means of preventing bone loss associated with immobilization.


Surgical management of atlantoaxial subluxation in 23 dogs.

Twenty-eight surgical procedures were performed in 23 dogs with atlantoaxial subluxation. Dorsal stabilization in seven dogs resulted in two recoveries and five failures of fixation. Ventral decompression and stabilization in 18 dogs resulted in eight recoveries and four failures of fixation. Six dogs died or were euthanatized within 7 days of ventral stabilization. Using either technique, four of seven nonambulatory dogs recovered.


Subdural hematoma in a dog.

A traumatic subacute subdural hematoma in a dog was diagnosed by computed tomography and treated successfully by craniectomy and surgical drainage.


Endoscopic examination of normal paranasal sinuses in horses.

The frontal, caudal maxillary, and rostral maxillary sinuses of 10 equine cadavers were examined endoscopically, and the findings were confirmed by sinusotomy. Similar endoscopic examinations were performed in five conscious, adult horses by using sedation and local anesthesia. Useful portals of entry for the arthroscope in adult horses were: for the frontal sinus, 60% of the distance in a lateral direction from midline to the medial canthus and 0.5 cm caudal to the medial canthus; for the caudal maxillary sinus, 2 cm rostral and 2 cm ventral to the medial canthus; and for the rostral maxillary sinus, 50% of the distance from the rostral end of the facial crest to the level of the medial canthus and 1 cm ventral to a line joining the infraorbital foramen and the medial canthus. The frontal sinus portal was most useful for examination of the frontal and caudal maxillary sinuses. The caudal maxillary sinus portal was most useful for examining the sphenopalatine sinus. Structures in the frontal and caudal maxillary sinuses could be approached surgically by viewing them through the frontal sinus portal and guiding an instrument to them through the caudal maxillary sinus portal. Tooth root identification was reliable for the second and third upper molars in animals older than 5 years, but was more difficult for the rostral teeth and in younger animals. Endoscopy was not difficult to perform and was well tolerated in standing, sedated horses. The only complication of this procedure was mild, local subcutaneous emphysema that resolved spontaneously within 14 days.


The isolated perfused equine skin flap. Preparation and metabolic parameters.

A model for the study of equine cutaneous physiology, pharmacology, and toxicology was developed. Four 4 x 12 cm and twenty-one 6 x 12 cm single-pedicle axial pattern skin flaps based on the caudal superficial epigastric artery, and eight 6 x 12 cm flaps based on the saphenous artery and medial saphenous vein, were raised and sutured in a tubed configuration. On day 2, each flap was removed, the artery was cannulated, and the flap was perfused with a modified Krebs-Ringer's albumin-based medium for at least 6 hours. Flap viability was assessed by glucose use, lactate production, and histologic examination at the end of the perfusion period. The 4 x 12 cm flaps had evidence of skin necrosis, but the 6 x 12 cm flaps remained histologically viable. Results were compared to those previously reported from perfusion of porcine skin flaps based on the caudal superficial epigastric artery. While the ratios of glucose use to lactate production were similar, equine flaps used less glucose and produced less lactate per gram of tissue than similar pig flaps. Equine skin flaps perfused by saphenous vessels used more glucose and produced more lactate than flaps perfused by caudal superficial epigastric vessels. These results indicate that conclusions drawn from cutaneous physiology studies should not be extrapolated across species lines and that site-specific skin should be used for cutaneous physiology, pharmacology, and toxicology studies. The identified skin flaps may have applications in equine reconstructive surgery.


Incorporation of fresh and cryopreserved bone in osteochondral autografts in the horse.

The structural integrity of subchondral bone in fresh and frozen osteochondral autografts was investigated at month 3 in 10 horses. Two osteochondral autografts were harvested from the lateral aspect of the lateral trochlear ridge of the left talus in each of 10 anesthetized horses. Grafts were frozen in 7.5% DMSO. After 14 days, the thawed grafts were press-fitted into drill holes in the trochlear ridges of the right stifles. A fresh graft from the right hock was implanted in each left stifle. To control for the effects of surgery, a fresh graft was transferred from the right stifle to the left stifle. The end result was two grafts in each femoropatellar joint. Fresh and frozen bone grafts maintained a structurally intact support for the cartilage surface. Graft stability and surface congruency were determining factors in the outcome of the grafts. Incorporation of both types of graft was complete at month 3, but remodeling of the fresh grafts was more active.


Heterotopic transfer of fresh and cryopreserved autogenous articular cartilage in the horse.

Two 10 mm thick osteochondral grafts were harvested from the lateral aspect of the lateral trochlear ridge of the left talus in each of 10 anesthetized horses. The grafts were frozen in a 7.5% DMSO solution and stored in liquid nitrogen. The horses were anesthetized again on day 14 and the thawed grafts were press-fitted into drill holes in the trochlear ridges of the right stifle. A fresh graft was transferred from the right hock to the left stifle. To control for the effects of surgery, another fresh graft was transferred from the right stifle to the left stifle. The result was two grafts in each femoropatellar joint. Fresh and frozen osteoarticular autografts appeared to maintain a durable weight-bearing surface for 3 months; however, the fresh grafts were clearly superior. Frozen grafts had fewer living chondrocytes, decreased safranin-O staining, and decreased SO435 uptake. Graft stability and articular surface congruency were determining factors in the outcome of all grafts. Since the availability of osteochondral autografts is limited, further work on the use of preserved allogeneic osteochondral tissue is warranted.


Femoral head ostectomy in horses and cattle.

Femoral head ostectomy was performed in six horses, three ponies, and four cattle for treatment of fractures of the femoral capital physis, coxofemoral luxation, fractured acetabulum, or severe degenerative joint disease. The procedures were performed via a cranial approach that did not involve osteotomy of the greater trochanter. A dorsal approach for femoral head ostectomy via osteotomy of the greater trochanter was evaluated in three healthy adult ponies. Three animals (2 ponies, 1 calf) were euthanatized within a month and one horse was euthanatized at year 2 due to postoperative complications. Nine animals were discharged to owners and six of them fulfilled their intended functions of breeding, milking, and being kept as companions. One horse was lost to follow-up and two horses died of causes unrelated to the surgery. All surviving animals had a residual lameness that was described by owners as mild to moderate. None of the horses were used as riding animals. The mean age and weight of 10 animals that regained weight-bearing locomotion was 3.1 months and 84 kg; for three unsuccessful cases it was 34 months and 174 kg. We concluded that femoral head ostectomy was a viable salvage procedure for large animals with capital femoral physeal fracture, chronic coxofemoral luxation, or acetabular fracture. Surgical prognosis appeared to be favorable in young cattle and fair in young horses or ponies weighing less than 100 kg. Osteotomy of the greater trochanter resulted in superior exposure of the intact coxofemoral joint and allowed easier, less traumatic surgical luxation of the joint to facilitate femoral head ostectomy.


Epidural vs. intramuscular oxymorphone analgesia after thoracotomy in dogs.

Oxymorphone was administered epidurally (0.1 mg/kg) or intramuscularly (IM) (0.2 mg/kg) to 16 dogs undergoing thoracotomy, to compare the analgesic effectiveness. Heart rate, respiratory rate, systolic and diastolic blood pressure, and pain score were measured hourly. Arterial blood gases were measured at hour 1. A single dose of oxymorphone injected epidurally provided analgesia for up to 10 hours, whereas the IM route provided a comparable effect for less than 2 hours. There were statistically significant increases in heart rate, and systolic and diastolic blood pressures at hour 2 in the dogs treated IM over the dogs treated epidurally. We conclude that epidurally administered oxymorphone is highly effective in alleviating pain after thoracotomy in dogs and provides longer lasting analgesia than the IM route.


The influence of detomidine and epinephrine on heart rate, arterial blood pressure, and cardiac arrhythmia in horses.

Detomidine (10 micrograms/kg and 20 micrograms/kg) was administered to seven horses with and without epinephrine infusion (0.1 microgram/kg/min) from 5 minutes before to 5 minutes after detomidine injection. One or more single supraventricular premature heartbeats were observed in three horses after detomidine administration. Epinephrine infusion did not modify the incidence of cardiac arrhythmias in detomidine-treated horses at the doses tested. Relatively high momentary peak systolic pressures were registered in some horses after detomidine administration during epinephrine infusion. The highest systolic arterial blood pressure was 290 mm Hg, but this value was not higher than that reported in horses during maximum physical exercise. Epinephrine infusion did not alter blood gases, arterial pH, or base excess.


Soap gets in your eyes.

We present a previously unreported series of five cases of acute angle closure glaucoma associated with watching the Australia soap opera "Neighbours". Two cases were bilateral and associated with watching two episodes of "Neighbours" on the same day. The pathogenesis, and possible role of watching soap operas in the causation of primary angle closure glaucoma is discussed.


[Possible role of sexual and gastrointestinal hormones on cancer of the digestive system].

This is a review of the most interesting studies on the possible role of sex and gut hormones in gastrointestinal (GI) carcinomas. Many experimental and some human studies suggest that sex and gut peptides may have a growth effect on normal or neoplastic gut cells. It may be possible in the future to develop strategies for patients with GI cancers that will be based upon hormonal manipulation in a manner similar to current strategies that are at present employed in the treatment of patients with breast cancer.


[Comparative study of adriamycin, epirubicin and mitoxantrone in cancer of the breast. Review of the literature].

An overview of all published randomized trials which compared mitoxantrone or epirubicine to adriamycin was performed to analyse tolerance, toxicity and efficacy of these drugs, related to adriamycin. Mitoxantrone confirms its better tolerance: nausea and vomiting are less frequent (P < 10(-9)) and alopecia less intensive (P < 10(-9)). There is a significant decrease in cardiotoxicity occurrence with mitoxantrone (P < 0.01) but a significantly higher degree of leucopenia (P < 10(-4)). As far a response rate is concerned, mitoxantrone is somewhat less effective than adriamycin (P < 0.001). As compared to adriamycin, epirubicine does not reduce side effects incidence, nevertheless, their intensity is less important: nausea and vomiting (P < 0.04) and alopecia (P < 0.01). Leucopenia is less frequent following epirubicine administration as compared to adriamycin, documented course by course (P < 0.01) or on the overall treatment (P < 0.01). Epirubicine is noted to be less cardiotoxic than adriamycin (P = 0.001) with a decreased incidence of heart failures (P < 0.05). No difference can be observed in response rate between these two treatments, for objective as well as for complete responses.


[Catheter rupture at the site of implantation: a rare accident? Apropos of 2 observations].

Seventy three venous access ports of the same type have been implanted recently in our hospital. We have observed two cases of catheter's rupture. Both accidents show a lot of similarities: material of the same trade mark; same way of implantation: right subclavian vein; rupture before one year; rupture at the point of entry of the subclavian vein; migration of the distal part of catheter; withdrawal of this part by right femoral vein's catheterism. Both broken catheters and another not broken (implanted during more than one year) were tested. It appears that those catheters were weakened, especially around the rupture. The reason is the long-standing compression of the catheter at the narrow space between clavicle and first rib. Then, the rupture of this weakened catheter could have been produced by the high pressure due to injection with small syringes.


[Angiosarcoma of the breast. Apropos of 4 cases with a review of the literature].

Four cases of angiosarcoma of the breast were treated at Centre Léon-Bérard in Lyon. It is a rare malignant vascular tumor, constituting < 1% of all primary breast lesions. Radical local surgery is the treatment of choice; however, local recurrences and metastases are frequently observed. The 3-year disease-free survival rate is < 15%. The role of irradiation and adjuvant chemotherapy remains to be evaluated. Doxorubicin-ifosfamide-GM CSD gave a positive response in one case with metastases.


[Measurement of the kinetics of cell proliferation of cancer of the oropharynx in vivo by the incorporation of bromodeoxyuridine and flow cytometry].

Forty-six tumors from patients with oropharyngeal carcinoma were analysed by flow cytometry after injection of bromodeoxyuridine (Budr) for the labelling index, the duration of S phase and the potential doubling time (Tpot). The results show large variations in Tpot (from 2.6 to 16.7 days) among these tumors from the same site and with the same histology. The variations in Tpot were not significantly related to TNM status and differentiation grade. However, aneuploid tumors had statistically significant shorter Tpot. The predictive value of Tpot regarding the response to radiotherapy is currently under investigation.


[Characterization of a human cell line from an anaplastic carcinoma of the thyroid gland].

A new cell line derived from a thyroid anaplastic carcinoma, CAL 62, has been established in culture. This line is constituted by highly tumorigenic cells. Their epithelial phenotype is stable in culture. Immunochemical staining for human thyroglobulin is negative. Cytogenetic analysis showed a gain of chromosome 20, the translocation i (14q), and breakpoints of centrometric chromatine. These results are similar to those previously reported by other investigators. CAL 62 radiosensibility has been studied. The survival curve of the in vitro irradiated cells has been adjusted with a linear-quadratic model. This cell line is thus showed to be radioresistant. Cell line CAL 62 constitutes an appropriate model for in vitro studies of thyroid anaplastic carcinoma.