Production of a recombinant industrial protein using barley cell cultures.

The use of recombinant DNA-based protein production using genetically modified plants could provide a reproducible, consistent quality, safe, animal-component free, origin-traceable, and cost-effective source for industrial proteins required in large amounts (1000s of metric tons) and at low cost (below US$100/Kg). The aim of this work was to demonstrate the feasibility of using barley suspension cell culture to support timely testing of the genetic constructs and early product characterization to detect for example post-translational modifications within the industrial protein caused by the selected recombinant system. For this study the human Collagen I alpha 1 (CIa1) chain gene encoding the complete helical region of CIa1 optimized for monocot expression was fused to its N- and C-terminal telopeptide and to a bacteriophage T4 fibritin foldon peptide encoding sequences. The CIa1 accumulation was targeted to the endoplasmic reticulum (ER) by fusing the CIa1 gene to an ER-directing signal peptide sequence and an ER retention signal HDEL. The construct containing the CIa1 gene was then introduced into immature barley half embryos or barley cells by particle bombardment. Transgenic barley cells resulting from these transformations were grown as suspension cultures in flasks and in a Wave bioreactor producing CIa1 similar to CIa1 purified from the yeast Pichia pastoris based on Western blotting, pepsin resistance, and mass spectroscopy analysis. The barley cell culture derived-CIa1 intracellular accumulation levels ranged from 2 to 9 microg/l illustrating the need for further process improvement in order to use this technology to supply material for product development activities.

F4 (K88) fimbrial adhesin FaeG expressed in alfalfa reduces F4+ enterotoxigenic Escherichia coli excretion in weaned piglets.

Transgenic plants are attractive bioreactors to large-scale production of recombinant proteins because of their relatively low cost. This study reports for the first time the use of transgenic plants to reduce enterotoxigenic Escherichia coli (ETEC) excretion in its natural host species. The DNA sequence encoding the major subunit and adhesin FaeG of F4+ ETEC was transformed into edible alfalfa plants. Targeting of FaeG production to chloroplasts led to FaeG levels of up to 1% of the total soluble protein fraction of the transgenic alfalfa. Recombinant plant-produced FaeG (pFaeG) remained stable for 2 years when the plant material was dried and stored at room temperature. Intragastric immunization of piglets with pFaeG induced a weak F4-specific humoral response. Co-administration of pFaeG and the mucosal adjuvant cholera toxin (CT) enhanced the immune response against FaeG, reflected a better induction of an F4-specific immune response. In addition, the intragastric co-administration of CT with pFaeG significantly reduced F4+ E. coli excretion following F4+ ETEC challenge as compared with pigs that had received nontransgenic plant material. In conclusion, transgenic plants producing the FaeG subunit protein could be used for production and delivery of oral vaccines against F4+ ETEC infections.

Quercetin derivatives are deconjugated and converted to hydroxyphenylacetic acids but not methylated by human fecal flora in vitro.

By using a batch in vitro anaerobic fecal fermentation model, we have shown that the fecal microflora can rapidly deconjugate rutin, isoquercitrin, and a mixture of quercetin glucuronides. High levels of beta,D-glucosidase, alpha,L-rhamnosidase, and beta,D-glucuronidase were present. Rutin underwent deglycosylation, ring fission, and dehydroxylation. The main metabolite, 3,4-dihydroxyphenylacetic acid, appeared rapidly (2 h) and was dehydroxylated to 3-hydroxyphenylacetic acid within 8 h. The pattern of in vitro fermentation of rutin was not changed by changing the pH (6.0 or 6.9), fermentation scale (10 or 1000 mL), or donors of the inoculum. Hydroxyphenylacetic acids were not methylated by colon flora in vitro. The colonic microflora has enormous potential to transform flavonoids into lower molecular weight phenolics, and these might have protective biological activities in the colon. The site of absorption of flavonoids and the form in which they are absorbed are critical for determining their metabolic pathway and consequent biological activities in vivo.

Measuring Gene Flow in the Cultivation of Transgenic Barley.

Genetic engineering is becoming a useful tool in the improvement of plants and plant-based raw materials. Varieties with value-added traits are developed for nonfood use in industrial and medical production, and different production lines must be kept separate. For good management practices, knowledge of relevant gene flow parameters is required. In the present study, pollen-mediated dispersal of transgenes via cross-fertilization was examined. A transgenic barley (Hordeum vulgare L.) line carrying a marker gene coding for neomycin phosphotransferase II (nptII) was used as a pollen donor. For maximum resolution, a cytoplasmically male-sterile barley line was utilized as recipient and the flow of nptII transgene was monitored at distances of 1, 2, 3, 6, 12, 25, 50, and 100 m from the donor plots of 225 and 2000 m(2). Male-fertile plots at a distance of 1 m were included to measure the transgene flow in normal barley. The number of seeds obtained from male-sterile heads diminished rapidly with distance and only a few seeds were found at distances of 50 and 100 m. Molecular genetic analysis (polymerase chain reaction-PCR) revealed that all seeds obtained from male-sterile heads at a distance of 1 m were transgenic, as anticipated. However, only 3% of the distant seeds (50 m) actually carried the transgene, whereas most of them resulted from fertilization with nontransgenic background pollen. This background pollen was mainly due to pollen leakage in some male-sterile heads. In normal male-fertile barley, the cross-fertilization frequency with transgenic pollen varied from 0 to 7% at a distance of 1 m, depending on weather conditions on the heading day. We conclude that, because of competing self-produced and nontransgenic background pollen, the possibility of cross-pollination is very low between a transgenic barley field and an adjacent field cultivated with normal barley. However, adequate isolation distances and best management practices are needed for cultivation of transgenic barley.

Transgenic oat plants via visual selection of cells expressing green fluorescent protein.

New selectable markers and selection systems are needed to increase the efficiency and flexibility of plant transformation. The objective of this research was to determine if the green fluorescent protein (gfp) gene could be utilized as a visual selectable marker for transformation of oat (Avena sativa L.). A modified gfp gene was delivered into oat cells by microprojectile bombardment. Cell clusters expressing gfp were visually identified using fluorescence microscopy and physically isolated at each subculture. Eleven independent transgenic cell lines were obtained, and fertile plants regenerated from all lines. Transgene integration and expression were confirmed in transgenic plants and progeny. Transgene expression segregated in a 3 : 1 ratio in progeny of the majority of the transgenic lines.

Expression of fungal thermotolerant endo-1,4-beta-glucanase in transgenic barley seeds during germination.

The malting quality of two barley cultivars, Kymppi and Golden Promise, was modified to better meet the requirements of the brewing process. The egl1 gene, coding for fungal thermotolerant endo-1,4-beta-glucanase (EGI, cellulase), was transferred to the cultivars using particle bombardment, and transgenic plants were regenerated on bialaphos selection. Integration of the egl1 gene was confirmed by Southern blot hybridization. The transgenic seeds were screened for the expression of the heterologous EGI. Under the high-pI alpha-amylase promoter, the egl1 gene was expressed during germination. The heterologous enzyme was thermotolerant at 65 degrees C for 2 h, thus being suitable for mashing conditions. The amount of heterologous EGI produced by the seeds (ca. 0.025% of soluble seed protein), has been shown to be sufficient to reduce wort viscosity by decreasing the soluble beta-glucan content. A decrease in the soluble beta-glucan content in the wort improves the filtration rate of beer.

West syndrome: individualized ACTH therapy.

Individualized ACTH treatment of the West syndrome (WS) was assessed in a prospective multicenter study, in which each patient's dosage was increased stepwise according to response. Our series included six patients with cryptogenic and 24 with symptomatic infantile spasms. During the treatment period the total ACTH dose ranged from 58 to 373 i.u./kg. In the cryptogenic group one patient responded to pre-ACTH pyridoxine and four to the lowest dosage of ACTH (3 i.u./kg daily) with cessation of spasms and good outcome; one patient needed the highest dosage (12 i.u./kg daily) for cessation of seizures and became developmentally retarded. In the symptomatic group, 21 of the 24 patients needed 6-12 i.u./kg daily; 12 became seizure-free or having infrequent non-IS fits. Complications such as arterial hypertension, cerebral ventricle dilatation, cardiac hypertrophy, and prolonged adrenocortical hyporesponsiveness were related to the dose. The individualization provides all the benefits of ACTH treatment with minimal side effects and cost.

Correlation between the clinical symptoms and the proportion of mitochondrial DNA carrying the 8993 point mutation in the NARP syndrome.

We describe a four-generation family with a maternally inherited mitochondrial disorder. The symptoms were restricted to the CNS and muscle, the most common features being subacute necrotizing encephalomyopathy, cognitive impairment, ataxia, retinitis pigmentosa, infantile spasms, and optic atrophy. A point mutation at the nucleotide 8993 of the gene encoding subunit 6 of the ATP synthase, associated with the neurogenic muscle weakness, ataxia, retinitis pigmentosa (NARP) syndrome, was shown to be inherited maternally in this family, and a clear correlation was found between the clinical severity of the disease and the proportion of mutant mtDNA. Analysis of oxidative phosphorylation in mitochondria carrying 80% mutant mitochondrial DNA showed a reduction of the ATP generation rate coupled to substrate oxidation.

Infection of hairy roots of strawberry (Fragaria x Ananassa Duch.) with arbuscular mycorrhizal fungus.

Hairy root cultures of strawberry (Fragaria x ananassa Duch.) were induced with the Agrobacterium rhizogenes strain A4. Cultures were maintained on B50 medium but could also grow on a minimal medium, which did not inhibit the growth of arbuscular mycorrhizal fungi. The growth and nutrient uptake were characterized in shake flasks and in a bioreactor. Spores of the native Finnish arbuscular mycorrhizal fungus Glomus fistulosum V128 were used to infect strawberry (Fragaria x ananassa Duch. 'Senga Sengana') hairy roots in vitro. During cultivation, vegetative spore formation was observed. At the end of the cultivation, hyphae and arbuscules were observed in the stained roots.

Scaleup of ajmalicine production by plant cell cultures of Catharanthus roseus.

The effect of scaleup on he production of ajmalicine by a Catharanthus roseus cell suspension culture in a selected induction medium were studied. In preliminary experiments it was observed that the culture turned brown and the production was inhibited upon transfer from a shake flask to a stirred bioreactor with forced aeration. Two factors were recognized as the potential origin of the differences between shake flask and bioreactor cultures: gas composition and mechanical shear forces. These factors were studied separately.By recirculating a large part of the exhaust gas, a comparable gas regime was obtained in a bioreactor as occurred in a shake flask cultures. This resulted in the absence of browning and a similar pattern of ajmalicine production as observed in shake flasks. The effect of shear forces could not be demonstrated. However, the experiments showed that the culture may be very sensitive to liquid phase concentrations of gaseous compounds. The effects of k(L)a, aeration rate, CO(2) production rate, and influent gas phase CO(2) concentration on the liquid phase CO(2) concentration are discussed.

Jansky-Bielschowsky variant disease: CT, MRI, and SPECT findings.

Six patients with a variant type of Jansky-Bielschowsky (JBVD) disease were examined using 3 different imaging methods. Five of the patients underwent computed tomography, 4 magnetic resonance imaging, and 5 single photon emission computed tomography. All patients had brain atrophy that was most severe in the cerebellum. Magnetic resonance imaging demonstrated the parenchymal abnormalities well; all patients had hyperintense periventricular white matter, especially around the bodies and atria of the lateral ventricles, and a significant decrease in signal intensity in the thalami and/or putamina. Single photon emission computed tomography disclosed hypoperfusion of the cerebellum in all patients. Neuroimaging examinations are valuable in the diagnosis of JBVD. It may be difficult to divide patients with neuronal ceroid-lipofuscinosis disorders into clinical subtypes in the early stage of the disease. Magnetic resonance imaging, especially when combined with a typical clinical pattern, makes the diagnosis of JBVD highly likely. Radiologic examinations of the brain may also prove important in following the progression, as well as in investigating the pathophysiology, of the disease.

The spectrum of Jansky-Bielschowsky disease.

A series of five patients with a variant type of Jansky-Bielschowsky disease is presented. The disease initiated between four and a half and seven years with mental and slight motor symptoms. The additional neurological symptoms and signs, i.e. visual failure, retinal degeneration, ataxia, myclonia and epilepsy developed in all children before the age of ten years. The present series differs from our previous series of 16 cases especially in regard of neurophysiological findings (photic spikes, high visual evoked potential, VEP and high somatosensory evoked potential, SEP). VEP became abnormally high between 8.0 and 9.5 years instead of being an early finding as in the previous series. Photic spikes appeared also later in the present series. Electromicroscopic investigation revealed cytosomes with fingerprint profiles (FP) in the autonomic ganglion cells and cytosomes with both FP and curvilinear (CP) profiles in many extraneural cells including smooth muscle, Schwann cells, capillary endothelium and macrophages. In the light of our 21 Finnish patients and the literature, the spectrum of Jansky-Bielschowsky disease seems to be much wider than previously assumed. The diagnosis should be based on clinical, ophthalmological, neurophysiological and ultrastructural findings. Repeated neurophysiological studies may be necessary.

Neurologic sequelae following interferon therapy of juvenile laryngeal papilloma.

Human leukocyte interferon at doses from 2 million units every two days to 3 million units daily was given to a 2-year-old boy for the treatment of recurrent juvenile laryngeal papilloma. After 7 months of treatment the child developed spastic diplegia, which persisted despite the discontinuation of interferon therapy. The clinical picture was consistent with an upper motor neuron lesion; no evidence of peripheral neuropathy was found.

The inheritance of fragile sites: apparent absence of fra(2)(q13) in the parents of three unrelated probands.

We describe the inherited folate sensitive fragile site, fra(2)(q13), in three unrelated mentally retarded children, two of them with different forms of epilepsy. Fra(2)(q13) was detected in one healthy sib of one of the probands. Except for one cell in one of the fathers, fra(2)(q13) could not be detected in any of the six parents, who were repeatedly studied using methods known to induce fragile sites of this type. These findings suggest that fra(2)(q13) is not associated with the clinical features of our patients and can be transmitted by persons not expressing it. The expression of fra(2)(q13) may be age dependent.

Cerebrospinal fluid monoamine metabolite levels in male arsonists.

Cerebrospinal fluid (CSF) monoamine metabolite levels were studied in 20 arsonists, 20 habitually violent offenders, and ten healthy inpatient volunteers. The arsonists and violent offenders had been in prison an average of six months before the study. Both the raw data and data adjusted by analysis of covariance for group differences in age, height, sex, and season of the lumbar puncture showed significantly lower concentrations of 3-methoxy-4-hydroxyphenylglycol (MHPG) and 5-hydroxyindoleacetic acid (5-HIAA) in the arsonists than in the other groups. The finding remained the same when arsonists with violent suicide attempts were excluded from the analysis. Although CSF concentrations of MHPG or 5-HIAA did not correlate with the severity of repeated fire-setting behavior, low blood glucose nadir in the oral glucose tolerance test (a measure of the tendency toward hypoglycemia) did. These results support the hypothesis that poor impulse control in criminal offenders is associated with low levels of certain CSF monoamine metabolites and with a hypoglycemic tendency.

Benzodiazepines, but not antidepressants or neuroleptics, induce dose-dependent development of tolerance to lorazepam in psychiatric patients.

Psychomotor effects of oral lorazepam 3 mg were studied in psychiatric patients stratified into four groups: 1) a group of six patients with no previous use of psychotrophic drugs (NoD), 2) a group of 12 patients treated with antidepressants and/or neuroleptics (PsyD), 3) a group of 10 patients treated with low doses of benzodiazepines (BZs) (lowBZ), and 4) a group of nine patients treated with high doses of BZs (high BZ). Similar objective psychomotor tests and subjective assessments were administered under single-blind conditions to all treatment groups at baseline, after intake of placebo, and after intake of 3 mg lorazepam. Both lorazepam (CGC) and total BZs (bioassay) in serum were assayed. The results demonstrate that treatment with BZs induce dose-dependent development of tolerance to psychomotor effects of lorazepam. Antidepressants and neuroleptics failed to induce cross-tolerance to lorazepam. The rise in serum lorazepam concentrations after lorazepam intake was similar (about 28 micrograms/l) in all treatment groups, suggesting a functional, not dispositional, tolerance. However, the initial learning effect in psychomotor performance was poorer among BZ users than among others.