Engineering direct fructose production in processed potato tubers by expressing a bifunctional alpha-amylase/glucose isomerase gene complex.

Manipulation of starch biosynthesis/degradation and formation of novel molecules in storage organs of plants through genetic engineering is an attractive but technically challenging goal. We report here, for the first time, that starch was degraded and glucose and fructose were produced directly when crushed potato tubers expressing a starch degrading bifunctional gene were heated for 45 minutes at 65 degrees C. To achieve this, we have constructed a fusion gene encoding the thermostable enzymes: alpha-amylase (Bacillus stearothermophilus) and glucose isomerase (Thermus thermophilus). The chimeric gene was placed under the control of the granule-bound-starch synthase promoter. This enzymatic complex produced in transgenic tubers was only active at high temperature (65 degrees C). More than 100 independent transgenic potato plants were regenerated. Molecular analyses confirmed the stable integration of the chimeric gene into the potato genome. The biochemical analyses performed on young and old tubers after high-temperature treatment (65 degrees C) revealed an increase in the formation rate of fructose and glucose by a factor of 16.4 and 5. 7, respectively, in the transgenic tubers as compared to untransformed control tubers. No adverse discernible effect on plant development and metabolism including tuber formation and starch accumulation was observed in the transgenic plants before heat treatment. Our results demonstrate that it is possible to replace starch degradation using microbial enzymes via a system where the enzymes are produced directly in the plants, but active only at high temperature, thus offering novel and viable strategies for starch-processing industries.

The metabolic effects of native and transgenic hemoglobins on plants.

The strictly aerobic bacterium Vitreoscilla expresses a hemoglobin-like protein, VHb, when subjected to oxygen stress. When expressed in plants, this has several intriguing physiological effects, such as improving the overall growth rate, speeding germination and flowering, and increasing the productivity of certain oxygen-requiring metabolic pathways. Although the mechanisms behind the effects of VHb in heterologous hosts are not yet fully characterized, it has been suggested that VHb facilitates oxygen transport and/or storage. This hypothesis is supported by the kinetic properties of VHb, which allow very rapid dissociation of oxygen from the protein.

Transgenic tobacco expressing Vitreoscilla hemoglobin exhibits enhanced growth and altered metabolite production.

The gene for Vitreoscilla hemoglobin (VHb) has been introduced and expressed in Nicotiana tabaccum (tobacco). Transgenic tobacco plants expressing VHb exhibited enhanced growth, on average 80-100% more dry weight after 35 days of growth compared to wild-type controls. Furthermore, germination time is reduced from 6-8 days for wild-type tobacco to 3-4 days and the growth phase from germination to flowering was 3-5 days shorter for the VHb-expressing transgenes. Transgenic plants contained, on average, 30-40% more chlorophyll and 34% more nicotine than controls. VHb expression also resulted in an altered distribution of secondary metabolites: In the trangenic tobacco plants anabasine content was decreased 80% relative to control plants.

Ethanolic fermentation of xylose with Saccharomyces cerevisiae harboring the Thermus thermophilus xylA gene, which expresses an active xylose (glucose) isomerase.

The Thermus thermophilus xylA gene encoding xylose (glucose) isomerase was cloned and expressed in Saccharomyces cerevisiae under the control of the yeast PGK1 promoter. The recombinant xylose isomerase showed the highest activity at 85 degrees C with a specific activity of 1.0 U mg-1. A new functional metabolic pathway in S. cerevisiae with ethanol formation during oxygen-limited xylose fermentation was demonstrated. Xylitol and acetic acid were also formed during the fermentation.

Metabolic studies on Saccharomyces cerevisiae containing fused citrate synthase/malate dehydrogenase.

We have constructed two different fusion proteins consisting of the C-terminal end of CS1 fused in-frame to the N-terminal end of MDH1 and HSA, respectively. The fusion proteins were expressed in mutants of Saccharomyces cerevisiae in which CS1 and MDH1 had been deleted and the phenotypes of the transformants characterized. The results show that the fusion proteins are transported into the mitochondria and that they restore the ability for the yeast mutants CS1-, MDH1-, and CS1-/MDH1- to grow on acetate. Determination of CS1 activity in isolated mitochondria showed a 10-fold increase for the strain that expressed native CS1, relative to the parental. In the transformant with CS1/MDH1 fusion protein, parental levels of CS1 were observed, while one-fifth this amount was observed for the strain expressing the CS1/HSA conjugate. Oxygen consumption studies on isolated mitochondria did not show any significant differences between parental-type yeast and the strains expressing the different fusion proteins or native CS1. [3(-13)C]Propionate was used to study the Krebs TCA cycle metabolism of yeast cells containing CS1/MDH1 fusion constructs. The 13C NMR study was performed in respiratory-competent parental yeast cells and using the genetically engineered yeast cells consisting of CS1- mutants expressing native CS1 and the fusion proteins CS1/MDH1 and CS1/HSA, respectively. [3(-13)C]Propionate is believed to be metabolized to [2(-13)C]succinyl-CoA before it enters the TCA cycle in the mitochondria. This metabolite is then oxidized through two symmetrical intermediates, succinate and fumarate, followed by conversion to malate, oxalacetate, and other metabolites such as alanine.(ABSTRACT TRUNCATED AT 250 WORDS)

Artificial antifreeze proteins can improve NaCl tolerance when expressed in E. coli.

A chemically synthesized DNA fragment encoding an artificial antifreeze protein was expressed in E. coli as a translational fusion with a truncated protein A. Two constructions were made, with two and four antifreeze domains, respectively. The fusion proteins stimulated the growth of their bacterial host cells at inhibitory NaCl concentrations. The fusion protein carrying four antifreeze domains also conferred improved tolerance towards freezing.

[Rehabilitation of back problems in Finland--effect of illness certificate and early disability retirement]. / Ryggrehabilitering i Finland--effekt på sjukskrivning och förtidspensionering.

The effectiveness of rehabilitation has been studied in large series of Finnish patients with chronic low back pain. If the criterion of efficacy is reduction in the extent of sick leave and the incidence of disability pensions, the results are modest. Simple out-patient treatment yielded results comparable to those of costly treatment programmes at rehabilitation centres. Although intensive physical training increased mobility, muscular strength and endurance test values, it had no beneficial effect on figures for sick leave and disability retirement. To achieve better results in rehabilitation, the psychosocial factors involved in low back pain need to be taken into account as important components of the illness.

Clefts with associated anomalies and syndromes in Finland.

Probands with clefts born during an 11-year period, 1975-85, were evaluated; 1,586 probands were found of whom 345 (171 males and 174 females, 21.8%) had an associated anomaly. More male patients had cleft lips, with or without cleft palate (CL(P)) and more female patients had cleft palates (CP). The anomalies were subdivided according to anatomical site, and the biggest category was that of the extremities (29.7%) followed by cardiovascular (14.8%) and other facial anomalies (13.0%). The smallest category was chromosomal anomalies (2.7%) followed by miscellaneous anomalies (4.1%). A total of 560 malformations were found. Most anomalies per proband with clefts were found in the bilateral cleft lip and palate subgroup (mean 1.04). The lowest (0.14) was found in the subgroup with the least severe cleft deformity, the cleft lip with or without cleft alveolus. In the CP groups a similar trend was found with 0.21 in the subgroup of submucous cleft palate and 0.33 for the subgroup cleft palate, submucous clefts excluded. A total of 133 probands with 39 different recognised syndromes was delineated, 25 in the CL(P) group and 108 in the CP group (8.4% of the total 1,586 patients with clefts). There was no difference in parental age between probands with an associated anomaly and those with a solitary cleft. Anomalies were more than three times as frequent among probands with clefts as among the general population.

Birth weight and placental weight in cleft probands.

The mean birth weight of the 1,568 probands with clefts who were born in Finland between 1975 and 1985 was 3,346 g, which is lower than the mean of all live born babies in 1980 (3,541 g). The 345 who had an associated anomaly or syndrome had a mean birth weight of 3,027 g and placental weight of 538 g, compared with 3,435 g and 609 g, respectively, for those with solitary clefts. The more severe the cleft the lower the birth weight, but not the placental weight. The percentage of low birthweight (less than or equal to 2,500 g) infants was higher than in the general population (8.5% compared with 3.8%). The highest percentage of low birthweight infants was in the subgroup with bilateral cleft lip and palate (16.8%).

Metal affinity precipitation of proteins carrying genetically attached polyhistidine affinity tails.

In this study, galactose dehydrogenase (EC 1.1.1.48) was chosen as a prototype target protein to investigate the capability of metal affinity precipitation to facilitate the purification of genetically engineered proteins. A DNA fragment encoding five histidine residues was fused to the 3'-terminal end of the galactose dehydrogenase gene from Pseudomonas fluorescens and thereafter expressed in Escherichia coli. The additional five histidines functioned as an affinity tail and the modified enzyme could be purified using metal affinity precipitation when the metal-chelate complex with ethylene glycol-bis-(beta-aminoethyl ether) N,N,N',N'-tetra-acetic acid, EGTA(Zn)2, was added to the protein solution. The affinity tail could also be applied for the purification of the fusion protein utilising immobilised metal affinity chromatography. After purification, the pentahistidine affinity tail could be removed enzymatically by carboxypeptidase A. Furthermore, growth rate experiments demonstrated that the expression of the metal-binding affinity tail in E. coli cells enhanced the tolerance to zinc ions when added to the growth medium.

Chronic unilateral low-back pain. Predictors of outcome of facet joint injections.

One hundred nine patients with chronic (3-36 months; mean, 13.4 months) unilateral low-back pain and no signs of sciatica were subjected to facet joint injection, randomized in three therapy groups: cortisone and local anesthetic injected intra-articularly, the same mixture injected pericapsularly, and physiologic sodium hydrochloride injected intra-articularly into two facet joints. To evaluate the results, three outcome variables were formed: work, subjective, and disability outcome. The inappropriate signs (IAS) recorded before injections had the best predictability for a good outcome. The mode of injection or duration of symptoms had no significance as a predictor. It was concluded that the outcome after facet joint injection depends on the patient's biopsychosocial chances of self-facilitated improvement. If abnormal illness behavior and distress are found, it helps to estimate the response for treatment and to choose a realistic method of treatment.

Lumbar facet joint syndrome. A randomised clinical trial.

A group of 109 patients with unilateral low back pain for over three months were randomised to receive one of three types of injection treatment: cortisone and local anaesthetic injected into two facet joints (28), the same mixture around two facet joints (39), or physiological saline into two facet joints (42). The effect of the treatment was evaluated in relation to work attendance, pain, disability and movements of the lumbar spine. Patients were examined one hour and two and six weeks after treatment and also completed a questionnaire after three months. A significant improvement was observed in work attendance, pain and disability scores, but this was independent of the treatment given and movements of the lumbar spine were not improved. Of the 70 patients with initial pain relief after injection, 36% reported persisting benefit at the three month follow-up, independent of the mode of treatment given. We conclude that facet joint injection is a non-specific method of treatment and the good results depend on a tendency to spontaneous regression and to the psychosocial aspects of back pain.

[Lumbar facet joint syndrome. Significance of non-organic signs. A randomized placebo-controlled clinical study]. / Le syndrome des articulaires. Signification des signes de non-organicité. Une étude clinique randomisée et contrôlée par placebo.

One hundred and nine patients with chronic (greater than 3 months) unilateral low back pain had less than or equal to 2/5 or greater than or equal to 3/5 inappropriate signs (IAS) in 65 and 44 cases, respectively. The patients were randomized in three therapy groups: cortison and local anaesthetic injected intra-articularly into two facet joints (28 patients), the same mixture injected pericapsularly around two facet joints as well (39 patients) and injection of physiologic sodium hydrochloride intra-articularly into two facet joints (42 patients). The effect of the treatment was evaluated within an hour, two and six weeks after the treatment with work status, pain scale, disability score and movements of the lumbar spine. In addition, the patients returned a questionnaire three months after the treatment. Although similar improvement was observed during the follow-up the pain scales and disability scores were, however, in the beginning and at the end of the study more pathological in the group scoring greater than or equal to 3/5 IAS. It is suggested that persistent high levels of IAS depend more on psychosocial factors than on anatomical disorders and therefore explain why the somatic treatment does not work. Identification of these patients may also prevent the doctor from a burn-out syndrome after many failed treatments. This study also shows that if a biological effect of a treatment is to be studied the patients with multiple IAS should be excluded from the material. There was no difference in the results when either intra-articular or pericapsular cortisone and local anaesthetic or saline intra-articularly was used.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue expander injection dome leakage.

Injection dome leakage was found to be the cause for partial disappearance of the saline solution injected into some tissue expanders. When the injection domes of 22 consecutively removed tissue expanders were checked, we found that the average leakage pressure of these was 32 mmHg, with a range of 8 to 110 mmHg. When the patients were lying on their expanders, the weight of the corresponding body part caused intraluminal pressures of up to 90 mmHg in their expanders. Following removal of the expanders, the total leakage was calculated quantitatively and found to be maximally over 50 percent of the injected volume in some instances. The leakage flow through the injection dome in seven consecutively removed expanders was checked at pressures of 25, 50, and 75 mmHg over 30 minutes and was found to be slow. The possibility of injection dome leakage should be kept in mind, however, as a potential complication under unfavorable conditions. At the same time, we want to emphasize that in most instances tissue expanders function quite well and in predictable ways.