Targeted expression of a synthetic codon optimized gene, encoding the spruce budworm antifreeze protein, leads to accumulation of antifreeze activity in the apoplasts of transgenic tobacco.

A synthetic gene based on the primary sequence of the mature spruce budworm antifreeze protein (sbwAFP) was constructed by primer overlap extension. The amino acid codons were chosen to mimic those of a highly expressed tobacco nuclear gene. A DNA sequence encoding the amino-terminal leader sequence from the tobacco pathogen related protein 1b (PR), which targets the protein to the apoplastic space, was fused in frame to the synthetic sbwAFP gene. This fusion was placed downstream of the cauliflower mosaic virus 35S promoter and upstream of the nopaline synthase terminator in a T-DNA binary vector. Transgenic tobacco lines transcribing PR-sbwAFP were selected by RT-PCR. The apoplastic protein fractions of sbwAFP expressing tobacco lines exhibited enhanced antifreeze activity as demonstrated by the ability to inhibit ice re-crystallization and increased thermal hysteresis.

Error-prone PCR of Vitreoscilla hemoglobin (VHb) to support the growth of microaerobic Escherichia coli.

Expression of the gene encoding bacterial hemoglobin (VHb) from Vitreoscilla has been previously used to improve recombinant cell growth and enhance product formation under microaerobic conditions. It is very likely that the properties of VHb are not optimized for foreign hosts; therefore, we used error-prone PCR to generate a number of randomly mutated vhb genes to be expressed and studied in Escherichia coli. In addition, the mutated VHb proteins also contained an extension of eight residues (MTMITPSF) at the amino terminus. VHb mutants were screened for improved growth properties under microaerobic conditions and 15 clones expressing mutated hemoglobin protein were selected for further characterization and cultivated in a microaerobic bioreactor to analyze the physiological effects of novel VHb proteins on cell growth. The expression of four VHb mutants, carried by pVM20, pVM50, pVM104, and pVM134, were able to enhance microaerobic growth of E. coli by approximately 22%, 155%, 50%, and 90%, respectively, with a concomitant decrease of acetate excretion into the culture medium. The vhb gene in pVM20 contains two mutations substituting residues Glu19(A17) and Glu137(H23) to Gly. pVM50 expresses a VHb protein carrying two mutations: His36(C1) to Arg36 and Gln66(E20) to Arg66. pVM104 and pVM134 express VHb proteins carrying the mutations Ala56(E10) to Gly and Ile24(B5) to Thr, respectively. Our experiments also indicate that the positive effects elicited by mutant VHb-expression from pVM20 and pVM50 are linked to the peptide tail. Removal of the N-terminal sequence reduced cell growth approximately 23% and 53%, respectively, relative to wild-type controls. These results clearly demonstrate that it is possible to obtain mutated VHb proteins with improved characteristics for improving microaerobic growth of E. coli by using combined mutation techniques, addition of a peptide tail, and random error-prone PCR.

Redesign of the coenzyme specificity in L-lactate dehydrogenase from bacillus stearothermophilus using site-directed mutagenesis and media engineering.

L-lactate dehydrogenase (LDH) from Bacillus stearothermophilus is a redox enzyme which has a strong preference for NADH over NADPH as coenzyme. To exclude NADPH from the coenzyme-binding pocket, LDH contains a conserved aspartate residue at position 52. However, this residue is probably not solely responsible for the NADH specificity. In this report we examine the possibilities of altering the coenzyme specificity of LDH by introducing a range of different point mutations in the coenzyme-binding domain. Furthermore, after choosing the mutant with the highest selectivity for NADPH, we also investigated the possibility of further altering the coenzyme specificity by adding an organic solvent to the reaction mixture. The LDH mutant, I51K:D52S, exhibited a 56-fold increased specificity to NADPH over the wild-type LDH in a reaction mixture containing 15% methanol. Furthermore, the NADPH turnover number of this mutant was increased almost fourfold as compared with wild-type LDH. To explain the altered coenzyme specificity exhibited by the D52SI51K double mutant, molecular dynamics simulations were performed.

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.

Directed evolution of a type I antifreeze protein expressed in Escherichia coli with sodium chloride as selective pressure and its effect on antifreeze tolerance.

Both freezing tolerance and NaCl tolerance are improved when antifreeze proteins are expressed as fusion proteins with two domains of staphylococcal protein A (SPA) in Escherichia coli. To characterize these properties further we created a randomly mutated expression library in E. coli, based on the winter flounder antifreeze protein HPLC-8 component gene. Low-fidelity PCR products of this gene were fused to the spa gene encoding two domains of the SPA. The library was screened for enhanced NaCl tolerance and four clones were selected. The freezing tolerance of each of the selected clones was enhanced to varying extents. DNA sequencing of the isolated mutants revealed that the amphiphilic properties of the native antifreeze protein were essentially conserved. Furthermore, by studying the primary sequence of the randomly mutated clones, in comparison with the degree of freezing tolerance, we have identified clues which help in understanding the relationship between salt and freezing tolerance.

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.

Identification of calmodulin-sensitive Ca(2+)-transporting ATPase in the plasma membrane of bovine corneal epithelial cell.

ATP-dependent Ca2+ uptake was characterized in a plasma membrane enriched fraction obtained from the bovine corneal epithelium. This uptake essentially represented intravesicular accumulation because 72% of the Ca2+ content was releasable following exposure to 10(-6) M A23187. The substrate and Ca2+ requirements for maximal transport activity were similar to those described in the red blood cell because: (1) exogenous calmodulin (3 microM) significantly decreased the apparent Km for Ca2+ to 0.31 microM and increased the rate of Ca2+ uptake; (2) a hydroxylamine labile Ca(2+)-dependent phosphoenzyme intermediate was identified with an apparent molecular size of 140 kDa; (3) Ca(2+)-dependent binding of 125I-labelled calmodulin to this protein was demonstrated which could be antagonized with a calmodulin antagonist, trifluoperazine. These results show that the plasma membrane contains an ATP-dependent Ca2+ transporter. However, its relationship to a previously described high affinity form of Ca(2+)-stimulated Mg(2+)-dependent ATPase is not apparent because their [Mg2+] requirements to elicit maximal activity differed by two orders of magnitude.

Development of an electronic prescription processing option: an aid for general practice.

An interactive computer-supported prescription processing system has been developed as an add-on to existing general practitioner information systems. The aim of the system is to improve the clarity, efficiency and economy of drug treatment choices and prescription writing. It enables the doctor to choose the best treatment from the system's formulary according to the patient's complaint, symptom or diagnosis. The selections are based on complaints and diagnoses from the International classification of primary care (ICPC). A prescription is printed and the potential exists for individualized patient instruction leaflets to be printed. Furthermore, the system may prove useful for retrospective and prospective statistical and epidemiological studies. This implies continuous adaptation, which is also necessary to keep the system updated. As well as an aid in daily general practice, the system is also designed to serve the needs of graduate and postgraduate training programmes.

Characterization of soluble vs membrane-bound human placental 5'-nucleotidase.

Three forms of 5'-nucleotidase purified from human placenta (two membrane-bound forms, one sensitive and one resistant to cleavage by phosphatidylinositol-specific phospholipase C, as well as a soluble form) had the same molecular weight before (73,000 Da) and after (56,000 Da) digestion with N-glycosidase F and showed similar amino acid compositions, N-terminal amino acid sequences, and KMs for IMP (9.6 to 11.9 microM). Thus, these three forms of 5'-nucleotidase appear to have very similar structures. The form sensitive to phosphatidylinositol-specific phospholipase C contained nearly 1 mol myo-inositol/mol of protein as determined by mass spectrometry, indicating a glycosyl phosphatidylinositol membrane anchor. Soluble 5'-nucleotidase contained a similar quantity of myo-inositol, suggesting that it was previously membrane-anchored via glycosyl phosphatidylinositol. The form resistant to phosphatidylinositol-specific phospholipase C contained less myo-inositol, leaving open the possibility of a third form of 5'-nucleotidase with a conventional transmembrane anchor.

Heterophilic antibodies against rabbit serum causing falsely elevated gonadotropin levels.

Two cases are presented in which previously acquired antibodies against rabbits in the patient's serum caused falsely elevated hormonal levels in radio-immuno assays (RIA) based on rabbit antibodies to follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG). The erroneous results led to unnecessary operative intervention and inconvenience for the patients.

Inhibition of calcium of beta adrenoceptor mediated cAMP responses in isolated bovine corneal epithelial cells.

The increases in adenosine 3',5' monophosphate (cAMP) content were measured in isolated bovine corneal epithelial cells in response to either adrenergic agonists or adenylate cyclase stimulation. The beta selective adrenergic agonist, isoproterenol, and the adrenergic agonists, norepinephrine as well as epinephrine elicited large increases in cAMP accumulation. At their maximum effective concentrations, the respective increases were 16-fold, 6.6-fold and 4.7-fold. These stimulatory effects were completely inhibited by the beta selective adrenergic antagonist, propranolol. Similarly forskolin increased cAMP content more than 3-fold. These increases and the previous identification of beta adrenoceptors in fresh intact bovine corneas as well as cells in culture indicate that this enzymatic dissociation procedure does not affect the cAMP responses to either adrenergic agonists or forskolin. The relationship was considered between increases in Ca2+ concentration and the effects of either isoproterenol or forskolin, on cAMP accumulation. There were no changes in any of the cAMP responses at bathing solution Ca2+ concentrations between 0.01 microM and 1 microM. However, in cells permeabilized to Ca2+ with 10 microM ionomycin, increases within this concentration range depressed the baseline levels of cAMP content. Furthermore, the stimulatory effects of both forskolin and isoproterenol on cAMP accumulation were significantly blunted in this concentration range. These blunting effects by Ca2+ were not the result of any measurable decrease in ATP content. This negative relationship between increases in Ca2+ concentration and increases in cAMP content indicates that changes in intracellular Ca2+ concentration could modulate the second messenger function of cAMP linked to these agents.

Ca-stimulated Mg dependent ATPase activity in a plasma membrane enriched fraction of bovine corneal epithelium.

In a subcellular plasma membrane enriched fraction of bovine corneal epithelium, Ca2+ stimulated Mg2+ dependent ATPase activity was characterized. This membrane fraction was more than 5-fold and 4-fold enriched with 5'-nucleotidase and alkaline phosphatase activities, respectively, relative to the 100,000 X g pellet. With 250 microM ATP, maximum stimulation of a high affinity form of Ca2+ stimulated Mg2+ dependent ATPase activity was obtained with 1.7 microM free Ca2+. This activation required no exogenously added Mg2+ and was unaffected by either 0.1 mM ouabain, 3 microM ruthenium red, 20 mM sodium azide or 0.2 microgram/ml oligomycin. Exogenous calmodulin (6 microM) elicited a 53% increase in this activity which was completely inhibited by 300 microM trifluoperazine (TFP). These effects of calmodulin and TFP are consistent with the notion of a plasma membrane origin for this activity and also suggest that this activity could be a basis for the regulation of intracellular Ca2+ activity in the submicromolar range.

Prediction of size of infants at birth by measurement of symphysis fundus height.

Symphysis fundus heights (SF) were measured approximately 15 times during pregnancy in a consecutive series of 2941 women with regular menstrual cycles and known last menstrual period. A reference SF chart from 17 to 40 weeks of pregnancy was derived from measurements in 1350 of these women who were healthy, and heights and pre-pregnancy weights within the 10th and 90th centiles and were delivered vaginally of healthy infants with a birthweight/length ratio within +/- 2 SD. The reference chart was used to predict fetal growth deviations in the unselected series of pregnancies. The effectiveness of SF measures to detect fetuses with an infant birthweight/length ratio below -2 SD or a birthweight below the 10th centile was low; the sensitivity was only 16.7 and 26.6% and the predictive value of positive screening result was 1.8 and 18.0%, respectively. Corresponding values for fetuses with an infant birthweight/length ratio above + 2 SD or a birthweight above the 90th centile were 31.8 and 37.5% and 3.3 and 24.5%, respectively. Symphysis fundus (SF) measurement has thus been found to be of limited value as a screening method to detect abnormal size at birth.

Prostaglandin and thromboxane synthesis by microsomes of inflamed rabbit ciliary body--iris.

Microsomes of albino rabbit ciliary body--iris were prepared 6 hr, 24 hr, 3 days, 7 days, and 28 days after intravitreal injection of 10 micrograms of Shigella endotoxin. The microsomal preparations were incubated for 15 min with [1-14C]arachidonic acid. Prostaglandin and thromboxane products (cyclo-oxygenase products) were identified by thin-layer chromatography and quantified by scintillation counting. Synthesis of prostaglandin F2 alpha (PGF2, PGD2, 6-keto-PGF1 alpha (a stable metabolite of PGI2) and thromboxane B2 (TXB2) (a stable metabolite of TXA2) was increased 24 hr, 3 days, and 7 days after endotoxin injection. The greatest increase was in TXB2 synthesis. Cyclo-oxygenase product synthesis returned to normal levels by 28 days. Ciliary body--iris microsomes prepared 15 min after paracentesis synthesized increased amounts of all cyclo-oxygenase products assayed, most notably TXB2 and PGE2. Ciliary body--iris microsomes from albino rabbit treated with topical 1% nitrogen mustard or pigmented rabbits treated with subcutaneous alpha-melanocyte--stimulating hormone (20 micrograms/kg) synthesized normal amounts of cyclo-oxygenase products.

Prostaglandin and thromboxane synthesis by microsomes of rabbit ocular tissues.

Microsomes of albino rabbit ocular tissues were incubated with (1-14C)-arachidonic acid for 15 min at 37 degrees C. Thin-layer chromatography revealed that ciliary body-iris microsomes were capable of synthesizing prostaglandin F2alpha (PGF2alpha), PGE2, PGD2, thromboxane B2(TXB2), and 6-keto-PGF1alpha. Indomethacin 14 micrometer in the incubation medium essentially abolished all prostaglandin synthesis detectable by this method. Imidazole 10 mM in the incubation medium inhibited only TXB2 synthesis. Ciliary body-iris microsomes were incubated for 2 min at 0 degrees C with PGH2. The products of this reaction were superfused over spiral strips of rabbit aorta and produced the strong contractions typical of TXA2. Addition to imidazole to the incubation medium blocked the formation of the contracting substance. Incubation of ciliary body-iris microsomes with (1-14C)--8,11,14-eicosatrienoic acid produced PGF1alpha, PGD1, and PGE1 but no evidence of any thromboxane product or 6-keto-PGF1alpha. Conjunctival and corneal microsomes synthesized prostaglandins, although less effectively than ciliary body-iris microsomes, when incubated with (1-14C)-arachidonic acid. Microsomes of sclera, retina-choroid, and lens synthesized little, if any, prostaglandins.

Increased intraocular pressure following topical azide or nitroprusside.

In rabbits the topical administration of sodium azide (NaNs) or sodium nitroprusside (SNP) increased intraocular pressure in a dose-response manner. These agents, which activate guanylate cyclase, elevated cyclic GMP in the aqueous humor. Systemic blood pressure and pulse were not altered. Tonographic outflow facility was unchanged, suggesting an increase in aqueous humor flow as the mechanism for the elevation of intraocular pressure. Posterior chamber aqueous humor ascorbate concentration was decreased in the eye receiving the NaN3 or SNP. Systemic pretreatment with phenoxybenzamine, an alpha-adrenergic blocking agent, prevented the elevation of intraocular pressure observed following NaN3 and SNP. Pretreatment with systemic indomethacin, propranolol, or acetazolamide or the topical application of atropine or epinephrine failed to alter the elevation of intraocular pressure by either NaN3 or SNP.

The influence of the bony pelvis in persistent occiput posterior position.

Roentgenologic pelvimetry has been performed in 68 consecutive cases of occiput posterior position. The results point to an influence from a transverse narrowing of the pelvis or a tendency towards large dimensions. In many cases, however, no clear correlation to pelvic dimensions is found. A significant difference exists in the rate of obstetrical complications between nulliparous and parous women. The reasons for this and for the persistent OP position are discussed.

Serum factors precipitating with bovine lens extract in cataract patients and noncataract patients.

The present study tested the assertion that there exists exclusively in persons with senile cataracts a serum factor capable of precipitating with gamma crystallin extract from bovine lens. The results of this work do not support this assertion without the imposition of rigid specifications regarding test solution concentrations and observation time. Among the 84 subjects tested, 2 individuals had posterior subcapsular cataracts. The precipitates resulting from incubation with serum from these individuals were similar to those resulting from incubations with serum from senile cataract patients. The present study did not confirm the existence of exclusive serum factors capable of precipitating with gamma crystallin extract in patients with senile cataracts.