An engineered Escherichia coli having a high intracellular level of ATP and enhanced recombinant protein production

Artificial amplification of gluconeogenic phosphoenolpyruvate carboxykinase (PCK) under glycolytic conditions enables Escherichia coli to maintain a greater intracellular ATP concentration during its growth phase. To demonstrate the biotechnological benefit of E. coli harboring a high intracellular ATP concentration, we compared the recombinant protein synthesis of a soluble protein (enhanced green fluorescence protein, GFP) with that of a secretory protein (alkaline protease, AP), under control of the T7 promoter in E. coli BL21(DE3) overexpressing PCK. According to the batch fermentations, the strain overexpressing PCK produced more GFP and AP with a lower increase in biomass than the control strain. In a chemostat culture (D = 0.7 h⁻¹), the GFP production in the PCK overexpressing strain was 99.0 ± 4.31 mg/g cell, with a biomass of 0.22 g/L, while that of the control strain was 53.5 ± 3.07 mg/g cell, with a biomass of 0.35 g/L. These results indicate that the PCK overexpressing E. coli strain harboring high intracellular levels of ATP can be useful as a protein-synthesizing host. The potential uses of the strain and associated rationale are discussed.     

Comparison of separate hydrolysis and fermentation and simultaneous saccharification and fermentation processes for ethanol production from wheat straw by recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> strain FBR5

Ethanol production by recombinant Escherichia coli strain FBR5 from dilute acid pretreated wheat straw (WS) by separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) was studied. The yield of total sugars from dilute acid (0.5% H₂SO₄) pretreated (160 °C, 10 min) and enzymatically saccharified (pH 5.0, 45 °C, 72 h) WS (86 g/l) was 50.0 ± 1.4 g/l. The hydrolyzate contained 1,184 ± 19 mg furfural and 161 ± 1 mg hydroxymethyl furfural per liter. The recombinant E. coli FBR5 could not grow at all at pH controlled at 4.5 to 6.5 in the non-abated wheat straw hydrolyzate (WSH) at 35 °C. However, it produced 21.9 ± 0.3 g ethanol from non-abated WSH (total sugars, 44.1 ± 0.4 g/l) in 90 h including the lag time of 24 h at controlled pH 7.0 and 35 °C. The bioabatement of WS was performed by growing Coniochaeta ligniaria NRRL 30616 in the liquid portion of the pretreated WS aerobically at pH 6.5 and 30 °C for 15 h. The bacterium produced 21.6 ± 0.5 g ethanol per liter in 40 h from the bioabated enzymatically saccharified WSH (total sugars, 44.1 ± 0.4 g) at pH 6.0. It produced 24.9 ± 0.3 g ethanol in 96 h and 26.7 ± 0.0 g ethanol in 72 h per liter from bioabated WSH by batch SSF and fed-batch SSF, respectively. SSF offered a distinct advantage over SHF with respect to reducing total time required to produce ethanol from the bioabated WS. Also, fed-batch SSF performed better than the batch SSF with respect to shortening the time requirement and increase in ethanol yield.     

Heterologous expression and characterization of Choline Oxidase from the soil bacterium Arthrobacter nicotianae

In the course of a microbial screening of soil samples for new oxidases, different enrichment strategies were carried out. With choline as the only carbon source, a microorganism was isolated and identified as Arthrobacter nicotianae. From this strain, a gene coding for a choline oxidase was isolated from chromosomal DNA. This gene named codA was cloned in Escherichia coli BL21-Gold and the protein (An_CodA) heterologously overexpressed as a soluble intracellular protein of 59.1 kDa. Basic biochemical characterization of purified protein revealed a pH optimum of 7.4 and activity over a broad temperature range (15–70 °C). Specific activities were determined toward choline chloride (4.70 ± 0.12 U/mg) and the synthetic analogs bis(2-hydroxyethyl)-dimethylammonium chloride (0.05 ± 0.45 × 10^–2 U/mg) and tris-(2-hydroxyethyl)-methylammonium methylsulfate (0.01 ± 0.12 × 10^–2 U/mg). With increasing number of oxidizable groups, a significant decrease in activity was noted. Determination of kinetic parameters in atmorspheric oxygen resulted in K _M = 1.51 ± 0.09 mM and V _max = 42.73 ± 0.42 mU/min for choline chloride and K _M = 4.77 ± 0.76 mM and V _max = 48.40 ± 2.88 mU/min for the reaction intermediate betaine aldehyde respectively. Nuclear magnetic resonance spectroscopic analysis of the products formed during the enzyme reaction with choline chloride showed that in vitro the intermediate betaine aldehyde exists also free in solution.     

Large-scale expression,purification, and glucose uptake activity of recombinant human FGF21 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

As a novel important regulator of glucose and lipid metabolism homeostasis, human fibroblast growth factor 21 (hFGF21) has become a potential drug candidate for the treatment of metabolic diseases including obesity, and type 2 diabetes, as well as non-alcoholic fatty liver disease. To improve the production of recombinant hFGF21 to meet the increasing demand in clinical applications, an artificial gene encoding its mature peptide sequence was constructed, cloned into vector pET-3c and then expressed in Escherichia coli Origami B (DE3). Under optimal conditions in a 50-L fermentor, the average bacterial yield and the soluble expression level of recombinant hFGF21 of six batches attained 1750 ± 185 g and 32 ± 1.5%, respectively. The target protein was purified by the combination of nickel-nitrilotriacetic acid affinity chromatography and Sephadex S-100 resin. 5% (w/v) trehalose solution was able to prevent rhFGF21 from degradation effectively. The purity of rhFGF21 was higher than 97%, and the yield was 213 ± 17 mg/L. The preliminary biochemical characterization of rhFGF21 was confirmed using Western blot and peptide map finger analysis. Based on the glucose oxidase–peroxidase assay, the EC50 of glucose uptake activity of the purified rhFGF21 was 22.1 nM.     

Urine Iodine Levels in Preeclamptic and Normal Pregnant Women

The aim of this study was to investigate the urine iodine concentration in women with severe preeclampsia and in healthy women in Erzurum, Turkey. Urine specimens were obtained from 40 severe preeclampsia and 18 healthy pregnant women. Urinary iodine levels were determined by the Foss method based on the Sandell–Kolthoff reaction. The urinary iodine level for women with severe preeclampsia was 4.25 ± 2.7 μg/dL, lower than 20.89 ± 6.4 μg/dL of urinary iodine for healthy pregnant women (p < 0.001). Blood magnesium concentration was found to be 1.63 ± 0.05 mg/dL for women with severe preeclampsia, which is lower than that of healthy pregnant women (1.87 ± 0.05 mg/dL; p < 0.001). There was a positive correlation between urinary iodine level and blood magnesium level in pregnant women with preeclampsia (Pearson correlation coefficient = 0.43; p < 0.01). However, there was no correlation between urinary iodine level and blood magnesium level in healthy pregnant women. There was no difference in thyroid hormone levels (T4, TSH, FT4) between women with severe preeclampsia and healthy pregnant women. However, there was a difference in T3 thyroid hormone levels between women with severe preeclampsia (1.86 ± 0.4 μg/dL) and healthy pregnant women (1.45 ± 0.3 μg/dL; p < 0.001). There was also a difference in FT3 between women with severe preeclampsia (2.77 ± 0.4 pg/mL) and healthy pregnant women (2.41 ± 0.5 μg/dL; p < 0.01). Urinary iodine excretion is currently the most convenient laboratory marker of iodine deficiency. The method is useful for the rapid and low-cost assessment of iodine deficiency. Our results suggested that urinary iodine concentration might be a useful marker for prediagnosing preeclamptic women. In addition, iodine supplementation may also be considered for preeclamptic therapy.     

Optimization of the solubilization and renaturation of fish growth hormone produced by Escherichia coli

Growth hormone (GH) enhances the growth rate of aquacultured fish and shellfish, but it is difficult to extract native GH from fish pituitary glands. However, fish recombinant GH (rGH) can be efficiently synthesized by Escherichia coli cells, although it exists in denatured form in inclusion bodies (IB). We studied the solubilization of IB and the renaturation of rGH to help facilitate the production of a large amount of biologically active rGH. A 100-ml sample of rGH-producing E. coli produced 73.43 ± 5.47 mg IB (dry weight, n = 3) after 20 h induction by 1 mM isopropyl β-o-thiogalactopyranoside. Interestingly, if the bacteria were induced by 0.1 mM β-lactose, 95.3 ± 3.43 mg of IB was obtained. The optimal conditions for denaturation and renaturation of rGH were when IB were solubilized in 6 M guanidine hydrochloride and then dialysed against pH 10 dialysis buffer (50 mM ammonium bicarbonate and 2 mM EDTA) containing 100 mM l-arginine, 2 mM oxidized glutathione and 2 mM reduced glutathione for 24 h at 4 °C in a volume ratio of 3 to 500. At least 20% of the denaturated rGH in IB was renatured. Juvenile black sea bream injected with 0.05 μg/g resultant rGH once every 2 weeks exhibited significant increases (P < 0.05) in weight gain (84%) relative to fish in the control group over a 16-week period. This process is an economical and effective way to obtain an active form of rGH biosynthesized by a prokaryotic system. Received: 18 November 1996 / Received revision: 5 March 1997 / Accepted: 7 March 1997     

High-level production of a kringle domain variant by high-cell-density cultivation of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Human kringle domains (KDs) are ubiquitously expressed binding modulators that fold into seven flexible loops and it has been previously demonstrated that KDs can be engineered toward target-specific binding proteins as a non-antibody protein scaffold. Here, we report a method for efficient expression of a KD derivative (KD548)—a promising anti-cancer agent—by high-cell-density culture of Escherichia coli at a preparative scale production. The correct folding of KD548 requires three disulfide bonds. Nevertheless, cytoplasmic expression of KD548 in E. coli led to good yields of highly soluble proteins with high activity. For efficient expression, four sets of expression systems consisting of different promoters (lac or T7) and fusion tags (His or FLAG) were examined. Of these, the expression system using a combination of the T7 promoter with the FLAG tag resulted in the highest production in shake flask cultivation as well as in high-cell-density cultivation performed in a 6.6-L jar bioreactor. When protein expression was induced at high-cell density (optical density [OD] = 100) and when complex feeding solutions were supplemented, cell density (maximum OD = 184) and production yield (∼5.4 g/L) were significantly enhanced to values that were much higher than those found previously with Pichia cultivation (<8 mg/L).     

Influence of Therapy with Metformin on the Concentration of Certain Divalent Cations in Patients with Non-insulin-Dependent Diabetes Mellitus

Research was performed on a group of 30 patients with non-insulin-dependent diabetes mellitus (NIDDM), who never received antidiabetic medication before, and on a group of 17 healthy adults. The patients were administered treatment with metformin, 1,000 mg/day. Plasmatic and urinary concentration of magnesium have been measured, copper and zinc along with the concentrations of glucose, HDL, LDL, cholesterol, tryglicerides, HbA1c, and total erythrocyte magnesium, in advance and after 3 months of treatment. Data showed significant differences in the NIDDM group vs the control group: for plasma magnesium—1.95 ± 0.19 vs 2.20 ± 0.18 mg/dl, p < 0.001; urine magnesium—237.28 ± 34.51 vs 126.25 ± 38.22 mg/24 h, p < 0.001; erythrocyte magnesium—5.09 ± 0.63 vs 6.38 ± 0.75 mg/dl, p < 0.001; plasma zinc—67.56 ± 6.21 vs 98.41 ± 20.47 μg/dl, p < 0.001; urine zinc—1,347.54 ± 158.24 vs 851.65 ± 209.75 μg/24 h, p < 0.001; plasma copper—111.91 ± 20.98 vs 96.33 ± 8.56 μg/dl, p < 0.001; and urine copper—51.70 ± 23.79 vs 36.00 ± 11.70 μg/24 h, p < 0.05. Treatment with metformin for 3 months modified significant erythrocyte magnesium—5.75 ± 0.61 vs 5.09 ± 0.63 mg/dl, p < 0.001 and urine magnesium—198.27 ± 27.07 vs 237.28 ± 34.51 mg/24 h, p < 0.001, whereas it did not modify significant the plasmatic and urinary concentration of the other cations. The erythrocyte magnesium concentration was inversely correlated with HbA1c (r = −0.438, p = 0.015). The plasma level of copper was positively correlated with HbA1c (r = 0.517, p < 0.003), tryglicerides (r = 0.534, p < 0.003), and cholesterol (r = 0.440, p < 0.05), and the plasma level of zinc was inversely correlated with glycemia (r = −0.399, p = 0.029). Our data show a significant action of metformin therapy, by increasing the total intraerythrocyte magnesium concentration and decreasing the urinary magnesium elimination, positively correlated with the decrease of glycemia and HbA1c in NIDDM patients.     

Aortic Elasticity is Impaired in Patients with Endemic Fluorosis

Sixty-three patients with endemic fluorosis (36 males/27 females; mean age 33.9 ± 8.6 years) and 45 age-, sex-, and body mass index-matched healthy controls (30 males/15 females; mean age 32.7 ± 8.8 years) were included in this study. Aortic stiffness indices, aortic strain (AS), aortic distensibility (AD), and aortic strain index (ASI) were calculated from the aortic diameters measured by echocardiography and blood pressure obtained by sphygmomanometry. The urine fluoride levels of fluorosis patients were significantly higher than control subjects as expected (1.9 ± 0.1 mg/l vs. 0.4 ± 0.1 mg/l, respectively; P < 0.001). AS and AD were significantly lower in fluorosis patients than in the controls (for AS 5.3 ± 3.6 vs. 8.0 ± 3.4%; P < 0.001 and for AD 0.2 ± 0.1 vs. 0.3 ± 0.1 cm² dyn⁻¹ 10⁻³; P < 0.001, respectively). In contrast, signicantly higher ASI was observed in fluorosis patients than in the controls (3.4 ± 0.6 vs. 3.0 ± 0.4; P < 0.001, respectively). The results of our study demonstrate that elastic properties of ascending aorta are impaired in patients with endemic fluorosis.     

Effective expression of soluble aglycosylated recombinant human Fcγ receptor I by low translational efficiency in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Human FcγRI (CD64) is an integral membrane glycoprotein functioning as a high-affinity receptor binding to monomeric IgG. In this study, the extracellular region of FcγRI, which is the actual part that interacts with IgG, was expressed as aglycosylated recombinant human FcγRI (rhFcγRI) in Escherichia coli. The soluble form of aglycosylated rhFcγRI was expressed in the periplasm of E. coli. The production of soluble aglycosylated rhFcγRI was increased by low induction levels. Furthermore, this production was increased by low translational efficiency, controlled by modification of the putative region between the ribosome binding site and initiation codon of rhFcγRI fusing signal peptide (MalE, PelB, or TorT) of the expression vector. By the optimization of induction and translational efficiency, the production of soluble aglycosylated rhFcγRI was up to approximately 0.8 mg/l of culture medium. Surface plasmon resonance analysis revealed that the binding affinities of aglycosylated rhFcγRI for human IgG1 (equilibrium dissociation constant K _D = [1.7 ± 0.2] × 10⁻¹⁰ M) and IgG3 (K _D = [1.1 ± 0.2] × 10⁻¹⁰ M) were similar to those of glycosylated rhFcγRI.     

Biotechnological production of <Emphasis Type="SmallCaps">l</Emphasis>-ribose from <Emphasis Type="SmallCaps">l</Emphasis>-arabinose

l-Ribose is a rare and expensive sugar that can be used as a precursor for the production of l-nucleoside analogues, which are used as antiviral drugs. In this work, we describe a novel way of producing l-ribose from the readily available raw material l-arabinose. This was achieved by introducing l-ribose isomerase activity into l-ribulokinase-deficient Escherichia coli UP1110 and Lactobacillus plantarum BPT197 strains. The process for l-ribose production by resting cells was investigated. The initial l-ribose production rates at 39°C and pH 8 were 0.46 ± 0.01 g g⁻¹ h⁻¹ (1.84 ± 0.03 g l⁻¹ h⁻¹) and 0.27 ± 0.01 g g⁻¹ h⁻¹ (1.91 ± 0.1 g l⁻¹ h⁻¹) for E. coli and for L. plantarum, respectively. Conversions were around 20% at their highest in the experiments. Also partially purified protein precipitates having both l-arabinose isomerase and l-ribose isomerase activity were successfully used for converting l-arabinose to l-ribose.     

Beneficial effects of banana (<Emphasis Type="Italic">Musa</Emphasis> sp. var. elakki bale) flower and pseudostem on hyperglycemia and advanced glycation end-products (AGEs) in streptozotocin-induced diabetic rats

Diabetes is a chronic health problem and major cause of death in most of the countries. Diet management plays an important role in controlling diabetes and its complications along with insulin and drugs. We have examined the effect of banana (Musa sp. var. elakki bale) flower and pseudostem on hyperglycemia and advanced glycation end-products (AGEs) in streptozotocin-induced diabetic rats. Our results indicated that banana flower and pseudostem have low glycemic index and have a high content of dietary fiber and antioxidants. Diabetic symptoms like hyperglycemia, polyuria, polyphagia, polydipsia, urine sugar, and body weight were ameliorated in banana flower- and pseudostem-treated rats. Increased glomerular filtration rate in the diabetic group (5.1 ± 0.22 ml/min) was decreased in banana flower-fed (2.5 ± 0.37 ml/min) and pseudostem-fed (3.0 ± 0.45 ml/min) groups and were significant at P < 0.001 and P < 0.01, respectively. Fructosamine and AGEs formed during diabetes were inhibited in treated groups when compared with the diabetic group. The diabetic group showed 11.5 ± 0.64 μg of AGEs/mg protein in kidney, whereas, in banana flower- and pseudostem-fed groups, it was reduced to 9.21 ± 0.32 and 9.29 ± 0.24 μg/mg protein, respectively, and were significant at P < 0.01. These findings suggest that banana flower and pseudostem have anti-diabetic and anti-AGEs properties and are beneficial as food supplements for diabetics.     

Serum Copper,Zinc, and Magnesium Levels in Patients with Chronic Fluorosis

Although there are many studies on effect of fluoride on trace elements in experimental animals, few studies exist on serum trace elements levels in patients with endemic fluorosis. We aimed to determine the serum levels of trace elements including serum copper (Cu), zinc (Zn), and serum levels of minerals including calcium (Ca), phosphorus (P), magnesium (Mg), sodium (Na), potassium (K) in patients with endemic fluorosis. The study group consisted of 30 patients with endemic fluorosis (17 females, 13 males, mean age 33.53 ± 9.85 years). An age, gender, and body mass index matched 30 healthy volunteers comprised control group (21 females, ten males with a mean age 33.93 ± 7.39 years). Urine fluoride levels of chronic fluorosis patients were significantly higher than that of control subjects as expected (1.92 ± 0.10 mg/l vs. 0.41 ± 0.09 mg/l, respectively; P < 0.001). Serum Cu levels (89.14 ± 16.77 μg/dL vs. 102.69 ± 25.04 μg/dL, respectively, P = 0.017), serum Zn levels (77.98 ± 20.58 μg/dL vs. 94.57 ± 35.87μg/dL, respectively, P = 0.032), and serum Mg levels (1.92 ± 0.18 mg/dL vs. 2.07 ± 0.31 mg/dL, respectively, p = 0.022) was significantly lower in chronic fluorosis patients than in controls. There were no statistically significant differences between the fluorosis group and control group with respect to serum levels of Na, K, Ca, and P. We concluded that chronic fluorosis is associated with reduced serum levels of Cu, Zn, and Mg.     

Selenium Levels in First-Degree Relatives of Diabetic Patients

The present study was conducted to evaluate the serum selenium levels in first-degree relatives of diabetic patients (FDR) according to controls. Insulin resistance, serum lipid levels, inflammation markers, and blood pressure were also studied in these patients. Serum levels of selenium in FDR were significantly lower than control group (74.65 ± 5.9 vs 88.7 ± 8.7 μg/dl, p < 0.0001). HsCRP, HOMA-IR, insulin, homocysteine levels were significantly higher in FDR according to the control group (1.32 ± 0.9 vs 0.63 ± 0.4 mg/dL, p < 0.0001; 2.07 ± 0.84 vs 1.51 ± 0.69, p < 0.0001; 9.26 ± 3.8 vs 6.8 ± 2.98 μU/MI, p < 0.0001; 15.7 ± 7.4 vs 11.5 ± 5.1 μmol/L, p < 0.0001, respectively). There was significant correlation between selenium levels and hsCRP (r = − 0.450, p < 0.0001). There was also weak significant correlation also between HOMA-IR and selenium levels (r = −0.227, p = 0.003). There was a correlation between systolic blood pressure and BMI (r = 0.365, p < 0.0001). But there was no correlation between selenium levels and blood pressure or other parameters. HsCRP, HOMA-IR, homocysteine levels in individuals with selenium levels < 80 μg/L (n = 78) was significantly higher than hsCRP HOMA-IR, homocysteine levels in individuals with selenium levels ≥ 80 (n = 91; 1.23 ± 0.98 vs 0.81 ± 0.76 mg/dL, p < 0.003; 1.99 ± 0.88 vs 1.64 ± 0.74, p < 0.005; 15.0 ± 7.6 vs 12.9 ± 5.7 μmol/L, p < 0.049, respectively). Selenium deficiency may contribute to cardiovascular disease risk in FDR.     

Haematological and biochemical studies in tigers (<Emphasis Type="Italic">Panthera tigris tigris</Emphasis>)

Haematological and biochemical studies were conducted on 12 clinically healthy tigers of Central India. The range and mean (with one standard deviation), respectively for the parameters examined were: red blood cells, 4.66 to 9.15, 7.9 ± 1.42, 10⁶/μl; haemoglobin, 7.8 to 13.8, 12.8 ± 1.65 g/dl; packed cell volume, 36 to 45, 38 ± 2.54; icterus index, 2 to 5, 2 ± 1.51 U; erythrocyte sedimentation rate, 14 to 26, 21 ± 4.21 mm at 1 h; white blood cells, 6.2 to 11.05, 8.5 ± 1.49, 10³/μl; neutrophils, 57 to 75, 60 ± 5.08%; lymphocytes, 18 to 35, 30 ± 4.56%; monocytes, 2 to 6, 5 ± 1.21%; eosinophils, 2 to 6, 4 ± 1.3; basophils, 0 to 4, 1 ± 1.21; plasma albumin, 2.1 to 4.6, 3.5 ± 0.99 mg/dl; total protein, 3.7 to 8.7, 6.4 ± 1.88 mg/dl; total bilirubin, 0.4 to 3.2, 1.9 ± 1.21 mg/dl; creatinine, 1.6 to 4.6, 2.90 ± 1.03 mg/dl; blood urea nitrogen, 6.5 to 48.2, 27.90 ± 13.77 mg/dl; glutamic pyruvic transaminase, 21.2 to 109.0, 67.88 ± 27.84 IU/L and glutamic oxaloacetate transaminase, 14.4 to 84, 57.96 ± 17.27 IU/L; index conspicuous erythrocyte sedimentation rate; absence of reticulocytes and predominance of neutrophils.     

Effects of deoxynivalenol (DON) in the lactation diet on the feed intake and fertility of sows

A diet contaminated with 2.8 mg deoxynivalenol (DON)/kg was fed at 6 kg per day to 32 mycotoxin-exposed pluriparous sows (M) during lactation. The 31 control sows (C) received 6 kg of an uncontaminated diet. Although more contaminated diet was refused (P = 0.05), DON exposure had no effect (P > 0.1) on body weight loss of the sows during lactation (M: 27.9 ± 12.3 kg; C: 29.7 ± 10.2 kg), the number of weaned piglets (M: 9.8 ± 1.4; C: 9.7 ± 1.6) and their daily weight gain (M: 266 ± 70 g; C: 272 ± 64 g). Several sows were culled after weaning for reasons unrelated to the experiment. Compared with the remaining 21 C sows, the remaining 26 M sows had an identical interval between weaning and the next farrowing (M: 120 ± 1 days; C: 120 ± 1 days) and a similar litter size (M: 14.5 ± 2.7; C: 14.9 ± 3.0; P > 0.10). The daily intake of 17 mg DON during lactation thus did not affect the reproductive performance of the sows.     

Effect of Different Iodine Nutrition on Cerebellum Pcp-2 in Rat Offspring During Lactation

The thyroid functions of breastfed infants, as well as (indirectly) the development of their central nervous system, are dependent on the iodine status of the lactating mother. Purkinje cell protein-2 is a cell-specific marker of the cerebellum Purkinje cell and is a suitable indicator for observing the postnatal development of the cerebellum after birth. We measured the Purkinje cell protein-2 mRNA and protein levels in the rat cerebellum in the critical postnatal (14 days after birth) and maturation periods (28 days after birth) to determine the effect of different nutritional iodine levels on cerebellum growth in the offspring during lactation. We found that severe iodine deficiency resulted in thyroid dysfunction in lactating rats and their offspring on both 14 and 28 days, showing maternal total T₄ 16.7 ± 12.0 vs 36.4 ± 15.0, P < 0.05 (14 days) and 22.6 ± 18.7 vs 53.4 ± 9.4, P < 0.01 (28 days), and neonatal total T₄ 10.6 ± 2.3 vs 16.4 ± 4.7, P < 0.01(14 days) and 12.8 ± 2.9 vs 16.7 ± 3.4, P < 0.05 (28 days), respectively. The Purkinje cell protein-2 mRNA and its protein levels in offspring rats were significantly reduced that showed Purkinje cell protein-2 mRNA 1.12 ± 0.04 vs 2.25 ± 0.53, P < 0.05 (14 days) and 1.74 ± 0.94 vs 8.69 ± 2.71, P < 0.01 (28 days). However, mild iodine deficiency and excessive iodine maintained almost normal thyroid function in maternal and neonatal rats and normal Purkinje cell protein-2 mRNA and protein levels in offspring’s cerebellum. We conclude that severe iodine deficiency could significantly reduce Purkinje cell protein-2 mRNA and its protein levels, indicating that the cerebellum development was retarded, but mild iodine deficiency and excessive iodine could maintain them at an approximately normal level by the mother’s and offspring’s compensations, especially by the mother’s mammary glands.     

Mercury and cadmium concentrations in the tissues of three species of southern albatrosses

Cadmium and mercury concentrations were measured in the tissues of 64 individual albatrosses [23 wandering albatrosses (Diomedea exulans), 9 royal albatrosses (Diomedea epomophora) and 32 shy albatrosses (Thalassarche cauta)] which were killed as by-catch in longline fishing activities between 1991 and 1994. Mercury concentrations were also determined for 33 shy albatross eggs (excluding shells). The birds were all sexed and assigned to one of two age classes (immature and adult). The three species exhibited differences both in overall concentrations of cadmium and mercury, and also in the pattern of accumulation of metals with age and sex. Wandering albatrosses exhibited the highest mercury concentrations with a mean concentration in adult liver samples of 920.0 ± 794.1 μg g⁻¹ dry weight. Shy albatrosses had the lowest mercury concentrations with mean concentrations in adult livers of 36.3 ± 21.4 mg g⁻¹ dry weight. The highest mercury concentration was 1800 μg g⁻¹ for an adult female wandering albatross. Cadmium concentrations were less variable, with adult royal albatrosses having the highest average concentrations (180.0 ± 165.0 in adult kidneys) and adult shy albatrosses the lowest (40.1 ± 20.0 in adult kidney). The highest individual cadmium concentration was 287 μg g⁻¹ for a juvenile wandering albatross. There was no evidence of increased accumulation of cadmium with age in any of the species, but wandering albatrosses showed higher mercury concentrations in adults than juveniles. Female wandering albatrosses also had significantly higher mercury concentrations than males. The mercury contents of the shy albatross eggs were very low, with a maximum concentration of 5.4 μg g⁻¹. The results of this study are consistent with the findings of previous work on albatrosses and support the notion that the life-history strategy of these species (i.e. long-lived with low reproductive output) may be an important determinant in the concentrations of some metals found in their tissues. Accepted: 15 February 1999     

Micropropagation of <Emphasis Type="Italic">Vitex agnus-castus</Emphasis>, (Verbenaceae)—a valuable medicinal plant

This study describes in vitro shoot induction and plant regeneration from a mature apical meristem and nodal explants of the endangered medicinal shrub Vitex agnus-castus. Multiple shoots were induced directly from the axis of nodal and apical meristem explants on Murashige and Skoog (MS) medium containing 3% sucrose and different concentrations (1.0, 1.5, 2.0, and 2.5 mg/l) of 6-benzyl aminopurine (BAP) in combination with Kinetin (Kin) and α-naphthalene acetic acid (NAA), both at 0.1 mg/l. BAP and Kinetin were used as supplements to MS basal medium, either individually or in combination with auxins. The optimal concentration of BAP for inducing bud break was found to be 2.0 mg/l when Kinetin was at 0.1 mg/l. Regeneration frequency was highest for both apical meristem and nodal explants (94.5% and 90.3%, respectively) when explants were cultured on MS medium supplemented with BAP (2.0 mg/l) and Kin (0.1 mg/l). A maximum of 7.7 ± 0.4 and 6.7 ± 0.2 shoots were obtained per explant for apical meristem and nodal explants, respectively. Regenerated shoots, transferred to MS medium supplemented with either 1.0 or 1.5 mg/l BAP combined with 0.1 mg/l GA₃, showed maximum elongation of 6.7 ± 0.4 and 6.0 ± 1.3 cm in apical meristem and nodal explants, respectively. In vitro regenerated shoots transferred to half-strength MS medium supplemented with 0.1 mg/l IBA induced 90.4% of the shoots to form roots after 30–35 d of culture. Up to 80% of the regenerated shoots were successfully established in soil in the greenhouse.     

Comparison of metabolite levels in callus of <Emphasis Type="Italic">Tecoma stans</Emphasis> (L.) Juss. ex Kunth. cultured in photoperiod and darkness

Tecoma stans is a tropical plant from the Americas. Antioxidant activity and both phenolic compound and flavonoid total content were determined for callus tissue of T. stans cultured in either a set photoperiod or in darkness. Callus lines from three explant types (hypocotyls, stem, and leaf) were established on B5 culture medium supplemented with 0.5 μM 2,4-D and 5.0 μM kinetin. While leaf-derived callus grew slower under a 16-h photoperiod (specific growth rate, μ = 0.179 d⁻¹, t _D = 3.9 d) than in darkness (μ = 0.236 d⁻¹, t _D = 2.9 d), it accumulated the highest amount (p < 0.05) of both phenolics (86.6 ± 0.01 mg gallic acid equivalents/g) and flavonoids (339.6 ± 0.06 mg catechin equivalents/g). Similarly, antioxidant activity was significantly higher (p < 0.05) when callus was cultured in period light than when grown in extended darkness. Antioxidant activity measured with a 2,20-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS)-based assay was 350.5 ± 15.8 mmol Trolox/g extract for callus cultured under a defined photoperiod compared to 129.1 ± 7.5 mmol Trolox/g extract from callus cultured in darkness. Content of phenolic compounds and flavonoids was in agreement with a better antioxidant power (EC₅₀ = 450 μg extract/mg 1,1-diphenyl-2-picrylhydrazyl) and antiradical efficiency. Results of the present study show that calli of T. stans are a source of compounds with antioxidant activity that is favored by culture under a set photoperiod.