Generation and antitumor effects of an engineered and energized fusion protein VL-LDP-AE composed of single-domain antibody and lidamycin

Type IV collagenase plays a pivotal role in invasion, metastasis and angiogenesis of tumor. Single domain antibodies are attractive as tumor-targeting vehicle because of their much smaller size compared with antibody molecules produced by conventional methods. Lidamycin (LDM) is a potent enediyne-containing antitumor antibiotic. In this study an engineered and energized fusion protein VL-LDP-AE composed of lidamycin and VL domain of mAb 3G11 directed against type IV collagenase was prepared using a novel two-step method. First a VL-LDP fusion protein was constructed by DNA recombination. Secondly VL-LDP-AE was obtained by molecular reconstitution. In MTT assay, VL-LDP-AE showed potent cytotoxicity to HT-1080 cells and KB cells with IC ₅₀ values of 8.55×10⁻¹² and 1.70×10⁻¹¹ mol/L, respectively. VL-LDP-AE showed antiangiogenic activity in chick chrorioallantoic membrane (CAM) assay and tube formation assay. In in vivo experiments, VL-LDP-AE was proved to be more effective than free LDM against the growth of subcutaneously transplanted hepatoma 22 in mice. Drugs were given intravenously on day 3 and 10 after tumor transplantation. Compared in terms of maximal tolerated doses, VL-LDP-AE at 0.25 mg/kg suppressed the tumor growth by 89.5%, LDM at 0.05 mg/kg by 69.9%, and mitomycin at 1 mg/kg by 35%. Having a molecular weight of 25.2 kDa, VL-LDP-AE was much smaller than other reported antibody-based drugs. The results suggested that VL-LDP-AE would be a promising candidate for tumor targeting therapy. And the 2-step approach could serve as a new technology platform for making a series of highly potent engineered antibody-based drugs for a variety of cancers.     

Generation and antitumor effects of an engineered and energized fusion protein VL-LDP-AE composed of single-domain antibody and lidamycin

Type IV collagenase plays a pivotal role in invasion, metastasis and angiogenesis of tumor. Single domain antibodies are attractive as tumor-targeting vehicle because of their much smaller size com-pared with antibody molecules produced by conventional methods. Lidamycin (LDM) is a potent enediyne-containing antitumor antibiotic. In this study an engineered and energized fusion protein VL-LDP-AE composed of lidamycin and VL domain of mAb 3G11 directed against type IV collagenase was prepared using a novel two-step method. First a VL-LDP fusion protein was constructed by DNA recombination. Secondly VL-LDP-AE was obtained by molecular reconstitution. In MTT assay, VL-LDP-AE showed potent cytotoxicity to HT-1080 cells and KB cells with IC50 values of 8.55×10-12 and 1.70×10-11 mol/L, respectively. VL-LDP-AE showed antiangiogenic activity in chick chrorioallantoic membrane (CAM) assay and tube formation assay. In in vivo experiments, VL-LDP-AE was proved to be more effective than free LDM against the growth of subcutaneously transplanted hepatoma 22 in mice. Drugs were given intravenously on day 3 and 10 after tumor transplantation. Compared in terms of maximal tolerated doses, VL-LDP-AE at 0.25 mg/kg suppressed the tumor growth by 89.5%, LDM at 0.05 mg/kg by 69.9%, and mitomycin at 1 mg/kg by 35%. Having a molecular weight of 25.2 kDa, VL-LDP-AE was much smaller than other reported antibody-based drugs. The results suggested that VL-LDP-AE would be a promising candidate for tumor targeting therapy. And the 2-step approach could serve as a new technology platform for making a series of highly potent engineered antibody-based drugs for a variety of cancers.     

Phytoextraction of Cd and Zn from agricultural soils by Salix ssp. and intercropping of Salix caprea and Arabidopsis halleri

Contamination of agricultural topsoils with Cd above guideline values is of concern in many countries throughout the world. Extraction of metals from contaminated soils using high-biomass, metal-accumulating Salix sp. has been proposed as a low-cost, gentle remediation strategy, but reasonable phytoextraction rates remain to be demonstrated. In an outdoor pot experiment we assessed the phytoextraction potential for Cd and Zn of four willow species (Salix caprea, S. fragilis, S. × smithiana, S. × dasyclados) and intercropping of S. caprea with the hyperaccumulator Arabidopsis halleri on three moderately contaminated, agricultural soils. Large concentrations of Cd (250 mg kg⁻¹) and Zn (3,300 mg kg⁻¹) were determined in leaves of Salix × smithiana grown on a soil containing 13.4 mg kg⁻¹ Cd and 955 mg kg⁻¹ Zn, resulting in bioaccumulation factors of 27 (Cd) and 3 (Zn). Total removal of up to 20% Cd and 5% Zn after three vegetation periods were shown for Salix × smithiana closely followed by S. caprea, S. fragilis and S. × dasyclados. While total Cd concentrations in soils were reduced by up to 20%, 1 M NH₄NO₃-extractable metal concentrations did not significantly decrease within 3 years. Intercropping of S. caprea and A. halleri partly increased total removal of Zn, but did not enhance total Cd extraction compared to single plantings of S. caprea after two vegetation periods.     

Arsenic hyperaccumulation and localization in the pinnule and stipe tissues of the gold-dust fern (Pityrogramma calomelanos (L.) Link var. austroamericana (Domin) Farw.) using quantitative micro-PIXE spectroscopy

Spatial distribution patterns of arsenic (As) in the tissues of a lesser-known As hyperaccumulating fern Pityrogramma calomelanos (L.) Link var. austroamericana (Domin) Farw. (Pteridaceae) have been studied. Quantitative micro-proton-induced X-ray emission (micro-PIXE) spectroscopy was employed to examine As localization in pinnule and stipe cross-sections of this species. In addition, As hyperaccumulation status of P. calomelanos var. austroamericana was compared with the well-known As hyperaccumulating fern Pteris vittata L. Both species were grown in pots under controlled conditions and exposed to four levels of As (0–500 mg As kg⁻¹) for 20 weeks. Pityrogramma calomelanos var. austroamericana accumulated up to 16 415 mg As kg⁻¹ dry weight (DW), however, phytotoxicity symptoms such as necrotic pinnule tips and margins, appeared in fronds with concentrations >3,008 mg As kg⁻¹ DW. Arsenic was readily translocated to fronds, with concentrations up to 75 times greater in fronds than in roots. Quantitative elemental maps of As generated using micro-PIXE analysis revealed that As concentrations in pinnule cross-sections were higher than in stipe cross-sections with concentrations of 3.7 × 10³ and 1.6 × 10³ mg As kg⁻¹ DW, respectively (as determined by region selection analysis; RSA). In pinnules, RSA revealed variable concentrations of As, however did not resolve a clear pattern of compartmentalization across different anatomical regions. In stipe tissues, As concentrations followed the order vascular bundle > cortex > epidermis (as determined by RSA). Our results show that P. calomelanos var. austroamericana is an As hyperaccumulator and has the potential for use in phytoremediation of soils with low levels (up to 50 mg kg⁻¹) of As contamination.     

Characterization of an ecotype of brake-fern, Pteris vittata, for arsenic tolerance and accumulation in plant biomass

An ecotype of brake fern (Pteris vittata) was assessed for arsenic tolerance and accumulation in its biomass under in vivo and in vitro condition; using soil, and agar-gelled Murashige and Skoog (MS) medium supplemented with different concentrations of arsenic. The plants were raised in soil amended with 100–1000 mg arsenic kg⁻¹ soil, and MS medium was supplemented with 10–300 mg arsenic 1⁻¹ medium using Na₂HAsO₄ · 7H₂O. The spores and haploid gametophytic-prothalli were raised in vitro on MS medium supplemented with arsenic. The field plants showed normal growth and biomass formation in arsenic amended soil, and accumulated 1908–4700 mg arsenic kg⁻¹ dry aerial biomass after 10 weeks of growth. Arsenic toxicity was observed above >200 mg arsenic kg⁻¹ soil. The concentrations of arsenic accumulated in the plant biomass were statistically significant (p < 0.05). Normal plants were developed from spores and gametophyte prothalli on the MS media supplemented with 50–200 mg arsenic 1⁻¹ medium. The in vitro raised plants were tolerant to 300 mg arsenic kg⁻¹ of soil and accumulated up to 3232 mg arsenic kg⁻¹ dry aerial biomass that showed better growth performance, biomass generation and arsenic accumulation in comparison to the field plants. The text was submitted by the authors in English.     

ABE production from corn: a recent economic evaluation

This article details an economic assessment of butanol production from corn using the newly developed hyper-butanol-producing strain of Clostridium beijerinckii BA101. Butanol is produced in batch reactors and recovered by distillation. For a plant with 153,000 metric tons of acetone, butanol, and ethanol (ABE) production capacity, the production equipment cost and total working capital cost is US$33.47×10⁶ and US$110.46×10⁶, respectively. Based on a corn price (C _p) of US$79.23 ton⁻¹ (US$2.01 bushel⁻¹), an ABE yield of 0.42 (g ABE/g glucose) butanol price is projected to be US$0.34 kg⁻¹. An improved yield of 0.50 will reduce this price to US$0.29 kg⁻¹. Assumptions, such as by-product credit for gases and complete conversion of corn steep liquor (CSL) to fermentation by-products, have been taken into consideration. An increased price of corn to US$197.10 ton⁻¹ would result in a butanol price of US$0.47 kg⁻¹. A grass-rooted plant would result in a butanol price of US$0.73 kg⁻¹ (C _p US$79.23 ton⁻¹). In a worst case scenario, the price of butanol would increase to US$1.07 kg⁻¹ (C _p 197.10 ton⁻¹ for a grass-rooted plant and assuming no credit for gases). This is based on the assumption that corn price would not increase to more than US$197.10 ton⁻¹. Journal of Industrial Microbiology & Biotechnology (2001) 27, 292–297. Received 12 September 2000/ Accepted in revised form 12 January 2001     

Natural Occurrence of Mycotoxins in Staple Cereals from Ethiopia

The occurrence of mycotoxins in barley, sorghum, teff (Eragrostis tef) and wheat from Ethiopia has been studied. Samples were analyzed for aflatoxin B₁ (AFB1), ochratoxin A (OTA), deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEN) using high performance liquid chromatography (HPLC) and for fumonisins (FUM) using enzyme linked immunosorbent assay (ELISA). AFB1 and OTA were detected in samples of all the four crops. AFB1 was detected in 8.8% of the 352 samples analyzed at concentrations ranging from trace to 26 μg kg⁻¹. OTA occurred in 24.3% of 321 samples at a mean concentration of 54.1 μg kg⁻¹ and a maximum of 2106 μg kg⁻¹. DON occurred in barley, sorghum and wheat at 40–2340 μg kg⁻¹ with an overall incidence of 48.8% among the 84 mainly ‘suspect’ samples analyzed; NIV was co-analyzed with DON and was detected at 40 μg kg⁻¹ in a wheat sample and at 50, 380, and 490 μg kg⁻¹ in three sorghum samples. FUM and ZEN occurred only in sorghum samples with low frequencies at concentrations reaching 2117 and 32 μg kg⁻¹, respectively. The analytical results indicate higher mycotoxin contamination in sorghum, which could be related to the widespread storage of sorghum grain in underground pits leading to elevated seed moisture contents. This is the first report on the occurrence of OTA in teff.     

Co-expression of Oct-4 and Nestin in human breast cancers

The aim is to investigate the clinical implications of the Oct-4 and Nestin protein in human breast cancers. A total of 346 cases including 26 fresh and 320 paraffin-embedded tumor tissues were selected for characterizing the frequency of CD44⁺CD24⁻ tumor cells by flow cytometry and the differential expression of the stem cell-related genes between CD44⁺CD24⁻ and non-CD44⁺CD24⁻ tumor cells was analyzed by PCR Array and immunofluorescence. In comparison with the non-CD44⁺CD24⁻ tumor cells, the CD44⁺CD24⁻, particularly for those with high percentage of Oct-4⁺ and Nestin⁺, tumor cells had higher tumorigenicity by forming mammospheres in vitro. More importantly, 42 (13.125%) out of 320 tumor tissues were positive for Oct-4 and Nestin staining. Universal analysis and multivariate analysis revealed that the expression of Oct-4 and Nestin was associated significantly with younger age, pathogenic degrees, lymph node metastasis and triple-negative breast cancer independently (P < 0.05) as well as shorter survival (P = 0.001). Oct-4 and Nestin were important regulators of the development of breast cancer, and Oct-4 and Nestin may be used as predictors for the prognosis of breast cancers.     

Characteristics of the phytoplankton community and bioaccumulation of heavy metals during algal blooms in Xiangjiang River (Hunan,China)

The frequency of algal blooms has increased in the mid and downstream reaches of the Xiangjiang River (Hunan, China), one of the most heavily polluted rivers in China. We identified the bloom-forming species in a bloom that occurred mid-late September 2010. In addition, we determined the extent of metal bioaccumulation in the algae and measured the toxicity of the algae using a mouse bioassay. Water samples were collected at upstream (Yongzhou), midstream (Hengyang), and downstream (Zhuzhou, Xiangtan, and Changsha) sites. The dominant species was Aulacoseira granulata, formerly known as Melosira granulata. The heaviest bloom occurred at Xiangtan and Changsha, where the number of A. granulata peaked at 1.3×10⁵ filaments L⁻¹ and chlorophyll a at 0.04 mg L⁻¹. Concentrations of Al, Fe, and Mn were 4.4×10³, 768.4, and 138.7 mg kg⁻¹ dry weight in the phytoplankton. The bioaccumulation factor was 4.0×10⁵, 7.7×10⁵, and 3.2×10³, respectively. The heavy metal Pb had the greatest tendency to bioaccumulate among the highly toxic heavy metals, with a concentration of 19.2 mg kg⁻¹ dry weight and bioaccumulation factor of 9.6×10³. The mouse bioassay suggested the bloom was toxic. The LD₅₀ was 384 mg kg⁻¹ and all surviving mice lost weight during the first 72 h after exposure. Our results demonstrate that blooms of A. granulata in rivers contaminated with heavy metals pose a threat to freshwater ecosystems and human health. Thus, measures should be taken to control eutrophication and heavy metal pollution in such rivers.     

Comparative analysis of the digestive efficiency and nitrogen and energy requirements of the phyllostomid fruit-bat (Artibeus jamaicensis) and the pteropodid fruit-bat (Rousettus aegyptiacus)

Nitrogen (N) and energy (E) requirements of the phyllostomid fruit bat, Artibeus jamaicensis, and the pteropodid fruit bat Rousettus aegyptiacus, were measured in adults that were fed on four experimental diets. Mean daily food intake by A. jamaicensis and R. aegyptiacus ranged from 1.1–1.6 times body mass and 0.8–1.0 times body mass, respectively. Dry matter digestibility and metabolizable E coefficient were high (81.1% and 82.4%, respectively) for A. jamaicensis and (77.5% and 78.0%, respectively) for R. aegyptiacus. Across the four diets, bats maintained constant body mass with mean metabolizable E intakes ranging from 1357.3 kJ · kg^−0.75 · day⁻¹ to 1767.3 kJ · kg^−0.75 · day⁻¹ for A. jamaicensis and 1282.6–1545.2 kJ · kg^−0.75 · day⁻¹ for R. aegyptiacus. Maintenance E costs were high, in the order of 3.6–5.4 times the basal metabolic rate (BMR). It is unlikely that the E intakes that we observed represent a true measure of maintenance E requirements. All evidence seems to indicate that fruit bats are E maximizers, ingesting more E than required and regulating storage by adjusting metabolic output. We suggest that true maintenance E requirements are substantially lower than what we observed. If it follows the eutherian norm of two times the BMR, fruit bats must necessarily over-ingest E on low-N fruit diet. Dietary E content did affect N metabolism of A. jamaicensis. On respective low- and high-E diets, metabolic fecal N were 0.492 mg N · g⁻¹ and 0.756 mg N · g⁻¹ dry matter intake and endogenous urinary N losses were 163.31 mg N · kg^−0.75 · day⁻¹ and 71.54 mg N · kg^−0.75 · day⁻¹. A. jamaicensis required 332.3 mg · kg^−0.75 · day⁻¹ and 885.3 mg · kg^−0.75 · day⁻¹ of total N on high- and low-E diets, respectively, and 213.7 mg · kg^−0.75 · day⁻¹ of truly digestible N to achieve N balance. True N digestibilities were low (29% and 49%) for low- and high-E diets, respectively. For R. aegyptiacus, metabolic fecal N and endogenous urinary N losses were 1.27 mg N · g⁻¹ dry matter intake and 96.0 mg N · kg^−0.75 · day⁻¹, respectively, and bats required 529.8 mg · kg^−0.75 · day⁻¹ (total N) or 284.0 mg · kg^−0.75 · day⁻¹ (truly digestible N). True N digestibility was relatively low (50%). Based on direct comparison, we found no evidence that R. aegyptiacus exhibits a greater degree of specialization in digestive function and N retention than A. jamaicensis. When combined with results from previous studies, our results indicate that all fruit bats appear to be specialized in their ability to retain N when faced with low N diet. Accepted: 24 November 1998     

Inhibitory effect of curcumin on liver injury in a murine model of endotoxemic shock

The effect of curcumin on lipopolysaccharide/d-galactosamine (LPS/GalN)-induced acute shock model of liver injury was examined in mice. The simultaneous administration of LPS (5–20 μg kg⁻¹, i.p.) and GalN (700 mg kg⁻¹, i.p.) markedly increased the serum tumor necrosis factor-α (TNF-α), glutamic oxaloacetic transaminase/glutamic pyruvic transaminase (GOT/GPT), and massive hepatic necrosis and inflammation, leading to 100% lethality. Pre-administration of curcumin (100 mg kg⁻¹, i.p.) 3 h before induction with LPS/GalN imparted a large extent of protection against acute elevation in serum TNF-α and serum GOT/GPT. Hepatic necrosis and lethality caused by LPS/GalN was also greatly reduced by curcumin treatment. The results demonstrated that curcumin could protect mice from LPS/GalN-induced hepatic injury and inflammation through blockading TNF-α production, eventually raising the survival rate of septic-shock-induced mice.     

The development of an isokinetic squat device: reliability and relationship to functional performance

The aims of the present study were: (1) to assess aerobic metabolism in paraplegic (P) athletes (spinal lesion level, T4–L3) by means of peak oxygen uptake (V˙O_2peak) and ventilatory threshold (VT), and (2) to determine the nature of exercise limitation in these athletes by means of cardioventilatory responses at peak exercise. Eight P athletes underwent conventional spirographic measurements and then performed an incremental wheelchair exercise on an adapted treadmill. Ventilatory data were collected every minute using an automated metabolic system: ventilation (l · min⁻¹), oxygen uptake (V˙O₂, l · min⁻¹, ml · min⁻¹ · kg⁻¹), carbon dioxide production (V˙CO₂, ml · min⁻¹), respiratory exchange ratio, breathing frequency and tidal volume. Heart rate (HR, beats · min⁻¹) was collected with the aid of a standard electrocardiogram. V˙O_2peak was determined using conventional criteria. VT was determined by the breakpoint in the V˙CO₂−V˙O₂ relationship, and is expressed as the absolute VT (V˙O₂, ml · min⁻¹ · kg⁻¹) and relative VT (percentage of V˙O_2peak). Spirometric values and cardioventilatory responses at rest and at peak exercise allowed the measurement of ventilatory reserve (VR), heart rate reserve (HRr), heart rate response (HRR), and O₂ pulse (O₂ P). Results showed a V˙O_2peak value of 40.6 (2.5) ml · min⁻¹ · kg⁻¹, an absolute VT detected at 23.1 (1.5) ml · min⁻¹ · kg⁻¹ V˙O₂ and a relative VT at 56.4 (2.2)% V˙O_2peak. HRr [15.8 (3.2) beats · min⁻¹], HRR [48.6 (4.3) beat · l⁻¹], and O₂ P [0.23 (0.02) ml · kg⁻¹ · beat⁻¹] were normal, whereas VR at peak exercise [42.7 (2.4)%] was increased. As wheelchair exercise excluded the use of an able-bodied (AB) control group, we compared our V˙O_2peak and VT results with those for other P subjects and AB controls reported in the literature, and we compared our cardioventilatory responses with those for respiratory and cardiac patients. The low V˙O_2peak values obtained compared with subject values obtained during an arm-crank exercise may be due to a reduced active muscle mass. Absolute VT was somewhat comparable to that of AB subjects, mainly due to the similar muscle mass involved in wheelchair and arm-crank exercise by P and AB subjects, respectively. The increased VR, as reported in patients with chronic heart failure, suggested that P athletes exhibited cardiac limitation at peak exercise, and this contributed to the lower V˙O_2peak measured in these subjects. Accepted: 22 April 1997     

Element Concentrations in Some Species of Seaweeds from La Paz Bay and La Paz Lagoon, South-Western Baja California, Mexico

La Paz Bay and La Paz Lagoon are water bodies of the Gulf of California that are influenced by waste water discharges from the City of La Paz and from activities of the phosphorite mining company “Rofomex”. Because seaweeds concentrate elements from the water and are used as effective indicators of contamination by metals, we investigated their usefulness in this region. Concentrations of certain major elements (Ca, Fe, K and Na) and trace elements (As, Ba, Co, Cr, Cs, Hf, Rb, Sb, Sc, Se, Sr, Ta, Th, U, Zn and Zr) were determined in 12 species of seaweeds from La Paz Bay and La Paz Lagoon using instrumental neutron activation analysis. The contents of trace elements of environmental importance (As, Co, Cr, Fe, Sb, Se and Zn) in all studied samples are within the range of typical levels for a pristine environment not subjected to anthropogenic impact. Somewhat higher concentrations of Cr (81mg kg⁻¹), Hf (4mg kg⁻¹), Rb (48mg kg⁻¹), Sc (6.3mg kg⁻¹), Ta (0.95mg kg⁻¹), Th (6.8mg kg⁻¹), U (33mg kg⁻¹) and Zn (90mg kg⁻¹) were found in the green seaweed species Ulva (formerly Enteromorpha) intestinalis, whereas such elements as As (77mg kg⁻¹), Sb (1.4mg kg⁻¹) and Se (1.8mg kg⁻¹) were mainly concentrated in the species Sargassum sinicola, Codium cuneatum and Padina mexicana respectively. Because of their higher abundance and heterogeneity in elemental composition the seaweeds species Ulva intestinalis and Caulerpa sertularioides seem to be more suitable for further biomonitoring of heavy metal pollution of the coastal waters in this zone.     

Recognition by human Vγ9/Vδ2 T cells of melanoma cells upon fusion with Daudi cells

 Daudi Burkitt’s lymphoma cells, unlike other tumor cell lines, stimulate human T cells coexpressing the variable (V) region genes TCRG-V9 and V TCRD-V2 to proliferate and secrete lymphokines. Hybrids, derived by the fusion of Daudi cells with the human melanoma cell line MZ2-MEL 2.2, retain the morphology of melanoma cells. Unlike the parental melanoma cell line, these Daudi × MZ2-MEL 2.2 hybrids stimulate secretion of tumor necrosis factor (TNF) and granulocyte/macrophage colony stimulating factor (GM-CSF) by CD4-positive Vγ9/Vδ2 T-cell clones. Whereas the stimulator phenotype of Daudi cells behaves as a dominant trait in Daudi × melanoma hybrids, the expression of B-cell differentiation markers is suppressed. Thus, the γ/δ T-cell ligand expressed by Daudi cells behaves as a dominant tumor antigen in Daudi × melanoma hybrids and is unrelated to the differentiated B-cell phenotype. Dominant expression of the Daudi ligand for human Vγ9/Vδ2 T cells in these hybrids may provide a basis for defining the stimulatory principle at the molecular level. Received: 2 May 1996 / Revised: 15 July 1996     

Tumor-derived Fas ligand induces toxicity in lymphoid organs and plays an important role in successful chemotherapy

 Recent studies have suggested that Fas ligand (FasL⁺) tumor cells can induce apoptosis in Fas⁺ T cells. However, the effect of growth of FasL⁺ tumors in vivo, on lymphoid tissues of the host is not clear and therefore was the subject of this investigation. Injection of FasL⁺ LSA tumor caused a significant decrease in cellularity of the thymus and spleen, resulting from marked apoptosis, in syngeneic C57BL/6+/+ (wild-type) but not C57BL/6-lpr/lpr (Fas-deficient) mice. The tumor-induced toxicity resulted from tumor-derived rather than host-derived FasL, inasmuch as LSA tumor growth in C57BL/6-gld/gld (FasL-defective) mice, induced marked apoptosis and toxicity in the thymus and spleen. The LSA tumor growth induced a significant decrease in the percentage of CD4⁺CD8⁺ T cells in the thymus of C57BL/6+/+ mice and an increase in the percentage of CD4⁺, CD8⁺ and CD4⁻CD8⁻ T cells. Of the four subpopulations tested, the CD4⁺CD8⁺ T cells showed maximum apoptosis. The LSA (FasL⁺) but not P815(FasL⁻) tumor cell lysates and culture supernatants induced marked apoptosis in Fas⁺ thymocytes, when tested both in vitro and in vivo. The LSA-tumor-induced apoptosis in vitro was inhibited by antibodies against FasL or by caspase and other inhibitors of apoptosis. Chemotherapy of LSA-tumor-bearing C57BL/6+/+ mice at advanced stages of tumor growth failed to cure the mice, whereas, more than 80% of LSA-tumor-bearing C57BL/6-lpr/lpr mice, similarly treated, survived. Together, the current study demonstrates that FasL produced by LSA tumor cells is functional in vivo and can cause severe toxicity in lymphoid organs of the host. Also, Fas/FasL interactions may play an important role in the successful chemotherapy of FasL-bearing tumor. Received: 31 August 1999 / Accepted: 12 November 1999     

Soy molasses as fermentation substrate for production of butanol using Clostridium beijerinckii BA101

Spray-dried soy molasses (SDSM) contains the sugars dextrose, sucrose, fructose, pinitol, raffinose, verbascose, melibiose, and stachyose. Of the 746 g kg⁻¹ total sugars in SDSM, 434 g kg⁻¹ is fermentable using Clostridium beijerinckii BA101. SDSM was used to produce acetone, butanol, and ethanol (ABE) by C. beijerinckii BA101 in batch cultures. Using 80 g l⁻¹ SDSM, 10.7 g l⁻¹ ABE was produced in P2 medium. Higher concentrations of SDSM resulted in poor solvent production due to the presence of excessive salt and inhibitory components. C. beijerinckii BA101 in SDSM at 80 g l⁻¹ concentration produced 22.8 g l⁻¹ ABE when supplemented with 25.3 g l⁻¹ glucose. SDSM contains 57.4 g kg⁻¹ mineral ash and 2% tri-calcium phosphate. Tri-calcium phosphate up to 43.1 g l⁻¹ was not inhibitory and at a tri-calcium phosphate concentration of 28.8 g l⁻¹, the culture produced more solvents (30.1 g l⁻¹) than the control experiment (23.8 g l⁻¹). In contrast, sodium chloride was a strong inhibitor of C. beijerinckii BA101 cell growth. At a concentration of 10 g l⁻¹ sodium chloride, a maximum cell concentration of 0.6 g l⁻¹ was achieved compared to 1.7 g l⁻¹ in the control experiment. The effects of two salts on specific growth rate constant (μ) and specific rate of ABE production (ν) for C. beijerinckii BA101 were examined. Journal of Industrial Microbiology & Biotechnology (2001) 26, 290–295. Received 20 September 2000/ Accepted in revised form 16 February 2001     

A simple and efficient protocol for cryopreservation of embryogenic calli of the medicinal plant <Emphasis Type="Italic">Anemarrhena asphodeloides</Emphasis> Bunge by vitrification

Somatic embryogenesis and whole plant regeneration was achieved in callus cultures derived from immature zygotic embryos of Prosopis laevigata (Humb. & Bonpl. ex Willd.) M.C. Johnst., recently identified as chromium (Cr), cadmium (Cd), lead (Pb) and nickel (Ni) accumulator. Embryogenic calli were induced on Murashige and Skoog (MS) medium added with a mixture of organic components plus N-6 benzyladenine (BA) (6.62 μM) and 2,4-dichlorophenoxyacetic acid (2,4-D) (2.26 μM) or thidiazuron (4.54–9.08 μM) and indole-3-acetic acid (1.42 μM). Embryogenic calli transferred onto half-strength MS medium without plant growth regulators developed globular embryos, of which 20% matured when treated with 3.75% (w/v) polyethylene glycol (PEG), and of these 50% fully differentiated into plantlet embryo. Regenerated plants were successfully acclimatized (90%), while in vitro seedlings transferred to MS medium containing 0.5 mM Cd, Cr, Ni or Pb, exhibited high heavy metals accumulation (627 mg Cr kg⁻¹, 5,688 mg Cd kg⁻¹, 1,148 mg Ni kg⁻¹, and 3,037 mg Pb kg⁻¹ dry weight) and efficient roots to shoots translocation (42–73%).     

Effect of manipulation of the renin-angiotensin system in control of drinking in juvenile Atlantic salmon (Salmosalar L) in fresh water and after transfer to sea water

Drinking in Atlantic salmon (Salmo salar) juveniles was investigated in fresh water and following transfer to sea water. There was a significant effect of fish size on drinking, and smolts (20–30 g) imbibed about ten times less water than alevins of 0.2–0.3 g. Freshwater smolts drank at a rate of 0.15 ± 0.03 ml · kg⁻¹ · h⁻¹ and administration of doses of 10 or 20 mg · kg⁻¹ of papaverine (stimulator of the renin- angiotensin system RAS) or [Asn¹, Val⁵]-Angiotensin II (0.4 μmol · kg⁻¹) resulted in significant increases in drinking, while administration of the angiotensin converting enzyme inhibitor, enalapril (50 mg · kg⁻¹) had no effect on drinking. Transfer of Atlantic salmon smolts to 1/3, 2/3 and full strength sea water resulted in significant increases in drinking to 1.06 ± 0.12, 1.24 ± 0.0.16 and 3.89 ± 0.28 ml · kg⁻¹ · h⁻¹, respectively. In sea water, stimulation of the endogenous RAS by administration of papaverine (20 mg · kg⁻¹) resulted in a 20% increase in drinking, while administration of enalapril to doses of 50 and 200 mg · kg⁻¹ lowered drinking to 1.99 ± 0.48 and 0.32 ± 0.06 ml · kg⁻¹ · h⁻¹, respectively. All treatments were without effect on blood plasma levels of Na⁺ and Cl⁻ in fresh water, while in sea water smolts both stimulation and inhibition of drinking resulted in hemoconcentration of Na⁺ and Cl⁻. The role of the renin angiotensin system in control of drinking and hydromineral balance in Atlantic salmon is discussed. Accepted: 27 February 1997     

Body and scale growth of wild Atlantic salmon smolts during seaward emigration

The relationship between scale and body growth for emigrating Atlantic salmon, Salmo salar, smolts was previously not understood and therefore was examined in this study using mark-recapture techniques. The size of smolts at time of recapture was significantly greater than when marked (P = 0.0002). The growth in length of smolts emigrating 5 km over an average of 20 days was 7.7 ± 6.1 mm per day. Instantaneous somatic growth (G _body) ranged from 7.0 × 10⁻⁴ to 5.1 × 10⁻³ (mean = 2.7 × 10⁻³ ± 1.3 × 10⁻³). The mean number of plus growth circuli present per scale was significantly greater for smolts when recaptured compared to when marked (P = 0.0014). The instantaneous growth rate of scales (G _scale) ranged from 1.4 × 10⁻³ to 11.5 × 10⁻³ (mean = 6.6 × 10⁻³ ± 4.3 × 10⁻³). The relationship between body size and scale radius showed positive allometry rather than isometry. The relationship of G _scale with G _body showed positive allometry indicating that scales grew at a slightly faster rate than the body during the emigratory period.     

Detrimental effect of increasing sugar concentrations on ethanol production from maize or decorticated sorghum mashes fermented with <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> or <Emphasis Type="Italic">Zymomonas mobilis</Emphasis>

The efficiency of ethanol fermentation, as affected by grain source (maize and decorticated red sorghum), total sugar concentration (13 or 20° Plato) and type of microorganism (Saccharomyces cerevisiae or Zymomonas mobilis) was studied. Maize mashes yielded 0.32 l ethanol kg⁻¹ ground grain whereas mashes prepared with decorticated red sorghum produced 0.28 l ethanol kg⁻¹. Both microorganisms yielded similar amounts of ethanol. However, high-gravity mashes (20° Plato) yielded lower amounts of ethanol compared to counterparts adjusted to 13° Plato (0.28 vs. 0.22 l ethanol kg⁻¹ ground grains). In decorticated sorghum mashes adjusted to 20° P, Z. mobilis produced 40 ml kg⁻¹ more ethanol compared to S. cerevisiae. In addition, Z. mobilis had a lower dependency on nitrogenous compounds.