High-level expression of a functional humanized anti-CTLA4 single-chain variable fragment antibody in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The administration of antibodies against the cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is a promising approach in the upregulation of immune responses in many cancers and infectious diseases. The single-chain variable fragment of antibody against CTLA4 is also useful in developing immunotoxins that might be used in the treatment of cancer, transplant rejection, and autoimmune diseases. Here, we report the production of a soluble and functional scFv antibody against CTLA4 by using Pichia pastoris as the expression system. The gene encoding scFv hS83 with an additional 6His-tag at the 5’-end was inserted into the expression vector pPIC9K. Then, the transformants were double-screened on plates containing 0.25 mg/mL and 1.5 mg/mL of neomycin G418 and many clones with different levels of G418-resistance were selected for further studies on expression. After induction by the addition of methanol, various levels of hS83 were detected in the supernatant of P. pastoris containing pPIC9K-hS83. Clones with low G418-resistance produced more hS83 than those with higher G418-resistance. Under the optimized conditions (initial inoculum, 40 A_600nm AU/mL; pH 6.0; methanol concentration, 3.0%; induction time, 72 h), approximately 16–20 mg protein could be recovered from 1 L of the culture. The purified hS83 had a stronger binding ability towards CTLA4-positive Raji cells than CTLA4-negative ECV304 cells. This finding indicates that the antibody produced by P. pastoris is functional and may be used in immunotherapy for cancer, infection, transplant rejection, and autoimmune diseases. Huawei Cai and Lihong Chen contributed equally to this work.     

Movements of brown smoothhounds, <Emphasis Type="Italic">Mustelus henlei</Emphasis>, in Tomales Bay,California

Ultrasonic telemetry was used to analyze the effects of environmental variables on movement directions and movement rates of brown smoothhounds, Mustelus henlei, in Tomales Bay, California. Ultrasonic transmitters were surgically implanted in the peritoneal cavities of one male and five female brown smoothhounds and tracked during the period of 29 June to 15 July 2004. Coarse-scale tracking consisted of locating all tagged individuals multiple times during a single session, while fine-scale tracking consisted of following a single individual continuously during a session. Coarse-scale tracking suggested movement toward the inner bay with incoming and high tides and toward the outer bay with outgoing and low tides (P = 0.01), whereas the diel cycle had no apparent effect on their movement directions. Mean shark movement rate was 0.09 m s⁻¹ (range: 0.01–0.34 m s⁻¹), with diel and tidal cycles both having significant effects on their rates of movement (P = 0.02 and P < 0.01), respectively. We tracked two female sharks on a fine scale over three tracking sessions in July 2004. Both individuals exhibited higher rates of movement during the night compared to the day (P < 0.01). While one shark’s rate of movement was not significantly affected by tidal stage, the other’s was (P < 0.001).     

Heterologous expression of polyhydroxyalkanoate depolymerase from <Emphasis Type="Italic">Thermobifida</Emphasis> sp. in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and catalytic analysis by surface plasmon resonance

A polyhydroxyalkanote depolymerase gene from Thermobifida sp. isolate BCC23166 was cloned and expressed as a C-terminal His₆-tagged fusion in Pichia pastoris. Primary structure analysis revealed that the enzyme PhaZ-Th is a member of a proposed new subgroup of SCL-PHA depolymerase containing a proline–serine repeat linker. PhaZ-Th was expressed as two glycosylated forms with apparent molecular weights of 61 and 70 kDa, respectively. The enzyme showed esterase activity toward p-nitrophenyl alkanotes with V _max and K _m of 3.63 ± 0.16 μmol min⁻¹ mg⁻¹ and 0.79 ± 0.12 mM, respectively, on p-nitrophenyl butyrate with optimal activity at 50–55°C and pH 7–8. Surface plasmon resonance (SPR) analysis demonstrated that PhaZ-Th catalyzed the degradation of poly-[(R)-3-hydroxybutyrate] (PHB) films, which was accelerated in (R)-3-hydroxyvalerate copolymers with a maximum degradation rate of 882 ng cm⁻² h⁻¹ for poly[(R)-3-hydroxybutyrate-co-3-hydroxyvalerate] (12 mol% V). Surface deterioration, especially on the amorphous regions of PHB films was observed after exposure to PhaZ-Th by atomic force microscopy. The use of P. pastoris as an alternative recombinant system for bioplastic degrading enzymes in secreted form and a sensitive SPR analytical technique will be of utility for further study of bioplastic degradation.     

High-level heterologous expression of an alkaline lipase gene from <Emphasis Type="Italic">Penicillium cyclopium</Emphasis> PG37 in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A 777-bp cDNA fragment encoding a mature alkaline lipase (LipI) from Penicillium cyclopium PG37 was amplified by RT–PCR, and inserted into the expression plasmid pPIC9 K. The recombinant plasmid, designated as pPIC9 K-lipI, was linearized with SalI and transformed into Pichia pastoris GS115 (his4, Mut⁺) by electroporation. MD plate and YPD plates containing G418 were used for screening of the multi-copy P. pastoris transformants (His⁺, Mut⁺). One transformant resistant to 4.0 mg/ml of G418, numbered as P. pastoris GSL4-7, expressing the highest recombinant LipI (rLipI) activity was chosen for optimizing expression conditions. The integration of the gene LipI into the P. pastoris GS115 genome was confirmed by PCR analysis using 5′- and 3′-AOX1 primers. SDS–PAGE and lipase activity assays demonstrated that the rLipI, a glycosylated protein with an apparent molecular weight of about 31.5 kDa, was extracellularly expressed in P. pastoris. When the P. pastoris GSL4-7 was cultured under the optimized conditions, the expressed rLipI activity was up to 407 U/ml, much higher than that (10.5 U/ml) expressed with standard protocol. The rLipI showed the highest activity at pH 10.5 and 25°C, and was stable at a broad pH range of 7.0–10.5 and at a temperature of 30°C or below.     

Co-expression of the lipase and foldase of Pseudomonas aeruginosa to a functional lipase in Escherichia coli

The lipA gene, a structural gene encoding for protein of molecular mass 48 kDa, and lipB gene, encoding for a lipase-specific chaperone with molecular mass of 35 kDa, of Pseudomonas aeruginosa B2264 were co-expressed in heterologous host Escherichia coli BL21 (DE3) to obtain in vivo expression of functional lipase. The recombinant lipase was expressed with histidine tag at its N terminus and was purified to homogeneity using nickel affinity chromatography. The amino acid sequence of LipA and LipB of P. aeruginosa B2264 was 99–100% identical with the corresponding sequence of LipA and LipB of P. aeruginosa LST-03 and P. aeruginosa PA01, but it has less identity with Pseudomonas cepacia (Burkholderia cepacia) as it showed only 37.6% and 23.3% identity with the B. cepacia LipA and LipB sequence, respectively. The molecular mass of the recombinant lipase was found to be 48 kDa. The recombinant lipase exhibited optimal activity at pH 8.0 and 37°C, though it was active between pH 5.0 and pH 9.0 and up to 45°C. K _m and V _max values for recombinant P. aeruginosa lipase were found to be 151.5 ± 29 μM and 217 ± 22.5 μmol min⁻¹ mg⁻¹ protein, respectively.     

<Emphasis Type="Italic">Dipcadi goaense</Emphasis> (Hyacinthaceae), a new species from the foothills of the Western Ghats,India

A new species of Dipcadi (Hyacinthaceae) that is allied to D. concanense (Dalzell) Baker but differs in its small flowers (13 – 18 mm long vs 35 – 47 mm long) and funnel shaped perianth tube (5 – 6  ×  5 – 6 mm vs 18 – 27  ×  4 – 5.5 mm) is described as D. goaense. The new species is apparently endemic, because it is known only from the type locality in Goa state of India. The type locality is at the foothills of Western Ghats and the habitat is a soil covered, lateritic, open area.     

Reproductive and Life History Parameters of Wild Female <Emphasis Type="Italic">Macaca assamensis</Emphasis>

Information on basic reproductive parameters and life-history traits is crucial for the understanding of primate evolution, ecology, social behavior, and reproductive strategies. Here, we report 4 yr of data on reproductive and life-history traits for wild female Assamese macaques (Macaca assamensis) at Phu Khieo Wildlife Sanctuary, northeastern Thailand. During 2 consecutive reproductive seasons, we investigated reproductive behavior and sexual swelling size in 16 females and collected 1832 fecal samples. Using enzyme immunoassays, we measured fecal estrogen and progesterone metabolites to assess ovarian activity and timing of ovulation and to ascertain conceptions and pregnancies. Timing of reproduction was strictly seasonal (births: April–July, 86% in April–June, 4 yr, n = 29; conceptions: October–February, 65% in December–January, 2 yr, n = 17). Females showed no cyclic ovarian activity outside the mating season and conceived in their first or second cycle (mean: 1.2 cycles to conception, n = 13). Gestation length was on average 164.2 d (range: 158–170, n = 10), and females had their first infant at an age of 5 yr (n = 4). Interbirth intervals were bimodally distributed, with females giving birth on average every 13.9 or 23.2 mo. Shorter interbirth intervals were linked to early parturition within the birth season. Most females displayed subcaudal sexual swellings which, however, did not reliably indicate female reproductive status or fertility. Overall, our results fall within the range of findings reported for other macaque species. These results thus add to the growing body of information available for wild macaques, facilitating comparative studies for a better understanding of interspecific differences in social and reproductive patterns.     

Effect of Diets Supplemented with Different Levels of Manganese,Zinc, and Copper from their Organic or Inorganic Sources on Egg Production and Quality Characteristics in Laying Hens

This experiment was conducted to evaluate the effect of zinc, manganese, and copper sources (inorganic vs. organic) in the diet on laying performance and eggshell quality characteristics. One hundred and eighty Hy-Line W-36 layers at 38 weeks of age were allocated to 36-layer cages of five hens each. Each six cages were randomly assigned to one of the six experimental diets fed from 38 to 53 week of age. In three experimental treatments, the basal diet was supplemented with 65–75–7 or 65–75–7 or 40–40–7 mg/kg of Zn, Mn, and Cu, respectively, from their oxide or sulfate sources. Three other groups were fed diets supplemented with 20–20–3.5 or 40–40–7.5 or 60–60–10.5 mg/kg of organic forms of Zn, Mn, and Cu, respectively. Dietary treatments significantly did affect feed intake (P < 0.001), feed conversion ratio (P < 0.001) and percentage of broken eggs (P < 0.05). Substitution of Zn and Mn oxides (65 and 75 mg kg⁻¹, respectively) with equal amounts of their sulfate forms significantly improved feed intake, feed conversion ratio, percentage of broken eggs, and Haugh Unit (P < 0.05). In addition, laying hens maintained their performance when substitution of Zn and Mn oxides and Cu sulfate (65, 75, and 7 mg kg⁻¹, respectively) reduced up to 20, 20, and 3.5 mg kg⁻¹ by amino acid complexes of the microelements. The results showed that a corn–soybean diet supplemented with the organic forms of Zn, Mn, and Cu at a dosage 50% to 75% lower than NRC recommendation is sufficient to maintain laying performance and can improve eggshell and albumen qualities of the egg in laying hens.     

High-level expression of the <Emphasis Type="Italic">Penicillium notatum</Emphasis> glucose oxidase gene in <Emphasis Type="Italic">Pichia pastoris</Emphasis> using codon optimization

The glucose oxidase (GOD) gene from Penicillium notatum was expressed in Pichia pastoris. The 1,815 bp gene, god-w, encodes 604 amino acids. Recombinant GOD-w had optimal activity at 35–40°C and pH 6.2 and was stable, from pH 3 to 7 maintaining >75% maximum activity after incubation at 50°C for 1 h. GOD-w worked as well as commercial GODs to improve bread making. To achieve high-level expression of recombinant GOD in P. pastoris, 272 nucleotides involving 228 residues were mutated, consistent with the codon bias of P. pastoris. The optimized recombinant GOD-m yielded 615 U ml⁻¹ (2.5 g protein l⁻¹) in a 3 l fermentor—410% higher than GOD-w (148 U ml⁻¹), and thus is a low-cost alternative for the bread baking industry.     

Simple and efficient expression of <Emphasis Type="Italic">Agaricus meleagris</Emphasis> pyranose dehydrogenase in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Pyranose dehydrogenase (PDH) is a fungal flavin-dependent sugar oxidoreductase that is highly interesting for applications in organic synthesis or electrochemistry. The low expression levels of the filamentous fungus Agaricus meleagris as well as the demand for engineered PDH make heterologous expression necessary. Recently, Aspergillus species were described to efficiently secrete recombinant PDH. Here, we evaluate recombinant protein production with expression hosts more suitable for genetic engineering. Expression in Escherichia coli resulted in no soluble or active PDH. Heterologous expression in the methylotrophic yeast Pichia pastoris was investigated using two different signal sequences as well as a codon-optimized sequence. A 96-well plate activity screening for transformants of all constructs was established and the best expressing clone was used for large-scale production in 50-L scale, which gave a volumetric yield of 223 mg L⁻¹ PDH or 1,330 U L⁻¹ d⁻¹ in space–time yield. Purification yielded 13.4 g of pure enzyme representing 95.8% of the initial activity. The hyperglycosylated recombinant enzyme had a 20% lower specific activity than the native enzyme; however, the kinetic properties were essentially identical. This study demonstrates the successful expression of PDH in the eukaryotic host organism P. pastoris paving the way for protein engineering. Additionally, the feasibility of large-scale production of the enzyme with this expression system together with a simplified purification scheme for easy high-yield purification is shown.     

Comparative studies on the metabolism of linoleic acid by rumen bacteria,protozoa, and their mixture in vitro

Linoleic acid was differentially catabolized by the various rumen microbial fractions, such as rumen bacteria (B), protozoa (P), and their mixture (BP). The predominant isomer of conjugated linoleic acids (CLA) synthesized by B, P, and BP from linoleic acid was 9c11t-CLA. The formation of 9c11t-CLA was higher (P < 0.05) in P suspension (53.6 μg/mg microbial nitrogen) compared with B (38.3 μg/mg microbial nitrogen) and BP (28.8 μg/mg microbial nitrogen) suspensions by 12 h of incubation. The second most abundant CLA isomer was 10t12c. The accumulation of 10t12c-CLA in BP suspension was 2.3 times lower (P < 0.05) than that in B suspension (84.8 μg/mg microbial nitrogen) by 12 h of incubation. The accumulation of 10t-18:1 in BP suspension during 6- and 12-h incubation periods were not different (P > 0.05) than that in B suspension (6.8 and 14.0 μg/mg microbial nitrogen, respectively). However, the accumulation of 11t-18:1 in BP suspension at 6- and 12-h incubations were 2.7 and 3.3 times higher (P < 0.05), respectively, than that in B suspension. There were no significant accumulations of 11t-18:1, 10t-18:1, and 18:0 in P suspension throughout the incubation period. It was concluded that B, P, and BP metabolized linoleic acid to different isomers of CLA, whereas B, including BP, was only capable of biohydrogenating the CLA isomers to 18:0 by the reduction of 18:1 isomers. P was incapable of biohydrogenating LA, but its association with B in the BP suspension altered the biohydrogenation of LA significantly compared with B alone.     

Impact of seropositivity to <Emphasis Type="Italic">Chlamydia pneumoniae</Emphasis> and anti-hHSP60 on cardiovascular events in hemodialysis patients

Autoimmunity to heat shock protein 60 (HSP60) has been related to atherosclerosis. Chlamydia pneumoniae (CP), the most studied infectious agent implicated in promoting atherosclerosis, produces a form of HSP60, which can induce an autoimmune response, due to high antigenic homology with human HSP60 (hHSP60). In this study, we evaluated the correlations among anti-hHSP60 antibodies, CP infection, and cardiovascular disease (CVD) in a high-risk population, such as patients undergoing hemodialysis (HD). Thirty-two patients (67.9 ± 13.9 years; male/female, 23:9) on regular HD were enrolled. Global absolute cardiovascular risk (GCR) was assessed using the Italian CUORE Project’s risk charts, which evaluate age, gender, smoking habits, diabetes, systolic blood pressure, and serum cholesterol. The occurrence of cardiovascular events during a 24-month follow-up was recorded. Seropositivity to CP and the presence of anti-hHSP60 antibodies were tested by specific enzyme-linked immunosorbent assays. Inflammation was assessed by measurement of C-reactive protein (CRP) serum levels. Fifteen healthy sex and age-matched (61.9 ± 9.5 years; male/female, 11:4) subjects were the control group. Fifteen of 32 patients resulted seropositive for CP. CP + patients were older than CP−, while they did not differ for GCR, CRP, and dialytic parameters. CVD incidence was significantly higher in CP+ (9 CP+ vs 2 CP−, p < 0.05). Cox analysis recognized that the incidence of CVD was independently correlated with seropositivity to CP (HR, 7.59; p = 0.01; 95% CI = 1.63–35.4). On the other hand, there were no significant differences in anti-hHSP60 levels among CP+, CP− and healthy subjects: 18.11 μg/mL (14.8–47.8), 31.4 μg/mL (23.2–75.3), and 24.72 μg/mL (17.7–41.1), respectively. Anti-hHSP60 did not correlate to GCR, CRP, and incidence of CVD. In conclusion, our data suggest that anti-hHSP60 autoimmune response is not related to CP infection and CP-related CVD risk in HD patients.     

Age composition,growth, and reproductive biology of yellow catfish (<Emphasis Type="Italic">Peltobagrus fulvidraco</Emphasis>, Bagridae) in Ce Lake of Hubei Province,Central China

This study presents fundamental information on the reproductive biology, size and age composition, and growth of yellow catfish, Peltobagrus fulvidraco. Sampling was carried out monthly from Ce Lake in Hubei province from January 2004 and December 2004. Overall female/male (F/M) sex ratio was 1.08, not significantly different from expected 1.0 ratio. But sex ratio (F/M) of the spawning group was 1.14:1 with skewness towards females (p < 0.05). The smallest sexually mature male and female massed 20.51 and 16.93 g, and measured 100.1 and 94.3 mm in total length, respectively. Reproductive period for this species is from April to August when GSI is considerably higher than the rest months (p < 0.05). The highest GSI values were measured from May to July for both sexes and this period was presumably the peak spawn. First maturation occurred at age 1⁺ for both sexes. Fish of age 1⁺ and 2⁺ comprised the greatest portion of spawners. The absolute fecundity (AF) of P. fulvidraco ranged from 657 to 11,696 eggs per fish, with mean AF value of 3,570 ± 122 (S.D.) eggs per fish, varying considerably at given length, weight and age. The fecundity relative to total length (F/L) fluctuated from 58 to 680 eggs/mm, with a mean value of 246 ± 34 (SD) eggs/mm, and fecundity relative to total weight (F/W) varied from 29–133 eggs/g, with a mean value of 67 ± 7 (SD) eggs/g. P. fulvidraco is a single spawner, with low fecundity compared with other Bagrid fish.     

Ovarian and Uterine Ultrasonography in <Emphasis Type="Italic">Aotus azarai infulatus</Emphasis>

We evaluated the uterus and ovaries of owl monkeys (Aotus azarai infulatus) via gynecological ultrasound examination. We evaluated the subjects in 2 different time periods. The first period (P1) was characterized by the absence of mating, with daily examinations, during 4 mo (n = 10). At the end of P1, we paired the subjects for 30 d, but without ultrasonographic evaluation. The second period (P2) was characterized by the presence of mating, with examinations once a week, during 7 consecutive months (n = 9). We evaluated the uterus and ovaries in sagittal and transverse scans, using a 5–12 MHz linear array probe. The uterine volume (UV) was directly proportional to the number of previous parturitions. The right ovary volume (RtOV) is greater than the left (LtOV) in P1 and P2. There is a positive correlation (p < 0.05) between the females’ mass, RtOV (r = 0.28) and LtOV (r = 0.16).     

Protection of Selenium on Hepatic Mitochondrial Respiratory Control Ratio and Respiratory Chain Complex Activities in Ducklings Intoxicated with Aflatoxin B<Subscript>1</Subscript>

To investigate the protection of selenium on hepatic mitochondrial functions, 90 7-day-old ducklings were randomly divided into three groups (groups I–III). Group I was used as a blank control. Group II was administered with aflatoxin B₁ (0.1 mg/kg body weight). Group III was administered with aflatoxin B₁ (0.1 mg/kg body weight) plus selenium (sodium selenite, 1 mg/kg body weight). All treatments were given once daily for 21 days. The results showed that the activities of hepatic mitochondrial complexes I–IV in group II ducklings significantly decreased when compared with group I (P < 0.01). Furthermore, the activities of hepatic mitochondrial complexes I–IV in group III significantly increased when compared with group II (P < 0.05). The hepatic mitochondrial respiratory control ratio (RCR) in group II ducklings significantly decreased when compared with group I (P < 0.01). In addition, the hepatic mitochondrial RCR in group III significantly increased when compared with group II (P < 0.05). These results revealed that the aflatoxin B₁ significantly induced hepatic mitochondrial dysfunction in the activities of hepatic mitochondrial respiratory chain complexes I–IV and the RCR in ducklings. However, sodium selenite could significantly ameliorate the negative effect induced by aflatoxin B₁.     

Heterologous expression, characterization and site-directed mutagenesis of cutinase CUTAB1 from Alternaria brassicicola

The cutinase CUTAB1 was cloned from a cutin induced culture of Alternaria brassicicola and heterologously expressed in Pichia pastoris under the control of the methanol-inducible AOX1 promoter. From a 400-ml culture, 36 mg of purified recombinant enzyme were obtained. Biochemical characterization revealed highest catalytic activity of the enzyme at 40°C and pH 7-9 using p-nitrophenyl palmitate (p-NPP) as substrate. Among several fatty acid methyl and ethyl esters, glycerol esters and p-nitrophenyl esters tested, CUTAB1 showed highest activity towards tributyrin (3,302 ± 160 U mg⁻¹) and the activity decreased with increase in chain length of the investigated esters. Lowest activity was found for p-NPP. Replacing Leu80, Leu181 and Ile183, respectively, by the smaller alanine in the hydrophobic binding loop of CUTAB1, drastically reduced the overall activity of the enzyme. On the other hand, mutation A84F located in the small helical flap of CUTAB1 significantly increased the activity of the enzyme towards longer chain substrates like p-NPP.     

Redescription of <Emphasis Type="Italic">Bregmaceros mcclellandi</Emphasis> Thompson, 1840 (Gadiformes: Bregmacerotidae)

 This study redescribes Bregmaceros mcclellandi Thompson, 1840, based on one specimen (74.4 mm SL) from the Bay of Bengal and 66 specimens (30.0–84.7 mm SL) from Mumbai (Bombay), India, because the type specimens have apparently been lost. The present specimens are characterized by having black dorsal, pectoral, and caudal fins and show the following morphology: caudal fin slightly forked; body chromatophores present mainly at the dorsal part; no scales on cheek; vertebrae 52–55 (13–15 + 38–41); dorsal rays 52–59; anal rays 54–60; pectoral rays 18–20; caudal rays 27–31 (principal rays 14); transverse scales 14–15. In the 66 Mumbai specimens, it was confirmed that the distinctive black fin pigmentation developed sequentially with growth, with complete pigmentation first on the anterior lobe of the dorsal fin, then simultaneously on the posterior lobe of the dorsal fin, the caudal fin, and the pectoral fin, and last, on the anal fin. This species is known only from the Arabian Sea, Bay of Bengal, and Gulf of Thailand. A review of 16 nominal Bregmaceros species indicates that, besides B. mcclellandi, the distinctive dark fin pigmentation is found in B. atripinnis (Tickell), B. atlanticus Goode and Bean, B. japonicus Tanaka, and B. lanceolatus Shen. B. atripinnis is considered a junior synonym of B. mcclellandi, and the others are clearly distinct from B. mcclellandi. Comments are made on some of the characters to more fully characterize the species and for reference in future revisionary and phylogenetic studies. Received: June 17, 2002 / Revised: December 2, 2002 / Accepted: December 24, 2002     

The viability to a wall shear stress and propagation of <Emphasis Type="Italic">Bifidobacterium longum</Emphasis> in the intensive membrane bioreactor

Bifidobacterium longum grew at 65 L pilot scale of the membrane bioreactor (MBR), externally fitted with ceramic membrane (0.7 m²). Cell mass at the MBR reached 22.18 g L⁻¹ as dry cell weight in 12 h, which is 8.44 times higher than cell mass attained at the vial culture. The growth rate in the vial culture was μ = 0.385 h⁻ and at the batch culture was μ = 1.13 h⁻ in the exponential period and μ = 0.31 h⁻¹ in the stationary period. In the fed-batch mode was μ = 1.102 h⁻¹ for 6 h with inoculation and declined to μ = 0.456 h⁻¹ with feeding of feed medium. The growth rate at the MBR was μ = 0.134 h⁻¹. The number of viable cells was 6.01 × 10¹² cfu L⁻¹ at the batch culture, but increased to 1.15 × 10¹⁴ cfu L⁻¹ at the MBR culture. The specific growth rate of viable cell number (colony-forming units per liter, per hour) improved by 6.01 times from the batch to the MBR culture. The wall shear stress mainly generated by the pump, and the membrane incorporated into the MBR was controlled during the cultivation at the MBR. The viability of B. longum declined to under 10% in the first 2 weeks of the 4-week stability test (40°C) as B. longum was exposed to over wall shear stress 713 Pa, but the viability improved to 30–40% in wall shear stress of 260 Pa or STR culture. The loss in the cell viability can be saved by managing with wall shear stress during the cultivation at the MBR.     

Inhibition of degradation and aggregation of recombinant human consensus interferon-α mutant expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis> with complex medium in bioreactor

The methylotrophic yeast Pichia pastoris has been used for the expression of many proteins. However, limitations such as protein degradation and aggregation became obvious when secreting heterologous protein-recombinant human consensus interferon-α mutant. Here, we investigate the effect of induction temperature on the yield and stability of interferon mutant expressed by P. patoris with buffered complex medium. The best results in terms of interferon mutant bioactivity and specific bioactivity were obtained when the microorganism was induced at 15°C, which were 2.91 × 10⁸ ± 0.3 × 10⁸ and 2.26 × 10⁸± 0.23 × 10⁸ IU mg⁻¹, respectively. At the same time, the cells grew fast owing to high AOX1-specific activity, and interferon mutant expression level reached 1.23 g l⁻¹, which was almost 30 times higher than that in the flask. Also, the proteolytic degradation of interferon mutant was inhibited completely because of lower protease bioactivity probably due to a reduced cell death rate at lower temperatures as well as protection of yeast extract and peptone in complex medium. In addition, interferon mutant aggregation was repressed significantly by the addition of Tween-80, and a specific bioactivity of 7.35 × 10⁸ ± 0.56 × 10⁸ IU mg⁻¹ was obtained. These results should be applicable to other low-stability recombinant proteins expressed in P. pastoris.     

Aflatoxin B<Subscript>1</Subscript> in chilies from the Punjab region,Pakistan

The occurrence of aflatoxin B1 (AFB1) in chilies from Pakistan was determined by using HPLC in work undertaken in Pakistan. Whole (n = 22) and powdered (n = 22) chilies were analyzed. Sixteen (73.0%) and 19 (86.4%) samples of whole and ground chilies, respectively, were contaminated. The mean concentration in powdered chilies (32.20 μg/kg) was higher statistically than in whole chilies (24.69 μg/kg). Concentrations ranged from 0.00 to 89.56 μg/kg for powdered chilies, compared with 0.00–96.3 μg/kg for whole chilies. The limits of detection and quantification were 0.05 μg/kg and 0.53 μg/kg, respectively. The concentrations were high in general and greater than the statutory limit set by the European Union. There is considerable scope for improvements in chili production in Pakistan.