Cell lines from diamondback moth exhibiting differential susceptibility to baculovirus infection and expressing midgut genes

Abstract Six new cell lines were established from embryonic tissues of the diamondback moth, Plutella xylostella (L.). The cell lines showed differential characteristics, including growth in attachment or in suspension, susceptibility to a baculovirus infection and expression of genes involved in the glucosinolate detoxification pathway in R xylostella larvae. Five of the cell lines grew attached to the culture flask and one cell line grew unattached as a suspension cell line. The cell lines had population doubling times ranging from IS to 23 h. Among five of the P. xylostella cell lines examined for infection of a nucleopolyhe. drovirus from Autographa californica, AcMNPV four cell lines were highly susceptible to AcMNPV infection, but one was only semi-permissive to AcMNPV infection. The production of two recombinant proteins, a β-galactosidase of bacterial origin and a secreted alkaline phosphatase of eukaryotic origin, in the R xylostella cell lines was examined in comparison with that in the cell line Sf9 which is commonly used for recombinant protein production. In the P. xylostella cell lines, expression of three important midgut genes involved in the glucosinolate detoxification pathway, including the glucosinolate sulfatase genes GSS1 and GSS2 and the sulfatase modifying factor gene SUMF1、was detected. The R xylostella cell lines developed in this study could be useful in in vitro research systems for studying insec-virus interactions and complex molecular mechanisms in glucosinolate detoxification and insect-plant interactions.     

Diversity of culturable sodium dodecyl sulfate (SDS) degrading bacteria isolated from detergent contaminated ponds situated in Varanasi city, India

In the present investigation, an attempt has been made to isolate and identify SDS-degrading bacteria from different detergent contaminated ponds situated in Varanasi city, UP, India. Initial survey of ponds indicated that these ponds were contaminated with detergents. Employing enrichment technique in minimal medium (PBM) with SDS as a sole carbon source, a total of 24 isolates were recovered from 7 detergent contaminated ponds. Studies on rates of SDS degradation indicated that the rate of SDS degradation varied from 97.2% to 19.6% after 12h incubation under identical conditions. An estimation of alkyl sulfatase activity indicated that the activity varied from 0.168 ± 0.004 to 0.024 ± 0.005 μmol SDS/mg protein/min. Molecular characterization of these isolates was performed on the basis of ARDRA and ERIC PCR, which indicated that these isolates were broadly divided in 8 groups. Some selected isolates were identified on the basis of 16S rDNA sequencing. It was found that these isolates belonged to Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas stutzeri, Pseudomonas alcaligenes, Pseudomonas pseudoalcaligenes, Pseudomonas putida and Pseudomonas otitidis respectively. Among these isolates P. aeruginosa, P. putida and P. otitidis have been previously shown to degrade and metabolize SDS, the rest of the isolates appear to be new.     

Long-term Study of Group Dynamics and Female Reproduction in Venezuelan Pithecia pithecia

White-faced sakis (Pithecia pithecia) are well known for their specialized feeding strategy and dependence on seed-eating, but we know less about social dynamics and reproduction. I summarize data from 1990 to 2001 on sakis inhabiting a small island in Lago Guri, Venezuela that includes changes in group composition, aggressive behavior, and female reproductive histories. Though the characteristic group size and typical mating system are still uncertain for wild, free-ranging Pithecia spp., size of the study group ranged from 2–3 adult females and 1–4 adult males. My group and I collected fecal samples from adult females, which we dried and assayed for estrogen (E₁C) and progesterone (PdG) conjugates to document reproductive cycles and female development. During the study, as many as 3 females were either cycling or pregnant at the same time. Ten infants were born during the study, 4 of which survived to adulthood. Five interbirth intervals after weaning an infant ranged from 12 to 36 mo. Though researchers often assume Pithecia spp. are monogamous, multiple breeding females can coexist in groups without evidence of reproductive suppression. Within the pitheciine clade, Pithecia spp. exhibit a mosaic of behavioral and morphological traits intermediate between bearded sakis and uakaris on the one hand and their sister-group, the pair-bonded titis, on the other.     

Diurnal variations of serum testosterone levels in intact and gonadectomized male and female rhesus monkeys.

A radioimmunoassay for serum testosterone which does not require chromatographic separation was used to measure the diurnal variations in intact and orchidecomized males and intact and ovariectomized females. The intact male rhesus monkey shows a distinctive diurnal variation in serum levels of testosterone characterized by lower values during the day and a marked increase in the early evening (1900-2200 hr). The testosterone levels remain high throughout most of the lights-off period in the intact male. In contrast to the intact male, the markedly lowered serum levels of testosterone in the orchidectomized male were higher during the day and consistently showed a nadir during the early evening (2000-2200 hr). The evening nadir of testosterone levels was 51.0% lower than the 24-hr mean whereas the maximum serum level was 46.4% higher. A similar circadian pattern of testosterone was seen in both the intact and ovariectomized females. The testosterone values were higher during the day and consistently showed a nadir during the early evening. These results suggest that the adrenal secretion of testosterone varies in a diurnal pattern characterized by an early evening nadir. This adrenal pattern is overshadowed by a much larger gonadal rhythm in the intact male.     

Role of transmembrane domain 10 for the function of organic anion transporting polypeptide 1B1

The liver‐specific organic anion transporting polypeptides OATP1B1 and OATP1B3 are highly homologous and share numerous substrates. However, at low concentrations OATP1B1 shows substrate selectivity for estrone‐3‐sulfate. In this study, we investigated the molecular mechanism for this substrate selectivity of OATP1B1 by constructing OATP1B1/1B3 chimeric transporters and by site‐directed mutagenesis. Functional studies of chimeras showed that transmembrane domain 10 is critical for the function of OATP1B1. We further identified four amino acid residues, namely L545, F546, L550, and S554 in TM10, whose simultaneous mutation caused almost complete loss of OATP1B1‐mediated estrone‐3‐sulfate transport. Comparison of the kinetics of estrone‐3‐sulfate transport confirmed a biphasic pattern for OATP1B1, but showed a monophasic pattern for the quadruple mutant L545S/F546L/L550T/S554T. This mutant also showed reduced transport for other OATP1B1 substrates such as bromosulfophthalein and [D ‐penicillamine^2,5]enkephalin. Helical wheel analysis and molecular modeling suggest that L545 is facing the substrate translocation pathway, whereas F546, L550, and S554 are located inside the protein. These results indicate that L545 might contribute to OATP1B1 function by interacting with substrates, whereas F546, L550, and S554 seem important for protein structure. In conclusion, our results show that TM10 is critical for the function of OATP1B1.     

A superfamily of metalloenzymes unifies phosphopentomutase and cofactor-independent phosphoglycerate mutase with alkaline phosphatases and sulfatases

Sequence analysis of the probable archaeal phosphoglycerate mutase resulted in the identification of a superfamily of metalloenzymes with similar metal-binding sites and predicted conserved structural fold. This superfamily unites alkaline phosphatase, N-acetylgalactosamine-4-sulfatase, and cerebroside sulfatase, enzymes with known three-dimensional structures, with phosphopentomutase, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase, phosphoglycerol transferase, phosphonate monoesterase, streptomycin-6-phosphate phosphatase, alkaline phosphodiesterase/nucleotide pyrophosphatase PC-1, and several closely related sulfatases. In addition to the metal-binding motifs, all these enzymes contain a set of conserved amino acid residues that are likely to be required for the enzymatic activity. Mutational changes in the vicinity of these residues in several sulfatases cause mucopolysaccharidosis (Hunter, Maroteaux-Lamy, Morquio, and Sanfilippo syndromes) and metachromatic leucodystrophy.     

Androgen receptor in human skin fibroblasts. Characterization of a specific 17beta-hydroxy-5alpha-androstan-3-one-protein complex in cell sonicates and nuclei.

Cultured human skin fibroblasts were shown to contain an androgen binding activity (receptor) which was heat-labile and destroyed by trypsin. Specific binding was seen after incubations of these cells with 1,2-3-H-testosterone, 1,2-3-H17beta-hydroxy-5alpha-androstan-3-one (dihydrotestosterone, DHT) and 1,2-3-H-5alpha-androstane-3alpha, 17beta-diol. This receptor had a high affinity (Kd=0,2-1.6 nM) and a high degree of specificity for DHT. It was measured as a 3-H-DHT-protein complex by gel filtration chromatography using a method which distinguishes specific from nonspecific binding. Receptor activity was distributed about equally between nuclear and extranuclear components at all times studied and was present in both compartments when cell incubations were carried out at 4 degrees and 37 degrees. Saturation analysis indicated that there were 1250-18,600 binding sites per whole cell. By sucrose gradient centrifugation the receptor had a sedimentation coefficient (S20,w) of about 4. Cells grown for 8 days without serum in the medium maintained the same levels of 3-H-DHT binding. Within 15 hours puromycin (20 mug/ml) in serum-free medium caused a 40-60 percent decrease in binding for the same cell lines. Although the highest levels of 3-H-DHT binding were observed in fibroblasts from newborn foreskin, appreciable cytosol and nuclear binding were seen in cells from forearm, neck and abdominal skin. Receptor activity was stable during prolonged culture. Fibroblasts from several skin sites from patients with the androgen insensitivity syndrome (testicular feminization) had no detectable specific DHT binding. In this study it was demonstrated that skin fibroblasts can rapidly convert testosterone to its active form, DHT, bind DHT to a specific receptor protein and transport this complex to their nuclei. Therefore this may prove to be a convenient system for studying androgen action in vitro.     

Blood production rates of estrogens in women with differing ratios of urinary estrogen conjugates.

On the basis of the ratios of the estrogen conjugates in their urine (estriol/estrone + estradiol: E3/[E1+E2]), 19 women were divided into two groups: 9 women had ratios less than 0.6 and 10 women had ratios greater than 1.3. All women had measurements made of endogenous estrogens in their plasma by radioimmunoassay. They were then given constant infusions of 3H-estrone, 3H-estradiol and 14C-estriol during days 5-7 and days 20-22 of their cycles, and metabolic clearance rates (MCR) and blood production rates (PB) of estrone, estradiol and estriol were determined. Despite the wide disparity in their ratios of urinary estrogens, no differences could be found between the groups for the MCR's and PB's for all estrogens at either time of the cycle. The mean ratios of PB's (PB3/[PB2+PB1]) of estrone, estradiol and estriol ranged from 0.07 to 0.10 for each group during the cycle. The amounts of estriol entering the blood are small compared to the amounts of estrone and estradiol and do not correlate with the ratios of their urinary conjugates.     

Effect of sulfur supply on sulfate uptake,and alkaline sulfatase activity in free-living and symbiotic bradyrhizobia

The effect of sulfur limitation on sulfate transport and metabolism was studied in four bradyrhizobia strains using sulfur-limited and sulfur-excess chemostat cultures. Characteristics of bradyrhizobia associated with sulfurlimitation were determined and these parameters used to bioassay the sulfur status of bacteroids in nodules on sulfur adequate or sulfur deficient soybean and peanut plants. Sulfur-limited cells took up sulfate 16- to 100-fold faster than sulfur-rich cells. The sulfate-uptake system appeared similar in all strains with apparent K _m values ranging from 3.1 M to 20 M sulfate with maximum activities between 1.6 and 10 nmol·min^-1·mg^-1 protein of cells. Sulfate-limited cells of all strains derepressed the enzyme alkaline sulfatase in parallel with the derepression of the sulfate transport system. Similarly, the initial enzyme of sulfate assimilation (ATP sulfurylase) was fully derepressed in sulfur-limited cultures. Bacteroids isolated from sulfur adequate and sulfur deficient soybean and peanut possessed very limited sulfate uptake activity and low levels of activity of ATP sulfurylase as well as lacking alkaline sulfatase activity. These results indicate bacteriods have access to adequate sulfur to meet their requirements even when the host plant is sulfur-deficient.Abbreviations CCCP Carbonyl cyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexyl carbodiimide     

Contribution of ovarian steroid production to urinary estrone conjugate concentrations in Macaca mulatta

This study was designed to test the hypothesis that basal estrone conjugate (E1C) profiles do not accurately detect ovarian function when ovarian estrogen production is low or absent. We employed surgical removal of active ovaries from laboratory rhesus macaques to simulate an acute decline in ovarian estrogen production. In the first experiment, urine samples collected prior to and following ovariectomy (Ovx) were subjected to high-performance liquid chromatography (HPLC) separation. Eluates were then assayed for E1C immunoreactive components. The results indicated a modest decrease in total immunoreactive polar conjugates following ovariectomy, with no substantial change in the overall retention profile. In the second experiment, estradiol (E2) cypionate injections were used to replace the E2 component of ovarian estrogen production in the treated (Tx) group, while the control group (C) received only vehicle. Urine samples were hydrolyzed and individual estrogens were separated by celite chromatography prior to immuno-assay. Both the Tx and C groups exhibited similar urinary excretion levels of estrone (E1), E2, and E1C prior to Ovx (Pre-Ovx) and after Ovx (Post-Ovx), but there were significant differences between groups after treatment (Post-Tx). Significant differences were observed in the Tx group's excretion of E1, E2, and E1C in the Pre- vs. Post-Ovx samples and in the Post-Ovx and Post-Tx samples. The C group also showed the expected significant differences in the Pre- vs. Post-Ovx samples, as well as in the Pre-Ovx and Post-Tx samples. The results indicate that the use of E1C measurements is clearly a suitable method for monitoring ovarian function in intact, cycling animals, but urinary E2 measurements are required to verify loss of follicular activity.