Human erythroid cells produced ex vivo at large scale differentiate into red blood cells in vivo

New sources of red blood cells (RBCs) would improve the transfusion capacity of blood centers. Our objective was to generate cells for transfusion by inducing a massive proliferation of hematopoietic stem and progenitor cells, followed by terminal erythroid differentiation. We describe here a procedure for amplifying hematopoietic stem cells (HSCs) from human cord blood (CB) by the sequential application of specific combinations of growth factors in a serum-free culture medium. The procedure allowed the ex vivo expansion of CD34+ progenitor and stem cells into a pure erythroid precursor population. When injected into nonobese diabetic, severe combined immunodeficient (NOD/SCID) mice, the erythroid cells were capable of proliferation and terminal differentiation into mature enucleated RBCs. The approach may eventually be useful in clinical transfusion applications.     

Homoisoflavonoids from Ophiopogon japonicus Ker-Gawler

From the ethyl acetate extract of the tuberous roots of Ophiopogon japonicus (Liliaceae) eight known and five new homoisoflavonoidal compounds were isolated. The new compounds are 5,7-dihydroxy-8-methoxy-6-methyl-3-(2'-hydroxy-4'-methoxybenzyl)chroman-4-one (1), 7-hydroxy-5,8-dimethoxy-6-methyl-3-(2'-hydroxy-4'-methoxybenzyl)chroman-4-one (2), 5,7-dihydroxy-6,8-dimethyl-3-(4'-hydroxy-3'-methoxybenzyl)chroman-4-one (3), 2,5,7-trihydroxy-6,8-dimethyl-3-(3',4'-methylenedioxybenzyl)chroman-4-one (4) and 2,5,7-trihydroxy-6,8-dimethyl-3-(4'-methoxybenzyl)chroman-4-one (5). Their structures have been elucidated by mass and NMR spectroscopy. Compounds 4 and 5 are the first isolated homoisoflavonoids with a hemiacetal function at position 2.     

Expression in E. coli and purification of Thermus thermophilus translation initiation factors IF1 and IF3

The initiation of protein translation in bacteria requires in addition to mRNA, fMet-tRNA, and ribosomal subunits three protein factors, the initiation factor 1 (IF1), initiation factor 2 (IF2), and initiation factor 3 (IF3). The genes coding for IF1 and IF3 from Thermus thermophilus have been identified and cloned into pET expression vector and were expressed as soluble proteins in Escherichia coli. IF1 was purified by a DEAE-cellulose chromatography, followed by heat denaturation, chromatography on Hydroxylapatit, and gel permeation chromatography using Sephacryl 200HR. For the purification of IF3, a heat denaturation step is followed by anion-exchange chromatography on Q-Sepharose FF and gel permeation chromatography on Sephacryl 200HR. Using these procedures we obtained chromatographically pure and biologically active preparations of both T. thermophilus IF1 and IF3.     

The effects of Eucalyptus terpenes on hepatic cytochrome P450 CYP4A, peroxisomal Acyl CoA oxidase (AOX) and peroxisome proliferator activated receptor alpha (PPARalpha) in the common brush tail possum (Trichosurus vulpecula)

Eucalyptus leaves contain a high proportion of essential oils comprising of a complex mixture of monoterpenes such as 1,8-cineole, alpha-pinene, d-limonene and p-cymene. In this study, hepatic levels of microsomal lauric acid hydroxylase and peroxisomal cyanide-intensive palmitoyl coenzyme A oxidative activities were examined in livers of possums given an artificial diet consisting of the above monoterpenes for 10 days. These values were compared with those of possums fed a control diet containing only fruits and cereals. Peroxisomal cyanide-intensive palmitoyl coenzyme A oxidative activity was significantly higher in livers of treated possums relative to that of control possums (2.96+/-0.93 vs. 0.98+/-0.88 nmol/mg protein per min, P<0.01) (mean+/-S.D., n=4). A small increase in microsomal lauric acid hydroxylase activity was observed in the treated possum in comparison with the control group (4.40+/-0.85 vs. 3.60+/-0.48 nmol/mg protein per min) (mean+/-S.D., n=4). A higher NAD/ NADP ratio was observed in treated possums as compared with control possums (4.73+/-0.65 vs. 3.51+/-0.64 nmol/mg protein per min, P<0.05) (mean+/-S.D., n=4). No other statistically significant differences in pyridine nucleotide contents were found between control and treated possums. Northern blot analysis of mRNA from rat, human, terpene treated and control possum livers, using the corresponding koala cDNA probes, detected a more intense acyl CoA oxidase (AOX) mRNA band in livers of terpene fed possums. Negligible differences in the intensity of CYP4A and PPARalpha mRNA bands were observed between the two groups. These data suggest that Eucalyptus terpenes elevate hepatic AOX expression in possums.     

Thermal comfort zones in the Vietnamese

Thermally comfortable zones in Vietnamese were investigated during winter in Hanoi. The subjects were 21 males (age: 19.7 +/- 0.4 yrs; height: 165 +/- 1.5 cm; body mass: 55.1 +/- 1.1 kg) and 19 females (age: 19.7 +/- 0.4 yrs; height; 155.6 +/- 1.7 cm; body mass: 45.6 +/- 1.3 kg). Each participant entered singly the climatic chamber controlled at 22 degrees C and 40% RH. After 20 min rest, the participant was requested to indicate on a 7-point scale (Table 1) how he or she felt to the room temperature given. Then, the room temperature increased by 1 degrees C over 10 min every 20 min. Just before the rise of the room temperature, the participant judged his or her thermal sensation. More than 90% of the participants felt 24-29 degrees C of the room temperature as "slightly cool", "neutral" and "slightly warm" (Table 2). We defined these sensations as "thermally comfort". These thermally comfortable zones were quite higher than those (20-24 degrees C) recommended by ISO-7730 (1994). We discussed these discrepancies in terms of higher establishment of thermoregulatory set-point in the Vietnamese.     

GTP enhances the degradation of the photosystem II D1 protein irrespective of its conformational heterogeneity at the Q(B) site

The light exposure history and/or binding of different herbicides at the Q(B) site may induce heterogeneity of photosystem II acceptor side conformation that affects D1 protein degradation under photoinhibitory conditions. GTP was recently found to stimulate the D1 protein degradation of photoinactivated photosystem II (Spetea, C. , Hundal, T., Lohmann, F., and Andersson, B. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6547-6552). Here we report that GTP enhances the cleavage of the D1 protein D-E loop following exposure of thylakoid membranes to either high light, low light, or repetitive single turnover flashes but not to trypsin. GTP does not stimulate D1 protein degradation in the presence of herbicides known to affect the accessibility of the cleavage site to proteolysis. However, GTP stimulates degradation that can be induced even in darkness in some photosystem II conformers following binding of the PNO8 herbicide (Nakajima, Y., Yoshida, S., Inoue, Y., Yoneyama, K., and Ono, T. (1995) Biochim. Biophys. Acta 1230, 38-44). Both the PNO8- and the light-induced primary cleavage of the D1 protein occur in the grana membrane domains. The subsequent migration of photosytem II containing the D1 protein fragments to the stroma domains for secondary proteolysis is light-activated. We conclude that the GTP effect is not confined to a specific photoinactivation pathway nor to the conformational state of the photosystem II acceptor side. Consequently, GTP does not interact with the site of D1 protein cleavage but rather enhances the activity of the endogenous proteolytic system.     

Impact of crop management on intraspecific diversity of Pseudomonas corrugata in bulk soil

Colonization at sugar beet root surfaces by seedling-inoculated biocontrol strain Pseudomonas fluorescens DR54 and native soil bacteria was followed over a period of 3 weeks using a combination of immunofluorescence (DR54-targeting specific antibody) and fluorescence in situ hybridization (rRNA-targeting Eubacteria EUB338 probe) techniques with confocal laser scanning microscopy. The dual staining protocol allowed cellular activity (ribosomal number) to be recorded in both single cells and microcolonies of strain DR54 during establishment on the root. After 2 days, the population density of strain DR54 reached a constant level at the root basis. From this time, however, high cellular activity was only found in few bacteria located as single cells, whereas all microcolony-forming cells occurring in aggregates were still active. In contrast, a low density of strain DR54 was observed at the root tip, but here many of the bacteria located as single cells were active. The native population of soil bacteria, comprising a diverse assembly of morphologically different forms and size classes, initiated colonization at the root basis only after 2 days of incubation. Hence the dual staining protocol allowed direct microscopic studies of early root colonization by both inoculant and native soil bacteria, including their differentiation into active and non-active cells and into single or microcolony-forming cells.     

Characterization of an unstable allele of the Arabidopsis HY4 locus.

The Arabidopsis HY4 gene encodes the nonessential blue light photoreceptor CRY1. Loss-of-function hy4 mutants have an elongated hypocotyl phenotype after germination under blue light. We previously analyzed 20 independent hy4 alleles produced by fast neutron mutagenesis. These alleles were grouped into two classes based on their genetic behavior and corresponding deletion size: (1) null hy4 alleles that were semidominant over wild type and contained small or moderate-sized deletions at HY4 and (2) null hy4 alleles that were recessive lethal and contained large HY4 deletions. Here we describe one additional fast neutron hy4 mutant, B144, that did not fall into either of these two classes. Mutant B144 was isolated as a heterozygote with an intermediate hy4 phenotype. One allele from this mutant, hy4-B144(Delta), contains a large deletion at HY4 and is recessive lethal. The other allele from this mutant, HY4-B144*, appears to be intact and functional but is unstable and spontaneously converts to a nonfunctional hy4 allele. In addition, HY4-B144* is lethal in homozygotes and suppresses local recombination. We discuss genetic and epigenetic mechanisms that may account for the unusual behavior of the HY4-B144* allele.     

Conformationally constrained opioid peptide analogs with novel activity profiles

Novel conformationally constrained opioid peptide analogs with antagonist, mixed agonist/ antagonist or agonist properties were developed. TIP(P)-related antagonists showed unprecedented antagonist potency and receptor selectivity, and may have potential for use in analgesia in combination with agonists. A definitive model of their receptor-bound conformation was developed. Three prototype mixed agonist/ antagonists were discovered. They represent the only known compounds with this pharmacological profile and, as expected, one of them was shown to be a potent analgesic and to produce no dependence and less tolerance than morphine. Novel dipeptide derivatives turned out to be potent and selective agonists. Because of their low molecular weight and lipophilic character, these compounds may cross the blood-brain barrier and, thus, may have potential as centrally acting analgesics.