L-selectin serves as an E-selectin ligand on cultured human T lymphoblasts

Previous studies reported that L-selectin (CD62L) on human peripheral blood neutrophils serves as an E-selectin ligand. This study shows that CD62L acquired E-selectin-binding activity following phorbol ester (PMA) treatment of the Jurkat T cell line and anti-CD3/IL-2-driven proliferation of human T lymphocytes in vitro. The recombinant porcine E-selectin/human Ig chimera P11.4 showed neuraminidase-sensitive and calcium-dependent attachment to PMA-stimulated human Jurkat T cells in a flow cytometry assay. The anti-CD62L mAb (DREG 56) blocked this binding interaction by approximately 60% and P11.4 precipitated CD62L from detergent lysates of PMA-activated Jurkat cells. In contrast, P11.4 precipitated minimal amounts of CD62L from detergent lysates of nonactivated human PBL. As reported previously, P-selectin glycoprotein ligand 1 and a distinct 130-kDa glycoprotein were the major species in these precipitates. However, T cell activation on plate-immobilized anti-CD3 and growth in low-dose IL-2 increased the percentage of CD62L molecules with E-selectin-binding activity. After two cycles of activation and culture, approximately 60-70% of the CD62L was precipitated with the P11.4 chimera. These cultured T lymphoblasts rolled avidly on both E-selectin and P-selectin at physiologic levels of linear shear stress. The DREG 56 Ab partially blocked rolling on the E-selectin substrate, whereas no effect was seen on P-selectin. Thus, CD62L on human cultured T lymphoblasts is one of several glycoproteins that interacts directly with E-selectin and contributes to rolling under flow.     

Metabolic profiling of saponins in Medicago sativa and Medicago truncatula using HPLC coupled to an electrospray ion-trap mass spectrometer

Triterpene saponins isolated from Medicago sativa (alfalfa) and Medicago truncatula roots were separated, profiled and identified using an optimized, reversed-phase HPLC with on-line photodiode array detection and electrospray ionization mass spectrometry method (HPLC/PDA/ESI/MS). ESI source polarity and solvent conditions were compared. The effects of these parameters on mass spectral attributes were determined. Ion structures were confirmed using tandem mass spectrometry (MS/MS). Fifteen saponins were identified in alfalfa (cultivars Apollo, Radius, and Kleszczewska) based upon negative-ion HPLC/PDA/ESI/MS, HPLC/PDA/ESI/MS/MS and literature data. In addition, the identification of two new malonated saponins in alfalfa are proposed. Negative-ion HPLC/PDA/ESI/MS and HPLC/PDA/ESI/MS/MS spectra were utilized along with HPLC retention times to profile and identify 27 saponins in M. truncatula (cultivar Jemalong, A17). M. truncatula yielded a much more complex mixture of saponins than observed for alfalfa. The authors are not aware of any previous reports identifying saponin glycosides in M. truncatula.     

Endophilin mutations block clathrin-mediated endocytosis but not neurotransmitter release

We have identified mutations in Drosophila endophilin to study its function in vivo. Endophilin is required presynaptically at the neuromuscular junction, and absence of Endophilin dramatically impairs endocytosis in vivo. Mutant larvae that lack Endophilin fail to take up FM1-43 dye in synaptic boutons, indicating an inability to retrieve synaptic membrane. This defect is accompanied by an expansion of the presynaptic membrane, and a depletion of vesicles from the bouton lumen. Interestingly, mutant larvae are still able to sustain release at 15%-20% of the normal rate during high-frequency stimulation. We propose that kiss-and-run maintains neurotransmission at active zones of the larval NMJ in endophilin animals.     

Ovarian expression of messenger RNA encoding the receptors for luteinizing hormone and follicle-stimulating hormone in a marsupial, the brushtail possum (Trichosurus vulpecula)

Both LH and FSH play a central role in controlling ovarian function in mammals. However, little is known about the type of ovarian cells that are responsive to LH and FSH in marsupials. We determined, using in situ hybridization, the localization of mRNA encoding the receptors (R) for LH and FSH in ovaries of brushtail possums. The mRNA encoding FSH-R was observed in granulosa cells of healthy follicles containing at least two complete layers of cells. The mRNA encoding LH-R was first observed in granulosa cells at the time of antrum formation. Cells of the theca interna expressed LH-R mRNA but not FSH-R mRNA. Neither FSH-R nor LH-R mRNA was detected in atretic follicles. Both FSH-R and LH-R mRNAs were observed in luteal tissue, but only LH-R mRNA was observed in interstitial cells. Granulosa cells from follicles of various sizes (0.5 to >2 mm in diameter) responded to LH and FSH treatment with an increase in cAMP synthesis. In contrast, luteal tissue did not respond to either FSH or LH treatment. In conclusion, expression of FSH-R in the brushtail possum ovary was similar to that observed in many eutherian mammals. However, active LH-R was expressed in granulosa cells much earlier in follicular development than has been previously observed. In addition, although mRNAs for both FSH-R and LH-R were observed, neither FSH nor LH treatment stimulated cAMP synthesis in luteal tissue.     

Self-designing two-stage trials to minimize expected costs

In the design of clinical trials, the sample size for the trial is traditionally calculated from estimates of parameters of interest, such as the mean treatment effect, which can often be inaccurate. However, recalculation of the sample size based on an estimate of the parameter of interest that uses accumulating data from the trial can lead to inflation of the overall Type I error rate of the trial. The self-designing method of Fisher, also known as the variance-spending method, allows the use of all accumulating data in a sequential trial (including the estimated treatment effect) in determining the sample size for the next stage of the trial without inflating the Type I error rate. We propose a self-designing group sequential procedure to minimize the expected total cost of a trial. Cost is an important parameter to consider in the statistical design of clinical trials due to limited financial resources. Using Bayesian decision theory on the accumulating data, the design specifies sequentially the optimal sample size and proportion of the test statistic's variance needed for each stage of a trial to minimize the expected cost of the trial. The optimality is with respect to a prior distribution on the parameter of interest. Results are presented for a simple two-stage trial. This method can extend to nonmonetary costs, such as ethical costs or quality-adjusted life years.     

Measurement of intact sulfate and glucuronide phytoestrogen conjugates in human urine using isotope dilution liquid chromatography-tandem mass spectrometry with [13C(3)]isoflavone internal standards

A method has been developed for the analysis of phytoestrogens and their conjugates in human urine using liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Stable isotopically labeled [13C(3)]daidzein and [13C(3)]genistein were synthesized and used as internal standards for isotope dilution mass spectrometry. Free aglycons and intact glucuronide, sulfate, diglucuronide, disulfate, and mixed sulfoglucuronide conjugates of isoflavones and lignans were observed in naturally incurred urine samples. Sample pretreatment was not necessary, other than addition of internal standards and pH adjustment. Urine was injected directly onto the analytical column. The limits of detection were generally <50ng/ml, precision was generally <10% CV for conjugates. Total hydrolyzed daidzein and genistein were measured against quality assurance urine sample and were accurate to within 12%. The accuracy of conjugate measurement can not be ascertained, as no reference samples are available. The mean sum of daidzein and its conjugates was within 20% of the hydrolyzed value. Concentrations of the free aglycons of up to 22% of genistein and 18% of daidzein were observed. The average pattern was ca. 54% 7-glucuronide, 25% 4(')-glucuronide, 13% monosulfates, 7% free daidzein, 0.9% sulfoglucuronides, 0.4% diglucuronide, and <0.1% disulfate. Selective enzymatic deconjugation with glucuronidase and mixed glucuronidase/sulfatase were used to validate the accuracy of the quantitation of the intact daidzein conjugates. There were no apparent sex differences, or conditioning effects on the conjugation profile of isoflavones after chronic dosing.     

Intrastrand DNA cross-links as tools for studying DNA replication and repair: two-, three-, and four-carbon tethers between the N(2) positions of adjacent guanines

A general protocol for preparation of oligonucleotides containing intrastrand cross-links between the exocyclic amino groups of adjacent deoxyguanosines has been developed. A series of 2, 3, and 4 methylene cross-links was incorporated site-specifically into an 11-mer (5'-GGCAGGTGGTG-3', cross-linked positions are underlined) via a reaction between oligonucleotide containing 2-fluoro-O(6)-trimethylsilylethyl deoxyinosines and the appropriate diamine (ethylenediamine, 1,3-diaminopropane, 1,4-diaminobutane). These cross-linked-oligonucleotides were studied for their ability to bend DNA by the method of Koo and Crothers [Koo, H. S., and Crothers, D. M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 1763-1767] in which the mobility of ligated oligomers in nondenaturing polyacrylamide gels is evaluated. It was found that all cross-links induced bending (2-carbon cross-link, 30.0 +/- 4.0 deg/turn; 3-carbon cross-link, 11.7 +/- 1.6 deg/turn; 4-carbon cross-link, 7.4 +/- 1.0 deg/turn). Despite the differing extent of helical distortion exhibited by the cross-links, all appeared to be equally blocking to replication by the Escherichia coli polymerases, pol I, pol II, and pol III. In contrast, when incision of the cross-links by the E. coli UvrABC nucleotide incision complex was studied, the extent of incision of the cross-link was found to correlate closely with the degree of bending measured in the gel mobility assay, i.e., the efficiency of incision was 2-carbon > 3-carbon > 4-carbon.     

Nitrite inhibits hydrogen production and kills the cattle parasite Tritrichomonas foetus

AIMS: To investigate the effects of NaNO2 on the microaerophilic flagellated protozoan, Tritrichomonas foetus KV1, an economically important cattle parasite that inhabits the vagina and can spread rapidly through herds of animals by sexual transmission and leads to abortion of foetal calves. METHODS AND RESULTS: Growth of the parasite was inhibited by 50% in the presence of 4 mm NaNO2; immediate killing occurred at 10 mm. Mass spectrometric monitoring of gases showed that H2 and CO2 evolution were inhibited by NaNO2, and electron paramagnetic resonance spectrometry revealed a signal similar to that of a thiolate-iron-NO complex. Growth with sublethal concentrations of NaNO2 yielded organisms that produced ethanol rather than H2. CONCLUSIONS: NaNO2 probably inactivates FeS protein(s) of hydrogenosomes so as to inhibit the conversion of pyruvate (derived from maltose in the growth medium) to H2 and acetate. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of NaNO2 as a topical antitrichomonal agent in veterinary practice is a possibility. At present, slaughter of infected animals is the favoured method of control.     

Limits to lifespan

It has long-been accepted that normal somatic cells have intrinsic mechanisms that limit their proliferative lifespan. Recent work has now challenged this view by demonstrating that extrinsic factors might be determining proliferative potential.     

A novel and efficient method for synthetic carbohydrate conjugate vaccine preparation: synthesis of sialyl Tn-KLH conjugate using a 4-(4-N-maleimidomethyl) cyclohexane-1-carboxyl hydrazide (MMCCH) linker arm

STn (NeuAc26GalNAc-O-Ser/Thr) is a carbohydrate epitope overexpressed in various human carcinomas. Clinical trials are underway using synthetic STn or STn trimeric glycopeptides [STn, cluster; STn(c) conjugated with keyhole limpet hemocyanin (KLH) as active specific immunotherapy for these cancers. These vaccines have been prepared by conjugating a crotyl ethyl amide derivative of STn or STn(c) to KLH by direct reductive amination after ozonolysis. In the case of STn(c) the conjugation efficiency and the resulting epitope ratios were low. This may be due to steric hinderance of the short spacer arm. To overcome these difficulties, without resynthesis, the STn(c) glycopeptide was modified by attachment of an MMCCH (4-(4-N-maleimidomethyl) cyclohexane-1-carboxyl hydrazide) spacer arm to the aldehyde derivative, and then conjugated with thiolated KLH. This method gave a higher epitope ratio and yield than the direct method. The STn(c)-MMCCH-KLH conjugate induced high titer antibodies in mice against STn(c). This method may be generally applicable for large synthetic oligosaccharides.