Simple liquid chromatographic method for the determination of physostigmine and its metabolite eseroline in rat plasma: application to a pharmacokinetic study

Physostigmine, an anticholinergic drug, and its metabolite eseroline can be quantitated by high-performance liquid chromatography (HPLC) with photodiode-array detection. After addition of the structurally related internal standard (-)-N-methylphysostigmine, rat plasma samples were extracted and cleaned using a Varian Bond Elut C(18) column. The methanol-ammonia (98:2) eluate was evaporated to dryness and reconstituted with 0.01 M sodium dihydrogenphosphate (pH 3). Physostigmine and eseroline were separated on an Alltech Ultrasphere Silica column (250x4.6 mm I.D.; particle size 5 micrometer) at a flow-rate of 1 ml/min, with a mobile phase of 0.01 M sodium dihydrogenphosphate (pH 3)-acetonitrile (85:15). The limits of detection were 10 and 25 ng/ml for physostigmine and eseroline, respectively; the signal-to-noise ratio for this concentration was approximately 3:1. Spiked rat plasma containing 0.1-2.5 microgram/ml of physostigmine and eseroline gives good linearity. The average percentage recovery from five spiked plasma samples was 88.0+/-2.9 and 61.1+/-5.6% for physostigmine and eseroline, respectively. Within the concentration range 0.1-2.5 microgram/ml, the within-day precision was 1.9-8.3 and 3.0-7.7% for physostigmine and eseroline, respectively, and the between-day precision was 4.1-9.3 and 3.7-11% for physostigmine and eseroline, respectively. The method is rapid, simple and reliable, thus it is suitable for pharmacokinetic studies in the rat.     

A defect in lineage fate decision during fetal thymic invariant NKT cell development may regulate susceptibility to type 1 diabetes

A numerical and functional deficiency in invariant NKT (iNKT) cells detectable by 3 wk of age in the thymus and spleen mediates the pathogenesis of type 1 diabetes in NOD mice, but the stage of T cell development at which this deficiency first occurs is unknown. We report in this study that this deficiency develops after the CD4(+)CD8(+) double-positive stage of thymic T cell development and is due to a lineage-specific depletion of CD4(-)CD8(-) double-negative alphabeta T cells and iNKT cells from the thymus between embryonic day 18 and day 1 after birth. Thus, an inheritable defect in a lineage fate decision that elicits a deficiency in fetal thymic iNKT cell development may predispose to susceptibility to type 1 diabetes.     

Chemokines in acute respiratory distress syndrome

A characteristic feature of all inflammatory disorders is the excessive recruitment of leukocytes to the site of inflammation. The loss of control in trafficking these cells contributes to inflammatory diseases. Leukocyte recruitment is a well-orchestrated process that includes several protein families including the large cytokine subfamily of chemotactic cytokines, the chemokines. Chemokines and their receptors are involved in the pathogenesis of several diseases. Acute lung injury that clinically manifests as acute respiratory distress syndrome (ARDS) is caused by an uncontrolled systemic inflammatory response resulting from clinical events including major surgery, trauma, multiple transfusions, severe burns, pancreatitis, and sepsis. Systemic inflammatory response syndrome involves activation of alveolar macrophages and sequestered neutrophils in the lung. The clinical hallmarks of ARDS are severe hypoxemia, diffuse bilateral pulmonary infiltrates, and normal intracardiac filling pressures. The magnitude and duration of the inflammatory process may ultimately determine the outcome in patients with ARDS. Recent evidence shows that activated leukocytes and chemokines play a key role in the pathogenesis of ARDS. The expanding number of antagonists of chemokine receptors for inflammatory disorders may hold promise for new medicines to combat ARDS.     

Identification of a novel family of proteins in snake venoms. Purification and structural characterization of nawaprin from Naja nigricollis snake venom

The three-dimensional structure of nawaprin has been determined by nuclear magnetic resonance spectroscopy. This 51-amino acid residue peptide was isolated from the venom of the spitting cobra, Naja nigricollis, and is the first member of a new family of snake venom proteins referred to as waprins. Nawaprin is relatively flat and disc-like in shape, characterized by a spiral backbone configuration that forms outer and inner circular segments. The two circular segments are held together by four disulfide bonds, three of which are clustered at the base of the molecule. The inner segment contains a short antiparallel beta-sheet, whereas the outer segment is devoid of secondary structures except for a small turn or 310 helix. The structure of nawaprin is very similar to elafin, a human leukocyte elastase-specific inhibitor. Although substantial parts of the nawaprin molecule are well defined, the tips of the outer and inner circular segments, which are hypothesized to be critical for binding interactions, are apparently disordered, similar to that found in elafin. The amino acid residues in these important regions in nawaprin are different from those in elafin, suggesting that nawaprin is not an elastase-specific inhibitor and therefore has a different function in the snake venom.     

The control of gene conversion properties and corresponding-site interference: the effects of conversion control factor 5 on conversion at locus w9 in Ascobolus immersus

The controls of various aspects and parameters of gene conversion at locus w-9 in the fungus Ascobolus immersus were investigated, along with positive and negative corresponding-site interference in meiotic chromatid pairing. When conversion control factor 5 alleles A and B were heterozygous, conversion frequencies at the closely linked target locus w9 were reduced to 3%, compared with 10.7% when A or B was homozygous, through effects on hybrid-DNA (h-DNA) formation parameters gamma1 and gamma2. In different ways, not related to whether ccf-5 alleles were homozygous or heterozygous, ccf-5 also affected parameters relating to the relative frequencies of asymmetric and symmetric h-DNA, the frequency with which the chromatid bearing the wild-type allele was the invading chromatid in asymmetric h-DNA, and h-DNA correction parameters for the frequency and direction of correction of mispairs. Corresponding-site interference is interference between the two pairs of non-sister chromatids of a bivalent in h-DNA formation at exactly corresponding sites. This interference was positive in the high conversion frequency crosses homozygous for ccf-5 alleles but was strongly negative in the low conversion frequency crosses heterozygous for ccf-5 alleles, through differential effects on parameters gamma1 and gamma2. Models of chromatid pairing are discussed.     

Interaction of light quality and benzimidazole on pigment contents and on photochemical activity of chloroplasts isolated from detached senescing wheat (Triticum aestivum) leaves

The possibilities of an interaction between light and the growth regulator benzimidazole in retarding senescence-induced changes in activity of chloroplasts isolated from detached wheat ( Triticum aestivum L. cv. Kalyansona) leaves have been investigated. The effect of benzimidazole on the rate of degradation of chlorophylls in light depended on the quality of light used. Far-red irradiation given to detached leaves in the presence of benzimidazole retarded the pigment loss more significantly than did white or red light of similar intensity. Senescence-induced loss in chloroplast photochemical activity was higher than the loss of chlorophylls. Loss of photosystem I activity was greater than photosystem II activity. Benzimidazole maintained the gramicidin-mediated enhancement in whole chain electron transport uniformly throughout the incubation period irrespective of the light quality. There was no effect of light and benzimidazole in retarding the loss in photochemical activity, although the same preserved the chlorophyll contents as well as characteristics of the chloroplast absorption spectrum.     

Monoclonal antibodies directed against the galactose-binding lectin of Entamoeba histolytica enhance adherence.

The Entamoeba histolytica galactose-binding lectin is a surface glycoprotein composed of 170- and 35-kDa subunits. Inhibition of this lectin with galactose or anti-170 kDa subunit polyclonal antibody blocks amebic adherence to target cells and colonic mucin glycoproteins. We describe the properties of 10 mAb with specificity for the 170-kDa subunit. Based on competitive binding studies, six nonoverlapping antigenic determinants on the lectin were identified. The effect of the mAb on adherence of amebic trophozoites to both Chinese hamster ovary (CHO) cells and human colonic mucins was measured. Antilectin antibodies directed against epitopes 1 and 2 enhanced adherence, with the number of amebae having at least three adherent CHO cells increasing with the addition of epitope 1 mAb from 26 +/- 9 to 88 +/- 2% and the binding of colonic mucins increasing from 34 +/- 1 to 164 +/- 3 pg/10(5) amebae. Antibody-enhanced adherence remained 90 to 100% galactose inhibitable, occurred at 4 degrees C and was not Fc mediated. Univalent Fab fragments of epitope 1 mAb augmented mucin binding by 238% and CHO cell adherence by 338%. The binding of purified lectin to CHO cells was increased from 1.1 +/- 0.1 to 2.4 +/- 0.3 ng/10(3) CHO cells by mAb directed to epitope 1, demonstrating that enhanced adherence was due to direct activation of the lectin. mAb to epitope 3 bound to the lectin only upon its solubilization from the membrane and had no effect on adherence. Adherence to CHO cells and mucins was inhibited from 50 to 75% by mAb to epitopes 4 and 5; epitope 6 mAb inhibited amebic adherence to CHO cells but not mucins. The pooled sera from 10 patients with amebic liver abscess blocked the binding to the 170-kDa subunit of mAb directed to all six epitopes. Striking individual variations in the effects of immune sera on adherence were observed. Although the sera of all 44 South African patients with amebic liver abscess had high titer anti-lectin antibodies, 16 patients' sera significantly (more than 3 SEM) enhanced adherence whereas 25 patients' sera significantly inhibited adherence. Antilectin antibodies exert profound functional effects on the interaction of E. histolytica with target cells and human colonic mucins. Exploration of the clinical consequences of adherence-enhancing and inhibitory antibody responses may give insight into the role of antilectin antibodies in immunity to invasive amebiasis.     

Inhibition of oligopeptide transport in S. cerevisiae by a peptide-poly (ethylene glycol) conjugate

A soluble macromolecule-peptide conjugate, [(Met)₃-OPEG] inhibited the uptake of Met-Met-[${}^{14}\text{C}$] Met into $\text{S.}\text{cerevisiae}$. Uptake of leucine into this strain was not affected by Met₃-OPEG under identical conditions. Inhibition by the macromolecular inhibitor was competitive (K_I = 5.1 × 10^?5M)and followed the structural requirments of the peptide transport systems in $\text{S.}\text{cerevisiae}$ and $\text{C.}$$\text{albicans}$. These findings give the first example of inhibition of metabolite transport by a synthetic macromolecular competitor.     

Uptake and release of ferritin iron. Surface effects and exchange within the crystalline core

The uptake and subsequent release of iron by apoferritin and ferritin was studied by using labelled iron ((59)Fe). The experimental results are consistent with predictions arising from a model system developed in the interpretation of previous experiments. In this model, uptake and release of ferritin iron is controlled by the available surface area of the small crystalline particles of hydrous ferric oxide found within the ferritin molecule. Evidence is also presented for the exchange of Fe(3+) ions among the various cation sites within these crystallites.     

Investigation on the potential of Mueller matrix imaging for digital staining

Digital staining based on Mueller matrix measurements and their derivatives was investigated. Mueller matrix imaging was performed at the microscopic level on gastric tissue sections. Full Mueller matrices (4 × 4) were reconstructed using recorded images, followed by the extraction of polarization parameters. The most effective parameters and their combinations were extracted from Mueller matrix elements, principal component scores and polarization parameters respectively to classify samples into three categories – i.e. cancer, dysplasia and intestinal metaplasia/normal glands for various regions of interest sizes. It was observed that two‐step classification yielded higher classification accuracy than the traditional one‐step classification and that pixel classification based on Mueller matrix elements yielded higher accuracy than that based on polarization parameters and derived principal components. Moreover, Mueller matrix images with a lower spatial resolution generated higher classification accuracy but those with a higher spatial resolution revealed more morphological details.ns.

The original stained image (top) and the digital staining image (bottom).