Interactions of α1–proteinase inhibitor with small ligands of therapeutic potential: binding with retinoic acid

Human α₁–proteinase inhibitor (α₁–PI), also known as α₁-antitrypsin, is the most abundant plasma serine protease inhibitor (serpin). It is best recognized for inhibition of neutrophil elastase. The α₁–PI interactions with non-protease ligands were investigated mainly in regards to those molecules that may block the aggregation of α₁–PI Z mutant. The objective of this study was to evaluate the potential of α₁–PI to bind small non-peptide ligands of pharmaceutical interest that may attain additional properties to currently available α₁–PI therapeutic preparations. Among putative ligands of bio-medical interest examined in this study, all-trans retinoic acid (RA) was selected due to its recently proposed roles in the lungs, and as an efficient optical probe. The results of this study, including absorption spectroscopy data, fluorescence quenching and the protein-induced chirality of the visible circular dichroism strongly suggest that α₁–PI does bind RA in vitro to non-covalent complexes of up to two moles of RA per one mole of the protein. To our knowledge, this is the first report that provides experimental evidence of the α₁–PI potential towards bi-functional drugs via a combination with RA, or potentially other molecules of pharmaceutical interest, that ultimately, may enhance currently available α₁–PI therapies.     

Reactivity of penicillin-binding proteins with peptidoglycan-mimetic beta-lactams: what's wrong with these enzymes?

Beta-lactams exert their antibiotic action through their inhibition of bacterial DD-peptidases (penicillin-binding proteins). Bacteria, in general, carry several such enzymes localized on the outside of their cell membrane to catalyze the final step in cell wall (peptidoglycan) synthesis. They have been classified into two major groups, one of high molecular weight, the other of low. Members of the former group act as transpeptidases in vivo, and their inhibition by beta-lactams leads to cessation of bacterial growth. The latter group consists of DD-carboxypeptidases, and their inhibition by beta-lactams is generally not fatal to bacteria. We have previously shown that representatives of the former group are ineffective at catalyzing the hydrolysis/aminolysis of peptidoglycan-mimetic peptides in vitro [Anderson et al. (2003) Biochem. J. 373, 949-955]. The theme of these experiments is expanded in the present paper where we describe the synthesis of a series of beta-lactams (penicillins and cephalosporins) containing peptidoglycan-mimetic side chains and the kinetics of their inhibition of a panel of penicillin-binding proteins spanning the major classes (Escherichia coli PBP 2 and PBP 5, Streptococcus pneumoniae PBP 1b, PBP 2x and PBP 3, the Actinomadura R39 DD-peptidase, and the Streptomyces R61 DD-peptidase). The results of these experiments mirror and expand the previous results with peptides. Neither peptides nor beta-lactams with appropriate peptidoglycan-mimetic side chains react with the solubilized constructs of membrane-bound penicillin binding proteins (the first five enzymes above) at rates exceeding those of generic analogues. Such peptides and beta-lactams do react at greatly enhanced rates with certain soluble low molecular weight enzymes (R61 and R39 DD-peptidases). The former result is unexpected and interesting. Why do the majority of penicillin-binding proteins not recognize elements of local peptidoglycan structure? Possible answers are discussed. That this question needs to be asked casts fascinating shadows on current studies of penicillin-binding proteins for new drug design.     

Interaction of human α-lactalbumin with fatty acids: Determination of binding parameters

The interaction of holo- and apo-forms of human alpha-lactalbumin with fatty acids was studied by a partition equilibrium method. Apo-alpha-lactalbumin, obtained by treatment with EDTA, displays one binding site for fatty acids, the association constants for oleic and palmitic acids being 1.9.10(6) and 4.2.10(5) M(-1), respectively. However, holo-alpha-lactalbumin was unable to bind fatty acids as measured by this technique. Likewise, no fatty acids bound to holo-alpha-lactalbumin, isolated using nondenaturing conditions, were detected by gas chromatography. These results demonstrate that the conformational change induced in alpha-lactalbumin by the removal of calcium enables the protein to interact with fatty acids.     

Treatment of Chronic Insomnia with Yoga: A Preliminary Study with Sleep–Wake Diaries

There is good evidence for cognitive and physiological arousal in chronic insomnia. Accordingly, clinical trial studies of insomnia treatments aimed at reducing arousal, including relaxation and meditation, have reported positive results. Yoga is a multicomponent practice that is also known to be effective in reducing arousal, although it has not been well evaluated as a treatment for insomnia. In this preliminary study, a simple daily yoga treatment was evaluated in a chronic insomnia population consisting of sleep-onset and/or sleep-maintenance insomnia and primary or secondary insomnia. Participants maintained sleep–wake diaries during a pretreatment 2-week baseline and a subsequent 8-week intervention, in which they practiced the treatment on their own following a single in-person training session with subsequent brief in-person and telephone follow-ups. Sleep efficiency (SE), total sleep time (TST), total wake time (TWT), sleep onset latency (SOL), wake time after sleep onset (WASO), number of awakenings, and sleep quality measures were derived from sleep–wake diary entries and were averaged in 2-week intervals. For 20 participants completing the protocol, statistically significant improvements were observed in SE, TST, TWT, SOL, and WASO at end-treatment as compared with pretreatment values.     

Pelizaeus-Merzbacher disease: identification of heterozygotes with magnetic resonance imaging?

Summary We report magnetic resonance imaging (MRI) findings in two obligate and four facultative carriers for the classical X-linked from of Pelizaeus-Merzbacher disease (PMD). In T2-weigthed images MR revealed bilateral multiple areas with signal hyperintensity in the periventricular and subcortical white matter in five women. Until suitable and closely linked DNA probes are found for heterozygote determination, MRI may represent a suitable means for carrier detection in individuals at risk in PMD families.Presented in part at the meeting (recent result session) of European Federation of Child Neurology Societies, June 1987, Hyvinkää, Finland, and the fall meeting of the Clinical Genetics Society, November 1987, London, UK     

Review of Mexican Species of Podogaster Brullé (Hymenoptera: Ichneumonidae: Anomaloninae) with Description of Two New Species

Two new species of Podogaster Brullé, Podogaster brunneus n. sp. and Podogaster lagartensis n. sp., are described. The material was collected with Malaise traps operated for a year in the Ría Lagartos Biosphere Reserve, a dry tropical area of Southeast Mexico. Podogaster rosteri Gauld & Bradshaw is synonymized with Podogaster mexicanus (Cresson). A key to the Mexican species is also provided.     

Attracting carnivorous arthropods with plant volatiles: The future of biocontrol or playing with fire?

Herbivore-induced plant volatiles (HIPVs) are potent attractants for entomophagous arthropods and researchers have long speculated that HIPVs can be used to lure natural enemies into crops, reestablishing predator–prey relationships that become decoupled in disturbed agricultural habitats. This speculation has since become reality as the number of field trials investigating HIPV-mediated attraction and its consequences for pest suppression has risen dramatically over the past 10 years. Here, I provide an overview of recent field efforts to augment natural enemy populations using HIPVs, with emphasis on those studies manipulating synthetic compounds in controlled-release dispensers, and outline a prospectus for future research needs. Specifically, I review and discuss: (i) choice of compounds and release rates; (ii) functional changes in predator and parasitoid communities; (iii) non-target effects; (iv) mechanisms of attraction and prey suppression; (v) spatial- and landscape-level considerations; (vi) context-dependent responses; and (vii) temporal stability of attraction.     

Interaction of Tumor with Its Micro-environment: A Mathematical Model

This paper is concerned with early development of transformed epithelial cells (TECs) in the presence of fibroblasts in the tumor micro-environment. These two types of cells interact by means of cytokines such as transforming growth factor (TGF-β) and epidermal growth factor (EGF) secreted, respectively, by the TECs and the fibroblasts. As this interaction proceeds, TGF-β induces fibroblasts to differentiate into myofibroblasts which secrete EGF at a larger rate than fibroblasts. We monitor the entire process in silico, in a setup which mimics experiments in a Tumor Chamber Invasion Assay, where a semi-permeable membrane coated by extracellular matrix (ECM) is placed between two chambers, one containing TECs and another containing fibroblasts. We develop a mathematical model, based on a system of PDEs, that includes the interaction between TECs, fibroblasts, myofibroblasts, TGF-β, and EGF, and we show how model parameters affect tumor progression. The model is used to generate several hypotheses on how to slow tumor growth and invasion. In an Appendix, it is proved that the mathematical model has a unique global in-time solution.     

Cancer: a single disease with a multitude of manifestions?

The relationships of critical nutrients such as plant phenolics, vitamins, minerals and lipids are considered with respect to the incidence of a variety of cancers, and analyzed in terms of how these nutrient deficiencies alter immune function, DNA integrity and cell proliferation. With a significant correlation found between cancer and these nutrient deficiencies, the hypothesis is presented here that nutrition could provide a unifying perception of cancer and recast it as a single disease. This further suggests that a coordinated administration of specific, critical nutrients to cancer patients could lead to the reversal of the disease. It is also proposed that the concurrent presence of a variety of nutritional deficiencies in cancer patients requires a multilevel, systemic approach to this disease as opposed to the single active therapeutic agent approach that is the cornerstone of contemporary research and pharmacology.     

Genetic regulation of γ gene expression: Study of the interaction of β-thalassemia with heterocellular HPFH

Summary A family has been observed in which a gene for heterocellular herediatry persistence of fetal hemoglobin (HPFH), probably identical to that previously described as Swiss type HPFH, has been inherited together with -thalassemia. The interaction of these two genes resulted in -thalassemia heterozygotes with unusually high levels of fetal hemoglobin (3.6–6.15), heterogeneously distributed. Globin synthesis studies showed a similar degree of chain imbalance in the heterocellular HPFH- thalassemia compound heterozygotes and in the heterozygous -thalassemia member of the family. On the basis of the pattern of genetic transmission of these two characters it can be concluded that the HPFH determinant does not behave as an allele of the complex.     

Upregulation of the plant protein remorin correlates with dehiscence and cell maturation: A link with the maturation of plasmodesmata?

Remorins are plant-specific proteins found associated with plasma membrane microdomains, called lipid rafts. Recently, we have shown that this lipid raft marker also accumulated at plasmodesmata, likely within the plasma membrane lining these structures. Here, we have investigated the gene expression and protein accumulation patterns of remorin at the organ and cell type levels. We show that remorin level is significantly increased in dehiscent, mature and ageing tissues, as well as in source parts of the leaves, where mature branched plasmodesmata are in majority. these results suggest that remorin predominantly associates with mature branched plasmodesmata.Key words: plasma membrane, lipid rafts, plant protein remorin, plasmodesmata     

Inhibition of the β-carbonic anhydrases from Mycobacterium tuberculosis with C-cinnamoyl glycosides: Identification of the first inhibitor with anti-mycobacterial activity

A small series of C-cinnamoyl glycoside containing the phenol moiety was tested for the inhibition of the three Mycobacterium tuberculosis β-carbonic anhydrases (CAs, EC 4.2.1.1) with activities in the low micromolar range detected. The compounds were also tested for the inhibition of growth of M. tuberculosis H₃₇Rv strain, leading to the identification of (E)-1-(2′,3′,4′,6′-tetra-O-acetyl-β-d-glucopyranosyl)-4-(3-hydroxyphenyl)but-3-en-2-one (1) as the first carbonic anhydrase inhibitor with anti-tubercular activity.     

Some properties of KCl-filled microelectrodes: Correlation of potassium “leakage” with tip resistance

Summary This study was undertaken in order to determine directly the rates of K leakage (J _K) out of the tips of microelectrodes into a solution of 100 mM KCl (approximating the K concentration of the cell interior) and to relate these rates to the concentration of the filling solution and the tip resistance. The values ofJ _K for electrodes filled with 3m KCl having resistances of 16 and 30 M (when measured in 3m KCl) were 10 and 5.5 fmol/sec, respectively. When the same electrodes were filled with 0.5m KCl, the resistances (measured in 0.5m KCl) increased to 62 and 115 M, respectively, andJ _K fell to 1.8 and 1.0 fmol/sec, respectively. These values are in reasonable agreement with what would be expected from theoretical considerations if leakage of KCl were the result of diffusion plus convective flow due to the hydrostatic pressure of the filling solution.We conclude that K leakage out of microelectrodes filled with 3m KCl is unnecessarily high; leakage can be reduced fivefold by filling electrodes with 0.5m KCl without incurring significant increases in tip or diffusion potentials or unmanageable tip resistances.Finally, the lowest rate of K leakage observed (1 fmol/sec) is still very considerable for the case of animal cells with an intracellular volume of approximately 1 pl and a K content of approximately 100 fmol. The finding of stable intracellular potentials, often for many minutes, in some tissues suggests that K which enters the cell rapidly diffuses into neighboring cells via high conductance intercellular communications.     

Müllerian adenosarcoma of the uterus: report of a case with imprint cytology

BACKGROUND: Müllerian adenosarcoma is a rare morphologic variant of uterine sarcoma that, although well described histologically, is scarcely mentioned in the cytologic literature. CASE: A 75-year-old female was suspected of having atypical endometrial hyperplasia on an endometrial smear. However, subsequent imaging techniques revealed the presence of a bulky, polypoid mass filling the uterine cavity. On pathologic examination of the hysterectomy specimen, the polypoid tumor was diagnosed as mullerian adenosarcoma, homologous, with sarcomatous overgrowth, in which the sarcomatous component was compatible with high grade endometrial stromal sarcoma. Imprint smears of the tumor consisted of two morphologic patterns, sarcomatous and glandular. The sarcomatous tumor cells, with coarse chromatin and relatively scant cytoplasm, formed small aggregates or appeared alone. These cells were semiround or oval and had conspicuous nucleoli. In addition to these observations, small and large clusters of glandular cells with mild atypism were interspersed with the sarcomatous cells. CONCLUSION: Cytologic examination of müllerian adenosarcoma well reflects its pathologic features.     

Development of phosphocellulose paper-based screening of inhibitors of lipid kinases: Case study with PI3Kβ

The phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases that regulate the cellular signal transduction pathways involved in cell growth, proliferation, survival, apoptosis, and adhesion. Deregulation of these pathways are common in oncogenesis, and they are known to be altered in other metabolic disorders as well. Despite its huge potential as an attractive target in these diseases, there is an unmet need for the development of a successful inhibitor. Unlike protein kinase inhibitors, screening for lipid kinase inhibitors has been challenging. Here we report, for the first time, the development of a radioactive lipid kinase screening platform using a phosphocellulose plate that involves transfer of radiolabeled [γ-³²P]ATP to phosphatidylinositol 4,5-phosphate forming phosphatidylinositol 3,4,5-phosphate, captured on the phosphocellulose plate. Enzyme kinetics and inhibitory properties were established in the plate format using standard inhibitors, such as LY294002, TGX-221, and wortmannin, having different potencies toward PI3K isoforms. ATP and lipid apparent K_m for both were determined and IC₅₀ values generated that matched the historical data. Here we report the use of a phosphocellulose plate for a lipid kinase assay (PI3Kβ as the target) as an excellent platform for the identification of novel chemical entities in PI3K drug discovery.