Plant-Derived Natural Products for Parkinson's Disease Therapy.

Plant-derived natural products have made their own niche in the treatment of neurological diseases since time immemorial. Parkinson's disease (PD), the second most prevalent neurodegenerative disorder, has no cure and the treatment available currently is symptomatic. This chapter thoughtfully and objectively assesses the scientific basis that supports the increasing use of these plant-derived natural products for the treatment of this chronic and progressive disorder. Proper considerations are made on the chemical nature, sources, preclinical tests and their validity, and mechanisms of behavioural or biochemical recovery observed following treatment with various plants derived natural products relevant to PD therapy. The scientific basis underlying the neuroprotective effect of 6 Ayurvedic herbs/formulations, 12 Chinese medicinal herbs/formulations, 33 other plants, and 5 plant-derived molecules have been judiciously examined emphasizing behavioral, cellular, or biochemical aspects of neuroprotection observed in the cellular or animal models of the disease. The molecular mechanisms triggered by these natural products to promote cell survivability and to reduce the risk of cellular degeneration have also been brought to light in this study. The study helped to reveal certain limitations in the scenario: lack of preclinical studies in all cases barring two; heavy dependence on in vitro test systems; singular animal or cellular model to establish any therapeutic potential of drugs. This strongly warrants further studies so as to reproduce and confirm these reported effects. However, the current literature offers scientific credence to traditionally used plant-derived natural products for the treatment of PD.

A Computational Approach Using Ratio Statistics for Identifying Housekeeping Genes from cDNA Microarray Data.

We predict housekeeping genes from replicate microarray gene expression data of human lymphoblastoid cells and liver tissue with outliers removed using a scoring scheme, by an algorithm based on statistical hypothesis testing, assuming that such genes are constitutively expressed. A few predicted genes were examined and found to be housekeeping.

Enstrophy-based proper orthogonal decomposition for reduced-order modeling of flow past a cylinder.

Here proper orthogonal decomposition (POD) modal decomposition are performed for flow past a circular cylinder at supercritical Reynolds numbers by projecting this onto instability modes. The important task of modeling a cylinder wake by Stuart-Landau (SL) and the Stuart-Landau-Eckhaus (SLE) equation for instability modes is discussed, with the latter shown to be more consistent with multimodal pictures of POD and instability modes. The difficult task of finding the coefficients of the SLE equation is reported by taking a least squares approach for the reduced order model (ROM). The important aspect of the ROM is the choice of initial condition for the developed SLE equations, as these are stiff ordinary differential equations which are very sensitive to the choice of initial conditions. An accurate representation of enstrophy-based POD also reveals the presence of modes which occur in isolation (in comparison to modes that come in pairs) and the traditional approach of treating instability modes by SL or SLE equations does not work directly, which also reveals higher frequency variations. Quantifying effects of this mode by time-averaged Navier-Stokes equation (NSE) fail to show the variation of the phase of these isolated time-varying modes and this is captured here using direct numerical simulation (DNS) data by a multitime scale approach. A reconstructed 3-mode ROM solution and the disturbance vorticity from DNS match globally in the flow. The agreement between 3-mode SLE reconstruction and DNS also proves the consistency of the proposed method and helps explain the physical nature of the ensuing Hopf bifurcation following an instability.

Drug Utilization Study in Ophthalmology Out-patient Department of a Medical College in India.

BACKGROUND: Drug utilization studies provide a pharmacoeconomic basis for making evidence-based health-care decisions. In ophthalmology practice, rational prescribing plays a crucial role in reducing the ocular disease burden. AIM: The aim of the study was to investigate the drug utilization pattern in ophthalmology out-patient department (OPD) of a Medical College in India. SUBJECTS AND METHODS: A prospective, cross-sectional study was conducted for a period of 2 months. The prescriptions for all consecutive patients attending the OPD for the first time (first time encounter) were included and audited using a pre-designed form to record information from the OPD prescription cards of each patient. Data analysis was carried out using the descriptive statistical methods: Frequencies, percentage, mean and standard deviation. RESULTS: A total of 640 prescriptions were analyzed with the average number of drugs per prescription being 2.4 (0.9). The most common disorders diagnosed were refractive errors (31.6% [202/640]) followed by cataract, glaucoma and others. Drugs were prescribed in different dosage forms with eye drops being the most common (70.8% [1073/1516]) followed by tablets (15.9% [241/1516]), ointment (6.1% [93/1516]), syrup (1.1% [16/1516]) and others; injections contributed 2.1% (30/1516) of all dosage forms. The frequency of drug administration and duration of treatment was recorded in 96% (614/640) and 75% (480/640) of all prescriptions respectively. Antimicrobials were most commonly prescribed (36.4% [552/1516]) followed by anti-inflammatory and anti-allergic (24.2% [367/1516]), anti-glaucoma medications (21.4% [323/1516]), mydriatic and cycloplegics (7.2% [109/1516]), miotics (6.2% [94/1516]), multivitamins (4.6% [70/1516]). Drugs were predominantly prescribed in brand name 83% (1258/1516) instead of generic name. A total of 62% (940/1516) of drugs were prescribed from national essential medicine list. CONCLUSION: The present study revealed certain lacunae in the prescribing practices of the Ophthalmologists of the institute as evidenced by low generic prescribing, inadequate information about frequency of administration and duration of therapy in many prescriptions. This can be addressed through proper sensitization of clinicians in the art of rational prescribing.

Precursor of transition to turbulence: spatiotemporal wave front.

To understand transition to turbulence via 3D disturbance growth, we report here results obtained from the solution of Navier-Stokes equation (NSE) to reproduce experimental results obtained by minimizing background disturbances and imposing deterministic excitation inside the shear layer. A similar approach was adopted in Sengupta and Bhaumik [Phys. Rev. Lett. 107, 154501 (2011)], where a route of transition from receptivity to fully developed turbulent stage was explained for 2D flow in terms of the spatio-temporal wave-front (STWF). The STWF was identified as the unit process of 2D turbulence creation for low amplitude wall excitation. Theoretical prediction of STWF for boundary layer was established earlier in Sengupta, Rao, and Venkatasubbaiah [Phys. Rev. Lett. 96, 224504 (2006)] from the Orr-Sommerfeld equation as due to spatiotemporal instability. Here, the same unit process of the STWF during transition is shown to be present for 3D disturbance field from the solution of governing NSE.

Direct numerical simulation of two-dimensional wall-bounded turbulent flows from receptivity stage.

Deterministic route to turbulence creation in 2D wall boundary layer is shown here by solving full Navier-Stokes equation by dispersion relation preserving (DRP) numerical methods for flow over a flat plate excited by wall and free stream excitations. Present results show the transition caused by wall excitation is predominantly due to nonlinear growth of the spatiotemporal wave front, even in the presence of Tollmien-Schlichting (TS) waves. The existence and linear mechanism of creating the spatiotemporal wave front was established in Sengupta, Rao and Venkatasubbaiah [Phys. Rev. Lett. 96, 224504 (2006)] via the solution of Orr-Sommerfeld equation. Effects of spatiotemporal front(s) in the nonlinear phase of disturbance evolution have been documented by Sengupta and Bhaumik [Phys. Rev. Lett. 107, 154501 (2011)], where a flow is taken from the receptivity stage to the fully developed 2D turbulent state exhibiting a k(-3) energy spectrum by solving the Navier-Stokes equation without any artifice. The details of this mechanism are presented here for the first time, along with another problem of forced excitation of the boundary layer by convecting free stream vortices. Thus, the excitations considered here are for a zero pressure gradient (ZPG) boundary layer by (i) monochromatic time-harmonic wall excitation and (ii) free stream excitation by convecting train of vortices at a constant height. The latter case demonstrates neither monochromatic TS wave, nor the spatiotemporal wave front, yet both the cases eventually show the presence of k(-3) energy spectrum, which has been shown experimentally for atmospheric dynamics in Nastrom, Gage and Jasperson [Nature 310, 36 (1984)]. Transition by a nonlinear mechanism of the Navier-Stokes equation leading to k(-3) energy spectrum in the inertial subrange is the typical characteristic feature of all 2D turbulent flows. Reproduction of the spectrum noted in atmospheric data (showing dominance of the k(-3) spectrum over the k(-5/3) spectrum in Nastrom et al.) in laboratory scale indicates universality of this spectrum for all 2D turbulent flows. Creation of universal features of 2D turbulence by a deterministic route has been established here for the first time by solving the Navier-Stokes equation without any modeling, as has been reported earlier in the literature by other researchers.

Onset of turbulence from the receptivity stage of fluid flows.

The traditional viewpoint of fluid flow considers the transition to turbulence to occur by the secondary and nonlinear instability of wave packets, which have been created experimentally by localized harmonic excitation. The boundary layer has been shown theoretically to support spatiotemporal growing wave fronts by Sengupta, Rao, and Venkatasubbaiah [Phys. Rev. Lett. 96, 224504 (2006)] by a linear mechanism, which is shown here to grow continuously, causing the transition to turbulence. Here, we track spatiotemporal wave fronts to a nonlinear turbulent state by solving the full 2D Navier-Stokes equation, without any limiting assumptions. Thus, this is the only demonstration of deterministic disturbances evolving from a receptivity stage to the full turbulent flow. This is despite the prevalent competing conjectures of the event being three-dimensional and/or stochastic in nature.

Antiparkinsonian effects of aqueous methanolic extract of Hyoscyamus niger seeds result from its monoamine oxidase inhibitory and hydroxyl radical scavenging potency.

Hyoscyamus species is one of the four plants used in Ayurveda for the treatment of Parkinson's disease (PD). Since Hyoscyamus niger was found to contain negligible levels of L-DOPA, we evaluated neuroprotective potential, if any, of characterized petroleum ether and aqueous methanol extracts of its seeds in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD in mice. Air dried authenticated H. niger seeds were sequentially extracted using petroleum ether and aqueous methanol and were characterized employing HPLC-electrochemistry and LCMS. Parkinsonian mice were treated daily twice with the extracts (125-500 mg/kg, p.o.) for two days and motor functions and striatal dopamine levels were assayed. Administration of the aqueous methanol extract (containing 0.03% w/w of L-DOPA), but not petroleum ether extract, significantly attenuated motor disabilities (akinesia, catalepsy and reduced swim score) and striatal dopamine loss in MPTP treated mice. Since the extract caused significant inhibition of monoamine oxidase activity and attenuated 1-methyl-4-phenyl pyridinium (MPP+)-induced hydroxyl radical (·OH) generation in isolated mitochondria, it is possible that the methanolic extract of Hyoscyamus niger seeds protects against parkinsonism in mice by means of its ability to inhibit increased ·OH generated in the mitochondria.

2-Phenylethylamine, a constituent of chocolate and wine, causes mitochondrial complex-I inhibition, generation of hydroxyl radicals and depletion of striatal biogenic amines leading to psycho-motor dysfunctions in Balb/c mice.

Behavioral and neurochemical effects of chronic administration of high doses of 2-phenylethylamine (PEA; 25-75 mg/kg, i.p. for up to 7 days) have been investigated in Balb/c mice. Depression and anxiety, as demonstrated respectively by increased floating time in forced swim test, and reduction in number of entries and the time spent in the open arms in an elevated plus maze were observed in these animals. General motor disabilities in terms of akinesia, catalepsy and decreased swimming ability were also observed in these animals. Acute and sub-acute administration of PEA caused significant, dose-dependent depletion of striatal dopamine, and its metabolites levels. PEA caused dose-dependent generation of hydroxyl radicals in vitro in Fenton's reaction in test tubes, in isolated mitochondrial fraction, and in vivo in the striatum of mice. A significant inhibition of NADH-ubiquinone oxidoreductase (complex-I; EC: 1.6.5.3) activity suggests the inhibition in oxidative phosphorylation in the mitochondria resulting in hydroxyl radical generation. Nissl staining and TH immnunohistochemistry in brain sections failed to show any morphological aberrations in dopaminergic neurons or nerve terminals. Long-term over-consumption of PEA containing food items could be a neurological risk factor having significant pathological relevance to disease conditions such as depression or motor dysfunction. However, per-oral administration of higher doses of PEA (75-125 mg/kg; 7 days) failed to cause such overt neurochemical effects in rats, which suggested safe consumption of food items rich in this trace amine by normal population.

Spatiotemporal growing wave fronts in spatially stable boundary layers.

In fluid dynamical systems, it is not known a priori whether disturbances grow either in space or in time or as spatiotemporal structures. For a zero pressure gradient boundary layer (also known as the Blasius boundary layer), it is customary to treat it as a spatial problem, and some limited comparison between prediction and laboratory experiments exist. In the present work, the two-dimensional receptivity problem of a Blasius boundary layer excited by a localized harmonic source is investigated under the general spatiotemporal framework, by using the Bromwich contour integral method. While this approach is seen to be equivalent to the spatial study for unstable systems, here we show for the first time how spatially stable systems show spatiotemporally growing wave fronts.

Protein-protein interaction conferring stability to an extracellular acetyl (xylan) esterase produced by Termitomyces clypeatus.

Acetyl esterase (AE) activity present in the culture filtrate of Termitomyces clypeatus was separated into lower molar mass (LMM) and higher molar mass (HMM) protein fractions during BioGel P-200 gel chromatography. AE was purified as a 30 kDa nonglycosylated protein from LMM fractions by CM-Sepharose ion exchange chromatography and HPGPLC. Although the HMM fraction had a number of enzyme activities (sucrase, beta-xylosidase, beta-glucosidase, and alpha-L-arabinofuranosidase) other than AE, protein present in the fraction was eluted as a single protein peak in HPGPLC and gave a single band in native PAGE. The fraction, subsequently purified by DEAE-Sephadex chromatography, was a SDS-PAGE homogeneous 80 kDa glycoprotein, but with both AE and cellobiase activities. The aggregate dissociated during ConA-Sepharose chromatography and 30 kDa AE and 56 kDa glycosylated cellobiase were purified separately. The dissociation caused significant loss of cellobiase activity but not that of AE. AE purified from both HMM and LMM fractions was characterized to be the same enzyme in terms of molar masses, pI (7.3), and other physicochemical properties. AE as an aggregate with cellobiase showed higher thermostability, temperature optimum, and resistance toward chemical denaturants than those of purified AE. Compared to cellobiase purified earlier from the same fungus, the enzyme present with AE in the aggregate also showed higher catalytic activity, thermostability, and temperature optimum. The study indicated that the formation of such SDS-resistant enzyme aggregate was associated with significant changes in the physicochemical properties of the enzymes, mainly toward improvement of rigidity of enzymes, and sometimes with the improvement of catalytic activity.

Lateral phase separation in adsorbed binary protein films at the air-water interface.

Lateral phase separation in two-dimensional mixed films of soy 11S/beta-casein, acidic subunits of soy 11 (AS11S)/beta-casein, and alpha-lactalbumin/beta-casein adsorbed at the air-water interface has been studied using an epifluorescence microscopy method. No distinct lateral phase separation was observed in the mixed protein films when they were examined after 24 h of adsorption from the bulk phase. However, when the soy 11S/beta-casein and AS11S/beta-casein films were aged at the air-water interface for 96 h, phase-separated regions of the constituent proteins were evident, indicating that the phase separation process was kinetically limited by a viscosity barrier against lateral diffusion. In these films, beta-casein always formed the continuous phase and the other globular protein the dispersed phase. The morphology of the dispersed patches was affected by the protein composition in the film. In contrast with soy 11S/beta-casein and AS11S/beta-casein films, no lateral phase separation was observed in the alpha-lactalbumin/beta-casein film at both low and high concentration ratios in the film. The results of these studies proved that proteins in adsorbed binary films exhibit limited miscibility, and the deviation of competitive adsorption behavior of proteins at the air-water interface from that predicted by the ideal Langmuir model (Razumovsky, L.; Damodaran, S. J. Agric. Food Chem. 2001, 49, 3080-3086) is in fact due to thermodynamic incompatibility of mixing of the proteins in the binary film. It is hypothesized that phase separation in adsorbed mixed protein films at the air-water and possibly oil-water interfaces of foams and emulsions might be a source of instability in these dispersed systems.

Characterisation of the gene encoding type II DNA topoisomerase from Leishmania donovani: a key molecular target in antileishmanial therapy.

The gene encoding type II DNA topoisomerase from the kinetoplastid hemoflagellated protozoan parasite Leishmania donovani (LdTOP2) was isolated from a genomic DNA library of this parasite. DNA sequence analysis revealed an ORF of 3711 bp encoding a putative protein of 1236 amino acids with no introns. The deduced amino acid sequence of LdTOP2 showed strong homologies to TOP2 sequences from other kinetoplastids, namely Crithidia and Trypanosoma spp. with estimated identities of 86 and 68%, respectively. LdTOP2 shares a much lower identity of 32% with its human homologue. LdTOP2 is located as a single copy on a chromosome in the 0.7 Mb region in the L.donovani genome and is expressed as a 5 kb transcript. 5'-Mapping studies indicate that the LdTOP2 gene transcript is matured post-transcriptionally with the trans-splicing of the mini-exon occurring at -639 from the predicted initiation site. Antiserum raised in rabbit against glutathione S-transferase fusion protein containing the major catalytic portion of the recombinant L.donovani topoisomerase II protein could detect a band on western blots at approximately 132 kDa, the expected size of the entire protein. Use of the same antiserum for immunolocalisation analysis led to the identification of nuclear, as well as kinetoplast, antigens for L.donovani topoisomerase II. The in vitro biochemical properties of the full-length recombinant LdTOP2 when overexpressed in E.coli were similar to the Mg(II) and ATP-dependent activity found in cell extracts of L.donovani.

Magnetic field effect on the exciplex between all-s-trans- 1,4-diphenylbuta- 1,3-diene and 1,4-dicyanobenzene: a comparative study with other alpha,omega-diphenyl polyenes.

The exciplex between all-s-trans-1,4-diphenylbuta-1,3-diene and 1,4-dicyanobenzene has been studied by steady state fluorescence along with the magnetic field effect (MFE) and compared with the other alpha,omega-diphenyl polyenes. The exciplex formation and magnetic field effect are dictated by the chain length of the polyene rather than the electronic requirement of these phenomena. The wavelength dependence of the MFE confirms the presence of two different charge-transfer complexes.

RegA proteins from phage T4 and RB69 have conserved helix-loop groove RNA binding motifs but different RNA binding specificities.

The RegA proteins from the bacteriophage T4 and RB69 are translational repressors that control the expression of multiple phage mRNAs. RegA proteins from the two phages share 78% sequence identity; however, in vivo expression studies have suggested that the RB69 RegA protein binds target RNAs with a higher affinity than T4 RegA protein. To study the RNA binding properties of T4 and RB69 RegA proteins more directly, the binding sites of RB69 RegA protein on synthetic RNAs corresponding to the translation initiation region of two RB69 target genes were mapped by RNase protection assays. These assays revealed that RB69 RegA protein protects nucleotides -9 to -3 (relative to the start codon) on RB69 gene 44, which contains the sequence GAAAAUU. On RB69 gene 45, the protected site (nucleotides -8 to -3) contains a similar purine-rich sequence: GAAAUA. Interestingly, T4 RegA protein protected the same nucleotides on these RNAs. To examine the specificity of RNA binding, quantitative RNA gel shift assays were performed with synthetic RNAs corresponding to recognition elements (REs) in three T4 and three RB69 mRNAs. Comparative gel shift assays demonstrated that RB69 RegA protein has an approximately 7-fold higher affinity for T4 gene 44 RE RNA than T4 RegA protein. RB69 RegA protein also binds RB69 gene 44 RE RNA with a 4-fold higher affinity than T4 RegA protein. On the other hand, T4 RegA exhibited a higher affinity than RB69 RegA protein for RB69 gene 45 RE RNA. With respect to their affinities for cognate RNAs, both RegA proteins exhibited the following hierarchy of affinities: gene 44 > gene 45 > regA. Interestingly, T4 RegA exhibited the highest affinity towards RB69 gene 45 RE RNA, whereas RB69 RegA protein had the highest affinity for T4 gene 44 RE RNA. The helix-loop groove RNA binding motif of T4 RegA protein is fully conserved in RB69 RegA protein. However, homology modeling of the structure of RB69 RegA protein reveals that the divergent residues are clustered in two areas of the surface, and that there are two large areas of high conservation near the helix-loop groove, which may also play a role in RNA binding.

Incompatibility and Phase Separation in a Bovine Serum Albumin/beta-Casein/Water Ternary Film at the Air-Water Interface.

Thermodynamic incompatibility and two-dimensional phase separation in a bovine serum albumin (BSA)/beta-casein/water ternary film at the air-water interface has been studied using an epifluorescence microscopy technique. The incompatibility between BSA and beta-casein at the air-water interface was deduced from deviation of the experimental equilibrium composition of the proteins in the mixed saturated monolayer film from that predicted by the Langmuir-type competitive adsorption model at various bulk concentration ratios. Fluorescence microscopy of the mixed monolayer film showed distinct phase-separated BSA-rich and beta-casein-rich regions coexisting with inhomogeneous mixed regions. BSA always tended to be the dispersed phase and beta-casein the continuous phase. It is suggested that because the free energy at the "interfaces" between the phase-separated regions is generally higher than at other regions of the film, they may act as zones of instability in protein-stabilized foams and possibly emulsions. Copyright 2000 Academic Press.

Intraperoxisomal localization of very-long-chain fatty acyl-CoA synthetase: implication in X-adrenoleukodystrophy.

X-adrenoleukodystrophy (X-ALD) is a demyelinating disorder characterized by the accumulation of saturated very-long-chain (VLC) fatty acids (>C(22:0)) due to the impaired activity of VLC acyl-CoA synthetase (VLCAS). The gene responsible for X-ALD was found to code for a peroxisomal integral membrane protein (ALDP) that belongs to the ATP binding cassette superfamily of transporters. To understand the function of ALDP and how ALDP and VLCAS interrelate in the peroxisomal beta-oxidation of VLC fatty acids we investigated the peroxisomal topology of VLCAS protein. Antibodies raised against a peptide toward the C-terminus of VLCAS as well as against the N-terminus were used to define the intraperoxisomal localization and orientation of VLCAS in peroxisomes. Indirect immunofluorescent and electron microscopic studies show that peroxisomal VLCAS is localized on the matrix side. This finding was supported by protease protection assays and Western blot analysis of isolated peroxisomes. To further address the membrane topology of VLCAS, Western blot analysis of total membranes or integral membranes prepared from microsomes and peroxisomes indicates that VLCAS is a peripheral membrane-associated protein in peroxisomes, but an integral membrane in microsomes. Moreover, peroxisomes isolated from cultured skin fibroblasts from X-ALD patients with a mutation as well as a deletion in ALDP showed a normal amount of VLCAS. The consequence of VLCAS being localized to the luminal side of peroxisomes suggests that ALDP may be involved in stabilizing VLCAS activity, possibly through protein-protein interactions, and that loss or alterations in these interactions may account for the observed loss of peroxisomal VLCAS activity in X-ALD.

Identification of the RNA binding domain of T4 RegA protein by structure-based mutagenesis.

The T4 translational repressor RegA protein folds into two structural domains, as revealed by the crystal structure (Kang, C.-H. , Chan, R., Berger, I., Lockshin, C., Green, L., Gold, L., and Rich, A. (1995) Science 268, 1170-1173). Domain I of the RegA protein contains a four-stranded beta-sheet and two alpha-helices. Domain II contains a four-stranded beta-sheet and an unusual 3/10 helix. Since beta-sheet residues play a role in a number of protein-RNA interactions, one or both of the beta-sheet regions in RegA protein may be involved in RNA binding. To test this possibility, mutagenesis of residues on both beta-sheets was performed, and the effects on the RNA binding affinities of RegA protein were measured. Additional sites for mutagenesis were selected from molecular modeling of RegA protein. The RNA binding affinities of three purified mutant RegA proteins were evaluated by fluorescence quenching equilibrium binding assays. The activities of the remainder of the mutant proteins were evaluated by quantitative RNA gel mobility shift assays using lysed cell supernatants. The results of this mutagenesis study ruled out the participation of beta-sheet residues. Instead, the RNA binding site was found to be a surface pocket formed by residues on two loops and an alpha-helix. Thus, RegA protein appears to use a unique structural motif in binding RNA, which may be related to its unusual RNA recognition properties.

IL-4 selectively inhibits IL-2-triggered Stat5 activation, but not proliferation, in human T cells.

IL-2 activates several distinct signaling pathways that are important for T cell activation, proliferation, and differentiation into both Th1 and Th2 phenotypes. IL-4, the major cytokine that promotes differentiation of Th2 cells, has been shown to block signaling of the Th1-promoting cytokine IL-12. As IL-2 synergizes with IL-12 in promoting Th1 differentiation, the effects of IL-4 on IL-2 signal transduction were investigated. IL-4 suppressed activation of DNA binding and tyrosine phosphorylation of the transcription factor Stat5 by IL-2, and suppressed the expression of the IL-2-inducible genes CD25, CIS, the PGE2 receptor, and cytokine responsive (CR) genes CR1 and CR8. Activation of Stat5 by cytokines that share a common gamma receptor subunit, IL-2, IL-7, and IL-15, was suppressed by preculture in IL-4. Activation of the Jak1 and Jak3 kinases that are proximal to Stat5 in the IL-2-Jak-STAT signaling pathway was suppressed, and this correlated with inhibition of IL-2Rbeta subunit expression. In contrast to suppression of Stat5, proliferative responses to IL-2 were augmented in IL-4-cultured cells, and activation of proliferative pathways leading to activation of mitogen activated protein kinases, induction of expression of Myc, Fos, Pim-1, and cyclin D3, and decreased levels of the cyclin-dependent kinase inhibitor p27 were intact. These results identify molecular mechanisms underlying interactions between IL-4 and IL-2 in T cells and demonstrate that one mechanism of regulation of IL-2 activity is selective and differential modulation of signaling pathways.

A New Methodology for Studying Protein Adsorption at Oil-Water Interfaces.

A new methodology has been developed for studying the adsorption behavior of proteins at oil-water interfaces. This technique employs the radiotracer method for monitoring adsorption of 14C-labeled proteins at the oil-water interface. The uniqueness of the new method lies in the formation of a 1000 Å thick triglyceride oil film on the water surface. beta-casein was used to generate a standard curve for relating interfacial radioactivity (µCi/m2) to cpm at the oil-water interface. Adsorption isotherm of beta-casein was determined in the bulk protein concentration range 1.5 x 10(-5)-3.8 x 10(-3)% by weight of solution. The saturated monolayer coverage was found to be about 7.3 mg/m2. This value was quite different from other values reported in the literature. Adsorption studies with another protein, lysozyme, at the oil-water interface also revealed a high surface concentration of 3.0 mg/m2. The most significant difference between the adsorption of beta-casein at the oil-water and air-water interfaces was the lack of an induction period for the development of interfacial pressure in the former. This difference may be attributed to the attractive dispersion interaction between protein molecules and the oil phase. Copyright 1998 Academic Press.