Acute exercise adjustments of cardiovascular autonomic control in diabetic rats

Introduction: We evaluated the role of cardiovascular autonomic changes in hemodynamics at rest and in response to exercise in streptozotocin-induced diabetic rats. Methods: Male Wistar rats were divided into nondiabetic (ND, n = 8) and diabetic (D, n = 8) groups. Arterial pressure signals were recorded in the basal state and after atropine or propranolol injections at rest, during exercise and during recovery. Results: At rest, vagal tonus was reduced in D (37 +/- 3 bpm) in comparison with the ND group (61 +/- 9 bpm). Heart rate during exercise was lower in D in relation to ND rats associated with reduced vagal withdrawal in the D group. The D rats had an increase in vagal tonus in the recovery period (49 +/- 6 bpm). Conclusions: Exercise-induced hemodynamic adjustment impairment in diabetic rats was associated with reduced cardiac vagal control. The vagal dysfunction was attenuated after aerobic exercise, reinforcing the positive role of this approach in the management of cardiovascular risk in diabetics. Muscle Nerve 46: 96101, 2012

Expression of NADPH Oxidase by Trophoblast Cells: Potential Implications for the Postimplanting Mouse Embryo

Cytochemical localization of hydrogen peroxide-generating sites suggests NADPH (nicotinamide adenine dinucleotide 3-phosphate [ reduced form]) oxidase expression at the maternal-fetal interface. To explore this possibility, we have characterized the expression and activity of the NADPH oxidase complex in trophoblast cells during the postimplantation period. Implantation sites and ectoplacental cones (EPCs) from 7.5-gestational day embryos from CD1 mice were used as a source for expression analyses of NADPH oxidase catalytic and regulatory subunits. EPCs grown in primary culture were used to investigate the production of superoxide anion through dihydroxyethidium oxidation in confocal microscopy and immunohistochemical assays. NADPH subunits Cybb (gp91phox), Cyba (p22phox), Ncf4 (p40phox), Ncf1 (p47phox), Ncf2 (p67phox), and Rac1 were expressed by trophoblast cells. The fundamental subunits of membrane CYBB and cytosolic NCF2 were markedly upregulated after phorbol-12-myristate-13-acetate (PMA) treatment, as detected by quantitative real-time PCR, Western blotting, and immunohistochemistry. Fluorescence microscopy imaging showed colocalization of cytosolic and plasma membrane NADPH oxidase subunits mainly after PMA treatment, suggesting assembly of the complex after enzyme activation. Cultured EPCs produced superoxide in a NADPH-dependent manner, associating the NADPH oxidase-mediated superoxide production with postimplantation trophoblast physiology. NADPH-oxidase cDNA subunit sequencing showed a high degree of homology between the trophoblast and neutrophil isoforms of the oxidase, emphasizing a putative role for reactive oxygen species production in phagocytic activity and innate immune responses.

Increased Glyceride-Glycerol Synthesis in Liver and Brown Adipose Tissue of Rat: In-Vivo Contribution of Glycolysis and Glyceroneogenesis

We have previously shown that a high-protein, carbohydrate-free diet can decrease the production of glycerol-3-phosphate (G3P) from glucose and increase glyceroneogenesis in both brown (BAT) and epididymal (EAT) adipose tissue. Here, we utilized an in-vivo approach to examine the hypothesis that there is reciprocal regulation in the G3P synthesis from glucose (via glycolysis) and glyceroneogenesis in BAT, EAT and liver of fasted rats and cafeteria diet-fed rats. Glyceroneogenesis played a prominent role in the generation of G3P in the liver (similar to 70 %) as well as in BAT and EAT (similar to 80 %) in controls rats. The cafeteria diet induced an increase in the total glyceride-glycerol synthesis and G3P synthesis from glucose and a decrease in glyceroneogenesis in BAT; this diet did not affect either the total glyceride-glycerol synthesis or G3P generation from glyceroneogenesis or glycolysis in the liver or EAT. Fasting induced an increase in total glyceride-glycerol synthesis and glyceroneogenesis and a decrease in G3P synthesis from glucose in the liver but did not affect either the total glyceride-glycerol synthesis or G3P synthesis from glyceroneogenesis in BAT and EAT, despite a reduction in glycolysis in these tissues. These data demonstrate that reciprocal changes in the G3P generation from glucose and from glyceroneogenesis in the rat liver and BAT occur only when the synthesis of glycerides-glycerol is increased. Further, our data suggest that this increase may be essential for the systemic recycling of fatty acids by the liver from fasted rats and for the maintenance of the thermogenic capacity of BAT from cafeteria diet-fed rats.

Signature of the scattering between dark sectors in large scale cosmic microwave background anisotropies

We study the interaction between dark sectors by considering the momentum transfer caused by the dark matter scattering elastically within the dark energy fluid. Describing the dark scattering analogy to the Thomson scattering which couples baryons and photons, we examine the impact of the dark scattering in CMB observations. Performing global fitting with the latest observational data, we find that for a dark energy equation of state w < -1, the CMB gives tight constraints on dark matter-dark energy elastic scattering. Assuming a dark matter particle of proton mass, we derive an elastic scattering cross section of sigma(D) < 3.295 x 10(-10)sigma(T) where sigma(T) is the cross section of Thomson scattering. For w > -1, however, the constraints are poor. For w = -1, sigma(D) can formally take any value.

A Low-Protein, High-Carbohydrate Diet Increases Fatty Acid Uptake and Reduces Norepinephrine-Induced Lipolysis in Rat Retroperitoneal White Adipose Tissue

A low-protein, high-carbohydrate (LPHC) diet for 15 days increased the lipid content in the carcass and adipose tissues of rats. The aim of this work was to investigate the mechanisms of this lipid increase in the retroperitoneal white adipose tissue (RWAT) of these animals. The LPHC diet induced an approximately two- and tenfold increase in serum corticosterone and TNF-alpha, respectively. The rate of de novo fatty acid (FA) synthesis in vivo was reduced (50%) in LPHC rats, and the lipoprotein lipase activity increased (100%). In addition, glycerokinase activity increased (60%), and the phosphoenolpyruvate carboxykinase content decreased (27%). Basal [U-C-14]-glucose incorporation into glycerol-triacylglycerol did not differ between the groups; however, in the presence of insulin, [U-C-14]-glucose incorporation increased by 124% in adipocytes from only control rats. The reductions in IRS1 and AKT content as well as AKT phosphorylation in the RWAT from LPHC rats and the absence of an insulin response suggest that these adipocytes have reduced insulin sensitivity. The increase in NE turnover by 45% and the lack of a lipolytic response to NE in adipocytes from LPHC rats imply catecholamine resistance. The data reveal that the increase in fat storage in the RWAT of LPHC rats results from an increase in FA uptake from circulating lipoproteins and glycerol phosphorylation, which is accompanied by an impaired lipolysis that is activated by NE.

Modeling the gluon propagator in Landau gauge: Lattice estimates of pole masses and dimension-two condensates

We present an analytic description of numerical results for the Landau-gauge SU(2) gluon propagator D(p(2)), obtained from lattice simulations (in the scaling region) for the largest lattice sizes to date, in d = 2, 3 and 4 space-time dimensions. Fits to the gluon data in 3d and in 4d show very good agreement with the tree-level prediction of the refined Gribov-Zwanziger (RGZ) framework, supporting a massive behavior for D(p(2)) in the infrared limit. In particular, we investigate the propagator's pole structure and provide estimates of the dynamical mass scales that can be associated with dimension-two condensates in the theory. In the 2d case, fitting the data requires a noninteger power of the momentum p in the numerator of the expression for D(p(2)). In this case, an infinite-volume-limit extrapolation gives D(0) = 0. Our analysis suggests that this result is related to a particular symmetry in the complex-pole structure of the propagator and not to purely imaginary poles, as would be expected in the original Gribov-Zwanziger scenario.

Constipation Is More Frequent Than Diarrhea in Patients Fed Exclusively by Enteral Nutrition: Results of an Observational Study

Background: Digestive complications in enteral nutrition (EN) can negatively affect the nutrition clinical outcome of hospitalized patients. Diarrhea and constipation are intestinal motility disorders associated with pharmacotherapy, hydration, nutrition status, and age. The aim of this study was to analyze the frequency of these intestinal motility disorders in patients receiving EN and assess risk factors associated with diarrhea and constipation in hospitalized patients receiving exclusive EN therapy in a general hospital. Materials and Methods: The authors performed a sequential and observational study of 110 hospitalized adult patients fed exclusively by EN through a feeding tube. Patients were categorized according to the type of intestinal transit disorder as follows: group D (diarrhea, 3 or more watery evacuations in 24 hours), group C (constipation, less than 1 evacuation during 3 days), and group N (absence of diarrhea or constipation). All prescription drugs were recorded, and patients were analyzed according to the type and amount of medication received. The authors also investigated the presence of fiber in the enteral formula. Results: Patients classified in group C represented 70% of the study population; group D comprised 13%, and group N represented 17%. There was an association between group C and orotracheal intubation as the indication for EN (P<.001). Enteral formula without fiber was associated with constipation (logistic regression analysis: P<.001). Conclusion: Constipation is more frequent than diarrhea in patients fed exclusively by EN. Enteral diet with fiber may protect against medication-associated intestinal motility disorders. The addition of prokinetic drugs seems to be useful in preventing constipation. (Nutr Clin Pract. XXXX;xx:xx-xx)

Design and synthesis of new (E)-cinnamic N-acylhydrazones as potent antitrypanosomal agents

We report herein the synthesis and trypanocidal profile of new (E)-cinnamic N-acylhydrazones (NAHs) designed by exploiting molecular hybridization between the potent cruzain inhibitors (E)-1-(benzo[d] 11,3)dioxol-5-yl)-3-(4-bromophenyl)prop-2-en-1-one and (E)-3-hydroxy-N'-((2-hydroxynaphthalen-1-yl)methylene)-7-methoxy-2-naphthohydrazide. These derivatives were evaluated against both amastigote and trypomastigote forms of Trypanosoma cruzi and lead us to identify two compounds that were approximately two times more active than the reference drug, benznidazole, and with good cytotoxic index. Although designed as cruzain inhibitors, the weak potency displayed by the best cinnamyl NAH derivatives indicated that another mechanism of action was likely responsible for their trypanocide action. (C) 2012 Elsevier Masson SAS. All rights reserved.

RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies

Abstract Background The development of protocols for RNA extraction from paraffin-embedded samples facilitates gene expression studies on archival samples with known clinical outcome. Older samples are particularly valuable because they are associated with longer clinical follow up. RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue is problematic due to chemical modifications and continued degradation over time. We compared quantity and quality of RNA extracted by four different protocols from 14 ten year old and 14 recently archived (three to ten months old) FFPE breast cancer tissues. Using three spin column purification-based protocols and one magnetic bead-based protocol, total RNA was extracted in triplicate, generating 336 RNA extraction experiments. RNA fragment size was assayed by reverse transcription-polymerase chain reaction (RT-PCR) for the housekeeping gene glucose-6-phosphate dehydrogenase (G6PD), testing primer sets designed to target RNA fragment sizes of 67 bp, 151 bp, and 242 bp. Results Biologically useful RNA (minimum RNA integrity number, RIN, 1.4) was extracted in at least one of three attempts of each protocol in 86–100% of older and 100% of recently archived ("months old") samples. Short RNA fragments up to 151 bp were assayable by RT-PCR for G6PD in all ten year old and months old tissues tested, but none of the ten year old and only 43% of months old samples showed amplification if the targeted fragment was 242 bp. Conclusion All protocols extracted RNA from ten year old FFPE samples with a minimum RIN of 1.4. Gene expression of G6PD could be measured in all samples, old and recent, using RT-PCR primers designed for RNA fragments up to 151 bp. RNA quality from ten year old FFPE samples was similar to that extracted from months old samples, but quantity and success rate were generally higher for the months old group. We preferred the magnetic bead-based protocol because of its speed and higher quantity of extracted RNA, although it produced similar quality RNA to other protocols. If a chosen protocol fails to extract biologically useful RNA from a given sample in a first attempt, another attempt and then another protocol should be tried before excluding the case from molecular analysis.

Chemical resistance of the gram-negative bacteria to different sanitizers in a water purification system

Abstract Background Purified water for pharmaceutical purposes must be free of microbial contamination and pyrogens. Even with the additional sanitary and disinfecting treatments applied to the system (sequential operational stages), Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas alcaligenes, Pseudomonas picketti, Flavobacterium aureum, Acinetobacter lowffi and Pseudomonas diminuta were isolated and identified from a thirteen-stage purification system. To evaluate the efficacy of the chemical agents used in the disinfecting process along with those used to adjust chemical characteristics of the system, over the identified bacteria, the kinetic parameter of killing time (D-value) necessary to inactivate 90% of the initial bioburden (decimal reduction time) was experimentally determined. Methods Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas alcaligenes, Pseudomonas picketti, Flavobacterium aureum, Acinetobacter lowffi and Pseudomonas diminuta were called in house (wild) bacteria. Pseudomonas diminuta ATCC 11568, Pseudomonas alcaligenes INCQS , Pseudomonas aeruginosa ATCC 15442, Pseudomonas fluorescens ATCC 3178, Pseudomonas picketti ATCC 5031, Bacillus subtilis ATCC 937 and Escherichia coli ATCC 25922 were used as 'standard' bacteria to evaluate resistance at 25°C against either 0.5% citric acid, 0.5% hydrochloric acid, 70% ethanol, 0.5% sodium bisulfite, 0.4% sodium hydroxide, 0.5% sodium hypochlorite, or a mixture of 2.2% hydrogen peroxide (H2O2) and 0.45% peracetic acid. Results The efficacy of the sanitizers varied with concentration and contact time to reduce decimal logarithmic (log10) population (n cycles). To kill 90% of the initial population (or one log10 cycle), the necessary time (D-value) was for P. aeruginosa into: (i) 0.5% citric acid, D = 3.8 min; (ii) 0.5% hydrochloric acid, D = 6.9 min; (iii) 70% ethanol, D = 9.7 min; (iv) 0.5% sodium bisulfite, D = 5.3 min; (v) 0.4% sodium hydroxide, D = 14.2 min; (vi) 0.5% sodium hypochlorite, D = 7.9 min; (vii) mixture of hydrogen peroxide (2.2%) plus peracetic acid (0.45%), D = 5.5 min. Conclusion The contact time of 180 min of the system with the mixture of H2O2+ peracetic acid, a total theoretical reduction of 6 log10 cycles was attained in the water purified storage tank and distribution loop. The contact time between the water purification system (WPS) and the sanitary agents should be reviewed to reach sufficient bioburden reduction (over 6 log10).

Palmitoleic acid (n-7) increases white adipocyte lipolysis and lipase content in a PPARα-dependent manner

We investigated whether palmitoleic acid, a fatty acid that enhances whole body glucose disposal and suppresses hepatic steatosis, modulates triacylglycerol (TAG) metabolism in adipocytes. For this, both differentiated 3T3-L1 cells treated with either palmitoleic acid (16:1n7, 200 μM) or palmitic acid (16:0, 200 μM) for 24 h and primary adipocytes from wild-type or PPARα-deficient mice treated with 16:1n7 (300 mg•kg(-1)•day(-1)) or oleic acid (18:1n9, 300 mg•kg(-1)•day(-1)) by gavage for 10 days were evaluated for lipolysis, TAG, and glycerol 3-phosphate synthesis and gene and protein expression profile. Treatment of differentiated 3T3-L1 cells with 16:1n7, but not 16:0, increased basal and isoproterenol-stimulated lipolysis, mRNA levels of adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) and protein content of ATGL and pSer(660)-HSL. Such increase in lipolysis induced by 16:1n7, which can be prevented by pharmacological inhibition of PPARα, was associated with higher rates of PPARα binding to DNA. In contrast to lipolysis, both 16:1n7 and 16:0 increased fatty acid incorporation into TAG and glycerol 3-phosphate synthesis from glucose without affecting glyceroneogenesis and glycerokinase expression. Corroborating in vitro findings, treatment of wild-type but not PPARα-deficient mice with 16:1n7 increased primary adipocyte basal and stimulated lipolysis and ATGL and HSL mRNA levels. In contrast to lipolysis, however, 16:1n7 treatment increased fatty acid incorporation into TAG and glycerol 3-phosphate synthesis from glucose in both wild-type and PPARα-deficient mice. In conclusion, palmitoleic acid increases adipocyte lipolysis and lipases by a mechanism that requires a functional PPARα

De Novo Design of secondary structures: Synthesis and conformational studies

Chemists have long sought to extrapolate the power of biological catalysis and recognition to synthetic systems. These efforts have focused largely on low molecular weight catalysts and receptors; however, biological systems themselves rely almost exclusively on polymers, proteins and RNA, to perform complex chemical functions. Proteins and RNA are unique in their ability to adopt compact, well-ordered conformations, and specific folding provides precise spatial orientation of the functional groups that comprise the “active site”. These features suggest that identification of new polymer backbones with discrete and predictable folding propensities (“foldamers”) will provide a basis for design of molecular machines with unique capabilities. The foldamer approach complements current efforts to design unnatural properties into polypeptides and polynucleotides. The aim of this thesis is the synthesis and conformational studies of new classes of foldamers, using a peptidomimetic approach. Moreover their attitude to be utilized as ionophores, catalysts, and nanobiomaterials were analyzed in solution and in the solid state. This thesis is divided in thematically chapters that are reported below. It begins with a very general introduction (page 4) which is useful, but not strictly necessary, to the expert reader. It is worth mentioning that paragraph I.3 (page 22) is the starting point of this work and paragraph I.5 (page 32) isrequired to better understand the results of chapters 4 and 5. In chapter 1 (page 39) is reported the synthesis and conformational analysis of a novel class of foldamers containing (S)-β3-homophenylglycine [(S)-β3-hPhg] and D- 4-carboxy-oxazolidin-2-one (D-Oxd) residues in alternate order is reported. The experimental conformational analysis performed in solution by IR, 1HNMR, and CD spectroscopy unambiguously proved that these oligomers fold into ordered structures with increasing sequence length. Theoretical calculations employing ab initio MO theory suggest a helix with 11-membered hydrogenbonded rings as the preferred secondary structure type. The novel structures enrich the field of peptidic foldamers and might be useful in the mimicry of native peptides. In chapter 2 cyclo-(L-Ala-D-Oxd)3 and cyclo-(L-Ala-DOxd) 4 were prepared in the liquid phase with good overall yields and were utilized for bivalent ions chelation (Ca2+, Mg2+, Cu2+, Zn2+ and Hg2+); their chelation skill was analyzed with ESI-MS, CD and 1HNMR techniques and the best results were obtained with cyclo-(L-Ala-D-Oxd)3 and Mg2+ or Ca2+. Chapter 3 describes an application of oligopeptides as catalysts for aldol reactions. Paragraph 3.1 concerns the use of prolinamides as catalysts of the cross aldol addition of hydroxyacetone to aromatic aldeydes, whereas paragraphs 3.2 and 3.3 are about the catalyzed aldol addition of acetone to isatins. By means of DFT and AIM calculations, the steric and stereoelectronic effects that control the enantioselectivity in the cross-aldol addition of acetone to isatin catalysed by L-proline have been studied, also in the presence of small quantities of water. In chapter 4 is reported the synthesis and the analysis of a new fiber-like material, obtained from the selfaggregation of the dipeptide Boc-L-Phe-D-Oxd-OBn, which spontaneously forms uniform fibers consisting of parallel infinite linear chains arising from singleintermolecular N-H···O=C hydrogen bonds. This is the absolute borderline case of a parallel β-sheet structure. Longer oligomers of the same series with general formula Boc-(L-Phe-D-Oxd)n-OBn (where n = 2-5), are described in chapter 5. Their properties in solution and in the solid state were analyzed, in correlation with their attitude to form intramolecular hydrogen bond. In chapter 6 is reported the synthesis of imidazolidin-2- one-4-carboxylate and (tetrahydro)-pyrimidin-2-one-5- carboxylate, via an efficient modification of the Hofmann rearrangement. The reaction affords the desired compounds from protected asparagine or glutamine in good to high yield, using PhI(OAc)2 as source of iodine(III).

Gleichmäßig dünne Basen zweiter Ordnung

Eine Menge B nicht negativer ganzer Zahlen heißt Basis h-ter Ordnung, wenn jede nicht negative ganze Zahl Summe von höchstens h Elementen von B ist. Eine der großen Fragen der additiven Zahlentheorie ist die nach der effektivsten Basis h-ter Ordnung für ein gegebenes h>=2. Im Fokus des Interesses steht dabei der immer noch offene Fall h=2. Bezeichnet B(x) die Anzahl der Elemente b aus B mit 0= af(x), wobei f die Wurzelfunktion bezeichne. Andererseits gibt es Basen B zweiter Ordnung mit B(x) <= cf(x). Daher kann man den Limes superior S(B), den Limes inferior s(B) sowie im Falle der Existenz den Limes d(B) des Quotienten B(x) / f(x) als Dichtefunktionen von Basen zweiter Ordnung betrachten. J. W. S. Cassels konstruierte 1957 eine Basis C zweiter Ordnung mit d(C)=5,196…. G. Hofmeister gab 2001 eine Basis H zweiter Ordnung mit asymptotischer Wurzeldichte d(H)=4,638… an. In der vorliegenden Arbeit wird eine Basis S zweiter Ordnung mit asymptotischer Wurzeldichte d(S)=3,464… konstruiert. Darüber hinaus wird für die von J. W. S. Cassels, für die von G. Hofmeister und für die in dieser Arbeit verwendete Klasse von Basen zweiter Ordnung gezeigt, dass die asymptotische Wurzeldichte innerhalb der jeweiligen Klasse nicht mehr zu verbessern ist. Bisher war die Frage nach möglichen Verbesserungen innerhalb der jeweiligen Konstruktionsprinzipien offen geblieben.

Preparation of new crystal forms via photochemical, mechanochemical and sol-gel methods

This work of thesis involves various aspects of crystal engineering. Chapter 1 focuses on crystals containing crown ether complexes. Aspects such as the possibility of preparing these materials by non-solution methods, i.e. by direct reaction of the solid components, thermal behavior and also isomorphism and interconversion between hydrates are taken into account. In chapter 2 a study is presented aimed to understanding the relationship between hydrogen bonding capability and shape of the building blocks chosen to construct crystals. The focus is on the control exerted by shape on the organization of sandwich cations such as cobalticinium, decamethylcobalticinium and bisbenzenchromium(I) and on the aggregation of monoanions all containing carboxylic and carboxylate groups, into 0-D, 1-D, 2-D and 3-D networks. Reactions conducted in multi-component molecular assemblies or co-crystals have been recognized as a way to control reactivity in the solid state. The [2+2] photodimerization of olefins is a successful demonstration of how templated solid state synthesis can efficiently synthesize unique materials with remarkable stereoselectivity and under environment-friendly conditions. A demonstration of this synthetic strategy is given in chapter 3. The combination of various types of intermolecular linkages, leading to formation of high order aggregation and crystalline materials or to a random aggregation resulting in an amorphous precipitate, may not go to completeness. In such rare cases an aggregation process intermediate between crystalline and amorphous materials is observed, resulting in the formation of a gel, i.e. a viscoelastic solid-like or liquid-like material. In chapter 4 design of new Low Molecular Weight Gelators is presented. Aspects such as the relationships between molecular structure, crystal packing and gelation properties and the application of this kind of gels as a medium for crystal growth of organic molecules, such as APIs, are also discussed.

Charakterisierung spezieller pflanzlicher Gamma-Tubulin-Sequenzbereiche und Detektion potentieller Interaktionspartner mittels Yeast-two-Hybrid-System

Mitotische und postmitotische Vorgänge pflanzlicher Zellen basieren auf der Funktion von Mikrotubuli. Es liegen nur wenige gesicherte Erkenntnisse zur Organisation dieser Multifunktionalität vor. Eine zentrale Bedeutung wird bei der Nukleation der Mikrotubuli an MTOCs durch γ-Tubulin zugeschrieben. Deren Zusammenlagerung an MTOCs ist jedoch noch nicht richtig verstanden. Domänen, die an der Proteinoberfläche exponiert werden, könnten in Interaktionen involviert sein. Hier werden im Besonderen der γ-A und γ-B-Peptivmotiv diskutiert. Es wurde das γ-A- und γ-B-Peptidmotiv des γ-Tubulins hinsichtlich einer Konservierung innerhalb des Pflanzenreiches untersucht. Die beiden Bereiche sind bei den grünen Landpflanzen stark konserviert. Sie divergieren stark zu den einzelligen Grünalgen Chlamydomonas reinhardtii und Chlorella spec. Es wurden daher in der bestehenden phylogentischen Lücke weitere Organismen hinsichtlich des γ-A und γ-B Peptidmotivs untersucht. Auswahlkriterien der Organismen waren Ein-/Mehrzelligkeit, Besitz/Abwesenheit von Centriolen und Besitz/Abwesenheit von Geißeln. Des weiteren wurde mit verschiedenen γ-Tubulin-Konstrukten um das γ-A- und γ-B-Peptidmotiv, gewonnen aus Nicotiana tabacum (BY2) mittels Y2H-System nach Interaktionspartnern gesucht. Bei den Sequenzuntersuchungen des γ-A- und γ-B-Peptidmotivs konnte festgestellt werden, dass die Konservierung innerhalb der Streptophytenlinie erfolgt. Interessant erweist sich die Tatsache, dass dieses Motiv bei den Jochalgen, welche ebenfalls den Streptophyten angehören, nur im γ-A-Peptidmotiv auftritt. Es besteht die Möglichkeit, dass die beiden potentiellen Interaktionspartner verschiedene Proteine als Interaktions-partner besitzen. Durch eine Anwendung eines auf dem GAL4-Protein basierenden Y2H-Systems mit vier unterschiedlichen Konstrukten des γ-Tubulin-A/B-Peptidbereichs als Köder-konstrukt und einer cDNA-Bibliothek als Beutekonstrukt, wurden diverse Sequenzen identifiziert. Identifiziert wurden das Poly(A)-Bindeprotein, Glycerin-aldehyd-3-phosphatdehydrogenase, die S-adenosyl-L-methionine-Synthetase, diverse Proteasom-Untereinheiten, eine sekretorische Peroxidase, eine Ascorbat-Peroxidase, die NtPOX1-Peroxidase und verschiedene Peroxidasen aus Nicotiana tabacum, Sequenzen des Chloroplastengenoms, ein Myosin-ähnliches Protein und eine Sequenz auf dem 5. Chromosom des Medicago truncatula-Klons mth2-16f8 und diverse humane Sequenzen der Proteine DKFZp68 und DKFZp77. Die Ergebnisse weisen auf eine komplexe Funktionsweise der unterschiedlichen Komponenten des pflanzlichen Cytoskeletts und des γ-Tubulins hin. Zur Aufklärung müsste dies in Zukunft mittels anderer genetischer, biochemischer oder funktioneller Methoden untersucht werden. Hypothesen über Interaktionen der Cytoskelettkomponenten können wahrscheinlich nicht allein durch die Anwendung des Y2H-Systems aufgeklärt werden.