A model of the complex between human β-microseminoprotein and CRISP-3 based on NMR data

β-Microseminoprotein (MSP), a 10 kDa seminal plasma protein, forms a tight complex with cysteine-rich secretory protein 3 (CRISP-3) from granulocytes. The 3D structure of human MSP has been determined but there is as yet no 3D structure for CRISP-3. We have now studied the complex between human MSP and CRISP-3 with multidimensional NMR. ¹⁵N-HSQC spectra show substantial differences between free and complexed hMSP. Using several 3D-NMR spectra of triply labeled hMSP in complex with a recombinant N-terminal domain of CRISP-3, most of the backbone of hMSP could be assigned. The data show that only one side of hMSP, comprising β-strands 1, 4, 5, and 8 are affected by the complex formation, indicating that β-strands 1 and 8 form the main binding surface. Based on this we present a tentative structure for the hMSP-CRISP-3 complex using the known crystal structure of triflin as a model of CRISP-3.     

The effects of ambient temperature,nature and temperature of the floor and radiant heat on the metabolic rate of the new—born pig

Using a closed circuit technique, the oxygen consumption of 108 piglets below one week of age have been measured while modifying the physical attributes of the thermal environment. Moving the piglets onto a wooden floor from concrete reduced the oxygen consumption rate from 37.8 ml to 34.5 ml/kg. min) at 10°C, and from 33.7 to 31.4 ml/(kg. min) on a rubber floor at the same temperature. Oxygen consumption was also significantly reduced when the piglets were placed on a straw floor, were supplied with radiant heat and when the floor temperature was raised independently of chamber temperature. It is concluded that the provision of supplementary heating either in the floor or by overhead radiation can greatly reduce the effects of a cold environment on the new-born pig and so enhance the animal's viability.

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Zusammenfassung Der Sauerstoff-Umsatz von 108 Ferkeln, die jünger als eine Woche waren, wurde unter wechselnden Bedingungen der thermischen Umwelt gemessen. Das Verlegen der Ferkel von einem Zement- auf einen Holzboden führte zur Senkung des 0₂-Umsatzes von 37,8 auf 34,5 ml O₂/kg. min) und von 33,7 auf 31,4 ml O₂/(kg. min) bei 10°C auf einem Gummiboden. Auf einem strohbedecktem Boden war der O₂-Umsatz ebenfalls vermindert. Den gleichen Effekt hatte die Anwendung von Strahlungswärme oder die Erhöhung der Bodentemperatur. Danach kann zusätzliche Erwärmung vom Boden oder von der Decke die Kälteeinwirkung auf das Ferkel aus der Umgebung stark reduzieren und die Lebensfähigkeit der Tiere erhöhen.

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Resume Au moyen d'un appareil à circuit fermé, on a mesuré la consommation en oxygène le 108 porcelets âgés de moins d'une semaine soumis à divers régimes thermiques et cela dans une température ambiante constante. En transférant les porcelets d'un sol de béton à un plancher de bois, leur consommation d'oxygène a passé de 37,8 à 34,5 ml/(kg. min), et de 33,7 à 31,4 ml/(kg. min) en passant du béton à un sol recouvert de caoutchouc. La consommation d'oxygène a également été réduite de façon significative pour un sol recouvert de paille. Il en est allé de même pour des lampes à infra-rouge et si le sol était chauffé indépendamment du local. On en conclut qu'un chauffage supplémentaire soit dans le sol soit par rayonnement peut réduire sensiblement les effets néfastes d'une ambiance froide sur lesporcelets nouveau-nés et rehausser leurs chances de survie.

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Presented during the Fifth International Biometeorological Congress, 1–6 September 1969, Montreux, Switzerland.     

Co-localization of CD9 and GPIIb-IIIa (·IIb ·3 integrin) on activated platelet pseudopods and ·-granule membranes

Summary CD9 is a 24-kDa membrane glycoprotein expressed on the surface of human platelets and potentially involved in cellular activation and adhesion functions. This protein belongs to a recently delineated family of cell-surface antigens that span the membrane four times, called tetraspans, and found mainly in leucocytes and tumour cells. As a first approach to clarify the function of CD9, we used immunoelectron microscopy to determine the localization of this antigen in human platelets, and compared its distribution with that of the GPIIb-IIIa integrin, the platelet receptor for fibrinogen. Monoclonal antibodies against CD9 (MAb7) and GPIIb-IIIa (HP1-1D) coupled to colloidal gold of different sizes (5 and 15 nm) were incubated with intact platelets in suspension or on ultrathin sections of platelets embedded in LR white. CD9 was found in association with GPIIb-IIIa on the inner face of ·-granule membranes. These two antigens also co-localized on pseudopods of activated platelets and in contact regions between adjacent platelets. CD63, another member of the tetraspan family, was absent from ·-granules but was associated with lysosomal structures. Flow cytometric analysis of platelet CD9 with a series of monoclonal antibodies revealed an increased expression upon thrombin stimulation, confirming the presence of an intracellular granular pool. The observation that CD9 and GPIIb-IIIa are stored in the same intracellular structures and migrate to the same activation zones after platelet stimulation lends support to previous suggestions of a close association between CD9 and GPIIb-IIIa in human platelets and of a possible involvement of CD9 in adhesive functions of platelets. This revised version was published online in November 2006 with corrections to the Cover Date.     

Backbone dynamics of (1–71)- and (1–36)bacterioopsin studied by two-dimensional 1H-15N NMR spectroscopy

Summary The backbone dynamics of uniformly ¹⁵N-labelled fragments (residues 1–71 and 1–36) of bacterioopsin, solubilized in two media (methanol-chloroform (1:1), 0.1 M ²HCO₂NH₄, or SDS micelles) have been investigated using 2D proton-detected heteronuclear ¹H-¹⁵N NMR spectroscopy at two spectrometer frequencies, 600 and 400 MHz. Contributions of the conformational exchange to the transverse relaxation rates of individual nitrogens were elucidated using a set of different rates of the CPMG spin-lock pulse train and were essentially suppressed by the high-frequency CPMG spin-lock. We found that most of the backbone amide groups of (1–71)bacterioopsin in SDS micelles are involved in the conformational exchange process over a rate range of 10³ to 10⁴ s^-1. This conformational exchange is supposed to be due to an interaction between two -helixes of (1–71)bacterioopsin, since the hydrolysis of the peptide bond in the loop region results in the disappearance of exchange line broadening. ¹⁵N relaxation rates and ¹H-¹⁵N NOE values were interpreted using the model-free approach of Lipari and Szabo [Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc., 104, 4546–4559]. In addition to overall rotation of the molecule, the backbone N-H vectors of the peptides are involved in two types of internal motions: fast, on a time scale <20 ps, and intermediate, on a time scale close to 1 ns. The intermediate dynamics in the -helical stretches was mostly attributed to bending motions. A decrease in the order parameter of intermediate motions was also observed for residues next to Pro⁵⁰, indicating an anisotropy of the overall rotational diffusion of the molecule. Distinctly mobile regions are identified by a large decrease in the order parameter of intermediate motions and correspond to the N- and C-termini, and to a loop connecting the -helixes of (1–71)bacterioopsin. The internal dynamics of the -helixes on the millisecond and nanosecond time scales should be taken into account in the development of a model of the functioning bacteriorhodopsin.Abbreviations BO bacterioopsin - 2D two-dimensional - CPMG Carr-Purcell-Meiboom-Gill (Carr and Purcell, 1954) - SDS sodium dodecyl(²H₂₅) sulfate - R(S_x), R(S_z) ¹⁵N transverse and longitudinal relaxation rates, respectively     

New data on the composition and distribution of the genus Leptomona Bechyné, 1958 (Coleoptera, Chrysomelidae: Galerucinae)

The leaf-beetle Leptomona russica (Gmelin, 1790) known from the Caucasus, Kazakhstan, and Middle Asia was found in Orenburg Province for the first time. It is the first record of this species from Europe and the first record of the genus Leptomona from European Russia. The diagnostic characters of the two Russian Leptomona species are included.     

Effect of (–)-epicatechin-3-gallate and (–)-epigallocatechin-3-gallate on the binding of tegafur to human serum albumin as determined by spectroscopy,isothermal titration calorimetry,and molecular docking

Green tea has attracted great interest as a cancer prevention agent. Interactions of tea polyphenols with serum albumin may influence the efficacy of drugs. The interactions of (–)-epigallocatechin-3-gallate (EGCG), (–)-epicatechin-3-gallate (ECG), and tegafur (TF) alone or in combination with human serum albumin (HSA) at pH 7.4 and different temperatures were investigated by spectroscopic methods, isothermal titration calorimetry (ITC), and molecular docking. The binding affinities to HSA were ranked in the order of EGCG?>?ECG?>?TF, and the interactions were spontaneous and exothermic. Ternary system studies showed that the presence of one component hindered the binding of another component to HSA. The secondary structures of HSA were slightly altered in the presence of the ligands. Site marking experiments and molecular docking showed that EGCG and ECG mainly bound to subdomain IIA and ΙΙΙA while TF bound to subdomain ΙΙA and ΙB. Results indicated that the existence of ECG and EGCG would influence the binding of TF to HSA and can increase the free concentration of TF. Obtained results would provide beneficial information about possible interference upon simultaneous co-administration of the tea components and drugs.

Communicated by Ramaswamy H. Sarma     

Description of the female and new data on the distribution of Annitella apfelbecki Klapálek, 1899 (Insecta: Trichoptera)

Annitella apfelbecki is one of three Annitella species with distribution ranges restricted to the Balkan Peninsula. In this paper, we describe the hitherto unknown female of A. apfelbecki and give the most important morphological features to enable its identification and separation from the other Annitella females. Additionally, we provide new data on distribution and discuss zoogeography, life cycle and ecology of this species.     

New data on the fauna and systematics of Palaearctic species of the click-beetle subfamily Agrypninae Candèze, 1857 (Coleoptera,Elateridae)

New data on the distribution and taxonomy of some Palaearctic species of click-beetles of the subfamily Agrypninae are given. The external morphology and genitalia of the previously unknown females of Compsolacon korotjaevi Gur. and C. agrestis (Vats et Kash.) comb. n. are described. Several taxa are recorded from the following territories for the first time: the genus Eumoeus Cand. from the Palaearctic Region, the species Compsolacon agrestis comb. n. and C. himalayanus Jag. from Afghanistan, Compsolacon turkestanicus (Schw.) from Kyrgyzstan and Afghanistan, Danosoma conspersum (Gyll.) from Kazakhstan, Lacon quadrinodatus Lew. from Russia, and L. unicolor (Cand.) from Iraq. Several Northern Indian species of Agrypnus Esch. are transferred to Compsolacon Rtt., and their systematic positions are briefly discussed: C. agrestis, C. inlustris (Vats et Kash.), C. jacksoni (Vats et Kash.), C. kuluensis (Vats et Kash.), C. maisus (Vats et Kash.), C. planocorpus (Vats et Kash.), C. rameshi (Vats et Kash.), C. repercussus (Vats et Kash.), C. solanensis (Vats et Kash.), C. souslapisus (Vats et Kash.), C. subargillus (Vats et Kash.), C. subfaenum (Vats et Kash.) and C. sublapideus (Vats et Kash.) (all comb. n.). The following new combinations are also established: Compsolacon aequalis (Cand.) comb. n. and Compsolacon brachychaetus (Kollar) comb. n. (both ex Lacon). The following new synonymies are established: Lacon funebris (Sols.) = Adelocera grisea Schw., syn. n.; = Adelocera incompta Kr., syn. n.; Eumoeus murrayi Cand. = Tetralobus quadrifoveatus Vats et Kash., syn. n.     

Does progressive nuclear staining with hemalum (alum hematoxylin) involve DNA,and what is the nature of the dye-chromatin complex?

Previous investigators have disagreed about whether hemalum stains DNA or its associated nucleoproteins. I review here the literature and describe new experiments in an attempt to resolve the controversy. Hemalum solutions, which contain aluminum ions and hematein, are routinely used to stain nuclei. A solution containing 16 Al³⁺ ions for each hematein molecule, at pH 2.0–2.5, provides selective progressive staining of chromatin without cytoplasmic or extracellular “background color.” Such solutions contain a red cationic dye-metal complex and an excess of Al³⁺ ions. The red complex is converted to an insoluble blue compound, assumed to be polymeric, but of undetermined composition, when stained sections are blued in water at pH 5.5–8.5. Staining experiments with DNA, histone and DNA + histone mixtures support the theory that DNA, not histone, is progressively colored by hemalum. Extraction of nucleic acids, by either a strong acid or nucleases at near neutral pH, prevented chromatin staining by a simple cationic dye, thionine, pH 4, and by hemalum, with pH adjustments in the range, 2.0–3.5. Staining by hemalum at pH 2.0–3.5 was not inhibited by methylation, which completely prevented staining by thionine at pH 4. Staining by hemalum and other dye-metal complexes at pH ≤ 2 may be due to the high acidity of DNA-phosphodiester (pK_a ~ 1). This argument does not explain the requirement for a much higher pH to stain DNA with those dyes and fluorochromes not used as dye-metal complexes. Sequential treatment of sections with Al₂(SO₄)₃ followed by hematein provides nuclear staining that is weaker than that attainable with hemalum. Stronger staining is seen if the pH is raised to 3.0–3.5, but there is also coloration of cytoplasm and other materials. These observations do not support the theory that Al³⁺ forms bridges between chromatin and hematein. When staining with hematein is followed by an Al₂(SO₄)₃ solution, there is no significant staining. Taken together, the results of my study indicate that the red hemalum cation is electrostatically attracted to the phosphate anion of DNA. The bulky complex cation is too large to intercalate between base pairs of DNA and is unlikely to fit into the minor groove. The short range van der Waals forces that bind planar dye cations to DNA probably do not contribute to the stability of progressive hemalum staining. The red cation is precipitated in situ as a blue compound, insoluble in water, ethanol and water-ethanol mixtures, when a stained preparation is blued at pH > 5.5.     

Further studies on the copper(II) complexes of lysine: [Cu(II)(H3N·C5H10·CH·NH2·COO)2] [HgI3]2

Spontaneous resolution in the formation of the [HgI₃]⁻ salts of the copper complex of racemic lysine was previously reported. X-ray and IR studies were used to support this conclusion. Gas chromatographic studies using a chiral phase on the crystals originally studied, and on newly formed crystals using D,L-lysine, do not substantiate the suggestion that spontaneous resolution occurs.     

The effects of structural variations of thiophene-containing Ru(II) complexes on the acid–base and DNA binding properties

A phenylthiophenyl-bearing Ru(II) complex of [Ru(bpy)₂(Hbptip)](PF₆)₂ {bpy?=?2,2′-bipyridine, Hbptip?=?2-(4-phenylthiophen-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline} was synthesized and characterized by elemental analysis, ¹H NMR spectroscopy, and electrospray ionization mass spectrometry. The ground- and excited-state acid–base properties of the complex were studied by UV–visible absorption and photoluminescence spectrophotometric pH titrations and the negative logarithm values of the ground-state acid ionization constants were derived to be pK _a1?=?1.31?±?0.09 and pK _a2?=?5.71?±?0.11 with the pK _a2 associated deprotonation/protonation process occurring over 3 pK _a units more acidic than thiophenyl-free parent complex of [Ru(bpy)₂(Hpip)]²⁺ {Hpip?=?2-phenyl-1H-imidazo[4,5-f][1,10]phenanthroline}. The calf thymus DNA-binding properties of [Ru(bpy)₂(Hbptip)]²⁺ in Tris–HCl buffer (pH 7.1 and 50?mM NaCl) were investigated by DNA viscosities and density functional theoretical calculations as well as UV–visible and emission spectroscopy techniques of UV–visible and luminescence titrations, steady-state emission quenching by [Fe(CN)₆]^4?, DNA competitive binding with ethidium bromide, DNA melting experiments, and reverse salt effects. The complex was evidenced to bind to the DNA intercalatively with binding affinity being greater than those for previously reported analogs of [Ru(bpy)₂(Hip)]²⁺, [Ru(bpy)₂(Htip)]²⁺, and [Ru(bpy)₂(Haptip)]²⁺ {Hip?=?1H-imidazo[4,5-f][1,10]phenanthroline, Htip?=?2-thiophenimidazo[4,5-f][1,10]phenanthroline, Haptip?=?2-(5-phenylthiophen-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline}.     

The leucocyte β2 (CD18) integrins: the structure, functional regulation and signalling properties

Leucocytes are highly motile cells. Their ability to migrate into tissues and organs is dependent on cell adhesion molecules. The integrins are a family of heterodimeric transmembrane cell adhesion molecules that are also signalling receptors. They are involved in many biological processes, including the development of metazoans, immunity, haemostasis, wound healing and cell survival, proliferation and differentiation. The leucocyte-restricted β2 integrins comprise four members, namely αLβ2, αMβ2, αXβ2 and αDβ2, which are required for a functional immune system. In this paper, the structure, functional regulation and signalling properties of these integrins are reviewed.     

Solution NMR and CD spectroscopy of an intrinsically disordered,peripheral membrane protein: evaluation of aqueous and membrane-mimetic solvent conditions for studying the conformational adaptability of the 18.5 kDa isoform of myelin basic protein (MBP)

The stability and secondary structure propensity of recombinant murine 18.5 kDa myelin basic protein (rmMBP, 176 residues) was assessed using circular dichroic and nuclear magnetic resonance spectroscopy ((1)H-(15)N HSQC experiments) to determine the optimal sample conditions for further NMR studies (i.e., resonance assignments and protein-protein interactions). Six solvent conditions were selected based on their ability to stabilise the protein, and their tractability to currently standard solution NMR methodology. Selected solvent conditions were further characterised as functions of concentration, temperature, and pH. The results of these trials indicated that 30% TFE-d(2) in H(2)O (v/v), pH 6.5 at 300 K, and 100 mM KCl, pH 6.5 at 277 K were the best conditions to use for future solution NMR studies of MBP. Micelles of DPC were found to be inappropriate for backbone resonance assignments of rmMBP in this instance.     

The effect of the 3′ → 5′ exonuclease of T7 DNA polymerase on frameshifts and deletions

Both spontaneous frameshift mutation and deletion mutation were measured in a T7 phage deficient in the 3′ → 5′ exonuclease of T7 DNA polymerase. It was found that the absence of this exonuclease caused a marked increase in the revision of both plus one and minus one mutations. The exonuclease deficiency caused essentially no effect on the frequency of deletion between 10-bp direct repeats even when the segment between the direct repeats contained a 25-bp palindrome.     

A comparative theoretical investigation into the strength of the trigger-bond in the Na+, Mg2+ and HF complexes involving the nitro group of R–NO2 (R = –CH3, –NH2 and –OCH3) or the C = C bond of (E)-O2N–CH = CH–NO2

A comparative theoretical investigation into the change in strength of the trigger-bond upon formation of the Na⁺, Mg²⁺ and HF complexes involving the nitro group of RNO₂ (R?=? –CH₃, –NH₂, –OCH₃) or the C?=?C bond of (E)-O₂N–CH?=?CH–NO₂ was carried out using the B3LYP and MP2(full) methods with the 6-311++G**, 6-311++G(2df,2p) and aug-cc-pVTZ basis sets. Except for the Mg²⁺?π system with (E)-O₂N–CH?=?CH–NO₂ (i.e., C₂H₂N₂O₄?Mg²⁺), the strength of the trigger-bond X–NO₂ (X?=?C, N or O) was enhanced upon complex formation. Furthermore, the increment of bond dissociation energy of the X–NO₂ bond in the Na⁺ complex was far greater than that in the corresponding HF system. Thus, the explosive sensitivity in the former might be lower than that in the latter. For C₂H₂N₂O₄?Mg²⁺, the explosive sensitivity might also be reduced. Therefore, it is possible that introducing cations into the structure of explosives might be more efficacious at reducing explosive sensitivity than the formation of an intermolecular hydrogen-bonded complex. AIM, NBO and electron density shifts analyses showed that the electron density shifted toward the X–NO₂ bond upon complex formation, leading to a strengthened X–NO₂ bond and possibly reduced explosive sensitivity.

Daily tracking of the locomotion of the nudibranch Tritonia tetraquetra (Pallas 1788 = Tritonia diomedea Bergh 1894) in nature and the influence of water flow on orientation,crawling, and drag

We observed orientation and locomotion of the nudibranch Tritonia tetraquetra in its natural habitat using SCUBA over many sequential days, in three different months. The slugs oriented significantly headfirst to tidal currents. Nevertheless, the direction of locomotion of the slugs over hours was not usually correlated with tidal flow direction (i.e. not indicative of consistent rheotaxis). We did not find evidence of consistent body axis orientation to the geomagnetic field, but the direction of locomotion of some groups of slugs over hours was significantly correlated with geomagnetic direction. Independent of direction, each slug changed position by an average of ～2?m during a single tidal phase (～6?h), and changed position by an average of ～4?m over a full tidal cycle (～25?h). Orientation to flow reduced drag, and reduced the probability that a slug will be dislodged from the soft bottom, in laboratory experiments. Slugs deprived of olfactory and flow cues exhibit a search-like pattern of multiple and frequent turns.     

Combined effects of experimental heavy-metal contamination (Cu, Zn, and CH3Hg) and starvation on quail’s body condition

Combined effects of heavy-metal contamination (Cu, Zn, and CH₃Hg) and starvation were tested on common quails (Coturnix coturnix japonica) and used as a model for comparison with a wild common guillemot (Uria aalge) population found stranded at the Belgian coast. Appropriate heavy-metal levels were given to the quails to obtain concentrations similar to those found in the seabirds’s tissues. The contaminated animals were then starved for 4 d to simulate the evident malnutrition symptoms observed at the guillemot’s level. In such conditions, food intake and total-body weight are shown to decrease in contaminated individuals with simultaneous significant hepatic and renal increase of the heavy-metal concentrations. Like guillemots, higher heavy-metal levels were observed in those contaminated quails that had also developed a cachectic status characterized by a general atrophy of their pectoral muscle and complete absence of subcutaneous and/or abdominal fat depots. Although likely the result of a general protein catabolism during starvation, it is suggested that these higher metal levels could as well enhance a general muscle wasting process (cachectic status).     

Chromosomal locations of the gene coding for the CD3 (T3) γ subunit of the human and mouse CD3/T-cell antigen receptor complexes

The gene coding for the M _r 26000 chain of the human CD3 (T3) antigen/T-cell antigen receptor complex was mapped to chromosome band 11q23 by using a cDNA clone (pJ6T3 -2), by in situ hybridization to metaphase chromosomes and by Southern blot analysis of a panel of human-rodent somatic cell hybrids. The mouse homolog, here termed Cdg-3, was mapped to chromosome 9 using the mouse cDNA clone pB10.AT3 -1 and a panel of mouse-hamster somatic cell hybrids. Similar locations for the CD3 genes have been described previously. Thus, the corporate results indicate that the CD3 and genes have remained together since they duplicated about 200 million years ago.