bluuebunny/crossref_metadata_2025 huggingface dataset:
Il sinkhole di Marcellina del 2001 (Monti Lucretili, Lazio)
Authors: Argentieri Alessio , Di Filippo Michele
Published: December 2020

Nel gennaio 2001 si verificò presso Marcellina (area metropolitana di Roma), alle pendici occidentali dei Monti Lucretili, un fenomeno di sprofondamento catastrofico improvviso. Il sinkhole è collocato in località Pozzo Grande, area pianeggiante posta allo sbocco di diversi valloni che solcano profondamente la dorsale carbonatica (Fig. 1). Dopo molti decenni, fu il primo caso documentato in tempo reale nel territorio provinciale di Roma; la memoria storica porta infatti agli analoghi collassi osservati nella vicina bassa Valle del Tevere, presso Leprignano (l’odierna Capena), ripetutisi ciclicamente tra la fine del XIX secolo e le prime decadi del Novecento (Console e Nisio 2020), in un’area che di recente ha dato segni di attività con l’evento sismico di bassa magnitudo del 4 ottobre 2020 e una serie di boati percepiti nei giorni successivi [...].
Methane Emission Factor in Italian Mediterranean Buffalo According to Production Management
Authors: Ciriello Nunzia , Albano Lorenza , Auriemma Giuseppe , Palomba Raffaele , Grazioli Giuseppe , Sarubbi Fiorella
Published: December 2020

The environmental impact of greenhouse gases caused by livestock farms plays a fundamental role due to the implications and environmental consequences that livestock practices entail, affecting the stability of the entire ecosystem connected to them, especially as a consequence of the growing demand for products of animal origin.

The aim of this work was to quantify the CH4 emissions factor in lactating buffaloes by comparing four different types of livestock management: family, conventional, organic and sustainable.

To determine the enteric CH4 emissions from buffalo, information about animal production and farm management was analyzed, and the CH4 emission factor was calculated using the IPCC Tier 2 model.

ANOVA was conducted to evaluate significant differences between the farms; Pearson’s correlation was used to evaluate the relationship between parameters.

In a conventional farm, the CH4 emission factor for buffalo was 27.69 kg CH4/head/yr compared to 22.77 and 21.61 kg CH4/head/yr respectively for organic and family-run. These data may also depend on the higher protein and fiber content in the administered unifeed. Furthermore, the ratio of enteric emissions factor of CH4 / gross energy intake ratio reflected these data (12.04 vs 10.93 vs 10.16 vs 10.65 for conventional, organic, sustainable, and family-run farms, respectively).
Prevalence of Anemia in Pregnant Women under Antenatal Care at the Rivers State University Teaching Hospital, Port-Harcourt, Nigeria and Association to Knowledge and Attitude on Anemia and Its Prevention
Authors: Awoyesuku P. A. , Ohaka C. , Ngeri B.
Published: December 2020

Background: Anemia in pregnancy is an important public health problem worldwide. Despite the efforts of government and health bodies, maternal anemia continues to be a common cause of morbidity and mortality. This suggests that other factors contribute to the high prevalence of anemia in pregnancy despite the intervention efforts.

Objective: This study sought to determine the prevalence and anemic status of pregnant women at booking, to assess their knowledge of anemia and attitude to anemia prevention measures. And to determine associated risk factors for anemia and if there is a correlation between anemic status and level of knowledge/attitude to anemia prevention

Methodology: An institutional-based, cross-sectional study was carried out. 322 consenting participants between 18-48 years were interviewed at booking using a structured questionnaire. Information on socio-demographic characteristics; their knowledge on causes, symptoms, prevention, and complications of anemia in pregnancy; and information on attitude towards anemia prevention strategies were collected. The PCV and HIV results of the women were retrieved. Data were entered into an Excel spreadsheet and analyzed with SPSS version 20. Associations between different variables were determined using Fisher's exact test or Chi-square test, as appropriate, and logistic regression was used to test statistical significance at P<0.05.

Results: Their mean age±SD was 31.65±4.72 years and the median parity was 1.Of the 322 women, 194(60.2%) were anemic, with 186(57.8%) having mild anemia. There was no significant association between anemia and age, marital status, education, employment, parity, pregnancy interval and socio-economic status, but there was a significant association between gestational age and HIV status, with only gestational age remaining significant after logistic regression. The relationship between anemia and knowledge and attitude was not significant, but the higher educational status was significantly related to knowledge of anemia and its prevention.

Conclusion: The prevalence of anemia in this study was high. Despite good knowledge and attitude to anemia prevention, late booking for ANC caused a significant association with the occurrence of anemia. Efforts are needed to encourage early booking and early commencement of iron and folic acid supplements.
Analyse electrophoretique des proteines de l'enveloppe des chloroplastes d'epinard
Authors: Pineau B.
Published: July 2002

The polypeptide heterogeneity of the chloroplast envelope has been compared with these of other fractions of Spinach chloroplasts: thylakoïds and stroma by means of SDS‐polyacrylamide gel electrophoresis. The envelope electrophoretic pattern shows many polypeptides whose mobility differ from those of the thylakoïds. The possible identity of the small subunit of RudPcarboxylase with a polypeptide of the chloroplast external membrane is discussed.
Mn‐preserving extraction of 33‐, 24‐ and 16‐kDa proteins from O2‐evolving PS II particles by divalent salt‐washing
Authors: Ono Taka-Aki , Inoue Yorinao
Published: July 2002

Divalent salt‐washing of O2‐evolving PS II particles caused total liberation of 33‐, 24‐ and 16‐kDa proteins, but the resulting PS II particles retained almost all amounts of Mn present in initial particles. The retained Mn was EPR‐silent when the particles were kept in high concentrations of divalent salt. By divalent salt‐washing, the activity of diphenylcarbazide (DPC) photooxidation was not affected at all, neither suppressed nor enhanced, while O2 evolution was totally inactivated. These results indicate that Mn can be kept associated with PS II particles even after liberation of the 33‐kDa protein, and suggest that the 33‐kDa protein is probably not responsible for binding Mn onto membranes, but is possibly responsible for maintaining the function of Mn atoms in the O2‐evolving center.
Immunochemical evidence for the presence of 5mC, 6mA and 7mG in human, Drosophila and mealybug DNA
Authors: Achwal Chhaya W. , Iyer Chitra A. , Chandra H.Sharat
Published: July 2002

We have reported that production and characterization of antibodies highly specific to 5‐methyl‐cytosine (5mC) and the development of a sensitive immunochemical method for the detection of 5mC in DNA [FEBS Lett. (1982) 150, 469]. Extension of this method to two other modified bases, 6‐methyladenine (6mA) and 7‐methylguanine (7mG), is reported here. By use of this immunochemical approach, we are able to detect 5mC, 6mA and 7mG in human and Drosophila DNA and confirm their presence in the DNA of two mealybug species.
The significance of the kinetic analysis of fluorescence induction in DCMU‐inhibited chloroplasts in terms of photosystem 2 connectivity and heterogeneity
Authors: Hodges M. , Barber J.
Published: July 2002

A study has been made on the slow component (βmax) of chlorophyll fluorescence induction curves exhibited by DCMU‐inhibited pea thylakoids. In the absence of high levels of screening cations, βmax is high and the fluorescence yield low, while the addition of K+, Mg2+ or Tris(ethylenediamine) cobaltic(III) cation (TEC3+) decreases βmax and increases fluorescence yield. These changes are inhibited when the thylakoids are fixed with glutaraldehyde. By comparing cation‐ and light‐harvesting chlorophyll a/b‐protein (LHCP) phosphorylation‐induced changes it can be seen that βmax correlates with changes in the overall kinetics of photosystem (PS) 2 photoreduction, as indicated from the normalised area over the induction curve (Anorm). When the plastoquinone (PQ) pool is chemically reduced by dithionite, prior to the initiation of the curve, both F
o and F
m are increased and the slow component removed. These observations are used to question the concept of two structurally distinct PS2 centres [Arch. Biochem. Biophys. (1978) 190, 523–530] and to discuss the use of βmax in monitoring PS2 organisation.
Sulfhydryl group modification of photoreceptor G‐protein prevents its light‐induced binding to rhodopsin
Authors: Reichert J. , Hofmann K.P.
Published: July 2002

The effect of sulfhydryl modification on the light‐induced interaction between rhodopsin and the peripheral GTP‐binding protein of the photoreceptor membrane (G‐protein) has been investigated by time‐resolved near‐infrared light‐scattering and polyacrylamide gel electrophoresis. It has been found that the modification of rhodopsin with the alkylating agent N‐ethylmaleimide (NEM) does not affect its light‐induced interaction with the G‐protein. Modification of G‐protein with NEM or other sulfhydryl agents prevents any light‐induced binding to rhodopsin. Dark‐associatiion of G to the membrane as well as the light‐induced complex with rhodopsin (once formed) is insensitive to NEM.
Ca2+‐dependent restoration of O2‐evolving activity in CaCl2‐washed PS II particles depleted of 33, 24 and 16 kDa proteins
Authors: Ono Taka-aki , Inoue Yorinao
Published: July 2002

CaCl2‐washing of O2‐evolving PS II particles liberated 33, 24 and 16 kDa proteins concomitant with inactivation of O2 evolution, whereas almost all Mn remained associated with membranes [FEBS Lett. 164 (1983) 252–260], and the lost O2 evolution was significantly restored when 33 kDa protein rebound to the washed membranes [FEBS Lett. 166 (1984) 381–384]. Half of the Mn atoms retained in CaCl2‐washed particles were unstably associated with the membrane, being gradually released during incubation in the absence of Ca2+, whereas in the presence of Ca2+ the release of Mn was suppressed concomitant with partial reactivation of O2 evolution. These results were interpreted as indicating that Ca2+ as well as 33 kDa protein maintains the conformation around the Mn‐binding sites as required for O2 evolution.
The release of a 10‐kDa polypeptide from everted photosystem II thylakoid membranes by alkaline tris
Authors: Ljungberg Ulf , Åkerlund Hans-Erik , Andersson Bertil
Published: July 2002

The release of polypeptides from inside‐out thylakoid vesicles and photosystem II by alkaline Tris treatment was reinvestigated, using SDS‐polyacrylanude gel electrophoresis (PAGE) in the presence of urea, with highly increased resolution in the low molecular mass region. In addition to the 33‐, 23‐, and 16‐kDa proteins of the oxygen‐evolving complex, a 10‐kDa polypeptide was released. This 10‐kDa polypeptide is an entrinsic polypeptide located at the inner grana thylakoid surface and with a likely role in photosynthetic oxygen evolution.
Degradation of soluble collagen by ozone or hydroxyl radicals
Authors: Curran Samantha F. , Amoruso Marie A. , Goldstein Bernard D. , Berg Richard A.
Published: July 2002

Collagen exposed to ozone or hydroxyl radicals was degraded in a time‐ and dose‐dependent manner. This degradation was inhibited by free radical scavengers. Furthermore, lower levels of these oxidants did not degrade the molecule, but caused it to become susceptible to proteolytic degradation. We suggest an alternative mechanism by which oxygen‐derived free radicals participate in the destruction of extracellular matrix observed during acute lung injury by oxidant gas, in addition to the commonly accepted proteinase‐antiproteinase theory of lung injury.
Light‐induced binding of 48‐kDa protein to photoreceptor membranes is highly enhanced by phosphorylation of rhodopsin
Authors: Kühn H. , Hall S.W. , Wilden U.
Published: July 2002

The 48‐kDa protein, a major protein of rod photoreceptor cells, is soluble in the dark but associates with the disk membranes when some (5–10%) of their rhodopsin has absorbed light and if this rhodopsin is additionally phosphorylated by ATP and rhodopsin kinase. If rhodopsin has been phosphorylated and regenerated prior to the protein binding experiment, the binding of 48‐kDa protein depends on light but no longer on the presence of ATP. Another photoreceptor protein, GTP‐binding protein, associates with both phosphorylated and unphosphorylated rhodopsin upon illumination. Excess GTP‐binding protein thereby displaces 48‐kDa protein from phosphorylated disks; this indicates competition between these two proteins for binding sites on illuminated phosphorylated rhodopsin molecules.
Primary structure of the β‐subunit of bovine transducin deduced from the cDNA sequence
Authors: Sugimoto Katsunori , Nukada Toshihide , Tanabe Tsutomu , Takahashi Hideo , Noda Masaharu , Minamino Naoto , Kangawa Kenji , Matsuo Hisayuki , Hirose Tadaaki , Inayama Seiichi , Numa Shosaku
Published: July 2002

DNA complementary to the bovine retinal mRNA coding for the β‐subunit of transducin has been cloned by screening a cDNA library with oligodeoxyribonucleotide probes. Nucleotide sequence analysis of the cloned cDNA has revealed that this polypeptide consists of 340 amino acid residues (including the initiating methionine). Furthermore, cDNA hybridizable with a transducin β‐subunit cDNA probe has been cloned from a library derived from bovine brain poly(A)+ RNA. Comparison of the cloned cDNAs, in conjunction with blot hybridization analysis and S1 nuclease mapping of poly(A)+ RNA from bovine retina, brain and liver, suggests that the mRNAs coding for the β‐subunits of transducin and other guanine nucleotide binding proteins have the same protein‐coding sequence but partly different 5'‐noncoding sequences.
Penicillin‐binding proteins of β‐lactam‐resistant strains of Staphylococcus aureus
Authors: Reynolds Peter E. , Brown Derek F.J.
Published: July 2002

Methidllin‐resistant clinical isolates of Staphylococcus aureus are intrinsically resistant to β‐lactam antibiotics in that the resistance mechanism is unrelated to the possession of β‐lactamases. We have demonstrated that a new, high‐molecular‐mass penicillin‐binding protein (PBP) is present in these strains with a low affinity for β‐lactams and that its amount is regulated by the growth conditions. The new PBP from all strains that have been examined has an identical mobility on SDS gel electrophoresis and is the only PBP still present in an uncomplexed state with β‐lactams (and therefore the only functional PBP) when these strains are grown in media containing concentrations of β‐lactam antibiotics sufficient to kill sensitive strains.
The oxygen‐evolving photosystem II core complex
Authors: Tang Xiao-Song , Satoh Kimiyuki
Published: July 2002

Digitonin extracts of spinach thylakoid membranes obtained as 100000 × g supernatant have been shown to retain part of the oxygen‐evolving activity if the extraction is carried out at acidic pH. The photosystem II reaction center complex with a uniform particle size of 300–500 kDa was purified from the extracts by ion‐exchange/gel‐permeation chromatography performed at pH 6.0 using DEAE‐Toyopearl‐650. The purified materials contained only two polypeptides of about 22 (not 24) and 33 kDa, in addition to those of the reaction center core complex, i.e., 47, 43, 30 and 10 kDa. The purified complex was fairly active in the photochemical oxygen evolution supported by phenyl‐p‐benzoquinone (~150μmol oxygen evolved·mg chl−1·h−1) and DCIP photoreduction with water. From these results it is concluded that the principal site of water oxidation is associated rather with the photosystem II core complex than with the water soluble peripheral complex consisting of 18,24 and 33 kDa polypeptides and that the thylakoid membrane structure is not essential for manifestation of oxygen‐evolving activity.
1H‐NMR study of gramicidin A transmembrane ion channel
Authors: Arseniev A.S. , Barsukov I.L. , Bystrov V.F. , Lomize A.L. , Ovchinnikov Yu.A.
Published: July 2002

The structure of[Val1]gramicidin A incorporated into sodium dodecyl‐d
25 sulphate micelles has been studied by two‐dimensional proton NMR spectroscopy. Analysis of nuclear Overhauser effects, spin‐spin couplings and solvent accessibility of NH groups show that the conformation of the Na+ complex of gramicidin A in detergent micelles, which in many ways mimic the phospholipid bilayer of biomembranes, is an N‐terminal to N‐terminal (head‐to‐head) dimer formed by two right‐handed, single‐stranded β6.3 helices with 6.3 residues per turn, differing from Urry's structure by handedness of the helices.
Rapid modulation of transcription of nuclear genes encoding chloroplast proteins by light
Authors: Gallagher T.F. , Jenkins G.I. , Ellis R.J.
Published: July 2002

We have investigated the effects of light on transcription of the nuclear genes encoding the small subunit (SSU) of ribulose‐1,5‐biphosphate carboxylase‐oxygenase, and the major apoprotein (LHCP) of the lightharvesting chlorophyll a/b complex in Pisumsativum. Light treatments were given in vivo and transcription was assayed subsequently in isolated nuclei using specific cloned cDNA probes. We have identified three different temporal effects of light on transcription of these genes: an initial increase when dark‐grown seedlings are first illuminated, a slow increase in the ability to transcribe these genes at maximal rates, and a rapid modulation of specific gene transcription in fully greened plants.
Site specificity of iron removal from transferrin by α‐ketohydroxypyridine chelators
Authors: Kontoghiorghes George J. , Evans Robert W.
Published: July 2002

Transferrin
Iron mobilisation
Site specificity
α‐Ketohydroxypyridine chelators
Mimosine
Dimeric enzyme IImtl of the E. coli phosphoenolpyruvate‐dependent phosphotransferase system
Authors: Roossien F.F. , van Es-Spiekman W. , Robillard G.T.
Published: July 2002

The occurrence of intermolecular dithiols on EIImtl has been studied with a number of thiol‐specific crosslinking reagents. The reaction of EIImtl with bifunctional maleimide derivatives inactivates the enzyme. At the same time the enzyme is irreversibly cross‐linked to a dimeric species. Under optimal conditions 50% of the protein is cross‐linked upon reaction with the dimaleimides. The enzyme is also cross‐linked under oxidizing conditions in the presence of CuCl2, presumably by oxidizing an intermolecular dithiol to a disulfide. This oxidation can be reversed by the addition of the reducing agent dithiothreitol. The reaction of phosphorylated EIImtl with the same sulfhydryl‐specific bifunctional reagents does not lead to any crosslinked product. The results are discussed in terms of the association state of the purified protein and the distribution of its thiol groups.
A monoclonal antibody recognizes a phosphorylated epitope shared by proteins of the cell nucleus and the erythrocyte membrane skeleton
Authors: Turner Bryan M. , Davies Suzanne
Published: July 2002

Monoclonal antibody 3C5 recognizes a family of proteins in the nuclei of cultured cells [(1985) Eur. J. Cell Biol. 38, 344]. This antibody has now been shown to recognize equivalent proteins in liver nuclei and in the Triton‐insoluble fraction of tissue extracts. In human erythrocytes the antibody recognized a single protein, present in the membrane skeleton fraction and with the molecular mass and extraction properties of β‐spectrin. The epitope recognized by 3C5 was destroyed by alkaline phosphatase. We conclude that this antibody recognizes a phosphorylation site shared by nuclear proteins and a protein of the erythrocyte membrane skeleton, probably β‐spectrin.
Inositol phosphate production following α1‐adrenergic, muscarinic or electrical stimulation in isolated rat heart
Authors: Poggioli J. , Sulpice J.C. , Vassort G.
Published: July 2002

A possible participation of polyphosphoinositide metabolism in the excitation‐contraction coupling in heart was investigated. Isolated rat ventricles prelabelled with myo‐[2‐3H]inositol were stimulated by conditions that increase mechanical activity. Both noradrenaline and carbachol increased the basal level of IP3 IP2 and IP by the activation of α1‐adrenergic and muscarinic receptors, respectively. Electrical stimulation accelerated inositol lipid degradation by phospholipase C thus enhancing the IP3 level as compared to quiescent ventricles. It is proposed that IP3 may be involved in excitation‐contraction coupling in cardiac tissue.
Moss cell cultures as sources of arachidonic and eicosapentaenoic acids
Authors: Hartmann E. , Beutelmann P. , Vandekerkhove O. , Euler R. , Kohn G.
Published: July 2002

Lipid classes from tissue cultures of the moss Leptobryum pyriforme (Hedw.) Wils. were analyzed. In the total lipid fraction, this species contained 20% arachidonic acid and about 7% eicosapentaenoic acid. The distribution of these fatty acids showed a preference for the phospholipid fraction. In particular, the phosphatidylethanolamine fraction was enriched in arachidonic acid. The arachidonic acid content of Leptobryum could be altered by transferring the cultures to different culture conditions. Mosses show high organic mass production in tissue cultures in relatively simple media. The great potential of using mosses as sources for the production of polyunsaturated fatty acids is evident.
Hydrophobic cluster analysis: An efficient new way to compare and analyse amino acid sequences
Authors: Gaboriaud C. , Bissery V. , Benchetrit T. , Mornon J.P.
Published: July 2002

A new method for comparing and aligning protein sequences is described. This method, hydrophobic cluster analysis (HCA), relies upon a two‐dimensional (2D) representation of the sequences. Hydrophobic clusters are determined in this 2D pattern and then used for the sequence comparisons. The method does not require powerful computer resources and can deal with distantly related proteins, even if no 3D data are available. This is illustrated in the present report by a comparison of human haemoglobin with leghaemoglobin, a comparison of the two domains of liver rhodanese (thiosulphate sulphurtransferase) and a comparison of plastocyanin and azurin.
Evidence for the identity of anti‐proteinase pulmonary protein CCSP and uteroglobin
Authors: de Haro M.S.López , Alvarez L. , Nieto A.
Published: July 2002

Purified Clara cell secretory protein (CCSP) from rabbit lung was analyzed by SDS gel electrophoresis, and immunoblotting with a specific anti‐uteroglobin antibody as well as for its ability to bind [3H]progesterone. The results obtained indicate that proteins CCSP and uteroglobin are identical.
Energy transfer from carotenoid to bacteriochlorophyll a in the B800–820 antenna complexes from Rhodopseudomonas acidophila strain 7050
Authors: Gillbro Tomas , Cogdell Richard J. , Sundström Villy
Published: July 2002

We have resolved the excitation energy transfer from carotenoids to bacteriochlorophyll a in the B800–820 antenna complex of Rhodopseudomonas acidophila and found it to be about 3 ps. This efficient transfer can best be explained by the electron‐exchange mechanism with a distance of about 4.5 Å between the molecules.
Stable and transient expression of mouse submaxillary gland renin cDNA in AtT20 cells: Proteolytic processing and secretory pathways
Authors: Ladenheim Ruth G. , Seidah Nabil , Lutfalla Georges , Rougeon François
Published: July 2002

Apart from kidney, where renin synthesis takes place in all mammals, the submaxillary gland (SMG) of most mouse strains constitutes an important source of an isoenzyme, renin‐2, that is highly homologous to renal renin, but unglycosylated [(1982) Nature 298, 90–92]. This unique phenotype is due to the presence of an extra copy of the renin gene. A puzzling observation is that (pro)renin‐2 cannot be detected in the kidney of these animals, although both mRNAs accumulate at similar levels [(1985) Proc. Natl. Acad. Sci. USA 82, 6196–6200]. In order to investigate whether (pro)renin‐2 expression is detectable in mouse heterologous cell lines we transfected the renin‐2 cDNA into AtT20 (pituitary corticotrope) and BTG9A (hepatoma) cells. Stable clones expressing renin were obtained in both cases. BTG9A cells secreted only prorenin while AtT20 cells secreted prorenin and active renin. In addition, in AtT20 cells the secretion of active renin was stimulated by 8‐Br cAMP. Our results show that unglycosylated (pro)renin‐2 can be expressed and secreted in two murine cell lines. Moreover, it is correctly processed to active renin and secreted upon stimulation in AtT20 cells.
Characterization of excitation energy trapping in photosynthetic purple bacteria at 77 K
Authors: Bergström H. , van Grondelle R. , Sundström V.
Published: July 2002

We have studied the energy‐transfer dynamics in chromatophores of Rhodobacter sphaeroides and Rhodospirillum rubrum at 77 K, with functional charge separation. Using low‐intensity picosecond absorption recovery, we determined that transfer between the energetically low‐lying antenna component BChl896 and the special pair of the reaction center occurs with a time constant of 37 ps in Rb. sphaeroides and 75 ps in R. rubrum. Assuming that a Förster energy‐transfer mechanism applies to the process, this allows us to estimate the distance between BChl896 in the B875 complex and the special pair P870 in the reaction center to range between 26 and 39 Å in Rb. sphaeroides. Such a distance indicates that the BChl896 pigment and the special pair of the reaction center are at the minimum separation allowed by the size and shape of the reaction center and the light‐harvesting polypeptides.
The inner mitochondrial membrane contains ion‐conducting channels similar to those found in bacteria
Authors: Petronilli Valeria , Szabò Ildiko , Zoratti Mario
Published: July 2002

Patch‐clamp experiments were performed on rat liver mitochondria inner membranes. Application of voltage gradients of either polarity revealed the presence of several different conductances, ranging up to 1.3 nS in symmetrical 150 mM KCl. Evidence is presented that at least those higher than 0.3 nS are substates of the highest conductance channel. Increasing matrix‐side‐positive (unphysiological) transmembrane voltage gradients favored the switch of the 1.3 nS channel to operation in lower conductance states. The size of these conductances, the presence of substates and the channel behavior are strongly reminiscent on one hand of the observations on the membrane of protoplasts from the gram‐positive bacterium Streptococcus faecalis, [Zoratti M. and Petronilli V. (1988) FEBS Lett. 240, 105‐109], and on the other of some properties of previously described channels of mitochondrial origin.
Factor Xa cleavage of fusion proteins
Authors: Wearne Steven J.
Published: July 2002

A method is described for the elimination of non‐specific cleavage of fusion proteins by factor Xa. Putative non‐specific cleavage sites C‐terminal to lysyl residues are blocked by reversible acylation by 3,4,5,6‐tetrahydrophthalic anhydride prior to cleavage. After cleavage, the acyi groups are removed quantitatively by exposure to slightly acidic conditions. This method employs no harsh reagents or conditions, and may be generally applicable to factor Xa cleavage of fusion proteins.
Hydrogen peroxide production during experimental protein glycation
Authors: Jiang Zhen-Yue , Woollard Alison C.S. , Wolff Simon P.
Published: July 2002

The accumulation of hydrogen peroxide (H2O2) during incubations of protein with glucose (experimental glycation) has previously been too low for direct measurement although it is suggested to be the precursor of protein‐damaging hydroxylating agents. We have thus developed a simple H2O2‐measuring technique which relies upon the rapid peroxide‐mediated oxidation of Fe2+ to Fe3+ (catalysed by sorbitol) under acidic conditions followed by reaction of the latter cation with the dye, xylenol orange. We have used the method to demonstrate that incubation mixtures of protein and glucose generates nanomolar levels of hydrogen peroxide in the presence of protein under physiological conditions of pH and temperature.
Characterization of the transcript for a depressant insect selective neurotoxin gene with an isolated cDNA clone from the scorpion Buthotus judaicus
Authors: Gurevitz Michael , Zlotkin Eliahu , Zilberberg Noam
Published: July 2002

The poly(A)+ mRNA isolated from the venomous terminal segments of the scorpion Buthotus judaicus was reversed transcribed into cDNA. PCR amplification of the cDNA in presence of oligonucleotide primers prepared on basis of the known amino acid sequence of the depressant insect toxin II yielded a 125 bp long product. This fragment was cloned and its sequence determined. The deduced amino acid sequence has revealed a complete homology with the amino acid sequence of the toxin. This clone was used to probe a Northern blot resolving the poly(A)+ and poly(A)− fractions derived from the scorpion. An organ specific 360 nucleotide transcript which might be the processed product of a 4̃.0 kb precursor was elucidated. This cDNA clone may pave the way for a molecular genetic approach to study the structure‐function relationship of scorpion selective insect toxins.
Proteins under extreme physical conditions
Authors: Jaenicke Rainer , Závodszky Péter
Published: July 2002

Life on earth is ubiquitous within the limits from −5 to 110°C for temperature, 0.1 to 120 MPa for hydrostatic pressure. 1.0 to 0.6 for water activity and pH 1 to 12. In general, mutative adaptation of proteins to changing environmental conditions tends to maintain ‘corresponding states’ regarding overall topology, flexibility and hydration. Due to the minute changes in the free energy of stabilization responsible for enhanced stability, nature provides a wide variety of different adaptive strategies. In the case of thermophilic proteins, improved packing densities are crucial. In halophilic proteins, decreased hydrophobicity and clustered surface charges serve to increase water and salt binding required for solubilization at high salt concentration. In the case of barophiles, high‐pressure adaptation is expected to be less important than adaptation to low temperatures governing the deep sea. Nothing is known with respect to the mechanisms underlying psychrophilic and acidophilic alkalophilic adaptation.
Human milk bile‐salt stimulated lipase Sequence similarity with rat lysophospholipase and homology with the active site region of cholinesterases
Authors: Christie David L. , Cleverly Douglas R. , O'Connor Charmian J.
Published: July 2002

To determine the active site residue, human milk bile‐salt stimulated lipase (BSSL) was labelled with [3H]diisopropyl fluorophosphate (DFP). Partial sequence analysis or cyanogen bromide fragments (a total of 146 residues from 6 peptides) revealed 84% sequence identity with a putative rat lysophospholipase. Sequence analysis of a [3H]DFP‐labelled peptide indicated that the active site serine was contained in the sequence Gly‐Glu‐Scr‐Ala‐Gly. In addition to similarity with rat lysophospholipase, this sequence showed homology with regions of human butyrylcholinesterase and electric ray acetylcholinesterase (68% identity). It is concluded that these proteins are members of a new supergene family.
Molecular cloning of cDNA encoding adipogenesis inhibitory factor and identity with interleukin‐11
Authors: Kawashima Ichiro , Ohsumi Jun , Mita-Honjo Katsuko , Shimoda-Takano Kumi , Ishikawa Hiroko , Sakakibara Sachiko , Miyadai Kenji , Takiguchi Yo
Published: July 2002

A cDNA encoding a novel adipogenesis inhibitory factor (AGIF) that inhibits the process of adipogenesis in mouse 3T3‐L1 preadipocytes was cloned from a cDNA library of the human bone marrow‐derived stromal cell line KM‐102. The cloned cDNA contains an open reading frame coding for an AGIF precursor of 199 amino acids. Analysis of the sequence of this cDNA revealed identity of this factor with a recently reported novel cytokine, designated interleukin‐11 (IL‐11). AGIF/IL‐11 may play an important role in stromal cell‐associated hematopoiesis through its regulatory action on adipocyte differentiation in the bone marrow microenvironment.
Mapping the site of block by tetrodotoxin and saxitoxin of sodium channel II
Authors: Terlau Heinrich , Heinemann Stefan H. , Stühmer Walter , Pusch Michael , Conti Franco , Imoto Keiji , Numa Shosaku
Published: July 2002

The SS2 and adjacent regions of the 4 internal repeats of sodium channel II were subjected to single mutations involving, mainly, charged amino acid residues. These sodium channel mutants, expressed in Xenopus oocytes by microinjection of cDNA‐derived mRNAs, were tested for sensitivity to tetrodotoxin and saxitoxin and for single‐channel conductance. The results obtained show that mutations involving 2 clusters of predominantly negatively charged residues, located at equivalent positions in the SS2 segment of the 4 repeats, strongly reduce toxin sensitivity, whereas mutations of adjacent residues exert much smaller or no effects. This suggests that the 2 clusters of residues, probably forming ring structures, take part in the extracellular mouth and/or the pore wall of the sodium channel. This view is further supported by our finding that all mutations reducing net negative charge in these amino acid clusters cause a marked decrease in single‐channel conductance.
A novel P‐type ATPase from yeast involved in sodium transport
Authors: Haro Rosario , Garciadeblas Blanca , Rodriguez-Navarro Alonso
Published: July 2002

The gene ENA1 was cloned by its ability to complement the Li+ sensitivity of a low Li+‐efflux strain. The nucleotide sequence of the cloned DNA fragment showed that there are two almost identical genes in tandem, and predicts that they encode P‐ATPases. Disruption of both genes originated a strain defective in Na+ and Li+ effluxes, and sensitive to Na+, to Li+ and to alkaline pH. By transformation with ENA1 the defective effluxes and tolerances were repaired.
Opioid peptides derived from wheat gluten: Their isolation and characterization
Authors: Fukudome Shin-ichi , Yoshikawa Masaaki
Published: July 2002

Four opioid peptides were isolated from the enzymatic digest of wheat gluten. Their structures were Gly‐Tyr‐Tyr‐Pro‐Thr, Gly‐Tyr‐Tyr‐Pro,Tyr‐Gly‐Gly‐Trp‐Leu and Tyr‐Gly‐Gly‐Trp, which were named gluten exorphins A5, A4, B5 and B4, respectively. The gluten exorphin A5 sequence was found at 15 sites in the primary structure of the high molecular weight glutenin and was highly specific for δ‐receptors. The structure—activity relationships of gluten exorphins A were unique in that the presence of Gly at their N‐termini increased their activities. Gluten exorphin B5, which corresponds to [Trp4,Leu5]enkephalin, showed the most potent activity among these peptides. Its IC50 values were 0.05 μM and 0.017 μM, respectively, on the GPI and the MVD assays.
Interleukin‐8 receptor‐mediated chemotaxis of normal human epidermal cells
Authors: Michel Günter , Kemény Lajos , Peter Ralf U. , Beetz Axel , Ried Christine , Arenberger Petr , Ruzicka Thomas
Published: July 2002

Normal human keratinocytes show chemotactic behavior towards interleukin‐8 (IL‐8). Under physiological conditions this cylokine seems to be present in an equilibrium between monomeric and dimeric forms, as indicated by Western blotting data. Radioligand binding studies suggest that keratinocyte chemotaxis is mediated by receptors specific for IL‐8 dimers. IL‐8 rcceptor‐specific mRNA can be detected in a keratinocyte cell line by polymerase chain reaction.
Activin A increases intracellular free calcium concentrations in rat pancreatic islets
Authors: Shibata Hiroshi , Yasuda Hiroshi , Sekine Nobuo , Mine Tetsuya , Totsuka Yasuo , Kojima Itaru
Published: July 2002

Activin A stimulated insulin secretion in rat pancreatic islets, an effect that was attenuated by reduction of extracellular Ca2+ and abolished by either nitrendipine or verapamil. Activin A increased intracellular the free Ca2+ concentration, [Ca2+]i in fura‐2‐loaded islets. Activin A‐mediated elevation of [Ca2+]i was abolished by the reduction of extracellular Ca2+ or the addition of nifedipine. In addition, activin A did not increase [Ca2+]i in the presence of diazoxide, an opener of ATP‐sensitive K+ channels. These results suggest that activin A increases insulin secretion by stimulating Ca2+ entry.
Activation of snake venom metalloproteinases by a cysteine switch‐like mechanism
Authors: Grams Frank , Huber Robert , Kress Lawrence F. , Moroder Luis , Bode Wolfram
Published: July 2002

The cDNAs of several snake venom zinc endopeptidases code for a putative propeptide, which includes the conserved cysteine‐containing sequence PKMCGVT. It has been suggested that binding of the cysteine thiol function to the active‐site zinc, resulting in inactivation of the catalytic domain, occurs in a mode similar to the ‘cysteine switch’ mechanism proposed for matrix metalloproteinases. In order to confirm this hypothesis, inhibition kinetics have been performed on the metalloproteinase adamalysin II of the venom of the snake Crotalus adamanteus using several cysteine peptides. Among these the synthetic hexapeptide PKMCGV‐NH2, corresponding to the conserved sequence portion of the known propeptides, was found to be by far the strongest inhibitor of this proteinase with a K
i of 3.4 μM. The inhibitory potencies of an equivalent peptide with the l‐Cys replaced by a d‐Cys or by an l‐Ser as well as of reduced glutathione, cysteine and two unrelated cysteine peptides were by one to two orders of magnitudes lower. These findings strongly support a cysteine switch‐like mechanism even for activation of the snake venom metalloproteinases.
Characterization of the P4 promoter region of the human insulin‐like growth factor II gene
Authors: Hyun Sang Won , Kim Seong-Jin , Park Keunchil , Rho Hyune Mo , Lee Young Ik
Published: July 2002

The human insulin‐like growth factor II (IGF‐II) gene contains four promoters (P1, P2, P3 and P4). In order to determine the mechanism by which the P4 promoter is controlled, the human IGF‐II P4 promoter was analyzed in cell lines. DNA sequence analysis of the human IGF‐II P4 promoter gene showed that the P4 promoter region contains a TATA‐like sequence and several G + C rich regions which are essential for transcription. Analysis of the transcription initiation site by S1 nuclease mapping revealed two transcription start sites; both are located immediately behind TATA‐like sequence. To determine the location of sites that may be important for the function of the human IGF‐II P4 promoter, we constructed chimeric genes of the human IGF‐II P4 promoter fused to the coding region for chloramphenicol acetyltransferase (CAT). These constructs were transfected into HepG2, PLC/PRF/5, G401 and A549 cells, and were examined for CAT activity. All transfected cells showed a similar profile of CAT activity. Sequences responsible for putative enhancer and silencer regions were identified and the 5' flanking sequences of the human IGF‐II P4 promoter contain negative regulatory regions (−213 to −174). The 53‐base pair fragment located between 111 and 59 base pairs upstream of the start site contains positive regulatory activity. Gel mobility shift assay showed that Spl and another proteins might be involved in positive regulation of the human IGF‐II P4 promoter.
A left‐handed crossover involved in amidohydrolase catalysis
Authors: Miller Maria , Rao J.K.Mohana , Wlodawer Alexander , Gribskov Michael R.
Published: July 2002

The crystal structure of l‐asparaginase from Erwinia chrysanthemi in the presence and absence of l‐aspartate was determined at 1.8 Å resolution. Conserved residues in a left‐handed crossover (a rare occurrence in protein structures) link pairs of dimers into the catalytically active tetrameric form of the enzyme. The structure of ErA containing bound aspartic acid shows that this unusual strand connectivity is an essential part of the active site architecture, responsible for releasing the product of the enzymatic hydrolysis. The orientation of the bound aspartate indicates for the first time a threonine residue as a catalytic nucleophile.
Heterogeneous lipoprotein (a) size isoforms differ by their interaction with the low density lipoprotein receptor and the low density lipoprotein receptor‐related protein/α2‐macroglobulin receptor
Authors: März Winfried , Beckmann Angela , Scharnagl Hubert , Siekmeier Rüdiger , Mondorf Uli , Held Irmtraud , Schneider Wolfgang , Preissner Klaus T. , Curtiss Linda K. , Groß Werner , Hüttinger Manfred
Published: July 2002

Lipoprotein (a) (Lp(a)) is a complex of low density lipoprotein (LDL) with apolipoprotein (apo) (a). To examine the size distribution of Lp(a), plasma was separated by fast flow gel filtration and Lp(a): B complexes were determined in the eluate by enzyme immunoassays, in which detection was performed with monoclonal antibodies specific for apoB Lp(a): B particles displayed apparent molecular masses (Mr
) of 2 × 106 to at least 10 × 106. Lp(a) size isoforms differed by the expression of apoB epitopes and their interaction with cultured human skin fibroblasts. LDL was more effective in inhibiting binding, uptake, and degradation of low Mr
, Lp(a) than of high Mr
Lp(a). In contrast, Glu‐plasmmogen, α2‐macroglobulin and tissue‐type plasminogen activator were more effective in competing for the cellular degradation of high Mr
Lp(a) than of low Mr
Lp(a). Ligand blotting revealed that Lp(a) bound to the low density lipoprotein receptor, the low density lipoprotein receptor‐related protein/α2‐macroglobulin receptor (LRP) and to two other endosomal membrane proteins. We propose that the LDL receptor preferentially internalizes low Mr
Lp(a), whereas LRP may have a role in the clearance of high Mr
Lp(a).
Protein folding and stability: the pathway of folding of barnase
Authors: Fersht Alan R.
Published: July 2002

The pathway of folding of a protein will be completely solved when the structures and energetics of the initial unfolded states, all folding intermediates, all transition states and the final folded state, have been determined. The ultimate goal is to analyse, at the detail of individual residues, the non‐covalent interactions that are primarily responsible for dictating secondary and tertiary structure. Until recently, the tools for tackling such a daunting task were quite inadequate, but recent developments in NMR and protein engineering have made it possible to determine crucial features in the folding process. It now seems feasible that sufficient experimental detail will be obtained to provide general principles that govern protein folding and provide the basis for its rigorous theoretical analysis. This lecture will outline the progress and prospects in attainment of the goals as applied to the small ribonuclease, barnase.
Chemical crosslinking with disuccinimidyl tartrate defines the relative positions of the two antiparallel coiled coils of the desmin protofilament unit
Authors: Geisler Norbert
Published: July 2002

Filaments formed by desmin, the myogenic intermediate filament protein, were crosslinked with the lysine specific crosslinker DST (disuccinimidyl tartrate; 0.64 nm span) and three DST crosslinked peptides were characterized. Two correspond to crosslinks previously obtained with the longer crosslinker EGS (ethylene glycol bis(succinimidylsuccinate), 1.61 nm span) which defined the antiparallel on‐stagger relationship of neighbouring coiled coils. The two DST crosslinks now provide the relative positions of the coiled coils within a limit of about 9 α‐helical residues. The third DST crosslink most likely connecting two helices of a single coiled coil gives a direct measure of the distance spanned in DST crosslinks.
Okadaic acid‐sensitive protein phosphatases dephosphorylate MARCKS, a major protein kinase C substrate
Authors: Clarke Paul R. , Siddhanti Suresh R. , Cohen Philip , Blackshear Perry J.
Published: July 2002

The myristoylated alanine‐rich C kinase substrate (MARCKS) undergoes a rapid and, in certain circumstances, transient increase in phosphorylation in response to stimuli that activate protein kinase C. We have investigated the protein‐serine/threonine phosphatase activity responsible for reversing the phosphorylation of MARCKS. In cell‐free assays, protein phosphatases 1, 2A and 2C (PP1, PP2A and PP2C) all dephosphorylate recombinant MARCKS or a synthetic peptide based on its phosphorylation site domain. In intact Swiss 3T3 cells, okadaic acid, a specific inhibitor of PP1 and PP2A, had little effect on MARCKS phosphorylation on its own, but largely prevented the dephosphorylation of MARCKS that occured following activation of protein kinase C by bombesin with subsequent receptor blockade. These results indicate that although the dephosphorylation of MARCKS can be mediated by PP2C in vitro, this protein is dephosphorylated by okadaic acid‐sensitive phosphatases in the intact cell.
‘Channelled’ pathways can be more sensitive to specific regulatory signals
Authors: Kholodenko Boris N. , Demin Oleg V. , Westerhoff Hans V.
Published: July 2002

In ‘simple’ metabolic pathways the response to an external signal is readily described in terms of the effect of the signal on its receptor enzyme and the control exerted by that enzyme. We show here that in the response of ‘channelled’ pathways to such a signal, additional terms appear that reflect the direct enzyme‐enzyme interactions. They tend to enhance the responsiveness of the pathway. The normalized value of the response is called the signal transduction coefficient. We show that in channelled pathways these coefficients are usually larger than in corresponding non‐channelled (simple) pathways.
Gluten exorphin C
Authors: Fukudome Shin-ichi , Yoshikawa Masaaki
Published: July 2002

A novel opioid peptide, Tyr‐Pro‐Ile‐Ser‐Leu, was isolated from the pepsin‐trypsin‐chymotrypsin digest of wheat gluten. Its IC50 values were 40 μM and 13.5 μM in the GPI and MVD assays, respectively. This peptide was named gluten exorphin C. Gluten exorphin C had a structure quite different from any of the endogenous and exogenous opioid peptides ever reported in that the N‐terminal Tyr was the only aromatic amino acid. The analogs containing Tyr‐Pro‐X‐Ser‐Leu were synthesized to study its structure‐activity relationship. Peptides in which X was an aromatic amino acid or an aliphatic hydrophobic amino acid had opioid activity.
The formation of symmetrical GroEL‐GroES complexes in the presence of ATP
Authors: Llorca Oscar , Marco Sergio , Carrascosa José L. , Valpuesta José M.
Published: July 2002

The incubation of chaperonins cpn60 (GroEL) and cpn10 (GroES) from E. coli in the presence of Mg‐ATP and KCl generates the formation, as revealed by electron microscopy, of GroEL—GroES complexes with a symmetrical shape in which one toroidal GroES oligomer is bound to each end of the tetradecameric GroEL aggregate (1:2 GroEL:GroES oligomer molar ratio). The symmetrical complexes are not observed in the presence of ADP or the non‐hydrolyzable ATP analog, ATPγS, where only asymmetrical complexes (1:1 GroEL:GroES oligomer molar ratio) are formed. These results suggest that ATP hydrolysis is required for the formation of symmetrical complexes.