pH-dependent equilibrium between long lived near-UV intermediates of photoactive yellow protein

The long lived intermediate (signaling state) of photoactive yellow protein (PYP(M)), which is formed in the photocycle, was characterized at various pHs. PYP(M) at neutral pH was in equilibrium between two spectroscopically distinct states. Absorption maxima of the acidic form (PYP(M)(acid)) and alkaline form (PYP(M)(alkali)) were located at 367 and 356 nm, respectively. Equilibrium was represented by the Henderson-Hasselbalch equation, in which apparent pK(a) was 6.4. Content of alpha- and/or beta-structure of PYP(M)(acid) was significantly greater than PYP(M)(alkali) as demonstrated by the molar ellipticity at 222 nm. In addition, changes in amide I and II modes of beta-structure in the difference Fourier transform infrared spectra for formation of PYP(M)(acid) was smaller than that of PYP(M)(alkali). The vibrational mode at 1747 cm(-1) of protonated Glu-46 was found as a small band for PYP(M)(acid) but not for PYP(M)(alkali), suggesting that Glu-46 remains partially protonated in PYP(M)(acid), whereas it is fully deprotonated in PYP(M)(alkali). Small angle x-ray scattering measurements demonstrated that the radius of gyration of PYP(M)(acid) was 15.7 Angstroms, whereas for PYP(M)(alkali) it was 16.2 Angstroms. These results indicate that PYP(M)(acid) assumes a more ordered and compact structure than PYP(M)(alkali). Binding of citrate shifts this equilibrium toward PYP(M)(alkali). UV-visible absorption spectra and difference infrared spectra of the long lived intermediate formed from E46Q mutant was consistent with those of PYP(M)(acid), indicating that the mutation shifts this equilibrium toward PYP(M)(acid). Alterations in the nature of PYP(M) by pH, citrate, and mutation of Glu-46 are consistently explained by the shift of the equilibrium between PYP(M)(acid) and PYP(M)(alkali).     

Book reviews

Book reviewed in this article:
C oncepts in V iral P athogenesis III (1989). Edited by A.L. Notkins & M.B.A. Old-stone.
A C olour A tlas of M eat I nspection (1990). By J. Infante Gil & J. Costa Durao.
P romiscuous P lasmids of G ram -N egative B acteria (1989). Edited by Christopher M. Thomas.
S hort P rotocols in M olecular B iology (1989). Edited by F.M. Ausubel et al.
G enetics of B acterial D iversity (1989). Edited by D.A. Hopwood & K.F. Chater.
Y east G enetics . A M anual of M ethods (1989). By J.F.T. Spencer, D.M. Spencer & I.J. Burce.
M etals and M icro -O rganisms (1989). By M.N. Hughes & R.K. Poole.
S eed -B orne D isease and S eed H ealth T esting of R ice (1989). By P. C. Agarwal, C.N. Mortensen & S.B. Mathur.     

Book reviews

Book reviewed in this article:
T he R umen E cosystem : T he M icrobial M etabolism and its R egulation (1990). Edited by S. Hoshino, R. Onodera, H. Minato & H. Itabashi.
M olecular B iological M ethods for B acillus : M odern M icrobiological M ethods (1990). Edited by C.R. Harwood & S.M. Cutting.
B acillus subtilis : M olecular B iology and I ndustrial A pplication (1989). Edited by B. Maruo & H. Yoshikawa.
M olecular P arasitology (1990). By J.E. Hyde.
F undamental V irology (1990). Second edition. Edited by B.N. Fields, D.M. Knipe, R.M. Chanock, M.S. Hirsch, J.L. Melnick & T.P. Monath.     

High prevalence of Mycoplasma infections among European chronic fatigue syndrome patients. Examination of four Mycoplasma species in blood of chronic fatigue syndrome patients

Prevalence of Mycoplasma species infections in chronic fatigue syndrome (CFS) has been extensively reported in the scientific literature. However, all previous reports highlighted the presence of Mycoplasmas in American patients. In this prospective study, the presence of Mycoplasma fermentans, M. penetrans, M. pneumoniae and M. hominis in the blood of 261 European CFS patients and 36 healthy volunteers was examined using forensic polymerase chain reaction. One hundred and seventy-nine (68.6%) patients were infected by at least one species of Mycoplasma, compared to two out of 36 (5.6%) in the control sample (P<0.001). Among Mycoplasma-infected patients, M. hominis was the most frequently observed infection (n=96; 36.8% of the overall sample), followed by M. pneumoniae and M. fermentans infections (equal frequencies; n=67; 25.7%). M. penetrans infections were not found. Multiple mycoplasmal infections were detected in 45 patients (17.2%). Compared to American CFS patients (M. pneumoniae>M. hominis>M. penetrans), a slightly different pattern of mycoplasmal infections was found in European CFS patients (M. hominis>M. pneumoniae, M. fermentansz.Gt;M. penetrans).     

Effect of bradykinin on airway neural responses in vitro.

We investigated the effects of bradykinin (BK) on airway excitatory nonadrenergic noncholinergic (e-NANC) and cholinergic nerves in vitro. Neural responses were elicited by electrical field stimulation in guinea pig airways in vitro before and after the addition of BK (10(-10)-10(-7) M). Captopril (10(-5) M) and phosphoramidon (10(-6) M) were added to prevent degradation of BK, and all neural responses were measured in the presence of indomethacin (10(-5) M) and propranolol (10(-6) M). BK potentiated e-NANC responses in bronchi in a concentration-dependent manner (10(-10)-10(-7) M) without changing concentration-response curves to exogenously applied substance P (10(-10)-10(-5) M). BK significantly potentiated e-NANC neural constrictor responses by 22 +/- 7% at 10(-8) M (mean +/- SE, n = 5, P < 0.05) and 32 +/- 7% at 10(-7) M (n = 8, P < 0.01), compared with changes in time-matched control tissues (7 +/- 2%, n = 8). The potentiation of e-NANC responses by BK was abolished by pretreatment with a specific B2-receptor antagonist, HOE 140 (10(-7) M). Cholinergic constrictor responses elicited to electrical field stimulation were not affected by the addition of BK (up to 10(-7) M). These results suggest that BK potentiates e-NANC bronchoconstrictor responses prejunctionally via a B2-receptor.     

Books Reviews

S cience W riting for B eginners
M icrobiology . E ssentials and A pplications (1985). L. McKane & J. Kandel.
P rogress in C linical and B iological R esearch Volume 178 B luetongue and R elated O rbiviruses (1985). Edited by T. Lynwood Barber, M. M. Jochim & B. I. Osburn.
A ntimicrobial D rug R esistance (1984) Edited by L. E. Bryan.
A spects of M icrobial M etabolism and E cology (1984). Edited by G. A. Codd.
P rogress in L eukocyte B iology Volume 1 V iral M echanisms of I mmunosuppression (1985). Edited by N. Gilmore & M. A. Wainberg.
T he S ociety for A pplied B acteriology T echnical S eries No. 19 M icrobiological M ethods for E nvironmental B iotechnology (1984). Edited by J. M. Grainger & J. M. Lynch.
S crapie D isease in S heep (1983). H. B. Parry. Edited by D. R. Oppenheimer.     

Book reviews

Book reviewed in this article:
I ndustrial M icrobiological T esting (1987). Edited by J.W. Hopton & E.G. Hill.
D rinking W ater M icrobiology (1987). Edited by D.O. Cliver & R.A. Newman.
S urvival and D ormancy of M icroorganisms (1987). Edited by Y. Henis.
B rewing M icrobiology (1987). Edited by F.G. Priest & I. Campbell.
T he P athogenesis of B acterial I nfections (1985). Edited by G.G. Jackson & H. Thomas.
A dvances in M icrobial P hysiology Volume 28 (1986). Edited by A.M. Rose & D.W. Tempest.
A dvances in M icrobial P hysiology Volume 29 (1988). Edited by A.M. Rose & D.W. Tempest.
M icrobial Q uality A ssurance in P harmaceuticals C osmetics and T oiletries (1988). Edited by S.F. Bloomfield, R. Baird, R.E. Leak & R. Leech.     

The design of novel methoctramine-related tetraamines as muscarinic receptor subtype selective antagonists

Several novel methoctramine-related tetraamines were designed, and their biological profiles at muscarinic receptor subtypes were assessed by functional experiments in isolated guinea pig and rat atria (M2) and smooth muscle (ileum and trachea, M3) and by binding assays in rat cortex (M1), heart (M2), and submaxillary gland (M3) homogenates and NG 108-15 cells (M4). Tripitramine, a nonsymmetrical tetraamine, resulted in the most potent and the most selective muscarinic M2 receptor antagonist of the series (pA2 = 9.14-9.85; pKi = 9.54). Spirotramine (FC 15-94), a symmetrical tetraamine, was able to differentiate between muscarinic M1 receptors (pKi = 7.88) and the other subtypes (M2, pKi = 6.20; M3, pKi = 5.81; M4, pKi = 6.27). Thus, tripitramine and spirotramine could be valuable tools for the pharmacological classification and characterization of muscarinic receptor subtypes.     

A simple preparative method for the isolation of amylose and amylopectin from potato starch

The defatted starch was dispersed in NaOH (1 M) and neutralized with HCl (1 M). The amylose 1-butanol complex is adsorbed on defatted cellulose powder in the solvent system containing acetate buffer (pH 4.8,0.1 M) + urea (2 M) + 1-butanol (8.5%, v/v). The complex adsorbed on cellulose powder is separated by centrifugation (2418 g). The sediment is washed with the solvent system-I to obtain the intermediate fraction. The adsorbed amylose is eluted with urea (2 M) in acetate buffer (pH 4.8, 0.1 M). The amylose, intermediate fraction and amylopectin were precipitated with ethanol, washed free of urea and air dried. They were characterized by determining their blue value and beta -amylolysis limit.     

中国木兰属部分种的核型分析

对我国木兰属（Ｍａｇｎｏｌｉａ）１２个种的核型进行了分析,ｘ＝１９,厚朴、凹叶厚朴、红花山玉兰、山玉兰、天目木兰、夜合为二倍体,２ｎ＝２ｘ＝３８;紫玉兰、玉兰、玉堂春为四倍体,２ｎ＝４ｘ＝７６;乐东木兰、荷花玉兰、狭叶荷花玉兰为六倍体,２ｎ＝６ｘ＝１１４,该属具有属内多倍体（次生多倍化）。核型分析结果表明：木兰属种间核型差异较小,大部分为中部着丝粒染色体（ｍ）,少数为具近中部着丝粒染色体（ｓｍ）,二倍体种中只有一对具近端部着丝粒染色体（ｓｔ）,其核型均为２Ｂ型。     

Three different M1 RNA-containing viruslike particle types in Saccharomyces cerevisiae: in vitro M1 double-stranded RNA synthesis.

Killer strains of Saccharomyces cerevisiae bear at least two different double-stranded RNAs (dsRNAs) encapsidated in 39-nm viruslike particles (VLPs) of which the major coat protein is coded by the larger RNA (L-A dsRNA). The smaller dsRNA (M1 or M2) encodes an extracellular protein toxin (K1 or K2 toxin). Based on their densities on CsCl gradients, L-A- and M1-containing particles can be separated. Using this method, we detected a new type of M1 dsRNA-containing VLP (M1-H VLP, for heavy) that has a higher density than those previously reported (M1-L VLP, for light). M1-H and M1-L VLPs are present together in the same strains and in all those we tested. M1-H, M1-L, and L-A VLPs all have the same types of proteins in the same approximate proportions, but whereas L-A VLPs and M1-L VLPs have one dsRNA molecule per particle, M1-H VLPs contain two M1 dsRNA molecules per particle. Their RNA polymerase produces mainly plus single strands that are all extruded in the case of M1-H particles but are partially retained inside the M1-L particles to be used later for dsRNA synthesis. We show that M1-H VLPs are formed in vitro from the M1-L VLPs. We also show that the peak of M1 dsRNA synthesis is in fractions lighter than M1-L VLPs, presumably those carrying only a single plus M1 strand. We suggest that VLPs carrying two M1 dsRNAs (each 1.8 kilobases) can exist because the particle is designed to carry one L-A dsRNA (4.5 kilobases).     

Effect of nutrition and superovulation on oocyte morphology, follicular fluid composition and systemic hormone concentrations in ewes

The objective was to determine the effect of dietary intake on follicle and oocyte morphology in unstimulated and superovulated ewes. Fifty-four ewes were fed grass meal at 0.5, 1.0 or 2.0 times maintenance energy requirements (M) for 32 days. Oestrous cycles were synchronized using progestagen pessaries and either unstimulated or superovulated with 200 mg pig FSH. The ewes were killed and ovaries were collected either 36 or 12 h before the anticipated LH surge. Serum progesterone concentrations in ewes on day 10 after withdrawal of the pessary were lower in ewes fed 2.0M than in ewes fed 0.5M or 1.0M (P < 0.05). LH pulse frequency tended to be higher in ewes fed 2M than 1M (1.0 +/- 0.3 versus 0.3 +/- 0.2 pulses per 8 h) on day 6 after removal of the pessary but the effect was not significant. In unstimulated ewes, more follicles (>/= 3 mm) were observed when the animals were killed in ewes fed 2.0M (3.5 +/- 0.3) than in ewes fed 0.5M (2.4 +/- 0.3) or 1.0M (2.4 +/- 0.5; P < 0. 05). Fewer follicles were observed in superovulated ewes on 0.5M (7. 5 +/- 1.2) than in ewes on 1.0M (12.0 +/- 0.5) or 2.0M (12.3 +/- 1. 4; P < 0.05). Follicular fluid progesterone concentrations were higher in ewes fed 0.5M compared with those fed 1M or 2M (P < 0.05). Insulin-like growth factor (IGF)-I concentrations were higher in follicular fluid from ewes on 1M compared with either those on 0.5M or 2M (P < 0.05), whereas IGF-II concentrations were lower in follicular fluid from ewes on 2M compared with those on 1M or 0.5M (P < 0.05). Superovulation increased follicular fluid progesterone, oestradiol, IGF-I and IGF-II concentrations (P < 0.01). Concentrations of the 34, 22 and 20 kDa IGF binding proteins were lower in follicles from superovulated ewes compared with unstimulated ewes (P < 0.05). Oocytes from superovulated ewes showed abnormalities such as premature activation of cumulus expansion and vacuolation of the nucleolus and increased frequency of detachment of interchromatin-like granules from the nucleolar remnant. Collectively, these results indicate that both high and low dietary intakes can alter systemic and follicular fluid hormone concentrations. Relative to dietary effects, the effects of superovulation were greater and involved substantial increases in follicular fluid hormone concentrations and abnormal oocyte morphology.     

Reviews

Book reviewed in this article:
Bhandari, M. M. 1990. Flora of the Indian Desert (Revised 2nd edition).
Nayar, M. P., Thothatri, M. & Sanjappa, M. (eds). 1988. Fascicles of Flora of India.     

Book reviews

Book reviewed in this article:
HACCP: A P ractical A pproach (1994). By S. Mortimore and C. Wallace.
M olecular B iology of A rchaea (1994). Edited by F. Pfeiffer, P. Palm and K.-H. Schleifer.
R otaviruses (1994). Edited by R.F. Ramig.
P ower U nseen : H ow M icrobes R ule the W orld (1994). By B. Dixon.
V iruses and C ancer (1994). Edited by A. Minson, J. Niel and M. McCrae.
W ater M icrobiology (1994). By G. Bitton.
H andbook for R hizobia : M ethods in L egume - Rhizobium T echnology (1994). By P. Somasegaran and H.J. Hoben.
S tatistics and E xperimental D esign : A n I ntroduction for B iologists and B iochemists (1994). 3rd Edition. By G.M. Clarke
P rinciples of G ene M anipulation : A n I ntroduction to G enetic E ngineering (1994). 5th Edition. By R.W. Old and S.B. Primrose.
R espiratory I nfections : D iagnosis and M anagement (1994). Third edition. Edited by J.E. Pennington.
O bstetric and G ynecologic I nfectious D isease (1994). Edited by J.G. Pastorek II.
M olecular G enetics of B acteria (1994). 2nd Edition. By J.W. Dale.
B acterial P athogenesis : P art A: I dentification and R egulation of V irulence F actors (1994). Methods in Enzymology, Volume 235. Edited by V.L. Clark and P.M. Bavoil
B acterial P athogenesis : P art B: I nteraction of P athogenic B acteria with H ost C ells (1994). Methods in Enzymology, Volume 236. Edited by V.L. Clark and P.M. Bavoil.
M ononuclear P hagocytes : B iology of M onocytes and M acrophages (1992). Edited by R. van Furth.     

A Simple Preparative Method for the Isolation of Amylose and Amylopectin from Potato Starch

The defatted starch was dispersed in NaOH (1 M) and neutralized with HCl (1 M). The amylose 1-butanol complex is adsorbed on defatted cellulose powder in the solvent system containing acetate buffer (pH 4.8, 0.1 M) ± urea (2 M) ± 1-butanol (8.5 %, v/v). The complex adsorbed on cellulose powder is separated by centrifugation (2418 g). The sediment is washed with the solvent system-I to obtain the intermediate fraction. The adsorbed amylose is eluted with urea (2 M) in acetate buffer (pH 4.8, 0.1 M). The amylose, intermediate fraction and amylopectin were precipitated with ethanol, washed free of urea and air dried. They were characterized by determining their blue value and β -amylolysis limit.     

BOOKS REVIEWS

M aintenance OF M icroorganisms . A M anual OF L aboratory M ethods (1984). Edited by B. E. Kirsop & J. J. S. Snell.
P rogress IN C linical AND B iological R esearch , V olume 181, G ermfree R esearch : M icroflora C ontrol AND ITS A pplication TO THE B iomedical S ciences (1985). Edited by B. S. Wostmann, J. R. Pleasants, M. Pollard
S pecial P ublications OF THE S ociety FOR G eneral M icrobiology , N umber 15, C omputer -A ssisted B acterial S ystematics (1985). Edited by M. Goodfellow, D. Jones & F. G. Priest.
R epairable L esions IN M icroorganisms (1984). Edited by A. Hurst & A. Nasim.
T he V irulence OF E scherichia C oli . R eviews AND M ethods (1985). Edited by M. Sussman.     

Genetic divergence and analysis of the relationships between species of Mosla (Labiatae)

Seven species were recognized in Mosla in China. M. pauciflora (C. Y. Wu) C. Y. Wu et H. W. Li is an allotetraploid (2n=36 ), while the other six species are diploids (2n=18). Cluster analysis based on allozyme data from 28 loci of 15 enzyme systems reveals that the six diploid species formed three species pairs. M. cavaleriei Lévl.is closely related to M. dianthera (Buch.-Ham. ex Roxb. ) Maxim., M. chinensis and M. hangchouensis Matsuda are sibling species, and M. scabra (Thunb.) C. Y. Wu et H. W. Li is allied to M. soochouensis. Although M. cavaleriei and M. dianthera are close relatives, considerable genetic divergence has been detected between them. One third of alleles are unique to either of them, and 28.6 % of their loci have different alleles fixed. The average genetic identity ( 1 ) between populations of these two species is 0.770, and the average genetic distance (D) is 0.261. M. scabra and M. soochouensis are the least divergent species pair (I =0.979, D＝0.025). No completely divergent locus was detected, and the percentages of unique alleles are 11.1% to M. scabra and 16.7 % to M. soochouensis. This finding indicates that a high level of genetic differentiation is unnecessarily a prerequisite of speciation. A moderate divergence is detected between M. chinensis and M. hangchouensis (I＝0.899, D=0.107, and 7.1% of completely diverged loci) yet the latter harbors four times as many unique alleles (45.1% ) as the former does(11.8 % ). Compared to the genetic divergence between M. scabra and M. soochouensis, M. dianthera and M. hangchouensis and may well been undergoing active speciation have the high genetic distance betweenpopulations (0.034 and 0.026 respectively).     

Somatic Embryogenesis in Asparagus densiflorus (Kunth) Jessop cv Sprengeri

An efficient protocol has been developed for plant regeneration in Asparagus densiflorus (Kunth) Jessop cv Sprengeri (Asparagaceae) through somatic embryogenesis from spear sections. Callus culture was initiated on Murashige and Skoog (MS) medium formulation containing α-naphthaleneacetic acid (5.37 μM), indole-3-acetic acid (5.71 μM) and 6-benzylaminopurine (2.22 μM). The callus became embryogenic by transferring to 2, 4-dichlorophenoxyacetic acid (4.52 μM) containing MS medium. Somatic embryos developed and matured vigorously on MS medium with NAA (1.07 μM), 6-γ-γ- dimethylaminopurine (9.84 μM) and abscisic acid (1.89 μM). Mature bipolar embryos were converted efficiently into plants on MS medium in the presence of low level of kinetin (2.32 μM). Regenerated plants showed 80% survival after transfer to field. These plants were all diploid (2n=60). Peroxidase activity was maximum in the embryogenic callus as documented from the gel as well as spectrophotometric analysis.     

Reduction of methemoglobins M Hyde Park, M Saskatoon, and M Milwaukee by ferredoxin and ferredoxin-nicotinamide adenine dinucleotide phosphate reductase system

The reduction of hemoglobins (Hb) M such as Hb M Iwate, Hb M Boston, Hb M Hyde Park, Hb M Saskatoon, and Hb M Milwaukee by the ferredoxin and ferredoxin-NADP reductase system was studied systematically under anaerobic conditions. The enzyme system could not reduce the abnormal chains in methemoglobin M with an alpha chain anomaly but effectively converted the methemoglobin M with a beta chain anomaly to the fully reduced form. During the reduction of the methemoglobin M with a beta chain anomaly, the spectra showed a shift of the initial isosbestic points, indicating the possible formation of intermediate hemoglobins in the partially reduced state. On the reduction mode of the methemoglobin M, however, it was classified into three types. 1) Only normal chains were reduced (Hb M Iwate and Hb M Boston). 2) Sequential reduction from normal to abnormal chains occurred (Hb M Milwaukee and Hb M Hyde Park). 3) Normal chains were preferentially reduced, but the reduction of abnormal chains also started at the same rate when the reduction of normal ones had proceeded halfway (Hb M Saskatoon). These differences are discussed in relation to the redox potential of each abnormal chain in methemoglobin M.     

Conformational peculiarities of polynucleotides with a nonrandom base sequence according to the 1H----3H exchange rate in C8H groups of purinic residues

We have determined the 1H----3H exchange rate constants between water and C8H groups of purinic residues of alternating polynucleotides poly(dA-dT).poly(dA-dT), poly(dG-dC).poly(dG-dC) and poly(dA-dC).poly(dG-dT) as well as homopolynucleotides poly(dA).poly(dT) and poly(dG).poly(dC) in aqueous solutions with high-salt concentrations (3 M NaCl and 4-6 M CsF), in water-ethanol (60%) solution and in 0.15 M NaCl at 25 degrees C. The rate constants for adenine (kA) and guanine (kG) of polynucleotides were compared with corresponding constants for E. coli DNA. dGMP nd dAMP at the same conditions. The relation between exchange rates and conformations of polynucleotides permits the study of their conformational peculiarities in solution. Of three alternating polynucleotides examined in 0.15 M NaCl the exchange retardation was observed only for poly(dA-dT).poly(dA-dT) as compared with that in B-DNA, which is in good agreement with the B-alternating "wrinkled" DNA model. The conformations of poly(dG-dC).poly(dG-dC) and poly(dA-dC).poly(dG-dT), according to the exchange data obtained are within the B form. For homopolynucleotides in 0.15 M NaCl, the KA value for poly(dA).poly(dT) is nearly the same as kA for B-DNA, which indicates the similarity of their conformations, whereas the kG value for poly(dG).poly(dC) is 1.7-fold lower in comparison with the kG value in B-DNA. This seems to be connected with the existence of B = A conformation equilibrium for poly(dG).poly(dC) in solution. The increase of NaCl concentration to 3 M results in a B----Z transition in the case of poly(dG-dC).poly(dG-dC) and in the shift of B = A equilibrium towards the A-form in the case of poly(dG).poly(dC) as is evidenced by alterations of their KG values. Poly(dA-dT).poly(dA-dT) in 6 M CsF and poly(dA-dC).poly(dG-dT) in 4.3 M CsF maintain their inherent conformations in 0.15 M NaCl in spite of the fact that they are characterised by the "X-type" CD-spectrum at these conditions. According to the exchange data the conformation of poly(dA).poly(dT) in 6 M CsF corresponds to the "heteronomous" DNA model or some other structure with lower accessibility of C8H groups of adenylic residues.