A rationally designed monomeric variant of anthranilate phosphoribosyltransferase from Sulfolobus solfataricus is as active as the dimeric wild-type enzyme but less thermostable

The anthranilate phosphoribosyltransferase from Sulfolobus solfataricus (ssAnPRT) forms a homodimer with a hydrophobic subunit interface. To elucidate the role of oligomerisation for catalytic activity and thermal stability of the enzyme, we loosened the dimer by replacing two apolar interface residues with negatively charged residues (mutations I36E and M47D). The purified double mutant I36E+M47D formed a monomer with wild-type catalytic activity but reduced thermal stability. The single mutants I36E and M47D were present in a monomer-dimer equilibrium with dissociation constants of about 1 μM and 20 μM, respectively, which were calculated from the concentration-dependence of their heat inactivation kinetics. The monomeric form of M47D, which is populated at low subunit concentrations, was as thermolabile as monomeric I36E+M47D. Likewise, the dimeric form of I36E, which was populated at high subunit concentrations, was as thermostable as dimeric wild-type ssAnPRT. These findings show that the increased stability of wild-type ssAnPRT compared to the I36E+M47D double mutant is not caused by the amino acid exchanges per se but by the higher intrinsic stability of the dimer compared to the monomer. In accordance with the negligible effect of the mutations on catalytic activity and stability, the X-ray structure of M47D contains only minor local perturbations at the dimer interface. We conclude that the monomeric double mutant resembles the individual wild-type subunits, and that ssAnPRT is a dimer for stability but not for activity reasons.     

REVIEWS

Books Review in this article:
Forest Trees and Timbers of the British Empire. Part I. Some East African Coniferae and Leguminosae. By L. C halk , M.A., D.Phil., J. B urtt D avy , M.A., Ph.D. and H. E. D esch , B.SC.
Part II. Twenty West African Timber Trees. By L. C halk , M.A., D.Phil., J. B urtt D avy , M.A., Ph.D., H. E. D esch , B.SC, M.A. and A. C. H oyle , B.Sc, M.A.
Part III. Fifteen South African High Forest Timber Trees. By L. C halk , M.A., D.Phil., M. M. C hattaway , B.SC, M.A., J. B urtt D avy , M.A., Ph.D., F. S. L aughton , B.SC. and M. H. S cott , B.SC.
Colloids in Agriculture. By C. E. M arshall .
Flore Laurentienne. By F rère M arie -V ictorin , D.SC.
Plant Viruses. By K enneth M. S mith .
Plant Life: A Text-book of Botany. By D. B. S wingle .
Gardening in East Africa: A Practical Handbook. Edited by A. J. J ex -B lake , with a foreword by Sir A rthur W. H ill .
British Stem- and Leaf-Fungi (Coelomycetes). Vol. I. Sphaeropsidales. By W. B. G rove , M.A.     

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.     

REVIEWS

Book reviewed in this article
Plant Physiology. By N. A. M aximov , edited by R. B. H arvey and A. E. M urneek .
The Physiology of Plants. By W illiam S eifriz .
A Textbook of General Botany. By R. M. H olman and W. W. R obbins .
Plant Form and Function. By F. E. F ritsch and E. J. S alisbury .
Plant Physiology, with Reference to the Green Plant. By E. C. M iller , Ph.D.
The Vegetation of South Africa. By R. S. A damson , M.A., D.Sc.     

Behavioral and neuroendocrine effects of the selective CRF2 receptor agonists urocortin II and urocortin III

We compared the in vivo efficacy of two selective CRF2 agonists, mouse urocortin II (mUcn II) and human urocortin III (hUcn III), using food intake, anxious behavior, or ACTH release in CD-1 or Balb/c mice as indices of biological stress responses. All three peptides produced anorexia (Minimal Effective Dose (M.E.D.) for CRF and mUcn II = 0.03 nmol; M.E.D. for hUcn III = 0.3 nmol). Only mUcn II and CRF appeared to increase anxious behaviors in the elevated plus maze test (M.E.D. = 0.3 and 0.01 nmol, respectively). CRF increased the release of plasma ACTH (M.E.D. of 0.3 nmol), while mUcn II and hUcn III had no effect on ACTH release. These data suggest that the CRF2 receptor subtype plays a primary role in the activation of behavioral, but not neuroendocrine, stress responses.     

THE EVOLUTION OF THE CONIFER CONE

Die Koniferen des Oberkarbons und des unteren Perms , Parts 1–7. By R udolf F lorin .
Plant Life of the Pacific World. By E lmer D. M errill
Merrilleana , A Selection from the General Writings of E lmer D rew M errill , Sc.D., LL.D.
Forest Soils and Forest Grozuth. By S. A. W ilde , F.E., D.T ech .S c .
The Natural Vegetation of Trinidad. By J. S. B eard .
Dating the Past: An Introduction to Geochronology. By F rederick E. Z euner .
La Culture des Tissits. By R.-J. G autheret .     

Book Reviews

ELECTRON TRANSPORT AND PHOTOPHOS-PHORYLATION, TOPICS IN PHOTOSYNTHESIS , VOL. 4. Edited by J. B arber
THE MATHEMATICS OF PHOTOSYNTHESIS AND PRODUCTIVITY . Experimental Botany. An International Series of Monographs, Vol. 16. By D. A. C harles -E dwards
DIFFERENTIATION IN VITRO . Proceedings of the Fourth Symposium of the British Society for Cell Biology. Edited by M. M. Y eoman & D. E. S. T ruman
PLASMALEMMA AND TONOPLAST: THEIR FUNCTIONS IN THE PLANT CELL . Edited by D. M armè , E. M arrè & R. H ertel
THE PLANT COMMUNITY AS A WORKING MECHANISM , Special Publications Series of the British Ecological Society 1. Edited by E. I. N ewman
THE NATURAL COUMARINS: OCCURRENCE, CHEMISTRY AND BIOCHEMISTRY . By R D. H. M urray , J. M endez & S. A. B rown
BIOLOGICAL CONTROL IN CROP PRODUCTION . Edited by G. C. P apavizas     

Symmetrical stabilization of bound Ca2+ ions in a cooperative pair of EF-hands through hydrogen bonding of coordinating water molecules in calbindin D(9k)

Water molecules are found to complete the Ca2+ coordination sphere when a protein fails to provide enough ligating oxygens. Hydrogen bonding of these water molecules to the protein backbone or side chains may contribute favorably to the Ca2+ affinity, as suggested in an earlier study of two calbindin D(9k) mutants [E60D and E60Q; Linse et al. (1994) Biochemistry 33, 12478-12486]. To investigate the generality of this conclusion, another side chain, Gln 22, which hydrogen bonds to a Ca2+-coordinating water molecule in calbindin D(9k), was mutated. Two calbindin D(9k) mutants, (Q22E+P43M) and (Q22N+P43M), were constructed to examine the interaction between Gln 22 and the water molecule in the C-terminal calcium binding site II. Shortening of the side chain, as in (Q22N+P43M), reduces the affinity of binding two calcium ions by a factor of 18 at low ionic strength, whereas introduction of a negative charge, as in (Q22E+P43M), leads to a 12-fold reduction. In 0.15 M KCl, a 7-fold reduction in affinity was observed for both mutants. The cooperativity of Ca2+ binding increases for (Q22E+P43M), while it decreases for (Q22N+P43M). The rates of Ca2+ dissociation are 5.5-fold higher for the double mutants than for P43M at low ionic strength. For both mutants, reduced strength of hydrogen bonding to calcium-coordinating water molecules is a likely explanation for the observed effects on Ca2+ affinity and dissociation. In the apo forms, the (Q22E+P43M) mutant has lower stability toward urea denaturation than (Q22N+P43M) and P43M. 2D (1)H NMR and crystallographic experiments suggest that the structure of (Q22E+P43M) and (Q22N+P43M) is unchanged relative to P43M, except for local perturbations in the loop regions.     

13种石斛属植物遗传多样性的AFLP分析

采用AFLP技术对13种石斛属植物的遗传多样性进行了分析。选择性扩增引物组合E ACT/M CAC、E AAC/M CAC和E ACA/M CAC分别对这13种材料进行扩增,得到丰富的条带。在100-300bp共得到346条带,多态性带342条,多态性百分率为98.8%。聚类分析结果表明,在相似系数0.54处,可将13种材料分为Ⅰ、Ⅱ、Ⅲ类。Ⅰ类包括:串珠石斛、铁皮石斛、广东石斛、重唇石斛、晶帽石斛、细叶石斛、滇桂石斛、报春石斛、玫瑰石斛、球花石斛;Ⅱ类包括:鼓槌石斛;Ⅲ类包括:美花石斛(花,浅红)、美花石斛(花,淡白)。AFLP分析结果与传统分类学的结果基本一致,表明该标记技术对石斛属植物的遗传多样性和分类研究是可行的。     

REVIEWS

Book reviewed in this article
Growth Regulators in Plant Science, Monograph 8. Ed. by M. B. J ackson , B. G rout and I. A. M ackenzie .
An Introduction to Plant Taxonomy. By C. J effrey .
Pest and Diseases of Tropical Crops , Vol. 1. By D. S. H ill and J. M. W aller .
Flora of Turkey , Vol. 7 ( Orobanchaceae to Ceratophyllaceae and Ruhiaceae ). Ed. by P. H. D avis , assisted by J. R. E dmondson , R. R. M ill and K. T an .
Plant Growth Curves. The Functional Approach to Plant Growth Analysis. By R oderick H unt .
Bryophyte Ecology. Ed. by A. J. E. S mith . Bryophyte Ecology. Ed. by A. J. E. S mith . Bryophyte Ecology. Ed. by A. J. E. S mith . Bryophyte Ecology. Ed. by A. J. E. S mith .
Forest Succession: concepts and application. Ed. by D. C. W est , M. H. S hugart and D. B otkin .     

Books reviews

M olecular B iology of P lant -P athogen I nteractions (1989). Edited by B. Staskawicz, P. Ahlquist & O. Yoder.
A dvances in B iochemical E ngineering /B iotechnology . Volume 36. E nzyme S tudies . (1988). Edited by A. Fiechter.
Microbial Proteinases: H.M. Kalisz The Amidases from a Bacterium Strain: M. Maestracci, K. Bui, A. Thiery, A. Arnaud and P. Glazy
L aboratory D iagnosis of I nfectious D iseases : P rinciples and P ractice (1988).
S afety A ssurance for E nvironmental I ntroductions of G enetically -E ngineered O rganisms (1988). Edited by J. Fiksel & V.T. Covello.
C oncise E ncyclopedia of S cience and T echnology (1989). 2nd Edition. Edited by S.P. Parker.
N ew C oncepts in V iral H eart D isease : V irology , I mmunology and C linical M anagement (1988).     

Reviews

Books reviewed in this article:
Molecular Biology of Photosynthesis. Ed. By G ovindjee , H. J. B ohnert , W. B ottomley , D. A. B ryant , J. E. M ullet , W. L. O gren , H. P akrasi and C. R. S omerville .
Biochemical and Physiological Aspects of Ethylene Production in Lower and Higher Plants. Ed. by H. C lijsters , M. de P roft , R. M arcelle and M. V an P oucke .
Nitrogen, Phosphorus and Sulphur Utilisation by Fungi. By L. B oddy , R. M archant and D. J. R ead .
Fungi and Ecological Disturbance. Ed. by L. B oddy , R. W atling and A. J. E. L yon .
Phytoplankton , 2nd Edn. By A. D. B oney .
The Holocene: An Environmental History. By N eil R oberts .
Chambers Biology Dictionary. Ed. by P. M. B. W alker , C.B.E., F.R.S.E.     

Dehydrogenase activity of the pineal gland during the estrous cycle

An investigation of the relationship of succinic dehydrogenase (SDH) and lactate dehydrogenase (LDH) in the pineal gland of the adult female albino rats was carried out during seven definitive stages of the estrous cycle: Early Estrus (E1), Late Estrus (E2), Metestrus (ME), Early Diestrus (D1), Late Diestrus (D2), Preproestrus (PPE) and Proestrus (PE). Controlled housing conditions were maintained on 7:00 A.M. to 7:00 P.M. light-dark cycle. The animals were necropsied at precise times after the onset of estrus and subsequent stages of the cycle. Comparative histochemical and biochemical analysis revealed the following for SDH activity: E2 greater than E1 greater than D2 greater than D1 greater than PPE greater than PE greater than ME; and for LDH activity: PE greater than E2 greater than ME greater than D1 greater than PPE greater than E1 greater than D2. Interrelationships are postulated between pineal SDH, GOT, GPT, and estrogen concentrations in the rat.     

Effects of daily administration of tetrahydrocannabinol on rat preovulatory follicles. A quantitative electron-microscopic analysis

Using morphometric techniques, we analyzed the effects of delta 9-tetrahydrocannabinol (THC) on the ultrastructure of rat preovulatory follicles. Rats were injected intraperitonally once daily with vehicle alone, or with vehicle plus THC on diestrus (D), metestrus (M) and diestrus (D), or on estrus (E), M and D. All animals were killed on proestrus. Nuclear, mitochondrial, lipid droplet and lysosomal volume density in the membrana granulosa were analyzed. There was no change in nuclear volume, but there were significant THC-induced decreases in mitochondrial and lipid droplet volumes in the peripheral region of the granulosa. There were increases in dense-body volume in all regions of the granulosa from rats given THC either on D alone, or M and D. Dense-body volume returned to control levels in rats injected on E, M and D. It appears that THC can affect follicular ultrastructure and the effects change with the number of injections.     

Comparative study on the effectiveness of modified Kato's cellophane thick smear and Stoll's dilution egg counting technique for quantitative fecal examination of helminth eggs]

A total of 197 fecal specimens was prepared for quantitative examination of helminth eggs by modified Kato's cellophane thick smear (M.C.T.S.) and Stoll's dilution egg counting technique (D.E.C.T.). The comparative effectiveness of two techniques was evaluated and conversion function was deduced. The average time required for the microscopic examination on one slide by M.C.T.S. was 12.6 minutes and that of D.E.C.T. was 14.6 minutes. M.C.T.S. showed lower false negative rate than D.E.C.T. in light worm burden cases. Functions to convert the counts obtained by M.C.T.S. to E.P.G. by Stoll's dilution egg counting technique were 47.86 x 10(0.87) logM.C.T.S. in A. lumbricoides, 41.69 x 10(0.82) logM.C.T.S. in T. trichiura and 63.10 x 10(0.85) logM.C.T.S. in C. sinensis. It was suggested M.C.T.S. be better than D.E.C.T. for the quantitative examination of intestinal helminthiases such as A. lumbricoides, T. trichiura, and C. sinensis infections even in the cases with low worm burden.     

Book Reviews

Book reviewed in this article:
Winter Garden Glory. Adrian Bloom
Gardening with Perennials Month by Month. Joseph Hudek
Flora Europaea. Volume 1, second edition, Psilotaceae to Platanaceae. Edited by T. G. Tutin, N. A. Burgess, A. O. Chater, J. R. Edmondson, V. H. Heywood, D. M. Moore, D. H. Valentine, S. M. Walters and D. A. Webb, assisted by J. R. Akeroyd and M. E. Newton
Bark. The Formation, Characteristics and Usefulness of Bark around the World. Ghillean Tolmie Prance and Anne E. Prance     

Functional and biochemical analysis of the type 1 inositol (1,4,5)-trisphosphate receptor calcium sensor

Modulation of the type 1 inositol (1,4,5)-trisphosphate receptors (InsP(3)R1) by cytosolic calcium (Ca(2+)) plays an essential role in their signaling function, but structural determinants and mechanisms responsible for the InsP(3)R1 regulation by Ca(2+) are poorly understood. Using DT40 cell expression system and Ca(2+) imaging assay, in our previous study we identified a critical role of E2100 residue in the InsP(3)R1 modulation by Ca(2+). By using intrinsic tryptophan fluorescence measurements in the present study we determined that the putative InsP(3)R1 Ca(2+)-sensor region (E1932-R2270) binds Ca(2+) with 0.16 micro M affinity. We further established that E2100D and E2100Q mutations decrease Ca(2+)-binding affinity of the putative InsP(3)R1 Ca(2+)-sensor region to 1 micro M. In planar lipid bilayer experiments with recombinant InsP(3)R1 expressed in Spodoptera frugiperda cells we discovered that E2100D and E2100Q mutations shifted the peak of the InsP(3)R1 bell-shaped Ca(2+) dependence from 0.2 micro M to 1.5 micro M Ca(2+). In agreement with the biochemical data, we found that the apparent affinities of Ca(2+) activating and inhibitory sites of the InsP(3)R1 were 0.2 micro M for the wild-type channels and 1-2 micro M Ca(2+) for the E2100D and E2100Q mutants. The results obtained in our study support the hypothesis that E2100 residue forms a part of the InsP(3)R1 Ca(2+) sensor.     

Books

Book reviews in this article:
C ommunication and B ehavior of W hales . R. Payne (ed.).
S trandings : W ays T o S ave W hales . F. D. r obson .
O rders and F amilies of R ecent M ammals of T he W orld . S. Anderson and J. Knox Jones, Jr. (eds.).
S oviet -A merican C ooperative R esearch on M arine M ammals . V olume l—P innipeds . F. H. Fay and G. A. Fedoseev (eds.).
P athobiology of M arine M ammal D iseases . E. B. Howard (ed.).
S eals of T he W orld . (Second Edition.) J. E. Ring. British Museum and Cornell University Press.
T he G ray W hale : E schrichtius R obustus . M. L. Jones, S. L. Swartt and S. Leatherwood.
T he S ierra C lub H andbook of W hales and D olphins . S. Leatherwood and R. R. Reeves.     

Assignment of molecular properties of a superactive coagulation factor VIIa variant to individual amino acid changes.

The most active factor VIIa (FVIIa) variants identified to date carry concurrent substitutions at positions 158, 296 and 298 with the intention of generating a thrombin-mimicking motif, optionally combined with additional replacements within the protease domain [Persson, E., Kjalke, M. & Olsen, O. H. (2001) Proc. Natl Acad. Sci. USA98, 13583-13588]. Here we have characterized variants of FVIIa mutated at one or two of these positions to assess the relative importance of the individual replacements. The E296V and M298Q mutations gave an increased intrinsic amidolytic activity (about two- and 3.5-fold, respectively) compared with wild-type FVIIa. An additive effect was observed upon their combination, resulting in the amidolytic activity of E296V/M298Q-FVIIa being close to that of the triple mutant. The level of amidolytic activity of a variant was correlated with the rate of inhibition by antithrombin (AT). Compared with wild-type FVIIa, the Ca2+ dependence of the intrinsic amidolytic activity was significantly attenuated upon introduction of the E296V mutation, but the effect was most pronounced in the triple mutant. Enhancement of the proteolytic activity requires substitution of Gln for Met298. The simultaneous presence of the V158D, E296V and M298Q mutations gives the highest intrinsic activity and is essential to achieve a dramatically higher relative increase in the proteolytic activity than that in the amidolytic activity. The N-terminal Ile153 is most efficiently buried in V158D/E296V/M298Q-FVIIa, but is less available for chemical modification also in the presence of the E296V or M298Q mutation alone. In summary, E296V and M298Q enhance the amidolytic activity and facilitate salt bridge formation between the N-terminus and Asp343, E296V reduces the Ca2+ dependence, M298Q is required for increased factor X (FX) activation, and the simultaneous presence of the V158D, E296V and M298Q mutations gives the most profound effect on all these parameters.     

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.