The complete amino acid sequence of the low molecular weight cytosolic acid phosphatase

This paper presents the complete amino acid sequence of the low molecular weight acid phosphatase from bovine liver. This isoenzyme of the acid phosphatase family is located in the cytosol, is not inhibited by L-(+)-tartrate and fluoride ions, but is inhibited by sulfhydryl reagents. The enzyme consists of 157 amino acid residues, has an acetylated NH2 terminus, and has arginine as the COOH-terminal residue. All 8 half-cystine residues are in the free thiol form. The molecular weight calculated from the sequence is 17,953. The sequence was determined by characterizing the peptides purified by reverse-phase high performance liquid chromatography from tryptic, thermolytic, peptic, Staphylococcus aureus protease, and chymotryptic digests of the carboxymethylated protein. No sequence homologies were found with the two known acylphosphatase isoenzymes or the metalloproteins porcine uteroferrin and purple acid phosphatase from bovine spleen (both of which have acid phosphatase activity). Two half-cystines at or near the active site were identified through the reaction of the enzyme with [14C] iodoacetate in the presence or in the absence of a competitive inhibitor (i.e. inorganic phosphate). Ac-A E Q V T K S V L F V C L G N I C R S P I A E A V F R K L V T D Q N I S D N W V I D S G A V S D W N V G R S P N P R A V S C L R N H G I N T A H K A R Q V T K E D F V T F D Y I L C M D E S N L R D L N R K S N Q V K N C R A K I E L L G S Y D P Q K Q L I I E D P Y Y G N D A D F E T V Y Q Q C V R C C R A F L E K V R-OH.     

The complete amino acid sequence of coagulogen isolated from Southeast Asian horseshoe crab, Carcinoscorpius rotundicauda

The complete amino acid sequence of coagulogen purified from the hemocytes of the horseshoe crab Carcinoscorpius rotundicauda was determined by characterization of the NH2-terminal sequence and the peptides generated after digestion of the protein with lysyl endopeptidase, Staphylococcal aureus protease V8 and trypsin. Upon sequencing the peptides by the automated Edman method, the following sequence was obtained: A D T N A P L C L C D E P G I L G R N Q L V T P E V K E K I E K A V E A V A E E S G V S G R G F S L F S H H P V F R E C G K Y E C R T V R P E H T R C Y N F P P F V H F T S E C P V S T R D C E P V F G Y T V A G E F R V I V Q A P R A G F R Q C V W Q H K C R Y G S N N C G F S G R C T Q Q R S V V R L V T Y N L E K D G F L C E S F R T C C G C P C R N Y Carcinoscorpius coagulogen consists of a single polypeptide chain with a total of 175 amino acid residues and a calculated molecular weight of 19,675. The secondary structure calculated by the method of Chou and Fasman reveals the presence of an alpha-helix region in the peptide C segment (residue Nos. 19 to 46), which is released during the proteolytic conversion of coagulogen to coagulin gel. The beta-sheet structure and the 16 half-cystines found in the molecule appear to yield a compact protein stable to acid and heat. The amino acid sequences of coagulogen of four species of limulus have been compared and the interspecies evolutionary differences are discussed.     

Pages of memoirs

My studies at the Biological Faculty of Moscow State University were particularly influenced by R.L. Berg, Academician I.I. Schmalhausen, Professors B.S. Matveev and V.G. Geptner, Docents A.N. Druzhinin, and N.V. Shibanov. In my first years at the Paleontological Institute, I learnt a great deal from B.P. Vjuschkov, Professors I.A. Efremov, K.K. Flerov, and P.G. Danilchenko, and Academician Yu.A. Orlov. These memoirs describe a meeting of the Scientific Council of the Biological Faculty of Moscow State University dedicated to discussion of intraspecific competition, and the consequences of this meeting after the Session of All-Union Academy of Agricultural Sciences (VASKhNIL) in August of 1948.     

A Three-Step Method for Teaching the Principles of Evolution to Non-Biology Major Undergraduates

A method for teaching the principles of evolution in a 50-minute lecture for undergraduate non-biology majors is described. The method “unpacks” evolution into three observable, factual occurrences: replication (R, reproduction), variation (V, differences between parent and offspring and siblings), and selection (S, nonrandom differential survival of offspring). This method has been particularly effective in demonstrating to students that evolution is the factual, unintended consequence of three independent phenomena (R, V, S).     

Isolation, characterization and biological activity of a CRF-related diuretic peptide from Periplaneta americana L.

A diuretic peptide (Periplaneta-DP) has been isolated from extracts of whole heads of the cockroach, Periplaneta americana. The purified peptide increases cyclic AMP production and the rate of fluid secretion by isolated Malpighian tubules in vitro. In the fluid secretion assay, the response to native Periplaneta-DP is comparable to that obtained with crude extracts of cockroach corpora cardiaca, and the EC50 lies between 10(-8) and 10(-9) M. The primary structure of Periplaneta-DP was established as a 46-residue amidated peptide: T G S G P S L S I V N P L D V L R Q R L L L E I A R R R M R Q S Q D Q I Q A N R E I L Q T I-NH2. Periplaneta-DP is a further member of the recently established family of CRF-related insect diuretic peptides.     

Comparative phylogenetic analysis of aquaporins provides insight into the gene family expansion and evolution in plants and their role in drought tolerant and susceptible chickpea cultivars

Aquaporins (AQPs) are water channel proteins that play a significant role in drought stress. Although the AQPs identified in multiple plant species, there is no detailed evolutionary and comparative study of AQPs regarding chickpea plant. The current study involved evolutionary analyses coupled with promoter and expression analyses of chickpea AQPs (CaAQPs). A total of 924 non-redundant AQPs were studied in 24 plant species including algae, mosses, lycophytes, monocots and dicots. Phylogenetic analysis demonstrated a clear divergence of eight AQP subfamilies (LIPs, SIPs, GIPs, NIPs, XIPs, PIPs, HIPs and TIPs). The comparative phylogenetic trees of AQP subfamilies among Arabidopsis, soybean, common bean, maize and chickpea demonstrated that the AQPs were highly species-specific. Interestingly, the dual NPA motif was conserved in all species. However, the ar/R selectivity filter signatures [W/T/S/N/G/A]-[V/S/L/I/A]-[S/G/A]-R (in NIPs), F-H-T-R (in PIPs), [H/N/Q/S]-[A/I/L/S/V]-[A/G]-[A/C/L/M/R/V] (in TIPs) and [V/I/L/M]-[V/I/A/F/M]-[A/S/F/C]-[N/F/L/I/A/S (in SIPs) were found in five species. Moreover, the Froger's positions (P1-P5) were found as [F/L/Y]-[S/T]-A-Y-[L/I/M/V/F] (in NIPs), [Q/E/M]-S-A-F-W (in PIPs), [A/L/S/T/V]-[A/C/N/S/T/V]-[P/R/S]-[Y/N/F]-[W/Q] (in TIPs) and [I/M/F]-[A/V]-[A/V]-Y-W (in SIPs). The MEME motif analyses showed that most of the motifs were specific to subfamily and subgroups. Tissue-specific expression profiling of CaAQPs revealed that CaTIPs and CaPIPs are highly expressed in most of the tissues, while CaNIPs and CaSIPs have low expression. In promoter analysis of CaAQPs, multiple stress-related cis-acting elements e.g. MYB, MYC, ABRE, etc. were found. Semi-quantitative RT-PCR analysis showed that CaPIP2;3 and CaNIP3;1 are positive regulator, while CaSIP1;1 and CaPIP2;1 have a negative role in drought tolerance. The findings and implications of this study are discussed in detail.     

C. botulinum neurotoxin types A and E: isolated light chain breaks down into two fragments. Comparison of their amino acid sequences with tetanus neurotoxin

The flaccid paralysis in the neuromuscular disease botulism appears to depend on the coordinated roles of the approximately 50 kDa light and approximately 100 kDa heavy chain subunits of the approximately 150 kDa neurotoxic protein produced by Clostridium botulinum (J. Biol. Chem. (1987) 262, 2660 and Eur. J. Biochem. (1988) 177, 683). We observed that the light chain after separation from its conjugate heavy chain, in the presence of dithiothreitol and 2 M urea, begins to split into approximately 28 and approximately 18 kDa fragments. The other subunit-the approximately 100 kDa heavy chain following its isolation-and the parent approximately 150 kDa dichain neurotoxin do not break down under comparable conditions. This cleavage was examined in the neurotoxin serotypes A and E. The cleavage does not appear to be due to a protease. Partial amino acid sequences established that: i) the approximately 28-kDa and approximately 18-kDa fragments comprise the N- and C-terminal regions of the light chain, respectively; ii) the light chain of the neurotoxin serotypes A and E break down at precise peptide bonds; iii) the peptide bonds cleaved in serotypes A and E are five residues apart; and iv) the portions of the approximately 18 kDa fragments of serotype A and E neurotoxin sequenced so far are highly homologous to the corresponding region of tetanus neurotoxin produced by Clostridium tetani. The partial N-terminal sequence of the approximately 28 kDa fragment matches with the N-terminal sequence of the intact L chain. The 47 residues of the approximately 18-kDa fragment of type A sequenced from its N-terminal are: -Y.E.M.S.G.L.E.V.S.F.E.E.L.R.T.F.G.G.H.D.A.K.F.I.D.S.L.Q.E.N.E.F.R.L.Y.Y .Y. N.K.F.K. D.I.A.S.T.L.-. These align with those of tetanus neurotoxin beginning at its residue #259 (Tyr); the 18 underlined residues of the above 47 residues (i.e. 38%) are identical in positions between the two proteins. The 41 residues sequenced from the approximately 18 kDa fragment of type E botulinum neurotoxin are: -K.G.I.N.I.E.E.F.L. T.F.G.N.N.D.L.N.I.I.T.V.A.Q.Y.N.D.I.Y.T.N.L.L.N.D.Y.R. K.I.A.X.K. L.-.(ABSTRACT TRUNCATED AT 250 WORDS)     

Book Reviews

Book reviewed in this article:
Buning, J.: The insect ovary. Ultrastructure, previtello-genic growth and evolution.
Robinson, G. S. Nielsen, E. S.: Tineid Genera of Australia.
Common, I. F. B.: Oecophorine genera of Australia I. The Wingia group.
REAL, L. A. (ed.): Ecological genetics. 95–195
KRIMBAS, C. B.: Drosophila subobscura. Biology, Genetics and Inversion Polymorphism.
BENECKE, N.: Der Mensch und seine Haustiere.     

Overall Prevalence and Distribution of Knockdown Resistance (kdr) Mutations in Aedes aegypti from Mandalay Region,Myanmar

Knockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC) of mosquitoes confer resistance to insecticides. Although insecticide resistance has been suspected to be widespread in the natural population of Aedes aegypti in Myanmar, only limited information is currently available. The overall prevalence and distribution of kdr mutations was analyzed in Ae. aegypti from Mandalay areas, Myanmar. Sequence analysis of the VGSC in Ae. aegypti from Myanmar revealed amino acid mutations at 13 and 11 positions in domains II and III of VGSC, respectively. High frequencies of S989P (68.6%), V1016G (73.5%), and F1534C (40.1%) were found in domains II and III. T1520I was also found, but the frequency was low (8.1%). The frequency of S989P/V1016G was high (55.0%), and the frequencies of V1016G/F1534C and S989P/V1016G/F1534C were also high at 30.1% and 23.5%, respectively. Novel mutations in domain II (L963Q, M976I, V977A, M994T, L995F, V996M/A, D998N, V999A, N1013D, and F1020S) and domain III (K1514R, Y1523H, V1529A, F1534L, F1537S, V1546A, F1551S, G1581D, and K1584R) were also identified. These results collectively suggest that high frequencies of kdr mutations were identified in Myanmar Ae. aegypti, indicating a high level of insecticide resistance.     

Lorenz,K. 1992: Denkwege. Ein Lesebuch. Herausgegeben von Beatrice Lorenz. Piper Verlag,München,Zürich

Book Reviewed in this article: Lorenz, K. 1992: Die Naturwissenschaft vom Menschen. Eine Einführung in die vergleichende Verhaltensforschung. Das russische Manuskript 1944–1948. Bateson, P. P. G., Klopfer, P. H. & Thompson, N. S. (eds) 1993: Perspectives in ethology. Vol. 10, Behavior and evolution.     

Biodiversity and spatial arrangement of Siberian flora

Algae, lichens, mushrooms, Bryophyta, and higher vascular plants that grow in West and Central Siberia are quite diverse. Their spatial arrangement is presented at levels of plain and mountainous territories, vegetation zones and subzones, altitudinal belts, and the most widespread ecosystems. Original Russian Text ∉ V.P. Sedel’nikov, Yu.V. Naumenko, N.V. Sedel’nikova, I.A. Gorbunova, O.Yu. Pisarenko, D.N. Shaulo, 2007, published in Sibisrkii Ekologicheskii Zrurnal, 2007, Vol. 14, No. 2, pp. 159–168.     

Purification and N-Terminal Amino Acid Sequence of Dextranicin 24, a Bacteriocin of Leuconostoc sp.

Leuconostoc mesenteroides subsp. dextranicum strain J24 synthesized a bacteriocin named Dextranicin 24 (Dex-24), which inhibited only other Leuconostoc sp. strains. It was purified by a two-step procedure from the fraction of the bacteriocin bound to the producer cells at the end of the growth: desorption form the cells at acidic pH, followed by reserve phase HPLC. The N-terminal sequence of Dex-24 was the following: NH₂₋ K G V L G W L S M A S S A L T G P Q Q . . . Received: 26 January 1996 / Accepted: 28 March 1996     

Fabry disease: thirty-five mutations in the alpha-galactosidase A gene in patients with classic and variant phenotypes.

BACKGROUND: Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from mutations in the alpha-galactosidase A (alpha-Gal A) gene located at Xq22.1. To determine the nature and frequency of the molecular lesions causing the classical and milder variant Fabry phenotypes and for precise carrier detection, the alpha-Gal A lesions in 42 unrelated Fabry hemizygotes were determined. MATERIALS AND METHODS: Genomic DNA was isolated from affected probands and their family members. The seven alpha-galactosidase A exons and flanking intronic sequences were PCR amplified and the nucleotide sequence was determined by solid-phase direct sequencing. RESULTS: Two patients with the mild cardiac phenotype had missense mutations, I9IT and F113L, respectively. In 38 classically affected patients, 33 new mutations were identified including 20 missense (MIT, A31V, H46R, Y86C, L89P, D92Y, C94Y, A97V, R100T, Y134S, G138R, A143T, S148R, G163V, D170V, C202Y, Y216D, N263S, W287C, and N298S), two nonsense (Q386X, W399X), one splice site mutation (IVS4 + 2T-->C), and eight small exonic insertions or deletions (304del1, 613del9, 777del1, 1057del2, 1074del2, 1077del1, 1212del3, and 1094ins1), which identified exon 7 as a region prone to gene rearrangements. In addition, two unique complex rearrangements consisting of contiguous small insertions and deletions were found in exons 1 and 2 causing L45R/H46S and L120X, respectively. CONCLUSIONS: These studies further define the heterogeneity of mutations causing Fabry disease, permit precise carrier identification and prenatal diagnosis in these families, and facilitate the identification of candidates for enzyme replacement therapy.     

Dependencies of Substitution Steps Number on Hamming Distance Are Identical For One-parameter Discrete Models of Both Direct and Parallel Genetic Diversity

Abstract

With the help of previously introduced enumeration procedure (M.Yu. Shchelkanov, A.N. Yudin, A.V Antonov, N.S. Starikov, A.A. Vedenov, E.V. Karamov, J. Biomol. Struct. Dyn. 15, 217–229 (1997)) and probability distribution function for the enumeration after some substitution steps (M.Yu. Shchelkanov, L.A. Soinov, V.V. Zalunin, D.A. Gumennyi, A.N. Yudin, A.A. Natan, V.B. Kireev, E.V. Karamov, J. Biomol. Struct. Dyn. 15, N 4, (1998)) we have demonstrated that dependencies of replication acts number on Hamming distance are identical for one-parameter discrete models of both direct and parallel genetic diversity.     

What Capabilities for the Animal?

In this essay, I defend a bi-constructivist approach to ethology—a constructivist ethology assuming that each animal adopts constructivist strategies. I put it in opposition to what I call a realist-Cartesian approach, which is currently the dominant approach to ethology and comparative psychology. The starting point of the bi-constructivist approach can be formulated as a shift from the classical Aristotelian question “What is an animal?” to the Spinozean question, which is much less classical but which seems to me to be much stronger: “What are the capacities of the animal?”. Is it possible to conceptualize an ethology which insists on interpretation and therefore on invention, innovation and creativity, rather than on causality, the monotony of behavioural routines, and/or genetic or environmental determination? Such an ethology would be based not on the fiction of an absent observer but on fully recognizing the necessity of an observer, who is effectively present in order to get an observation. A pluralistic ethology does not dissociate itself from the marginal epistemologies of practitioners like animal trainers, hunters, stockbreeders etc., or, moreover, non-western experts. An ethology of this kind is not clamped within the boundaries of purely academic epistemology, obsessed by demarcation lines between the human and the animal. My work on the bi-constructivist approach represents a contribution towards the elaboration of an authentically biosemiotic ethology, one which is significantly different from the mechanical ethology of today.     

Structural and kinetic analysis of drug resistant mutants of HIV-1 protease.

Mutants of HIV-1 protease that are commonly selected on exposure to different drugs, V82S, G48V, N88D and L90M, showed reduced catalytic activity compared to the wild-type protease on cleavage site peptides, CA-p2, p6pol-PR and PR-RT, critical for viral maturation. Mutant V82S is the least active (2-20% of wild-type protease), mutants N88D, R8Q, and L90M exhibit activities ranging from 20 to 40% and G48V from 50 to 80% of the wild-type activity. In contrast, D30N is variable in its activity on different substrates (10-110% of wild-type), with the PR-RT site being the most affected. Mutants K45I and M46L, usually selected in combination with other mutations, showed activities that are similar to (60-110%) or greater than (110-530%) wild-type, respectively. No direct relationship was observed between catalytic activity, inhibition, and structural stability. The mutants D30N and V82S were similar to wild-type protease in their stability toward urea denaturation, while R8Q, G48V, and L90M showed 1.5 to 2.7-fold decreased stability, and N88D and K45I showed 1.6 to 1.7-fold increased stability. The crystal structures of R8Q, K45I and L90M mutants complexed with a CA-p2 analog inhibitor were determined at 2.0, 1.55 and 1.88 A resolution, respectively, and compared to the wild-type structure. The intersubunit hydrophobic contacts observed in the crystal structures are in good agreement with the relative structural stability of the mutant proteases. All these results suggest that viral resistance does not arise by a single mechanism.     

Structural basis for type I and type II deficiencies of antithrombotic plasma protein C: Patterns revealed by three-dimensional molecular modelling of mutations of the protease domain

Familial deficiency of protein C is associated with inherited thrombophilia. To explore how specific missense mutations might cause observed clinical phenotypes, known protein C missense mutations were mapped onto three-dimensional homology models of the protein C protease domain, and the implications for domain folding and structure were evaluated. Most Type I missense mutations either replaced internal hydrophobic residues (I201T, L223F, A259V, A267T, A346T, A346V, G376D) or nearby interacting residues (I403M, T298M, Q184H), thus disrupting the packing of internal hydrophobic side chains, or changed hydrophilic residues, thus disrupting ion pairs (N256D, R178W). Mutations (P168L, R169W) at the activation site destabilized the region containing the activation peptide structure. Most Type II mutations involved solvent-exposed residues and were clustered either in a positively charged region (R147W, R157Q, R229Q, R352W) or were located in or near the active site region (S252N, D359N, G381S, G391S, H211Q). The cluster of arginines 147, 157, 229, and 352 may identify a functionally important exosite. Identification of the spatial relationships of natural mutations in the protein C model is helpful for understanding manifestations of protein C deficiency and for identification of novel, functionally important molecular features and exosites. © 1994 John Wiley & Sons, Inc.     

In vivo and in vitro characterization of TEV protease mutants

Tobacco etch virus protease (TEVp) is frequently applied in the cleavage of fusion protein. However, production of TEV protease in Escherichia coli is hampered by low yield and poor solubility, and auto-cleavage of wild type TEVp gives rise to the loss-of-function. Previously it was reported that TEVp S219V displayed more stability, and TEVp variant containing T17S/N68D/I77V and double mutant L56V/S135G resulted in the enhanced production and solubility, respectively. Here, we introduced T17S/N68D/I77V in TEVp S219V to generate TEVpM1 and combined five amino acid mutations (T17S/L56V/N68D/I77V/S135G) in TEVp S219V to create TEVpM2. Among TEVp S219V, and two constructed variants, TEVpM2 displayed highest solubility and catalytic activity in vivo, using EmGFP as the solubility reporter, and the designed fusion protein as in vivo substrate containing an N-terminal hexahistidine tagged GST, a peptide sequence for thrombin and TEV cut and E. coli diaminopropionate ammonia-lyase. The purified TEVp mutants fused with double hexahistidine-tag at N and C terminus showed highest yield, solubility and cleavage efficiency. Mutations of five amino acid residues in TEVpM2 slightly altered protein secondary structure conformed by circular dichroism assay.     

Familial Mediterranean fever in Syrian patients: <Emphasis Type="Italic">MEFV</Emphasis> gene mutations and genotype–phenotype correlation

Familial Mediterranean fever is an autosomal recessive disorder characterized by recurrent attacks of abdominal pain, synovitis and pleuritis. MEFV gene mutations are responsible for the disease. The objective of this study was to identify the frequency and distribution of 12 MEFV mutations in 153 Syrian patients and perform a genotype–phenotype correlation in the patients’ cohort. Of the 153 unrelated patients investigated, 97 (63.4%) had at least one mutation. The most frequent mutation was M694V (36.5%), followed by V726A (15.2%), E148Q (14.5%), M680I (G/C) (13.2%), and M694I (10.2%) mutations. Rare mutations (R761H, A744S, M680I (G/A), K695R, P369S, F479L and I692del) were also detected in the patients. M694V was associated with the severe form of the disease. The identification of a significant number of FMF patients with no mutations or only one known mutation identified indicates the presence of new mutations in the MEFV gene which will be investigated in the future.     

Kinetic, stability, and structural changes in high-resolution crystal structures of HIV-1 protease with drug-resistant mutations L24I, I50V, and G73S

The crystal structures, dimer stabilities, and kinetics have been analyzed for wild-type human immunodeficiency virus type 1 (HIV-1) protease (PR) and resistant mutants PR(L24I), PR(I50V), and PR(G73S) to gain insight into the molecular basis of drug resistance. The mutations lie in different structural regions. Mutation I50V alters a residue in the flexible flap that interacts with the inhibitor, L24I alters a residue adjacent to the catalytic Asp25, and G73S lies at the protein surface far from the inhibitor-binding site. PR(L24I) and PR(I50V), showed a 4% and 18% lower k(cat)/K(m), respectively, relative to PR. The relative k(cat)/K(m) of PR(G73S) varied from 14% to 400% when assayed using different substrates. Inhibition constants (K(i)) of the antiviral drug indinavir for the reaction catalyzed by the mutant enzymes were about threefold and 50-fold higher for PR(L24I) and PR(I50V), respectively, relative to PR and PR(G73S). The dimer dissociation constant (K(d)) was estimated to be approximately 20 nM for both PR(L24I) and PR(I50V), and below 5 nM for PR(G73S) and PR. Crystal structures of the mutants PR(L24I), PR(I50V) and PR(G73S) were determined in complexes with indinavir, or the p2/NC substrate analog at resolutions of 1.10-1.50 Angstrom. Each mutant revealed distinct structural changes relative to PR. The mutated residues in PR(L24I) and PR(I50V) had reduced intersubunit contacts, consistent with the increased K(d) for dimer dissociation. Relative to PR, PR(I50V) had fewer interactions of Val50 with inhibitors, in agreement with the dramatically increased K(i). The distal mutation G73S introduced new hydrogen bond interactions that can transmit changes to the substrate-binding site and alter catalytic activity. Therefore, the structural alterations observed for drug-resistant mutations were in agreement with kinetic and stability changes.