Structural studies on aspartate aminotransferase from Escherichia coli. Covalent structure

The amino acid sequence of aspartate aminotransferase from Escherichia coli was established by sequence analysis and alignment of 39 tryptic peptides and 7 cyanogen bromide peptides. The total number of amino acid residues of the subunit was 396, and the molecular weight was calculated to be 43,573. A comparison of the primary structure of the E. coli enzyme with all known sequences of the two types of isoenzyme (mitochondrial and cytosolic enzymes) in vertebrates revealed that approximately 25% of all residues are invariant. The amino acid residues which were proposed from crystallographic studies on the vertebrate enzymes to be essential for the enzymic action are well conserved in the E. coli enzyme. The E. coli enzyme shows a similar degree of sequence homology to both the mitochondrial and cytosolic isoenzymes (close to 40%). The finding that the positions of deletions introduced into the sequence of E. coli enzyme to give the maximum homology agree well with those of the mitochondrial enzymes supports the endosymbiotic hypothesis of mitochondrial origin.     

Covalent and three-dimensional structure of the cyclodextrinase from Flavobacterium sp. no. 92.

Starting with oligopeptide sequences and using PCR, the gene of the cyclodextrinase from Flavobacterium sp. no. 92 was derived from the genomic DNA. The gene was sequenced and expressed in Escherichia coli; the gene product was purified and crystallized. An X-ray diffraction analysis using seleno-methionines with multiwavelength anomalous diffraction techniques yielded the refined 3D structure at 2.1 A resolution. The enzyme hydrolyzes alpha(1,4)-glycosidic bonds of cyclodextrins and linear malto-oligosaccharides. It belongs to the glycosylhydrolase family no. 13 and has a chain fold similar to that of alpha-amylases, cyclodextrin glycosyltransferases, and other cyclodextrinases. In contrast with most family members but in agreement with other cyclodextrinases, the enzyme contains an additional characteristic N-terminal domain of about 100 residues. This domain participates in the formation of a putative D2-symmetric tetramer but not in cyclodextrin binding at the active center as observed with the other cyclodextrinases. Moreover, the domain is located at a position quite different from that of the other cyclodextrinases. Whether oligomerization facilitates the cyclodextrin deformation required for hydrolysis is discussed.     

Bio-histopathological studies of endometrium in repeat breeding buffaloes (Bubalus bubalis)

A total of 24 uterine biopsies were successfully obtained from 40 repeat-breeding buffaloes. The endometrium of 15 buffaloes showed marked neutrophilic infiltration in the epithelium, stroma and uterine glands. In some cases, lymphocyte and plasma cell infiltration, cystic dilatation of the glands and periglandular fibrosis indicated chronicity of the endometritis. Most of the animals (85.0%) had uterine cultures positive for Streptococci, Staphylococci, Gram positive variable cocci, Gram negative bacilli, Anthracoids, and Gram positive bacilli with a predominance of Corynebacteria. With the treatment based on in vitro drug sensitivity tests, 57.1% repeat-breeding buffaloes conceived.     

Induction of ovulatory oestrus in true anoestrous buffaloes during low breeding season

A fertility treatment trial of anoestrous buffaloes was undertaken on a well managed dairy farm in the low breeding season. Forty lactating buffaloes, 12 each from first and second parity and 16 from third or higher parity, not seen in oestrus for at least 3 to 5 months were diagnosed anoestrous by rectal examination. A subcutaneous ear implant containing 3 mg of norgestomet was inserted, accompanied by an intramuscular injection of 3 mg norgestomet and 5 mg of oestradiol valerate (Syncro-mate B, Intervet). After 9 days the implant was removed and 600 IU of PMSG (Intervet) were given intramuscularly. Irrespective of oestrous behaviour, the animals were inseminated with frozen semen at two fixed times: 48 and 72 h after removal of the implant. Thirty animals having calved in the same period as the experimental animals and not seen in oestrus since then served as controls. Milk samples for progesterone analysis were taken on the day of insertion of the implant, the day of removal and on days 10 and 22 after the second A.I. Progesterone determination by means of an enzyme immunoassay (EIA) revealed that 33 buffaloes out of 40 (82.5%) were in true anoestrus. Ovulation occurred in 21 buffaloes out of 30 (70%). Three buffaloes could not be followed up further. The overall conception rate was 53.3%. The parity-wise conception rate was 40%, 44.4% and 72.2% in first, second and third or higher parity, respectively. None of the control animals displayed oestrus during the course of the study.     

Covalent structure of the extracellular polysaccharide from Xanthomonas campestris: evidence from partial hydrolysis studies.

Partial, acid hydrolysis of the extracellular polysaccharide from Xanthomonas campestris gave products that were identified as cellobiose, 2-O-(β-d-glucopyranosyluronic acid)-d-mannose, O(β-d-glucopyranosyluronic acid)-(1→2)-O-α-d-mannopyranosyl-(1→3)-d-glucose, O-(β-d-glucopyranosyluronic acid)-(1→2)-O-α-d-mannopyranosyl-(1→3)-[O-β-d-glucopyranosyl-(1→4)]-d-glucose, and O-(β-d-glucopyranosyluronic acid)-(1→2)-O-α-d-mannopyranosyl-(1→3)-[O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranosyl-(1→4)-d-glucose. This and other evidence supports the following polysaccharide structure (1) which has been proposed independently by Jansson, Kenne, and Lindberg:

     

Covalent adducts of lactate oxidase. Photochemical formation and structure identification.

Lactate oxidase forms tight complexes with a variety of mono- and dicarboxylic acids. Most of these undergo facile photoreactions involving decarboxylation of the carboxylic acid and formation of covalent adducts at position N(5) of the flavin, characterized by absorption maxima from 325 to 365 nm and fluorescence emission in the range 440 to 490 nm. The properties of the adducts are strongly dependent on the nature of the substituent. Enzyme-bound N(5)-acyl adducts and N(5)-CH2-R derivatives are stable in the dark. Glycollyl- and alpha-lactyl adducts, however, decay to oxidized enzyme with half-lives in the order of minutes. Upon denaturation of the enzyme, the N(5)-alkyl adducts decay rapidly or are oxidized by oxygen. Reduced lactate oxidase is also photoalkylated in the presence of halogenated carboxylic acids. Bromoacetate yields an N(5)-carboxymethyl adduct; with beta-bromopropionate, a C(4a)-beta-propionyl derivate is formed. The N(5) adduct is identical with that from the photochemical reaction of oxidized enzyme and malonic acid. When the native coenzyme FMN is substituted by 2-S-FMN, qualitatively the same photoproducts are formed. The adducts obtained with the 2-S-FMN enzyme show the expected bathochromic shifts in absorption spectra. The results indicate that the photoreactivity of the enzyme is restricted to the positions C(4a) and N(5) of the flavin.     

Induction of ovulatory estrus and fertility in non-cycling buffaloes with norgestomet during low breeding season

Field trials were designed to evaluate use of norgestomet treatment to induce ovulatory estrus in non-cycling buffalo cows and heifers during low breeding season. Twenty-five buffalo cows and 50 heifers under village management were given a 9-day treatment with a polymer implant containing 6 mg norgestomet with IM injections of 5 mg estradiol valerate + 3 mg norgestomet at the time of implantation and 600 IU PMSG when the implant was removed. Fifty animals served as controls without any treatment. Seventy-four treated animals showed estrus during the period between 36 to 80 hours after removal of the implant. Twenty-five buffalo cows and 40 heifers that could be further followed up were inseminated twice at 8-hour intervals, 12 hrs after induction of estrus with chilled semen by recto-vaginal method. Of these, 15 (23.1%) conceived, 9 (36%) among buffalo cows and 6 (15%) among heifers. Fourteen buffalo cows and 30 heifers that did not conceive manifested cyclic estrus at an interval of 22.4 and 20.6 days. The conception rate in the cyclic estrus was 57% and 23.3%, respectively, for buffalo cows and heifers. The overall conception rate over two inseminations was 46.2%, 68% in buffalo cows and 32.5% in heifers. In the control group, five (10%) showed spontaneous estrus and two (40%) conceived during the period of the experiment.     

Covalent structure of the minor monomeric beta-lactoglobulin II component from donkey milk

The complete primary structure of the minor beta-lactoglobulin II component from donkey milk is presented. It has been established by amino-acid sequencing and mass-spectrometry analysis of intact protein and peptides obtained after enzymatic and chemical cleavages. The molecular mass and the pI of the protein are calculated to be 18,261 Da and 4.5 respectively. Despite the close structural similarity of the donkey and horse major beta-lactoglobulin I components, their minor beta-lactoglobulin II components show substantial differences in sequence. Most observed exchanges are clustered at residues 78-106 where only 6 amino-acid residues are conserved. The primary structure of donkey beta-lactoglobulin II reveals some unusual features of minor beta-lactoglobulins II and gives new light to the evolution of beta-lactoglobulins and other lipocalins involved in retinol binding or reproductive functions.     

Taxonomic status of the Oystercatcher Haematopus ostralegus breeding in Italy

The eastern limit of H. o. longipes has moved 700 km westwards.     

Phenology and synchrony of roe deer breeding in northern Italy

The timing and synchrony of births of roe deerCapreolus capreolus Linnaeus, 1758 population were examined for 7 consecutive years in northern Italy. From 1997 to 2003, 136 fawns, 65 females and 71 males, from 117 litters were captured. The average date of fawning (30th of May) was constant among years and between sexes. The onset of parturition was 19th of May while the total length of the parturition season was 25 days (from 19th of May to 13th of June). Births were highly synchronous, with 80% of births occurring in 17 days. Synchrony of birth did not exhibit any significant interannual or intersexual variation. The stability of the birth pattern was confirmed by the parturition date of 13 radiocollared females who all gave birth within 4 days in consecutive fawning seasons. In agreement with previous findings, our data support the hypothesis that the roe deer is constrained at both ends of the fawning season by the short time between the fawning season and rut and by the need to match optimal climate conditions and forage quality to face late gestation and lactation. All these factors result in a relatively stable and synchronous birth season.     

Covalent structure of soybean seed coat peroxidase

Peroxidase from soybean seed coat (SBP) is very stable at high temperature, extremes of pH, and in organic solvent. At the same time, it is highly reactive towards both organic and inorganic substrates, similar to horseradish peroxidase. SBP has a wide range of potential applications, and its structure is of particular interest for engineering purposes and as a model for stable heme peroxidases. The covalent structure of SBP has been determined by Edman sequencing and MALDI-TOF MS. SBP is a highly heterogeneous glycoprotein with MS determined masses from 39 to 41 kDa. The mature protein consists of 306 residues starting with pyrrolidone carboxylic acid. Seven glycosylation sites have been observed, although some sites were only partially glycosylated. No putative plant peroxidases were orthologous to SBP. However, SBP showed greater than 70% amino acid sequence identity to peroxidases from other legumes recruited in various defense responses.     

Influence of sugar beet breeding on populations of Beta vulgaris ssp. maritima in Italy

Abstract. It is highly probable that transgenic cultivars of sugar beet may influence wild beets in the seed-production-area of northern Italy. For this reason a survey of the local wild beet populations and their habitat characteristics was conducted in 1994/1995, i.e. before transgenic beets and their off spring could have become established. Wild beets (Beta vulgaris ssp. maritima) were found at 21 locations between Trieste and Cesenatico, as part of the natural littoral vegetation classified as Atriplicetum tatarici (Cakiletea maritimae) and Crithmetum (Crithmo-Staticetea). The analysis of phenotypic attributes leads to a division into three different sub-populations. Greenhouse studies on the morphology and life-cycle attributes demonstrated actual gene flow between conventional seed beet and the examined wild beet population.