Biomedical subjects

D C Shaw

Publications and source records attributed to D C Shaw.

At least 19 recordsLinked to original sources

PubMed2006

Dynamics of water transport and storage in conifers studied with deuterium and heat tracing techniques.

The volume and complexity of their vascular systems make the dynamics of long-distance water transport in large trees difficult to study. We used heat and deuterated water (D2)) as tracers to characterize whole-tree water transport and storage properties in individual trees belonging to the coniferous species Pseudotsuga menziesii (Mirb.) Franco and Tsuga heterophylla (Raf.) Sarg. The trees used in this study spanned a broad range of height (13.5-58 m) and diameter (0.14-1.43 m). Sap flow was monitored continuously with heat dissipation probes near the base of the trunk prior to, during and following injection of D2O. The transit time for D2O transport from the base of the trunk to the upper crown and the tracer residence time were determined by measuring hydrogen isotope ratios in water extracted from leaves sampled at regular intervals. Transit times for arrival of D2O in the upper crown ranged from 2.5 to 21 d and residence times ranged from 36 to 79 d. Estimates of maximum sap velocity derived from tracer transit times and path length ranged from 2.4 to 5.4 m d(-1). Tracer residence time and half-life increased as tree diameter increased, independent of species. Species-independent scaling of tracer velocity with sapwood-specific conductivity was also observed. When data from this study were combined with similar data from an earlier study of four tropical angiosperm trees, species-independent scaling of tracer velocity and residence time with sapwood hydraulic capacitance was observed. Sapwood capacitance is an intrinsic tissue-level property that appears to govern whole-tree water transport in a similar manner among both tracheid- and vessel-bearing species.

Biological Transport↗

PubMed2001

Secretion of whey acidic protein and cystatin is down regulated at mid-lactation in the red kangaroo (Macropus rufus).

Milk collected from the red kangaroo (Macropus rufus) between day 100 and 260 of lactation showed major changes in milk composition at around day 200 of lactation, the time at which the pouch young begins to temporarily exit the pouch and eat herbage. The carbohydrate content of milk declined abruptly at this time and although there was only a small increase in total protein content, SDS PAGE analysis of milk revealed asynchrony in the secretory pattern of individual proteins. The levels of alpha-lactalbumin, beta-lactoglobulin, serum albumin and transferrin remain unchanged during lactation. In contrast, the protease inhibitor cystatin, and the putative protease inhibitor whey acidic protein (WAP) first appeared in milk at elevated concentrations after approximately 150 days of lactation and then ceased to be secreted at approximately 200 days. In addition, a major whey protein, late lactation protein, was first detected in milk around the time whey acidic protein and cystatin cease to be secreted and was present at least until day 260 of lactation. The co-ordinated, but asynchronous secretion of putative protease inhibitors in milk may have several roles during lactation including tissue remodelling in the mammary gland and protecting specific proteins in milk required for physiological development of the dependent young.

Amino Acid Sequence↗

PubMed2001

Characterization of baboon (Papio hamadryas) milk proteins.

The major proteins of baboon milk were identified as beta-lactoglobulin (beta LG), alpha-lactalbumin (alpha LA), lysozyme, lactoferrin, casein, and albumin by immobiline isoelectric focusing, SDS-PAGE, immunoblotting of gels with rabbit antisera to human alpha LA, lysozyme, and albumin and bovine beta LG and casein, and N-terminal sequencing of proteins blotted from gels. The first 30 N-terminal residues of baboon beta LG are identical to those of macaque (Macaca fasicularis) beta LG except for a (D/N) polymorphism at residue 2. The complete cDNA sequence and derived amino acid composition of beta LG were elucidated using RT-PCR amplification of poly(A)+ mRNA purified from lactating mammary gland. Baboon beta LG consists of 168 amino acid residues (M(r) 20,750) and is the longest beta LG identified to date. beta LG and alpha LA polymorphisms with three (A, B, and C) and two (A and B) variants, respectively, were detected by immobiline IEF, pH 4-6, of individual baboon milk samples at varying stages of lactation.

Albumins↗

PubMed2000

The gene for a novel member of the whey acidic protein family encodes three four-disulfide core domains and is asynchronously expressed during lactation.

Secretion of whey acidic protein (WAP) in milk throughout lactation has previously been reported for a limited number of species, including the mouse, rat, rabbit, camel, and pig. We report here the isolation of WAP from the milk of a marsupial, the tammar wallaby (Macropus eugenii). Tammar WAP (tWAP) was isolated by reverse-phase HPLC and migrates in SDS-polyacrylamide gel electrophoresis at 29.9 kDa. tWAP is the major whey protein, but in contrast to eutherians, secretion is asynchronous and occurs only from approximately days 130 through 240 of lactation. The full-length cDNA codes for a mature protein of 191 amino acids, which is comprised of three four-disulfide core domains, contrasting with the two four-disulfide core domain arrangement in all other known WAPs. A three-dimensional model for tWAP has been constructed and suggests that the three domains have little interaction and could function independently. Analysis of the amino acid sequence suggests the protein belongs to a family of protease inhibitors; however, the predicted active site of these domains is dissimilar to the confirmed active site for known protease inhibitors. This suggests that any putative protease ligand may be unique to either the mammary gland, milk, or gut of the pouch young. Examination of the endocrine regulation of the tWAP gene showed consistently that the gene is prolactin-responsive but that the endocrine requirements for induction and maintenance of tWAP gene expression are different during lactation.

Amino Acid Sequence↗

PubMed2000

Non-oxidative modification of lens crystallins by kynurenine: a novel post-translational protein modification with possible relevance to ageing and cataract.

In humans, the crystallin proteins of the ocular lens become yellow-coloured and fluorescent with ageing. With the development of senile nuclear cataract, the crystallins become brown and additional fluorophores are formed. The mechanism underlying crystallin colouration is not known but may involve interaction with kynurenine-derived UV filter compounds. We have recently identified a sulphur-linked glutathionyl-3-hydroxykynurenine glucoside adduct in the lens and speculated that kynurenine may also form adducts with GSH and possibly with nucleophilic amino acids of the crystallins (e.g. Cys). Here we show that kynurenine modifies calf lens crystallins non-oxidatively to yield coloured (365 nm absorbing), fluorescent (Ex 380 nm/Em 450-490 nm) protein adducts. Carboxymethylation and succinylation of crystallins inhibited kynurenine-mediated modification by approx. 90%, suggesting that Cys, Lys and possibly His residues may be involved. This was confirmed by showing that kynurenine formed adducts with GSH as well as with poly-His and poly-Lys. NMR studies revealed that the novel poly-Lys-kynurenine covalent linkage was via the epsilon-amino group of the Lys side chain and the betaC of the kynurenine side chain. Analysis of tryptic peptides of kynurenine-modified crystallins revealed that all of the coloured peptides contained either His, Cys or an internal Lys residue. We propose a novel mechanism of kynurenine-mediated crystallin modification which does not require UV light or oxidative conditions as catalysts. Rather, we suggest that the side chain of kynurenine-derived lens UV filters becomes deaminated to yield an alpha,beta-unsaturated carbonyl which is highly susceptible to attack by nucleophilic amino acid residues of the crystallins. The inability of the lens fibre cells to metabolise their constituent proteins results in the accumulation of coloured/fluorescent crystallins with age.

Aging↗

PubMed1999

The whey acidic protein family: a new signature motif and three-dimensional structure by comparative modeling.

Whey acidic proteins (WAP) from the mouse, rat, rabbit, camel, and pig comprise two "four-disulfide core" domains. From a detailed analysis of all sequences containing this domain, we propose a new PROSITE motif ([KRHGVLN]-X-¿PF¿-X-[CF]-[PQSVLI]-X(9,19)-C-¿P¿-X-[DN]-X-¿N¿ -[CE]-X(5)-C-C) to accurately identify new four-disulfide core proteins. A consensus model for the WAP proteins is proposed, based on the human mucous proteinase inhibitor crystal structure. This article presents a detailed atomic model for the two-domain porcine WAP sequence by comparative modeling. Surface electrostatic potential calculations indicate that the second domain of the pig WAP model is similar to the functional human mucous proteinase inhibitor domains, whereas the first domain may be nonfunctional.

Amino Acid Sequence↗

PubMed1997

Characterisation of basic proteins from Spiroplasma melliferum using novel immobilised pH gradients.

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has become the method of choice for efficient separation of complex protein mixtures. Previously, analysis of the Spiroplasma melliferum proteome (protein complement of a genome) has been performed with pH 3-10 and narrow range pH 4-7 IPG gel strips. We report here on the use of novel 18 cm basic (pH 6-11) immobilised pH gradients (IPG) to increase the resolution of protein spots visible within 2-D gels. These gradients were synthesised to emulate the gradient of commercially available IPG gel strips in a 5 cm region of overlap so as to attempt construction of a more complete map of cellular protein expression. Approximately 50 additional gene products were detected from S. melliferum that were not previously well-resolved or visible using wide-range pH 3-10 IPG gel strips. Twenty-seven of these were electrotransferred to polyvinylidene difluoride (PVDF) membrane and analysed by N-terminal protein microsequencing. Protein spots with an initial peak yield of as little as 100 femtomoles (fm) were sequenced to 5-10 amino acid residues, demonstrating the importance of improved sample handling procedures and analytical technologies. Many essential metabolic enzymes were shown to have basic pI, including: glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, carbamate kinase and lactate dehydrogenase. A very basic protein (pI approximately 11.0) was identified as uridylate kinase, an enzyme indirectly associated with pyrimidine biosynthesis and thought be absent in some members of the bacterial class Mollicutes. The advent of novel basic (pH 6-11) IPGs has allowed the visualisation of a significantly greater percentage of the 'functional proteome', that portion of the total protein complement of a genome actively translated within a specific time frame, on 2-D electrophoresis gels. This will aid in the characterisation of translated gene products in conjunction with genome sequencing initiatives.

Amino Acid Sequence↗

PubMed1997

Lactose synthesis in a monotreme, the echidna (Tachyglossus aculeatus): isolation and amino acid sequence of echidna alpha-lactalbumin.

alpha-Lactalbumin and lysozyme were each isolated from echidna (Tachyglossus aculeatus) milk by gel permeation and ion exchange chromatography. The alpha-lactalbumin modified the action of echidna milk galactosyltransferase to promote the synthesis of lactose but had very little effect on bovine galactosyltransferase. Echidna alpha-lactalbumin is a glycosylated protein with an apparent molecular weight of 20,000 (SDS-PAGE) whose concentration in the milk is very low compared with the concentrations of alpha-lactalbumin in the milk of other species. Its amino acid sequence is more similar to that of another monotreme, the platypus (Ornithorhynchus anatinus), than to the sequences of eutherian or marsupial alpha-lactalbumins. Echidna milk lysozyme, even at high concentrations, did not promote the synthesis of lactose by either echidna or bovine galactosyltransferase. We conclude that lactose synthesis in the echidna occurs by the same mechanism as that found in the platypus and other mammals.

Amino Acid Sequence↗

PubMed1996

Primary structure of trypsin inhibitors from Sicyos australis.

Three trypsin inhibitors from Sicyos australis, have been isolated, purified and sequenced. Following protein extraction with ammonium sulphate, the mixture of inhibitors was separated from other proteins by trypsin-affinity chromatography. Subsequent purification of the individual inhibitors was accomplished by reversed-phase HPLC. The primary structures of each inhibitor were elucidated by a combination of protein sequencing and electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS-MS) on both the untreated and the reduced and S-carboxymethylated inhibitors. All three inhibitors show extensive sequence similarity with inhibitors from cultivated Cucurbitaceae species, although there are a number of novel residues present. One of the inhibitors has a blocked N-terminus (pyroglutamic acid) and the use of MS-MS was crucial to the elucidation of its primary structure. ESI-MS was further used to characterize the non-covalent complex between one of the inhibitors and trypsin.

Amino Acid Sequence↗

PubMed1996

Platypus insulin: indications from the amino acid sequence of significant differences in structure from porcine insulin.

Insulin from a monotreme, the platypus (Ornithorhynchus anatinus), was isolated and the amino acid sequence determined. It differs from pig insulin at eleven amino acid sites, mainly on the surface of the monomer. Substitutions relative to pig insulin occur in the monomer-monomer interface, the dimer-dimer interface and the receptor binding region. The residues A5 Glu, A8 Lys and A13 Met have not been reported before in any insulin. Multiple sequence comparison studies reveal a relatively close relationship with the nearest group of relatives to the platypus, the mammals. The relationship of the platypus sequence to reptilian insulin sequences (and amphibian and avian insulin sequences in this case) is sufficiently close to support the observation that platypus has retained some ancient reptilian characteristics over the course of evolution. Model building the platypus insulin sequence on the structure of porcine insulin indicates that there may be some interesting differences.

Amino Acid Sequence↗

PubMed1995

Evidence that some dinoflagellates contain a ribulose-1,5-bisphosphate carboxylase/oxygenase related to that of the alpha-proteobacteria.

The ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit from several dinoflagellates has a structure similar to that of the Form II enzyme from Rhodospirillum and Rhodobacter species rather than the Form I Rubisco of eukaryotic algae and higher plants. The dinoflagellate Rubisco was identified on native polyacrylamide gels by autoradiographic detection of the stable Rubisco-[2'-14C]-2-carboxy-D-arabinitol 1,5-bisphosphate complex. The antibody to the Symbiodinium sp. large subunit cross reacts with both the Rhodospirillum rubrum and Rhodobacter sphaeroides Form II enzyme whereas antibodies to the R. rubrum Rubisco cross react with a range of dinoflagellate Rubisco large subunits. The N-terminal amino acid sequence of the large subunit from both Symbiodinium sp. and Amphidinium carterae confirmed this relation. The lack of inhibition of the dinoflagellate Rubisco by 6-phosphogluconate is consistent with this structure.

Amino Acid Sequence↗

PubMed1994

Amino-acid sequence of a trypsin/chymotrypsin inhibitor from giant taro (Alocasia macrorrhiza).

Giant taro (Alocasia macrorrhiza) contains a protein which inhibits both trypsin and chymotrypsin. This trypsin/chymotrypsin inhibitor exists as a dimer of two identical monomers each with slight polymorphism and is an attractive candidate for conferring insect resistance in transgenic plants. The 184 amino-acid sequence (molecular mass of 19774 Da for the Met-24, Glu-50 form) has been determined and is compared with those of other Kunitz-type trypsin, chymotrypsin and subtilisin inhibitors. There appears to be greater 'homology' between the giant taro inhibitor and those inhibitors from other monocotyledons than inhibitors from dicotyledons. The P1 loop region is different from that of other Kunitz-type inhibitors and contains a sequence Leu-Ala-Phe-Phe-Pro at residues 56-60. This section of sequence differs only by a Leu/Ile replacement to a tight binding inhibitor of neutrophil elastase, recently produced by genetic engineering. The most likely candidate for the P1 residue in the giant taro trypsin/chymotrypsin inhibitor is Leu-56.

Amino Acid Sequence↗

PubMed1994

The complete primary structure of late lactation protein from quokka (Setonix brachyurus).

The complete primary structure of the late lactation protein from the milk of quokka (Setonix brachyurus) is presented. The amino acid sequence was established by N-terminal sequence analysis of high-performance liquid chromatography purified intact protein and peptides isolated from chemical and enzymatic digests of the protein. The protein contains 158 residues including four cysteines. The sequence comparison with the tamar wallaby (Macropus eugenii) late lactation protein shows only five differences. The protein is identified as a new member of a novel late lactation protein family present in the milk of marsupials.

Amino Acid Sequence↗

PubMed1994

Suppression of Ixodes scapularis (Acari: Ixodidae) nymphs in a large residential community.

To determine the feasibility of suppressing Ixodes scapularis Say populations in a large, hyperendemic residential community, several rates of granular carbaryl were applied by ground and air to the shrub layer and wooded buffers of a forested residential community during the peak activity period of nymphs. Granular carbaryl significantly reduced the abundance of I. scapularis nymphs on Peromyscus leucopus Raphinesque. Control nymphal ticks ranged between 70.0 and 90.3%. The use of properly timed acaricide applications to I. scapularis habitat within residential communities can provide an effective means of reducing exposure to I. scapularis nymphs, which are chiefly responsible for transmitting Borrelia burgdorferi to humans.

Animals↗

PubMed1994

Progressive changes in milk protein gene expression and prolactin binding during lactation in the tammar wallaby (Macropus eugenii).

Changes in milk protein gene expression and specific prolactin binding were quantified in mammary tissue from the tammar wallaby (Macropus eugenii) at different stages of lactation. The transition from early (phase 2) lactation to late (phase 3) lactation was characterized by the induction of the gene for late lactation protein, a novel whey protein. During the same period, the levels of beta-lactoglobulin and beta-casein gene expression increased, whereas there was no change in the levels of expression of alpha-lactalbumin and alpha-casein genes. Prolactin binding in the mammary gland doubled during the latter half of phase 2 of lactation but declined significantly during the transition to phase 3 of lactation. These changes in prolactin binding resulted from changes in the number of receptors and not from a change in the affinity of the receptor for prolactin. Treatment of membranes with concanavalin A increased the number of prolactin-binding sites by 40% in membranes from phase 2 mammary tissue but decreased binding by 40% in membranes from phase 3 tissue, indicating that significant changes had occurred in the membranes of cells during this period. The tammar wallaby can secrete phase 2 and phase 3 milk from adjacent mammary glands (asynchronous concurrent lactation) and the developmental changes in milk protein gene expression and prolactin binding observed during lactation were reflected in these individual glands. Taken collectively, these findings suggest that mammary development and milk secretion in the tammar wallaby are regulated by both endocrine and local (intramammary) mechanisms.

Animals↗

PubMed1993

Isolation, partial characterisation, and amino acid sequence of alpha-lactalbumin from platypus (Ornithorhynchus anatinus) milk.

alpha-Lactalbumin was isolated from the whey fraction of platypus (Ornithorhynchus anatinus) milk by successive ion-exchange, hydrophobic interaction and gel-permeation chromatography. The purified protein modified the action of partially-purified galactosyltransferase from platypus milk to promote the synthesis of lactose, but had very little modifier effect on bovine galactosyltransferase. Platypus alpha-lactalbumin has 126 amino-acid residues (molecular mass about 14.3 kDa), including a three-residue insertion not found in other alpha-lactalbumins or c-type lysozymes. It appears to have two sites of post-translational modification, of which at least one is N-glycosylated, to give an apparent molecular mass of 23 kDa on SDS-PAGE. The platypus sequence shows a high degree of positional identity (41-48%) with the alpha-lactalbumins of other species. Although it has no lysozyme activity, platypus alpha-lactalbumin is more similar to mammalian lysozymes than is any eutherian or marsupial alpha-lactalbumin, suggesting that this monotreme protein has evolved more slowly than other alpha-lactalbumins.

Amino Acid Sequence↗

PubMed1993

Feline and canine milk lysozymes.

1. The electrophoretic characteristics of feline (Felis catus) and canine (Canis familiaris) milk lysozymes were studied using starch gel electrophoresis and isoelectric focusing. 2. Feline milk lysozyme was found to be polymorphic (two variants, designated A and B with frequencies of 0.13 and 0.87, respectively). Canine milk lysozyme was not polymorphic. 3. The lytic activities of feline and canine milk lysozymes were examined in buffers of varying pH and ionic strength. Preliminary kinetics studies were done. 4. Maximal lytic activity for both lysozymes was found in imidazole-HCl buffer pH 7.4 and both exhibited second order reaction kinetics. 5. Amino acid compositions of both lysozymes were determined.

Amino Acids↗

PubMed1993

Comparison of amino acid sequences of the trypsin inhibitors from taro (Colocasia esculenta), giant taro (Alocasia macrorrhiza) and giant swamp taro (Cyrtosperma chamissonis).

The amino acid sequences of the trypsin inhibitors from taro Colocasia esculenta var. esculenta and giant swamp taro Cyrtosperma chamissonis have been determined and are compared with the protein sequence of the trypsin/chymotrypsin inhibitor from giant taro Alocasia macrorrhiza. Both inhibitors display polymorphism and there is evidence of two components in the giant swamp taro. The positional identity between the proteins is highest at 73-75% for the comparison of the giant taro (GT) with the polymorphic forms of the taro (T) inhibitors and lowest at 56-58% for the pairs of taro and giant swamp taro (GST) proteins. The comparisons show that the inhibitors from T and GT are more related to each other than to GST, which supports their taxonomic classification into different tribes. Location of the P1 site for the trypsin inhibitors of aroids is different from that of other Kunitz-type inhibitors and could be at Leu56.

Amino Acid Sequence↗