Adenovirus type 5 induces progression of quiescent rat cells into S phase without polyamine accumulation.

Adenovirus type 5 induces cellular DNA synthesis and thymidine kinase in quiescent rat cells but does not induce ornithine decarboxylase. We now show that unlike serum, adenovirus type 5 fails to induce S-adenosylmethionine decarboxylase or polyamine accumulation. The inhibition by methylglyoxal bis(guanylhydrazone) of the induction of thymidine kinase by adenovirus type 5 is probably unrelated to its effects on polyamine biosynthesis. Thus, induction of cellular thymidine kinase and DNA replication by adenovirus type 5 is uncoupled from polyamine accumulation.

Physicochemical characterization of lens proteins of the squid Nototodarus gouldi and comparison with vertebrate crystallins.

The main water-soluble proteins of squid lens (S-crystallins) have a molecular weight of 60 000, a sedimentation coefficient s020,w of 5.2 S, 20-30% alpha-helical secondary structure, and an unusually high methionine content (12%). The subunits of Mr 30 000 (major) and Mr 27 000 (minor) have related N-terminal amino acid sequences, but a very heterogeneous charge distribution with predominantly basic isoelectric points. Higher-Mr aggregates have similar secondary/tertiary structure and amino acid composition, but contain additional acidic subunits and Mr 35 000-40 000 subunits. S-crystallins resemble vertebrate beta-crystallins in their quaternary structure, and their N-terminal sequence shows analogy with the first 19 residues of calf beta/gamma-crystallin folding units. In the urea-soluble and urea-insoluble lens fractions polypeptides of Mr 58 000 and 80 000, respectively, predominate, which presumably correspond to the main cytoskeleton and membrane proteins. Water-soluble lens components of less than Mr 2000 were isolated which have ultraviolet absorption maxima at 327 and 370 nm.

Purification, properties, partial sequence and evolutionary relationships of marsupial erythrocyte carbonic anhydrase.

Carbonic anhydrase (EC 4.2.1.1) has been purified from the erythrocytes of the tammar wallaby (Macropus eugenii desmarest). The enzyme was separated into four zones of activity. The three major individual forms were isolated as discrete entities. Comparison of substrate specificity, specific activities, kinetic constants and inhibition characteristics indicated that these heteromorphs represented minor post-translational modifications of a single gene product of carbonic anhydrase II type. Double-immunodiffusion and peptide mapping confirmed this proposition. The marsupial enzyme exists as a monomer with a molecular weight of about 29 000 containing one atom of zinc per mole which is much more tightly bound to the enzyme than it is in either human carbonic anhydrase I or II. The wallaby enzyme was, like human carbonic anhydrase II, partially inactivated by p-hydroxymercuribenzoate under conditions not inhibitory for human carbonic anhydrase I. The partial sequence of 51 residues of cyanogen-bromide peptides was sufficiently homologous to allow unambiguous overlap with the sequence of both human carbonic anhydrase I and II isozymes as well as with the recently published sequence of an apparent type I-like enzyme from the turtle. It is clear that the single wallaby erythrocyte carbonic anhydrase belongs to the class of separately evolving type II isozymes which have previously been defined only for placental mammals.

The complete amino acid sequence of feline beta-lactoglobulin II and a partial revision of the equine beta-lactoglobulin II sequence.

The amino acid sequence of feline beta-lactoglobulin (designated II) has been determined. The protein chain is 163 amino acids long with a relative molecular mass of 18,558. The primary structure was determined by sequencing of native protein (residues 1-25), BPNS-skatole cleavage fragments and the peptides obtained by proteolytic cleavage with V8 proteinase and TPCK-trypsin. Feline beta-lactoglobulin II has 53 and 57% positional identities with bovine beta-lactoglobulin A and equine beta-lactoglobulin I, respectively, and approx. 68% with a revised sequence of equine beta-lactoglobulin II. The equine beta-lactoglobulin II sequence was re-examined between positions 78 and 122 resulting in a major revision in this area with only a single insertion to give a total of 163 residues.

Human muscle glutathione S-transferase (GST-4) shows close homology to human liver GST-1.

Human muscle specific glutathione S-transferase (RX: glutathione R-transferase, EC 2.5.1.18) (GST-4) and liver GST-1 have been purified and subjected to N-terminal sequence analysis. These two isozymes show close homology and only differ in 3 residues within the first 24. The N-terminal sequences of GST-1 and GST-4 differ significantly from those of GST-2 and GST-3. Although antiserum raised against native GST-1 did not cross-react with GST-4, cross-reactivity was obtained with antiserum raised against denatured GST-1. The homology between GST-1 and GST-4 indicates that they are both members of the mu evolutionary class.

The amino acid sequence of cytochrome c from the blowfly Lucilia cuprina.

The amino acid sequence of cytochrome c isolated from the sheep blowfly Lucilia cuprina has been determined by comparison of the compositions of the tryptic peptides to those predicted from the published sequences of cytochromes c from other insects. Cytochrome c from L. cuprina differs at a single residue when compared to cytochrome c from the screw worm fly Haematobia irritans, a species belonging to the same order as the blowfly. This substitution, proline for alanine, has been located at position 44 in the protein chain.

The structure of human thrombin in relation to autolytic degradation.

Human thrombin was obtained by activation of human prothrombin with venom of the Australian Taipan (Oxyuranus scutellatus scutellatus). This thrombin was precipitated with ammonium sulphate (75% saturation) and subsequently purified by gel-filtration (Sephadex G-75), ion-exchange (CM-Sephadex C-50) and affinity (aminobenzamidine-CH-Sepharose) chromatography. The final preparation (affinity thrombin) had a specific activity of 2340 Iowa units per absorbance unit (A1cm280). Thrombin proteins focused between 5 and 7, while prothrombin proteins focused to pH values less than 5. SDS-acrylamide gel electrophoresis indicated molecular weights of greater than 70 000 for prothrombin and 39 000, 28 000, 25 000-23 000 and 15 000-13 000 for affinity thrombin proteins. The 39 000-dalton species predominated (greater than 90%) when the enzyme was inhibited with phenylmethanesulphonyl fluoride prior to dialysis for SDS electrophoresis. Lack of such inhibition reduced the amount of the 39 000-dalton species to less than 60% with concomitant increase of the smaller species. Peptide mapping studies indicated that the smaller species were structurally related to the 39 000-dalton species. The amino acid compositions of the histidine and/or tyrosine containing peptides indicated a high degree of homology with bovine thrombin. It has been established that human thrombin can exist in at least two secondary structural forms, of different molecular weights, probably due to autolytic degradation of the largest (39 000-dalton) form.

Purification of a marsupial insulin: amino-acid sequence of insulin from the eastern grey kangaroo Macropus giganteus.

Insulin has been purified from kangaroo pancreas by acidic ethanol extraction, diethyl ether precipitation and gel filtration. The amino-acid sequence of this, the first marsupial insulin to be studied, is reported. It differs from human insulin by only four amino-acid substitutions, all in regions of the molecule previously known to be variable. However, it should be noted that one of these, asparagine for threonine at A8, has not been reported before. Computer comparisons of all 43 insulin sequences reported to date with kangaroo insulin show it to be most closely related to a group of mammalian insulins (dog, pig, cow, human) known to be of high biological potency. The measurement of blood glucose lowering in the rabbit by kangaroo insulin is consistent with this conclusion. Comparisons of amino-acid sequences of other proteins with their kangaroo counterparts show a greater difference, in line with the time of divergence of marsupials. The limited differences observed in insulin and cytochrome c suggest that their structures need to be closely conserved in order to maintain function.

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.

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.

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.

A mild method for the preparation of disulfide-linked hybrids of immunoglobulin light chains.

A method is described for the hybridization of immunoglobulin light chains (Bence-Jones proteins) from different patients. The interchain half-cystine residues in the light chains from one subject are converted into mixed disulfides with 2,2'-dithiodipyridine. In the Bence-Jones dimer from a second patient the interchain disulfide bond is reduced with dithiothreitol. A covalently linked hybrid molecule is produced by the reaction of the mixed disulfide with the reduced thiol. In favorable cases the mild treatment yields heterodimers which can be crystallized for X-ray diffraction studies. The procedure can also be employed for converting a monomer into a covalent dimer. The engineered dimer of one kappa chain (Jen) crystallizes in the same space group as an aggregate of monomers, but the unit cell is only one-third as large.

Introduced and Native Parasitoid Wasps Associated With Larch Casebearer (Lepidoptera: Coleophoridae) in Western Larch.

The larch casebearer [Coleophora laricella (Hubner)], a non-native insect, continues to impact western larch (Larix occidentalis Nutt.) through defoliation events in the Pacific Northwest. Biological control programs starting in the 1960s released seven species of parasitoid wasps to control C. laricella outbreaks. However, information about current population dynamics of C. laricella and associated parasitoids remains lacking. Therefore, the goal of this study was to document the presence, current distributions, densities, and parasitism rates of introduced and native parasitoid wasps occurring on C. laricella throughout the Northwestern U.S. range of L. occidentalis. We sampled L. occidentalis trees at multiple sites in Oregon, Washington, Idaho, and Montana. C. laricella was present at all sites with average state densities ranging from 6.2 to 13.1 moths/100 buds. We recovered two introduced hymenopteran biological control agents; Agathis pumila (Ratzeburg: Braconidae) at 79% of the sites, and Chrysocharis laricinellae (Ratzeburg: Eulophidae) at 63% of the sites. Fourteen species of native parasitic wasps were also recovered. The most common species were: Bracon sp., Spilochalcis albifrons, and Mesopolobus sp. The average native species parasitism rate across the four states was 9.0%, which was higher than the introduced species Ch. laricinellae (2.9%), but not as high as A. pumila (19.3%). While survey results suggest that native species may be more important for the control of C. laricella than previously thought, A. pumila remains the major source of regional control. However, further research is needed to better understand how introduced and native parasitoids interact to control invasive pest populations.

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.

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.

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.

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.

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.

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.

Evidence for the participation of alpha B-crystallin in human age-related nuclear cataract.

The aim of this study was to determine if the unusual coloured species characteristic of age-related nuclear cataract could be localised to specific residues of the crystallins. The insoluble, crosslinked and coloured cataract protein fraction (CPF) was isolated from cataract human lenses. Using a combination of tryptic digestion, gel filtration and multiple reversed phase high performance liquid chromatography (RP-HPLC), coloured peaks were isolated and subjected to amino acid sequence analysis. With these techniques, it was hoped to identify and locate the modified residues. Sequence information was obtained on 16 'coloured' peptides. Many of the peptides were found to be derived from alpha B-crystallin. When redundancies are taken into account, six distinctive peptides were found to be derived from alpha B-crystallin; one from beta B1-crystallin, two from beta A3/A1-crystallin and three from gamma S-crystallin. Three sites of possible crystallin residue isomerisation to modification were detected in the alpha B- and beta A3/beta A1-crystallins, including probable asp isomerisation at residues 25 and 36 in alpha B-crystallin. Since the CPF is unique to nuclear cataract lenses, these data suggest that alpha-crystallin, and alpha B-crystallin in particular, may be implicated in the cataract process. This finding supports that of a recent study on cataract proteins using pronase digestion [Chen YC, Reid GE, Simpson RJ, Truscott RJW. Exp Eye Res 1997;65:835.]