Costly parasite resistance: a genotype-dependent handicap in sand lizards?

Male sand lizards (Lacerta agilis) with a specific restriction fragment length polymorphism fragment in their major histocompatibility complex (MHC) genotype ('O-males') are more resistant to ectoparasites (a tick, Ixodes ricinus) than are males that lack this fragment ('NO-males'). However, emerging evidence suggests that such adaptive immune responses are costly, here manifested by reduced body condition and a compromised defence against secondary infections by haemoprotid parasites that use the ticks as vectors. Subsequent to tick encounter, O-males suffer from a higher leucocyte-erythrocyte ratio, and higher haemoprotid parasitaemia, in particular in relation to vector encounter rate. Furthermore, O-males (i.e. successful tick defenders) with more haemoprotid parasites remaining in their blood stream were in better body condition, whereas this did not apply in NO-males, demonstrating that the adaptive immunoreaction can--in the short term--be energetically even more costly than being moderately parasitized. In agreement with Zahavian handicap theory, O-males had a (marginally) higher reproductive success than males that lacked this fragment.     

Isochromanone-based urotensin-II receptor agonists

A series of analogues of the selective non-peptide urotensin II (UII) receptor agonist 3-(4-chlorophenyl)-3-(2-dimethylaminoethyl)-isochroman-1-one (AC-7954, 1) was synthesized and evaluated for UII agonist activity using a functional cell-based assay. The introduction of a methyl group in the 4-position resulted in a complete loss of activity, whereas substituents in the aromatic rings were beneficial. Sterically demanding amino groups were also detrimental to the activity. Several potent agonists were identified, six compounds being equally or more potent than 1. The most potent compound in the series was the 6,7-dimethyl analogue of 1 (16, pEC50 6.87). The racemate of 16 was resolved into the pure enantiomers using preparative straight phase HPLC. It was shown that the potency resides in the (+)-enantiomer (pEC50 7.11). The synthesized compounds seem to be selective for the UII receptor as no activities were observed at the closely related SSTR3 and 5 receptors.     

Ejaculates from the common marmoset (Callithrix jacchus) contain semenogelin and beta-microseminoprotein but not prostate-specific antigen

Human seminal plasma contains high concentrations of prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), beta-microseminoprotein (MSP), semenogelin I (SgI), and semenogelin II (SgII), whereas only PAP and MSP are present in rodents. In order to gain a better understanding of the evolution and function of semen proteins, we have studied ejaculates from the common marmoset (Callithrix jacchus)-a New World monkey. Semen samples were analyzed with SDS-PAGE, Western blotting, and isoelectric focusing. Under reducing conditions the dominating protein components appear as heterogeneous material of 55-70 kDa and distinct protein bands of 85, 17, 16, and 15 kDa. The heterogeneous material contains glycosylated material detected by an antiserum recognizing both human SgI and SgII. Southern blotting indicates that the common marmoset has genes for both SgI and SgII. There are several marmoset MSP genes, but the strong immunoreactivity against one 15 kDa semen component with pI 7.3 suggests preferential expression of one gene in the prostate. Expression of two other genes cannot be excluded as indicated by weak reaction to isoforms with pI 6.6 and 4.9. Unexpectedly, PSA was not detected by either immunological methods or activity measurements. This is in agreement with results from Southern blotting suggesting that the common marmoset might not have a PSA gene. Thus, in this study we have shown that semen coagulum proteins are present in marmoset seminal plasma, but the lack of PSA precludes a similar liquefaction as of human semen.     

Organization and evolution of the glandular kallikrein locus in Mus musculus

The gene of tissue kallikrein and closely related genes constitute the glandular kallikrein (GK) gene family. The number of members varies between species, ranging from three human to 25 murine. Recently, the gene family was extended with 12 new members, KLK4-KLK15, that were identified adjacent to the classical GK genes on human chromosome 19. In this report, the structure and phylogeny of the mouse GK gene locus are described. A comparison of the human and murine loci shows that the locations of the tissue kallikrein gene and KLK4-KLK15 are conserved. The region between the tissue kallikrein gene and KLK15, devoid of genes in human, is expanded and contains 23 classical GK genes in mouse. Downstream of KLK15, where the genes encoding PSA and hK2 are located in human, mouse carries the pseudogene PsimGK25. Phylogenetic analyses show that classical GK genes emerged after the separation of the primate and rodent lineages, forming a subgroup within the newly extended GK family.     

Binding of mouse nidogen-2 to basement membrane components and cells and its expression in embryonic and adult tissues suggest complementary functions of the two nidogens

Nidogen-1 binds several basement membrane components by well-defined, domain-specific interactions. Organ culture and gene targeting approaches suggest that a high-affinity nidogen-binding site of the laminin gamma1 chain (gamma1III4) is important for kidney development and for nerve guidance. Other proteins may also bind gamma1III4, although human nidogen-2 binds poorly to the mouse laminin gamma1 chain. We therefore characterized recombinant mouse nidogen-2 and its binding to basement membrane proteins and cells. Mouse nidogen-1 and -2 interacted at comparable levels with collagen IV, perlecan, and fibulin-2 and, most notably, also with laminin-1 fragments P1 and gamma1III3-5, which both contain the gamma1III4 module. In embryos, nidogen-2 mRNA was produced by mesenchyme at sites of epithelial-mesenchymal interactions, but the protein was deposited on epithelial basement membranes, as previously shown for nidogen-1. Hence, binding of both nidogens to the epithelial laminin gamma1 chain is dependent on epithelial-mesenchymal interactions. Epidermal growth factor stimulated expression of both nidogens in embryonic submandibular glands. Both nidogens were found in all studied embryonic and adult basement membranes. Nidogen-2 was more adhesive than nidogen-1 for some cell lines and was mainly mediated by alpha3beta1 and alpha6beta1 integrins as shown by antibody inhibition. These findings revealed extensive coregulation of nidogen-1 and -2 expression and much more complementary functions of the two nidogens than previously recognized.     

In vivo and in vitro studies of Bacillus subtilis ferrochelatase mutants suggest substrate channeling in the heme biosynthesis pathway

Ferrochelatase (EC 4.99.1.1) catalyzes the last reaction in the heme biosynthetic pathway. The enzyme was studied in the bacterium Bacillus subtilis, for which the ferrochelatase three-dimensional structure is known. Two conserved amino acid residues, S54 and Q63, were changed to alanine by site-directed mutagenesis in order to detect any function they might have. The effects of these changes were studied in vivo and in vitro. S54 and Q63 are both located at helix alpha3. The functional group of S54 points out from the enzyme, while Q63 is located in the interior of the structure. None of these residues interact with any other amino acid residues in the ferrochelatase and their function is not understood from the three-dimensional structure. The exchange S54A, but not Q63A, reduced the growth rate of B. subtilis and resulted in the accumulation of coproporphyrin III in the growth medium. This was in contrast to the in vitro activity measurements with the purified enzymes. The ferrochelatase with the exchange S54A was as active as wild-type ferrochelatase, whereas the exchange Q63A caused a 16-fold reduction in V(max). The function of Q63 remains unclear, but it is suggested that S54 is involved in substrate reception or delivery of the enzymatic product.     

ATP-dependent unwinding of a minimal origin of DNA replication by the origin-binding protein and the single-strand DNA-binding protein ICP8 from herpes simplex virus type I

The Herpes simplex virus type I origin-binding protein, OBP, is encoded by the UL9 gene. OBP binds the origin of DNA replication, oriS, in a cooperative and sequence-specific manner. OBP is also an ATP-dependent DNA helicase. We have recently shown that single-stranded oriS folds into a unique and evolutionarily conserved conformation, oriS*, which is stably bound by OBP. OriS* contains a stable hairpin formed by complementary base pairing between box I and box III in oriS. Here we show that OBP, in the presence of the single-stranded DNA-binding protein ICP8, can convert an 80-base pair double-stranded minimal oriS fragment to oriS* and form an OBP-oriS* complex. The formation of an OBP-oriS* complex requires hydrolysable ATP. We also demonstrate that OBP in the presence of ICP8 and ATP promotes slow but specific and complete unwinding of duplex minimal oriS. The possibility that the OBP-oriS* complex may serve as an assembly site for the herpes virus replisome is discussed.     

Glandular kallikreins of the cotton-top tamarin: molecular cloning of the gene encoding the tissue kallikrein

The glandular kallikrein family is composed of structurally related serine proteases. Studies show that the mouse family encompasses at least 14 highly conserved functional genes, but of these only the tissue kallikarein has a human ortholog. In man, the tissue kallikrein display high sequence similarity with prostate specific antigen and human glandular kallikrein 2, suggesting that they evolved after the separation of primates and rodents. A phylogenetic study of the genes encoding glandular kallikreins in species evolutionarily located between rodents and man may reveal interesting details on how the gene family evolved, which in turn could yield information about the function of the proteins. Therefore, we have initiated a study of the glandular kallikreins of the cotton-top tamarin (Saguinus oedipus), a New World Monkey. Here, we report the cloning and nucleotide sequence of one of these, the tissue kallikrein gene. The gene of 4.4 kb is composed of five exons, and the structure is 90% similar to that of the orthologous human gene. It gives rise to a polypeptide of 261 amino acids, including a signal peptide of 17 residues, a pro-piece of 7 residues, and the mature protein of 237 residues with an estimated molecular mass of 26.3 kD. The similarity to the human prostate specific antigen and human glandular kallikrein 2 genes is 73% and 72%, respectively, including introns and flanking regions. The lower similarity to these genes compared with the human tissue kallikrein gene indicates that they, or a progenitor to them, arose in primates prior to the separation of New and Old World monkeys. Genomic Southern blots also show that the cotton-top tamarin genome encompasses at least one more glandular kallikrein gene.     

Adenosine is not responsible for local metabolic control of coronary blood flow in dogs during exercise

The purpose of this investigation was to quantitatively evaluate the role of adenosine in coronary exercise hyperemia. Dogs (n = 10) were chronically instrumented with catheters in the aorta and coronary sinus, and a flow probe on the circumflex coronary artery. Cardiac interstitial adenosine concentration was estimated from arterial and coronary venous plasma concentrations using a previously tested mathematical model. Coronary blood flow, myocardial oxygen consumption, heart rate, and aortic pressure were measured at rest and during graded treadmill exercise with and without adenosine receptor blockade with either 8-phenyltheophylline (8-PT) or 8-p-sulfophenyltheophylline (8-PST). In control vehicle dogs, exercise increased myocardial oxygen consumption 4.2-fold, coronary blood flow 3.8-fold, and heart rate 2.5-fold, whereas mean aortic pressure was unchanged. Coronary venous plasma adenosine concentration was little changed with exercise, and the estimated interstitial adenosine concentration remained well below the threshold for coronary vasodilation. Adenosine receptor blockade did not significantly alter myocardial oxygen consumption or coronary blood flow at rest or during exercise. Coronary venous and estimated interstitial adenosine concentration did not increase to overcome the receptor blockade with either 8-PT or 8-PST as would be predicted if adenosine were part of a high-gain, negative-feedback, local metabolic control mechanism. These results demonstrate that adenosine is not responsible for local metabolic control of coronary blood flow in dogs during exercise.