Comparative metabolism of alpha- and beta-peptides in the insect Heliothis virescens and in plant cells of black Mexican sweet maize

The tripeptide H-Val-Ala-Leu-OH and the N-Ac-tetrapeptide amide Ac-Thr-Lys-Trp-Phe-NH2, and their beta-peptidic counterparts H-beta(3)hVal-beta(3)hAla-beta(3)hLeu-OH and Ac-beta(3)hThr-(S)beta(2)hLys-beta(3)hTrp-beta(3)hPhe-NH2, respectively, have been injected into Heliothis virescens larvae and added to cell cultures of black mexican sweet maize. The body liquids of the larvae and the supernatant of the plant cell cultures were sampled 0, 2, 3, 6, 17, and/or 24 h after application and analyzed by LC/MS. While the two alpha-peptides were degraded rapidly in these environments, the concentration of the beta-peptides was found to decrease very slowly. Thus, ca. 60% of the original amount of the beta-tetrapeptide was detected in the liquids of the insect after 24 h. The plant cells did not seem to make use of the beta-peptides at all, whereas, the alpha-tripeptide completely disappeared from the supernatant after 3 h. Thus, we have demonstrated, for the first time, the high stability of beta-peptides against degradation and metabolism in an insect and a plant. Especially remarkable is the persistence of the beta-tetrapeptide with its functionalised and, thus, 'metabolisable' side chains of Thr, Lys, Trp, and Phe in the insect larvae, which are known to have a high level of activity of oxidizing enzymes. The results described here match those of ADME investigations with radioactively labeled beta-peptides in rats, where essentially complete stability has been observed, while environmental microorganisms have been found to biodegrade beta-peptides, albeit slowly. Possible implications of these findings for biomedical and pest-control applications are proposed.     

The expansion of conservation genetics

The 'crisis discipline' of conservation biology has voraciously incorporated many technologies to speed up and increase the accuracy of conservation decision-making. Genetic approaches to characterizing endangered species or areas that contain endangered species are prime examples of this. Technical advances in areas such as high-throughput sequencing, microsatellite analysis and non-invasive DNA sampling have led to a much-expanded role for genetics in conservation. Such expansion will allow for more precise conservation decisions to be made and, more importantly, will allow conservation genetics to contribute to area- and landscape-based decision-making processes.     

Fluorescent ligands for the histamine H2 receptor: synthesis and preliminary characterization

3-[3-(Piperidinomethyl)phenoxy]alkyl, N-cyano-N′-[ω-[3-(1-piperidinylmethyl)phenoxy]alkyl]guanidine and 2-(5-methyl-4-imidazolyl)methyl thioethyl derivatives containing fluorescent functionalities were synthesized and the histamine H₂ receptor affinity was evaluated using the H₂ antagonist [¹²⁵I]-aminopotentidine. The compounds exhibited weak to potent H₂ receptor affinity with pK_i values ranging from <4 to 8.85. The highest H₂ receptor affinity was observed for N-cyano-N′-[ω-[3-(1-piperidinylmethyl)phenoxy]alkyl]guanidines substituted with methylanthranilate (13), cyanoindolizine (6) and cyanoisoindole (11) moieties via an ethyl or propyl linker.     

Functional expression and characterization of a purine nucleobase transporter gene from Leishmania major

Leishmania major, like all the other kinetoplastid protozoa, are unable to synthesize purines and rely on purine nucleobase and nucleoside acquisition across the parasite plasma membrane by specific permeases. Although, several genes have been cloned that encode nucleoside transporters in Leishmania and Trypanosoma brucei, much less progress has been made on nucleobase transporters, especially at the molecular level. The studies reported here have cloned and expressed the first gene for a L. major nucleobase transporter, designated LmaNT3. The LmaNT3 permease shows 33% identity to L. donovani nucleoside transporter 1.1 (LdNT1.1) and is, thus, a member of the equilibrative nucleoside transporter (ENT) family. ENT family members identified to date are nucleoside transporters, some of which also transport one or several nucleobases. Functional expression studies in Xenopus laevis oocytes revealed that LmaNT3 mediates high levels of uptake of hypoxanthine, xanthine, adenine and guanine. Moreover, LmaNT3 is an high affinity transporter with K(m) values for hypoxanthine, xanthine, adenine and guanine of 16.5 +/- 1.5, 8.5 +/- 0.6, 8.5 +/- 1.1, and 8.8 +/- 4.0 microM, respectively. LmaNT3 is, thus, the first member of the ENT family identified in any organism that functions as a nucleobase rather than nucleoside or nucleoside/nucleobase transporter.     

A basal membrane-like structure surrounding tumour nodules may prevent intraepithelial leucocyte infiltration in colorectal cancer

Epithelial tumours consist of an epithelial compartment and a stromal compartment, which are sometimes separated by a basal membrane-like structure. We sought to determine whether these factors have prognostic value in 84 curatively resected stage II and III colorectal cancer by immunohistochemically staining tumours for leucocytes (CD45) and extracellular matrix, and to assess the presence of a basal membrane-like structure. Leucocyte infiltration was also assessed in hematoxylin-eosin (HE) stained sections. Most leucocytes were located in the tumour stroma. A relatively high intraepithelial leucocyte infiltration was significantly correlated with a lower level of tumour recurrence (P=0.03) and a longer disease-free survival (P=0.05), whereas leucocytes located in the tumour stroma (P=0.92) or at the advancing margin (p=0.06) were not. Intraepithelial leucocyte infiltration was also significantly correlated with leucocyte infiltration in the tumour stroma (P=0.02) and at the advancing tumour margin (P=0.005), and as assessed in HE-stained tumour sections (P=0.05), but each of these parameters on its own did not have a prognostic value in predicting disease-free survival. Moreover, the presence of a basal membrane-like structure surrounding the tumour epithelium was inversely correlated with the number of intraepithelial leucocytes (P=0.05), suggesting that this membrane-like structure functions as a barrier to intraepithelial leucocyte infiltration. We conclude that leucocytes must be in the direct vicinity of tumour cells to affect tumour growth. The presence of an extracellular matrix barrier seems to prevent this interaction.     

Estimating average cellular turnover from 5-bromo-2'-deoxyuridine (BrdU) measurements

Cellular turnover rates in the immune system can be determined by labelling dividing cells with 5-bromo-2'-deoxyuridine (BrdU) or deuterated glucose ((2)H-glucose). To estimate the turnover rate from such measurements one has to fit a particular mathematical model to the data. The biological assumptions underlying various models developed for this purpose are controversial. Here, we fit a series of different models to BrdU data on CD4(+) T cells from SIV(-) and SIV(+) rhesus macaques. We first show that the parameter estimates obtained using these models depend strongly on the details of the model. To resolve this lack of generality we introduce a new parameter for each model, the 'average turnover rate', defined as the cellular death rate averaged over all subpopulations in the model. We show that very different models yield similar estimates of the average turnover rate, i.e. ca. 1% day(-1) in uninfected monkeys and ca. 2% day(-1) in SIV-infected monkeys. Thus, we show that one can use BrdU data from a possibly heterogeneous population of cells to estimate the average turnover rate of that population in a robust manner.     

The structure of bovine lysosomal alpha-mannosidase suggests a novel mechanism for low-pH activation

Lysosomal alpha-mannosidase (LAM: EC 3.2.1.24) belongs to the sequence-based glycoside hydrolase family 38 (GH38). Two other mammalian GH38 members, Golgi alpha-mannosidase II (GIIAM) and cytosolic alpha-mannosidase, are expressed in all tissues. In humans, cattle, cat and guinea pig, lack of lysosomal alpha-mannosidase activity causes the autosomal recessive disease alpha-mannosidosis. Here, we describe the three-dimensional structure of bovine lysosomal alpha-mannosidase (bLAM) at 2.7A resolution and confirm the solution state dimer by electron microscopy. We present the first structure of a mammalian GH38 enzyme that offers indications for the signal areas for mannose phosphorylation, suggests a previously undetected mechanism of low-pH activation and provides a template for further biochemical studies of the family 38 glycoside hydrolases as well as lysosomal transport. Furthermore, it provides a basis for understanding the human form of alpha-mannosidosis at the atomic level. The atomic coordinates and structure factors have been deposited in the Protein Data Bank (accession codes 1o7d and r1o7dsf).     

Integration site for Streptomyces phage phiBT1 and development of site-specific integrating vectors

Despite extensive similarities between the genomes of the Streptomyces temperate phages phiC31 and phiBT1, the attP-int loci are poorly conserved. Here we demonstrate that phiBT1 integrates into a different attachment site than phiC31. phiBT1 attB lies within SCO4848 encoding a 79-amino-acid putative integral membrane protein. Integration vectors based on phiBT1 integrase were shown to have a broad host range and are fully compatible with those based on the phiC31 attP-int locus.     

Mapping of synergistic components of weakly interacting protein-protein motifs using arrays of paired peptides

Protein-protein recognition usually involves multiple interactions among different motifs that are scattered over protein surfaces. To identify such weak interactions, we have developed a novel double peptide synthesis (DS) method. This method allows us to map protein-protein interactions that involve two linear dis- continuous components from a polypeptide by the use of spatially addressable synergistic pairs of synthetic peptides. The DS procedure is based on the "SPOT" membrane-bound peptide synthesis technique, but to synthesize a mixture of two peptides, it uses both Fmoc (N-(9-fluorenyl)methoxycarbonyl))-alanine and Alloc-alanine at the first cycle. This allows their selective deprotection by either piperidine or tributyltin/palladium treatment, respectively. Using SPOT DS, we confirmed as a proof of principle that Elk-1 Ser(383) phosphorylation by ERK-2 kinase is stimulated by the presence of the Elk-1-docking domain. SPOT DS can also be used to dissect protein-protein motifs that define phosphatase substrate affinity. Using this technique, we identified three new regions in the insulin receptor that stimulate the dephosphorylation of the receptor by protein-tyrosine phosphatase (PTP) 1B and presumably increase the selectivity of PTP for this substrate. These data demonstrate that the SPOT DS technique allows the identification of non-linear weakly interacting protein motifs, which are an important determinant of protein kinase and phosphatase substrate specificity and of protein-protein interactions in general.