Microsatellite loci in the Canary Islands endemic ground beetle Trechus flavocinctus and their applicability to cave‐dwelling related species

Six polymorphic microsatellite markers have been developed for the Canary endemic ground beetle Trechus flavocinctus, and the performance of primer pairs amplifying these loci in closely related taxa (including two cave‐obliged species) has been tested. The number of alleles in T. flavocinctus samples taken from three distant localities in two islands ranged from four to 14 per locus. Observed heterozygosities were from 0.333 to 0.933. These markers could be useful tools to study the population structure and the genetic consequences of island and cave colonization by ground beetles.     

High Nothofagusflower consumption and pollen emptying in the southern South American austral parakeet (Enicognathus ferrugineus)

Abstract We describe extraordinary aspects of the feeding ecology of the austral parakeet, Enicognathus ferrugineus– the most southerly distributed psittacid in the world – that allow this endemic species to inhabit and become a common bird in relatively species‐poor temperate and subantarctic Nothofagus forests of South America. We used two sources of information to analyse temporal and spatial dietary changes of austral parakeets in subalpine forests near Lake Distric of southern Argentina: (i) relative abundance of parakeet foraging on the forest ?oor along an altitudinal transect from 1000 to 1420 m; and (ii) faeces analyses of seasonal collections. Austral parakeets largely relied on a protein‐rich pollen‐based spring–early summer diet by destructively harvesting large quantities of wind‐pollinated Nothofagus pumilio?owers and efficiently emptying pollen grains – specializations previously described only in pollinating nectarivorous vertebrates. Pollen emptying rates (c. 65%) were the highest reported for psittacids and among the highest for vertebrates in general. Parakeets made extended use of short‐lived N. pumilio?owers by tracking the altitudinal shifts in ?owering phenology. Additionally, parakeets complemented their diet with carbohydrates from N. pumilio insect exudates. By late summer, parakeets switched to a lipid‐rich diet based on N. pumilio seeds. This resource remained available through mid‐autumn because parakeets also followed in altitude the phenological delays in fruiting. In winter, parakeets fed on N. pumilio parasitic Misodendrum mistletoe buds and leaves and Cyttaria sp. parasitic canopy fungi. These results suggest that stringent food availability in these relatively high latitudes may have led to behavioural and physiological specializations of austral parakeets to obtain year‐round food resources efficiently from Nothofagus trees.     

Reproductive performance of a lacertid lizard at the core and the periphery of the species' range

The range boundaries of organisms are frequently interpreted in terms of a decline in the extent to which the life histories of outer populations are able to adapt to local environmental conditions. To test this hypothesis, we compared the reproductive characteristics of two Iberian populations of the lizard Psammodromus algirus (Linnaeus, 1758). One of them (Lerma) is close to the northern edge of the species' range, whereas the other one (El Pardo) occupies a typical core habitat 200 km further south. Gravid females were captured in the field and transported to the lab for egg laying. Second clutches were less frequent at Lerma (where clutch size and clutch mass were larger for first than for second clutches) than at El Pardo. The total mass of both clutches combined was similar at both sites. Thus, the higher frequency of second clutches at El Pardo appeared to balance the between-sites difference in energy allocation to the first clutch. Females from Lerma laid more but smaller eggs than those from El Pardo. When incubated at the same temperature, eggs from Lerma hatched sooner even when controlling for between-sites differences in mean egg size. These differences are interpreted in the light of the advantages of early hatching and high fecundity in the northern population, as opposed to large offspring size in the core population. We conclude that the life-history traits studied show enough variation, presumably of an adaptive nature, to cope with environmental challenges at the edge of the species' range.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 92 , 87–96.     

Comparison between morphometry and immunostaining of malignant cells in non-small cell lung cancer

OBJECTIVE: To investigate the morphometric properties of tumor cells in combination with the expression of 5 immunomarkers, quantitatively evaluated by image analysis, in a series of 60 non-small cell lung carcinomas (NSCLCs) and to assess their prognostic significance. STUDY DESIGN: Tissue sections from 60 NSCLCs were assessed for the expression of p53, Ki-67, bcl-2, bax and FasL using immunohistochemistry and antibodies. Correlations between morphometric features and clinicopathologic variables, including overall survival and the expression of the above markers, were statistically investigated. RESULTS: Major and minor axis nuclear values, as well as nuclear area and perimeter values, were positively interrelated. No statistically significant correlations were observed between nuclear morphometry and any of the clinicopathologic parameters. p53 Accumulation, when quantitatively estimated, displayed a positive correlation with major axis (P = .008), minor axis (P = .06), nuclear perimeter (P = .006) and mainly nuclear area values (P = .003). Bcl-2- and bax-quantified immunostaining demonstrated a weak negative correlation with shape factor values (P = .039 and P = .025, respectively). Univariate and multivariate analysis showed that stage was the only significant predictor of survival in all patients. In univariate statistical analysis there was a trend between worse survival and shape factor values < 0.8 (P = .0791); this trend almost reached statistical significance in the subgroup of squamous cell carcinomas (P = .0570). CONCLUSION: p53 Accumulation, when quantified, appears to be positively linked with nuclear morphometric values. The prognostic significance of shape factor in NSCLC warrants further investigation.     

Morphometric analysis of AgNORs in thin-layer, liquid-based liver specimens

OBJECTIVE: To detect argyrophilic nucleolar organizer regions (AgNORs) in ThinPrep (Cytyc Corp., Boxborough, Massachusetts, U.S.A.) liver fine needle aspiration (FNA) specimens and to define the diagnostic value of their quantitative analysis in the evaluation of hepatic lesions. STUDY DESIGN: ThinPrep liquid-based FNA biopsy specimens from 49 malignant and benign liver lesions were resampled, fixed in 95% ethanol and stained with the AgNOR technique in accordance with the 1-step colloid method. The specimens included 11 benign and 38 malignant lesions (23 poorly differentiated hepatocellular carcinomas [HCCs] and 15 poorly differentiated metastatic adenocarcinomas [MCs]). Morphometric analysis was performed using a Zeiss Axiolab microscope (Carl Zeiss GmbH, Jena, Germany) with a mechanical stage fitted with a Sony-iris CCD videocamera (Tokyo, Japan). The videocamera was connected to a Pentium III P/C (Intel Corp., Santa Clara, California, U.S.A.) loaded with the appropriate image analysis software. The measurements were performed with ImageScan software (Jandel Scientific, Erkrath, Germany). The number of AgNORs per nucleus (NN) and the total area per nucleus occupied by AgNORs (AR) were calculated semiautomatically. Statistical analysis was performed using the SPSS software package (Chicago, Illinois, U.S.A.). RESULTS: The least significant deviance test for multiple comparisons revealed that NN differed significantly between the 3 groups of samples examined (P < .0001). The mean NN values in HCCs and MCs were significantly different (P < .0001). Logistic regression model demonstrated that as NN increased, the probability of a MC diagnosis decreased (<4%). AR values were different at a statistically significant level only between benign and malignant specimens (P = .00006), not between HCCs and MCs (P = .933). CONCLUSION: Quantitative analysis of AgNORs in ThinPrep specimens could be a diagnostically useful method in liver disease.     

Modulation of the catalytic activity of neutrophil collagenase MMP-8 on bovine collagen I. Role of the activation cleavage and of the hemopexin-like domain

The cleavage of bovine collagen I by neutrophil collagenase MMP-8 has been followed at pH 7.4, 37 degrees C. The behavior of the whole enzyme molecule (whMMP-8), displaying both the catalytic domain and the hemopexin-like domain, has been compared under the same experimental conditions with that of the catalytic domain only. The main observation is that whMMP-8 cleaves bovine collagen I only at a single specific site, as already reported by many others (Mallya, S. K., Mookhtiar, K. A., Gao, Y., Brew, K., Dioszegi, M., Birkedal-Hansen, H., and van Wart, H. E. (1990) Biochemistry 29, 10628-10634; Kn?uper, V., Osthues, A., DeClerk, Y. A., Langley, K. A., Bl?ser, J., and Tschesche, H. (1993) Biochem. J. 291, 847-854; Marini, S., Fasciglione, G. F., De Sanctis, G., D'Alessio, S., Politi, V., and Coletta, M. (2000) J. Biol. Chem. 275, 18657-18663), whereas the catalytic domain lacks this specificity and cleaves the collagen molecule at multiple sites. Furthermore, a meaningful difference is observed for the cleavage features displayed by two forms of the catalytic domain, which differ for the N terminus resulting from the activation process (i.e. the former Met(80) of the proenzyme (MetMMP-8) and the former Phe(79) of the proenzyme (PheMMP-8)). Thus, the PheMMP-8 species is characterized by a much faster k(cat)/K(m), fully attributable to a lower K(m), suggesting that the conformation of the catalytic domain, induced by the insertion of this N-terminal residue in a specific pocket (Reinemer, P., Grams, F., Huber, R., Kleine, T., Schnierer, S., Piper, M., Tschesche, H., and Bode, W. (1994) FEBS Lett. 338, 227-233), brings about a better, although less discriminatory, recognition process of cleavage site(s) on bovine collagen I.     

ASSESSMENT OF AN ESCHERICHIA COLI METHIONINE AUXOTROPH GROWTH ASSAY FOR QUANTIFYING CRYSTALLINE METHIONINE SUPPLEMENTED IN POULTRY FEEDS

Estimating availability of methionine is relevant to feed formulation since diets can be supplemented with crystalline methionine to meet the minimum requirements of rapidly growing birds. Bacterial assays have been developed to measure the bioavailable levels of several essential amino acids in feeds, including methionine. The E. coli methionine auxotroph strain used in this study exhibited a linear extent of growth response to increasing concentrations of methionine added to the minimal test media, in the range of 0 to 4 μg/mL. In addition the growth rates of the E. coli auxotroph were significantly (P < 0.01) different when the methionine concentrations were varied (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 μg/mL) in minimal media. To assay feeds, feed grade methionine was added to poultry feed mixtures and samples were diluted with M9 media. Using this assay for estimating crystalline methionine added to feed, the extent of growth of the methionine auxotroph was correlated with the levels of crystalline methionine supplemented in the feed (R²= 0.9873). For all supplementation levels methionine recovery percentages ranged from 71 to 80% indicating that the bacterial assay response to crystalline methionine was relatively constant in the presence of the feed matrix. The overall results indicate that the rapid detection of crystalline methionine added to feeds is possible using this E. coli methionine auxotroph growth-based assay.     

The amino acid sequence of glutamate dehydrogenase fromPyrococcus furiosus, a hyperthermophilic archaebacterium

The complete amino acid sequence of glutamate dehydrogenase from the archaebacteriumPyrococcus furiosus has been determined. The sequence was reconstructed by automated sequence analysis of peptides obtained after cleavage with cyanogen bromide, Asp-N endoproteinase, trypsin, or pepsin. The enzyme subunit is composed of 420 amino acid residues yielding a molecular mass of 47,122 D. In the recently determined primary structure of glutamate dehydrogenase from another thermophilic archaebacterium,Sulfolobus solfataricus, the presence of some methylated lysines was detected and the possible role of this posttranslational modification in enhancing the thermostability of the enzyme was discussed (Maras, B., Consalvi, V., Chiaraluce, R., Politi, L., De Rosa, M., Bossa, F., Scandurra, R., and Barra, D. (1992),Eur. J. Biochem. 203, 81–87). In the primary structure reported here, such posttranslational modification has not been found, indicating that the role of lysine methylation should be revisited. Comparison of the sequence of glutamate dehydrogenase fromPyrococcus furiosus with that ofS. solfataricus shows a 43.7% similarity, thus indicating a common evolutionary pathway.     

Role of tyrosine residues in cytoplasmic aspartate aminotransferase from beef kidney

Summary Cytoplasmic aspartate aminotransferase from beef kindney loses 25% of its activity on nitration with tetranitromethane while the apoenzyme about 95%. In the holoenzyme 0.5 tyrosine residue and 1.0 tyrosine residue in the apoenzyme are nitrated per enzyme protomer. In addition 1 cysteine residue per protomer is oxidized in both. The presence of substrates,-ketoglutarate and glutamate, both at ten times their K_m values, does not change these results. Mercaptoethanol does not affect the residual activity of either the nitrated holo or apoenzyme. Dithionite abolishes the activity of the nitrated holoenzyme by reducing the coenzyme moiety. It has no effect on the native holoenzyme or on either the native or nitroapoenzyme.This work is part of a program supported by a grant from the Consiglio Nazionale delle Ricerche.