Role of the Dinaric Karst (western Balkans) in shaping the phylogeographic structure of the threatened crayfish Austropotamobius torrentium

1. This study examines phylogeography and phylogeny of the threatened stone crayfish, Austropotamobius torrentium, in order to elucidate the role of the Dinaric Karst geology in shaping the evolutionary history and genetic diversity of aquatic fauna in the western Balkans. Mitochondrial 16S rRNA and COI genes were partially sequenced from 188 and 159 crayfish, respectively, sampled from 70 localities. Phylogenetic relationships were reconstructed using four methods of phylogenetic inference. Divergence times between phylogroups were estimated in a Bayesian framework, and their demographic history was examined using neutrality tests and mismatch distribution analysis. 2. Seven geographically localised phylogroups separated by pronounced genetic gaps were found. Five of them have a distribution range within the northern‐central Dinaric (NCD) region, while the remaining two include populations from the southern Balkans (SB) and central and south‐eastern Europe (CSE). The oldest divergence event separated two NCD lineages from the rest of populations in the Late Miocene or Early Pliocene. Divergences amongst the five NCD phylogroups and SB + CSE occurred in the Pliocene. The most recent split separated SB and CSE phylogroups during the Late Pliocene. For both genes, uncorrected pairwise divergences between most of the phylogroups (4.1–8.7% for COI and 1.6–4.8% for 16S rRNA) were of the same range as, or higher than, some of the interspecific distances previously reported for the genus Austropotamobius. 3. Geographically isolated and deeply divergent cryptic monophyletic phylogroups within A. torrentium in the NCD region arose in the course of intensification of Neotectonic movements during the Pliocene and the beginning of the Pleistocene and the development of karstification that has heavily fragmented the palaeohydrography of the area. The results confirm a gradual north–south expansion of stone crayfish during the pre‐Pleistocene that preceded the rapid northward post‐glacial re/colonisation of central Europe (CSE phylogroup) through the Danube drainage. 4. Austropotamobius torrentium comprises morphologically cryptic but molecularly distinct taxa. Considering the relatively small geographical areas they inhabit, the NCD phylogroups of stone crayfish should be given the highest conservation priority.     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

Effects of general,specific and combined warm-up on explosive muscular performance

The purpose of this study was to compare the acute effects of general, specific and combined warm-up (WU) on explosive performance. Healthy male (n = 10) subjects participated in six WU protocols in a crossover randomized study design. Protocols were: passive rest (PR; 15 min of passive rest), running (Run; 5 min of running at 70% of maximum heart rate), stretching (STR; 5 min of static stretching exercise), jumping [Jump; 5 min of jumping exercises – 3x8 countermovement jumps (CMJ) and 3x8 drop jumps from 60 cm (DJ60)], and combined (COM; protocols Run+STR+Jump combined). Immediately before and after each WU, subjects were assessed for explosive concentric-only (i.e. squat jump – SJ), slow stretch-shortening cycle (i.e. CMJ), fast stretch-shortening cycle (i.e. DJ60) and contact time (CT) muscle performance. PR significantly reduced SJ performance (p =0.007). Run increased SJ (p =0.0001) and CMJ (p =0.002). STR increased CMJ (p =0.048). Specific WU (i.e. Jump) increased SJ (p =0.001), CMJ (p =0.028) and DJ60 (p =0.006) performance. COM increased CMJ performance (p =0.006). Jump was superior in SJ performance vs. PR (p =0.001). Jump reduced (p =0.03) CT in DJ60. In conclusion, general, specific and combined WU increase slow stretch-shortening cycle (SSC) muscle performance, but only specific WU increases fast SSC muscle performance. Therefore, to increase fast SSC performance, specific fast SSC muscle actions must be included during the WU.     

Out of Africa and back again: nested cladistic analysis of human Y chromosome variation

We surveyed nine diallelic polymorphic sites on the Y chromosomes of 1,544 individuals from Africa, Asia, Europe, Oceania, and the New World. Phylogenetic analyses of these nine sites resulted in a tree for 10 distinct Y haplotypes with a coalescence time of approximately 150,000 years. The 10 haplotypes were unevenly distributed among human populations: 5 were restricted to a particular continent, 2 were shared between Africa and Europe, 1 was present only in the Old World, and 2 were found in all geographic regions surveyed. The ancestral haplotype was limited to African populations. Random permutation procedures revealed statistically significant patterns of geographical structuring of this paternal genetic variation. The results of a nested cladistic analysis indicated that these geographical associations arose through a combination of processes, including restricted, recurrent gene flow (isolation by distance) and range expansions. We inferred that one of the oldest events in the nested cladistic analysis was a range expansion out of Africa which resulted in the complete replacement of Y chromosomes throughout the Old World, a finding consistent with many versions of the Out of Africa Replacement Model. A second and more recent range expansion brought Asian Y chromosomes back to Africa without replacing the indigenous African male gene pool. Thus, the previously observed high levels of Y chromosomal genetic diversity in Africa may be due in part to bidirectional population movements. Finally, a comparison of our results with those from nested cladistic analyses of human mtDNA and beta-globin data revealed different patterns of inferences for males and females concerning the relative roles of population history (range expansions) and population structure (recurrent gene flow), thereby adding a new sex-specific component to models of human evolution.      

Water loss from Jackass Penguin Spheniscus demersus eggs during natural incubation

The water budget of incubating Jackass Penguin eggs was studied on Marcus Island, South Africa, and complementary measurements were made in the laboratory. The mean ambient temperature was 16-5 "C and the mean humidity was 12-4 Torr (89% relative humidity). The temperature of incubated live and water-filled eggs ranged between 14^oCand 37 ^oC. The mean calculated egg temperature was 34-9' C. The mean brood patch temperature was 37-1 ^oC, slightly lower than the cloacal temperature (37.8 ^oC). The mean brood patch area was about 38 cm². The rate of water loss was 411 mg day^-1. The total diffusive water loss during 37 days of incubation was, as predicted, 15-2% of the initial 100-3 g egg mass. The total pore number was 6245 per egg and the shell thickness was 577 fira. It is suggested that the eggshell parameters, incubation length and nesting behaviour are compensated in such a way that an egg-to-nest water vapour pressure difference lower than commonly found is sufficient to bring about the normal total water loss.     

gamma-Aminobutyric acid(A) receptor subunit expression predicts functional changes in hippocampal dentate granule cells during postnatal development

Profound alterations in the function of GABA occur over the course of postnatal development. Changes in GABA(A) receptor expression are thought to contribute to these differences in GABAergic function, but how subunit changes correlate with receptor function in individual developing neurons has not been defined precisely. In the current study, we correlate expression of 14 different GABA(A) receptor subunit mRNAs with changes in the pharmacological properties of the receptor in individual hippocampal dentate granule cells over the course of postnatal development in rat. We demonstrate significant developmental differences in GABA(A) receptor subunit mRNA expression, including greater than two-fold lower expression of alpha1-, alpha4- and gamma2-subunit mRNAs and 10-fold higher expression of alpha5-mRNA in immature compared with adult neurons. These differences correlate both with regional changes in subunit protein level and with alterations in GABA(A) receptor function in immature dentate granule cells, including two-fold higher blockade by zinc and three-fold lower augmentation by type-I benzodiazepine site modulators. Further, we find an inverse correlation between changes in GABA(A) receptor zinc sensitivity and abundance of vesicular zinc in dentate gyrus during postnatal development. These findings suggest that developmental differences in subunit expression contribute to alterations in GABA(A) receptor function during postnatal development.     

The phylogeny of the hominoid primates: a statistical analysis of the DNA-DNA hybridization data

Sibley and Ahlquist compared the single-copy nuclear DNA sequences of the hominoid primates using DNA-DNA hybridization. From this data set they estimated a phylogeny that clusters man and chimpanzees using a distance Wagner procedure. However, no assessment of statistical confidence in this estimated phylogeny was made, despite the fact that their data set contains internal inconsistencies concerning the correct branching order. This paper presents a modification of Pielou's Q- statistic that allows one to make nonparametric tests of phylogenetic relationship from distance data. The results of this analysis indicate that the estimated phylogeny of Sibley and Ahlquist is without statistical significance owing to the internal inconsistencies of the data set. A survey and additional analyses of other types of molecular data indicate that the phylogeny that clusters chimpanzees and gorillas and has the human lineage splitting off earlier is statistically consistent with all the molecular data (including the DNA-DNA hybridization data), whereas the phylogeny estimated by Sibley and Ahlquist can be rejected at the 5% level using the data on restriction- endonuclease sites in the mitochondrial genome.      

Monoclonal antibodies to rabbit liver cytochrome P-448

Cytochrome P-448 (mol wt 55,000 Daltons) from rabbit liver was purified to a specific content of 16.6 nmol/mg. Mice were immunised with this preparation, their spleens removed and dissociated lymphocytes hybridised with myeloma cells. Four monoclonal antibodies against cytochrome P-448 were raised and partially characterised. All four antibodies interacted with cytochrome P-448 in intact microsomal fractions and selectively immunoadsorbed cytochrome P-448 from solubilised microsomal preparations. One of the antibodies inhibited benzo[a] pyrene hydroxylase activity in a reconstituted system, one had no effect on activity and two increased activity. The possible applications of such antibodies are discussed.