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.      

Diffusion coefficient of fluorescein-labeled tubulin in the cytoplasm of embryonic cells of a sea urchin: video image analysis of fluorescence redistribution after photobleaching

The diffusion coefficient of tubulin has been measured in the cytoplasm of eggs and embryos of the sea urchin Lytechinus variegatus. We have used brain tubulin, conjugated to dichlorotriazinyl-aminofluorescein, to inject eggs and embryos. The resulting distributions of fluorescence were perturbed by bleaching with a microbeam of light from the 488-nm line of an argon ion laser. Fluorescence redistribution after photobleaching was monitored with a sensitive video camera and photography of the television-generated image. With standard photometric methods, we have calibrated this recording system and measured the rates of fluorescence redistribution for tubulin, conjugated to dichlorotriazinyl-aminofluorescein, not incorporated into the mitotic spindle. The diffusion coefficient (D) was calculated from these data using Fick's second law of diffusion and a digital method for analysis of the photometric curves. We have tested our method by determining D for bovine serum albumin (BSA) under conditions where the value is already known and by measuring D for fluorescein-labeled BSA in sea urchin eggs with a standard apparatus for monitoring fluorescence redistribution after photobleaching. The values agree to within experimental error. Dcytoplasmtubulin = 5.9 +/- 2.2 X 10(-8) cm2/s; DcytoplasmBSA = 8.6 +/- 2.0 X 10(-8) cm2/s. Because DH2OBSA = 68 X 10(-8) cm2/s, these data suggest that the viscosity of sea urchin cytoplasm for protein is about eight times that of water and that most of the tubulin of the sea urchin cytoplasm exists as a dimer or small oligomer, which is unbound to structures that would impede its diffusion. Values and limitations of our method are discussed, and we draw attention to both the variations in D for single proteins in different cells and the importance of D for the upper limit to the rates of polymerization reactions.     

Interactions ofCandida albicans with bacteria and salivary molecules in oral biofilms

The yeastCandida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells.C. albicans andCandida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind toStreptococcus gordonii NCTC 7869 while two otherCandida species (Candida krusei andCandida kefyr) do not. Adherence ofC. albicans was greatest when the yeast had been grown at 30° C to mid-exponential growth phase. For 21 strains ofC. albicans there was a positive correlation between the ability to adhere toS. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence ofC. albicans toS. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins ofC. albicans are co-expressed.     

Microwave thawing of frozen kidneys: A theoretically based experimentally-effective design

Equipment was designed and fabricated for uniformly thawing frozen canine kidneys using single-frequency electromagnetic radiation. Complete and uniform warming of frozen kidneys from ?70 to +14 °C over periods ranging from 1.5 to 4.5 min was achieved without “cooking” or experiencing thermal runaway. Dielectric measurements of renal slices (medulla and cortex) were performed as a function of temperature at a frequency of 918 MHz for a Me₂SO cryoprotectant concentration of 5% (0.7 M). Results of these measurements were then employed as an input to analytical computer models which were used to predict the internal field intensities and power distribution results for both frozen and thawed kidneys. From these predictions, a 918-MHz EM illuminator for thawing canine kidneys was designed and fabricated. Twenty-seven kidneys were thawed using this illumination system. Of these, excellent uniformity of thawing was achieved for 17 kidneys, good uniformity for 8 kidneys, and for only 2 kidneys was thawing uniformity fair to poor.     

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.     

Functional preservation of the mammalian kidney. IV. Functional effects of perfusion with dimethyl sulfoxide (DMSO) at normothermia

Rabbit kidneys were perfused at 37 °C with various concentrations of DMSO in a K⁺-Mg²⁺-rich perfusate. The effects of DMSO on various functional parameters of the rabbit kidney perfused for 60 min were compared with the functional effects of perfusion without DMSO under the same conditions. DMSO produced deviations in vascular resistance and perfusate flow rate from control values. In kidneys perfused with 1.4 and 2.8 m DMSO these vascular changes resulted in changes in GFR at relatively unchanged filtration fractions. The closely parallel relationship between changes in GFR and urine flow rate in all groups indicates that perfusion per se or perfusion with DMSO may shift the regulation of urine flow rate from tubular reabsorption, which obtains in the in vivo situation, to glomerular filtration. This view was supported by the relatively unchanged parameters of Na⁺ reabsorption and fractional water excretion during perfusion with all concentrations of DMSO. Additionally, DMSO perfusion resulted in significantly greater weight gains than those observed in kidneys perfused without DMSO, and significantly depressed clearances of PAH, with 2.1 and 2.8 m DMSO.     

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.     

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.