Dynamics of adaptation and diversification: a 10,000-generation experiment with bacterial populations

We followed evolutionary change in 12 populations of Escherichia coli propagated for 10,000 generations in identical environments. Both morphology (cell size) and fitness (measured in competition with the ancestor) evolved rapidly for the first 2000 generations or so after the populations were introduced into the experimental environment, but both were nearly static for the last 5000 generations. Although evolving in identical environments, the replicate populations diverged significantly from one another in both morphology and mean fitness. The divergence in mean fitness was sustained and implies that the populations have approached different fitness peaks of unequal height in the adaptive landscape. Although the experimental time scale and environment were microevolutionary in scope, our experiments were designed to address questions concerning the origin as well as the fate of genetic and phenotypic novelties, the repeatability of adaptation, the diversification of lineages, and thus the causes and consequences of the uniqueness of evolutionary history. In fact, we observed several hallmarks of macroevolutionary dynamics, including periods of rapid evolution and stasis, altered functional relationships between traits, and concordance of anagenetic and cladogenetic trends. Our results support a Wrightian interpretation, in which chance events (mutation and drift) play an important role in adaptive evolution, as do the complex genetic interactions that underlie the structure of organisms.     

Fluorescent monitoring of Jurkatt cell intracellular magnesium during metabolic poisoning

Divalent cation movement characterizes the final common pathway of cellular death from ischemic or metabolic injury. The influx of calcium is an essential step in cellular death. We hypothesized that intracellular magnesium levels may change during the progression to cellular death. Verapamil-sensitive changes in free ionized intracellular Mg2+ ([Mg2+[i) and Ca2+ ([Ca2+]i) levels were estimated in transformed T-lymphocytes exposed to metabolic inhibitors. Separate experiments used a Mg(2+)-sensitive fluoroprobe, fura-2 (Ex 1,344, Ex 2,376, Em 500), and a Ca(2+)-sensitive fluoroprobe, fura-2 (Ex 1,340, Ex 2,380, Em 510). Chemical anoxia (sodium cyanide 1 mM, iodoacetic acid 10 mM) caused a gradual increase in [Ca2+]i (control 126 +/- 13 nM) to > 1 mM by 10 min. This increase in [Ca2+]i was not affected by verapamil treatment. In separate experiments, [Mg2+]i levels were monitored during chemical anoxia. The specificity of mag-fura for Mg2+ over Ca2+ was reflected in the absence of a response to the lymphocyte Ca2+ mobilizer OKT-3. Uncorrected control [Mg2+]i levels (.4 +/- .1 mM) were not affected by the combined cyanide-iodoacetate treatment. A small increase in mag-fura-2 fluorescence was noted, probably due to binding of Ca2+ to the fluoroprobe when [Ca2]i exceeded 1 mM. Elimination of Ca2+ from the extracellular buffer increased the resting estimate of intracellular [Mg2+] to 1.6 + .1 mM. These results indicate that 1) extracellular Ca2+ can interfere with the fluorescent determination of intracellular magnesium concentration, and 2) intracellular free Mg2+ concentrations do not change in this cell line during chemical anoxia.     

Toxoplasma gondii: a paraformaldehyde-insensitive diaphorase activity acts as a specific histochemical marker for the single mitochondrion

Paired-pulse facilitation (PPF) of CA3-CA1 excitatory postsynaptic potentials (EPSP) was compared in hippocampal slices from juvenile (postnatal day (P) 15-21) and young adult rats (P28-P35) following application of adenosine. Relative to juveniles, young adults expressed an increase in baseline synaptic strength that was accompanied by a decrease in PPF suggesting a developmental increase in transmitter release. While adenosine depressed the EPSP slope to a similar extent in juveniles and young adults, PPF increased during adenosine application only for young adults. The differential effect of adenosine on PPF was not due to differences in receptor function or in extracellular ligand levels, since the A1 antagonist cyclopentyltheophylline (CPT) did not differentially affect PPF across age. Adenosine could increase PPF in juvenile slices under conditions of enhanced transmitter release, through an increase in the bath Ca2+ concentration, or addition of forskolin to the bath. These data indicate that the ability to modify synaptic transmission through presynaptic adenosine A1 receptors increases across postnatal development with the maturation of release mechanisms.     

Expanded T cell populations in patients with Wegener's granulomatosis: characteristics and correlates with disease activity

Patients with Wegener's granulomatosis have a high prevalence of expanded populations of CD4+ and CD8+ T cells bearing different alpha/beta T cell receptors. To elucidate the role of these populations, we studied the phenotypic and functional characteristics of 13 expanded T cell populations in four patients for a period of 35-51 months. The expanded populations generally showed a persistently high expression of the activation markers HLA-DR and CD25. This expression was independent of the activity of the disease. The expanded populations also expressed CD45RO and/or CD45RA and most of them expressed CD57 but not CD28. Analysis of intracellular presence and secretion of IFN-gamma, IL-2, and IL-4 showed that most of the expanded cell populations contained and/or secreted more of these cytokines than the nonexpanded populations, with an especially high expression/secretion of IFN-gamma and IL-2. The expanded populations showed little proliferative response to Con A and OKT3. The proliferative response of the cells was partly restored after preincubation in medium alone. Some of the expanded populations were associated with disease activity, thus suggesting a link between expanded T cells and the disease. The activated status of the expanded populations and the tendency for certain populations to correlate in magnitude with disease activity suggest their involvement in the disease process. The relative stability of these cell populations indicates that the stimulus driving them is persistent, in agreement with the chronicity of the disease.     

Vaginal pH as a marker for bacterial pathogens and menopausal status

OBJECTIVE: Our purpose was to confirm the elevation of vaginal pH expected in patients with bacterial pathogens in premenopausal women and to examine the relationship of serum follicle-stimulating hormone and estradiol levels to vaginal pH in menopausal patients without and with hormone replacement therapy. STUDY DESIGN: Vaginal pH was determined by phenaphthazine (Nitrazine) pH paper in 253 patients seen in a solo private practice for routine speculum examination. None of the patients were pregnant. Measurements were made of serum levels of follicle-stimulating hormone and estradiol for 172 patients and vaginal cultures were taken from 82 patients. Vaginal pH was correlated with vaginal cultures and serum follicle-stimulating hormone and estradiol levels by use of statistical analysis. RESULTS: Vaginal pH was elevated in all premenopausal patients with documented bacterial pathogens. Serum estradiol levels showed an inverse and serum follicle-stimulating hormone levels a direct statistical correlation with vaginal pH in menopausal patients. CONCLUSIONS: Measurement of vaginal pH is useful, effective, and inexpensive for screening purposes. A vaginal pH of 4.5 is consistent with a premenopausal serum estradiol level and the absence of bacterial pathogens. An elevated vaginal pH in the 5.0 to 6.5 range suggests a diagnosis of either bacterial pathogens or decreased serum estradiol. In patients with an elevated pH, vaginal culture should establish the diagnosis. In the absence of bacterial pathogens, a vaginal pH of 6.0 to 7.5 is strongly suggestive of menopause. Titration of estradiol level by vaginal pH during estrogen replacement therapy may help menopausal women avoid side effects or cessation of therapy.     

Biological activity of bacterial cell wall components. Immunogenicity of an immunostimulating bacterial extract

An immunostimulating bacterial extract (IBE, Broncho-Vaxom) used for the prevention and treatment of recurrent respiratory tract infections consists of lyophilized fractions derived from 21 bacterial strains occurring in these infections. The immunogenic properties of IBE were determined in vivo in a murine model. It could be demonstrated that the extract constitutes an active immunogen in Balb/c mice. As shown by ELISA, IBE-specific antisera could be obtained after repeated i.p. immunizations; the antibodies were mainly of the IgG and IgM isotypes. The antibodies also bound - to a variable degree - to the bacterial strains used for the preparation of the extract. Additionally the antisera were shown to recognize the bacterial cell wall components porin, lipoprotein/lipopeptide and murein. Thus, IBE constitutes an active immunogen in vivo inducing antibodies directed against bacterial cell wall components; this could be of importance for understanding the therapeutic effect of the extract.     

Interferon induction as a quasispecies marker of vesicular stomatitis virus populations

The interferon (IFN)-inducing capacity of different isolates of vesicular stomatitis virus (VSV) of the Indiana (IN) and New Jersey (NJ) serotypes were measured to assess the extent of variability of this phenotype. Over 200 preparations of wild-type field isolates, laboratory strains, and plaque-derived subpopulations were examined. Marked heterogeneity was found in the ability of these viruses to induce IFN, covering a 10,000-fold range. A good fit to a normal distribution for the log of the IFN yields suggests a continuum of incremental changes in the viral genome may govern the IFN-inducing capacity of consensus populations derived from independently arising infections. A broad range in the magnitude of these changes, skewed towards inducers of high IFN yields, is consistent with a comparable series of ribonucleotide changes in the VSV genome, a sine qua non of a quasispecies population. Plaque- or vesicle-derived populations displayed standard deviations less than the mean IFN yields, though skewed to higher yielders, whereas populations from field and laboratory samples which differed widely in time and origin of isolation gave standard deviations greater than the means. The plaque isolation of IFN-inducing particles of VSV-IN, normally masked in populations by the predominance of non-IFN-inducing particles that suppress IFN induction, and the isolation of potent wild-type IFN-inducing VSV-IN from cows during an outbreak of vesicular stomatitis in a region that had yielded only virus expressing the non-IFN-inducing phenotype in prior and subsequent years, supports the view that genetic bottlenecks are operative in the natural transmission of this disease.     

Inhibition of natural killer cell activity in mice treated with tobacco specific carcinogen NNK

Among the different chemicals present in tobacco and tobacco smoke, 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK) is the most potent carcinogen. In the present study the immunosuppressive effect of NNK was investigated in laboratory animals by analyzing the antitumor immune responses. Mice of B6C3F1 strain were treated with different doses of NNK by IP and assayed for natural killer cell activity by the lysis of 51Cr-labeled YAC-1 lymphoma cells. The control mice received physiological saline. The results showed a significant inhibition of natural killer cell activity in the spleen cells of mice treated with 100 or 250 mg/kg NNK. In contrast to the high-dose NNK group, treatment of mice with lower doses of NNK like 10 or 50 mg/kg had no significant effect on the natural killer cell activity. In addition to spleen, the natural killer cell activity was also suppressed in the hilar lymph nodes and lung cells of NNK-treated mice. The clearance of 125I labeled YAC-1 tumor cells was also reduced from the lungs of mice injected with NNK. Further, the metastatic potential of B16F10 melanoma cells was significantly higher, as evidenced by the increased lung tumor nodules in the high-dose NNK-treated mice. The decreased antitumor immune response in the carcinogen-treated mice was not due to a decrease of NK cells, because flow cytometric analysis indicated no change in the frequency of NK 1.1+ cells between control and treated animals. However, there was an increased plasma cortisone levels in the carcinogen-treated mice compared to control animals. Injection of mice with poly I:C or interleukin-12 was able to restore natural killer cell activity in the tobacco carcinogen-treated mice.     

Ambulatory behavior map, physical activity and biosignal monitoring system

In this study, we have developed an ambulatory human behavior map and physical activity monitoring system. This was accomplished by equipping our portable digital biosignal memory device developed previously with GPS sensors and piezoresistive accelerometers. Using this new system, we can get a subject's behavior map, and estimate his physical activities and posture changes in daily life.     

CT number distribution and its association with local control and as a marker of lung tumor response to radiation

An early noninvasive indicator of tumor response to therapy and the ability to predict clinical outcome may potentially enhance disease management. Currently, however, tumor response to therapy is often delayed, potentially compromising disease management. We examined the computed tomography (CT) number or Hounsfield unit distribution to follow lung tumor response to radiation treatment. To help interpret the results, we examined whether the CT number distribution follows a simple two-component model. The CT number distribution was derived from a CT-simulator for 11 patients with lung cancer before and after the initial radiation treatment (1-1.5 months, average 3,407 cGy). Clinical outcomes were followed in 8 patients who received 5,580-6,660 cGy. All patients were scanned serially, using identical radiation imaging parameters (voltage, current, scan time, and slice thickness) in a CT-simulator. The lung tumors were digitally contoured, and software windows were applied to avoid inclusion of lung tissue in the analysis. Histograms and statistical analysis of the CT numbers for the tumor were generated. Radiation-induced CT number or Hounsfield unit (HU) shifts exceeding a threshold (13 HU) in lung tumors were associated with (P=0.04) local control (> or = 10 months). Initial lung tumor size (below 100 cm3) was less well-associated with local control (P=0.26). The change in standard deviation of the CT numbers (derived from the more careful contouring and using software windows) induced by radiation treatment correlated with the change in average CT number (R2=0.71). The change in standard deviation did not correlate with a change in tumor volume (R2=0.02). Radiation treatments reduced the average CT number (P < 0.001). In summary, radiation reduces the CT number and this reduction may be associated with local control at 10 months. A two-component model is consistent with lung tumor number distribution and its response to radiation.     

Detection of the Ki-ras mutation as a specific and sensitive marker of pancreatic carcinoma

The authors describe the procedure used for assessment of a Ki-ras mutation as a specific and sensitive marker of carcinoma of the pancreas. The reliability of the method was tested in five patients with carcinoma of the pancreas.     

Telomerase activity as a novel marker of lung cancer

Telomerase activity is found in more than 80% of human malignant neoplasms, including lung cancer. Markers with high incidence in malignant samples and very low incidence in benign samples are useful in clinical diagnosis of cancer. Thus, telomerase activity in clinical materials may become a novel tumor marker of existing lung cancer cells. Moreover, since activation of telomerase is associated with cellular immortality and its activity level is quite different between lung cancer tissues, the activity level may become an indicator of some biological features in lung cancer.     

Insulin-like growth factor-I modulation of cerebellar cell populations is developmentally stage-dependent and mediated by specific intracellular pathways

Although development of transgenic animals overexpressing insulin-like growth factor-I has allowed the establishment of a role of this trophic factor in brain growth, detailed knowledge of the action of insulin-like growth factor-I on different brain areas is still lacking. We now provide evidence for a pleiotrophic role of this growth factor on cerebellar development. Insulin-like growth factor-I produced by cerebellar cultures is a survival factor for Purkinje cells and a mitogen/differentiation factor for cerebellar glioblasts. Trophic effects of insulin-like growth factor-I were observed only during specific developmental stages. In addition, insulin-like growth factor-I increased intracellular Ca2+ levels in Purkinje cells and c-Fos in dividing glioblasts. Survival-promoting effects of insulin-like growth factor-I on Purkinje cells required activation of protein kinase C, while glioblast division induced by insulin-like growth factor-I depended on phosphatidylinosytol 3-kinase activation. We conclude that insulin-like growth factor-I is a paracrine/autocrine pleiotrophic factor for both glia and neurons in the cerebellum. Its effects are mediated by distinct intracellular signals and appear to be specific to the developmental stage of the target cell. Since development of the different cell populations that compose a specific brain territory is not synchronized, the pleiotrophic action of growth factors such as insulin-like growth factor-I may be essential to ontogenetic processes underlying normal brain growth.     

Toward tense as a clinical marker of specific language impairment in English-speaking children

A critical clinical issue is the identification of a clinical marker, a linguistic form or principle that can be shown to be characteristic of children with Specific Language Impairment (SLI). In this paper we evaluate, as candidate clinical markers, a set of morphemes that mark Tense. In English, this includes -s third person singular, -ed regular past, BE, and DO. According to the Extended Optional Infinitive Account (EOI) of Rice, Wexler, and Cleave (1995), this set of morphemes is likely to appear optionally in the grammars of children with SLI at a rate lower than the optionality evident in younger controls. Three groups of preschool children participated: 37 children with SLI, and two control groups, one of 40 MLU-equivalent children and another of 45 age-equivalent children. Three kinds of evidence support the conclusion that a set of morphemes that marks Tense can be considered a clinical marker: (a) low levels of accuracy for the target morphemes for the SLI group relative to either of the two control groups; (b) affectedness for the set of morphemes defined by the linguistic function of Tense, but not for morphemes unrelated to Tense; and (c) a bimodal distribution for Tense-marking morphemes relative to age peers, in which the typical children are at essentially adult levels of the grammar, whereas children in the SLI group were at low (i.e., non-adultlike) levels of performance. The clinical symptoms are evident in omissions of surface forms. Errors of subject-verb agreement and syntactic misuses are rare, showing that, as predicted, children in an EOI stage who are likely to mark Tense optionally at the same time know a great deal about the grammatical properties of finiteness and agreement in the adult grammar. The findings are discussed in terms of alternative accounts of the grammatical limitations of children with SLI and implications for clinical identification.     

Molecular pathology in basal cell cancer with p53 as a genetic marker

Human basal cell cancer (BCC) has unique growth characteristics with virtual inability to metastasize. We investigated clonality and genetic progression using p53 mutations as marker. Sampling was done through microdissection of frozen immunohistochemically stained 16 microm slices of tumors. From 11 BCC tumors 78 samples were analysed. Direct DNA sequencing of exons 5-8 was performed, haplotypes were determined after cloning of p53 exons and loss of heterozygosity (LOH) ascertained by microsatellite analysis. All tumors had p53 mutations and in a majority both p53 alleles were affected, commonly through missense mutations. Microdissection of small parts (50-100 cells) of individual tumors showed BCC to be composed of a dominant cell clone and prone to genetic progression with appearance of subclones with a second and even third p53 mutation. Samples from normal immunohistochemically negative epidermis always showed wild type sequence, except for a case of previously unknown germline p53 mutation. Our analysis also included p53 immunoreactive patches i.e. morphologically normal epidermis with a compact pattern of p53 immunoreactivity. Mutations within those were never the same as in the adjacent BCC. This detailed study of only one gene thus uncovered a remarkable heterogeneity within a tumor category famous for its benign clinical behavior.     

Birthdate and cell marker analysis of scrambler: a novel mutation affecting cortical development with a reeler-like phenotype

The reeler mutation in mice produces an especially well characterized disorder, with systematically abnormal migration of cerebral cortical neurons. The reeler gene encodes a large protein, termed Reelin, that in the cortex is synthesized and secreted exclusively in the Cajal-Retzius neurons of the cortical marginal zone (D'Arcangelo et al., 1995). In reeler mutant mice, loss of Reelin protein is associated with a systematic loss of the normal, "inside-out" sequence of neurogenesis in the cortex: neurons are formed in the normal sequence but become localized in the cortex in a somewhat inverted, although relatively disorganized "outside-in" pattern. Here we show that the scrambler mutant mouse exhibits a loss of lamination in the cortex and hippocampus that is indistinguishable from that seen in the reeler mouse. We use BrdU birthdating studies to show that scrambler cortex shows a somewhat inverted "outside-in" sequence of birthdates for cortical neurons that is similar to that previously described in reeler cortex. Finally, we perform staining with the CR-50 monoclonal antibody (Ogawa et al., 1995), which recognizes the Reelin protein (D'Arcangelo et al., 1997). We show that Reelin immunoreactivity is present in the scrambler cortex in a normal pattern, suggesting that Reelin is synthesized and released normally. Our data suggest that scrambler is a mutation in the same gene pathway as the reeler gene (Relnrl) and is most likely downstream of Relnrl.