Evidence for recruitment and synchronization in leukemia and solid tumors.

In malignant cell populations which are sensitive to drug therapy it does not seem necessary to potentiate treatment with perturbation of the proliferative characteristics. However, in some relatively drug-resistant populations significant advantages can be obtained by recruiting resting cells into the drug-sensitive cell cycle. Further studies of growth-regulatory mechanisms for malignant cell populations may help a great deal in designing better methods of recruiting the resting cells back into active cell division. Synchronization of the dividing cells in a specificially drug-sensitive phase of the cell cycle is another method of potentiating chemotherapeutic effectiveness in relatively drug-resistant cell populations. The biochemical changes induced by sequential drug administration may be equally important in sensitizing the malignant cell to subsequent drug administration.     

Evidence from molecular systematics for decreased avian diversification in the pleistocene Epoch.

Pleistocene glaciations have been suggested as major events influencing speciation rates in vertebrates. Avian paleontological studies suggest that most extant species evolved in the Pleistocene Epoch and that species' durations decreased through the Pleistocene because of heightened speciation rates. Molecular systematic studies provide another data base for testing these predictions. In particular, rates of diversification can be determined from molecular phylogenetic trees. For example, an increasing rate of speciation (but constant extinction) requires shorter intervals between successive speciation events on a phylogenetic tree. Examination of the cumulative distribution of reconstructed speciation events in mtDNA phylogenies of 11 avian genera, however, reveals longer intervals between successive speciation events as the present time is approached, suggesting a decrease in net diversification rate through the Pleistocene Epoch. Thus, molecular systematic studies do not indicate a pulse of Pleistocene diversification in passerine birds but suggest, instead, that diversification rates were lower in the Pleistocene than for the preceding period. Documented habitat shifts likely led to the decreased rate of diversification, although from molecular evidence we cannot discern whether speciation rates decreased or extinction rates increased.     

Membrane recruitment of autophagy proteins in selective autophagy

Autophagy is a stress response that is upregulated in response to signals such as starvation, growth factor deprivation, endoplasmic reticulum stress, and pathogen infection. Defects in this pathway are the underlying cause of a number of diseases, including metabolic aberrations, infectious diseases, and cancer, which are closely related to hepatic disorders. To date, more than 30 human ATG (autophagy) genes have been reported to regulate autophagosome formation. In this review, we summarize the current understanding of how ATG proteins behave during autophagosome formation in both non-selective and selective autophagy.     

Evidence for specific DNA sequences in the nuclear acceptor sites of the avian oviduct progesterone receptor.

Recent studies have shown that saturable high-capacity nuclear binding sites (termed acceptor sites) for the avian oviduct progesterone receptor can be reconstituted by rehybridizing a specific oviduct chromatin protein fraction (CP-3) to pure hen DNA to generate a reconstituted nucleoacidic protein (NAP). Only a limited number of acceptor sites can be generated on hen DNA even at high protein/DNA ratios. This suggests the existence of a limited number of specific sequences in the avian genome that can participate in the acceptor sites. The studies presented in this paper show a specificity as to the source of DNA that can generate acceptor sites using hen oviduct CP-3 protein. The acceptor protein binds to all DNAs but generates acceptor sites only on DNAs from certain animals. The acceptor sites for the progesterone receptor, generated with heterologous mammalian DNAs and the avian oviduct CP-3 fraction, show saturation not only in number of acceptor sites generated on the DNAs but also in progesterone receptor binding. Binding to these sites is also receptor dependent. Using oviduct receptors from particular physiological states of the birds wherein the receptors do not bind to nuclear sites in vivo, it was found that the cell-free binding to these heterologous complexes of hen CP-3 protein and DNA from another species, termed heterologous NAP, is similarly absent. Thus, the cell-free binding to the native oviduct NAP and the heterologous NAP markedly resembles the nuclear binding in vivo. Interestingly, synthetic DNAs rich in adenine and thymine, but not those rich in guanine and cytosine, are capable of generating acceptor sites. Species-specific DNA sequences, as well as specific chromatin proteins, therefore, appear to be involved in the nuclear acceptor sites for the avian oviduct progesterone receptor. The DNA sequences appear to be conserved throughout most of the vertebrates but not among nonvertebrates as are the steroid hormones and their receptors. The exact numbers and distributions of these sequences in the avian genome are not known.     

Evidence for the presence of a neutral insulinotrophic peptide in the porcine duodenum

A crude mixture of thermostable peptides extracted from porcine duodenum was fractionated by electrofocusing. A neutral fraction, different from the basic fractions of GIP, VIP, PHI, and CCK was found to promote insulin secretion when injected in vivo to normal rats. This neutral fraction, extracted from the crude mixture by chromatography, stimulated insulin output from an isolated rat pancreas and enhanced glucose-induced insulin release. The insulinotrophic effect of this partially purified duodeno-jejunal material disappeared following digestion with trypsin. The insulin-releasing activity was found to correspond to a compound of molecular weight higher than that of insulin (i.e. higher than 6000). No GIP-like immunoreactivity was found in this neutral fraction indicating that the active peptide(s) are not GIP related compounds. These observations suggest that porcine duodenum contains and incretin activity different from that of the insulinotrophic factors already reported.     

Evidence for tandem integration of avian myeloblastosis virus DNA with endogenous provirus in leukemic chicken cells.

The integration site of avian myeloblastosis virus (AMV) proviral DNA in DNA from leukemia chicken myeloblasts has been studied by three sequential nucleic acid hybridizations that can localize the proviral DNA according to the repetitiveness of the adjacent cellular DNA regions. First, large denatured cellular DNA fragments (2.1 x 10(6) daltons) were reassociated and fractionated according to sequence reiteration frequenct. Next, DNA remaining single-stranded in each fraction was immobilized on nitrocellulose filters hybridized with an excess of unlabeled 70S RNA from Rous-associated virus-0 to saturate the endogenous proviral DNA sequences.     

Evidence for avian influenza A infections among Iowa's agricultural workers

Background Identifying risk factors for zoonotic influenza transmission may aid public health officials in pandemic influenza planning. Objectives We sought to evaluate rural Iowan agriculture workers exposed to poultry for previous evidence of avian influenza virus infection. Methods In 2004, we enrolled 803 rural adult Iowans in a 2‐year prospective study of zoonotic influenza transmission. Their enrollment data and sera were compared with those of 66 adult controls enrolled at the University of Iowa in 2006 by using proportional odds modeling. Results Of the 803 participants 58·8% were male with a mean age of 55·6 years. Forty‐eight percent reported previous poultry exposure. Sera were studied by microneutralization techniques for antibodies against avian H4, H5, H6, H7 and H9 viruses. Touching live birds was associated (OR 1·2; 95% CI 1·02–1·8) with increased antibody titer against H5 virus. Similarly, participants who reported hunting wild birds had increased antibody titers against H7 virus (OR 2·8; 95%CI 1·2–6·5) and subjects who reported recent exposure to poultry had increased antibody titers against H6 (OR 3·4; 95% CI 1·4–8·5) and H7 viruses (OR 2·5, 95% CI 1·1–5·7). There was no evidence of elevated antibody against avian H4 or H9 viruses. Conclusions These data suggest that hunting and exposure to poultry may be important risk factors for avian influenza virus infection among rural US populations. Agriculture workers should be included in influenza pandemic plans.     

Evidence of homologous relationship between thermolysin and neutral protease A of Bacillus subtilis.

A comparison of the partial amino-acid sequence of neutral protease A from Bacillus subtilis with the structure of thermolysin (EC 3.4.24.4) from Bacillus thermoproteolyticus reveals that these two proteins are homologous. Of 171 residues placed in neutral protease (54% of the sequence), 83 residues (49%) occur in identical positions in thermolysin, and include nine of the 13 residues previously identified as components of the active site of thermolysin. This similarity provides support for the hypothesis that the two enzymes have similar three-dimensional structures and a common mechanism of action. Since these enzymes differ markedly in their resistance to heat inactivation, a comparison of their structures may eventually provide a chemical basis for explaining the differences in their thermal stability.     

Human eosinophil chemotaxis and selective in vivo recruitment by sphingosine 1-phosphate

Sphingosine 1-phosphate (S1P) is a sphingolipid mediator that is involved in diverse biological functions. Local administration of S1P causes inflammation coupled to a large eosinophil (EO) recruitment in the rat-paw tissue. The inflammatory response is accompanied by an increase in S1P receptors, namely S1P(1), S1P(2), S1P(3), and by an enhanced expression of CCR3, which is the main chemokine receptor known to be involved in EO function. Human EOs constitutively express S1P(1) and, at a lower extent, S1P(2), S1P(3) receptors. S1P in vitro causes cultured human EO migration and an increase in S1P receptor mRNA copies and strongly up-regulates CCR3 and RANTES (regulated on activation, normal T cell-expressed and secreted) message levels; in particular CCR3 is up-regulated 18,000-fold by S1P. A blocking anti-CCR3 Ab inhibits S1P-induced chemotaxis, implying that S1P acts as specific recruiting signal for EOs not only through its own receptors but also through CCR3. These results show that S1P is involved in EO chemotaxis and contribute to shed light on the complex mechanisms underlying EO recruitment in several diseases such as asthma and some malignancies.     

Evidence for a causal inverse model in an avian cortico-basal ganglia circuit

Learning by imitation is fundamental to both communication and social behavior and requires the conversion of complex, nonlinear sensory codes for perception into similarly complex motor codes for generating action. To understand the neural substrates underlying this conversion, we study sensorimotor transformations in songbird cortical output neurons of a basal-ganglia pathway involved in song learning. Despite the complexity of sensory and motor codes, we find a simple, temporally specific, causal correspondence between them. Sensory neural responses to song playback mirror motor-related activity recorded during singing, with a temporal offset of roughly 40 ms, in agreement with short feedback loop delays estimated using electrical and auditory stimulation. Such matching of mirroring offsets and loop delays is consistent with a recent Hebbian theory of motor learning and suggests that cortico-basal ganglia pathways could support motor control via causal inverse models that can invert the rich correspondence between motor exploration and sensory feedback.The brain has evolved diverse strategies for combining sensory and motor signals, a prerequisite for many complex behaviors including hunting, communication, and observational learning of motor skills. For example, to accurately sense the external world while simultaneously moving within it, the nervous system must be able to detect changes in its sensory inputs that are not a predictable consequence of self-motion. Indeed, many sensory neurons respond with high sensitivity to unpredictable stimuli during motor behavior despite self-caused sensory feedback (1–5). Such remarkable sensitivity can be achieved by circuit mechanisms that counteract sensory feedback associated with self-generated motor output (6). Such mechanisms are also known as corollary discharges (7) or forward models of the motor system (8), which are synaptic mappings from motor neurons onto sensory neurons that can either predict or suppress sensory feedback from self-generated motor output.In contrast to our understanding of how the brain cancels predictable, motor-induced sensory feedback, much less is known about neural mechanisms for computing motor codes that produce desired sensory targets. However, the ability to learn to produce desired behaviors by observing others is considered to be a key advantage of sociality and a main driver for the evolution of culture (9, 10). Learning by imitation occurs spontaneously when humans learn to speak, parrots imitate surrounding sounds (11), or songbirds learn to imitate a tutor’s song (12). However, insights into the neural implementation of sensory-guided motor learning remain sparse, largely because we lack empirical information about the principles underlying the flow of neural activity through synaptic mappings from sensory to motor areas. Such mappings are known as inverse models and flow in the opposite direction of forward models that direct motor activity to sensory areas.To learn about principles of sensorimotor integration in the zebra finch, a vocal learner, we focus our attention on cortical premotor nuclei necessary for song production and song learning. The premotor area HVC is involved in generating the stereotyped song motifs of adult birds (13), whereas the lateral magnocellular nucleus of the anterior nidopallium (LMAN) forms the output of a basal-ganglia pathway involved in generating subtle song variability (14–17). HVC neurons produce highly stereotyped firing patterns during singing (18, 19), whereas LMAN neurons produce highly variable patterns (15, 20, 21).To gain insights into LMAN’s role in motor control, we consider recent theoretical work that establishes a conceptual link between inverse models and vocal-auditory mirror neurons (22–24). We consider three (nonexhaustive) possibilities about the flow of sensory information into motor areas. First, auditory afferents with some feature sensitivity could map onto motor neurons involved in generating those same features (Fig. 1A). This mapping forms a causal inverse, in which a sensory target input generates a motor activity pattern required to cause, or generate that same sensory target. Second, auditory afferents with some feature sensitivity could map onto motor neurons that typically fire after the ones involved in generating those features (Fig. 1B). This mapping forms a predictive inverse, in which a sensory input at some point in a stereotyped acoustic sequence elicits a predictive motor activity pattern required to generate the next acoustic signal in that sequence. Third, the auditory-to-motor connections could be randomly wired (Fig. 1C), in which case there would be no regularity in the relationship between sensory and motor responses.Open in a separate windowFig. 1.Three hypothetical sensorimotor mappings and associated mirroring offsets. Sensory-to-motor mappings could implement a causal inverse of the motor plan (A), a predictive inverse (B), or be random (C). Under a causal inverse, generated by variable sequences of song features (ABC-CBA), a spike burst in a motor neuron (neuron 2) triggers the production (black arrow) of a song feature (feature B) after latency , and the neuron receives sensory feedback (thick green arrow) from that same feature after an additional latency . In such a neuron, we expect to see a cross-covariance (CC) peak (red arrow) between singing-related and playback-evoked spike bursts (black vertical bars) at a time lag (the so-called mirroring offset, red horizontal bar) given by the delay of the sensorimotor loop . Under a predictive inverse (B), generated by stereotyped sequences of song features (ABC-ABC), the motor neuron 2 again triggers song feature B, but at the same time receives reliable feedback from the previous song feature A (thick green arrow). Thus, we expect to see a CC peak at a time lag much smaller than the sensorimotor loop delay . Finally, under a random sensory-to-motor mapping (C), we expected no CC between the motor- and sensory-evoked firing.Rather than directly characterize the auditory-to-motor mapping (a daunting task), we probe this mapping indirectly by studying the neural responses it causes in experiments in which we compare auditory responses elicited by playback of the bird’s own song (BOS) to motor responses recorded during production of these songs. Indeed, the three possibilities in Fig. 1 make specific, testable predictions at the level of single neurons. Consider for example a motor neuron downstream of a causal inverse model (i.e., neuron 2 in Fig. 1A). This motor neuron generates song feature B after a motor latency . Now, when the bird is not singing, this motor neuron also has a sensory selectivity for song feature B (because it is downstream of a causal inverse model). Therefore, during song playback, this same motor neuron will fire after playback of song feature B with an auditory latency . Thus, if one temporally aligns both the playback-evoked spike train and the singing-related spike train with the onset of song, the playback train will lag, or mirror the singing related train by a mirroring offset equal to the sensorimotor loop delay . In more subtle scenarios in which the motor neuron has selectivity for multiple acoustic features, or is subject to greater degrees of noise, this temporal alignment between the motor and sensory responses of the neuron can still be detected through the position of a peak in the cross-covariance function (CC function) between the playback and singing related spike trains both time-aligned to song (Fig. 1A, Lower), even when the offset may not be visually apparent by simply looking at spike trains (see SI Methods and Fig. S1 for a theory of the CC function in the case of multiple latency auditory responses).Conversely, consider a motor neuron downstream of a predictive inverse model (i.e., neuron 2 in Fig. 1B). Just as before, the neuron generates song feature B with a latency . However, because it is downstream of a predictive inverse model, when the bird is not singing, this neuron now has a sensory selectivity for the previous song feature, A. This selectivity occurs because a predictive inverse model takes a sensory stimulus, in this case song feature A, and generates a motor command for the next feature, in this case song feature B. Thus, when aligned to song, the playback spike occurs after syllable A while the singing related spike occurs before song feature B. The result is that the mirroring offset will be much smaller than the total sensorimotor loop delay , and the peak in the CC function will be much closer to zero time lag. Finally, in the random scenario in Fig. 1C, we expect no pronounced peak in the CC function.In theory, whether to expect a causal or predictive inverse depends on the sequence stereotypy of produced song features (23, 24). What are those features? A song feature could be a song syllable, in which case the sequence of features is stereotyped because adult zebra finches sing stereotyped syllable sequences ABC-ABC (Fig. 1B). Stereotyped song sequences are generated mainly by stereotyped firing patterns in HVC, and according to previous theory (23, 24), we expect a predictive inverse and its associated signature of small mirroring offsets, to arise in HVC.Alternatively, a song feature could be a brief pitch increase or decrease, in which case the features and their variable sequences (ABC, DBA; Fig. 1A) are mainly generated by variable LMAN firing patterns. Accordingly, we expect to find a causal inverse upstream of LMAN (23, 24). Indeed, in HVC neurons the mirroring offset between motor-related spiking and song-playback evoked spiking is less than 10 ms (25), much less than the roughly 40-ms loop delay of HVC estimated using electrical stimulation of HVC and using auditory stimulation of the ear (26–31). By contrast, mirroring offsets in LMAN have not been quantified yet, leaving it open as to whether they provide evidence for causal inverses.     

Pathological and clinical relevance of selective recruitment of Langerhans cells in the respiratory bronchioles of smokers

BackgroundSmoking causes an influx of inflammatory cells including Langerhans cells (LCs) into the airways and lung parenchyma, thus inducing histological changes, such as emphysema and fibrosis. We examined the distribution and quantity of Langerhans cells in relation to clinical and pathological findings and explored the association between smoking and accumulation of Langerhans cells in the respiratory bronchioles.MethodsFifty-three patients who underwent lung resection for primary diseases, including lung cancer, were recruited. Histological and immunohistochemistry analyses were utilized to identify CD1a-positive Langerhans cells in peripheral lung specimens separated from primary lesions. Clinical characteristics, pathological changes, and distribution of CD1a-positive Langerhans cells distribution were assessed.ResultsOf the 53 patients, 35 were smokers and 18 were non-smokers. The number of Langerhans cells in the respiratory bronchioles was significantly increased in smokers as compared to that in non-smokers (p < 0.001). The number of Langerhans cells in smokers was significantly higher in patients with mild emphysema than in those without emphysema (p < 0.01). The high-LC group showed more frequent smoking-related histological changes, such as respiratory bronchiolitis, parenchymal fibrosis, accumulation of macrophages, and smoking-related interstitial fibrosis, than the low-LC group. However, there were no differences in the smoking indices and pulmonary functions of the groups.ConclusionsSelective accumulation of Langerhans cells in the respiratory bronchioles of smokers may lead to the development of smoking-related pathological changes.     

Glomerular selective permeability to macromolecular neutral dextrans in experimental diabetes

Summary Rats with streptozotocin-induced chronic diabetes mellitus develop a glomerulopathy functionally manifested by proteinuria. The ability of the glomerular capillary wall to retard filtration of macromolecules was examined in 5 chronically diabetic Munich-Wistar rats exhibiting excessive proteinuria (39±7mg/24h, mean±SEM) and 5 age-matched normal Munich-Wistar rats without increased proteinuria (4.7±0.2 mg/24 h). Urinary albumin excretion was not increased in the diabetic rats (2.0±0.6 mg/24h vs 1.6±0.3 mg/24h) suggesting that the normal net electronegative charge of the glomerular capillary wall was not altered. Fractional clearances of macromolecular neutral dextrans were similar in diabetic and normal rats throughout a wide range of molecular size (18–42 Å). Glomerular filtration rate was the same in the two groups of rats (2.77±0.16ml/min in diabetics and 2.72±0.11ml/ min in normals) suggesting that renal haemodynamic factors did not influence fractional clearances of neutral dextrans in diabetic rats. We conclude that the proteinuria exhibited by these chronically diabetic rats is not attributable to alterations of size-selective properties of the glomerular capillary wall, such as increases in the size or the number of pores.     

Evidence for early recruitment of granulocyte precursors during high- dose methotrexate infusions in mice

The effects of constant exposure to high concentrations of methotrexate in vivo on the committed stem cell (CFU-C) were studied by in vitro culture of mouse bone marrow. Bone marrow samples were obstained from animals receiving a continuous infusion, and were cultured in a methotrexate-free semisolid gel system. The effects of methotrexate infusion on the pluripotent stem cell population (CFU-S) were studied as well. Constant exposure to 10(-5) M methotrexate produced a rapid decrease in total nucleated cells per femur, reaching 35% of control at 12 hr and remaining at approximately this level throughout 48 hr of drug infusion. A decrease in the number of both CFU-C and CFU-S per femur was observed, which paralleled the drop in nucleated cells during the first 24 hr. However, in contrast to an additional drop in the number of CFU-S, an increase of CFU-C number per femur was observed from 24 to 48 hr. These data indicated a self-limited cell kill of nucleated bone marrow cells, and suggested recruitment of CFU-C from the CFU-S pool between 24 and 48 hr of infusion despite continued methotrexate infusion.     

Model of effectively neutral mutations in which selective constraint is incorporated

Based on the idea that selective neutrality is the limit when the selective disadvantage becomes indefinitely small, a model of neutral (and nearly neutral) mutations is proposed that assumes that the selection coefficient (s′) against the mutant at various sites within a cistron (gene) follows a Γ distribution; f(s′) = α^βe^-αs′s′^β-1/Γ(β), in which α = β/¯s′ and ¯s′ is the mean selection coefficient against the mutants (¯s′ > 0; 1 [unk] β > 0). The mutation rate for alleles whose selection coefficients s′ lie in the range between 0 and 1/(2N_e), in which N_e is the effective population size, is termed the effectively neutral mutation rate (denoted by v_e). Using the model of “infinite sites” in population genetics, formulas are derived giving the average heterozygosity (¯h_e) and evolutionary rate per generation (k_g) in terms of mutant substitutions. It is shown that, with parameter values such as β = 0.5 and ¯s′ = 0.001, the average heterozygosity increases much more slowly as N_e increases, compared with the case in which a constant fraction of mutations are neutral. Furthermore, the rate of evolution per year (k₁) becomes constant among various organisms, if the generation span (g) in years is inversely proportional to √N_e among them and if the mutation rate per generation is constant. Also, it is shown that we have roughly k_g = v_e. The situation becomes quite different if slightly advantageous mutations occur at a constant rate independent of environmental conditions. In this case, the evolutionary rate can become enormously higher in a species with a very large population size than in a species with a small population size, contrary to the observed pattern of evolution at the molecular level.     

Evidence for a regulatory protein involved in the increased activity of system A for neutral amino acid transport in osmotically stressed mammalian cells.

System A for neutral amino acid transport is increased by hypertonic shock in NBL-1 cells previously induced to express system A activity by amino acid starvation. The hypertonicity-mediated effect can be blocked by cycloheximide but is insensitive to tunicamycin. The activity induced may be inactivated irreversibly by the addition of system A substrates, by a rapid mechanism insensitive to cycloheximide. In CHO-K1 cells, hypertonicity increases system A activity, as has been shown in NBL-1 cells. This effect is additive to the activity produced by derepression of system A by amino acid starvation and is insensitive to tunicamycin. Furthermore, the alanine-resistant mutant CHO-K1 alar4, which bears a mutation affecting the regulatory gene R1, involved in the derepression of system A activity after amino acid starvation, is still able to respond to the hypertonic shock by increasing system A activity to a level similar to that described in hypertonicity-induced derepressed CHO-K1 (wild type) cells. These results suggest (i) that the hypertonicity-mediated increase of system A activity occurs through a mechanism other than that involved in system A derepression and (ii) that a regulatory protein coded by an osmotically sensitive gene is responsible for further activation of preexisting A carriers.     

Transfer across the human blood-brain barrier: Evidence for capillary recruitment and for a paradox glucose permeability increase in hypocapnia

The present study was undertaken to investigate whether there is intermittency of capillary flow in the human brain, i.e., whether more capillaries open up at high blood flow thus increasing the surface for diffusional exchange. Unidirectional transfer across the blood-brain barrier of labeled D-glucose, l-phenylalanine, l-leucine, thiourea, and propranolol was measured using the indicator diffusion method with labeled Na⁺, Cl^?, and chelated In as impermeable reference substances. Forty-three patients needing carotid angiography were studied in different situations, rest, hyperventilation, hypercapnia, hypo/hypertension (within the limits of autoregulation). Some older data on the seizure situation are included. Cerebral blood flow (CBF) was measured concomitantly. In situations with high blood flow, extraction (E) decreased and the permeability surface area product (PS) increased for both the lipophilic substances and for those transferred by carrier mechanism. With low CBF the reverse happened except for an unexpected PS increase for glucose in hyperventilation. Variations of PS in parallel with CBF are evidence of capillary recruitment which constitutes a more efficient way of increasing tissue supply. PS and E remained constant with a constant CBF even when arterial blood pressure was changed, indicating that autoregulatory mechanisms do not affect the diffusional exchange surface and probably take place at the arteriolar level. PS for glucose increased in hyperventilation perhaps as an expression of a pH dependence of its carrier.