A green to red photoconvertible protein as an analyzing tool for early vertebrate development.

Lineage labeling is one of the most important techniques in developmental biology. Most recently, a set of photoactivatable fluorescent proteins originating from marine cnidarians became available. Here, we introduce the application of the green to red photoconvertible protein EosFP as a novel technique to analyze early vertebrate development. Both injection of EosFP mRNA and purified, recombinant EosFP followed by a light-driven green to red conversion allow lineage labeling in virtually any temporal and spatial dimension during embryonic development for at least 2 weeks. Specific staining of cells from nonsurface layers is greatly facilitated by light-driven conversion of EosFP compared with traditional methods. Therefore, green to red photoactivatable proteins promise to be a powerful tool with the potential to satisfy the increasing demand for methods enabling detailed phenotypical analyses after manipulations of morphogenetic events, gene expression, or signal transduction.     

Cytogenetic and comparative genomic hybridization findings in four cases of breast cancer after neoadjuvant chemotherapy

To assess a potential common pattern of genetic alterations in chemotherapy-resistant tumors we analyzed four tumors from breast cancer patients (patients 1-4) after neoadjuvant chemotherapy, by comparative genome hybridization (CGH) and conventional chromosome banding analysis. All patients showed structural aberrations involving chromosomes 1, 5, 11, 16, and 17. In CGH analysis, the patients showed typical imbalances for ductal breast cancer: gains of 1q (3 patients), 5q (2 patients), 8q (3 patients), and X (4 patients) and losses of 1p33 approximately p36 (3 patients), 16q (3 patients), 17p (3 patients), 19 (4 patients), and 22q (4 patients). Other recurrent imbalances of atypical pattern for ductal breast cancer were gain of 4q21 approximately q32 (2 patients), 20q21 approximately q22 (2 patients), and 21 (2 patients) and loss of 20p (3 patients). Three patients showed involvement of several regions bearing genes of drug resistance (MDR1 [HUGO symbol: ABCB1], BCRP [HUGO symbol: ABCG2], MRP1 [HUGO symbol: ABCC1], RFC1); the fourth patient displayed an amplification in the region of MYC (alias c-myc), thus providing--at the level of the light microscope--an explanatory background for the ability of their tumors to survive anthracycline-, taxane- and cyclophosphamide-based chemotherapy. Conventional cytogenetic analysis and CGH displayed highly coincidental findings in the tumors of four patients after neoadjuvant chemotherapy for breast cancer.     

Habituation and adaptation of the vestibuloocular reflex: a model of differential control by the vestibulocerebellum

Summary We habituated the dominant time constant of the horizontal vestibuloocular reflex (VOR) of rhesus and cynomolgus monkeys by repeated testing with steps of velocity about a vertical axis and adapted the gain of the VOR by altering visual input with magnifying and reducing lenses. After baseline values were established, the nodulus and ventral uvula of the vestibulocerebellum were ablated in two monkeys, and the effects of nodulouvulectomy and flocculectomy on VOR gain adaptation and habituation were compared. The VOR time constant decreased with repeated testing, rapidly at first and more slowly thereafter. The gain of the VOR was unaffected. Massed trials were more effective than distributed trials in producing habituation. Regardless of the schedule of testing, the VOR time constant never fell below the time constant of the semicircular canals (5 s). This finding indicates that only the slow component of the vestibular response, the component produced by velocity storage, was habituated. In agreement with this, the time constant of optokinetic after-nystagmus (OKAN) was habituated concurrently with the VOR. Average values for VOR habituation were obtained on a per session basis for six animals. The VOR gain was adapted by natural head movements in partially habituated monkeys while they wore ×2.2 magnifying or ×0.5 reducing lenses. Adaptation occurred rapidly and reached about ±30%, similar to values obtained using forced rotation. VOR gain adaptation did not cause additional habituation of the time constant. When the VOR gain was reduced in animals with a long VOR time constant, there were overshoots in eye velocity that peaked at about 6–8 s after the onset or end of constant-velocity rotation. These overshoots occurred at times when the velocity storage integrator would have been maximally activated by semicircular canal input. Since the activity generated in the canals is not altered by visual adaptation, this finding indicates that the gain element that controls rapid changes in eye velocity in the VOR is separate from that which couples afferent input to velocity storage. Nodulouvulectomy caused a prompt and permanent loss of habituation, returning VOR time constants to initial values. VOR gain adaptation, which is lost after flocculectomy, was unaffected by nodulouvulectomy. Flocculectomy did not alter habituation of the VOR or of OKAN. Using a simplified model of the VOR, the decrease in the duration of vestibular nystagmus due to habituation was related to a decrement in the dominant time constant of the velocity storage integrator (1/h ₀). Nodulouvulectomy, which reversed habituation, would be effected by decreasing h ₀, thereby increasing the VOR time constant. Small values of h ₀ would cause velocity storage to approach an ideal integrative process, leading the system to become unstable. By controlling the VOR time constant through habituation, the nodulus and uvula can stabilize the slow component of the VOR. VOR gain adaptation was related to a modification of the direct vestibular path gain g ₁, without altering the coupling to velocity storage g ₀ or its time constant (1/h ₀). The mismatched direct- and indirect-pathway gains simulated the overshoots in the dynamic response to a step in velocity, that were observed experimentally. We conclude that independent distributed elements in the VOR modify its dynamic response, under control of separate parts of the vestibulocerebellum.     

Event-Related Potential Correlates of Two Stages of Information Processing in Physical and Semantic Discrimination Tasks

Event-related potentials were studied while subjects performed physical and semantic discrimination tasks. Two negative components, N_A and N2, were observed in both kinds of discriminations. The earlier component, N_A, had a constant onset latency, but its peak latency varied as a function of stimulus complexity. N2 latency varied in relation to changes in the peak of N_A. RT and P3 followed N2 by similar amounts of time across tasks. The N_A and N2 components were interpreted as reflecting partially overlapping sequential stages of processing associated with pattern recognition and stimulus classification, respectively.     

Proton magnetic resonance spectroscopy in ecstasy (MDMA) users

The popular recreational drug 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) has well-recognized neurotoxic effects upon central serotonergic systems in animal studies. In humans, the use of MDMA has been linked to cognitive problems, particularly to deficits in long-term memory and learning. Recent studies with proton magnetic resonance spectroscopy (1H MRS) have reported relatively low levels of the neuronal marker N-acetylaspartate (NAA) in MDMA users, however, these results have been ambiguous. Moreover, the only available 1H MRS study of the hippocampus reported normal findings in a small sample of five MDMA users. In the present study, we compared 13 polyvalent ecstasy users with 13 matched controls. We found no differences between the NAA/creatine/phosphocreatine (Cr) ratios of users and controls in neocortical regions, and only a tendency towards lower NAA/Cr ratios in the left hippocampus of MDMA users. Thus, compared with cognitive deficits, 1H MRS appears to be a less sensitive marker of potential neurotoxic damage in ecstasy users.     

Cytotoxicity of Hemolytic, Cytotoxic Necrotizing Factor 1-Positive and -Negative Escherichia coli to Human T24 Bladder Cells

Approximately one-half of Escherichia coli isolates from patients with cystitis or pyelonephritis produce the pore-forming cytotoxin hemolysin, a molecule with the capacity to lyse erythrocytes and a range of nucleated cell types. A second toxin, cytotoxic necrotizing factor 1 (CNF1), is found in approximately 70% of hemolytic, but rarely in nonhemolytic, isolates. To evaluate the potential interplay of these two toxins, we used epidemiological and molecular biologic techniques to compare the cytotoxicity of hemolytic, CNF1⁺, and CNF1⁻ cystitis strains toward human T24 bladder epithelial cells in vitro. A total of 29 isolates from two collections of cystitis-associated E. coli were evaluated by using methylene blue staining of bladder monolayers at 1-h intervals after inoculation with each strain. Most (20 of 29) isolates damaged or destroyed the T24 monolayer (less than 50% remaining) within 4 h after inoculation. As a group, CNF1⁺ isolates from one collection (11 strains) were less cytotoxic at 4 h than the CNF1⁻ strains in that collection (P = 0.009), but this pattern was not observed among isolates from the second collection (18 strains). To directly evaluate the role of CNF1 in cytotoxicity of hemolytic E. coli without the variables present in multiple clinical isolates, we constructed mutants defective in production of CNF1. Compared to the CNF1⁺ parental isolates, no change in cytotoxicity was detected in these cnf1 mutants. Our results indicate that CNF1 does not have a detectable effect on the ability of hemolytic E. coli to damage human bladder cell monolayers in vitro.     

A comparison of micropuncture and lithium clearance methods in the assessment of renal tubular function in rats with diabetes insipidus

The lithium clearance technique has been proposed as a non-invasive method whereby fluid delivery from the pars recta and pars convoluta of proximal tubules can be measured as C_Li and C_IN [0.78 C_Li/C_IN+0.22], respectively [12], C_Li being the clearance of lithium and C_IN that of inulin. In the present study, fluid delivery from proximal tubules was estimated simultaneously by micropuncture and lithium clearance techniques in anaesthetized Brattleboro rats with diabetes insipidus, under control conditions and following chronic treatment with hydrochlorothiazide. Absolute deliveries from the proximal convoluted tubules as determined by the micropuncture and lithium clearance methods were 437 and 427 μl/min, respectively, in untreated animals and 348 and 355 μl/min, respectively, in thiazide-treated animals. The individual results obtained by the two methods showed a high degree of correlation (r=0.85,P<0.001). In untreated Brattleboro rats, proximal fluid delivery as estimated by both the micropuncture and lithium clearance techniques showed significant (P<0.001) correlations with urine flow rate. These results provide further evidence for the acceptance of lithium clearance as a valid estimate of proximal tubular fluid delivery.     

Antibody to Mycoplasma pneumoniae in Nasal Secretions and Sputa of Experimentally Infected Human Volunteers

After experimental infection with Mycoplasma pneumoniae, 42% of 67 volunteers developed a threefold or greater rise in antibody in nasal secretions as measured by radioimmunoprecipitation. Development of an antibody increase in sputum was detected more often, i.e., in 73% of the volunteers. Each of the antibody increases involved immunoglobulin (Ig) A. Twelve rises in IgG antibody were detected in the specimens which exhibited a rise in IgA antibody. In almost every instance the rise in IgA antibody exceeded that seen with IgG antibody. Analysis of the response to experimental challenge with M. pneumoniae of volunteers with different levels of preexisting respiratory tract IgA antibody suggested that this secretory antibody was related to host resistance to M. pneumoniae disease. Further, respiratory tract IgA antibody appeared to be more directly related to host resistance than was antibody in serum.     

THE INTERACTION OF RESPONSES IN THE BRAIN TO SEMANTIC STIMULI

Long time-constant EEG recording during paired stimuli has led to the discovery of the contingent negative variation or expectancy wave (Walter, 1964). This effect is produced when a conditional stimulus signals that an imperative stimulus demanding action, decision, or attention will follow at a short, constant time interval. Symbolic and meaningful stimuli were presented to subjects tachistoscopically, and the evoked responses in the brain were electronically averaged. The cerebral evoked responses to such psychological stimuli are more complex than to flashes. A slow negative DC potential shift (CNV) was seen during the interval between an auditory ready signal and the visual exposure if recognition of the stimulus was required, or if it was interesting. Following the visual exposure, a slow positive DC shift occurred. The method has been developed to study the brain responses to psychological stimuli. The amplitude of the responses relates to the information content and subjective factors rather than to the physical strength of the stimulus.     

Placental pathologic changes in malaria. A histologic and ultrastructural study.

Placenta malarial changes (PMCs) related to maternal plasmodium infection were present in 33% (247 cases) of a series of 741 placentas collected from an unselected population living in an area of high malarial endemicity (Haut-Ogooué, Gabon, Africa). Plasmodia were found on material thick blood films taken at the time of delivery in 42% of the women with and 24% of women without associated PMCs. Plasmodium falciparum was the most frequent infecting organism. PMCs were more frequent and, in general, more marked in primiparas. The primiparas were significantly (P less than 0.001) more numerous in the group with PMCs than in the control group without such changes. The mean weight of term placentas with malarial changes was significantly (46 g; P less than 0.001) less than that of placentas without such changes. The morphologic changes were a combination of the following features: 1) presence of parasites in the intervillous spaces; 2) macrophage concentration in the intervillous spaces; 3) malarial pigment deposits; 4) excess of perivillous fibrinoid deposits; 5) syncytiotrophoblastic damage; and 6) trophoblastic basal lamina thickening. Plasmodia were found in placental intervillous spaces in 42% (105/247). Local parasitemia varied in magnitude; in a few cases, 30% or more of the maternal erythrocytes were infected. Macrophage concentration in the intervillous spaces was present in 29% (72/247) and was always associated with local parasitemia. Macrophages phagocytized red blood cells and malarial pigment, and their number varied inversely with that of the local parasites. It seems, therefore, that macrophages play an important role in local parasite clearance. Malarial brown pigment was observed in all cases from the series. It had characteristic ultrastructural features and occurred in perivillous deposits of fibrinoid, in macrophages, or free in intervillous spaces. Excessive perivillous fibrinoid deposits were a constant histologic finding and were usually associated with syncytiotrophoblastic necrosis or ultrastructural damage such as partial microvilli loss, filamentous material accumulation in intracytoplasmic vacuoles, and "podocytelike" cytoplasmic projections on the basal surface. At these sites the trophoblastic basal lamina was usually thickened. Previously reported morphologic data and our own findings suggest that the peculiar placental changes in malaria, restricted to intervillous spaces and to villous surfaces, may be related to an immunopathologic process.