Development of the gigantocerebellum of the weakly electric fish Pollimyrus

The morphogenesis of the "hypertrophied" mormyrid cerebellum was investigated in Pollimyrus (Pisces). Two adults and 36 larvae and young fish raised in captivity were used. Two Gnathonemus petersii adults were taken for comparison. The ontogenetic development of the various cerebellar structures was analysed in inverse chronological order with the aid of serial sagittal and frontal brain sections. Special attention was given to the trilobed corpus cerebelli (C1, C2, C3), the lobi transitorii et caudales, the valvula, the crista cerebelli, the eminentia granularis and the lobus lineae lateralis. 1. The cerebellar structures are of bilateral origin; they develop from the cerebellar and acoustico-lateral "anlage" of the rhombencephalon behind the rhombomesencephalic fissure, either through budding or individualisation and appear between the 4th and 11th day after spawning. The midline fusion of the symmetrical structures is accomplished somewhat later, between the 8th and 23rd days. 2. The cerebellar structures acquire their definitive spatial organisation within 38 days, except for the valvula whose development takes much longer. Recognisable from the 11th day, the valvula upon which ridges are visible from the beginning continues to grow after the 38th day beyond the mesencephalic ventricle, finally overlying the telencephalon frontally and the different rhombencephalic structures caudally. This development, which includes a large antero-lateral folding of the valvula, takes 240 days. 3. Cytological differentiation is just as complex as the general development of the cerebellar structures. Cortical stratification first begins on the 8th to the 11th day in the corpus cerebelli and in the valvula from day 21 to 23 onwards. This differentiation is characterised throughout almost the entire cerebellum by a downward migration of the superficial undifferentiated cells which then constitute the granular layer. In the valvula, the majority of the undifferentiated cells leave the ridges to form a continuous granular layer at the base of the ridges. 4. A differentiation gradient was observed on the antero-posterior axis. 5. In spite of its complexity, the mormyrid cerebellum develops much more rapidly than the cerebellum of the trout.     

Murine erythroleukemia cell variants: isolation of cells that have amplified the dihydrofolate reductase gene and retained the ability to be induced to differentiate

A series of murine erythroleukemia cell (MELC) variants was generated by selection for the ability to grow in increasing concentrations of the folate antagonist methotrexate (MTX). Growth of the parental MELC strain DS-19 was completely inhibited by 0.1 microM MTX. We isolated cells able to grow in 5, 40, 200, 400, and 800 microM MTX. Growth rates and yields were essentially the same in the presence or absence of the selective dose of MTX for all variants. MTX resistance was not the result of a transport defect. Dihydrofolate reductase (DHFR) from our variants and DS-19 was inhibited to the same extent by MTX. Variants had increased dihydrofolate reductase activities. The specific activity of DHFR was proportional to the selective concentration of MTX employed to isolate a given variant. DNA dot blotting established that the cloned variant (MR400-3) had a 160-fold increase in DHFR gene copy number relative to the parental strain (DS-19). Hybridization studies performed in situ established the presence of amplified DHFR genes on the chromosomes of the MTX-resistant but not the MTX-sensitive (parental) cells. Quantitation of DHFR mRNA by cytoplasmic dot blotting established that the amplified DHFR gene expression was proportional to gene copy number. Thus, MTX resistance was due to amplification of the DHFR gene. The variants retained the ability to be induced to differentiate in response to dimethyl sulfoxide and hexamethylenebis(acetamide) as evaluated by the criteria of globin mRNA accumulation, hemoglobin accumulation, cell volume decreases, and terminal cell division.(ABSTRACT TRUNCATED AT 250 WORDS)     

A time-resolved fluorescence study of azurin and metalloazurin derivatives

Nickel and cobalt derivatives of Pseudomonas fluorescens (ATCC 13525) azurin were prepared and their steady-state fluorescence and time-resolved fluorescence monitored. Like the copper-containing native protein, the fluorescence decay of both metallo derivatives was best fit to a sum of three exponentials, whereas the apoazurin from which they were prepared obeyed single-exponential decay kinetics. However, comparison of the lifetimes and fractional of each of the components in these derivatives to those in the oxidized and reduced native proteins revealed significant differences. These results suggest that the presence of a metal center in azurin imparts a conformational heterogeneity which is strongly dependent on the nature of the metal center. Further, the results are used to comment on current ideas concerning the geometry of the active site in this redox protein.     

Mobilization of intracellular calcium and stimulation of calcium fluxes by endothelin-2 in cultured mouse swiss 3T3 fibroblasts

Specific receptor-induced signal transduction mechanisms for the endothelin-2 isoform (ET-2), a potent vasoconstrictor of vascular smooth muscle, were examined in Swiss 3T3 cells. Half-maximal binding (EC₅₀) and maximal, saturable binding (B_max) were estimated from Scatchard analyses and were found to be 24.2 ± 3.3 pM and 56500 ± 1700 sites/cells, respectively. A saturating concentration of ET-2 (100 nM) increased intracellular free calcium (measured by Fura-2 fluorescence) from a resting level of 100 nM to a peak level of 600–800 nM. The initial increase in intracellular free calcium was transitory and was followed by a smaller maintained elevation (250 nM). In the absence of extracellular calcium, ET-2 induced a transitory response equal in size to the peak in the presence of extracellular calcium, but the maintained response was absent. ET-2 increased intracellular free calcium in a concentration-dependent manner with an EC₅₀ of 1 nM. In calcium free solution (2 mM EGTA), ET-2 increased the efflux of ⁴⁵Ca from cells loaded to isotopic equilibrium (3 h) with ⁴⁵Ca. The intracellular second messenger, IP₃, also increased the calcium efflux from saponin permeabilized 3T3 cells loaded with ⁴⁵Ca (pCa 6) in the presence of MgATP. In the presence of extracellular calcium, ET-2 significantly increased calcium uptake into 3T3 cells by 92 ± 36.6 pmoles/million cells/2 min (n = 8). It is suggested that ET-2 binds to specific, high affinity receptors in 3T3 cells and that this receptor interaction increases the intracellular free calcium by IP₃-induced mobilization of calcium from cellular stores and by increasing influx of extracellular calcium.     

Can Creutzfeldt-Jakob disease unravel the mysteries of Alzheimer?

Recent studies on iatrogenic Creutzfeldt-Jakob disease (CJD) raised concerns that one of the hallmark lesions of Alzheimer disease (AD), amyloid-β (Aβ), may be transmitted from human-to-human. The neuropathology of AD-related lesions is complex. Therefore, many aspects need to be considered in deciding on this issue. Observations of recent studies can be summarized as follows: 1) The frequency of iatrogenic CJD cases with parencyhmal and vascular Aβ deposits is statistically higher than expected; 2) The morphology and distribution of Aβ deposition may show distinct features; 3) The pituitary and the dura mater themselves may serve as potential sources of Aβ seeds; 4) Cadaveric dura mater from 2 examined cases shows Aβ deposition; and 5) There is a lack of evidence that the clinical phenotype of AD appears following the application of cadaveric pituitary hormone or dura mater transplantation. These studies support the notion that neurodegenerative diseases have common features regarding propagation of disease-associated proteins as seeds. However, until further evidence emerges, prions of transmissible spongiform encephalopathies are the only neurodegenerative disease-related proteins proven to propagate clinicopathological phenotypes.     

Folding of VemP into translation-arresting secondary structure is driven by the ribosome exit tunnel

The ribosome is a fundamental biomolecular complex that synthesizes proteins in cells. Nascent proteins emerge from the ribosome through a tunnel, where they may interact with the tunnel walls or small molecules such as antibiotics. These interactions can cause translational arrest with notable physiological consequences. Here, we studied the arrest caused by the regulatory peptide VemP, which is known to form α-helices inside the ribosome tunnel near the peptidyl transferase center under specific conditions. We used all-atom molecular dynamics simulations of the entire ribosome and circular dichroism spectroscopy to study the driving forces of helix formation and how VemP causes the translational arrest. To that aim, we compared VemP dynamics in the ribosome tunnel with its dynamics in solution. We show that the VemP peptide has a low helical propensity in water and that the propensity is higher in mixtures of water and trifluorethanol. We propose that helix formation within the ribosome is driven by the interactions of VemP with the tunnel and that a part of VemP acts as an anchor. This anchor might slow down VemP progression through the tunnel enabling α-helix formation, which causes the elongation arrest.