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B Freeman

Publications and source records attributed to B Freeman.

13 recordsLinked to original sources
PubMed1992

Mutations activating the yeast eIF-2 alpha kinase GCN2: isolation of alleles altering the domain related to histidyl-tRNA synthetases.

The protein kinase GCN2 stimulates expression of the yeast transcriptional activator GCN4 at the translational level by phosphorylating the alpha subunit of translation initiation factor 2 (eIF-2 alpha) in amino acid-starved cells. Phosphorylation of eIF-2 alpha reduces its activity, allowing ribosomes to bypass short open reading frames present in the GCN4 mRNA leader and initiate translation at the GCN4 start codon. We describe here 17 dominant GCN2 mutations that lead to derepression of GCN4 expression in the absence of amino acid starvation. Seven of these GCN2c alleles map in the protein kinase moiety, and two in this group alter the presumed ATP-binding domain, suggesting that ATP binding is a regulated aspect of GCN2 function. Six GCN2c alleles map in a region related to histidyl-tRNA synthetases, and two in this group alter a sequence motif conserved among class II aminoacyl-tRNA synthetases that directly interacts with the acceptor stem of tRNA. These results support the idea that GCN2 kinase function is activated under starvation conditions by binding uncharged tRNA to the domain related to histidyl-tRNA synthetase. The remaining GCN2c alleles map at the extreme C terminus, a domain required for ribosome association of the protein. Representative mutations in each domain were shown to depend on the phosphorylation site in eIF-2 alpha for their effects on GCN4 expression and to increase the level of eIF-2 alpha phosphorylation in the absence of amino acid starvation. Synthetic GCN2c double mutations show greater derepression of GCN4 expression than the parental single mutations, and they have a slow-growth phenotype that we attribute to inhibition of general translation initiation. The phenotypes of the GCN2c alleles are dependent on GCN1 and GCN3, indicating that these two positive regulators of GCN4 expression mediate the inhibitory effects on translation initiation associated with activation of the yeast eIF-2 alpha kinase GCN2.

Alleles↗
PubMed1992

Inflammation and oxygen free radical formation during pulmonary ischemia-reperfusion injury.

In a companion study, we showed that 2 h of warm unilateral lung ischemia followed by reperfusion resulted in bilateral tissue injury, indicated by increases in extravascular density (EVD) and permeability, measured as the pulmonary transcapillary escape rate (PTCER) for radiolabeled transferrin. EVD and PTCER measurements were obtained with the quantitative imaging technique of positron emission tomography (PET). In the current study, we evaluated this increase in EVD histologically and correlated EVD and PTCER with measurements of oxidant-reactive sulfhydryls (RSH) in plasma as a marker of oxygen free radical (OFR) formation. Histologically edema, leukocyte infiltration, and hemorrhage were all present on the ischemic side, but only after reperfusion, whereas only neutrophil infiltration was observed on the nonischemic side. Histology scores correlated with EVD (r = 0.81) and PTCER (r = 0.75), but permeability was abnormal at times even in the absence of neutrophil infiltration. Plasma RSH concentration from the ischemic lung decreased significantly (P less than 0.05) during pulmonary ischemia (i.e., before reperfusion) and returned to baseline on reperfusion. The degree of RSH oxidation did not correlate with the severity of injury as measured by PET or histology. Thus pulmonary ischemia-reperfusion injury is characterized by inflammation, hemorrhage, edema, and OFR formation. Injury occurred after reperfusion, not after ischemia alone. In addition, injury to the contralateral nonischemic lung suggests a neutrophil-independent circulating mediator of injury.

Animals↗
PubMed1992

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Audiology↗
PubMed1991

Patient-triggered synchronized assisted ventilation of newborns. Report of a preliminary study and three years' experience.

The safety, efficacy, and outcome of long-term patient-triggered synchronized assisted ventilation in infants (SAVI) was studied in very low birthweight infants (450 to 1250 g) with uncomplicated respiratory failure. Synchronization was accomplished by modified thoracic impedance technology using standard neonatal cardiorespiratory monitors with the output of the ventilation synchronizer triggering the exhalation solenoid of a standard neonatal respirator. In this nonrandomized study, the technique was found to be safe and reliable without any episodes of technical failure or adverse effects. The results indicated that in comparison with infants receiving conventional ventilation, infants receiving SAVI required (1) shorter duration of ventilation, (2) decreased duration of oxygen therapy, and (3) decreased progression of intraventricular hemorrhage to greater than or equal to grade III. Decreased duration of ventilation was statistically significant in infants weighing less than 850 g (P less than .02). These beneficial trends were ratified during a 3-year period in a total of 110 cases.

Equipment Safety↗
PubMed1988

Fluorescent glucagon derivatives. I. Synthesis and characterisation of fluorescent glucagon derivatives.

The synthesis of monofluorescein, monorhodamine, and mono-4-nitrobenz-2-oxa-1,3-diazole (NBD) derivatives of glucagon is reported. The fluorescent groups were introduced by converting tryptophan-25 to 2-thioltryptophan using thiol-specific fluorescent reagents. All derivatives retained the ability to activate adenylate cyclase when compared to glucagon and thus were considered full agonists. IC50 values of 6.8.10(-9), 1.7.10(-8), 1.8.10(-8) and 5.4.10(-9) M were measured in rat liver membranes for NBD-, fluorescein-, rhodamine-Trp25-glucagon and native glucagon, respectively. From the IC50 values Kd values of 2.16.10(-9), 4.10(-9), 2.10(-9) and 1.72.10(-9) M were calculated for the binding of NBD-, fluorescein-, rhodamine-Trp25-glucagon and native glucagon, respectively. The highest quantum yield (0.18) of the monomer derivatives was obtained with fluorescein-Trp25-glucagon in phosphate-buffered saline (pH 7.4). Difluorescein-glucagon was also prepared by reacting the amino groups of histidine-1 and lysine-12 with fluorescein isothiocyanate and dimer derivatives were prepared using fluorescein-labelled 2-thiolTrp25-glucagon. Difluorescein-glucagon bound only weakly to glucagon receptors and displayed antagonist properties. The dimer derivative formed from two difluorescein-2-thiolTrp25-glucagon molecules had similar poor binding qualities, whereas the dimer formed from difluorescein-2-thiolTrp25-glucagon and 2-thiolTrp25-glucagon exhibited, at low concentrations, properties similar to monofluorescein-glucagon. Both dimer derivatives were only sparingly soluble in aqueous medium. Specific binding of fluorescein-Trp25-glucagon and difluorescein-glucagon to rat hepatocytes was followed using flow cytometry.

Adenylyl Cyclases↗
PubMed1978

Myelin sheath thickness and conduction latency groups in the cat optic nerve.

In many animals, the optic nerve has multiple conduction latency groups of fibres yet displays a unimodal frequency distribution of fibre sizes. An attempt has been made to resolve this discrepancy in the cat. Examination of teased preparations of fixed cat optic nerve fibres by polarizing interference light microscopy indicated that a fibre's size may change abruptly along its length. Furthermore, the diameters of single nerve fibres followed for distances of up to 30 mum in serial transverse sections of cat optic nerve showed variations of up to 100% of a fibre's average diameter over that distance. On the other hand, the myelin sheath thickness of these fibres appeared to be relatively constant along any one fibre. A population of cat optic nerve fibres which had a unimodal axon diameter frequency distribution was found to have a myelin sheath thickness frequency distribution with five modes. These modes have been interpreted as indicating the existence of five groups of fibres in the cat optic nerve. Four of these groups may be related to four negative peaks seen in the antidromic compound action potential recorded at the margin of the cat optic disc following stimulation of the optic chiasm. The fifth myelin thickness group appears to represent the slowly conducting fibres which do not make an obvious contribution to the antidromic compound action potential. It is concluded that for the cat optic nerve, the conduction latency groups seen in the compound action potential may be more directly correlated with the frequency distribution of fixed nerve myelin thickness than with the frequency distribution of fixed nerve fibre diameter.

Action Potentials↗
PubMed1978

The optic nerve of the brush-tailed possum, Trichosurus vulpecula: fibre diameter spectrum and conduction latency groups.

The principal findings of this report on the morphology and electrophysiology of the possum optic nerve are: (i) There are about 230,000 fibres in the optic nerve. This fibre count, based on electron microscopy, is slightly less than a previously reported estimate of the total number of ganglion cells in the possum retina. (ii) The majority (greater than 98%) of the fibres of the optic nerve are myelinated axons of retinal ganglion cells. The diameters of these fibres range from 0.4--4.6 micrometer (axon diameter range: 0.3--3.8 micrometer) and the frequency distribution of the fibre diameters (and axon diameters) is positively skewed and unimodal. (iii) The antidromic compound action potential of the possum optic nerve shows four negative peaks following stimulation of the optic chiasm. These peaks are associated with four conduction latency groups of fibres which have been designated t1, t2, t3 and t4 in order of increasing conduction latency. (iv) The mean peak conduction velocities of the fibres in the conduction latency groups are 13.1 ms-1 (t1), 8.1 ms-1 (t2), 5.7 ms-1 (t3) and 3.1 ms-1 (t4). (v) There is no direct correlation between the frequency distribution of fibre (or axon) diameters as measured by electron microscopy of transverse sections of fixed optic nerve and the conduction latency groups. (vi) The reconstruction of the possum optic nerve compund action potential on the basis of either axon or fibre diameter frequency distribution does not provide an acceptable, indirect correlation between the morphology and the electrophysiology of this optic nerve.

Action Potentials↗
PubMed1978

The number and distribution of ganglion cells in the retina of the brush-tailed possum, Trichosurus vulpecula.

The distribution of ganglion cells in the retina of the adult brush-tailed possum was determined by light microscopy of Nissl stained retinal whole mounts. Qualitatively, the distribution in this marsupial retina shows features, such as an area centralis and a visual streak, which are found separately or together in eutherian mammals. The possum retina is avascular and the eye has a weak tapetum in the superior fundus.The retinal area is 260 mm2 and there are about 280,000 ganglion cells. The diameters of the ganglion cell somas range from 5 micrometer to 26 micrometer and the frequency distribution of soma size classes is skewed and unimodal (mean" 12.8 micrometer) with 62% of the cells falling in the class of diameters 7-13 micrometer. Maps of ganglion cell density were made for five retinae. These maps show that there is a band of high ganglion cell density (greater 2,000 cells mm-2) which extends across the retina about 0.6 mm above the optic disc in the tapetal region of the fundus and which lies in the plane of the animal's horizon when the eyes are in their primary position. By analogy with other species, this band is termed the visual streak. Near the temporal end of the visual streak, 2.9 mm from the optic disc, the ganglion cell density reaches a localized maximum of approximately 5,000 cells mm-2 thereby defining the centre of an area centralis (greater than 3,000 cells mm-2). The posterior nodal distance of the possum eye was estimated at 7.8 mm, which corresponds to a retinal magnification of 136 micrometer per degree of visual field. There are up to 30,000 glial cells which lie in, or slightly vitread to, the layer of the retinal ganglion cells.

Animals↗