Subunit location of the iron-sulfur clusters in fumarate reductase from Escherichia coli

The subunit location of the [2Fe-2S], [3Fe-4S], and [4Fe-4S] clusters in Escherichia coli fumarate reductase has been investigated by EPR studies of whole cells or whole cells extracts of a fumarate reductase deletion mutant with plasmid amplified expression of discrete fumarate reductase subunits or groups of subunits. The results indicate that both the [2Fe-2S] and [3Fe-4S] clusters are located entirely in the iron-sulfur protein subunit. Information concerning the specific cysteine residues that ligate these clusters has been obtained by investigating the EPR characteristics of cells of the deletion mutant amplified with a plasmid coding for the flavoprotein subunit and a truncated iron-sulfur protein subunit. While the results are not definitive with respect to the location of the [4Fe-4S] cluster, they are most readily interpreted in terms of this cluster being entirely in the flavoprotein subunit or bridging between the two catalytic domain subunits. These new results are discussed in light of the amino acid sequences of the two subunits and the sequences of structurally well characterized iron-sulfur proteins containing [2Fe-2S], [3Fe-4S], and [4Fe-4S] centers.     

Secretion of soluble functional insulin receptors by transfected NIH3T3 cells

In order to determine whether the human insulin receptor ectodomain can be expressed as a functional protein, the coding regions for the transmembrane and cytoplasmic domain of a full-length human insulin receptor cDNA were deleted by site-directed mutagenesis, and the resultant construct was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH3T3 cells, a cell line secreting an insulin binding protein was isolated. The insulin binding alpha subunit had an Mr of 138,000 and a beta subunit of Mr 48,000 (compared to 147,000 and 105,000 for the full-length human insulin receptor expressed in NIH3T3 cells). This difference in size of the alpha subunit was due to a difference in glycosylation as N-glycanase digestion reduced the apparent size of the alpha subunits of secreted and normal membrane-bound receptors to identical values. The secreted receptor formed disulfide-linked heterotetrameric structures with an Mr of 280,000. It was synthesized as an Mr 160,000 precursor which was cleaved into mature subunits with a t1/2 of 3 h. Increasing expression of the cDNA by induction with sodium butyrate lead to the appearance of an Mr 180,000 protein in the medium as well as the mature alpha and beta subunits. A Scatchard plot of insulin binding to the secreted receptor was curvilinear with a Kd of 7 X 10(-10) M for the high affinity sites and 10(-7) M for the low affinity site (compared to Kd values of 1.1 X 10(-9) M and 10(-7) M, respectively, for human insulin receptors expressed in these cells.     

Transition state analogs for protocatechuate 3,4-dioxygenase. Spectroscopic and kinetic studies of the binding reactions of ketonized substrate analogs

The binding reactions of two heterocyclic analogs of protocatechuate (PCA), 2-hydroxyisonicotinic acid N-oxide and 6-hydroxynicotinic acid N-oxide, to Brevibacterium fuscum protocatechuate 3,4-dioxygenase have been characterized. These analogs were synthesized as models for the ketonized tautomer of PCA which we have previously proposed as the form which reacts with O2 in the enzyme complex (Que, L., Jr., Lipscomb, J.D., Munck, E., and Wood, J.M. (1977) Biochim. Biophys. Acta 485, 60-74). Both analogs have much higher affinity for the enzyme than PCA. Repetitive scan optical spectra of each binding reaction show that at least one intermediate is formed. The spectra of the intermediates are red-shifted (lambda max = 500 nm) relative to that of native enzyme (lambda max = 435 nm) but are similar to that of the anaerobic enzyme-PCA complex. In contrast, the spectrum of the final, deadend complex formed by each analog is significantly blue-shifted (lambda max less than 340 nm) resulting in an apparent bleaching of the chromophore of the enzyme. A transient intermediate exhibiting a similar bleached spectrum has been detected in the enzyme reaction cycle immediately after O2 is added to the enzyme-PCA complex (Bull C., Ballou D.P., and Otsuka, S. (1981) J. Biol. Chem. 256, 12681-12686). Stopped flow measurements of the analog binding reactions show that a relatively weak enzyme complex is initially formed followed by at least two isomerizations leading to the bleached, high affinity complexes. EPR spectra of both the early and final complexes reveal only high spin Fe3+ with negative zero field splitting, showing that the optical bleaching is not due to Fe reduction. The studies show that the ketonized analogs are poor models for the enzyme-substrate complex but do successfully mimic many features of the first oxy complex of the reaction cycle. We propose that substrate ketonization occurs coincident with or after O2 binding and may be involved directly in the O2 insertion reaction.     

The subcellular distribution of [N-Me-3H]-acetylcholine synthesized by brain in vivo

1. Three forms of acetylcholine occur in subcellular fractions of brain tissue: free acetylcholine, present in the high-speed supernatant from eserinized sucrose homogenates; stable bound acetylcholine, present in synaptic vesicles; and labile bound acetylcholine, present in the cytoplasm of synaptosomes (detached presynaptic nerve terminals). 2. The relationship between these forms has been investigated by isolating the subcellular fractions from the cortical tissue of cats and guinea pigs excised 1hr. after infiltration of [N-Me-(3)H]choline into the cortex in vivo. 3. Since choline is a ubiquitous metabolite, means were devised for isolating the radioactive acetylcholine on columns of the weak acid ion-exchange resin IRF-97; control experiments with samples of extracts treated with acetylcholinesterase showed that the radioactivity attributed to acetylcholine migrated to the choline peak after cholinesterase treatment. 4. The specific radioactivities of the various forms of acetylcholine were different: labile bound (synaptosomal cytoplasmic) acetylcholine had the highest, stable bound (vesicular) acetylcholine the next highest, and the high-speed-supernatant form the lowest. 5. It is concluded that the various forms of acetylcholine could not have arisen during fractionation from a single pre-existing pool of acetylcholine.     

Variation in the skull of the Long-tailed field-mouse, Apodemus sylvaticus in mainland Britain

Nine skull measurements and a measure of age have been made on each of 613 specimens of Apodemus sylvaticus (L.). The mice were collected from 12 mainland and three island localities. From five of the localities they were obtained in more than one year; each year's collection was kept separate in the subsequent analysis.
Unadjusted and adjusted (for age) means have been calculated for each character. Differences in both space and time are small for the mainland populations. A discriminant function analysis was undertaken with a view to accounting for the larger part of the variation using a limited number of linear combinations of the adjusted measurements. It was found that the greater part of the variation was contained in the first two canonical variates. Finally, the generalized distances were obtained.
The multivariate analyses suggest that one island population (St Mary's) is quite distinct, the other two (Tresco, Mull) less so, that there is in any one locality little variation from year to year, that the Scottish mainland populations as a group display a small statistical divergence from the English mainland populations and that the former differ rather more from each other than do the latter.     

Age differences in responsiveness of brainstem chemosensitive neurons to extracellular pH changes

The sensitivity of neurons in the caudal chemosensitive area on the ventrolateral surface of the medulla oblongata (VMS) to extracellular pH changes was examined in newborn and young developing kittens and compared to that of adult cats. The pH was varied by superfusion of the VMS with mock cerebrospinal fluid (CSF) of pH 7.4 (control), 7.0 (acid) and 7.8 (alkaline). A total of 97 neuronal units in the three age groups changed their firing rates inversely in response to extracellular fluid (ECF) pH changes. The greatest sensitivity was found in the adult group where acid superfusion caused an increase in neuronal activity. The least sensitivity was observed in the newborn group (1-6 days old), whereas the young kitten group (4-6 weeks old) exhibited an intermediate sensitivity. Neurons of kittens older than 7 weeks of age demonstrated a response pattern characteristic of the adult group. Neurons of neonates older than seven days, exhibited a response pattern characteristic of the young kitten group.     

Confirmation of the Cholinergic Specificity of the Chol-1 Gangliosides in Mammalian Brain Using Affinity-Purified Antisera and Lesions Affecting the Cholinergic Input to the Hippocampus

An antiserum raised to Torpedo electromotor synaptosomal membranes (anti-TSM antiserum) induces a cholinergic-specific immune lysis of mammalian brain synaptosomes and recognizes a group of minor gangliosides appeared, therefore, to be specific to the cholinergic neuron and were designated Chol-1. To confirm the cholinergic specificity of the Chol-1 gangliosidic antigens, we have shown that not only does a mammalian ganglioside fraction that is enriched with respect to the Chol-1 gangliosides inhibit the cholinergic-specific immune lysis induced by the anti-TSM antiserum, but also it can be used to affinity-purify a subpopulation of immunoglobulins from the anti-TSM antiserum that also induce a cholinergic-specific lysis. Furthermore, we have demonstrated that fimbrial lesions, which cause a massive degeneration of cholinergic terminals in the ipsilateral hippocampus, lead to a loss of the Chol-1 gangliosides concomitant with that shown by choline acetyltransferase activity and that lesions to the entorhinal cortex, which cause a loss of mainly glutamergic synapses in the ipsilateral dentate gyrus leading to cholinergic sprouting from adjacent hippocampal areas and an increase in cholinergic markers in the dentate gyrus, produce concomitant increases in choline acetyltransferase activity and Chol-1 content. These results provide strong evidence in favour of the cholinergic specificity of the Chol-1 gangliosides.     

Somatization: Social control and illness production in a religious cult

The aim of this ethnographic work is twofold: first, to document the specifics of the illness experience within the highly controlling milieu of a religious cult; second, to explore the connection between somatization and social control in this particular context. Anthropologists have long realized that in order to comprehend disease etiology, one must examine both biological and sociocultural processes, as both are implicated in the production of illness. Illness experiences of those members of the cult described here appeared to be direct responses to extreme intrapsychic and social control: that is, members were required to be celibate, unmarried, and detached from their pre-cult identity and the emotional support structure of family and friends. Additionally, members were subjected to constant surveillance by peers and were often punished for expressing views that were in conflict with the ideology of the leader, thereby encouraging the somatization of distress. This research is based upon two years of participant observation within the milieu, during which time more than 100 participants were interviewed; however, this paper specifically discusses in-depth interviews with eight individuals whose health problems exemplify those experienced by other members of the cult.Here of late everybody is getting on my nerves so bad. It's no wonder my leg hurts all the time. I guess it knows my heart sure can't hold all the hurt in it. So I guess it's trying to help out... from the novelClover, by Dori Sanders     

Germline and somatic mosaicism in a female carrier of Duchenne muscular dystrophy

The family of a male with Duchenne muscular dystrophy (DMD) and a deletion within the dystrophin gene has been studied. Polymerase chain reaction analysis of ectopic mRNA from peripheral blood T+B lymphocytes and the use of (CA) _n repeat polymorphisms in and around the deleted region showed the proband's mother to be both a germline mosaic and a somatic mosaic for the deletion seen in her son. The mutation therefore occurred as a mitotic event early in embryogenesis.     

The 141-year period for Dr. Beal's seed viability experiment: A hybrid surprise

Premise

In 1879, Dr. William Beal buried 20 glass bottles filled with seeds and sand at a single site at Michigan State University. The goal of the experiment was to understand seed longevity in the soil, a topic of general importance in ecology, restoration, conservation, and agriculture, by periodically assaying germinability of these seeds over 100 years. The interval between germination assays has been extended and the experiment will now end after 221 years, in 2100.

Methods

We dug up the 16th bottle in April 2021 and attempted to germinate the 141-year-old seeds it contained. We grew germinants to maturity and identified these to species by vegetative and reproductive phenotypes. For the first time in the history of this experiment, genomic DNA was sequenced to confirm species identities.

Results

Twenty seeds germinated over the 244-day assay. Eight germinated in the first 11 days. All 20 belonged to the Verbascum genus: Nineteen were V. blattaria according to phenotype and ITS2 genotype; and one had a hybrid V. blattaria × V. thapsus phenotype and ITS2 genotype. In total, 20/50 (40%) of the original Verbascum seeds in the bottle germinated in year 141.

Conclusions

While most species in the Beal experiment lost all seed viability in the first 60 years, a high percentage of Verbascum seeds can still germinate after 141 years in the soil. Long-term experiments such as this one are rare and invaluable for studying seed viability in natural soil conditions.