A new DNA marker (D4S90) is located terminally on the short arm of chromosome 4, close to the Huntington disease gene

Genetic linkage studies have mapped Huntington's disease (HD) to the distal portion of the short arm of chromosome 4 (4p16.3), 4 cM distal to D4S10 (G8). To date, no definite flanking marker has been identified. A new DNA marker, D4S90 (D5), which maps to the distal region of 4p16.3, is described. The marker was used in a genetic linkage study in the CEPH reference families with seven other markers at 4p16. The study, together with knowledge of the physical map of the region, places D4S90 as the most distal marker, 6 cM from D4S10. A provisional linkage study with HD gave a maximum lod score of 2.14 at a θ of 0.00 and no evidence of linkage disequilibrium. As D4S90 appears to be located terminally, it should play an important role in the accurate mapping and cloning of the HD gene.     

Engineered herpes simplex virus DNA polymerase point mutants: the most highly conserved region shared among alpha-like DNA polymerases is involved in substrate recognition.

Eucaryotic, viral, and bacteriophage DNA polymerases of the alpha-like family share blocks of sequence similarity, the most conserved of which has been designated region I. Region I includes a YGDTDS motif that is almost invariant within the alpha-like family and that is similar to a motif conserved among RNA-directed polymerases and also includes adjacent amino acids that are more moderately conserved. To study the function of these conserved amino acids in vivo, site-specific mutagenesis was used to generate herpes simplex virus region I mutants. A recombinant virus constructed to contain a mutation within the nearly invariant YGDTDS motif was severely impaired for growth on Vero cells which do not contain a viral polymerase gene. However, three recombinants constructed to contain mutations altering more moderately conserved residues grew on Vero cells and exhibited altered sensitivities to nucleoside and PPi analogs and to aphidicolin. Marker rescue and DNA sequencing of one such recombinant demonstrated that the region I alteration confers the altered drug sensitivity phenotype. These results indicate that this region has an essential role in polymerase function in vivo and is involved directly or indirectly in drug and substrate recognition.     

A recombination event that redefines the Huntington disease region

We report both a recombination event that places the Huntington disease gene proximal to the marker D4S98 and an extended linkage-disequilibrium study that uses this marker and confirms the existence of disequilibrium between it and the HD locus. We also report the cloning of other sequences in the region around D4S98, including a new polymorphic marker R10 and conserved sequences that identify a gene in the region of interest.     

Calcium mobilization and muscle contraction induced by acetylcholine in swine trachealis

The roles of Ca²⁺ mobilization in development of tension induced by acetylcholine (ACh, 0.1–100 µM) in swine tracheal smooth muscle strips were studied. Under control conditions, ACh induced a transient increase in free cytosolic calcium concentration ([Ca²⁺]_i) that declined to a steady-state level. The peak increase in [Ca²⁺]_i correlated with the magnitude of tension at each [ACh] after a single exposure to ACh, while the steady-state [Ca²⁺]_i did not. Removal of extracellular Ca²⁺ had little effect on peak [Ca²⁺]_i but greatly reduced steady-state increases in [Ca²⁺]_i and tension. Verapamil inhibited steady-state [Ca²⁺]_i only at [ACh]<1 µM. After depletion of internal Ca²⁺ stores by 10 min exposure to ACh in Ca²⁺-free solution and then washout of ACh for 5 min in Ca²⁺-free solution, simultaneous re-exposure to ACh in the presence of 2.5 mM Ca²⁺ increased [Ca²⁺]_i to the control steady-state level without overshoot. The tension attained was the same as control for each [ACh] used. Continuous exposure to successively increasing [ACh] (0.1–100 µM) also reduced the overshoot of [Ca²⁺]_i at 10 and 100 µM ACh, yet tension reached control levels at each [ACh] used. We conclude that the steady-state increase in [Ca²⁺]_i is necessary for tension maintenance and is dependent on Ca²⁺ influx through voltage-gated calcium channels at 0.1 µM ACh and through a verapamil-insensitive pathway at 10 and 100 µM. The initial transient increase in calcium arises from intracellular stores and is correlated with the magnitude of tension only in muscles that have completely recovered from previous exposure to agonists.     

Investigation of the role of the disulphide bond in the activity and structure of staphylococcal enterotoxin C1

The goal of this study was to Investigate the role of the disulphide bond of staphylococcal enterotoxin C1 (SEC1) in the structure and activity of the toxin. Mutants unable to form a disulphide bond were generated by substituting alanine or serine for cysteine at positions 93 and/or 110. Although we did not directly investigate the residues between the disulphide linkage, tryptic lability showed that significant native structure in the cystine loop is preserved in the absence of covalent bonding between residues 93 and 110. Since no correlation was observed between the behaviour of these mutants with regard to toxin stability, emesis and T cell proliferation, we conclude that SEC1 -induced emesis and T cell proliferation are dependent on separate regions of the molecule. The disulphide bond itself is not an absolute requirement for either activity. However, conformation within or adjacent to the loop is important for emesis. Although mutants with alanine substitutions were not emetic, those with serine substitutions retained this activity, suggesting that the disulphide linkage stabilizes a crucial conformation but can be replaced by residues which hydrogen bond.     

Aurintricarboxylic Acid Protects Hippocampal Neurons from NMDA-and Ischemia-Induced Toxicity In Vivo

Abstract: The polymeric dye aurintricarboxylic acid (ATA) has been shown to protect various cell types from apoptotic cell death, reportedly through inhibition of a calcium-dependent endonuclease activity. Recent studies have indicated that there may be some commonalities among apoptosis, programmed cell death, and certain other forms of neuronal death. To begin to explore the possibility of common biochemical mechanisms underlying ischemia-or excitotoxin-induced neuronal death and apoptosis in vivo, gerbils or rats subjected to transient global ischemia or NMDA microinjection, respectively, received a simultaneous intracerebral infusion of ATA or vehicle. As a biochemical marker of neuronal death, spectrin proteolysis, which is mediated by activation of calpain I, was measured in hippocampus after 24 h. ATA treatment resulted in a profound reduction of both NMDA-and ischemia-induced spectrin proteolysis, consistent with the possibility of some common mechanism in apoptosis and other forms of neuronal death in vivo.     

Effects of elevated cytosolic calcium on ACh-induced swine tracheal smooth muscle contraction

Increased intracellular calcium concentration ([Ca²⁺]_i) is required for smooth muscle contraction. In tracheal and other tonic smooth muscles, contraction and elevated [Ca²⁺]_i are maintained as long as an agonist is present. To evaluate the physiological role of steady-state increases in Ca²⁺ on tension maintenance, [Ca²⁺]_i was elevated using ionomycin, a Ca²⁺ ionophore or charybdotoxin, a large-conductance calcium-activated potassium channel (K_Ca) blocker prior to or during exposure of tracheal smooth muscle strips to Ach (10^–9 to 10^–4 M). Ionomycin (5 µM) in resting muscles induced increases in [Ca²⁺]_i to 500±230 nM and small increases in force of 2.6±2.3 N/cm². This tension is only 10% of the maximal tension induced by ACh. Charybdotoxin had no effect on [Ca²⁺]_i or tension in resting muscle. After pretreatment of muscle with ionomycin, the concentration-response relationship for ACh-induced changes in tension shifted to the left (EC₅₀=0.07±0.05 µM ionomycin; 0.17±0.07 µM, control, p<0.05). When applied to the muscles during steady-state responses to submaximal concentrations of ACh, both ionomycin and charybdotoxin induced further increases in tension. The same magnitude increase in tension occurs after ionomycin and charybdotoxin treatment, even though the increase in [Ca²⁺]_i induced by charybdotoxin is much smaller than that induced by ionomycin. We conclude that the resting muscle is much less sensitive to elevation of [Ca²⁺]_i when compared to muscles stimulated with ACh. Steady-state [Ca²⁺]_i limits tension development induced by submaximal concentrations of ACh. The activity of K_Ca moderates the response of the muscle to ACh at concentrations less than 1 µM.     

The genetics of sex ratio distortion by cytoplasmic infection under maternal and contagious transmission: An epidemiological study

Nuclear and cytoplasmic determinants jointly influence the sex ratio in several organisms. A mathematical model of a maternally inherited extra-chromosomal agent that affects the fitness of its carriers and distorts the sex ratio in their broods is analyzed. The agent is transmitted through the cytoplasm from mother to daughter, or it may pass contagiously among females of the same generation. It is shown that under natural selection the deviation between the population sex ratio and Fisher's optimum value evolves to a minimum.