An extra disulfide bridge in the constant domain of Rana catesbeiana immunoglobulin light chains

The immunoglobulins of the bullfrog Rana catesbeiana are unusual in that, in all classes, the light chains are not disulfide bonded to heavy chains or to other light chains. Moreover, the light chains contain six, rather than the usual five, residues of half-cystine. As none of these half-cystines is in the sulfhydryl form or is alkylated after mild reduction, we suggested that the light chains probably contain three intrachain disulfide bridges. We have now carried out experiments to confirm the existence of an extra intrachain disulfide bridge in Rana catesbeiana light chains and to determine its location. Disulfide bridge assignments were based on 1) isolation and sequence analysis of S-(carboxymethyl)cysteine-containing peptides and 2) isolation, from unreduced light chains, of peptides containing a disulfide bridge. Half-cystine residues were found at positions 134 and 194, and these were shown to be joined in the conserved intradomain disulfide bridge. In addition, we found that a residue of half-cystine, located at the third position from the carboxy-terminus, forms a disulfide bridge with a half-cystine at position 119, near the amino-terminus of the domain, the latter residue replacing a proline that has been found at this position in all other light chains. An intrachain disulfide bridge has not been found at this location in any other light chain.     

Kinetic Effects of Terbium Ions on Muscarinic Acetylcholine Receptors of Murine Neuroblastoma Cells

Preincubation of murine neuroblastoma cells (clone N1E-115) with terbium chloride resulted in a significant potentiation of carbachol-mediated increase in cyclic GMP formation. This effect was accompanied by a shift of the peak response from 30 s to 120 s and a 6-fold decrease in carbachol concentration producing half-maximal responses, in addition to a significant increase in the Hill coefficient. Terbium ions also caused a significant decrease in the affinity and an increase in the maximum binding of [3H]quinuclidinyl benzilate to muscarinic receptors, the change in affinity being mainly due to a decrease in the association rate. Preincubation of cells with 1 mM carbachol for 4 h (the desensitized state of the muscarinic receptor) resulted in a decrease in the ability of terbium to alter [3H]quinuclidinyl benzilate binding. The effects of terbium reported here might be due to its affecting muscarinic receptor-effector coupling, which is considered to be lost upon receptor desensitization.     

Molecular organization of paramyosin in the core of molluscan thick filaments

An arrangement of paramyosin molecules in the polar part of molluscan thick filaments is proposed which accounts for the X-ray diffraction pattern of the smooth adductor muscle (other than the part ascribed to actin) and for the appearance of separated filaments in the electron microscope. The proposed structure is based on the PI arrangement of Cohen et al. (1971), and contains sets of parallel, equidistant molecules with successive molecules displaced along the molecular axis by 72 nm, which we call PI sheets. Every molecule belongs to two PI sheets which are nearly perpendicular. This array is not propagated throughout the filament, but is sheared periodically in the direction of the molecular (filament) axis by 2/5 X 72 nm. The shear occurs along parallel equidistant planes which are inclined to the PI sheets. The analysis of the X-ray data has been made possible by concentrating on those patterns from filaments in which the two sets of PI sheets appear to be mutually perpendicular, a condition brought about by bathing the muscle in aqueous acetone. In one set, there are four intermolecular spaces between shear planes (this appears to be true at least for the smooth adductors of Ostrea edulis, Crassostrea angulata and Mercenaria mercenaria). In the other set, the number varies with species and probably lies between eight and ten in the first two and appears to be six in the last named species. The known paracrystalline nature of paramyosin filaments suggests that this number, though dominant in one species, is not exactly constant.     

Cholesteryl hemisuccinate alters membrane fluidity, angiotensin receptors, and responses in adrenal glomerulosa cells

We examined the effects of cholesteryl hemisuccinate on membrane fluidity and angiotensin II (AII) actions in bovine adrenal glomerulosa cells. Incubating cells with cholesteryl hemisuccinate decreased membrane fluidity and markedly inhibited AII binding. The effect on binding was characterized by a decrease in AII receptor number. The effects of AII on phosphatidyl inositol turnover and calcium fluxes, proposed intermediaries of AII actions on aldosterone secretion, were less impaired than AII binding by cholesteryl hemisccinate. AII stimulation of aldosterone secretion was preserved despite the decrease in AII binding after cholesteryl hemisuccinate treatment. These results indicate that AII binding can be dissociated from its effects on aldosteronogenesis by a reagent that alters membrane fluidity.     

Effect of melatonin infusion duration and frequency on gonad, lipid, and body mass in pinealectomized male Siberian hamsters

The goal of this study was to discriminate between two hypotheses regarding how the circadian rhythm of pineal melatonin (MEL) production transmits photoperiodic information: (1) A circadian rhythm of sensitivity to MEL regulates the hormone's effect; (2) the duration of the MEL signal, rather than its circadian timing, is the critical parameter of the MEL rhythm. The experiment examined the response of pinealectomized (PINX) male Siberian hamsters to 10-hr (short-day-type) versus 6-hr (long-day-type) duration MEL infusions (10 ng/infusion) in cycles with period lengths (T) of 18, 24, 36, and 48 hr. After cannula implantation, animals were moved from LD 16:8 to LD 10:14 (lights-on from 0500 to 1500 hr, EST), where the timed infusions began. Additional T 24 cycles included as controls employed 18-hr MEL, 18-hr saline (SAL), and 10-hr SAL infusions: Body weight and food intake were measured weekly. After 6 weeks, animals were killed; blood samples were taken for radioimmunoassay (RIA) of serum follicle-stimulating hormone (FSH) and prolactin (PRL); and terminal body, epididymal white adipose tissue (EPIWAT), and paired testis weights were recorded. Six-hour MEL infusions failed to induce short-day-type effects, regardless of the period (T) of the infusion cycle. In contrast, compared to SAL and 6-hr MEL infusions, 10-hr MEL resulted in decreases in body, EPIWAT, and testis weights in T 24, but not in T 36 or T 48. In T 18, testis, body, and EPIWAT mass were decreased, but not to the same extent as in T 24. Similarly, daily 18-hr MEL infusions (T24) were less effective as a short-day stimulus than were 10-hr MEL infusions. The effectiveness of 10-hr, but not 6-hr, MEL infusions in T 18 and T 24 is consistent with the duration hypothesis and argues against the circadian hypothesis. Neither hypothesis could have predicted that all infusion cycles of T greater than or equal to 36 hr, regardless of the infusion durations, would fail to elicit short-day-type responses. This outcome suggests a need for relatively frequent (T less than 36 hr) MEL stimulation in addition to the requirement for adequate duration of each MEL infusion.     

Ventilatory control during exercise in calves with artificial hearts

To determine the role of cardiac reflexes in mediating exercise hyperpnea, we investigated ventilatory responses to treadmill exercise in seven calves with artificial hearts and seven controls. In both groups, the ventilatory responses were adequate for the metabolic demands of the exercise; this resulted in regulation of arterial PCO2 and pH despite the absence of cardiac output increase in the implanted group. In this group, there was a small but significant reduction of arterial PO2 by 4 +/- 3 Torr and a rise of blood lactate by 1.1 +/- 1 mmol/l. When cardiac output was experimentally increased in the implanted calves to a level commensurate with that spontaneously occurring in the control calves, ventilation was not affected. However, experimental reductions of cardiac output led to an immediate augmentation of exercise hyperpnea by 4.56 +/- 4.3 l/min and a further significant lactate increase of 1.2 +/- 1.22 mmol/l that was associated with a significant decrease in the exercise O2 consumption (0.32 +/- 0.13 l/min). These observations indicate that neither cardiac nor hemodynamic effects of increased cardiac output constitute an obligatory cause of exercise hyperpnea in the calf.     

Expression of A mating type genes of Coprinus cinereus in a heterologous basidiomycete host

The A mating factor of Coprinus cinereus determines compatibility in mating by regulating part of a developmental sequence that leads to dikaryon formation. The A genes that trigger development encode two different classes of homeodomain proteins, and for a successful mating, a protein of one class, HD 1, must interact with a protein of the other class, HD 2. In this report we show that C. cinereus A genes that encode HD 2 proteins, a2-1 and b2-1, can elicit A-regulated development in the heterologous host C. bilanatus. Transformation rates were very low, suggesting that the genes were poorly transcribed. The fact that the HD 2 genes are functionally expressed implies successful heteromultimeric association of putative DNA-binding proteins coded by the two Coprinus species. This interaction was sufficient to satisfy the need for different A factors in the formation of a fertile C. bilanatus dikaryon, but fertile dikaryons were more readily produced in matings with the a2-1 gene transformants. The C. cinereus A genes, b1-1 and d1-1, which encode HD1 proteins, were either not expressed or their proteins were non-functional in C. bilanatus. These experiments raise some interesting questions regarding HD1–HD2 protein interactions.     

Electron microscopic studies on liver 5-aminolaevulinate synthase

The structure of chick embryo liver 5-aminolaevulinate synthase has been examined by electron microscopic studies using negative staining. From the different projections of the enzyme particles observed in electron micrographs, a model for the enzyme molecule has been proposed. In this model, an enzyme molecule consists of two curved and identical subunits associated in opposite polarities. From the dimensions of an enzyme molecule subunit measured from electron micrographs, the relative molecular mass of each subunit is estimated to be 70 000.     

Proteinase-sensitive release of enzymes from pancreatic microsomal fraction.

Suspensions of rat pancreatic microsomal fraction release alpha-amylase and ribonuclease on incubation at 37 degrees C, but not at 2 degrees C. The release is abolished by proteolytic enzymes. Ribonuclease associated with the microsomal fraction is protected from subtilisin BPN' attack, but is sensitive after release.     

Aluminium toxicity during regular haemodialysis.

In the west of Scotland the incidence of dialysis encephalopathy has been confined to three geographical areas where the concentration of aluminium in the water supply is greatly increased owing to the addition of aluminium sulphate. Eight patients with encephalopathy who dialysed at home in these areas had greatly increased serum aluminium concentrations, and a significant correlation was found between serum aluminium concentrations and the concentrations of aluminium in the water supply. This study provides further evidence that the dialysis encephalopathy syndrome is due to aluminium intoxication, the major source of aluminium being the water supply from which dialysis fluid prepared.