The role of proteolysis in the processing and assembly of 11S seed globulins.

11S seed storage proteins are synthesized as precursors that are cleaved post-translationally in storage vacuoles by an asparaginyl endopeptidase. To study the specificity of the reaction catalyzed by this asparaginyl endopeptidase, we prepared a series of octapeptides and mutant legumin B and G4 glycinin subunits. These contained amino acid mutations in the region surrounding the cleavage site. The endopeptidase had an absolute specificity for Asn on the N-terminal side of the severed peptide bond but exhibited little specificity for amino acids on the C-terminal side. The ability of unmodified and modified subunits to assemble into hexamers after post-translational modification was evaluated. Cleavage of subunits in trimers is required for hexamer assembly in vitro. Products from a mutant gene encoding a noncleavable prolegumin subunit (LeBDeltaN281) accumulated as trimers in seed of transgenic tobacco, but products from the unmodified prolegumin B gene accumulated as hexamers. Therefore, the asparaginyl endopeptidase is required for hexamer assembly.     

Relating drug-induced changes in carotid artery mechanics to cardiovagal and sympathetic baroreflex control

Previous evidence indicates that sensitivity of the baroreflex cardiovagal and sympathetic arms is dissociated. In addition, pharmacologic assessment of baroreflex sensitivity (BRS) has revealed that cardiovagal, but not sympathetic, BRS is greater when blood pressure is increasing versus falling. The origin of this hysteresis is unknown. In this study, carotid artery distensibility and absolute distension (diameter) were assessed to test the hypothesis that vessel mechanics in barosensitive regions affect the BRS of cardiovagal, but not sympathetic, outflow. R-R interval (i.e. time between successive R waves), finger arterial blood pressure, muscle sympathetic nerve activity, and carotid artery dimensions (B-mode imaging) were measured during sequential infusions of sodium nitroprusside (SNP) and phenylephrine (PHE). Systolic and diastolic common carotid artery diameters and pulse pressure were recorded to calculate distensibility of this vessel under each drug condition. Cardiovagal BRS was greater when blood pressure was increasing versus decreasing (p < 0.01). Sympathetic BRS was not affected by direction of pressure change. Distensibility did not differ between SNP and PHE injections. However, compared with SNP, infusion of PHE resulted in larger absolute systolic and diastolic carotid diameters (p < 0.001). Therefore, cardiovagal reflex hysteresis was related to drug-induced changes in common carotid artery diameter but not distensibility. The lack of sympathetic hysteresis in this model suggests a relative insensitivity of this baroreflex component to carotid artery dimensions and provides a possible mechanism for the dissociation between cardiovagal and sympathetic BRS.     

Relocation or duplication of the helix A sequence of T4 lysozyme causes only modest changes in structure but can increase or decrease the rate of folding

In T4 lysozyme, helix A is located at the amino terminus of the sequence but is associated with the C-terminal domain in the folded structure. To investigate the implications of this arrangement for the folding of the protein, we first created a circularly permuted variant with a new amino terminus at residue 12. In effect, this moves the sequence corresponding to helix A from the N- to the C-terminus of the molecule. The protein crystallized nonisomorphously with the wild type but has a very similar structure, showing that the unit consisting of helix A and the C-terminal domain can be reconstituted from a contiguous polypeptide chain. The protein is less stable than the wild type but folds slightly faster. We then produced a second variant in which the helix A sequence was appended at the C-terminus (as in the first variant), but was also restored at the N-terminus (as in the wild type). This variant has two helix A sequences, one at the N-terminus and the other at the C-terminus, each of which can compete for the same site in the folded protein. The crystal structure shows that it is the N-terminal sequence that folds in a manner similar to that of the wild type, whereas the copy at the C-terminus is forced to loop out. The stability of this protein is much closer to that of the wild type, but its rate of folding is significantly slower. The reduction in rate is attributed to the presence of the two identical sequence segments which compete for a single, mutually exclusive, site.     

Inositol diphosphate signaling regulates telomere length

Activation of phospholipase C-dependent inositol polyphosphate signaling pathways generates distinct messengers derived from inositol 1,4,5-trisphosphate that control gene expression and mRNA export. Here we report the regulation of telomere length by production of a diphosphorylinositol tetrakisphosphate, PP-IP4, synthesized by the KCS1 gene product. Loss of PP-IP4 production results in lengthening of telomeres, whereas overproduction leads to their shortening. This effect requires the presence of Tel1, the yeast homologue of ATM, the protein mutated in the human disease ataxia telangiectasia. Our data provide in vivo evidence of a regulatory link between inositol polyphosphate signaling and the checkpoint kinase family and describe a third nuclear process modulated by phospholipase C activation.     

Effect of growth stage on mycolic acid structure in cell walls of Nocardia asteroides GUH-2

Mycolic acids were extracted from the cell walls of Nocardia asteroides GUH-2 during different phases of growth at 37 degrees C. These were subjected to structural analysis by combining thin-layer chromatography and gas-liquid chromatography with UV and infrared spectrophotometry and mass spectroscopy of both methyl esters and trimethyl silyl derivatives. By analyzing the fragmentation patterns of these derivatives by three different methods of mass spectroscopy combined with gas-liquid chromatographic separation, the different structural subclasses of mycolic acids were quantitated. Significant qualitative and quantitative modifications of specific mycolic acid subclasses occurred in the cell walls of N. asteroides GUH-2 that were growth stage dependent. The mycolic acids that were predominant in the log phase were polyunsaturated (greater than 2 double bonds per molecule), with long chain lengths and even carbon atom numbers (i.e., C54, C56). In contrast, those that were prominent in the stationary phase were more saturated (few or no double bonds) and of shorter overall carbon chain length (less than or equal to C52). Furthermore, stationary-phase cells had significantly increased amounts of mycolic acids with odd-numbered carbon chain lengths (i.e., C49, C51, C53).     

An approach to the unification of suppressor T cell circuits: a simplified assay for the induction of suppression by T cell-derived, antigen-binding molecules (T-ABM)

A system is presented in which the in vitro response to sheep red blood cells (SRBC) can be regulated using antigenic determinants coupled to SRBC and T cell-derived antigen-binding molecules (T-ABM) directed against the coupled determinants. T suppressor-inducer factors (TsiF's) are composed of two molecules, one of which is a T-ABM and one which bears I-J determinants (I-J+ molecule). Using two purified T-ABM which have not previously been shown to have in vitro activity, we produced antigen-specific TsiF's which were capable of inducing the suppression of the anti-SRBC response. Suppression was found to require both the T-ABM and the I-J+ molecule, SRBC conjugated with the antigen for which the T-ABM was specific, and a population of Ly-2+ T cells in the culture. Two monoclonal TsiF (or TsF1) were demonstrated to induce suppression of the anti-SRBC response in this system, provided the relevant antigen was coupled to the SRBC in culture. The results are discussed in terms of the general functions of T-ABM in the immune system. This model will be useful in direct, experimental comparisons of the function of T-ABM and suppressor T cell factors under study in different systems and laboratories.     

Mutant cell line demonstrating a block in insulin and insulin-like growth factor type 1 (IGF-1) induced mitogenesis

Recently, we have isolated a Chinese hamster cell variant (IV-A1-j) resistant to an insulin-diphtheria-A chain toxic conjugate (Leckett and Germinario: Cytotechnology [in press]. This cell line exhibited a decreased level of insulin binding, but normal growth in serum-containing medium when compared to the parental cell line (V-79). In this paper we further demonstrate that while IV-A1-j cells are capable of growing in serum-containing medium, they are insensitive to the mitogenic actions of either insulin or IGF-1. In contrast, epidermal growth factor (EGF) and/or α-thrombin (THR) generate a mitogenic effect in IV-A1-j cells comparable to that observed in the parental V-79 cells. The combination of EGF and/or THR with either insulin or IGF-1 results in an increase in V-79 cell growth above EGF and/or THR alone. On the other hand, insulin or IGF-1 in the presence of other mitogens did not stimulate further growth in IV-A1-j cells. While insulin binding was lower in IV-A1-j cells, internalization of ¹²⁵I-insulin was not different in the two cell types. Additionally, insulin-stimulated glycogen synthesis and protein synthesis were not different in the two cell types. These observations are consistent with insulin and IGF-1 sharing a mitogenic signalling pathway in Chinese hamster fibroblasts and that this pathway is distinct from other growth factor signalling pathways. The fact that this pathway is defective in the IV-A1-j cell line indicates the potential usefulness of these cells in identifying a key step(s) in the insulin (IGF-1) mitogenic pathway. © 1993 Wiley-Liss, Inc.     

Cu,Zn-Superoxide Dismutase Increases Toxicity of Mutant and Zinc-deficient Superoxide Dismutase by Enhancing Protein Stability

When replete with zinc and copper, amyotrophic lateral sclerosis (ALS)-associated mutant SOD proteins can protect motor neurons in culture from trophic factor deprivation as efficiently as wild-type SOD. However, the removal of zinc from either mutant or wild-type SOD results in apoptosis of motor neurons through a copper- and peroxynitrite-dependent mechanism. It has also been shown that motor neurons isolated from transgenic mice expressing mutant SODs survive well in culture but undergo apoptosis when exposed to nitric oxide via a Fas-dependent mechanism. We combined these two parallel approaches for understanding SOD toxicity in ALS and found that zinc-deficient SOD-induced motor neuron death required Fas activation, whereas the nitric oxide-dependent death of G93A SOD-expressing motor neurons required copper and involved peroxynitrite formation. Surprisingly, motor neuron death doubled when Cu,Zn-SOD protein was either delivered intracellularly to G93A SOD-expressing motor neurons or co-delivered with zinc-deficient SOD to nontransgenic motor neurons. These results could be rationalized by biophysical data showing that heterodimer formation of Cu,Zn-SOD with zinc-deficient SOD prevented the monomerization and subsequent aggregation of zinc-deficient SOD under thiol-reducing conditions. ALS mutant SOD was also stabilized by mutating cysteine 111 to serine, which greatly increased the toxicity of zinc-deficient SOD. Thus, stabilization of ALS mutant SOD by two different approaches augmented its toxicity to motor neurons. Taken together, these results are consistent with copper-containing zinc-deficient SOD being the elusive “partially unfolded intermediate” responsible for the toxic gain of function conferred by ALS mutant SOD.