Isolation by zinc-affinity chromatography of the histidine-proline-rich-glycoprotein molecule associated with rabbit skeletal muscle AMP deaminase. Evidence that the formation of a protein-protein complex between the catalytic subunit and the novel component is critical for the stability of the enzyme

The histidine-proline-rich glycoprotein (HPRG) component of rabbit skeletal muscle AMP deaminase under denaturing and reducing conditions specifically binds to a Zn(2+)-charged affinity column and is only eluted with an EDTA-containing buffer that strips Zn(2+) from the gel. The isolated protein is homogeneous showing an apparent molecular weight (MW) of 95000 and the N-terminal sequence L-T-P-T-D-X-K-T-T-K-P-L-A-E-K-A-L-D-L-I, corresponding to that of rabbit plasma HPRG. The incubation with peptide-N-glycosidase F promotes the reduction of the apparent MW of isolated HPRG to 70000, characterizing it as a N-glycosylated protein. The separation from AMP deaminase of an 85-kDa component with a blocked N terminus is observed when the enzyme is applied to the Zn-charged column under nondenaturing conditions. On storage under reducing conditions, this component undergoes an 85- to 95-kDa transition yielding a L-T-P-T-D-X-K-T-T-K-P-L N-terminal sequence, suggesting that the shift in the migration on SDS/PAGE as well as the truncation of the protein at its N terminus are promoted by the reduction of a disulfide bond present in freshly isolated HPRG. The separation of HPRG induces a marked reduction in the solubility of AMP deaminase, strongly suggesting a role of HPRG in assuring the molecular integrity of the enzyme.     

Repeated allergen inhalation induces phenotypic modulation of smooth muscle in bronchioles of sensitized rats

Repeated ovalbumin (OA) or saline exposure of sensitized Brown Norway rats was examined on agonist reactivity, airway smooth muscle (ASM) content, and contractile protein expression in small bronchioles at 24 h, 7 days, and 35 days after challenge. OA increased ASM content (P < 0.05 vs. saline) at 24 h, which resolved by 7 days. Maximum developed tension (T(max)) to carbachol, KCl, and 4-beta-phorbol 12,13-dibutyrate was increased (P < 0.05) by OA in bronchioles at 24 h but was abrogated after correction for ASM. Differences in T(max) were not present at 7 days. In contrast, at 35 days, T(max) was increased (P < 0.05) after correction for ASM. Smooth muscle (sm)-alpha-actin, sm-myosin heavy chain (MHC) isoform 1, calponin, smoothelin-A, and sm-myosin light chain kinase expression were reduced (P < 0.05) by OA at 24 h in bronchioles but not in trachealis. Consistent with contraction findings, no difference in expression of these proteins was detected at 7 days. At 35 days, however, with the exception of sm-alpha-actin, their abundance was again reduced (P < 0.05) by OA. Nonmuscle MHC and beta-actin were unchanged throughout by OA. These findings indicate persistent changes in contractile protein content, consistent with ASM phenotypic modulation in vivo, which occur in response to repeated OA inhalation. Thus, OA exposure induces structural changes in bronchiole ASM content and in agonist responsiveness ex vivo that resemble remodeling in asthma.     

Role of the gerI Operon of Bacillus cereus 569 in the Response of Spores to Germinants

Bacillus cereus 569 (ATCC 10876) germinates in response to inosine or to l-alanine, but the most rapid germination response is elicited by a combination of these germinants. Mutants defective in their germination response to either inosine or to l-alanine were isolated after Tn917-LTV1 mutagenesis and enrichment procedures; one class of mutant could not germinate in response to inosine as a sole germinant but still germinated in response to l-alanine, although at a reduced rate; another mutant germinated normally in response to inosine but was slowed in its germination response to l-alanine. These mutants demonstrated that at least two signal response pathways are involved in the triggering of germination. Stimulation of germination in l-alanine by limiting concentrations of inosine and stimulation of germination in inosine by low concentrations of l-alanine were still detectable in these mutants, suggesting that such stimulation is not dependent on complete functionality of both these germination loci. Two transposon insertions that affected inosine germination were found to be located 2.2 kb apart on the chromosome. This region was cloned and sequenced, revealing an operon of three open reading frames homologous to those in the gerA and related operons of Bacillus subtilis. The individual genes of this gerI operon have been named gerIA, gerIB, and gerIC. The GerIA protein is predicted to possess an unusually long, charged, N-terminal domain containing nine tandem copies of a 13-amino-acid glutamine- and serine-rich sequence.Bacillus species have the ability, under certain nutrient stresses, to undergo a complex differentiation process resulting in the formation of a highly resistant dormant endospore (6). These spores can then persist in the environment for prolonged periods until a sensitive response mechanism detects specific environmental conditions, initiating the processes of germination and outgrowth (9, 21, 25). Germination can be initiated by a variety of agents (12), including nutrients, enzymes, or physical factors, such as abrasion or hydrostatic pressure.The molecular genetics of spore germination has been most extensively studied in Bacillus subtilis 168 (21). B. subtilis spores can be triggered to germinate in response to either l-alanine or to a combination (29) of asparagine, glucose, fructose, and potassium ions (AGFK). Mutants of B. subtilis which are defective in germination responses to one or to both types of germinant have been isolated previously (20, 27). Analysis of these mutants suggests that the germinants interact with separate germinant-specific complexes within the spore (21). This in some way leads to activation of components of the germination apparatus common to both responses, such as germination-specific cortex lytic enzymes, leading in turn to complete germination of the spore (10, 22). The mutations within the gerA operon of B. subtilis specifically block germination initiated by l-alanine (34). The predicted amino acid sequences of the three GerA proteins encoded in the operon suggest that these proteins could be membrane associated, and they are the most likely candidates to represent the germinant receptor for alanine (21).The amino acid l-alanine has been identified as a common but not universal germinant in a variety of Bacillus species, often requiring the presence of adjuncts such as electrolytes and sugars. Ribosides, such as inosine, represent another type of common germinant, although many species are unable to germinate rapidly in response to these without the addition of l-alanine (9).The food-borne pathogen Bacillus cereus is a major cause of food poisoning of an emetic and diarrheal type (13, 16). The germination and growth of Bacillus cereus spores during food storage can lead to food spoilage and the potential to cause food poisoning (16). B. cereus has been shown to germinate in response to l-alanine and to ribosides (11, 18, 23). Spore germination can be triggered by l-alanine alone, but at high spore densities this response becomes inhibited by d-alanine, generated by the alanine racemase activity associated with the spores (8, 11). This auto-inhibition of l-alanine germination can be reduced by the inclusion of a racemase inhibitor (O-carbamyl-d-serine) with the germinating spores (11).Inosine is the most effective riboside germinant for B. cereus T, while adenosine and guanosine are less potent (28). The rate of riboside-triggered germination has been reported to be enhanced dramatically by the addition of l-alanine (18). It is unclear whether ribosides can act as a sole germinant, or whether there is an absolute requirement for l-alanine (28).An attempt has been made to analyze genetically the molecular components of the germination apparatus in B. cereus in order to dissect the germination responses of this species and to determine whether riboside-induced germination involves components related to those already described for amino acid and sugar germinants in B. subtilis.     

Species delimitation of the liquorice tribe (Leguminosae: Glycyrrhizeae) based on phylogenomic and machine learning analyses

The liquorice tribe Glycyrrhizeae is a leguminous herbaceous group of plants comprised of the genera Glycyrrhiza and Glycyrrhizopsis. Some Glycyrrhiza taxa contain glycyrrhizin, a pharmacologically significant sweet substance that also has applications in crafting industrial materials. Here, we utilized an expanded taxon sampling of Glycyrrhizeae to reconstruct the phylogenetic relationships in the tribe based on genome skimming data, including whole chloroplast genomes, nuclear ribosomal DNA, and low-copy nuclear DNA. We also launched machine learning analysis (MLA) for one species pair with controversial taxonomic boundary. The integrated results indicated Glycyrrhizopsis should be split from Glycyrrhiza, while the former genus Meristotropis should be treated as part of Glycyrrhiza. Glycyrrhizopsis includes two species, Glycyrrhizopsis asymmetrica and Glycyrrhizopsis flavescens, and we recognize 13 species in Glycyrrhiza: Glycyrrhiza acanthocarpa, Glycyrrhiza astragalina, Glycyrrhiza bucharica, Glycyrrhiza echinata, Glycyrrhiza foetida, Glycyrrhiza glabra, Glycyrrhiza gontscharovii, Glycyrrhiza lepidota, Glycyrrhiza macedonica, Glycyrrhiza pallidiflora, Glycyrrhiza squamulosa, Glycyrrhiza triphylla, and Glycyrrhiza yunnanensis. We propose a broader G. glabra that includes former Glycyrrhiza aspera, G. glabra s.s., Glycyrrhiza inflata, and Glycyrrhiza uralensis, and represents the glycyrrhizin-contained medicinal group. Our ancestral state inferences show the ancestor of Glycyrrhiza lacked glycyrrhizin, and the presence of glycyrrhizin evolved twice within Glycyrrhiza during the last one million years. Our integrative phylogenomics-MLA study not only provides new insights into long-standing taxonomic controversies of Glycyrrhizeae, but also represents a useful approach for future taxonomic studies on other plant taxa.     

Rearrangements of integral membrane components during in vitro aging of sheep erythrocyte membranes

In vitro aged sheep erythrocytes and sheep erythrocyte ghosts spontaneously release vesicles that consist of long protrusions affixed to flattened headlike structures. The intramembranous particles seen on the protoplasmic face of freeze fracture electron micrographs of vesicle protrusions are arranged in paired particle rows. On the equivalent fracture face of headlike structures, the particle density is low; if particles are present, they are clustered along the rim of the flattened headlike structure and at the junction with the protrusion. The released vesicles are depleted of the intramembranous particles seen on the exoplasmic face of ghost but retain almost exclusively particles of the protoplasmic face. Correspondingly, the exoplasmic face of ghosts that have released vesicles reveals a 28 percent higher density of intramembranous particles than that of fresh ghosts. Purified vesicles are depleted of spectrin but retain integral membrane proteins, with one of an apparent mol wt of 160,000 accounting for nearly 50 percent of the total protein (Lutz, H.U.,R. Barber, and R.F. McGuire. 1976. J. Biol. Chem. 251:3500-3510). When vesicles are modified with the cleavable cross-linking reagent [(35)S]dithiobis (succinimidyl propionate)at 0 degrees C, the 160,000 mol wt protein is rapidly converted to disulfide-linked dimers and higher oligomers. Exposure of intact ghosts to the reagent in the same way fails to yield equivalent polymers. A comparison of the morphological and biochemical aspects of ghosts and vesicles suggest that a marked rearrangement of membrane proteins accompanies the supramolecular redistribution of intramembranous particles during spontaneous vesiculation. The results also suggest that the paired particles of the protoplasmic face of vesicle protrusions are arranged in paired helices and contain the 160,000 mol wt protein as dimers.     

Human Brain βA4 Amyloid Protein Precursor of Alzheimer''s Disease: Purification and Partial Characterization

The major component of the amyloid deposition that characterizes Alzheimer's disease is the 4-kDa beta A4 protein, which is derived from a much larger amyloid protein precursor (APP). A procedure for the complete purification of APP from human brain is described. The same amino terminal sequence of APP was found in two patients with Alzheimer's disease and one control subject. Two major forms of APP were identified in human brain with apparent molecular masses of 100-110 kDa and 120-130 kDa. Soluble and membrane fractions of brain contained nearly equal amounts of APP in both humans and rats. Immunoprecipitation with carboxyl terminus-directed antibodies indicates that the soluble forms of APP are truncated. Carboxyl terminus truncation of membrane-associated forms of human brain APP was also found to occur during postmortem autolysis. The availability of purified human brain APP will facilitate the investigation of its normal function and the events that lead to its abnormal cleavage in patients with Alzheimer's disease.     

Cold-Sensitive Cell-Division-Cycle Mutants of Yeast: Isolation, Properties, and Pseudoreversion Studies

We isolated 18 independent recessive cold-sensitive cell-division-cycle (cdc) mutants of Saccharomyces cerevisiae, in nine complementation groups. Terminal phenotypes exhibited include medial nuclear division, cytokinesis, and a previously undescribed terminal phenotype consisting of cells with a single small bud and an undivided nucleus. Four of the cold-sensitive mutants proved to be alleles of CDC11, while the remaining mutants defined at least six new cell-division-cycle genes: CDC44, CDC45, CDC48, CDC49, CDC50 and CDC51.—Spontaneous revertants from cold-sensitivity of four of the medial nuclear division cs cdc mutants were screened for simultaneous acquisition of a temperature-sensitive phenotype. The temperature-sensitive revertants of four different cs cdc mutants carried single new mutations, called Sup/Ts to denote their dual phenotype: suppression of the cold-sensitivity and concomitant conditional lethality at 37°. Many of the Sup/Ts mutations exhibited a cell-division-cycle terminal phenotype at the high temperature, and they defined two new cdc genes (CDC46 and CDC47). Two cold-sensitive medial nuclear division cdc mutants representing two different cdc genes were suppressed by different Sup/Ts alleles of another gene which also bears a medial nuclear division function (CDC46). In addition, the cold-sensitive medial nuclear division cdc mutant csH80 was suppressed by a Sup/Ts mutation yielding an unbudded terminal phenotype with an undivided nucleus at the high temperature. This mutation was an allele of CDC32. These results suggest a pattern of interaction among cdc gene products and indicate that cdc gene proteins might act in the cell cycle as complex specific functional assemblies.     

Determination of the Order of Gene Function in the Yeast Nuclear Division Pathway Using cs and ts Mutants

Cold-sensitive (cs) and heat-sensitive (ts) conditional-lethal mutations that affect specifically the cell division cycle of budding yeast (Saccharomyces cerevisiae) were used to determine the order of gene function. Reciprocal temperature-shift experiments using cs-ts double mutants revealed a detailed order of function among genes whose execution points and mutant phenotypes are very similar. The data suggest that the nuclear branch of the overall cell-cycle pathway itself contains at least one branch.