Eukaryotic initiation factor 2 from rat liver: no apparent function for the β-subunit in the formation of initiation complexes

Eukaryotic protein synthesis initiation factor 2 (eIF-2) from rat liver has been resolved into two subfractions by anion-exchange chromatography on DEAE-cellulose. One of these contained all three components (eIF-2α, eIF-2β, eIF-2γ) characteristic of mammalian eIF-2, whilst the other fraction contained only two. By a number of criteria these were shown to be eIF-2α and eIF-2γ. The absence of eIF-2β from this fraction was not due to its proteolytic degradation during purification since it was unaffected by the inclusion of a range of proteinase inhibitors in the isolation media. The properties of eIF-2 containing or lacking eIF-2β have been directly compared. It was found that eIF-2β was not required for the binding of guanine nucleotides to eIF-2 or for formation of ternary initiation complexes with GTP and the initiator tRNA. eIF-2 lacking eIF-2β was able to form 40 S initiation complexes and the presence of eIF-2β was also unnecessary for the stimulation of eIF-2 activity by the recycling factor, eIF-2B. Some of these findings are at variance with previous reports in which eIF-2β was removed proteolytically. The role of eIF-2β in the overall physiological function of eIF-2 remains to be elucidated.     

Immunohistochemical localization of transforming growth factor β1 and β2 in mouse testes during postnatal development

We examined age-related changes in the expression of transforming growth factor-β(1) (TGF-β(1)) and transforming growth factor-β(2) in mouse testes. The mice were assigned to three age groups: 35, 50, and 75 days old. Paraffin embedded testis sections were processed for the standard streptavidin biotin peroxidase complex immunohistochemistry method. TGF-β(1) expression increased in aging round spermatids over the time studied. There was no expression in 35-day-old Leydig cells, whereas strong expression of TGF-β(1) was observed in 50-day-old Leydig cells. Expression decreased in 75-day-old Leydig cells. TGF-β(2) expression was weak in 35- and 50-day-old mouse spermatids, but expression was greater in 75-day-old elongated spermatids. In Leydig cells, TGF-β(2) expression was strong in both 35- and 50-day-old mice, whereas the expression of TGF-β(2) was less in 75-day-old Leydig cells. Our results suggest that TGF-β(1) and TGF-β(2) may play significant roles in testicular functions and germ cell development in mice.     

Heterogeneity and differential expression of the γ-aminobutyric acidA (GABAA)/benzodiazepine receptor in the avian brain during development

Summary 1. The changes in the GABA_A/benzodiazepine receptor in chicken brain during development has been studied by using³H-flunitrazepam as the probe for the benzodiazepine modulator site and the antibodies recognizing the receptor protein. In the telencephalon and optic tectum, the proteins of 48, 50, and 51 kD were markedly labeled by³H-flunitrazepam from embryonic day 18 to postnatal days, as revealed by photoaffinity labeling and SDS-PAGE of the brain membranes; the 51-kD protein appeared to be the predominant one in labeling intensity except at embryonic day 18 and postnatal days 14 and 28, whereas the 47- and 50-kD proteins were dominant in the cerebellum. However, the 47- and 48-kD proteins were faintly seen after postnatal day 28 in the three regions examined.2. Immunoblotting using a monoclonal antibody against the 50- and 51-kD proteins showed that the straining pattern in the developing telecephalon or optic tectum was similar to the 50 kD/51 kD pattern obtained from fluorography. The antibody also stained the 50- and 51-kD proteins in the cerebellum despite the fact that the 51-kD protein was barely seen in the fluorogram. Moreover, the 50-kD protein was recognized by an antiserum raised against a partial sequence of the 1 subunit of the receptor expressed in bacteria. The staining levels for the 50-kd protein by the antiserum on immunoblots of the brain regions were low in embryonic animals but higher during postnatal stages, consistent with that seen in fluorograms.3. Receptor binding autoradiography using³H-flunitrazepam exhibited that varying degrees of labeling intensity occurred among various brain areas at different ages. High densities of binding were present in the olfactory bulb, paleostriatum, optic tectum, and midbrain. These results support the diversity of the GABA_A/benzodiazepine receptor in the vertebrate CNS.     

Structure and phosphorylation of eukaryotic initiation factor 2. Casein kinase 2 and protein kinase C phosphorylate distinct but adjacent sites in the β-subunit

Eukaryotic initiation factor 2 (eIF-2) from rabbit reticulocytes can be phosphorylated on its β-subunit by two different protein kinases, protein kinase C and casein kinase 2. Phosphorylation by these kinases is additive, suggesting that they phosphorylate different sites (serine residues) in eIF-2β. Two-dimensional peptide mapping of the phosphopeptides generated from labelled eIF-2β by digestion with trypsin, cyanogen bromide or Staphylococcus aureus V8 proteinase showed that protein kinase C and casein kinase 2 phosphorylated distinct and different sites in this protein. This conclusion was supported by the results of analysis of the phosphopeptides on reverse-phase chromatography. Analysis of the phosphopeptides derived from eIF-2β labelled by both kinases together strongly suggested that the sites labelled by protein kinase C and casein kinase 2 are adjacent in the primary sequence. These data are discussed in the light of the present understanding of the sequence specificity of the kinases. Rat liver eIF-2β was also found to be a substrate for protein kinase C and casein kinase 2, which were again shown to label different serine residues.     

Heterogeneity of β2-Microglobulin in Human Urine

Purification and characterization of β₂-microglobulin from human urine was performed. The yield was 30.1%, and 150.4 mg of β₂-microglobulin was obtained. The final preparation of β₂-microglobulin obtained showed three bands on disc gel electrophoresis at pH 9.5, and all of them have immunological activity. However, these three bands migrated as a single band on disc gel electrophoresis at pH 4.3. It is concluded that the three bands observed on disc gel electrophoresis at pH 9.5 were charge isomers. The isoelectric points of isomers were determined by isotachophoresis and two of them were 5.4 and 5.9 respectively, while the other one was not determined.     

Expression of the β-subunit of β-conglycinin in seeds of transgenic plants

Soybean (Glycine max (L.) Merr.) seeds contain the storage protein -conglycinin, encoded by a multigene family. -Conglycinin consists of three subunits; , , and . A genomic clone for a -subunit of -conglycinin has been characterized by restriction-enzyme mapping and hybrid selected in-vitro translation followed by immunoprecipitation. In order to determine the developmental regulation of this -subunit gene, its expression was studied in seeds of transgenic petunia (Petunia hybrida) and tobacco (Nicotiana tabacum L.) plants. The -subunit expressed in seeds of petunia and tobacco was recognized by anti--conglycinin serum at a relative molecular mass of 53 000, equivalent to that of the native protein. Separation of the petunia-seed proteins by isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis showed that multiple isoelectric forms of the -subunit were produced. There was approximately a twofold variation in the accumulation of the -subunit protein in the mature seeds of transgenic petunia plants, each containing a single -subunit gene. However, the level of protein accumulation in mature seeds and the amount of -subunit mRNA in developing seeds was not correlated. Accumulation of the -subunit protein in transgenic seeds was less than the -subunit protein that accumulated in transgenic petunia seeds containing a single -subunit gene and less than the amount of the -subunit in mature soybean seeds which contain 8–13 -subunit genes. In transgenic tobacco plants, the accumulation of the -subunit protein in seeds was generally well correlated with the number of genes that were incorporated in the different transformants.Abbreviations kb kilobase - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

The NH2-terminal cleavage of Escherichia coli translational initiation factor IF3. A mechanism to control the intracellular level of the factor?

A short form of Escherichia coli translational initiation factor IF3, repeatedly found both in vivo and in vitro, lacking the positively charged N-terminal hexapeptide has been produced by mild trypsinization. The properties of this short form of IF3 have been studied. Compared to the long native form of the factor, the shortened IF3 displays a markedly decreased thermal stability and affinity for the 30 S ribosomal subunit, as well as a reduced biological activity in protein synthesis. Following the loss of the N-terminal hexapeptide, a second peptide bond (Lys-90-Val-91) becomes easily accessible to proteolytic attack suggesting that formation of the short IF3 may be the first step in the physiological degradation of the factor.     

Distinct patterns of expression of the β-1,4-galactosyltransferases during testicular development in the mouse

Glycosylation is one of the most important post-translational modifications and it is clear that the single step of -1,4-galactosylation is performed by a family of -1,4-galactosyltransferases (4-GalTs) and that each member of this family may play a distinct role in different tissues and cells. In this study, we characterized the gene expression of six 4-GalTs in mouse testis and analyzed the changes of galactosylation of testis glycoproteins during postnatal development. Northern blot analysis revealed that 4-GalT-I and 4-GalT-IV were expressed mainly in newborn mouse testis and that the expression of 4-GalT-II increased markedly and persisted at the highest levels in adult mouse testis. The expression of 4-GalT-III and 4-GalT-V, however, remained relatively at low levels during mouse testicular development. In contrast, the expression of 4-GalT-VI was undetectable in mouse testis. The gene expression of 4-GalT-II in mouse testis was further analyzed by in situ hybridization due to its unique expression pattern. Strong hybridization signals were detected in the seminiferous tubules and the expression varied among the different stages of spermatogenic differentiation. The distinct gene expression patterns of 4-GalTs in mouse testis could affect the differential galactosylation of testis glycoproteins, as revealed by lectin histochemistry analysis.     

Autophosphorylation of heme-regulated eukaryotic initiation factor 2α kinase and the role of the modification in catalysis

Heme-regulated eukaryotic initiation factor 2α (eIF2α) kinase (HRI), functions in response to heme shortage in reticulocytes and aids in the maintenance of a heme:globin ratio of 1:1. Under normal conditions, heme binds to HRI and blocks its function. However, during heme shortage, heme dissociates from the protein and autophosphorylation subsequently occurs. Autophosphorylation comprises a preliminary critical step before the execution of the intrinsic function of HRI; specifically, phosphorylation of Ser-51 of eIF2α to inhibit translation of the globin protein. The present study indicates that dephosphorylated mouse HRI exhibits strong intramolecular interactions (between the N-terminal and C-terminal domains) compared to phosphorylated HRI. It is therefore suggested that autophosphorylation reduces the intramolecular interaction, which induces irreversible catalytic flow to the intrinsic eIF2α kinase activity after heme dissociates from the protein. With the aid of MS, we identified 33 phosphorylated sites in mouse HRI overexpressed in Escherichia coli. Phosphorylated sites at Ser, Thr and Tyr were predominantly localized within the kinase insertion region (16 sites) and kinase domain (12 sites), whereas the N-terminal domain contained five sites. We further generated 30 enzymes with mutations at the phosphorylated residues and examined their catalytic activities. The activities of Y193F, T485A and T490A mutants were significantly lower than that of wild-type protein, whereas the other mutant proteins displayed essentially similar activity. Accordingly, we suggest that Tyr193, Thr485 and Thr490 are essential residues in the catalysis.     

Dissociation of the α-subunit Calf-2 domain and the β-subunit I-EGF4 domain in integrin activation and signaling

Integrin conformational changes mediate integrin activation and signaling triggered by intracellular molecules or extracellular ligands. Even though it is known that αβ transmembrane domain separation is required for integrin signaling, it is still not clear how this signal is transmitted from the transmembrane domain through two long extracellular legs to the ligand-binding headpiece. This study addresses whether the separation of the membrane-proximal extracellular αβ legs is critical for integrin activation and outside-in signaling. Using a disulfide bond to restrict dissociation of the α-subunit Calf-2 domain and β-subunit I-EGF4 domain, we were able to abolish integrin inside-out activation and outside-in signaling. In contrast, disrupting the interface by introducing a glycosylation site into either subunit activated integrins for ligand binding through a global conformational change. Our results suggest that the interface of the Calf-2 domain and the I-EGF4 domain is critical for integrin bidirectional signaling.     

The initiation of adult development in Sarcophaga argyrostoma by β-ecdysone

Sarcophaga argyrostoma pupae otherwise destined to diapause can be made to initiate prompt, complete adult development by injection of 0.2 to 0.3 μg β-ecdysone/g body weight, if the injection is given soon after head eversion at the time ecdysone levels would normally increase in developing animals. At these doses some animals show only the earliest signs of adult development and then enter diapause. After two weeks in diapause, a 40-fold higher dose (10 μg/g) is required to initiate development promptly. β-ecdysone can also accelerate the termination of diapause after a time delay which is dose-dependent. Doses of 6 to 10 μg/g lead to hyperhormonal abnormalities affecting antennae, genitalia, and bristle orientation.     

Tissue distribution of the laminin β1 and β2 chain during embryonic and fetal human development

Laminins are the major glycoproteins present in all basement membranes. Previously, we showed that perlecan is present during human development. Although an overview of mRNA-expression of the laminin β1 and β2 chains in various developing fetal organs is already available, a systematic localization of the laminin β1 and β2 chains on the protein level during embryonic and fetal human development is missing. Therefore, we studied the immunohistochemical expression and tissue distribution of the laminin β1 and β2 chains in various developing embryonic and fetal human organs between gestational weeks 8 and 12. The laminin β1 chain was ubiquitously expressed in the basement membrane zones of the brain, ganglia, blood vessels, liver, kidney, skin, pancreas, intestine, heart and skeletal system. Furthermore, the laminin β2 chain was present in the basement membrane zones of the brain, ganglia, skin, heart and skeletal system. The findings of this study support and expand upon the theory that these two laminin chains are important during human development.     

Histochemical demonstration of β-glucuronidase activity during sequential molar development in the Swiss albino mouse

Summary Using naphthol AS-BI -D glucuronide as a substrate and diazotized pararosanilin as a coupling reagent, the distribution of -glucuronidase was studied throughout mouse molar tooth development. A strongly positive reaction was not only observed in some of the cellular components of the stellate reticulum of the enamel organ, but also in certain cells of the subodontoblastic cell zone. A moderate reaction was noted in the distal cytoplasm of the odontoblasts particularly in the older ones which were found in the cuspal areas. A mild reaction was elicited in the stratum intermedium and a weak one in the ameloblasts and prospective dental pulp.Supported PHS. Grant No. 2800-02 National Institute of Dental Research. National Institutes of Health.     

Secretory expression of the α-subunit of human coagulation factor XIII in the yeast Pichia pastoris

Pro-FXIIIa (the -subunit of FXIII with activation peptide, which must be removed to produce the active form of FXIIIa), cloned from human placenta cDNA library, was overexpressed in the methylotrophic yeast Pichia pastoris GS115 (his4) and secreted into the culture medium to yield the recombinant pro-FXIIIa subunit with a predicted molecular mass of approximately 83 kDa. The gene was located immediately downstream of the strong yeast alcohol oxidase promoter (AOX1). In shake flask culture, recombinant pro-FXIIIa (rFXIIIa) was secreted into the culture medium at above 50 mg l^–1. The fibrin-stabilizing activity of the recombinant pro-FXIIIa, after thrombin activation, was confirmed using fibrin cross-linking patterns, and analyzed by SDS-PAGE.     

The role of TRPM2 in pancreatic β-cells and the development of diabetes

TRPM2 is a Ca²⁺-permeable non-selective cation channel that can be activated by adenosine dinucleotides, hydrogen peroxide, or intracellular Ca²⁺. The protein is expressed in a wide variety of cells, including neurons in the brain, immune cells, endocrine cells, and endothelial cells. This channel is also well expressed in β-cells in the pancreas. Insulin secretion from pancreatic β-cells is the primary mechanism by which the concentration of blood glucose is reduced. Thus, impairment of insulin secretion leads to hyperglycemia and eventually causes diabetes. Glucose is the principal stimulator of insulin secretion. The primary pathway involved in glucose-stimulated insulin secretion is the ATP-sensitive K⁺ (K_ATP) channel to voltage-gated Ca²⁺ channel (VGCC)-mediated pathway. Increases in the intracellular Ca²⁺ concentration are necessary for insulin secretion, but VGCC is not sufficient to explain [Ca²⁺]_i increases in pancreatic β-cells and the resultant secretion of insulin. In this review, we focus on TRPM2 as a candidate for a [Ca²⁺]_i modulator in pancreatic β-cells and its involvement in insulin secretion and development of diabetes. Although further analyses are needed to clarify the mechanism underlying TRPM2-mediated insulin secretion, TRPM2 could be a key player in the regulation of insulin secretion and could represent a new target for diabetes therapy.     

Hsp90 regulates protein synthesis by activating the heme-regulated eukaryotic initiation factor 2α (eIF-2α) kinase in rabbit reticulocyte lysates

Heat shock protein 90 (Hsp90), an abundant and ubiquitous cytoplasmic protein has recently been indicated to participate in the regulation of protein synthesis by interacting with the heme-regulated eukaryotic initiation factor 2α (eIF-2α) kinase, also known as the heme-regulated inhibitor (HRI). However, there exists an ambiguity on the exact nature of its action. In this investigation, the interaction of Hsp90 and HRI has been examined bothin vitro using purified proteins, andin situ in rabbit reticulocyte lysates subjected to heat shock and treatment with N-ethylmaleimide (NEM), a sulfhydryl reagent known to induce stress response. During heat shock or NEM-treatment of reticulocyte lysates, Hsp90 co-immunoprecipitated with activated HRI by anti-HRI monoclonal antibodies. Furthermore, the amount of Hsp90 being associated with HRI was a function of duration of heat shock and was correlated with the extent of HRI activation. Interestingly, simultaneous heat shock and NRM-treatment of reticulocyte lysates led to maximal association of HRI and Hsp90, leaving nearly no free HRI in the lysates.In vitro, with the purified proteins, the autokinase and the eIF-2α kinase activities of HRI were enhanced when HRI was pre-incubated with Hsp90, both in the presence and absence of hemin. These data, therefore, clearly demonstrate that Hsp90 interacts with HRI during stress, and that this association leads to activation of HRI and thereby inhibition of protein synthesis at the level of initiation. Considering the ubiquitous nature of Hsp90 and the presence of HRI or HRI-like eIF-2α kinase activity in a number of organisms, it is highly possible that Hsp90 may universally mediate down regulation of global protein synthesis during stress response.     

N-cadherin and β1-integrins cooperate during the development of the enteric nervous system

Cell adhesion controls various embryonic morphogenetic processes, including the development of the enteric nervous system (ENS). Ablation of β1-integrin (β1-/-) expression in enteric neural crest cells (ENCC) in mice leads to major alterations in the ENS structure caused by reduced migration and increased aggregation properties of ENCC during gut colonization, which gives rise to a Hirschsprung's disease-like phenotype. In the present study, we examined the role of N-cadherin in ENS development and the interplay with β1 integrins during this process. The Ht-PA-Cre mouse model was used to target gene disruption of N-cadherin and β1 integrin in migratory NCC and to produce single- and double-conditional mutants for these two types of adhesion receptors. Double mutation of N-cadherin and β1 integrin led to embryonic lethality with severe defects in ENS development. N-cadherin-null (Ncad-/-) ENCC exhibited a delayed colonization in the developing gut at E12.5, although this was to a lesser extent than in β1-/- mutants. This delay of Ncad-/- ENCC migration was recovered at later stages of development. The double Ncad-/-; β1-/- mutant ENCC failed to colonize the distal part of the gut and there was more severe aganglionosis in the proximal hindgut than in the single mutants for N-cadherin or β1-integrin. This was due to an altered speed of locomotion and directionality in the gut wall. The abnormal aggregation defect of ENCC and the disorganized ganglia network in the β1-/- mutant was not observed in the double mutant. This indicates that N-cadherin enhances the effect of the β1-integrin mutation and demonstrates cooperation between these two adhesion receptors during ENS ontogenesis. In conclusion, our data reveal that N-cadherin is not essential for ENS development but it does modulate the modes of ENCC migration and acts in concert with β1-integrin to control the proper development of the ENS.     

Developmental changes in β-subunit composition of Na,K-ATPase in the Drosophila eye

The Drosophila genome contains at least three loci for the Na,K-ATPase β-subunit; however, only the protein products of nrv1 and nrv2 have been characterized hitherto. Here, we provide evidence that nrv3 also encodes for a functional Na,K-ATPase β-subunit, as its protein product co-precipitates with the Na,K-ATPase α-subunit. Nrv3 expression in adult flies is restricted to the nervous system in which Nrv3 is enriched in selective types of sensory cells. Because Nrv3 expression is especially prominent in the compound eye, we have analyzed the subcellular and developmental distribution of Nrv3 within the visual cells and related this distribution to those of the α-subunit and of the β-subunits Nrv1 and Nrv2. Prospective visual cells express Nrv2 in the third larval instar stage and during the first half of pupal development. During the last third of pupal life, Nrv3 gradually replaces Nrv2 as the Na,K-ATPase β-subunit in the photoreceptor cells. Adult photoreceptors express Nrv3 as their major β-subunit; the visual cells R1–R6 co-express Nrv2 at a low level, whereas R7 and R8 co-express Nrv1. Notably, β-subunits do not co-distribute exactly with the α-subunit at some developmental stages, supporting the concept that the α-subunit and β-subunit can exist in the plasma membrane without being engaged in α/β heterodimers. The non-visual cells within the compound eye express almost exclusively Nrv2, which segregates together with the α-subunit to septate junctions throughout development.