Two Forms of GO Type G Proteins: Identification and Distribution in Various Rat Tissues and Cloned Cells

A G(o) type G protein distinct from the major species of G(o) was recently isolated from bovine brain and designated G(o)*. The cDNAs encoding two forms of mammalian G(o) alpha were also isolated and designated GoA alpha and GoB alpha. To recognize two forms of G(o) type G proteins, we raised antibodies in rabbits against two peptides with sequences found only in the respective proteins of murine GoA alpha (SNTYEDAAAYIQTQF) and GoB alpha (TEAVAHIQGQYWSK). Purified anti-GoA alpha antibodies reacted with the major species of G(o) alpha purified from bovine and rat brain, whereas anti-GoB alpha antibodies reacted only with rat G(o)*alpha, but not with the major species of G(o) alpha or bovine G(o)*alpha. These results indicate that the major species of G(o) alpha is encoded by GoA alpha cDNA and G(o)*alpha is encoded by GoB alpha cDNA. Using these antibodies, the distribution of GoA and GoB was studied in various rat tissues and cloned cells. Both GoA and GoB were present in many tissues, but their distribution in peripheral tissues was distinct. GoA alpha seemed to associate mainly with neural tissues. On the other hand, relatively high concentrations of GoB alpha were present in the brain, pituitary gland, adipose tissue, lung, and testis. The concentrations of both GoA and GoB in the brain increased during ontogenic development, but the increase in GoB was observed at a later age. Both GoA and GoB were found in such cloned cells as PC12, NG108-15, C6, GA-1, G8, and 3T3-L1 cells. Treatment of PC12 cells with nerve growth factor caused the extension of neuron-like processes and the increase in the level of GoA, but not in the level of GoB.     

Selective Patterns of Expression of G Protein α Subunits During In Vitro Development of Hypothalamic Neurons

Abstract: Expression of different α subunits of G proteins was studied in hypothalamic primary cultures grown in defined medium and enriched in either neurons or glial (astrocyte) cells. In parallel, the cellular distribution of Gi, Gs, and GoA subunits was visualized by in situ hybridization. Immunoblots using specific antisera and hybridization of mRNAs with specific oligonucleotide probes allowed us to characterize Gs, Gi2, and GoA as major neuronal G proteins in the hypothalamus, whereas the glial cells expressed mostly Gs, Gi2, and GoB forms. Gi was found to be expressed very early and transiently in the culture, whereas expression of Gs and GoA increased regularly with time.     

Inhibition of the omega-conotoxin-sensitive calcium current by distinct G proteins.

Leu-enkephalin (Leu-Enk), norepinephrine (NE), somatostatin (SS), and bradykinin (BK) decrease the voltage-dependent calcium current in NG108-15 cells. Here we have investigated whether distinct G proteins, or a G protein common to all of the pathways, mediates this inhibition. We found that pertussis toxin (PTX) reduced all of these transmitter actions, except that of BK. To examine which of the PTX-sensitive pathways is transduced by GoA, we constructed an NG108-15 cell line that stably expresses a mutant, PTX-resistant alpha subunit of GoA. After treatment with PTX, the mutant GoA alpha rescued the Leu-Enk and NE pathways but not the SS pathway. At least three different G proteins can transduce receptor-mediated inhibition of calcium currents in nerve cells. The effects of these G proteins appear to converge on the omega-conotoxin GVIA-sensitive calcium current.     

A coral reef refuge in the Red Sea

The stability and persistence of coral reefs in the decades to come is uncertain due to global warming and repeated bleaching events that will lead to reduced resilience of these ecological and socio‐economically important ecosystems. Identifying key refugia is potentially important for future conservation actions. We suggest that the Gulf of Aqaba (GoA) (Red Sea) may serve as a reef refugium due to a unique suite of environmental conditions. Our hypothesis is based on experimental detection of an exceptionally high bleaching threshold of northern Red Sea corals and on the potential dispersal of coral planulae larvae through a selective thermal barrier estimated using an ocean model. We propose that millennia of natural selection in the form of a thermal barrier at the southernmost end of the Red Sea have selected coral genotypes that are less susceptible to thermal stress in the northern Red Sea, delaying bleaching events in the GoA by at least a century.     

The Relationship of Goα Subunit Deamidation to the Tissue Distribution, Nucleotide Binding Properties, and βγ Dimer Interactions of Goα Subunit Isoforms

The distribution and properties in brain of the alpha subunits of the major bovine brain Go isoforms, GoA, GoB and GoC, were characterized. The alpha(o)A and alpha(o)B isoforms arise from alternative splicing of RNAs from a single alpha(o) gene, whereas alpha(o)C is a deamidated form of alpha(o)A. All three Go isoforms purify from brain with different populations of betagamma dimers. This variable subunit composition of Go heterotrimers is likely a consequence of their functional differences. This study examined the biochemical properties of the alpha(o) isoforms to see if these properties explain the variable betagamma composition of their heterotrimers. The brain distribution of alpha(o)B differed substantially from that of alpha(o)A and alpha(o)C, as did its guanine nucleotide binding properties. The unique subunit composition of GoB can be explained by its expression in different brain regions. The alpha(o)A and alpha(o)C showed slight differences in guanine nucleotide binding properties but no preference for particular betagamma dimers when reassociated with a heterogeneous betagamma pool. The alpha(o)C protein occurred in a constant ratio to alpha(o)A throughout the brain, but was a much larger percent of total brain alpha(o) than previously thought, approximately 35%. These results suggest that alpha(o)A is a precursor of alpha(o)C and that the association of G(o)alpha subunits with different betagamma dimers reflects the function of an adaptive, G-protein signaling mechanism in brain.     

ATP binding by glutamyl-tRNA synthetase is switched to the productive mode by tRNA binding

Aminoacyl-tRNA synthetases catalyze the formation of an aminoacyl-AMP from an amino acid and ATP, prior to the aminoacyl transfer to tRNA. A subset of aminoacyl-tRNA synthetases, including glutamyl-tRNA synthetase (GluRS), have a regulation mechanism to avoid aminoacyl-AMP formation in the absence of tRNA. In this study, we determined the crystal structure of the 'non-productive' complex of Thermus thermophilus GluRS, ATP and L-glutamate, together with those of the GluRS.ATP, GluRS.tRNA.ATP and GluRS.tRNA.GoA (a glutamyl-AMP analog) complexes. In the absence of tRNA(Glu), ATP is accommodated in a 'non-productive' subsite within the ATP-binding site, so that the ATP alpha-phosphate and the glutamate alpha-carboxyl groups in GluRS. ATP.Glu are too far from each other (6.2 A) to react. In contrast, the ATP-binding mode in GluRS.tRNA. ATP is dramatically different from those in GluRS.ATP.Glu and GluRS.ATP, but corresponds to the AMP moiety binding mode in GluRS.tRNA.GoA (the 'productive' subsite). Therefore, tRNA binding to GluRS switches the ATP-binding mode. The interactions of the three tRNA(Glu) regions with GluRS cause conformational changes around the ATP-binding site, and allow ATP to bind to the 'productive' subsite.     

Identification and isolation of common and tissue-specific geranylgeranylated gamma subunits of guanine-nucleotide-binding regulatory proteins in various tissues.

Heterotrimeric guanine-nucleotide-binding regulatory proteins (G proteins) have been classified into several subtypes on the basis of the properties of their alpha subunits, though a notable multiplicity of gamma subunits has also been demonstrated. To investigate whether each subtype of alpha subunit is associated with a particular gamma subunit, various oligomeric G proteins, purified from bovine tissues, were subjected to gel electrophoresis in a Tricine buffer system. All G proteins examined were shown to have more than two kinds of gamma subunit. Of the brain G proteins, GoA, GoB, and Gi1 contain the same set of three gamma subunits, but Gi2 contains only two of these subunits. Lung Gi1 and Gi2 and spleen Gi2 and Gi3 had similar sets of two gamma subunits, one of which was distinct from the gamma subunits of brain G proteins. These observations indicate that each subtype of alpha subunit is associated with a variety of beta gamma subunits, and that the combinations differ among cells. For analyses of the structural diversity of the gamma subunits, beta gamma subunits were purified from the total G proteins of each tissue and subjected to reverse-phase HPLC under denaturing conditions, where none of the beta subunits were eluted from the column. Three distinct gamma subunits were isolated in this way from brain beta gamma subunits. In contrast, lung and spleen beta gamma subunits contained at least five gamma subunits, the elution positions and electrophoretic mobilities of which were indistinguishable between the two tissues. Among several gamma subunits, two subspecies appeared to be common to the three tissues. In fact, in each case, the partial amino acid sequence of the most abundant gamma subunit in each tissue was identical, and the sequences coincided exactly with that of 'gamma 6' [Robishaw, J. D., Kalman, V. K., Moomaw, C. R. & Slaughter, C. A. (1989) J. Biol. Chem. 264, 15758-15761]. Fast-atom-bombardment mass spectrometry analysis indicated that this abundant gamma subunit in lung and spleen was geranylgeranylated and carboxymethylated at the C-terminus, as was 'gamma 6' from brain. In addition to abundant gamma subunits, other tissue-specific gamma subunits were also shown to be geranylgeranylated by gas-chromatography-coupled mass spectrometry analysis of Raney nickel-treated gamma subunits. These results suggest that most gamma subunits associated with many different subtypes of alpha subunit are geranylgeranylated in a variety of tissues, with the single exception being the retina where the G protein transducin has a farnesylated gamma subunit.     

Oligonucleotide directed mutagenesis of the human beta-globin gene: a general method for producing specific point mutations in cloned DNA.

A nonadecanucleotide has been used both as a site specific mutagen to introduce a T leads to A transversion mutation in the human beta-globin gene cloned in pBR322 as well as a probe to screen transformed colonies for the desired mutant. The specificity of the oligonucleotide as a mutagen and as a hybridization probe provide a general method for producing site specific mutations in DNA cloned in plasmid vectors such as pBR322.     

Changes in prostacyclin and thromboxane biosynthesis and their catabolic enzyme activity in kidneys of aging rats

The effects of aging on the prostacyclin and thromboxane biosynthesis and prostaglandin catabolic enzyme activity in rat kidney were investigated. The prostacyclin biosynthesis, using arachidonic acid as substrate, was the greatest in young kidneys (2 months old) and then progressively decreased in mature (12 months old) and old (24 months old) kidneys, while thromboxane biosynthetic activity showed no significant change as a function of age. When prostaglandin H2 was used as substrate, the prostacyclin and thromboxane biosynthesis showed similar results as when arachidonic acid was used as substrate; the prostacyclin biosynthesis progressively decreased and thromboxane biosynthesis showed no significant change as a function of age. The fatty acid cyclooxygenase in kidney was measured by a specific radioimmunoassay. No significant change in renal fatty acid cyclooxygenase as a function of age was found. Thus, we concluded that the progressive decrease in renal prostacyclin biosynthesis as a function of age is due to a defect in prostacyclin synthetase in aged kidneys. The prostaglandin catabolic enzyme, NAD+-dependent 15-hydroxyprostaglandin dehydrogenase, in kidneys was also investigated. The enzyme activity progressively decreased as a function of age, which suggested a decrease in the metabolism of thromboxane A2 in aged kidneys. The present results, indicating a decrease in renal prostacyclin biosynthesis and renal 15-hydroxyprostaglandin dehydrogenase activity with aging, might contribute to a plausible explanation of the progressive decrease in renal functions in the elderly.     

Biotechnological uses of cyanobacteria

Cyanobacteria (blue-green algae) are 0₂-evolving photosynthesizing prokaryotes that have an extensive history of use as a human food source and as a fertilizer in rice fields. They have also been recognized as an excellent source of vitamins and proteins and as such are found in health food stores in North America and elsewhere. Cyanobacteria have a great deal of potential as a source of fine chemicals, as a biofertilizer and as a source of renewable fuel. This potential is being realized as data from research in the areas of the physiology and chemistry of these organisms are gathered and as the knowledge of cyanobacterial genetics and genetic engineering increases. We review, here, the present (and possible future) uses of cyanobacteria and assess the state of the art with respect to the genetic manipulation of cyanobacteria.     

Age dependent changes in enzymes involved in macromolecular synthesis in Turbatrix aceti.

The specific activities of elongation factor 1, RNA and DNA polymerase, as well as fructose-1,6-diphosphate aldolase were determined as a function of age in the nematode Turbatrix aceti. It was found that the specific activities of both DNA polymerase and aldolase declined constantly as the animal aged. In contrast, both elongation factor 1 and RNA polymerase showed sharp increases in their specific activities at 5 and 15 days, respectively, before ultimately declining. It was also shown that elongation factor 1, which exists mainly as a high molecular weight species in the young animal, undergoes a conversion to a lower molecular weight species as the organism ages. In addition there is a progressive accumulation of inactive or partially active elongation factor 1 molecules in older animals. The addition of α-tocopherol to the growth medium of these nematodes resulted in an increased life-span as well as alterations in the patterns of the enzyme activities.     

Transforming growth factor beta1 and prostaglandin E2 concentrations are associated with bone formation markers in ulcerative colitis patients

Osteoporosis is a significant complication of a multifactoral etiology associated with inflammatory bowel disease. The aim of study was to evaluate the relationships between bone mineral density as well as bone turnover markers and inflammatory activity modulators (i.e., PGE2 and TGFbeta1) in ulcerative colitis (UC). Twenty-one active ulcerative colitis subjects and 14 healthy individuals were included into the study. We observed no significant differences in serum concentrations of osteoprotegerin and osteocalcin, as well as bone mineral density between UC patients and healthy individuals. Plasma concentrations of PGE2, TGFbeta1 and TNF-alpha were significantly higher in UC patients than in controls. Serum osteocalcin demonstrated a positive correlation with both serum PGE2 and plasma TGFbeta1. Moreover there was significant correlation between osteoprotegerin and TGFbeta1 as well as serum TNF-alpha concentrations. In conclusion a positive association between PGE2 and TGFbeta1 and bone formation markers-osteoprotegerin and osteocalcin, as well as a comparable BMD in UC patients and healthy individuals was shown. Our results may indicate that increase of PGE2 as well as TGFbeta1 concentrations may play a protective role against bone loss in ulcerative colitis patients.     

Hereditary defects in both germ cells and the blood-testis barrier system in as-mutant rats: evidence from spermatogonial transplantation and tracer-permeability analysis

The rat mutant allele as is located on chromosome 12. Homozygous (as/as) males show arrested spermatogenesis, mainly at the pachytene spermatocyte stage. It is not clear whether this defective spermatogenesis is caused by a failure in a somatic cell component that supports spermatogenesis or in the germ cell itself. Spermatogonial transplantation was performed to identify the genetically defective site in the as/as testis. In experiment 1, germ cells collected from as/as testes were transplanted into the testes of immunodeficient mice and normal rats. In experiment 2, normal rat germ cells were transplanted into as/as testes. The results of experiment 1 showed arrest of spermatogenesis at the pachytene spermatocyte stage, accompanied by a characteristic morphological feature, i.e., the formation of inclusion-like bodies in the cytoplasm, in both rat and mouse recipients. These results revealed the intrinsic effect of the mutant gene(s) on germ cells. In experiment 2, no restoration of spermatogenesis was detected in the recipient testes despite thorough histological examination. These results suggest that defects in a somatic cell component in as/as testes prevent the donor germ cells from colonizing and regaining their spermatogenetic ability. When the seminiferous epithelium of the as/as testis was examined by electron microscopy, no morphological abnormalities, including the formation of ectoplasmic specializations between adjacent Sertoli cells, were observed in the somatic cell components. However, when cytochrome c was applied as a tracer material, it penetrated the tight junctions between the Sertoli cells, indicating dysfunction of the blood-testis barrier in the as/as testis. The lack of restoration of spermatogenesis in the as/as testis after transplantation of normal germ cells may have been caused by the unfavorable environment in the seminiferous epithelium resulting from the incomplete barrier system between adjoining Sertoli cells. The gene(s) at the as locus may have a role in both germ cell differentiation and the establishment of the blood-testis barrier.     

The specific targeting of immune regulation: T-cell responses against Indoleamine 2,3-dioxygenase

Indoleamine 2,3-dioxygenase (IDO) is an immunoregulatory enzyme that is implicated in suppressing T-cell immunity in many settings including cancer. In recent years, we have described spontaneous CD8(+) as well as CD4(+) T-cell reactivity against IDO in the tumor microenvironment of different cancer patients as well as in the peripheral blood of both cancer patients and to a lesser extent in healthy donors. We have demonstrated that IDO-reactive CD8(+) T cells were peptide-specific, cytotoxic effector cells, which are able to recognize and kill IDO-expressing cells including tumor cells as well as dendritic cells. Consequently, IDO may serve as a widely applicable target for immunotherapeutic strategies with a completely different function as well as expression pattern compared to previously described antigens. IDO constitutes a significant counter-regulatory mechanism induced by pro-inflammatory signals, and IDO-based immunotherapy may consequently be synergistic with additional immunotherapy. In this regard, we have shown that the presence of IDO-specific T cells boosted immunity against CMV and tumor antigens by eliminating IDO(+) suppressive cells and changing the regulatory microenvironment. The current review summarizes current knowledge of IDO as a T-cell antigen, reports the initial results that are suggesting a general function of IDO-specific T cells in immunoregulation, and discusses future opportunities.     

FSI analysis of a human trachea before and after prosthesis implantation

In this work we analyzed the response of a stenotic trachea after a stent implantation. An endotracheal stent is the common treatment for tracheal diseases such as stenosis, chronic cough, or dispnoea episodes. Medical treatment and surgical techniques are still challenging due to the difficulties in overcoming potential complications after prosthesis implantation. A finite element model of a diseased and stented trachea was developed starting from a patient specific computerized tomography (CT) scan. The tracheal wall was modeled as a fiber reinforced hyperelastic material in which we modeled the anisotropy due to the orientation of the collagen fibers. Deformations of the tracheal cartilage rings and of the muscular membrane, as well as the maximum principal stresses, are analyzed using a fluid solid interaction (FSI) approach. For this reason, as boundary conditions, impedance-based pressure waveforms were computed modeling the nonreconstructed vessels as a binary fractal network. The results showed that the presence of the stent prevents tracheal muscle deflections and indicated a local recirculatory flow on the stent top surface which may play a role in the process of mucous accumulation. The present work gives new insight into clinical procedures, predicting their mechanical consequences. This tool could be used in the future as preoperative planning software to help the thoracic surgeons in deciding the optimal prosthesis type as well as its size and positioning.     

The dsbA-dsbB disulfide bond formation system of Burkholderia cepacia is involved in the production of protease and alkaline phosphatase, motility, metal resistance, and multi-drug resistance

In a previous study, we isolated a dsbB mutant of Burkholderia cepacia KF1 and showed that phenotypes of protease production and motility are dependent on DsbB, a membrane-bound disulfide bond oxidoreductase. We have now isolated a dsbA mutant by transposon mutagenesis, cloned the dsbA gene encoding a periplasmic disulfide bond oxidoreductase, and characterized the function of the DsbA-DsbB disulfide bond formation system in B. cepacia. The complementing DNA fragment had an open reading frame for a 212-amino acid polypeptide with a potential redox-active site sequence of Cys-Pro-His-Cys that is homologous to Escherichia coli DsbA. The dsbA mutant, as well as the previously isolated dsbB mutant, was defective in the production of extracellular protease and alkaline phosphatase, as well as in motility. In addition, mutation in the DsbA-DsbB system resulted in an increase in sensitivity to Cd2+ and Zn2+ as well as a variety of antibiotics including beta-lactams, kanamycin, erythromycin, novobiocin, ofloxacin and sodium dodecyl sulfate. These results suggested that the DsbA-DsbB system might be involved in the formation of a metal efflux system as well as a multi-drug resistance system.     

Enantioselective synthesis of amines via reductive amination with a dehydrogenase mutant from Exigobacterium sibiricum: Substrate scope,co-solvent tolerance and biocatalyst immobilization

In recent years, the reductive amination of ketones in the presence of amine dehydrogenases emerged as an attractive synthetic strategy for the enantioselective preparation of amines starting from ketones, an ammonia source, a reducing reagent and a cofactor, which is recycled in situ by means of a second enzyme. Current challenges in this field consists of providing a broad synthetic platform as well as process development including enzyme immobilization. In this contribution these issues are addressed. Utilizing the amine dehydrogenase EsLeuDH-DM as a mutant of the leucine dehydrogenase from Exigobacterium sibiricum, a range of aryl-substituted ketones were tested as substrates revealing a broad substrate tolerance. Kinetics as well as inhibition effects were also studied and the suitability of this method for synthetic purpose was demonstrated with acetophenone as a model substrate. Even at an elevated substrate concentration of 50?mM, excellent conversion was achieved. In addition, the impact of water-miscible co-solvents was examined, and good activities were found when using DMSO of up to 30% (v/v). Furthermore, a successful immobilization of the EsLeuDH-DM was demonstrated utilizing a hydrophobic support and a support for covalent binding, respectively, as a carrier.     

Social exchange and reciprocity: confusion or a heuristic?

We propose that a “social exchange heuristic” is as important as the cheater detection mechanism for attaining mutual cooperation in social exchange. The social exchange heuristic prompts people to perceive a mixed-motive situation, such as the Prisoner's Dilemma (PD), as an Assurance Game (AG) situation in which cooperation is a personally better choice than defection insofar as the partner is cooperating as well. We demonstrate the operation of the social exchange heuristic through a comparison of the ordinary one-shot, simultaneous PD with the one-shot, sequential PD. Participants in the current experiments, involving a total of 261 volunteers, committed a logical error in the direction of favoring mutual cooperation as the situation involved more serious consequences. This result strongly suggests the operation of a domain specific “bias” that encourages pursuit of mutual cooperation in social exchange.     

The Taxon as an Ontological Problem

Although the term taxon is one of the most common concepts in biology, a range of its meanings cannot be comprehended by an universal definition. Usually, biologists construe their knowledge of “the same” taxon by substantially different interpretations, so they find themselves in need either to justify this “multiplication of taxon essences”, or to surmount their plurality unifying its interpretations into a single explanation of what a taxon is. In both cases, an ontological status (“reality”) of that taxon is questioned. Therefore, discrepancy between universality of the taxon concept in biology and unavoidable plurality of its interpretations can be regarded as a source of problem of the taxon ontology. The present work aims to clarify the premises of this discrepancy using phenomenological approach. Three ways of the taxon positing (as a class, as a place in the world, and as a individualized body) have been distinguished. Taxon as a class is established by common essence that is shared by a set of living beings. These living beings are regarded as speculative objects beyond an idea of the world, i.e. as objects of the experimental science. A question about ontology of taxon as a class refers to the scholastic problem of universalia; its status can be defined within the scope of the nominalism/realism opposition. Taxon as a place of common appearance of the specimens is regarded in the context of the etiological relations unifying various entities into the entire world. Taxon as a place refers to a certain position in the Natural System that is construed as an etiological map of the world. In this case a specimen of a living being is known as a curiosity, i.e. representant of its relationships as well as of the place of its origin. Ontological status of a taxon as a place is to be clarified within the framework of the natural/artificial opposition. The positing of a taxon as a collective body marked off by limits of joint survival of living beings is characteristic for biology in the strict sense which arose in the very beginning of the 19 century. A taxon as a body established by the techniques of disciplinary power sensu M. Foucault extended from the human bodies to bodies of other living beings. The ontological status of a taxon as a collective body can be defined within the scope of the wild/domesticated opposition. Therefore, the discrepancy between the universality of the taxon concept and the plurality of its interpretations is underlayed by interpenetration of three distinct modi of taxon establishing. Distinguishing between these three modi can clarify sources of ontological problems concerned with the term taxon in each case when they arise.