14-3-3 protects against stress-induced apoptosis.

Expression of human Bax, a cardinal regulator of mitochondrial membrane permeabilization, causes death in yeast. We screened a human cDNA library for suppressors of Bax-mediated yeast death and identified human 14-3-3ß/α, a protein whose paralogs have numerous chaperone-like functions. Here, we show that, yeast cells expressing human 14-3-3ß/α are able to complement deletion of the endogenous yeast 14-3-3 and confer resistance to a variety of different stresses including cadmium and cycloheximide. The expression of 14-3-3ß/α also conferred resistance to death induced by the target of rapamycin inhibitor rapamycin and by starvation for the amino acid leucine, conditions that induce autophagy. Cell death in response to these autophagic stimuli was also observed in the macroautophagic-deficient atg1Δ and atg7Δ mutants. Furthermore, 14-3-3ß/α retained its ability to protect against the autophagic stimuli in these autophagic-deficient mutants arguing against so called 'autophagic death'. In line, analysis of cell death markers including the accumulation of reactive oxygen species, membrane integrity and cell surface exposure of phosphatidylserine indicated that 14-3-3ß/α serves as a specific inhibitor of apoptosis. Finally, we demonstrate functional conservation of these phenotypes using the yeast homolog of 14-3-3: Bmh1. In sum, cell death in response to multiple stresses can be counteracted by 14-3-3 proteins.

Expressing and functional analysis of mammalian apoptotic regulators in yeast.

The ease by which yeast can be manipulated in conjunction with their similarities to cells of more complex metazoans makes many yeast species, particularly Saccharomyces cerevisae, very attractive models for the study of conserved evolutionary processes that occur in eukaryotes. The ability to functionally express heterologous genes in these cells has allowed the development of countless new and elegant approaches leading to detailed structure-function analysis of numerous mammalian genes. Of these, the most informative have been the studies involving the analysis of regulators that have no direct or obvious sequence orthologue in yeast, including members of the Bcl-2 family of proteins, caspases and tumour suppressors. Here we review the field and provide evidence that these studies have served to further understand mammalian apoptosis.

Regulatory mechanisms involved in modulating RGS function.

Regulator of G-Protein Signaling (RGS) refers to a conserved 120-125 amino acid motif that was first identified by its ability to negatively regulate G-Protein-Coupled Receptor (GPCR) signalling. Mechanistically, RGSs were found to regulate GPCR responses by binding to and stimulating the GTPase activity of the receptor-activated GTP-bound G alpha subunits. There are now over 25 mammalian RGSs containing proteins that are reported to carry out a variety of functions, many of which are unrelated to GPCR signalling. RGS proteins range in size from small proteins that contain little more than an RGS box to very large proteins that contain a variety of domains. The selectivity of function of the RGS proteins is attributable to the divergence of the RGS sequences as well as the presence of a variety of functional motifs, which allow them to interact with other proteins. Here we focus on the RGSs that are involved in modulating GPCR signalling by reviewing the diversity of the mechanisms involved in regulating these RGSs.

Regulators of G-protein signaling (RGS) 1 and 16 are induced in response to bacterial lipopolysaccharide and stimulate c-fos promoter expression.

Regulators of G-protein signaling (RGS) are negative regulators of G-protein-coupled receptor (GPCR) signaling. Sepsis is a pathophysiological condition that is induced primarily in response to bacterial infection and is associated with decreased responsiveness to a number of vasoactive GPCR agonists. Using a degenerate RT-PCR screen, we report that RGS1 and RGS16 were amplified from the heart and aorta of septic animals. By Northern blot analysis, RGS1 and RGS16 were upregulated, with their respective levels increasing 6- and 50-fold in septic hearts. Using a yeast-based bioassay, both RGS1 and RGS16 were found to be equipotent in their ability to attenuate GPCR signaling. These results suggest that both RGS1 and RGS16 contribute to the sepsis-mediated decrease in GPCR signaling. Elevated levels of some RGSs may also lead to an increase in Gbetagamma-activated signaling pathways in the absence of GPCR agonists. Using a c-fos luciferase reporter gene that is responsive to Gbetagamma-activated signaling pathways, we observed a respective 8- and 7-fold increase in the basal luciferase in serum-deprived transfected mammalian cells overexpressing RGS1 or RGS16. This suggests that RGSs play a role in promoting the sepsis-mediated increases in the activation of intracellular signal transduction pathways.

Expression and regulation of protein inhibitor of neuronal nitric oxide synthase in ventilatory muscles.

In skeletal muscle fibers, nitric oxide (NO) is synthesized by neuronal NO synthase (nNOS) and regulates excitation-contraction coupling, glucose uptake, and mitochondrial respiration. Recently, a novel 89-amino acid protein, designated protein inhibitor of nNOS (PIN), has been shown to interact with and specifically inhibit nNOS activity. In this study, we investigated the distribution, localization, and regulation of PIN expression in ventilatory and limb muscles of various species. Amplified PIN cDNA from the rat diaphragm revealed an open reading frame identical to that of human PIN. Among muscles of adult rats, PIN mRNA was strongly expressed in muscles rich in type I fibers, whereas much weaker expression was evident in muscles rich in type II fibers. By comparison, PIN protein expression was not related to fiber-type distribution. Similarly, PIN protein was equally expressed among rat, mouse, and human diaphragms. Both PIN mRNA and PIN protein were expressed at much higher levels in the embryonic rat diaphragm than in adult muscle. Immunohistochemistry revealed that PIN protein was localized in close proximity to the sarcolemma and nuclei. PIN protein was also abundant in muscle spindles and axons of nerves supplying skeletal muscle fibers. We conclude that PIN is expressed in various skeletal muscle fibers and that its expression is regulated during muscle development. The localization of PIN in muscle regions containing abundant nNOS protein suggests that it plays a role in the regulation of NO synthesis in skeletal muscle fibers.

Ontogenesis of nitric oxide synthases in the ventilatory muscles.

Nitric oxide (NO) acts as an endogenous mediator in mature skeletal muscle. In this study, we investigated the regulation of the endothelial (eNOS) and neuronal (nNOS) isoforms of nitric oxide synthase (NOS) in skeletal-muscle development (rat diaphragm). Muscle NOS activity, nNOS and eNOS protein, and mRNA expressions were markedly increased during the late gestational and early postnatal periods. Expression of both isoforms, however, declined progressively thereafter. Similarly, argininosuccinate lyase and argininosuccinate synthetase, both involved in the recycling of L-citrulline to L-arginine, were expressed at high levels in rat embryonic and neonatal diaphragms, with gradual reduction in their expression during late postnatal development. Immunostaining revealed extensive nNOS expression at the sarcolemma in neonatal and mature diaphragms, whereas eNOS expression was limited to the endothelium. Both neonatal and adult diaphragms expressed an alternatively spliced nNOS isoform with an insert of 34 amino acids between exons 16 and 17. In vitro-generated muscle force rose significantly after NOS inhibition in both neonatal and adult diaphragms, but the magnitude of force augmentation was larger in adult than in neonatal diaphragm. These results indicate that constitutive NOS isoforms are developmentally regulated in skeletal muscles, suggesting multiple roles for NO in developing and mature skeletal-muscle fibers.

Ligand binding pocket of the human somatostatin receptor 5: mutational analysis of the extracellular domains.

The ligand binding domain of G protein-coupled receptors for peptide ligands consists of a pocket formed by extracellular and transmembrane domain (TM) residues. In the case of somatostatin (SRIF), however, previous studies have suggested that the binding cavity of the octapeptide analog SMS201-995 (SMS) is lined by residues in TMs III-VII. The additional involvement of the extracellular domains for binding SMS or the natural SRIF ligands (SRIF-14, SRIF-28) has not been clarified. Using a cassette construct cDNA for the human somatostatin 5 receptor (sst5R), we systematically examined the role of exofacial structures in ligand binding by creating a series of mutants in which the extracellular portions have been altered by conservative segment exchange (CSE) mutagenesis for the extracellular loops (ECLs) and by deletion (for the NH2-terminal segment) or truncation analysis (ECL3). CHO-K1 cells were stably transfected with wild type or mutant human sst5R constructs, and agonist binding was assessed using membrane binding assays with 125I-LTT SRIF-28 ligand. Deletion of the NH2 terminus or CSE mutagenesis of ECL1 and ECL3 produced minor 2-8-fold decreases in affinity for SRIF-14, SRIF-28, and SMS ligands. Truncation of ECL3 to mimic the size of this loop in sst1R and sst4R (the two subtypes that do not bind SMS) did not interfere with the binding of SMS, SRIF-14, or SRIF-28. In contrast, both ECL2 mutants failed to bind 125I-LTT SRIF-28. Immunocytochemical analysis of nonpermeabilized cells with a human sst5R antibody revealed that the mutant receptors were targeted to the plasma membrane. Labeled SMS (125I-Tyr3 SMS) also failed to bind to the mutant ECL2 receptors. These results suggest a potential contribution of ECL2 (in addition to the previously identified residues in TMs III-VII) to the SRIF ligand binding pocket.

Distribution of protein inhibitor of neuronal nitric oxide synthase in rat brain.

In neuronal cells, nitric oxide (NO) is synthesized by neuronal nitric oxide synthase (nNOS) and performs various functions including neurotransmission, modulation of nociception and long-term potentiation and memory. Recently, a novel 89-amino acid protein, designated PIN, has been shown to interact specifically with nNOS and inhibit nNOS dimerization. In this report, we investigated the distribution and the correlation of PIN with that of nNOS in various brain regions of rats. Amplified PIN cDNA from brain tissues revealed an open reading frame which is identical to that of human PIN. Northern blotting of brain RNA with PIN cDNA produced two transcripts, a major 0.9 kb and a minor 2.5 kb. Expression of PIN cDNA as a fusion protein in E. coli produced a 10 kDa protein which interacted specifically with pure nNOS in an overlay assay. Immunoblotting of rat brain regions with nNOS antibody demonstrated strong expression in the cerebellum, moderate expression in the cerebral cortex, midbrain, medulla and hippocampus with only weak expression in the spinal cord. By comparison, PIN expression was stronger in the cerebral cortex, midbrain, hippocampus and medulla compared with that of cerebellum and spinal cord. We conclude that PIN interacts strongly with nNOS and is constitutively expressed in various brain regions. The dissimilarity between nNOS and PIN expressions in various brain regions may explain the well known differences in NOS activity between these regions. Our results also suggest that PIN may serve other functions other than nNOS inhibition.

Expression of mRNA for somatostatin receptor (sstr) types 2 and 5 in individual rat pituitary cells. A double labeling in situ hybridization analysis.

To characterize cell specific expression of sstr subtypes in the pituitary we have analyzed mRNA for sstr1-5 in rat pituitary somatotrophs by reverse transcriptase polymerase chain reaction and determined the pattern and level of expression of mRNA for sstr subtypes 2 and 5 in individual pituitary cell subpopulations by double label in situ hybridization. Purified somatotrophs expressed mRNA for all 5 sstrs. In situ hybridization analysis revealed sstr5 mRNA in 70% of somatotrophs, 57% of thyrotrophs, 38% of corticotrophs, 33% of lactotrophs, and 21% of gonadotrophs. mRNA for sstr2 occurred in 40% of somatotrophs, 36% of thyrotrophs, 26% of lactotrophs, 3% of corticotrophs, and 8% of gonadotrophs. Not only were more cells positive for sstr5 mRNA but the average number of autoradiographic grains/cell was also higher for sstr5 than sstr2. These results show expression of multiple sstr genes in individual pituitary cells. mRNA for sstr2 and 5 occur in each of the 5 major pituitary cell subsets, sstr5 mRNA being more widely and more abundantly expressed than sstr2.

Cloning of the gene encoding human somatostatin receptor 2: sequence analysis of the 5'-flanking promoter region.

Using a cDNA probe, genomic clones were obtained for the 5' flanking promoter sequence of the human somatostatin receptor 2-encoding gene (SSTR2). A 3.8-kb sequence directly upstream from the start codon was analyzed. This sequence shares a number of characteristics with the promoters of other G-protein-coupled receptor (GPCR)-encoding genes including a number of G+C-rich regions, binding sites for several transcriptional factors, and the absence of coupled TATAA and CAAT sequences.

The somatostatin receptor family.

The diverse biological effects of somatostatin (SST) are mediated through a family of G protein coupled receptors of which 5 members have been recently identified by molecular cloning. This review focuses on the molecular biology, pharmacology, expression, and function of these receptors with particular emphasis on the human (h) homologs. hSSTRs are encoded by a family of 5 genes which map to separate chromosomes and which, with one exception, are intronless. SSTR2 gives rise to spliced variants, SSTR2A and 2B. hSSTR1-4 display weak selectivity for SST-14 binding whereas hSSTR5 is SST-28 selective. Based on structural similarity and reactivity for octapeptide and hexapeptide SST analogs, hSSTR2,3, and 5 belong to a similar SSTR subclass. hSSTR1 and 4 react poorly with these analogs and belong to a separate subclass. All 5 hSSTRs are functionally coupled to inhibition of adenylyl cyclase via pertussis toxin sensitive GTP binding proteins. Some of the subtypes are also coupled to tyrosine phosphatase (SSTR1,2), Ca2+ channels (SSTR2), Na+/H+ exchanger (SSTR1), PLA-2 (SSTR4), and MAP kinase (SSTR4). mRNA for SSTR1-5 is widely expressed in brain and peripheral organs and displays an overlapping but characteristic pattern that is subtype-selective, and tissue- and species-specific. Pituitary and islet tumors express several SSTR genes suggesting that multiple SSTR subtypes are coexpressed in the same cell. Structure-function studies indicate that the core residues in SST-14 ligand Phe6-Phe11 dock within a ligand binding pocket located in TMDs 3-7 which is lined by hydrophobic and charged amino acid residues.

Sequence analysis of the 5'-flanking promoter region of the human somatostatin receptor 5.

We have determined the sequence of 2.2 kb of 5' flanking promoter region of the human somatostatin receptor 5 (hsstr5) gene. A number of widely distributed promoter elements were identified including AP1, AP2, AP3, E2A, GCF, and SP1 consensus sequences. hsstr5/CAT gene fusions showed that the 0.9 kb of DNA immediately upstream of the ATG functions as a promoter in rat pituitary GH3 but not in CHO ovary cells. Insertion of this hsstr5 fragment in the anti-sense orientation led to a four fold reduction in CAT activity. Dibutyryl cAMP produced a three fold induction of CAT activity whereas estradiol and retinoic acid had no significant effect. These results indicate that we have identified a DNA fragment at the 5' end of the hsstr5 gene which contains both tissue-specific and regulated elements. The absence of CRE consensus sequence suggests that the cAMP effect is mediated by the multiple AP1 and AP2 sites.

Molecular cloning, functional characterization, and chromosomal localization of a human somatostatin receptor (somatostatin receptor type 5) with preferential affinity for somatostatin-28.

Using a combination of polymerase chain reaction and genomic library screening we have cloned a human gene for a subtype of the somatostatin (SST) receptor (SSTR) termed human SSTR5 (hSSTR5), which is located on chromosome 16. The predicted amino acid sequence of hSSTR5 displays 75% sequence identity with a recently identified rat SSTR [Mol. Pharmacol. 42:939-946 (1992)], suggesting that it is the human homologue of this receptor. hSSTR5 consists of a 363-residue polypeptide exhibiting a putative seven-transmembrane domain topology typical of G protein-coupled receptors. The receptor displays considerable sequence identity to hSSTR1 (42%), hSSTR2 (48%), hSSTR3 (47%), and hSSTR4 (46%). Membranes prepared from COS-7 cells transiently expressing the hSSTR5 gene bound 125I-Leu8,D-Trp22,Tyr25-SST-28 (125I-LTT-SST-28) with high affinity and in a saturable manner. SST-14, SST-28, and various synthetic SST peptide agonists produced dose-dependent inhibition of radioligand binding with the following rank order of potency: LTT-SST-28 > SST-28 > D-Trp8-SST-14 > SST-14 approximately RC-160 approximately BIM 23014 > MK-678 > SMS 201-995. hSSTR5 bound SST-28 with a 12.6-fold greater affinity (Ki = 0.19 nM), compared with SST-14 (Ki = 2.24 nM), indicating that the receptor is SST-28 selective. Addition of GTP, guanosine-5'-O-(3-thio)triphosphate, Na+ ions, or pertusis toxin greatly reduced 125I-LTT-SST-28 binding, thereby indicating that hSSTR5 is coupled to pertussis toxin-sensitive G proteins. Both SST-14 and SST-28 displayed dose-dependent inhibition of forskolin-stimulated cAMP accumulation, consistent with functional coupling of the receptor to adenylyl cyclase inhibition. Northern blot analysis of SSTR5 mRNA revealed a 2.4-kilobase transcript in normal rat pituitary and GH3 rat pituitary tumor cells and a 4.0-kilobase transcript in normal human pituitary. Reverse transcriptase polymerase chain reaction revealed expression of the hSSTR gene in fetal human pituitary and hypothalamus but not in human cerebral cortex. In situ hybridization of the rat pituitary showed that SSTR5 mRNA is selectively localized in the anterior lobe. SSTR5 mRNA was not expressed in four human pituitary tumors (somatotroph adenoma, prolactinoma, and chromophobe adenomas) or in a human insulinoma. Although hSSTR5 displays approximately 75% sequence identity with rat SSTR5, the two receptors display significantly different pharmacological profiles, especially with respect to their binding affinities for the SST analogue SMS 201-995.

All five cloned human somatostatin receptors (hSSTR1-5) are functionally coupled to adenylyl cyclase.

Recent reports have suggested that only some of the cloned somatostatin receptors (SSTRs) are coupled to adenylyl cyclase. These studies have used both stable and transiently transfected cells or cells lacking appropriate Gi alpha and are controversial. To investigate SSTR signalling mechanisms, we have established stably transfected CHO-K1 cells expressing human genes for SSTR1-5. The effect of 0.1-100 nM SST-14 and SST-28 on forskolin (1 microM) stimulated cAMP accumulation was determined and compared to their receptor binding affinities. The 5 expressed hSSTRs bound SST-14 and SST-28 with high affinity (IC50 1.1-2.1 nM for SST-14; IC50 0.25-5.4 nM for SST-28). hSSTR1-4 bound SST-14 > SST-28 whereas hSSTR5 bound SST-28 > SST-14. Radioligand binding to hSSTR1-5 was significantly inhibited by GTP, GTP gamma S and pertussis toxin. Both SST-14 and SST-28 inhibited forskolin-induced cAMP stimulation with ED50 values which paralleled their binding affinities for the individual hSSTR subtypes. These results demonstrate that all 5 human SSTRs are functionally coupled to inhibition of adenylyl cyclase in CHO-K1 cells via pertussis toxin sensitive G proteins.

Regulation of the gene encoding the p24 actin-binding protein in Dictyostelium discoideum.

We have isolated cDNA clones on the basis of sequence similarity to the gene encoding the cyclic cAMP-binding protein CABP1 of Dictyostelium discoideum. The predicted amino acid sequence of the cloned cDNAs shows that the homology to CABP1 is restricted to a region rich in proline, glycine, glutamine, and tyrosine. Sequence comparison indicates that the cloned cDNAs encode the actin-binding protein p24. We have examined by RNA blot hybridization the expression of the gene encoding p24. For cells developed in suspension, the levels of p24 mRNA increase rapidly during early development, reaching a peak at 3-4 h. Addition of high concentrations of exogenous cAMP during the first 4 h of development produced little or no effect on the accumulation of p24 mRNA. Treatment with cAMP during subsequent stages of development reduced the levels of p24 mRNA. We attempted to determine if the synthesis of new proteins during early development is a requirement for the reduction in p24 mRNA levels by treating the cells with protein synthesis inhibitor. Unexpectedly, the addition of the inhibitor cycloheximide resulted in an increase in the level of p24 mRNA. The roles of cycloheximide and cAMP on the expression of the p24 gene are discussed.

The louse Trinoton anserinum (Amblycera: Phthiraptera), an intermediate host of Sarconema eurycerca (Filarioidea: Nematoda), a heartworm of swans.

The role of the louse Trinoton anserinum (F) as an intermediate host of Sarconema eurycerca (Wehr) was investigated in swans. 8.3% of healthy swans carried one to twelve lice per bird, dispersed contagiously. Injured and lead-poisoned swans were more heavily infected. The mouthparts appear designed to penetrate the hosts' skin; the mandibles are robust and asymmetric, and the maxillae have a serrated intercutting surface. 22% fed exclusively on blood and 33% on both blood and feather. All life-cycle stages fed upon blood and the barbs and barbules from down feathers; hooklets from contour feathers were only found in adults. 9% of lice were infected with developing nematode larvae in the head, thorax or abdomen. Lice labelled with Technetium 99 m moved towards the scapulas and the wings. Lice were found to be highly active and were mobile.

Automatic recording of flea activity.

An Insect Activity Monitor was created to measure the behavioural responses of fleas (Siphonaptera). The apparatus allows for a range of visual, chemo- and mechanoreceptor cues to be presented. The jumping response is detected by counting amplified pulses produced as the fleas land on a stretched membrane held over a microphone. Horizontal movements are detected using a system of infra-red beams and phototransistors which, when broken, are counted as a measure of activity. The apparatus was tested using Ceratophyllus hirundinis (Curtis), Ceratophyllus farreni (Rothschild) and Ceratophyllus rusticus (Wagner), co-inhabiting species from the nests of the house martin (Delichon u.urbica L.). No unaided emigration or immigration has been demonstrated in these species (Clark, 1988) and much of their time is spent confined to the darkness of the nest. In addition to species contrasts, females were more active than males and fleas were more active at 25 degrees C than at 18 degrees C. The more active bird flea Ceratophyllus garei (Rothschild) from the nest of pheasant (Phasianus colchicus L), was tested for responses to light of varying intensity. Activity was stimulated by white light, but not proportional to light intensity.

Growth-rate-dependent regulation of the expression and inactivation of thymidylate synthase in Saccharomyces cerevisiae.

Thymidylate synthase activity fluctuated dramatically as cultures of Saccharomyces cerevisiae progressed through the different stages of batch culture growth. During logarithmic growth these yeast cultures each contained about 40 microU (1 microU is 1 pmol of 3H released per min) of thymidylate synthase activity per 10(8) haploid cells, but as cultures entered the stationary phase and during the stationary phase, activity dropped dramatically, eventually reaching undetectable levels. Stimulation of stationary-phase cells with fresh medium resulted in rapid reestablishment of log phase levels. Two mechanisms, the regulation of thymidylate synthase-specific transcripts and the irreversible inactivation of thymidylate synthase activity, acted in concert to regulate activity levels. These results suggested that thymidylate synthase represents a special subset of yeast proteins whose levels per cell vary quickly and dramatically in response to changes in proliferation rates.

Cell cycle-dependent expression of thymidylate synthase in Saccharomyces cerevisiae.

Synchronous populations of Saccharomyces cerevisiae cells, generated by two independent methods, have been used to show that thymidylate synthase, in contrast to the vast majority of cellular proteins thus far examined, fluctuates periodically during the S. cerevisiae cell cycle. The enzyme, as assayed by two different methods, accumulated during S period and peaked in mid to late S phase, and then its level dropped. These observations suggest that both periodic synthesis and the instability of the enzyme contribute to the activity profile seen during the cell cycle. Accumulation of thymidylate synthase is determined at the level of its transcript, with synthase-specific mRNA levels increasing at least 10-fold to peak near the beginning of S period and then falling dramatically to basal levels after the onset of DNA synthesis. This mRNA peak coincided with the time during the cell cycle when thymidylate synthase levels were increasing maximally and immediately preceded the peak of DNA synthesis, for which the enzyme provides precursor dTMP.