Cytoplasmic inclusions containing TAR DNA-binding protein of 43 kDa (TDP-43) or Fused in sarcoma (FUS) are a hallmark of amyotrophic lateral sclerosis (ALS) and several subtypes of frontotemporal lobar degeneration (FTLD). FUS-positive inclusions in FTLD and ALS patients are consistently co-labeled with stress granule (SG) marker proteins. Whether TDP-43 inclusions contain SG markers is currently still debated. We determined the requirements for SG recruitment of FUS and TDP-43 and found that cytoplasmic mislocalization is a common prerequisite for SG recruitment of FUS and TDP-43. For FUS, the arginine-glycine-glycine zinc finger domain, which is the protein's main RNA binding domain, is most important for SG recruitment, whereas the glycine-rich domain and RNA recognition motif (RRM) domain have a minor contribution and the glutamine-rich domain is dispensable. For TDP-43, both the RRM1 and the C-terminal glycine-rich domain are required for SG localization. ALS-associated point mutations located in the glycine-rich domain of TDP-43 do not affect SG recruitment. Interestingly, a 25-kDa C-terminal fragment of TDP-43, which is enriched in FTLD/ALS cortical inclusions but not spinal cord inclusions, fails to be recruited into SG. Consistently, inclusions in the cortex of FTLD patients, which are enriched for C-terminal fragments, are not co-labeled with the SG marker poly(A)-binding protein 1 (PABP-1), whereas inclusions in spinal cord, which contain full-length TDP-43, are frequently positive for this marker protein.

Proteins of the Jak family of non-receptor kinases play important roles in mammalian hematopoietic signal transduction. They mediate the cellular response to a wide range of cytokines and growth factors. A dominant mutation in a Drosophila Jak kinase, hopscotchTumorous-lethal (hopTum-l), causes hematopoietic defects. Here we conduct a molecular analysis of hopTum-l. We demonstrate that the hopTum-l hematopoietic phenotype is caused by a single amino acid substitution of glycine to glutamic acid at residue 341. We generate a true revertant of the hopTum-l mutation, in which both the molecular lesion and the mutant hematopoietic phenotype revert back to wild type. We also examine the effects of the G341E substitution in transgenic flies. The results indicate that a mutant Jak kinase can cause leukemia-like abnormalities.

The identification of encephalitis associated with antibodies against cell surface and synaptic proteins, although recent, has already had a substantial impact in clinical neurology and neuroscience. The target antigens are receptors and proteins that have critical roles in synaptic transmission and plasticity, including the NMDA receptor, the AMPA receptor, the GABA(B) receptor, and the glycine receptor. Other autoantigens, such as leucine-rich glioma-inactivated 1 and contactin-associated protein-like 2, form part of trans-synaptic complexes and neuronal cell adhesion molecules involved in fine-tuning synaptic transmission and nerve excitability. Syndromes resulting from these immune responses resemble those of pharmacologic or genetic models in which the antigens are disrupted. For some immune responses, there is evidence that the antibodies alter the structure and function of the antigen, suggesting a direct pathogenic effect. These disorders are important because they can affect children and young adults, are severe and protracted, occur with or without tumor association, and respond to treatment but may relapse. This review provides an update on these syndromes and autoantigens with special emphasis on clinical diagnosis and treatment.

Experiments were conducted to characterize the distribution of N compounds in the xylem sap of nodulated and nonnodulated soybean plants through development and to determine the effects of exogenous N on the distribution of N compounds in the xylem. Xylem sap was collected from nodulated and nonnodulated greenhouse-grown soybean plants (Glycine max [L.] Merr. "Ransom") from the vegetative phase to the pod-filling phase. The sum of the nitrogen in the amino acid, nitrate, ureide (allantoic acid and allantoin), and ammonium fractions of the sap from both types of plants agreed closely with total N as assayed by a Kjeldahl technique. Sap from nodulated plants supplied with N-free nutrient solution contained seasonal averages of 78 and 20% of the total N as ureide-N and amino acid-N, respectively. Sap from nonnodulated plants supplied with a 20 millimolar KNO(3) nutrient solution contained seasonal averages of 6, 36, and 58% of total N as ureide-N, amino acid-N, and nitrate-N, respectively. Allantoic acid was the predominant ureide in the xylem sap and asparagine was the predominant amino acid. When well nodulated plants were supplied with 20 millimolar KNO(3), beginning at 65 days, C(2)H(2) reduction (N(2) fixation) decreased relative to nontreated plants and there was a concomitant decrease in the ureide content of the sap. A positive correlation (r = 0.89) was found between the ureide levels in xylem sap and nodule dry weights when either exogenous nitrate-N or urea-N was supplied at 10 and 20 millimolar concentrations to inoculated plants. The results demonstrate that ureides play a dominant role in N transport in nodulated soybeans and that the synthesis of ureides is largely dependent upon nodulation and N(2) fixation.

Subtilases are members of the family of subtilisin-like serine proteases. Presently, greater than 50 subtilases are known, greater than 40 of which with their complete amino acid sequences. We have compared these sequences and the available three-dimensional structures (subtilisin BPN', subtilisin Carlsberg, thermitase and proteinase K). The mature enzymes contain up to 1775 residues, with N-terminal catalytic domains ranging from 268 to 511 residues, and signal and/or activation-peptides ranging from 27 to 280 residues. Several members contain C-terminal extensions, relative to the subtilisins, which display additional properties such as sequence repeats, processing sites and membrane anchor segments. Multiple sequence alignment of the N-terminal catalytic domains allows the definition of two main classes of subtilases. A structurally conserved framework of 191 core residues has been defined from a comparison of the four known three-dimensional structures. Eighteen of these core residues are highly conserved, nine of which are glycines. While the alpha-helix and beta-sheet secondary structure elements show considerable sequence homology, this is less so for peptide loops that connect the core secondary structure elements. These loops can vary in length by greater than 150 residues. While the core three-dimensional structure is conserved, insertions and deletions are preferentially confined to surface loops. From the known three-dimensional structures various predictions are made for the other subtilases concerning essential conserved residues, allowable amino acid substitutions, disulphide bonds, Ca(2+)-binding sites, substrate-binding site residues, ionic and aromatic interactions, proteolytically susceptible surface loops, etc. These predictions form a basis for protein engineering of members of the subtilase family, for which no three-dimensional structure is known.

The interactions of a steroid anaesthetic, alphaxalone, with the GABA receptor-ionophore complex were investigated by two different experimental approaches. In the rat cuneate nucleus slice, alphaxalone (0.1-10 microM) potentiated depolarizing responses to superfused GABA and muscimol, but not those to glycine. The potentiating effect of alphaxalone was unaltered by the benzodiazepine antagonist Ro 15-1788. Alphaxalone (0.1-30 microM) also enhanced [3H]muscimol binding to rat brain membranes in the presence of Cl-ions; the enhancing effect on [3H]muscimol binding was abolished by Triton X-100. Analysis of binding curves for [3H]muscimol indicated that the steroid anaesthetic increases the affinity for [3H]muscimol of low affinity binding sites; this property is shared by pentobarbitone. The physiologically inactive beta-hydroxy isomer of the steroid was without activity in either of the experimental situations at 30 microM. It is suggested that alphaxalone and pentobarbitone share a common mode of action on the GABA system, which may be relevant to the mechanisms by which these drugs produce anaesthesia.

The alpha1beta heteromeric receptors are likely to be the predominant synaptic form of glycine receptors in the adult. Their activation mechanism was investigated by fitting putative mechanisms to single-channel recordings obtained at four glycine concentrations (10-1000 microm) from rat alpha1beta receptors, expressed in human embryonic kidney 293 cells. The adequacy of each mechanism, with its fitted rate constants, was assessed by comparing experimental dwell time distributions, open-shut correlations, and the concentration-open probability (P(open)) curve with the predictions of the model. A good description was obtained only if the mechanism had three glycine binding sites, allowed both partially and fully liganded openings, and predicted the presence of open-shut correlations. A strong feature of the data was the appearance of an increase in binding affinity as more glycine molecules bind, before the channel opens. One interpretation of this positive binding cooperativity is that binding sites interact, each site sensing the state of ligation of the others. An alternative, and novel, explanation is that agonist binding stabilizes a higher affinity form of the receptor that is produced by a conformational change ("flip") that is separate from, and precedes, channel opening. Both the "interaction" scheme and the flip scheme describe our data well, but the latter has fewer free parameters and above all it offers a mechanism for the affinity increase. Distinguishing between the two mechanisms will be important for our understanding of the structural dynamics of activation in the nicotinic superfamily and is important for our understanding of mutations in these receptors.

Previous studies have suggested that osteopontin, an arginine-glycine-aspartate-containing secreted protein, might be important in macrophage accumulation during interstitial nephritis and wound healing. The current study investigated the role of osteopontin in intradermal macrophage infiltration using immunohistochemistry and neutralizing antibodies. Purified osteopontin induced macrophage accumulation after injection in rat dermis and facilitated adhesion and migration of cultured macrophage-like cells in vitro. Intradermal injection of N-formyl-met-leu-phe (FMLP), a potent macrophage chemotactic peptide, induced a macrophage-rich infiltrate at the site of injection. Most of these macrophages expressed high levels of osteopontin as shown by immunochemical analysis. To determine whether osteopontin expressed by macrophages was required for their infiltration, we treated rats with either anti-osteopontin neutralizing antibody (OP199) or nonimmune antibody. We found that FMLP-induced macrophage accumulation was largely inhibited (>60%) by anti-osteopontin antibody treatment compared with rats receiving nonimmune antibody. These data support the hypothesis that osteopontin may be a critical mediator of inflammation in specific disease and injury states, potentially by promoting macrophage adhesion and migration.

We analyzed 2502 patients with acute myeloid leukemia at diagnosis for NRAS mutations around the hot spots at codons 12, 13, and 61 and correlated the results to cytomorphology, cytogenetics, other molecular markers, and prognostic relevance of these mutations. Two hundred fifty-seven (10.3%) of 2502 patients had NRAS mutations (NRAS(mut)). Most mutations (112 of 257; 43.6%) were found at codon 12, mostly resulting in changes from glycine to asparagine. The history of AML did not differ significantly in association with NRAS mutations. The subgroups with inv(16)/t(16;16) and inv(3)/t(3;3) showed a significantly higher frequency of NRAS(mut) (50 of 133, 37.6% [P < .001], and 11 of 41, 26.8% [P = .004], respectively) than the total cohort. In addition, in these 2 subgroups, mutations of codon 61 were significantly overrepresented (both P < .001). In contrast, NRAS mutations were significantly underrepresented in t(15;17) (2 of 102; 2%; P = .005) in the subgroup with MLL/11q23 rearrangements (3 of 77; 3.9%; P = .061) and in the complex aberrant karyotype (4 of 258; 1.6%; P < .001). Overall, we did not find a significant prognostic impact of NRAS(mut) for overall survival, event-free survival, and disease-free survival. However, there was a trend to better survival in most subgroups, especially when other molecular markers (FLT3-LM, MLL-PTD, and NPM) were taken into account.

Use of the Von Heijne algorithm allowed the identification of 686 open reading frames (ORFs) in the genome of Saccharomyces cerevisiae that encode proteins with a potential N-terminal signal sequence for entering the secretory pathway. On further analysis, 51 of these proteins contain a potential glycosyl-phosphatidylinositol (GPI)-attachment signal. Seven additional ORFs were found to belong to this group. Upon examination of the possible GPI-attachment sites, it was found that in yeast the most probable amino acids for GPI-attachment as asparagine and glycine. In yeast, GPI-proteins are found at the cell surface, either attached to the plasma-membrane or as an intrinsic part of the cell wall. It was noted that plasma-membrane GPI-proteins possess a dibasic residue motif just before their predicted GPI-attachment site. Based on this, and on homologies between proteins, families of plasma-membrane and cell wall proteins were assigned, revealing 20 potential plasma-membrane and 38 potential cell wall proteins. For members of three plasma-membrane protein families, a function has been described. On the other hand, most of the cell wall proteins seem to be structural components of the wall, responsive to different growth conditions. The GPI-attachment site of yeast slightly differs from mammalian cells. This might be of use in the development of anti-fungal drugs.

A suspension culture of soybean (Glycine max L.) was grown on a defined medium in which the nitrogen sources were nitrate (25 mM) and ammonium (2 mM). The cells did not grow on nitrate unless the medium was supplemented with ammonium or glutamine. The l- and d-isomers of 12 amino acids tested singly could not replace ammonium. Most amino acids (4 mM) inhibited growth when the cells were cultured on nitrate and ammonium. Cells from five other plants (Reseda luteoli L.; Triticum monococcum L.; flax, Linum usitatissimum L.; horseradish, Amoracia lapathifolia Gilib; Haplopappus gracilis L.) grew on the defined medium with nitrate (25 mM) as the sole nitrogen source. Higher cell yields were obtained when ammonium (2 mM) or glutamine also was present. Supplementing the defined medium with high concentrations of ammonium (20 mM) inhibited growth of soybean, Haplopappus, and wheat cells. Addition of citrate (5 mM) relieved the inhibitory effects of ammonium in soybean and wheat cells but not in the Haplopappus cells.

Hereditary hyperekplexia, or familial startle disease (STHE), is an autosomal dominant neurologic disorder characterized by marked muscle rigidity of central nervous system origin and an exaggerated startle response to unexpected acoustic or tactile stimuli. Linkage analyses in several large families provided evidence for locus homogeneity and showed the disease gene was linked to DNA markers on the long arm of chromosome 5. Here we describe the identification of point mutations in the gene encoding the alpha 1 subunit of the glycine receptor (GLRA1) in STHE patients from four different families. All mutations occur in the same base pair of exon 6 and result in the substitution of an uncharged amino acid (leucine or glutamine) for Arg271 in the mature protein.

The Arg-Gly-Asp sequence resides in the cell attachment region of fibronectin. Arg-Gly-Asp-containing peptides support fibroblast attachment, inhibit fibroblast adhesion to fibronectin, and inhibit fibronectin binding to thrombin-stimulated platelets. In view of the similarities between the binding of fibronectin, fibrinogen, and von Willebrand factor to stimulated platelets, we have examined the effects of Arg-Gly-Asp-containing peptides on the interaction of these latter two adhesive proteins with platelets. Gly-Arg-Gly-Asp-Ser-Pro was used as a prototype peptide, and this hexapeptide inhibited fibrinogen binding to ADP and thrombin-stimulated platelets in the 10-200 microM range. The inhibition exceeded 90% at high concentrations of peptide and was observed in the presence of either calcium or magnesium. Platelet aggregation was also inhibited by the peptide in this dose range. The hexapeptide inhibited fibrinogen binding to platelets with receptors fixed in an exposed state, indicating direct interference with the ligand-platelet interaction. The peptide was 1/2 to 1/3rd as potent in inhibiting fibrinogen as fibronectin binding to platelets, but fibrinogen and von Willebrand factor binding were inhibited to an identical extent. Conservative amino acid substitutions for the arginine, glycine, or aspartic acid markedly reduced inhibitory activity and the Asp-Gly-Arg sequence was inactive. These results indicate that Arg-Gly-Asp-containing peptides can inhibit the binding of the three adhesive proteins to stimulated platelets, establishing a basic common feature between the interaction of these molecules with platelets.

The small intestine is not only responsible for terminal digestion and absorption of nutrients, but it also plays an important role in catabolism of arterial glutamine and dietary amino acids. Most of glutamine and almost all of glutamate and aspartate in the diet are catabolized by small intestinal mucosa, and CO2 accounts for 56-64% of their metabolized carbons. The small intestinal mucosa also plays an important role in degrading arginine, proline and branched-chain amino acids, and perhaps methionine, lysine, phenylalanine, threonine, glycine and serine in the diet, such that 30-50% of these dietary amino acids are not available to extraintestinal tissues. Dietary amino acids are major fuels for the small intestinal mucosa and are essential precursors for intestinal synthesis of glutathione, nitric oxide, polyamines, purine and pyrimidine nucleotides, and amino acids (alanine, citrulline and proline), and are obligatory for maintaining intestinal mucosal mass and integrity. Because intestinal amino acid catabolism plays an important role in modulating dietary amino acid availability to extraintestinal tissues, it has important implications for the utilization efficiency of dietary protein and amino acids in animals and humans.

We investigated the transferability of 31 soybean (Glycine max) simple sequence repeat (SSR) loci to wild congeners and to other legume genera. Up to 65% of the soybean primer pairs amplified SSRs within Glycine, but frequently, the SSRs were short and interrupted compared with those of soybeans. Nevertheless, 85% of the loci were polymorphic within G. clandestina. Cross-species amplification outside of the genus was much lower (3%-13%), with polymorphism restricted to one primer pair, AG81. AG81 amplified loci in Glycine, Kennedia, and Vigna (Phaseoleae), Vicia (Vicieae), Trifolium (Trifolieae), and Lupinus (Genisteae) within the Papilionoideae, and in Albizia within the Mimosoideae. The primer conservation at AG81 may be explained by its apparent proximity to the seryl-tRNA synthetase gene. Interspecific differences in allele size at AG81 loci reflected repeat length variation within the SSR region and indels in the flanking region. Alleles of identical size with different underlying sequences (size homoplasy) were observed. Our findings and the emerging patterns in other plant studies suggest that in contrast to animals, successful cross-species amplification of SSRs in plants is largely restricted to congeners or closely related genera. Because mutations in both the SSR region and the flanking region contribute to variation in allele size among species, knowledge of DNA sequence is essential before SSR loci can be meaningfully used to address applied and evolutionary questions.

Four genes encode electroneutral, Na+-independent, K-Cl cotransporters. KCC2, is exclusively expressed in neurons where it is thought to drive intracellular Cl- to low concentrations and shift the reversal potential for Cl- conductances such as GABA(A) or glycine receptor channels, thus participating in the postnatal development of inhibitory mechanisms in the brain. Indeed, expression of the cotransporter is low at birth and increases postnatally, at a time when the intracellular Cl- concentration in neurons decreases and gamma-aminobutyric acid switches its effect from excitatory to inhibitory. To assert the significance of KCC2 in neuronal function, we disrupted the mouse gene encoding this neuronal-specific K-Cl cotransporter. We demonstrate that animals deficient in KCC2 exhibit frequent generalized seizures and die shortly after birth. We also show upregulation of Fos, the product of the immediate early gene c-fos, and the significant loss of parvalbumin-positive interneurons, both indicative of brain injury. The regions most affected are the hippocampus and temporal and entorhinal cortices. Extracellular field potential measurements in the CA1 hippocampus exhibited hyperexcitability. Application of picrotoxin, a blocker of the GABA(A) receptor, further increased hyperexcitability in homozygous hippocampal sections. Pharmacological treatment of pups showed that diazepam relieved the seizures while phenytoin prevented them between postnatal ages P4-P12. Finally, we demonstrate that adult heterozygote animals show increased susceptibility for epileptic seizure and increased resistance to the anticonvulsant effect of propofol. Taken together, these results indicate that KCC2 plays an important role in controlling CNS excitability during both postnatal development and adult life.

Thioredoxin 1 (Trx1) has redox-sensitive cysteine residues and acts as an antioxidant in cells. However, the extent of Trx1 contribution to overall antioxidant mechanisms is unknown in any organs. We generated transgenic mice with cardiac-specific overexpression of a dominant negative (DN) mutant (C32S/C35S) of Trx1 (Tg-DN-Trx1 mice), in which the activity of endogenous Trx was diminished. Markers of oxidative stress were significantly increased in hearts from Tg-DN-Trx1 mice compared with those from nontransgenic (NTg) mice. Tg-DN-Trx1 mice exhibited cardiac hypertrophy with maintained cardiac function at baseline. Intraperitoneal injection of N-2-mercaptopropionyl glycine, an antioxidant, normalized cardiac hypertrophy in Tg-DN-Trx1 mice. Thoracic aortic banding caused greater increases in myocardial oxidative stress and enhanced hypertrophy in Tg-DN-Trx1 compared with NTg mice. In contrast, transgenic mice with cardiac-specific overexpression of wild-type Trx1 did not show cardiac hypertrophy at baseline but exhibited reduced levels of hypertrophy and oxidative stress in response to pressure overload. These results demonstrate that endogenous Trx1 is an essential component of the cellular antioxidant mechanisms and plays a critical role in regulating oxidative stress in the heart in vivo. Furthermore, inhibition of endogenous Trx1 in the heart primarily stimulates hypertrophy, both under basal conditions and in response to pressure overload through redox-sensitive mechanisms.

Oxidants such as reactive oxygen species (ROS) have been shown to participate in myocardial ischemia/reperfusion injury. While many studies report a burst of ROS at reperfusion, few reports have presented evidence of significant ROS generation during ischemia. Our previous studies of cultured cardiomyocytes indicated that antioxidants are most effective when given prior to reperfusion during ischemia. Therefore, we hypothesized that significant ROS generation may occur during ischemia prior to reperfusion. We tested this in a perfused isolated cardiomyocyte system (i.e. without neutrophils, endothelial cells, or xanthine/xanthine oxidase) during simulated ischemia/reperfusion while measuring oxidant generation using intracellular fluorescent probes. During ischemia, the ROS probes dihydroethidium and 2',7'-dichlorofluorescin were significantly oxidized, suggesting superoxide and H2O2 generation. At reperfusion following 1 h ischemia, these probes suggested a further burst of H2O2 and hydroxyl radicals. The antioxidants 2-mercaptopropionyl glycine and 1,10-phenanthroline used during ischemia attenuated oxidant generation, increased cell viability, and improved return of contraction after ischemia. To further evaluate the relationship between residual O2 and ROS generation, we administered O2 scavengers during ischemia and measured corresponding changes in oxidant generation, cell viability and contraction during reperfusion. Enzymatic scavenging of residual O2 during ischemia (reducing PO2 from 3.5 to 2.5 tau) paradoxically improved subsequent viability and contraction. These results indicate that cultured cardiomyocytes generate significant ROS during ischemia. This ROS generation is related to residual O2 present during ischemia and contributes significantly to the cellular injury seen at reperfusion.

Cancer cells adapt their metabolic processes to support rapid proliferation, but less is known about how cancer cells alter metabolism to promote cell survival in a poorly vascularized tumour microenvironment. Here we identify a key role for serine and glycine metabolism in the survival of brain cancer cells within the ischaemic zones of gliomas. In human glioblastoma multiforme, mitochondrial serine hydroxymethyltransferase (SHMT2) and glycine decarboxylase (GLDC) are highly expressed in the pseudopalisading cells that surround necrotic foci. We find that SHMT2 activity limits that of pyruvate kinase (PKM2) and reduces oxygen consumption, eliciting a metabolic state that confers a profound survival advantage to cells in poorly vascularized tumour regions. GLDC inhibition impairs cells with high SHMT2 levels as the excess glycine not metabolized by GLDC can be converted to the toxic molecules aminoacetone and methylglyoxal. Thus, SHMT2 is required for cancer cells to adapt to the tumour environment, but also renders these cells sensitive to glycine cleavage system inhibition.

Glycine is a major amino acid in mammals and other animals. It is synthesized from serine, threonine, choline, and hydroxyproline via inter-organ metabolism involving primarily the liver and kidneys. Under normal feeding conditions, glycine is not adequately synthesized in birds or in other animals, particularly in a diseased state. Glycine degradation occurs through three pathways: the glycine cleavage system (GCS), serine hydroxymethyltransferase, and conversion to glyoxylate by peroxisomal D-amino acid oxidase. Among these pathways, GCS is the major enzyme to initiate glycine degradation to form ammonia and CO2 in animals. In addition, glycine is utilized for the biosynthesis of glutathione, heme, creatine, nucleic acids, and uric acid. Furthermore, glycine is a significant component of bile acids secreted into the lumen of the small intestine that is necessary for the digestion of dietary fat and the absorption of long-chain fatty acids. Glycine plays an important role in metabolic regulation, anti-oxidative reactions, and neurological function. Thus, this nutrient has been used to: (1) prevent tissue injury; (2) enhance anti-oxidative capacity; (3) promote protein synthesis and wound healing; (4) improve immunity; and (5) treat metabolic disorders in obesity, diabetes, cardiovascular disease, ischemia-reperfusion injuries, cancers, and various inflammatory diseases. These multiple beneficial effects of glycine, coupled with its insufficient de novo synthesis, support the notion that it is a conditionally essential and also a functional amino acid for mammals (including pigs and humans).

Subsite interactions are considered to define the stringent specificity of proteases for their natural substrates. To probe this issue in the proteolytic pathways leading to apoptosis we have examined the P(4), P(1) and P(1)' subsite preferences of human caspases 1, 3, 6, 7 and 8, using internally quenched fluorescent peptide substrates containing o-aminobenzoyl (also known as anthranilic acid) and 3-nitro-tyrosine. Previous work has demonstrated the importance of the S(4) subsite in directing specificity within the caspase family. Here we demonstrate the influence of the S(1) and S(1)' subsites that flank the scissile peptide bond. The S(1) subsite, the major specificity-determining site of the caspases, demonstrates tremendous selectivity, with a 20000-fold preference for cleaving substrates containing aspartic acid over glutamic acid at this position. Thus caspases are among the most selective of known endopeptidases. We find that the caspases show an unexpected degree of discrimination in the P(1)' position, with a general preference for small amino acid residues such as alanine, glycine and serine, with glycine being the preferred substituent. Large aromatic residues are also surprisingly well-tolerated, but charged residues are prohibited. While this describes the general order of P(1)' subsite preferences within the caspase family, there are some differences in individual profiles, with caspase-3 being particularly promiscuous. Overall, the subsite preferences can be used to predict natural substrates, but in certain cases the cleavage site within a presumed natural substrate cannot be predicted by looking for the preferred peptide cleavage sites. In the latter case we conclude that second-site interactions may overcome otherwise sub-optimal cleavage sequences.

The 4 kb (8.5 % lambda units) EcoRI fragment harboring the tufA gene of Escherichia coli was cloned using plasmid pTUA1 (Shibuya et al., 1979) and its structure was analyzed. The nucleotide sequence of about 1500 base pairs, covering the C-terminal portion of elongation factor EF-G (fus gene), the intercistronic region between fus and tufA, the entire structural gene for tufA with the GUG initiation and UAA termination codons, and the 3' flanking region of tufA, was determined. Comparison of the tufA nucleotide sequence with the tufB sequence (An and Friesen, 1980) and the known amino acid sequence of EF-Tu (Arai et al., 1980) revealed that the products of genes tufA and tufB are identical except for one amino acid at the C-terminal, i.e., glycine for tufA and serine for tufB. Nucleotide differences between tufA and tufB were found at 13 positions. Among them, one in the initiation codon and the other one in the C-terminal amino acid codon had replacements at the first letter of the codons. The other eleven changes were in the third codon positions, which did not affect the amino acid coding. The pattern of codon usage in tufA and tufB is highly nonrandom, and remarkably similar to that in ribosomal protein genes, with the codons for the most abundant species of isoaccepting tRNAs being preferentially utilized (Post et al., 1979; Post and Nomura, 1980).

OBJECTIVE

It is thought that enterovirus infections cause beta-cell damage and contribute to the development of Type 1 diabetes by replicating in the pancreatic islets. We sought evidence for this through autopsy studies and by investigating known enterovirus receptors in cultured human islets.

METHODS

Autopsy pancreases from 12 newborn infants who died of fulminant coxsackievirus infections and from 65 Type 1 diabetic patients were studied for presence of enteroviral ribonucleic acid by in situ hybridisation. Forty non-diabetic control pancreases were included in the study. The expression and role of receptor candidates in cultured human islets were investigated with receptor-specific antibodies using immunocytochemistry and functional assays.

RESULTS

Enterovirus-positive islet cells were found in some of both autopsy specimen collections, but not in control pancreases. No infected cells were seen in exocrine tissue. The cell surface molecules, poliovirus receptor and integrin alphavbeta3, which act as enterovirus receptors in established cell lines, were expressed in beta cells. Antibodies to poliovirus receptor, human coxsackievirus and adenovirus receptor and integrin alphavbeta3 protected islets and beta cells from adverse effects of poliovirus, coxsackie B viruses, and several of the arginine-glycine-aspartic acid motifs containing enteroviruses and human parechovirus 1 respectively. No evidence was found for expression of the decay-accelerating factor which acts as a receptor for several islet-cell-replicating echoviruses in established cell lines.

CONCLUSIONS

The results show a definite islet-cell tropism of enteroviruses in the human pancreas. Some enteroviruses seem to use previously identified cell surface molecules as receptors in beta cells, whereas the identity of receptors used by other enteroviruses remains unknown.