A Tour de Force: The Discovery, Properties, and Function of Piezo Channels.

Mechanical transducers appear throughout cell biology and are used to convert mechanical stress into chemical or electrical signals that allow the cell to respond to environmental changes. In the past six years, a eukaryotic mechanical channel family with two members, Piezo1 and Piezo2, has been identified. Piezo1 was shown to be a cation-selective channel that does not require ancillary proteins for activity. Mouse Piezo1 is large, with over 2500 amino acids, and is not homologous to other ion channels. Both piezo channels have rapid voltage-dependent inactivation with a reversal potential near 0mV. The CryoEm structure of Piezo1 at 4.8Å shows trimer stoichiometry. Since the discovery of the piezo channels, their roles in the physiological response of cells have started to emerge. Significant progress has been made in understanding the intrinsic properties of the channels and how these properties are modulated by cytoskeletal elements. Specific diseases, such as hereditary xerocytosis affecting red blood cells, have mutations in Piezo1 that alter the cell's response to force, typically slowing inactivation and introducing a latency for activation. A number of physiological functions for piezo channels have been identified. These range from sensing the stiffness of surrounding substrate, to the response to light touch, to serotonin release from the gut. This review provides a general overview of the properties and roles of Piezo1 and Piezo2 in eukaryotic mechanotransduction.

The Kinetics and the Permeation Properties of Piezo Channels.

Piezo channels are eukaryotic, cation-selective mechanosensitive channels (MSCs), which show rapid activation and voltage-dependent inactivation. The kinetics of these channels are largely consistent across multiple cell types and different stimulation paradigms with some minor variability. No accessory subunits that associate with Piezo channels have been reported. They are homotrimers and each ∼300kD monomer has an N-terminal propeller blade-like mechanosensing module, which can confer mechanosensing capabilities on ASIC-1 (the trimeric non-MSC, acid-sensing ion channel-1) and a C-terminal pore module, which influences conductance, selectivity, and channel inactivation. Repeated stimulation can cause domain fracture and diffusion of these channels leading to synchronous loss of inactivation. The reconstituted channels spontaneously open only in asymmetric bilayers but lack inactivation. Mutations that cause hereditary xerocytosis alter PIEZO1 kinetics. The kinetics of the wild-type PIEZO1 and alterations thereof in mutants (M2225R, R2456K, and DhPIEZO1) are summarized in the form of a quantitative model and hosted online. The pore is permeable to alkali ions although Li+ permeates poorly. Divalent cations, notably Ca2+, traverse the channel and inhibit the flux of monovalents. The large monovalent organic cations such as tetramethyl ammonium and tetraethyl ammonium can traverse the channel, but slowly, suggesting a pore diameter of ∼8Å, and the estimated in-plane area change upon opening is around 6-20nm2. Ruthenium red can enter the channel only from the extracellular side and seems to bind in a pocket close to residue 2496.

A Microfluidic Approach for Studying Piezo Channels.

Microfluidics is an interdisciplinary field intersecting many areas in engineering. Utilizing a combination of physics, chemistry, biology, and biotechnology, along with practical applications for designing devices that use low volumes of fluids to achieve high-throughput screening, is a major goal in microfluidics. Microfluidic approaches allow the study of cells growth and differentiation using a variety of conditions including control of fluid flow that generates shear stress. Recently, Piezo1 channels were shown to respond to fluid shear stress and are crucial for vascular development. This channel is ideal for studying fluid shear stress applied to cells using microfluidic devices. We have developed an approach that allows us to analyze the role of Piezo channels on any given cell and serves as a high-throughput screen for drug discovery. We show that this approach can provide detailed information about the inhibitors of Piezo channels.

Development of Autoimmune-Mediated ß Cell Failure After Total Pancreatectomy With Autologous Islet Transplantation.

Total pancreatectomy with islet autotransplantation (TPIAT) is performed for definitive treatment of chronic pancreatitis; patients are not diabetic before surgery, or have C-peptide positive pancreatogenous diabetes. Thus, TPIAT recipients are not traditionally considered at risk for autoimmune loss of the islet graft. We describe a 43-year-old female who underwent TPIAT with high mass islet graft of 6031 IEQ/kg, with no evidence of presurgical ß cell autoimmunity who developed type 1 diabetes within the first year after TPIAT, resulting in complete loss of beta cell function. The patient had positive GAD and insulin autoantibodies at 1 year and 18 months after TPIAT, not present prior, and undetectable C-peptide after mixed meal and intravenous glucose tolerance testing at 18 months. Glucagon secretion was preserved, suggesting the transplanted alpha cell mass was intact. HLA typing revealed a DR3/DR4 class II haplotype. This case highlights the need to consider de novo type 1 diabetes in patients with unexpected islet graft failure after TPIAT.

Teplizumab treatment may improve C-peptide responses in participants with type 1 diabetes after the new-onset period: a randomised controlled trial.

AIMS/HYPOTHESIS: Type 1 diabetes results from a chronic autoimmune process continuing for years after presentation. We tested whether treatment with teplizumab (a Fc receptor non-binding anti-CD3 monoclonal antibody), after the new-onset period, affects the decline in C-peptide production in individuals with type 1 diabetes. METHODS: In a randomised placebo-controlled trial we treated 58 participants with type 1 diabetes for 4-12 months with teplizumab or placebo at four academic centres in the USA. A central randomisation centre used computer generated tables to allocate treatments. Investigators, patients, and caregivers were blinded to group assignment. The primary outcome was a comparison of C-peptide responses to a mixed meal after 1 year. We explored modification of treatment effects in subgroups of patients. RESULTS: Thirty-four and 29 subjects were randomized to the drug and placebo treated groups, respectively. Thirty-one and 27, respectively, were analysed. Although the primary outcome analysis showed a 21.7% higher C-peptide response in the teplizumab-treated group (0.45 vs 0.371; difference, 0.059 [95% CI 0.006, 0.115] nmol/l) (p = 0.03), when corrected for baseline imbalances in HbA(1c) levels, the C-peptide levels in the teplizumab-treated group were 17.7% higher (0.44 vs 0.378; difference, 0.049 [95% CI 0, 0.108] nmol/l, p = 0.09). A greater proportion of placebo-treated participants lost detectable C-peptide responses at 12 months (p = 0.03). The teplizumab group required less exogenous insulin (p < 0.001) but treatment differences in HbA(1c) levels were not observed. Teplizumab was well tolerated. A subgroup analysis showed that treatment benefits were larger in younger individuals and those with HbA(1c) <6.5% at entry. Clinical responders to teplizumab had an increase in circulating CD8 central memory cells 2 months after enrolment compared with non-responders. CONCLUSIONS/INTERPRETATIONS: This study suggests that deterioration in insulin secretion may be affected by immune therapy with teplizumab after the new-onset period but the magnitude of the effect is less than during the new-onset period. Our studies identify characteristics of patients most likely to respond to this immune therapy. TRIAL REGISTRATION: ClinicalTrials.gov NCT00378508 FUNDING: This work was supported by grants 2007-502, 2007-1059 and 2006-351 from the JDRF and grants R01 DK057846, P30 DK20495, UL1 RR024139, UL1RR025780, UL1 RR024131 and UL1 RR024134 from the NIH.

Immunology of Diabetes Society T-Cell Workshop: HLA class II tetramer-directed epitope validation initiative.

BACKGROUND: Islet-antigen-specific CD4+ T cells are known to promote auto-immune destruction in T1D. Measuring T-cell number and function provides an important biomarker. In response to this need, we evaluated responses to proinsulin and GAD epitopes in a multicentre study. METHODS: A tetramer-based assay was used in five participating centres to measure T-cell reactivities to DR0401-restricted epitopes. Three participating centres concurrently performed ELISPOT or immunoblot assays. Each centre used blind-coded, centrally distributed peptide and tetramer reagents. RESULTS: All participating centres detected responses to auto-antigens and the positive control antigen, and in some cases cloned the corresponding T cells. However, response rates varied among centres. In total, 74% of patients were positive for at least one islet epitope. The most commonly recognized epitope was GAD270-285. Only a minority of the patients tested by tetramer and ELISPOT were concordant for both assays. CONCLUSIONS: This study successfully detected GAD and proinsulin responses using centrally distributed blind-coded reagents. Centres with little previous experience using class II tetramer reagents implemented the assay. The variability in response rates observed for different centres suggests technical difficulties and/or heterogeneity within the local patient populations tested. Dual analysis by tetramer and ELISPOT or immunoblot assays was frequently discordant, suggesting that these assays detect distinct cell populations. Future efforts should investigate shared blood samples to evaluate assay reproducibility and longitudinal samples to identify changes in T-cell phenotype that correlate with changes in disease course.

Immunomodulation in type 1 diabetes by NBI-6024, an altered peptide ligand of the insulin B epitope.

NBI-6024 is an altered peptide ligand (APL) corresponding to the 9-23 amino acid region of the insulin B chain (B(9-23)), an epitope recognized by inflammatory interferon-gamma-producing T helper (Th)1 lymphocytes in type 1 diabetic patients. Immunomodulatory effects of NBI-6024 administration in recent-onset diabetic patients in a phase I clinical trial (NBI-6024-0003) were measured in peripheral blood mononuclear cells using the enzyme-linked immunosorbent spot assay. Analysis of the mean magnitude of cytokine responses to B(9-23) and NBI-6024 for each cohort showed significant increases in interleukin-5 responses (a Th2 regulatory phenotype) in cohorts that received APL relative to those receiving placebo. A responder analysis showed that Th1 responses to B(9-23) and NBI-6024 were observed almost exclusively in the placebo-treated diabetic population but not in nondiabetic control subjects and that APL administration (five biweekly subcutaneous injections) significantly and dose-dependently reduced the percentage of patients with these Th1 responses. The results of this phase I clinical study strongly suggest that NBI-6024 treatment shifted the Th1 pathogenic responses in recent-onset type 1 diabetic patients to a protective Th2 regulatory phenotype. The significance of these findings on the clinical outcome of disease is currently under investigation in a phase II multidose study.

Additional association of intra-MHC genes, MICA and D6S273, with Addison's disease.

Intra-MHC sequences including MHC class I chain-related genes (MICAs), D6S273 and D6S2223 are associated with autoimmune diseases in addition to HLA class II. In the current study, we ascertained the haplotypes of 57 Caucasian patients with Addison's disease composed of these genetic markers and compared them either with 72 general population controls or with 105 child controls carrying Addison's disease high-risk DR3-DQ2/DR4-DQ8 genotypes. The MICA-A5.1/A5.1 genotype as well as HLA DR3/4 especially with DRB1*0404 were the main susceptibility markers. The homozygous MICA-A5.1/A5.1 genotype was significantly more frequent in the patients with Addison's disease (61%) than in the healthy controls (6%). The MICA-A5.1 allele was increased on both the DR3 and DR4 haplotypes, independent of DQ and DRB1 subtyping, in the patients with Addison's disease compared with the controls. Furthermore, the D6S273*140 allele on the DR3 haplotype and the D6S273*134 allele on the DR4 haplotype in the DR3/4 heterozygotes influenced susceptibility relative to the DR3/4 controls. The risk for Addison's disease was increased for the DR3-D6S273*140-MICA-A5.1/DRB1*0404-D6S273*134-MICA-A5.1 genotypes compared with that conferred by the DR3/4 controls. Susceptibility to Addison's disease is influenced by the genes around MICA and D6S273 for both the HLA DR3-DQ2 and DR4-DQ8 haplotypes.

Structure in nascent RNA leads to termination of slippage transcription by T7 RNA polymerase.

T7 RNA polymerase presents a very simple model system for the study of fundamental aspects of transcription. Some time ago it was observed that in the presence of only GTP as a substrate, on a template encoding the initial sequence GGGA., T7 RNA polymerase will synthesize a 'ladder' of poly-G RNA products. At each step, the ratio of elongation to product release is consistently approximately 0.75 until the RNA reaches a length of approximately 13-14 nt, at which point this ratio drops precipitously. One model to explain this drop in complex stability suggests that the nascent RNA may be structurally hindered by the protein; the RNA may be exiting via a pathway not taken by normally synthesized RNA and therefore becomes sterically destabilized. The fact that the length of RNA at which this occurs is close to the length at which the transition to a stably elongating complex occurs might have led to other mechanistic proposals. Here we show instead that elongation falls off due to the cooperative formation of structure in the nascent RNA, most likely an intramolecular G-quartet structure. Replacement of GTP by 7-deaza-GTP completely abolishes this transition and G-ladder synthesis continues with a constant efficiency of elongation beyond the limit of detection. The polymerase-DNA complex creates no barrier to the growth of the nascent (slippage) RNA, rather termination is similar to that which occurs in rho-independent termination.

Becaplermin and necrobiosis lipoidicum diabeticorum: results of a case control pilot study.

Necrobiosis lipoidica diabeticorum (NLD) is a rare skin condition associated with diabetes, which characteristically occurs in the pretibial region of the lower limbs (Boulton et al., 1988). The lesions generally appear as well-circumscribed reddish plaques, which are most often asymptomatic, resulting primarily in cosmetic disability. Currently, there is no reliable form of treatment for NLD, although many regimens have been tried (Shall et al., 1990)

The mechanism of RNA binding to TRAP: initiation and cooperative interactions.

The trp RNA-binding Attenuation Protein (TRAP) from Bacillus subtilis is an 11-subunit protein that binds a series of 11 GAG and UAG repeats separated by two to three-spacer nucleosides in trp leader mRNA. The structure of TRAP bound to an RNA containing 11 GAG repeats shows that the RNA wraps around the outside of the protein ring with each GAG interacting with the protein in nearly identical fashion. The only direct hydrogen bond interactions between the protein and the RNA backbone are to the 2'-hydroxyl groups on the third G of each repeat. Replacing all 11 of these guanosines with deoxyriboguanosine eliminates measurable binding to TRAP. In contrast, a single riboguanosine in an otherwise entirely DNA oligonucleotide dramatically stabilizes TRAP binding, and facilitates the interaction of the remaining all-DNA portion with the protein. Studies of TRAP binding to RNAs with between 2 and 11 GAGs, UAGs, AAGs, or CAGs showed that the stability of a TRAP-RNA complex is not directly proportional to the number of repeats in the RNA. These studies also showed that the effect of the identity of the residue in the first position of the triplet, with regard to binding to TRAP, is dependent on the number of repeats in the RNA. Together these data support a model in which TRAP binds to RNA by first forming an initial complex with a small subset of the repeats followed by a cooperative interaction with the remaining triplets.

A disease-associated cellular immune response in type 1 diabetics to an immunodominant epitope of insulin.

The 9-23 amino acid region of the insulin B chain (B9-23) is a dominant epitope recognized by pathogenic T lymphocytes in nonobese diabetic mice, the animal model for type 1 diabetes. We describe herein similar (B9-23)-specific T-cell responses in peripheral lymphocytes obtained from patients with recent-onset type 1 diabetes and from prediabetic subjects at high risk for disease. Short-term T-cell lines generated from patient peripheral lymphocytes showed significant proliferative responses to (B9-23), whereas lymphocytes isolated from HLA and/or age-matched nondiabetic normal controls were unresponsive. Antibody-mediated blockade demonstrated that the response was HLA class II restricted. Use of the highly sensitive cytokine-detection ELISPOT assay revealed that these (B9-23)-specific cells were present in freshly isolated lymphocytes from only the type 1 diabetics and prediabetics and produced the proinflammatory cytokine IFN-gamma. This study is, to our knowledge, the first demonstration of a cellular response to the (B9-23) insulin epitope in human type 1 diabetes and suggests that the mouse and human diseases have strikingly similar autoantigenic targets, a feature that should facilitate development of antigen-based therapeutics.

Impact of insulin lispro on HbA1c values in insulin pump users.

AIM: To compare the therapeutic efficacy of the short-acting insulin analogue insulin lispro (Humalog) with that of buffered regular human insulin (Velosulin) in patients on insulin pump therapy. PATIENTS AND METHODS: Sixty-two (45 women and 17 men) young patients with type 1 diabetes using insulin pump therapy were compared while using buffered regular human insulin for a mean +/- s.e.m. of 20.1+/-1.2 months or insulin lispro for a mean +/- s.e.m. of 19.7+/-0.5 months. The initial mean +/- s.e.m. age and duration of diabetes were 29.1+/-0.9 and 17.7+/-0.9 years, respectively. The mean HbA1c values, basal insulin dosages, premeal insulin dosages and number of low blood sugars were recorded during treatment with both insulins. RESULTS: Mean +/- s.e.m. HbA1c values were significantly lower (p < 0.001; paired Wilcoxon t-test) during insulin lispro treatment (7.4+/-0.1%) as compared to treatment with buffered regular human insulin (7.9+/-0.1%). Total units of insulin (mean +/- s.e.m.)/kg/day was significantly (p = 0.03) lower (0.61+/-0.02) during the insulin lispro treatment period as compared to the buffered regular human insulin treated period (0.65+/-0.03). Total mean +/- s.e.m. (U/kg/day) of basal insulin administered per day was higher when patients received insulin lispro treatment (0.44+/-0.02 vs. 0.42+/-0.01 for buffered regular human insulin treated period; p = 0.002). The premeal insulin boluses (mean +/- s.e.m.) for the two treatment groups were significantly different with less insulin required for the insulin lispro treatment period for all three meals (p < 0.001, t-test). The number of mild/moderate and severe hypoglycaemic episodes were similar in the two groups. CONCLUSION: We conclude that use of insulin lispro in pump therapy significantly lowers HbA1c values in comparison to therapy with buffered regular human insulin insulin without increasing hypoglycaemic episodes.

Probing the TRAP-RNA interaction with nucleoside analogs.

The trp RNA-binding Attenuation Protein (TRAP) from Bacillus subtilis binds a series of GAG and UAG repeats separated by 2-3 nonconserved spacer nucleotides in trp leader mRNA. To identify chemical groups on the RNA required for stability of the TRAP-RNA complex, we introduced several different nucleoside analogs into each pentamer of the RNA sequence 5'-(UAGCC)-3' repeated 11 times and measured their effect on the TRAP-RNA interaction. Deoxyribonucleoside substitutions revealed that a 2'-hydroxyl group (2'-OH) is required only on the guanosine occupying the third residue of the RNA triplets for high-affinity binding to TRAP. The remaining hydroxyl groups are dispensable. Base analog substitutions identified all of the exocyclic functional groups and N1 nitrogens of adenine and guanine in the second and third nucleotides, respectively, of the triplets as being involved in binding TRAP. In contrast, none of the substitutions made in the first residue caused any detectable changes in affinity, indicating that elements of these bases are not necessary for complex formation and stability. Studies using abasic nucleotides in the first residue of the triplets and in the two spacer residues confirmed that the majority of the specificity and stability of the TRAP-RNA complex is provided by the AG dinucleotide of the triplet repeats. In addition to direct effects on binding, we demonstrate that the N7-nitrogen of adenosine and guanosine in UAG triplet and the 2'-OHs of (UAGCC)11 RNA are involved in the formation of an as yet undetermined structure that interferes with TRAP binding.

Heterophile anti-mouse immunoglobulin antibodies may interfere with cytokine measurements in patients with HLA alleles protective for type 1A diabetes.

Wilson and coworkers (Wilson SB, Kent SC, Patton KT, Orban T, Jackson RA, Exley M, Porcelli S, Schatz DA, Atkinson MA, Balk SP, Strominger JL, Hafler DA: Extreme Th1 bias of invariant V alpha24J alpha Q T-cells in type 1 diabetes. Nature 391:177-181, 1998) have recently reported raised serum levels of interleukin-4 (IL-4) in anti-islet autoantibody-positive first-degree relatives of patients with type 1A diabetes who did not progress to diabetes. Protection from diabetes has been noted for several human lymphocyte antigen (HLA) alleles, such as HLA DR2-DQA1*0102-DQB1*0602. We, therefore, wanted to determine whether this cytokine phenotype was associated with HLA genes protective for type 1A diabetes. We used a two-site fluoroimmunoassay with the same monoclonal antibodies as those reported by Wilson et al. Using this assay, we have found evidence for human heterophile antibodies mimicking serum IL-4: all serum IL-4 reactivity was lost if mouse serum or mouse immunoglobulin were added to the assay; serum IL-4 activity was bound and then eluted by protein A/G chromatography; and levels of anti-mouse antibodies correlated with apparent serum IL-4. This pseudo-IL-4 activity was found in a subset of control subjects, patients with type 1A diabetes, and their relatives and was primarily associated with specific HLA alleles protective for type 1A diabetes (e.g., DQB1*0602). After adjustment for HLA, positive levels of heterophile antibodies were not associated with protection from diabetes. The confounding effect of protective HLA alleles associated with heterophile antibodies could explain the previously reported association between raised serum IL-4 and protection from type 1A diabetes. The mechanism by which specific DQ alleles protect from diabetes and are associated with increased heterophile antibodies is currently unknown.

Autoreactive T cell responses in insulin-dependent (Type 1) diabetes mellitus. Report of the first international workshop for standardization of T cell assays.

Type 1 diabetes is thought to result from a T cell-mediated destruction of the pancreatic beta-cells. Multiple and sometimes conflicting studies have identified a variety of aberrations in the cellular immune response to autoantigens in persons with the disease. Potential explanations for these discrepancies include incomparable techniques or culture conditions, diversity in the populations of patients or controls tested, and differences in autoantigen preparations. A T cell workshop was organized by the Immunology of Diabetes Society with the aim of appreciating and identifying problems associated with autoreactive T cell assays in type 1 diabetes. As a first phase, a series of candidate autoantigens were analysed by reference laboratories for quality. Subsequently, these preparations, as well as control stimuli, were distributed in a blind fashion to 26 laboratories worldwide, including all experienced centres, for analysis of T cell proliferation assays in 10 recent onset type 1 diabetes and 10 non-diabetic controls. For this analysis, participants used their own assays and references. The islet autoantigen quality control analyses performed prior to the distribution indicate that the quality of recombinant autoantigen preparations requires improvement. For example, several T cell clones specific for glutamic acid decarboxylase (GAD65) were unable to cross-react with GAD65 expressed in baculovirus, yeast or bacteria. Moreover, autoantigens expressed in E. coli interfered with autoantigen-specific proliferation of both T cell clones and peripheral blood mononuclear cells. Nonetheless, responses could be measured to all autoantigen preparations evaluated in the workshop. During the blind phase of the study, all centres were able to reproducibly measure T cell responses to two identical samples of tetanus toxoid, but there was significant interlaboratory variation in sensitivity and extent of the proliferative response measured. Third, the results using candidate autoantigens indicated that although a few laboratories could distinguish type 1 diabetes patients from non-diabetic controls in proliferative responses to individual islet autoantigens, in general, no differences in T cell proliferation between the two groups could be identified. This first T cell workshop on T cell autoreactivity in type 1 diabetes confirms that this was a difficult area for interlaboratory investigations, but provided insight towards future efforts focused on standardizing autoreactive T cell measurements. Some previously reported conflicting results can in part be explained by the observed interlaboratory variability. The inability to discriminate normal controls from new onset type 1 diabetes patients suggests that measuring proliferative responses in PBMC represents an incomplete picture of the immune response, perhaps complicated by difficulties in identifying suitable antigens and assays for standardized use.

DRB1*04 and DQ alleles: expression of 21-hydroxylase autoantibodies and risk of progression to Addison's disease.

Of 957 patients with type 1 diabetes without known Addison's disease 1.6% (n = 15) were positive for 21-hydroxylase autoantibodies. Among DQ8/DQ2 heterozygous patients, the percentage expressing 21-hydroxylase autoantibodies was 5% (10 of 208) vs. less than 0.5% of patients with neither DQ8 nor DQ2. Three of the diabetic patients found to have 21-hydroxylase autoantibodies on screening were subsequently diagnosed with Addison's disease. Overall, the genotype DQ8/DQ2, consisting of DRB1*0404/DQ8 with DRB1*0301/DQ2, was present in 14 of 21 patients with Addison's disease (8 of 12 with diabetes and 6 of 9 without diabetes or antiislet autoantibodies) vs. 0.7% of the general population (109 of 15,547; P < 10(-6)) and 11% of patients with DM without Addison's disease (62 of 578; P < 10(-6)). Among patients with diabetes with DQ8, Addison's disease was strongly associated with the specific DRB1 subtype, DRB1*0404 (8 of 9 patients from 8 families, in contrast to only 109 of 408 DQ8 DM patients with diabetes without Addison's disease having DRB1*0404; P < 0.001). Among 21-hydroxylase autoantibody-positive DQ8 patients, 80% with DRB1*0404 (12 of 15) had Addison's disease, in contrast to 1 of 10 autoantibody-positive patients with DRB1*0401 or DRB1*0402 (P < 0.001). We conclude that patients with DRB1*0404 and 21-hydroxylase autoantibodies are at high risk for Addison's disease. Patients with DRB1*0401 and DRB1*0402 have more limited progression to Addison's disease despite the presence of 21-hydroxylase autoantibodies.

RNA structure inhibits the TRAP (trp RNA-binding attenuation protein)-RNA interaction.

TRAP (trp RNA-binding attenuation protein) regulates expression of the tryptophan biosynthetic genes in response to tryptophan in Bacillus subtilis by binding to two sites containing a series of 9 or 11 (G/U)AG triplet repeats that are generally separated by two or three spacer nucleotides. Previous mutagenesis experiments have identified three TRAP residues, Lys-37, Lys-56, and Arg-58 that are essential for RNA binding. The location of these residues on the TRAP oligomer supports the proposal that RNA binds TRAP by encircling the TRAP oligomer. In this work, we show that RNAs containing 11 GAG or UAG repeats separated by CC dinucleotide spacers (((G/U)AGCC)11) form stable structures that inhibit binding to TRAP. This conclusion is based on the effects of temperature and Mg2+ on the affinity of TRAP for RNAs with CC spacers combined with UV hyperchromicity and circular dichroism. Furthermore, introducing the base analogue 7-deazaguanosine in the ((G/U)AGCC)11 RNAs stabilized the TRAP-RNA interaction. This effect was associated with decreased stability of the RNA structure as measured by circular dichroism spectroscopy. The precise nature of the structure of the ((G/U)AGCC)11 RNAs is not known but evidence is presented that it involves noncanonical interactions. We also observed that substitution of Arg-58 with Lys further reduced the ability of TRAP to interact with structured RNAs. Since in vivo function of TRAP may involve binding to structured RNAs, we suggest a potential function for this residue, which is conserved in TRAP from three different bacilli.

Diagnosis and treatment of pre-insulin dependent diabetes.

The development of genetic and serological markers of autoimmune Type I diabetes has allowed us to begin to identify subjects at risk for the development of Type I diabetes. Assessment of these variables can assign risk to subjects and permit the implementation of immune intervention trials in an attempt to alter the course of pre-diabetes. This review discusses relevant aspects of the genetics and diagnosis of pre-diabetes, and both current and future clinical trials that are attempting to prevent the full expression of clinical diabetes in these individuals.

Enzymatic incorporation of 2'-thio-CTP into the HDV ribozyme.

We have synthesized the analogue 2'-deoxy-2'-thio-CTP (CTP-SH) and tested its ability to support RNA transcription in place of CTP. The modified nucleotide in a transcription reaction and in the absence of CTP generated the appropriately sized fragment when a mutant T7 polymerase (Y639F) was used. Wild-type polymerase was unable to generate RNA under the same conditions. Transcription was optimal around pH 7.5 and was dependent upon CTP-SH concentration. Transcripts containing the analogue were efficiently isolated using a thiol-activated sepharose column. Insertion of CTP-SH into the HDV ribozyme, replacing all cytidine residues with 2'-thiocytidine, appears to inhibit self-cleaving activity, even in the presence of manganese. The ability to introduce the CTP-SH analogue enzymatically into RNA opens the way for new structure-function studies where the 2'-hydroxyl can be efficiently replaced by a thiol group.