Extremely low vertical-emittance beam in the accelerator test facility at KEK.

Electron beams with the lowest, normalized transverse emittance recorded so far were produced and confirmed in single-bunch-mode operation of the Accelerator Test Facility at KEK. We established a tuning method of the damping ring which achieves a small vertical dispersion and small x-y orbit coupling. The vertical emittance was less than 1% of the horizontal emittance. At the zero-intensity limit, the vertical normalized emittance was less than 2.8 x 10(-8) rad m at beam energy 1.3 GeV. At high intensity, strong effects of intrabeam scattering were observed, which had been expected in view of the extremely high particle density due to the small transverse emittance.

Pre-operative intra-aortic balloon counterpulsation in stable patients with left main coronary disease.

OBJECTIVE: To determine whether prophylactic, pre-operative, intra-aortic balloon counterpulsation (IABC) improves clinical outcome in stable patients with severe left main coronary artery disease. METHODS: A post-hoc analysis of 457 prospectively tracked, non-randomized patients undergoing coronary artery bypass graft surgery (CABG) for left main stenoses 50% and multivessel coronary disease, but without any hemodynamic compromise or ongoing angina, was conducted. Patients with heart failure, shock, ongoing ischemia or previous CABG were excluded. In 287 patients, pre-operative IABC was not used (Group 1), while IABC was initiated in 170 patients for "prophylaxis" (Group 2). RESULTS: Groups 1 and 2 were similar in age (67 +/- 10 years versus 67 +/- 11 years, respectively), sex (72% male versus 71% male, respectively), and body mass index (28 +/- 5.5 versus 27 +/- 5.1, respectively). However, more Group 1 patients had peripheral vascular disease (PVD) (25% versus 11%), but more Group 2 patients had diabetes (37% versus 29%), and a lower left ventricular ejection fraction. The unadjusted 30-day mortality was significantly higher in Group 1 [16 (5.6%) versus 2 (1.2%); p = 0.02]. Cardiopulmonary bypass time and post-operative length of stay did not differ between the two groups. After adjusting for PVD in the multivariate analysis, the p-value for the no IABP versus IABP comparison in the presence of PVD was 0.10, even though 0/18 patients with PVD and IABC died. CONCLUSION: While unadjusted mortality appears lower with prophylactic IABC, confounding variables such as PVD mandate a larger, randomized clinical trial in order to establish the role of IABC in stable patients with left main disease.

Characterization of a novel H2A(-)E+ transgenic model susceptible to heterologous but not self thyroglobulin in autoimmune thyroiditis: thyroiditis transfer with Vbeta8+ T cells.

Recently we reported on a novel H2E transgenic, IA-negative model of experimental autoimmune thyroiditis (EAT) that excludes reactivity to self in its susceptibility pattern to heterologous thyroglobulin (Tg). In conventional, susceptible mouse strains, EAT is inducible with both homologous and heterologous Tg; e.g., human (h)Tg shares conserved thyroiditogenic epitopes with mouse (m)Tg. However, when an H2Ea(k) transgene is introduced into class II-negative B10.Ab(0) mice, which express neither surface IA (mutant Abeta-chain) nor surface IE (nonfunctional Ea gene), the resultant H2E(b) molecules are permissive for EAT induction by hTg, but not self mTg. Also, the hTg-primed cells do not cross-react with mTg. To explore this unique capacity of E+B10.Ab(0) mice to distinguish self from nonself Tg, we have developed T cell lines to examine the T cell receptor repertoire and observed a consistent Vbeta8+ component after repeated hTg stimulation. Enrichment and activation of Vbeta8+ T cells by either superantigen staphylococcal entertoxin B or anti-Vbeta8 in vitro enabled thyroiditis transfer to untreated A-E+ recipients, similar to hTg activation. Vbeta8+ T cells isolated by FACS from hTg-immunized mice also proliferated to hTg in vitro. These studies support the contribution of Vbeta8 genes to the pathogenicity of hTg in this H2A-E+ transgenic model.

Peptide-based analysis of amino acid sequences important to the biological activity of eosinophil granule major basic protein.

Synthetic peptides corresponding to amino acid sequences in eosinophil granule major basic protein (MBP) were evaluated for cytotoxic activity toward K562 cells and for ability to stimulate basophil mediator release. Results obtained using 14 peptides spanning the 117-amino acid sequence of MBP in overlapping fashion indicated that the activities mapped to peptide sequences near the amino and carboxy termini of MBP. The activity of these regions was confirmed using two peptides corresponding to MBP residues 18-45 and 89-117. A 20-h incubation with 5 microM peptide 18-45 or peptide 89-117 caused approximately the same levels (>60%) of cytotoxicity in K562 cells as 5 microM MBP. Similarly, a 30-min incubation with peptides 18-44 and 89-117 stimulated basophil histamine release in a concentration-dependent manner over the range of 5-20 microM. The level of release stimulated by 20 microM peptide 89-117 approached that stimulated by 2 microM MBP. A 20 microM concentration of peptide 89-117 also stimulated leukotriene C4 (LTC4) production by the basophils. Neither peptide 18-45 nor peptide 89-117 was cytotoxic for basophils under the experimental conditions for histamine and LTC4 release, as determined by 51Cr release. These results indicate that two MBP peptide sequences, including one (89-117) that contains a unique carbohydrate-binding region, share the biologic activities of MBP.

Abciximab for the treatment of acute distal embolization associated with internal carotid artery angioplasty.

Carotid artery angioplasty and stent placement (CAS) can be complicated by procedure-related distal embolization and thrombus formation, potentially resulting in neurological sequelae. Patient A had CAS of left internal carotid artery and had loss of vision in the left eye with no flow in the left ophthalmic artery. Patient B had CAS of right internal carotid artery and developed left sided hemiparesis with total occlusion of the right middle cerebral artery. A selective intra-arterial injection of 5 mg of abciximab locally followed by a bolus of 5 mg of abciximab intravenously resulted in complete resolution of the filling defect on repeat angiography after 10 min in both patients.

Altering behavioral responses and dopamine transporter protein with antisense peptide nucleic acids.

The dopamine transporter (DAT) plays a role in locomotion and is an obligatory target for amphetamines. We designed and synthesized an antisense peptide nucleic acid (PNA) to rat DAT to examine the effect of this antisense molecule on locomotion and on responsiveness to amphetamines. Rats were injected intraperitoneally daily for 9 days with either saline, an antisense DAT PNA, a scrambled DAT PNA, or a mismatch DAT PNA. On days 7 and 9 after initial motility measurements were taken, the animals were challenged with 10 mg/kg of amphetamine and scored for motility. On day 7, there was no significant difference between the baseline levels of activity of any of the groups or their responses to amphetamine. On day 9, the antisense PNA-treated rats showed a statistically significant increase in their resting motility (P < 0.01). When these rats were challenged with amphetamine, motility of the saline-, scrambled PNA-, and mismatch PNA-treated animals showed increases of 31-, 36-, and 20-fold, respectively, while the antisense PNA-treated animals showed increases of only 3.4-fold (P < 0.01). ELISA results revealed a 32% decrease in striatal DAT in antisense PNA-treated rats compared with the saline, scrambled PNA, and mismatch PNA controls (P < 0.001). These results extend our previous findings that brain proteins can be knocked down in a specific manner by antisense molecules administered extracranially. Additionally, these results suggest some novel approaches for the treatment of diseases dependent upon the function of the dopamine transporter.

Intraperitoneal injection of antisense peptide nucleic acids targeted to the mu receptor decreases response to morphine and receptor protein levels in rat brain.

To determine the effectiveness of peptide nucleic acids (PNAs) in vivo, we designed and synthesized PNAs antisense to the mu receptor, the molecular target of morphine for inducing antinociception. Responsiveness of rats to morphine and the levels of mu receptor expression after treatment was measured. We delivered intraperitoneal injections of antisense PNAs targeted to the mu receptor (AS-MOR), mismatch PNAs (AS-MOR MM), antisense PNAs targeted to the neurotensin receptor subtype 1 (AS-NTR1), or saline and then challenged the rats with 5 mg/kg morphine (intraperitonally) or neurotensin directly into the periaqueductal gray region of the brain. To avoid tolerance, separate groups of animals were tested at 24, 48, and 72 h post-PNA treatment. Only animals treated with the AS-MOR showed a reduction in their antinociceptive response to morphine. The lack of effect of morphine on the AS-MOR rats was profound at 24 and 48 h, but animals tested at 72 h were similar to control groups. At 24 h the AS-MOR rats had a significant 55% decrease in the levels of mu receptor in their periaqueductal gray region, while AS-MOR MM rats showed no significant change. Lastly, the AS-MOR rats continued to show a normal antinociceptive response to neurotensin. This study, therefore, provides additional support for the use of PNAs to target proteins within brain by systemically administered PNAs.

Right heart catheterization in the presence of an inferior vena cava filter.

Inferior vena cava filters are being inserted with increasing frequency. When such patients later require right heart catheterization, brachial or jugular vein access is usually attempted. We describe our experience in 10 consecutive patients using the standard femoral approach, first assessing filter patency and then carefully crossing the filter using a straight guidewire. The right heart chambers were successfully accessed in every case. There were no complications, and in no case did the filter migrate or become dislodged. This technique may prove useful when right heart catheterization is indicated in a patient who has a Greenfield inferior vena cava filter.

Rheolytic thrombectomy during percutaneous revascularization for acute myocardial infarction: experience with the AngioJet catheter.

BACKGROUND: Although balloon angioplasty and stenting are effective in the treatment of acute myocardial infarction (MI), reduced coronary flow and distal embolization frequently complicate interventions when thrombus is present. Adjunctive treatment with mechanical thrombectomy devices may reduce these complications. METHODS AND RESULTS: We evaluated the angiographic and clinical outcomes of 70 patients with acute MI (16% with cardiogenic shock) and with angiographically evident thrombus who were treated with AngioJet rheolytic thrombectomy followed by immediate definitive treatment. Procedure success (residual diameter stenosis <50% and Thrombolysis in Myocardial Infarction [TIMI] flow > or =2 after final treatment) was achieved in 93.8%. Clinical success (procedure success without major in-hospital cardiac events) was achieved in 87.5%, with an in-hospital mortality rate of 7.1%. Final TIMI 3 flow was achieved in 87.7%. AngioJet treatment resulted in a mean thrombus area reduction from 73.2 +/- 64.6 mm(2) at baseline to 15.5 +/- 30.1 post-thrombectomy (P <.001). Subsequent definitive treatment included stenting in 67% and balloon angioplasty alone in 26% of patients. Procedural complications included distal embolization in six patients and perforation in two patients. There were no further major adverse events during 30-day follow-up. CONCLUSION: Rheolytic thrombectomy can be performed safely and effectively in patients with acute MI, allowing for immediate definitive treatment in thrombus-containing lesions.

A single amino acid of the human and rat neurotensin receptors (subtype 1) determining the pharmacological profile of a species-selective neurotensin agonist.

The neurotensin (NT) receptor, subtype 1 (NTR1), is a 7-transmembrane-spanning receptor, forming 3 extracellular and 3 intracellular loops. Previously, we showed that the third outer loop (E3) is the binding site for NT and its analogs, several of which bind with higher affinity to rat NTR1 (rNTR1) than to human NTR1 (hNTR1). In particular, NT34 [3,1'-naphthyl-l-Ala(11)]NT(8-13) has greater than 60-fold higher affinity for rNTR1 (46 and 60 pM for transiently- and stably-transfected cells, respectively) than for hNTR1 (2.8 and 5.8 nM for transiently- and stably-transfected cells, respectively) isolated from transfected cell membranes. Previously, our molecular modeling studies of rNTR1 and hNTR1 showed that the binding pocket in the human receptor for NT34 is smaller in volume from the bulky residue Tyr(339) in the pocket center, as compared with the corresponding residue Phe(344) in the rat binding pocket. Therefore, with site-directed mutagenesis, we derived mutant forms of rNTR1(F344Y) and hNTR1(Y339F). Examination of the mutant receptors from membranal preparations of transfected cells in radioligand binding assays and with intact cells in functional assays (phosphatidyl-4,5-bisphosphate turnover) showed that the human-like rat receptor and the rat-like human receptor bound NT34 with a predicted reverse of binding compared with its binding to the wild-type receptors. These results strongly affirm our molecular modeling studies and demonstrate the importance of the study of even minor structural variations in proteins to determine the basis of significantly different drug responses, an area of focus for pharmacological research in the 21st century.

A novel neurotensin peptide analog given extracranially decreases food intake and weight in rodents.

Neurotensin decreases food intake in the rat when injected into the cerebral ventricles. We tested the effect of a novel neurotensin analog (NT69L), injected intra-peritoneally (i.p.), on weight gain and food intake in rats. Sprague-Dawley rats (270 g) were injected i. p. with either saline or NT69L at 0.001 or 0.010 mg/kg. In further experiments, larger rats at a more steady state on the growth curve (400 g) were injected with either saline or 0.010 or 1 mg/kg NT69L. Food intake, water consumption and body weight were recorded daily. Weight gain was significantly reduced in the smaller rats injected with 0.001 or 0.010 mg/kg, showing only a 8.5 and 9.0% increase in original weight, respectively, as compared to a 29% increase for the controls. The larger rats injected with 1 mg/kg, had a significant reduction in body weight with a 3.0% decrease in original body weight as compared to a 2.4% increase for the controls. Food intake was significantly reduced suggesting that the weight loss observed after injection of NT69L was attributable in part to a reduction in food intake. The genetically obese Zucker rats injected with NT69L (1 mg/kg) had a significant reduction in weight gain and food intake. NT69L significantly increased blood glucose and corticosterone levels and decreased TSH and T4 in Sprague-Dawley and Zucker rats, an effect that was only transitory. NT69L also caused a decrease in norepinephrine in both the hypothalamus and nucleus accumbens, and an increase in dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and serotonin. In this study, NT69L exhibited a consistent and dramatic effect on body weight and food intake in Sprague-Dawley and obese Zucker rats, and enabled us to study the role that NT plays in weight control and the functional interactions of NT with brain amines, and metabolic and endocrinological parameters.

Effects of a novel neurotensin peptide analog given extracranially on CNS behaviors mediated by apomorphine and haloperidol.

Neurotensin (NT) is a neuropeptide neurotransmitter in the central nervous system. It has been implicated in the therapeutic and in the adverse effects of neuroleptics. Activity of NT in brain can only be shown by direct injection of the peptide into that organ. However, we have developed a novel analog of NT(8-13), NT69L, which is active upon intraperitoneal (i.p.) injection. Like atypical neuroleptics, NT69L blocked the climbing behavior in rats, but not the licking and sniffing behaviors of a high dose (600 microgram/kg) of the non-selective dopamine agonist apomorphine. Its blockade of climbing was very potent with an ED(50) (effective dose at 50% of maximum) of 16 microgram/kg. Both apomorphine and NT69L caused a long-lasting hypothermia, which was greater with the peptide but not synergistic in combination with apomorphine. The ED(50) of NT69L for hypothermia was 390 microgram/kg. NT69L (up to 5 mg/kg i.p.) did not produce catalepsy. However, when given before haloperidol, NT69L, but not clozapine, completely prevented catalepsy. When given after haloperidol, NT69L, but not clozapine, reversed haloperidol's cataleptic effects with an ED(50) of 260 microg/kg. There was no significant difference between the ED(50)s for hypothermia and anticataleptic effects of NT69L. However, the ED(50) for blocking the effects of apomorphine was significantly lower than the other two. These data suggest that NT69L may have neuroleptic properties in humans and may be useful in the treatment of extrapyramidal side effects caused by typical neuroleptics such as haloperidol.

Highly potent neurotensin analog that causes hypothermia and antinociception.

The tridecapeptide neurotensin has long been proposed as an endogenous neuroleptic. However, for neurotensin [or neurotensin(8-13) [NT(8-13)], the active fragment] to cause its effects, it must be administered centrally. Here, we report on an analog of NT(8-13), (N-methyl-Arg),Lys,Pro,L-neo-Trp,tert-Leu,Leu (NT69L), which contains a novel amino acid, L-neo5 degrees C (rectal), with a significant effect persisting for over 7 h. NT69L also caused a rapid (within 15 min) and persistent (for over 5 h) antinociceptive effect, as determined by the hot plate test. NT69L was overall the most potent and longest lasting neurotensin analog that has been reported. These studies provide the background for further testing of a stable, potent and long lasting neurotensin analog as a potential neuroleptic.

Analysis of binding sites and efficacy of a species-specific peptide at rat and human neurotensin receptors.

We have developed a neurotensin analog, L-[3,1'-naphthylalanine11]NT(8-13), NT34, that can distinguish between rat and human neurotensin receptors, and exhibits more than a 100-fold difference in binding affinities and a 60-fold difference in functional coupling to phosphatidylinositol turnover. Using cells transfected with different numbers of the appropriate receptors, we measured the changes in phosphatidylinositol production, and then evaluated the efficiency of receptor-effector coupling based on Furchgott's design. The binding of NT34 at both rat and human neurotensin receptors stably expressed in CHO-K1 cells was to two sites, while the binding of NT was to one site. At the rat receptor the equilibrium dissociation constant (Kd) for NT34 at the high-affinity site was 0.058 nM, while that at the low-affinity site was 3.1 nM. For the human receptor at the high-affinity site, the Kd for NT34 was 18 nM, while that at the low-affinity site was 180 nM. For both species the percentage of receptors representing the high-affinity site was approximately 60-70% with 30-40% at the low-affinity site. We derived agonist dissociation constants (Ka) for NT and NT34, which suggest that for NT34, the low-affinity site is functionally coupled to phosphatidylinositol turnover. Finally, we compared the relative efficacies of both compounds and found that NT34 was about 2-fold and 4-fold more efficacious than NT in stimulating phosphatidylinositol turnover in rat and human NT receptors, respectively.

BALB/c mice produce blister-causing antibodies upon immunization with a recombinant human desmoglein 3.

Pemphigus vulgaris (PV) is an Ab-mediated autoimmune blistering disease of mucotaneous surfaces. Over 95% of the patients with PV express DR4 or DRw6, and the disease is characterized by the presence of autoantibodies directed against desmoglein 3 (Dsg 3), a protein expressed on keratinocytes. An appropriate animal model is required to understand immunoregulation and to address the role of immunogenetic components in the production of pathogenic Abs that are characteristic of PV. Therefore, we turned to the development of a mouse model. Four strains of female mice (BALB/c, DBA/1, SJL/J, and HRS/J) were screened for their ability to produce pathogenic anti-Dsg 3 Abs. We demonstrated that only BALB/c mice immunized with a full-length Dsg 3 can produce pathogenic Abs capable of causing acantholysis of human foreskin in culture and blistering in neonatal mice. This observation suggested that either H-2d or the BALB background contains the immunogenetic makeup necessary for the production of pathogenic anti-Dsg 3 Abs. No correlation was noted between a given isotype and the pathogenic potential of autoantibodies from different strains of mice. Similarly, the pattern of reactivity of Abs with a panel of 46 synthetic peptides that span the entire Dsg 3 failed to reveal any association between binding specificity and the pathogenic potential, and suggested that pathogenic Abs might recognize conformational epitopes. Moreover, our studies showed that the epitopes recognized by pathogenic Abs are contained within the extracellular Dsg 3.

H2-E transgenic class II-negative mice can distinguish self from nonself in susceptibility to heterologous thyroglobulins in autoimmune thyroiditis.

Susceptibility to experimental autoimmune thyroiditis (EAT) is linked to H2-A class II genes; k and s haplotypes are susceptible, while b and f are resistant. EAT is inducible with thyroglobulins (Tgs) from several mammalian species which share portions of identical sequences. But cross-activation and cross-tolerance studies with mouse (m), human (h), and porcine (p) Tg have indicated mTg-unique T-cell epitope(s), in addition to conserved, in EAT induction. The recent introduction of the HLA-DRB1*0301 (DR3) transgene rendered major histocompatibility complex (MHC) class II-negative (Ab(0)) mice susceptible to EAT induction by both hTg and mTg, suggesting usage of conserved epitopes. Here, we introduced the H2-Ea(k) transgene into resistant B10 (H2(b)) or Ab(0) mice with a defective Ea gene to provide functional surface H2E (b haplotype) expression. Surprisingly, both transgenic strains showed severe inflammation only after hTg, but not mTg, immunization, although the moderating influence of the A(b) gene in B10 was evident. In proliferative assays, hTg-primed cells did not respond to mTg, nor to conserved 12mer peptides from three primary hormonogenic sites, two of which can activate T cells for thyroiditis transfer and cytotoxicity. The vigorous response to hTg stimulation was reduced only by Ebeta(b)-specific monoclonal antibody. EAT induction with bovine and pTg showed responses similar to hTg, suggesting thyroiditogenic epitopes shared with hTg, but not mTg. This is the first demonstration of: (1) nonpermissiveness for EAT induction with mTg, normally the most thyroiditogenic Tg and the one with unique epitopes for susceptible mice, and (2) the separation of hTg from mTg in EAT induction in H2-E-transgenic mice.

Identification of the molecular genetic defect of patients with methemoglobin M-Kankakee (M-Iwate), alpha87 (F8) His --> Tyr: evidence for an electrostatic model of alphaM hemoglobin assembly.

We determined that the molecular defect of 2 patients with hemoglobin (Hb) M-Kankakee [Hb M-Iwate, alpha87 (F8) His --> Tyr] resides in the alpha1-globin gene. The proportion of Hb M observed is higher than that predicted for an alpha1-globin variant. Our evidence suggests that the greater-than-expected proportion of Hb M-Kankakee results from preferential association of the electronegative beta-globin chains with the alpha(M)-globin chains that are more electropositive than normal alpha-globin chains.

In vitro binding and CNS effects of novel neurotensin agonists that cross the blood-brain barrier.

Animal studies with neurotensin (NT) directly injected into brain suggest that it has pharmacological properties similar to those of antipsychotic drugs. Here, we present radioligand binding data for some novel hexapeptide analogs of NT(8-13) at the molecularly cloned rat and human neurotensin receptors (NTR-1), along with behavioral and physiological effects of several of these peptides after intraperitoneal (i.p.) administration in rats. One unique analog, NT66L, which had high affinity (0.85 nM) for the molecularly cloned rat neurotensin receptor (NTR-1), caused a drop in body temperature and antinociception at doses as low as 0.1 mg/kg after i.p. injection. At 30 min post-injection, the ED50 for NT66L-induced hypothermia (rectal temperature) and antinociception (hot plate test) was 0.5 and 0.07 mg/kg, respectively. At a dose of 1 mg/kg i.p., NT66L caused 100% of the maximum possible effect for antinociception for up to 2 h after administration. At this dose body temperature lowering was greater than -2.5 degrees C from 20 to 120 min after i.p. administration. These results in animals suggest that NT66L has agonist properties at NTR-1 in vivo after extracranial administration and provide support for its further study in behavioral tests predictive of neuroleptic activity.

Peptide nucleic acids targeted to the neurotensin receptor and administered i.p. cross the blood-brain barrier and specifically reduce gene expression.

Intraperitoneal injection of an unmodified antisense peptide nucleic acid (PNA) complementary to mRNA of the rat neurotensin (NT) receptor (NTR1) was demonstrated by a gel shift assay to be present in brain, thus indicating that the PNA had in fact crossed the blood-brain barrier. An i.p. injection of this antisense PNA specifically inhibited the hypothermic and antinociceptive activities of NT microinjected into brain. These results were associated with a reduction in binding sites for NT both in brain and the small intestine. Additionally, the sense-NTR1 PNA, targeted to DNA, microinjected directly into the brain specifically reduced mRNA levels by 50% and caused a loss of response to NT. To demonstrate the specificity of changes in behavioral, binding, and mRNA studies, animals treated with NTR1 PNA were tested for behavioral responses to morphine and their mu receptor levels were determined. Both were found to be unaffected in these NTR1 PNA-treated animals. The effects of both the antisense and sense PNAs were completely reversible. This work provides evidence that any antisense strategy targeted to brain proteins can work through i. p. delivery by crossing the normal blood-brain barrier. Equally important was that an antigene strategy, the sense PNA, was shown in vivo to be a potentially effective therapeutic treatment.

Hb Chile [beta28(B10)Leu-->Met]: an unstable hemoglobin associated with chronic methemoglobinemia and sulfonamide or methylene blue-induced hemolytic anemia.

Among the causes of life-long cyanosis are congenital methemoglobinemia due to M hemoglobins, congenital methemoglobinemia due to methemoglobin reductase deficiency, a small number of low oxygen affinity hemoglobins, and a small number of unstable hemoglobins that spontaneously form methemoglobin in vivo at an accelerated rate. We report an unstable hemoglobin with these characteristics that was observed in a family of indigenous (native American) origin living near Santiago, Chile. This variant has the substitution beta28(B10)Leu-->Met, unambiguously corresponding to the DNA mutation of CTG-->ATG in beta-globin gene codon 28.