Regional glucose andβ-Hydroxybutyrate use by developing rat brain

Rates of glucose andd--hydroxybutyrate use were determined in five brain regions of 20-day-old rats. The regions studied were cerebral cortex, thalamus, striatum, cerebellum, and brain stem. The tracers for determining rates of substrate use were [³H]fluorodeoxyglucose and [3-¹⁴C]-d--hydroxybutyrate. Two or five minutes after isotope administration the animals were sacrificed in a 6-kW, 2450-MHz focused microwave device. Ten minutes prior to isotope administration the animals were injected intraperitoneally with normal saline or DL--hydroxybutyrate (10mmol/kg). Bloodd--hydroxybutyrate levels averaged 0.21 mol/ml in saline-injected and 3.13 mol/ml in hyperketonemic rats. Rates of glucose utilization were significantly heterogeneous between regions in both groups: thalamus > cerebral cortex striatum > brain stem > cerebellum. These rates were 20–35% lower in hyperketonemic rats. Rates ofd--hydroxybutyrate use varied significantly between regions only in the saline group, with the brain stem rate being significantly lower than that in cortex or cerebellum. Regional rates ofd--hydroxybutyrate use did not correlate significantly with regional rates of glucose use in either the saline or the hyperketonemic groups. Regional rates of glucose use were strongly and positively correlated between conditions, as were regional rates ofd--hydroxybutyrate use. Thus, in 20-day-old rats, the regional heterogeneity of brain glucose use is similar to that in adult rats.d--Hydroxybutyrate use is much less regionally heterogeneous. When brain regional rates ofd--hydroxybutyrate use are increased seven- to ninefold in acute hyperketonemia, there are compensatory decreases in regional rates of glucose use sufficient to keep regional rates of energy production unchanged.     

Lipophilic 1-β-d-arabinofuranosyl cytosine derivatives in liposomal formulations for oral and parenteral antileukemic therapy in the murine L1210 leukemia model

TheN ⁴-alkylcytosine arabinoside derivativeN ⁴-octadecyl-AraC (AraC-Ocd, NOAC) and the (1-octadecylglycero-3-phospho)-AraC (Ocd-GroP-AraC, OPA) conjugate are new lipophilic derivatives of the cytostatic drug 1--d-arabinofuranosylcytosine (AraC) that produce high antileukemic effects in the L1210 murine leukemia model when administered orally or parenterally as liposomal formulations. Between 83% and 100% of the treated animals were cured after five consecutive daily oral drug applications with a total dose of 1 mmol/kg AraC-Ocd or Ocd-GroP-AraC. Corresponding results were obtained after parenteral therapy on days 2 and 6 after tumor inoculation with five- to ten-fold lower concentrations of these two compounds. A comparable cytotoxic activity was found with the orally active AraC-5-(n-stearyl phosphate). However, because of its strong hemolytic toxicity this derivative cannot be used for parenteral therapy. Another AraC conjugate, which was modified with two long-chain hydrocarbons, the (1-octadecylglycero-3-phospho)-N ⁴-hexadecyl-AraC was, probably because of poor oral bioavailability, only active when applied parenterally. The new lipophilic AraC derivatives AraC-Ocd and Ocd-GroP-AraC are compounds with a high potential for the oral treatment of leukemias and possibly also of solid tumors.Abbreviations AraC 1--d-arabinofuranosylcytosine - AraC-Ocd N ⁴-octadecyl-1--d-arabinofuranosylcytosine - Ocd-GroP-AraC 5-O-(1-octadecyl-rac-glycero-3-phospho)-1--d-arabinofuranosylcytosine - OcdP-AraC 1--d-arabinofuranosylcytosine-5-(n-stearylphosphate) - Ocd-GroP-AraC-Hxd 5-O-(1-octadecyl-rac-glycero-3-phospho)-N ⁴-hexadecyl-1--d-arabinofuranosylcytosine     

Oligosaccharides accumulated in the bovineβ-mannosidosis kidney

Summary The phenotype of bovine-mannosidosis (-mannosidase deficiency), recently identified in Salers cattle, is similar to the caprine form of the disease (Abbittet al., 1991). This investigation was designed to characterize accumulated kidney oligosaccharides in bovine-mannosidosis. Oligosaccharides were extracted from the kidney of an affected Salers calf and purified by chromatographic techniques. The amount of accumulating oligosaccharides in 1 g of wet tissue was about 21µmol. Structures of derivatized oligosaccharides were characterized by high-performance liquid chromatography, mass spectrometry, methylation analysis and sequential exoglycosidase digestions. The major accumulating oligosaccharides were Man1-4GlcNAc and Man1-4GlcNAc1-4GlcNAc. Oligosaccharides accumulating in minor amounts were Man1-4GlcNAc1-4Man1-4GlcNAc, Man1-6Man1-4GlcNAc1-4GlcNAc and Man1-4GlcNAc1-4Man1-4GlcNAc1-4GlcNAc. As in caprine-mannosidosis, oligosaccharides with terminal-mannose residues and cleaved as well as uncleaved chitobiose linkages were identified in bovine-mannosidosis kidney. The accumulating oligosaccharides in tissue were thus identical in bovine and caprine-mannosidosis; however, the source of the novel oligosaccharides remains to be determined.     

Variation of lysosomal enzyme activity with gestational age in chorionic villi

Summary The activities of 14 lysosomal enzymes in chorionic villi at gestational ages of 6–12 weeks were assayed.Arylsulphatases A and B, -glucosidase and -glucuronidase activities increased with advancing gestational age. When compared with the activity in cultured amniotic fluid cells, arylsulphatase A, -galactosidase, -glucosidase, heparan N-sulphatase, -l-iduronidase, -mannosidase, neuraminidase and sphingomyelinase showed significant differences. All except -glucuronidase showed lower activity in chorionic villi than in cultured amniotic fluid cells. Prenatal diagnosis using chorionic villi was possible except for -l-iduronidase.Storage at –20°C up to 42 days did not significantly affect activity. The results emphasize the importance of using fresh or frozen age-matched control tissue for diagnosis.     

A sensitive semi-automated kinetic assay of α-D-glucosidase for the prenatal diagnosis of type 2 glycogenosis (Pompe's disease)

Prenatal diagnosis of type 2 glycogenosis (Pompe's disease) has been done on cultured amniotic fluid cells, using a semi-automated fluorimetric kinetic assay for -D-glucosidase with 4-methylumbelliferyl--D-glucoside as substrate. The activity of the enzyme was related to that of-D-galactosidase, and found to be absent in cells from an affected fetus. The diagnosis was confirmed in fetal liver, where the same assay was used to show absence of-D-glucosidase activity with normal-D-galactosidase activity, and where increased glycogen deposition was demonstrated histologically. This type of assay is generally applicable to lysosomal enzymes, and to other fluorigenic enzyme reactions.     

Distribution pattern of α and β myosin in normal and diseased human ventricular myocardium

Summary All fibers in three normal, four dilated, and two ischemic human ventricles were classified according to their myosin content using three sets of monoclonal antibodies each specific for one myosin heavy chain isoform (, and ). Numerous fibers contained only myosin heavy chain (denoted as fibers), others contained either and , or and myosin heavy chain (denoted as and fibers, respectively). The percentages of fibers were systematically determined along the walls of seven homologous regions of the ventricular myocardium.In all ventricles, there was an -fiber transmural gradient, with less fiber in the subendocardium than in the subepicardium. More fibers were found in the right than in the left ventricular wall but there was no difference between the mid-portion and the apex of the free wall of each ventricle. The diseased ventricles contained a lower fiber percentage than the normal hearts. fibers were very rare in the normal ventricles (less than 5%) and almost inexistent in pathological hearts. The correlation between the mean fiber percentages of the diseased hearts and their cardiac indices (r=0.88, P<0.05) suggests that the small amount of myosin distributed in a large number of ventricular fibers could play a role in the contractile performance of the heart. In conclusion, this study provides evidence for 1) an fiber transmural gradient, and 2) a lower myosin ratio in discased than in normal human ventricle.This work was supported in part by L'Institut National de la Santé et de la Recherche Médicale 101 rue de Tolbiac, 75013 Paris     

Presence of Two Signaling TGF-β Receptors in Human Pancreatic Cancer Correlates with Advanced Tumor Stage

Transforming growth factor- (TGF-)signal transduction is mediated via specific cellsurface signaling TGF- receptors, most notably thetype I ALK5 (TR-I_ALK5)and the type II(TR-II). We evaluated TR-I_ALK5 andTR-II expression in 41 human pancreatic cancertissue samples and correlated these findings withclinical data of the patients. Northern blot analysisindicated that, in comparison with the normal pancreas,pancreatic adenocarcinomas exhibited 8.0-fold and4.5-fold increases (P < 0.01), respectively, in mRNAlevels encoding TR-I_ALK5 andTR-II. In situ hybridization showed that both TR-I_ALK5 mRNAwere highly expressed in the majority of pancreaticcancer cells. Immunohistochemical analysis ofTR-I_ALK5 and TR-II revealedpositive immunostaining in 73% and 56% of the tumors, respectively. Both receptorswere concomitantly present in 54% of the pancreaticcancer samples. The presence ofTR-I_ALK5 or TR-II and theconcomitant presence of TR-I_ALK5 and TR-II in the cancer cells was associatedwith advanced tumor stage (P < 0.01). These findingsshow that in many human pancreatic cancers, increasedlevels of the two signaling TRs are present. The presence of the signaling TRs inadvanced tumor stages indicates a role in diseaseprogression.     

Functionalβ-adrenergic receptors in breast cancer cells

Summary Usingl-[³H]dihydroalprenolol ([³H]DHA), a potent-adrenergic antagonist, we demonstrated in breast cancer cells the presence of-adrenergic receptors with high affinity (K _d 1–9 nM) as shown by Scatchard analyses. Natural and synthetic agonists inhibited the [³H] DHA binding in the following order of potency:l-isoproterenol=l epinephrine > > >l-norepinephrine, identical to the well-established order of potency for these compounds in producing-adrenergic responses. We verified that these compounds actually stimulated cAMP production in breast cancer cells. At the present time, the pathophysiological significance of-adrenergic receptors remains unclear. In view of the importance of cAMP in lactose production and in tumor growth mechanisms, it seems to be important to characterize the-adrenergic receptors in breast cancer cells in more detail and study their possible involvement in breast tumor growth.Abbreviations used DHA dihydroalprenolol - -AR -adrenergic receptor     

Die β-Laktamase-Inhibitoren und ihre klinische Bedeutung

Zusammenfassung Zu den verschiedenen Möglichkeiten der Überwindung einer -Laktamase-bedingten Resistenz von Mikroorganismen gehört der Einsatz von Enzyminhibitoren, die selbst keine nennenswerte eigene antimikrobielle Aktivität aufweisen, jedoch in Kombination mit einem Breitspektrumantibiotikum vom -Laktamtyp synergistisch wirken. Auf diese Weise gelangen -Laktam-resistente Bakterien erneut in das Wirkungsspektrum von Substanzen wie Penicillin G oder Ampicillin, die aufgrund steigender Resistenzentwicklung in den letzten Jahren ihre therapeutische Effizienz zu verlieren drohen. 6--Bromopenicillansäure und die sogenannten Olivansäuren weisen eine bemerkenswerte Hemmpotenz gegenüber verschiedenen -Laktamasen auf. Die mikrobiologischen und bisher vorliegenden pharmakokinetischen Daten eines Penicillansäuresulfons, das ebenfalls signifikante Hemmeigenschaften verschiedener klinisch relevanter -Laktamasen besitzt, werden diskutiert. Von Clavulansäure, einem Stoffwechselprodukt vonStreptomyces clavuligerus mit -Laktamstruktur konnte ebenfalls gezeigt werden, daß es ein progressiver Hemmstoff der -Laktamasen vom Richmond-Typ II-V ist. Neben den bisher vorliegendenIn-vitro-Untersuchungen werden auch Ergebnisse klinischer Studien mit der Kombination Clavulansäure und Amoxicillin erwähnt.

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Inhibitors of -lactamases and their clinical significance

Summary One of the various possibilities of overcoming bacterial resistance due to -lactamase production is with enzyme inhibitors. These have no remarkable intrinsic antimicrobial activity, but act synergistically in combination with a broad spectrum antibiotic of the -lactam type. Thus -lactam resistant bacteria are once again within the antibacterial spectrum of substances like penicillin G or ampicillin, which have been in danger of losing their therapeutical effectiveness in recent years due to an ever increasing development of resistance. 6--bromopenicillanic acid and the so-called olivanic acids exhibit remarkable inhibitory properties against several -lactamases. Microbiological and pharmacokinetic data published recently on a penicillanic acid sulfone, which also shows significant inhibitory properties against several clinically relevant -lactamases, are discussed in this paper. Clavulanic acid, a recently discovered product ofStreptomyces clavuligerus with a -lactam structure, acts as a progressive inhibitor of Richmond type II-V -lactamases. In addition to microbiological and enzyme-kineticin vitro data, results of clinical studies with the combination clavulanic acid and amoxicillin are summarized.

     

Lysosomal enzyme activities in serum and leukocytes from patients with carbohydrate-deficient glycoprotein syndrome type IA (phosphomannomutase deficiency)

From 10 patients with carbohydrate-deficient glycoprotein (CDG) syndrome due to phosphomannomutase (PMM) deficiency, out of 10 lysosomal enzymes, 7 enzyme activities were measured in serum and 9 in leukocytes. In serum there was a 2-fold to 4-fold increase in activity of -glucuronidase, -hexosaminidase, -galactosidase, and arylsulphatase A. In leukocytes, however, several enzymes had reduced activity, particularly -fucosidase, -glucuronidase and -mannosidase. These abnormalities could result from missorting, defective reuptake and/or reduced stability of the enzymes due to the defective glycosylation.     

Serum concentrations of resistin-like molecules β and γ are elevated in high-fat-fed and obese db/db mice, with increased production in the intestinal tract and bone marrow

Aims/hypothesis Resistin and the resistin-like molecules (RELMs) comprise a novel class of cysteine-rich proteins. Among the RELMs, RELM and RELM are produced in non-adipocyte tissues, but the regulation of their expression and their physiological roles are largely unknown. We investigated in mice the tissue distribution and dimer formation of RELM and RELM and then examined whether their serum concentrations and tissue expression levels are related to insulin resistance.Methods Specific antibodies against RELM and RELM were generated. Dimer formation was examined using COS cells and the colon. RELM and RELM tissue localisation and expression levels were analysed by an RNase protection assay, immunoblotting and immunohistochemical study. Serum concentrations in high-fat-fed and db/db mice were also measured using the specific antibodies.Results The intestinal tract produces RELM and RELM, and colonic epithelial cells in particular express both RELM and RELM. In addition, RELM and RELM were shown to form a homodimer and a heterodimer with each other, in an overexpression system using cultured cells, and in mouse colon and serum. Serum RELM and RELM levels in high-fat-fed mice were markedly higher than those in mice fed normal chow. Serum RELM and RELM concentrations were also clearly higher in db/db mice than in lean littermates. Tissue expression levels revealed that elevated serum concentrations of RELM and RELM are attributable to increased production in the colon and bone marrow.Conclusions/interpretation RELM and RELM form homo/heterodimers, which are secreted into the circulation. Serum concentrations of RELM and RELM may be a novel intestinal-tract-mediating regulator of insulin sensitivity, possibly involved in insulin resistance induced by obesity and a high-fat diet.     

Plasma lysosomal enzyme levels in patients with motor neuron disease

Summary -Hexosaminidase and acid--mannosidase were estimated in 17 adult patients with motor neuron disease. Normal plasma levels of -hexosaminidase ((A+B) and A) were found in all patients studied. Plasma acid -mannosidase levels were normal in all but two patients with the spinal muscular atrophy type of the disorder. In addition, altered biochemical properties of acid -mannosidase (i.e.K _m, thermal stability) were found in the low-activity cases.     

Role of β-Adrenergic Receptor Subtypes in Lipolysis

In vitro lipolysis stimulated by low (-)-isopre-naline concentrations (30 nM) in epididymal white adipo-cytes from Sprague-Dawley rats was inhibited at least 60–80% by the specific ₁-antagonists LK 204-545 and CGP 20712A (1 M), suggesting that at these low (10 nM) concentrations of (-)-isoprenaline lipolysis was primarily (80%) but not solely mediated via ₁-adrenergic receptors. Low concentrations (100 nM) of (-)-noradrenaline and formoterol also confirmed a role for ₁-adrenergic receptors in mediating lipolysis at low concentrations of these agonists. At higher agonist concentrations, ₃-adrenergic receptors were fully activated and were the dominant -adrenergic receptor subtype mediating the maximum lipolytic response, and the maximum response was not affected by the ₁-antagonists, demonstrating that the ₃-receptor is capable of inducing maximum lipolysis on its own. Studies of lipolysis induced by the relatively ₂-selective agonist formoterol in the presence of ₁-blockade (1 M CGP 20712A) demonstrated the inability of the ₂-selective antagonist ICI 118-551 to inhibit the residual lipolysis at concentrations of ICI 118-551 1 M. Higher concentrations of ICI 118-551 inhibited the residual formoterol-induced lipolysis competetively, but with low affinity (500-fold lower than its ₂-adrenergic receptor pA ₂, 7.80 ± 0.21), suggesting that formoterol was not acting via ₂-adrenergic receptors. These data are consistent with ₁-adrenergic receptors playing an important role in lipolysis at physiological but not pharmacological concentrations of catecholamines and that ₂-adrenergic receptors play no obvious direct role in mediating -adrenergic receptor agonist-induced lipolysis in vitro. Finally, racemic-SR 59230A, unlike the pure (S, S)-isomer (a ₃-selective antagonist), was found to be a non-selective antagonist at the three -adrenergic receptor subtypes, showing that the other enantiomers have different selectivity.     

β-Endorphin-like immunoreactivity in normal mucosa,muscle layer,adenocarcinoma, and polyp of the colon

-Endorphin-like immunoreactivity was detected in the mucosa and muscle layer of normal colon, adenocarcinomas derived from the colon mucosa, and colon polyps which were histologically confirmed to be adenoma without a focus of carcinoma or with in situ carcinoma. The contents of -endorphin-like immunoreactivity in adenocarcinomatous tissue (11.94± 1.77 pmollg wet wt) and colon polyps without focus of carcinoma (10.71 ±1.50 pmollg wet wt) were found to be significantly higher than those in the mucosal layer (6.86± 0.64 pmollg wet wt) and muscle layer (8.30 ±0.68 pmollg wet wt) of normal colon. These data suggest that the production of -endorphin-like immunoreactivity is specifically increased in some adenocarcinomas and adenomatous polyps and may be related to the alteration of bowel habits. Gel exclusion chromatography of - endorphinlike immunoreactivity revealed three peaks corresponding to -endorphin, -lipotropin, and an immunoreactive form between the two. In the mucosal layer and muscle layer of the colon, a broad major peak was eluted at the position of -endorphin, and minor peaks were eluted at the position of -lipotropin and between -endorphin and -lipotropin. In adenocarcinoma and polyp, the peak size corresponding to authentic -lipotropin was greater than that of -endorphin. This study demonstrated that -endorphin-like immunoreactivity existed at a high concentration in some colon adenocarcinomas and polyps whose elution patterns were different from those of normal colon tissue.     

Diagnosis of the mucopolysaccharidoses using cultured skin fibroblasts and amniotic fluid cells

Assay of-L-iduronidase, heparin sulphamidase,N-acetyl--D-glucosaminidase, arylsulphatase B,-L-fucosidase,-glucuronidase,-galactosidase and-D-mannosidase in cultured cells is described. Activities in deficient fibroblast strains are compared to control fibroblast strains. The first case of Sanfilippo B in the United Kingdom is reported. A comparison of enzyme activities in cultured fibroblasts and amniotic fluid cells is made.     

Heterophilic interactions between cell adhesion molecule L1 and α<Subscript>v</Subscript> β<Subscript>3</Subscript>-integrin induce HUVEC process extension <Emphasis Type="Italic">in vitro</Emphasis> and angiogenesis <Emphasis Type="Italic">in vivo</Emphasis>

Cell adhesion molecule L1 was implicated in angiogenic processes, tumor formation and metastasis. Here, we provide evidence that the sixth Ig-like domain of L1 (L1Ig6) interacts with _{v 3} to induce process extension of human umbilical vein endothelial cells (HUVECs) in vitro and angiogenesis in vivo. HUVECs formed network-like structures on full-length L1 or L1Ig6 substrates comparable to structures found on matrigel. In the presence of mab _{v 3} or cyclic RGD, apoptosis was induced. In fibrin matrices where L1Ig6 was covalently incorporated, HUVECs formed multicellular and hollow processes through interactions between cell-surface _{v 3} and RGD-sites of matrix-immobilized L1Ig6. No such processes were induced by L1Ig6 having non-functional RDG-sites, or in the presence of mab _{v 3} or cyclic RGD. In those matrices, increased apoptosis was found. Co-immunoprecipitation of L1 or L1Ig6 with _{v 3} suggests close interactions. Furthermore, L1Ig6 stimulated HUVECs showed increased tyrosine phosphorylation of _{v 3} and phosphorylation of MAP kinases (ERK1 and ERK2) but not AKT indicating specific activation of _v and _{v 3} followed by activation of downstream kinases. Application of L1Ig6-modified fibrin matrices on CAMs induced 50–60% increased _v and _v3 protein expression and in vivo angiogenesis indicated by ~50% increased mean vascular length density. The results demonstrate angiogenic potential of L1Ig6 involving ligation and activation of _v3     

Synergistic interactions between interferonβ and carboplatin on SK-MEL 28 human melanoma cell growth inhibition in vitro

Carboplatin and interferon (IFN ) were tested alone and in combination for their anti-proliferative activity on the human melanoma cell line SK-MEL 28 in vitro. Cells were incubated for 4 days in the presence of carboplatin (0.1 mM and 0.1 M) and interferon (5 pM and 5 nM) and cell growth inhibition was determined by the sulphorhodamin B assay. The antiproliferative effects of the drug combinations were analysed using Berenbaum's hyperplane theorem to determine additive, synergistic and antagonistic effects. IFN was found to be 10 000 times more active in inhibiting cell growth of SK-MEL 28 cells than carboplatin on the basis of IC₅₀ values (IFN: IC₅₀=1.24 nM, carboplatin: IC₅₀=18.2, M). The addition of IFN at 0.5 nM reduced the IC₅₀ value of carboplatin 18.0-fold; with IFN at 0.05 nM a dose reduction of 1.84 was measured. At the carboplatin: IFN molar concentration ratios of 2000:1 and 6000:1, interaction indices (I) of 0.66 and 0.83 were determined respectively, indicating synergistic interactions between the two drugs. At higher carboplatin: IFN molar ratios (20 000:1 and 60 000:1) an additive interaction was observed (I=1.07 and 1.20). However, further in vitro studies with serveral melanoma cell lines are necessary to evaluate the potential effectiveness of the drug combination of carboplatin and IFN for eventual clinical utilisation.     

Interleukin-1β effects on cyclic GMP and cyclic AMP in cultured rat islets of Langerhans — arginine — dependence and relationship to insulin secretion

Summary When islets were cultured with interleukin-1 (1 or 100 pmol/l) for 12 h in arginine-containing medium, cyclic GMP levels were increased 1.6- and 4.5-fold respectively. The arginine analogue, N--nitro-l-arginine methyl ester, which blocks nitric oxide formation and partially reverses inhibition of insulin secretion by 100 pmol/l interleukin-1, largely, but not completely, blocked generation of cyclic GMP. Treatment of islets with 100 pmol/l interleukin-1 for 12 h significantly decreased islet cyclic AMP generation in the absence of isobutylmethylxanthine (from 13.1±0.7 to 9.3±0.8 fmol/g islet protein), this fall was arginine-dependent and may have resulted from an effect on a cyclic AMP phosphodiesterase, since it was masked if isobutylmethylxanthine was present. Isobutylmethylxanthine (0.4 mmol/l) reduced the inhibitory potency of interleukin-1 in 15 h slightly but significantly from 80.5 to 59.0%. The morpholinosydnonimine SIN-1, which is a nitric oxide donor, inhibited insulin secretion, raised islet cyclic GMP and lowered cyclic AMP; its effects were similar to those of interleukin-1. However, 6-anilinoquinoline-5,8-quinone, [LY83583 (1–10 mol/l)], inhibited insulin secretion, and significantly decreased cyclic GMP while 8-bromocyclic GMP stimulated insulin secretion. Both low- and high-dose interleukin-1 treatment give a large arginine-dependent and a small, yet significant, arginine-independent increase in cyclic GMP. The inhibitory effect of SIN-1 or interleukin-1 on insulin secretion seems to depend to a small extent on decreased islet cyclic AMP, though sustained increases in nitric oxide or depleted islet GTP may directly affect the secretory process.     

Rapid molecular characterization of mutations leading to unstable hemoglobin β-chain variants

Summary Characterization of unstable hemoglobins by protein analysis is often difficult. However, it is facilitated by DNA analysis, especially in the case of hyperunstable -chain variants, which produce a -thalassemia phenotype. We have applied an efficient strategy to the detection of such variants at the DNA level, based on computer-designed denaturing gradient gel electrophoresis (DGGE) of amplified DNA fragments. This approach makes it possible to detect any anomaly in the -globin gene. We describe the use of the DGGE method for rapid characterization of -chain variants and report a new missense mutation in the -globin gene third exon, 127 CAG-CGG/Gln-Arg, which is responsible for the synthesis of a highly unstable hemoglobin.