[Differentiate between the reference interval and the clinical diagnosis and treatment decision threshold for alanine aminotransferase].

The debate about whether the upper limit of normal for ALT should be lowered has been going on for nearly 20 years. However, the concept, the establishment method, and the application scope of reference interval are different from those of the clinical diagnosis and treatment decision threshold. We will differentiate the two concepts and review the related publications to help clinicians better apply the reference interval and the decision threshold for disease diagnosis and treatment.

[The medical laboratory issues about recommendation on uniform cutoff values of "Normal" ALT in the ACG guidelines].

In the recent clinical guidelines dealing with laboratory tests for liver disease evaluation, the American College of Gastroenterology (ACG) recommends ALT upper reference limits of 33 U/L for males and 25 U/L for females, and individuals with results above these "normal" cutoffs should be further investigated. Considering the differences between laboratory assays measuring ALT in our country, the uniform ACG "normal" range may not be suitable for Chinese population. On the other hand, reference upper/lower limits should not be equated with clinical decision thresholds. Simply acting in accordance with the reference range from ACG guidelines for ALT may lead to overdiagnosis and unnecessary follow-up examinations.

Exacerbation of poststroke dementia by type 2 diabetes is associated with synergistic increases of beta-secretase activation and beta-amyloid generation in rat brains.

We examined the effect of type 2 diabetes on stroke-induced Alzheimer's disease-like pathological and behavioral changes in rats. Rats were treated for 2 months with high fat diet (HFD) followed by streptozotocin (STZ) injection to induce type 2 diabetes (HFD-STZ model). Middle cerebral artery occlusion (MCAO) was used to induce cerebral focal ischemia. Animals were divided into four groups: Sham-NPD, Sham-HFD-STZ, MCAO-NPD and MCAO-HFD-STZ. The results showed that HFD-STZ treatment induced obesity, hypertriglyceridemia, hypercholesterolemia, hyperinsulinemia, hyperglycemia and insulin resistance, characteristics of type 2 diabetes. The performance of rats in the Morris water maze test was impaired in MCAO-NPD and Sham-HFD-STZ rats, indicating cognitive deficits. Hippocampal caspase-3+ and beta amyloid (Abeta+) cell numbers, as well as beta-site amyloid precursor protein-cleaving enzyme (BACE1) levels and activity, increased in both groups. Moreover, HFD-STZ treatment exacerbated stroke-induced cognitive deficits, additively increased MCAO-induced activation of caspase-3, and increased levels of BACE1, C99 and Abeta. However, the level of insulin decreased in MCAO-HFD-STZ rats. These results suggested that type 2 diabetes deteriorated stroke-induced brain damage and cognitive impairment, which might be associated with increased Abeta generation and cytotoxicity. We concluded that type 2 diabetes exacerbated poststroke dementia possibly due to brain injury and synergistic generation of Abeta via activation of BACE1.

Lipid-lowering and LDL-oxidation inhibitory effects of aqueous extract of freshwater clam (Corbicula fluminea)--using tilapia as an animal model.

A previous study has demonstrated that tilapia able to exhibit hyperlipidemia and hypercholesterolemia is a good model for the evaluation of beneficial effects of nutraceuticals. In this study, tilapia were used to evaluate the in vitro and in vivo effects of a hot water extract (FC-HW) of freshwater clam (Corbicula fluminea). FC-HW prolonged the lag phase of Cu(2+)-induced human and tilapia LDL oxidation. The prolongation of the lag phase was concentration-dependent in human (r(2)= 0.94) and tilapia LDL (r(2)= 0.98). The antioxidative potential of FC-HW was 0.33% (on a weight basis) of Trolox, a positive control. Male tilapia (n= 24) were randomly divided into 2 groups and separately fed for 60 d with an isocaloric also isoprotein diet containing 2% (w/w) FC-HW or a control diet. Body length and body mass were significantly higher in fish fed FC-HW than those of the control group (P < 0.05). Total triacylglycerol, cholesterol, and LDL-C in plasma of the FC-HW group were significantly lower (-89.9%, -61.8%, and -54.5%, respectively), while plasma total antioxidant capacity of the FC-HW group was higher and the lag phase in Cu(2+)-induced LDL oxidation was longer than those of the control group (P < 0.05). FC-HW demonstrated hypolipidemia and hypocholesterolemia effects and inhibited human LDL oxidation in vitro and tilapia LDL both in vitro and ex vivo, indicative that FC-HW can be a potential nutraceutical to reduce the risk factors of atherosclerosis.

The intramolecular delta(15)N of lysine responds to respiratory status in Paracoccus denitrificans.

Presented here is the first experimental evidence that natural, intramolecular, isotope ratios are sensitive to physiological status, based on observations of intramolecular delta(15)N of lysine in the mitochondrial mimic Paracoccus denitrificans. Paracoccus denitrificans, a versatile, gram-negative bacterium, was grown either aerobically or anaerobically on isotopically-characterized ammonium as sole cell-nitrogen source. Nitrogen isotope composition of the biomass with respect to source ammonium was Delta(15)N(cell - NH4) = delta(15) - delta(15)N(NH4) = -6.2 +/- 1.2 per thousand for whole cells under aerobic respiration, whereas cells grown anaerobically produced no net fractionation (Delta(15)N(cell - NH4) = -0.3 +/- 0.23 per thousand). Fractionation of (15)N between protein nitrogen and total cell nitrogen increased during anaerobic respiration and suggests that residual nitrogen-containing compounds in bacterial cell membranes are isotopically lighter under anaerobic respiration. In aerobic cells, the lysine intramolecular difference between peptide and sidechain nitrogen is negligible, but in anaerobic cells was a remarkable Delta(15)N(p - s) = delta(15)N(peptide) - delta(15)N(sidechain) = +11.0 per thousand, driven predominantly by enrichment at the peptide N. Consideration of known lysine pathways suggests this to be likely due to enhanced synthesis of peptidoglycans in the anaerobic state. These data indicate that distinct pathway branching ratios associated with microbial respiration can be detected by natural intramolecular Deltadelta(15)N measurements, and are the first in vivo observations of position-specific measurements of nitrogen isotope fractionation.

Angiogenesis-research frontiers. A basic science conference of the New York Academy of Medicine.

Angiogenesis, the development of new blood vessels, is essential for both tumour growth and metastasis. Recent advances in our understanding of the molecular mechanisms underlying the angiogenic process and its regulation have led to the discovery of a variety of targets for therapeutic intervention. The potential application of these angiogenic inhibitors is currently under intense preclinical and clinical investigation. Compelling evidence suggests that vascular endothelial growth factors (VEGFs) and their receptors play critical roles in tumour-associated angiogenesis. Tumour homing factors will drive the growth of new vessels, neoangiogenesis, to satisfy the demands of the growing tumour. By attacking the angiogenic process the tumour will he starved for oxygen and nutrients, thus impairing its growth. This has been demonstrated in a variety of animal tumour models in which disabling the function of VEGF or its receptor was shown to inhibit both tumour growth and metastasis. The New York Academy of Medicine organised a day-long meeting to discuss emerging ideas, currently available in vitro and animal models and evaluation of these therapies during their preclinical development and in clinical trials.

Rapid photometric assay evaluating antioxidative activity in edible plant material.

The objective of the present study is to develop a rapid and convenient method to determine antioxidative activity. It was determined by the inhibition capacity on the hemoglobin-catalyzed peroxidation of linoleic acid. The appropriate conditions for reaction of 4 mM linoleic acid were 0.002% hemoglobin at 37 degrees C for 10 min. Adding methanol to the reaction mixture at <20% showed no significant effect on the peroxidation of linoleic acid. Products formed from hemoglobin-catalyzed peroxidation of linoleic acid were 9- and 13-hydroperoxyoctadecadienoic acid at a ratio of approximately 50:50. Eight synthetic antioxidants were assayed for their antioxidative activity; all of them showed linear response to the logarithm of their concentration. Antioxidative activity from different plant samples was also examined. Tea, ginger, chrysanthemum, and roselle showed higher antioxidative activity. Either hydrophobic or hydrophilic antioxidants were able to be assayed with this method within 15 min.

Effect of melatonin on production of hydroxyl radical and lactate dehydrogenase during hypoxia in rat cortical slices.

AIM: To study the effect of melatonin on the production of hydroxyl radical (.OH) and lactate dehydrogenase (LDH) following hypoxia in cortical slice. METHODS: Cortical slice was incubated with artificial cerebrospinal fluid (ACSF) in tube. Hypoxia was achieved by substituting 91.6% N2 and 8.4% O2. The salicylate trapping method was used to measure hydroxyl radicals generated. The content of LDH in medium after hypoxia was measured by International Federation of Clinic Chemistry (IFCC) method. RESULTS: The contents of dihydroxybenzoic acid (DHBA) were increased significantly during hypoxia and reoxygenation in cortical slice. The production of DHBA in reoxygenation was decreased concentration-dependently by melatonin, but not during hypoxia 30 min. The release of LDH during hypoxia was steadily elevated and melatonin decreased the content of LDH after hypoxia. CONCLUSION: Melatonin decreased the injury and production of .OH after hypoxia.

Effects of isoproterenol on twitch contraction of wild type and phospholamban-deficient murine ventricular myocardium.

Ablation of the gene for phospholamban (PLB), a transmembrane peptide regulator for the cardiac sarcoplasmic reticulum Ca2+ pump, in mice brings about a complete loss of the myocardial responses to beta-adrenergic agonists (e.g., Luo et al., Circ. Res. 1994; 75: 401). We have evaluated the functional significance of PLB-independent mechanisms in the myocardial responses to beta-adrenergic stimulation in isolated intact ventricular myocardium. We compared the effects of (-)-isoproterenol (ISO) on isometric twitch contraction of paced right ventricular muscle strips of wild type (WT) and PLB-deficient (PLBKO) mice. At 37 degrees C, frequent spontaneous contractions in both types of muscles required the inclusion of lidocaine, an antiarrhythmic, in the bathing medium. Thus the experiments were also performed at two lower temperatures, 30 degrees C and 25 degrees C, at which lidocaine was not needed. Under three conditions, in the absence of ISO, PLBKO ventricular muscles exhibited substantially shortened time to peak tension (TPT) and half relaxation time (TR1/2), compared with the WT muscles. In both WT and PLBKO muscles ISO increased the peak developed tension and decreased TPT and TR1/2 in a dose-dependent manner although the effects were generally smaller in PLBKO than in WT muscles. One micromolar ISO caused TPT and TR1/2 to decrease by 7.3+/-1.2% (mean+/-SEM) and 7.5+/-1.2% in PLBKO vs. 22.8+/-0.7% and 29.1+/-1.7% in WT at 37 degrees C; by 13.5+/-0.4% and 14.1+/-1.2% in PLBKO vs. 31.3+/-0.8%, and 44.8+/-1.3% in WT at 30 degrees C; by 15.0+/-2.3% and 21.1+/-4.9% in PLBKO vs. 25.8+/-1.9% and 54.0+/-1.9% in WT at 25 degrees C. These findings strongly suggest that PLB-independent mechanisms play a significant role in mediating the positive inotropic and lusitropic effects of beta-adrenergic agonists on ventricular myocardium.

Effects of protein kinase A phosphorylation on signaling between cardiac troponin I and the N-terminal domain of cardiac troponin C.

During beta-adrenergic stimulation of the heart, there is a decrease in myofilament Ca2+ sensitivity mediated by the protein kinase A-(PKA-) induced phosphorylation of troponin I (cTnI). Phosphorylation, which occurs at Ser 23 and Ser 24 in an amino-terminal extension unique to cTnI, decreases the Ca2+ affinity of the amino-terminal regulatory site of cardiac troponin C (cTnC). In view of the antiparallel organization of the cTnI-cTnC complex [Krudy, G. A., Kleerekoper, Q., Guo, X., Howarth, J. W., Solaro, R. J., and Rosevear, P. R. (1994) J. Biol. Chem. 269, 23731-23735], it is not clear how the phosphorylation signal at one end of the complex affects the Ca2+ binding site at the other end. To address this question, we probed the interaction between cTnI and cTnC fragments, cTnC1-89 and cTnC90-162 (recombinant peptides corresponding to the N- and C-domains of cTnC). cTnI-Cys 5 mutant (S5C/C81I/C98S) and cTnC1-89 were fluorescently labeled with IAANS. When cTnI was phosphorylated, the affinity of Ca2+ for the cTnI-cTnC1-89 complex decreased significantly as indicated by a shift in the pCa50 value from 6.65 to 5.25. Upon phosphorylation, the affinity of cTnI for cTnC1-89 decreased by 3.8-fold in the absence of Ca2+ and 1.7-fold in the presence of Ca2+. In contrast to the case with full-length cTnC, neither cTnC1-89 nor cTnC90-162 induced significant structural changes in cTnI-Cys 5 as determined from intersite distance measurements between Cys 5 and Trp 192. Moreover, neither fragment of cTnC could significantly restore Ca2+ regulation of force generation, when exchanged into fiber bundles from which cTnC had been extracted. Our findings indicate that the transduction of PKA-induced phosphorylation signal from cTnI to the regulatory site of cTnC involves a global change in cTnI structure.

[Neurosteroids].

The term "neurosteroids" applies to those steroids that are synthesized in nervous system independent of supply by peripheral endocrine glands. The neurosteroids biosynthetic pathway, the mechanisms of neurosteroids in regulating neuronal activities and the neuromodulatory effects of neurosteroids, particularly the bimodal regulation of GABAA receptors, have been reported.

Cerebral glucose metabolism during pharmacologic studies: test-retest under placebo conditions.

UNLABELLED: The reliability of serial [18F]fluorodeoxyglucose (FDG) PET scans for psychopharmacologic studies was tested by using placebo infusions. METHODS: FDG scans were obtained before and after a 30 min placebo infusion (n = 10; Group 1) or after each of two bolus infusions with placebo (n = 8; Group 2). Subjects performed a continuous performance task (CPT) during each scan. Cardiovascular measures and ratings of anxiety were obtained in all subjects. Samples for determination of plasma norepinephrine (NE) were taken at multiple time points in Group 1. RESULTS: A slight increase in apparent global metabolism occurred between scans in both Groups 1 and 2. A few regions significantly increased in both groups. While an apparent increase in sympathetic activity occurred during the placebo infusion, neither NE levels, anxiety ratings nor cardiovascular measures correlated with global or regional FD6 uptake. CONCLUSION: Test-retest differences of global and regional glucose metabolism were highly consistent across two experimental designs. While increases in cerebral glucose metabolism appeared to occur during the second scan, differences between scans were small. This method may offer advantages for selected psychopharmacologic studies.

Interaction of cardiotonic thiadiazinone derivatives with cardiac troponin C.

The cardiotonic effects of thiadiazinone derivative EMD 57033 are mediated by direct actions on myofilaments (Lues, I., Beier, N., Jonas, R., Klockow, M., and Haeusler, G. J. (1993) Cardiovasc. Pharmacol. 21, 883-892). Cardiac troponin C has been postulated to be a potential target of the drug (White, J., Lee, J. A., Shah, N., and Orchard, C. H. (1993) Circ. Res. 73, 61-70). This study tested whether EMD 57033 interacts directly with recombinant human cardiac TnC (hcTnC). EMD 57033 caused concentration-dependent quenching of tyrosine (Tyr) fluorescence of hcTnC in the presence of Ca2+ (100 microM) and little change of the fluorescence in the presence of Mg2+ (2 mM). Kd for the drug-hcTnC interaction in the presence of Ca2+, determined by Tyr fluorescence titrations, was approximately 40 microM. The binding of EMD 57033 was stereo-selective: the optical isomer of EMD 57033 bound hcTnC much more weakly. The Ca2+ dependence and stereo-selectivity of EMD 57033 binding were substantiated by a dialysis-based direct binding assay. EMD 57033 was found to interfere with Ca(2+)-dependent binding of hydrophobic probe 1,1'-bi-(4-anili-no)naphthalene-5,5'-disulfonate (bis-ANS) to hcTnC. The relationships between [Ca2+] and Tyr fluorescence of hcTnC and between [Ca2+] and bis-ANS fluorescence in the presence of hcTnC were substantially altered by EMD 57033 in the range of [Ca2+] where Ca2+/Mg2+ sites of hcTnC were titrated by Ca2+. EMD 57033 was found to bind as tightly to 2 Ca2+.hcTnC as to 3 Ca(2+).hcTnC. These observations were interpreted as indicating that a EMD 57033-binding site is induced by Ca2+ binding, but not Mg2+ binding, to the Ca2+/Mg2+ sites of hcTnC. The drug-binding site most likely resides in the carboxyl domain of hcTnC.

Alterations in cyclic AMP generation and G protein subunits following transient ischemia in gerbil hippocampus.

We examined alterations in the cyclic AMP generating system and G protein subunits in gerbil hippocampus following 10 min of transient ischemia. In hippocampal slices, basal and isoproterenol- and forskolin-stimulated cyclic AMP accumulations were markedly increased at 6 and 24 h after ischemia. Interestingly, both the inhibition of forskolin-stimulated cyclic AMP and the potentiation of beta-adrenoceptor-stimulated cyclic AMP by a gamma-aminobutyric acidB receptor agonist were attenuated at these time points. Ischemia did not affect the immunolabeling of any of the G protein alpha subunits; only that of beta subunits was significantly decreased, by 28.2%, 4 days after ischemia. In contrast, pertussis toxin-catalyzed [32P]ADP ribosylation declined progressively during the late recirculation period, reaching a significant reduction (25.4%) at 6 h after ischemia. These results suggest that ischemia affects the heterotrimeric conformation (alpha beta gamma) of Gi/Go during the recirculation period, thereby leading to increased cyclic AMP production. Because cyclic AMP-dependent protein kinase A modulates the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-kainate receptor channels, postischemic sensitization of the cyclic AMP generating system may contribute to neuronal degeneration in the hippocampus.

A direct regulatory role for troponin T and a dual role for troponin C in the Ca2+ regulation of muscle contraction.

Troponin (Tn), containing three subunits: Ca2+ binding (TnC), inhibitory (TnI), and tropomyosin binding (TnT), plays a crucial role in the Ca2+ regulation of vertebrate striated muscle contraction. These three subunits function by interacting with each other and with the other thin filament proteins. Previous studies suggested that the primary role of TnT is to anchor the TnI.TnC complex to the thin filament, primarily through its interactions with TnI and tropomyosin. We propose here a new role for TnT. Our results indicate that, when TnT is combined with the TnI.TnC complex, there is an activation of actomyosin ATPase that is Ca(2+)-dependent. To determine whether the latter results from a direct effect of TnC on TnT or indirectly from an effect of TnC on TnI which is transmitted to TnT, we prepared a deletion mutant (deletion of residues 1-57) of TnI, TnId57 (Sheng et al. (1992) J. Biol. Chem. 267, 25407-25413), which interacts with TnC but not TnT. Both wild type (TnI.TnC.TnT) and mutant (TnId57.TnC.TnT) Tn complexes demonstrated equivalent activity in the Ca2+ regulation of actomyosin-S1 ATPase activity. Similarly, both TnI and TnId57 could equally reconstitute TnI-depleted skinned muscle fibers. Therefore, since TnId57 does not interact with TnT, these results suggest that TnT reconstitutes native Ca2+ sensitivity via direct interaction with TnC. Thus Ca2+ binding to TnC would have a dual role: 1) release of the ATPase inhibition by TnI and 2) activation of the ATPase through interaction with TnT.

Laparoscopic assessment and orchidectomy for the undescended testis.

Cryptorchidism is the most common disorder of male sexual differentiation. Pre-operative assessment and localization of undescended testes by various investigatory modalities have been of limited clinical value. The recent use of diagnostic and therapeutic laparoscopy for undescended testis peri-operatively has been shown to be safe and informative in the planning of appropriate surgical management. The authors' initial experience with this procedure, for five patients with unilateral undescended testis, is presented.

Stimulation of brain pregnenolone synthesis by mitochondrial diazepam binding inhibitor receptor ligands in vivo.

Evidence that neurosteroids are potent modulators of the action of GABA at GABAA receptors has prompted the investigation of the mechanism that controls brain neurosteroid synthesis by glial cell mitochondria in vivo. In vitro studies suggest that the interaction of the diazepam binding inhibitor (DBI)--a polypeptide that is abundant in steroidogenic cells--with glial mitochondrial DBI receptors (MDRs) is a crucial step in the physiological regulation of neurosteroid biosynthesis. MDRs bind 4'-chlorodiazepam (4'-CD), N,N-di-n-hexyl-2-(4-fluorophenyl)-indol-3-acetamide (FGIN-1-27), and the isoquinoline carboxamide PK 11195 with high affinity, and these ligands have been used to investigate whether the stimulation of glial MDRs increases brain pregnenolone production in vivo. Adrenalectomized and castrated (A-C) male rats (to eliminate peripheral sources of pregnenolone) were pretreated with trilostane (to prevent pregnenolone metabolism to progesterone), and the pregnenolone content in brain regions dissected after fixation with a 0.8-s exposure to microwave irradiation focused to the head was determined by HPLC followed by specific radioimmunoassay. The forebrain and cerebellum of A-C rats contained 4-7 ng of pregnenolone/g of tissue, and the olfactory bulb contained 10-14 ng/g. These concentrations of brain pregnenolone are only 30-40% lower than those of sham-operated rats. In contrast, the plasma pregnenolone content of sham-operated rats was 2-3 ng/ml, but it was only 0.15-0.20 ng/ml in the plasma of A-C rats. In A-C rats, treatment with the MDR ligands 4'-CD and FGIN-1-27 increased the pregnenolone content in the brain but failed to change the plasma or peripheral tissue content of this steroid. The effect of 4'-CD on brain pregnenolone content was maximal (70-100% increase) at the dose of 18 mumol/kg, 5-10 min after intravenous injection. The effect of oral administration of FGIN-1-27 on brain pregnenolone content was maximal (80-150% increase) at doses of 400-800 mumol/kg and peaked at approximately 1 h. That this effect of FGIN-1-27 was mediated by the MDR was documented by pretreatment with the MDR partial agonist PK 11195 (100 mumol/kg, i.p.). PK 11195 did not affect basal brain pregnenolone content but prevented the accumulation of brain pregnenolone induced by FGIN-1-27. FGIN-1-27 and 4'-CD failed to increase the brain concentration of dehydroepiandrosterone in A-C rats. These data suggest that glial cell MDRs play a role in neurosteroid biosynthesis in vivo.

Isolation, expression, and mutation of a rabbit skeletal muscle cDNA clone for troponin I. The role of the NH2 terminus of fast skeletal muscle troponin I in its biological activity.

A cDNA for rabbit fast skeletal muscle troponin I (TnI) was isolated and sequenced. The clone contains a coding sequence predicting a 182-amino-acid protein with a molecular mass of 21,162 daltons. The translated sequence is different from that reported by Wilkinson and Grand (Wilkinson, J. M., and Grand, R. J. A. (1978) Nature 271, 31-35) in that Arg-153, Asp-154, and Leu-155 must be inserted into their original sequence. Amino acid sequencing of adult rabbit TnI confirmed this result. In order to investigate the role of the NH2 terminus of TnI in its biological activity, we have expressed a recombinant deletion mutant (TnId57), which lacks residues 1-57, in a bacterial expression system. Both wild type TnI (WTnI) and TnId57 inhibited acto-S1-ATPase activity and this inhibition could be fully reversed by troponin C (TnC) in the presence of Ca2+. Additionally both WTnI and TnId57 bound to an actin affinity column. Thus, both inhibitory actin binding and Ca(2+)-dependent neutralization by TnC were retained in TnId57. TnC affinity chromatography was used to compare the binding of TnI and TnId57 to TnC. Using this method, two types of interaction between TnC and TnI were observed: 1) one which is metal independent (or structural) and 2) one dependent on Ca2+ or Mg2+ binding to the Ca(2+)-Mg2+ sites of TnC. The same experiments with TnId57 demonstrated that the type 1 interaction was weakened, and type 2 binding was lost. This method also revealed an interaction between TnC and TnI which is dependent upon Ca2+ binding to the Ca(2+)-specific sites of TnC and which is retained in TnId57. Taken together, these results suggest that the NH2 terminus of TnI may constitute a Ca(2+)-Mg(2+)-dependent interaction site between TnC and TnI and play, in part, a structural role in maintaining the stability of the troponin complex while the COOH terminus of TnI contains a Ca(2+)-specific site-dependent interaction site for TnC as well as the previously demonstrated Ca(2+)-sensitive inhibitory and actin binding activities.

Two genetically expressed troponin T fragments representing alpha and beta isoforms exhibit functional differences.

The carboxyl-terminal isoforms of troponin T (TnT), alpha and beta, differing in the sequence of a region near the COOH terminus, arise from alternative splicing of a primary transcript of the TnT gene and are expressed in a tissue-specific and developmentally regulated manner (Medford, R. M., Nguyen, H. T., Destree, A. T., Summers, E., and Nadal-Ginard, B. (1984) Cell 38, 409-421). To date, the beta isoform has not been studied directly at the protein level. To explore the potential functional differences between the alpha and beta sequences, we isolated two rabbit skeletal TnT cDNA clones: a full-length cDNA for a beta isoform and a partial-length cDNA for an alpha isoform. Two restriction fragments derived from the cDNA clones were used to direct overexpression, in Escherichia coli, of two TnT fragments, T2p-alpha and T2p-beta, each containing the last 108 amino acid residues of the alpha or beta isoform of TnT. Using purified T2p-alpha and T2p-beta along with fluorescent derivatives of troponin C (TnC) and alpha alpha-tropomyosin (Tm), we showed that T2p-alpha bound more strongly to TnC than did T2p-beta both in the presence and absence of Ca2+, and exhibited a higher affinity for Tm than did T2p-beta. More interestingly, the Ca2+ affinities of the Ca(2+)-specific regulatory sites of TnC in the T2p-alpha. TnC complex were found to be 3-fold higher than in T2p-beta.TnC complex. These results support the hypothesis that the sequence divergence between the alpha and beta isoforms of TnT may have functional significance in possibly contributing to the determination of the Ca2+ sensitivity of muscle fibers.