Telomere length determined by the fluorescence in situ hybridisation distinguishes malignant and benign cells in cytological specimens.

BACKGROUND: Telomeres are tandem repeats of TTAGGG at the end of eukaryotic chromosomes that play a key role in preventing chromosomal instability. The aim of the present study is to determine telomere length using fluorescence in situ hybridisation (FISH) on cytological specimens. METHODS: Aspiration samples (n = 41) were smeared on glass slides and used for FISH. RESULTS: Telomere signal intensity was significantly lower in positive cases (cases with malignancy, n = 25) as compared to negative cases (cases without malignancy, n = 16), and the same was observed for centromere intensity. The difference in DAPI intensity was not statistically significant. The ratio of telomere to centromere intensity did not show a significant difference between positive and negative cases. There was no statistical difference in the signal intensities of aspiration samples from ascites or pleural effusion (n = 23) and endoscopic ultrasound-guided FNA samples from the pancreas (n = 18). CONCLUSIONS: The present study revealed that telomere length can be used as an indicator to distinguish malignant and benign cells in cytological specimens. This novel approach may help improve diagnosis for cancer patients.

Nerve growth factor induces development of connective tissue-type mast cells in vitro from murine bone marrow cells.

The effect of nerve growth factor (NGF) on proliferation/differentiation of mast cells was investigated in vitro. Although NGF alone neither supported colony formation of bone marrow-derived cultured mast cells (BMCMC) nor induced development of mast cell colonies from nonadherent bone marrow cells (NBMC), addition of NGF to the suboptimal dose of interleukin 3 (IL-3) significantly increased the numbers of mast cell colonies produced by BMCMC or NBMC in methylcellulose. When stimulated by IL-3 alone, cells in mast cell colonies were not stained by berberine sulfate, a fluorescent dye. In contrast, mast cells developing in methylcellulose cultures obtaining both IL-3 and NGF were stained by berberine sulfate. The fluorescence was abolished by the treatment of heparinase but not of chondroitinase ABC, suggesting that mast cells stimulated by IL-3 and NGF produced and stored heparin proteoglycan. The histamine content of BMCMC maintained by IL-3 was also increased by addition of NGF. Since BMCMC showed mucosal mast cell-like phenotype, NGF appeared to induce the phenotypic change to connective tissue-type mast cells (CTMC). In the culture containing BMCMC, 3T3 fibroblasts, and IL-3, the phenotypic change of BMCMC to CTMC was observed as well. Since NGF was detected in this coculture and since addition of anti-NGF monoclonal antibody suppressed the phenotypic change, NGF produced by fibroblasts appeared to induce the phenotypic change. Neither BMCMC alone nor IL-3 alone increased the concentration of NGF. Therefore, there is a possibility that BMCMC stimulated by IL-3 may induce the production and/or release of NGF by fibroblasts.

The effect of co-existing solutes on arsenate removal with hydrotalcite compound.

Hydrotalcite (HTAL-Cl), an inorganic anion exchanger, is of use as an adsorbent for the removal of arsenate (As(V)) in water systems. The adsorption properties of HTAL-Cl for As(V) and the effects of co-existing anions on the As(V) removal performance were investigated in this work. Under the conditions of pH>or=4, the adsorption capacity for As(V) gradually decreased with an increase of pH, but As(V) was removed effectively within the range of pH = 5-8. Co-existing anions interfered As(V) removal, and the effect decreased in the order of HPO(4)(2-) > HCO(3)(-) > SO(4)(2-) > Cl(-). In binary solute systems containing phosphate and As(V), the maximum adsorption capacity of HTAL-Cl was 0.95 mmol g(-1) for phosphate and 0.65 mmol g(-1) for As(V): the total of these values corresponded to the maximum adsorption capacity for As(V) in single solute systems. The adsorption isotherms in these binary solute systems were approximated by the following modified Langmuir equations:As(V): q(As) = 18.7 radicalC(As)/(1 + 21.5 radicalC(P) + 12.8 radicalC(As)), phosphate : q(P) = 33.1 radicalC(P)/(1 + 21.5 radicalC(P) + 12.8 radicalC(As)). The column adsorption experiments showed that the adsorbed As(V) was released by the phosphate adsorption, because phosphate was adsorbed more strongly on HTAL-CL than As(V).

Adiponectin gene single-nucleotide polymorphisms and treatment response to obesity.

BACKGROUND: In the adiponectin gene polymorphisms, single-nucleotide polymorphism (SNP)-45 and SNP276 have reportedly been associated with obesity, Type 2 diabetes, and other features of metabolic syndrome. AIM: Whether these adiponectin SNP affect obesity-related parameters during caloric restriction in obese subjects. SUBJECTS AND METHODS: Thirty- two obese Japanese women were treated by meal replacement with a low calorie diet for 8 weeks and asked to maintain their habitual lifestyle. Obesity-related parameters were measured before and after the treatment period. We determined four SNP (T45G, I164T, G276T, and C-11377G) using a fluorescent allele-specific DNA primer assay systemand FRET probe assay system. RESULTS: After the treatment, the extent of decrease in waist circumference was greater in the subjects with the G/G or G/T genotype of SNP276 than in those with the T/T genotype (p=0.026). As for SNP45, the extent of decrease in triglyceride levels was greater in the subjects with the T/T genotype than in those with the T/G genotype (p=0.003). For SNP-11377, the extent of decrease in systolic blood pressure and fasting plasma glucose was greater in the subjects with the C/G or G/G genotype than in those with the C/C genotype (p=0.044). CONCLUSION: Our findings indicate that each SNP in the adiponectin gene might modify the change in obesity-related parameters during meal replacement with a low calorie diet.

Elucidation of structural requirements of mastoparan for mast cell activation-toward the comprehensive prediction of cryptides acting on mast cells.

Mastoparan, a toxic peptide from wasp venom, induces various biological functions including histamine release from rat peritoneal mast cells. Here we report that, for the activation of mast cells by mastoparan, at least two positively charged side chains are required on the hydrophilic side of the amphiphilic structure of the peptide. The present results are expected to be utilized for the bioinformatic and comprehensive identification of endogenous mast cell-stimulating cryptides.

Oral administration of dihomo-gamma-linolenic acid prevents development of atopic dermatitis in NC/Nga mice.

Disorders of the metabolism of essential fatty acids (EFAs) are related to atopic dermatitis (AD). Concentrations of dihomo-gamma-linolenic acid (DGLA), an EFA, in the serum of AD patients are lower than those in healthy volunteers. Recently we developed a fermented DGLA oil, and examined whether oral administration of DGLA prevents development of dermatitis in NC/Nga mice, which spontaneously develop human AD-like skin lesions. NC/Nga mice were fed a diet either containing or not containing DGLA for 8 weeks under in air-uncontrolled conventional circumstances. Clinical skin severity scores were significantly lower in mice fed DGLA than in mice not fed it. Scratching behavior and plasma total IgE levels were also reduced in the DGLA group, in association with histological improvement. DGLA suppressed clinical severity of skin lesions dose-dependently, with an increase in DGLA contents in phospholipids of skin, spleen, and plasma. Discontinuation of DGLA administration resulted in the onset of dermatitis and a decrease in DGLA contents in skin, spleen, and plasma. These findings indicate that oral administration of DGLA effectively prevents the development of AD in NC/Nga mice, and that DGLA in phospholipids is a compound of key importance in the development and prevention of dermatitis.

Polyphenol-enriched oolong tea increases fecal lipid excretion.

OBJECTIVE: To assess possibility of polyphenol-enriched oolong tea to reduce dietary lipid absorption in humans. DESIGN: Twelve healthy adult subjects, three males and nine females, aged (mean+/-s.d.) 22.0+/-1.8 years, respectively, were randomly divided into two groups. The participants were followed a double-blind placebo-controlled crossover design, including 7-day washout periods and 10-day treatment periods. During the treatment periods, subjects were given about 38 g of lipids from potato chips (19 g each within 30 min after lunch and dinner) and total 750 ml beverages (placebo- or polyphenol-enriched oolong tea) at three meals. Blood samples were collected for biochemical examination at days 8, 18, 25 and 35 of the study period. On the last 3 days of each treatment period, feces were collected to measure the excretion of lipids. RESULTS: Lipid excretion into feces was significantly higher in the polyphenol-enriched oolong tea period (19.3+/-12.9 g/3 day) than in the placebo period (9.4+/-7.3 g/3 day) (P < 0.01). Cholesterol excretion tended to increase in polyphenol-enriched oolong tea period (1.8+/-1.2 g/3 day) compared with that of placebo (1.2+/-0.6 g/3 day) (P = 0.056). CONCLUSIONS: The results of this study indicated that polyphenol-enriched oolong tea could increase lipid excretion into feces when subjects took high-lipid diet.

Structure of HIV-1 protease with KNI-272, a tight-binding transition-state analog containing allophenylnorstatine.

BACKGROUND: HIV-1 protease (HIV PR), an aspartic protease, cleaves Phe-Pro bonds in the Gag and Gag-Pol viral polyproteins. Substrate-based peptide mimics constitute a major class of inhibitors of HIV PR presently being developed for AIDS treatment. One such compound, KNI-272, which incorporates allophenylnorstatine (Apns)-thioproline (Thp) in place of Phe-Pro, has potent antiviral activity and is undergoing clinical trials. The structure of the enzyme-inhibitor complex should lead to an understanding of the structural basis for its tight binding properties and provide a framework for interpreting the emerging resistance to this drug. RESULTS: The three-dimensional crystal structure of KNI-272 bound to HIV PR has been determined to 2.0 A resolution and used to analyze structure-activity data and drug resistance for the Arg8-->Gln and ILe84-->Val mutations in HIV PR. The conformationally constrained Apns-Thp linkage is favorably recognized in its low energy trans conformation, which results in a symmetric mode of binding to the active-site aspartic acids and also explains the unusual preference of HIV PR for the S, or syn, hydroxyl group of the Apns residue. The inhibitor recognizes the enzyme via hydrogen bonds to three bridging water molecules, including one that is coordinated directly to the catalytic Asp125 residue. CONCLUSIONS: The structure of the HIV PR/KNI-272 complex illustrates the importance of limiting the conformational degrees of freedom and of using protein-bound water molecules for building potent inhibitors. The binding mode of HIV PR inhibitors can be predicted from the stereochemical relationship between adjacent hydroxyl-bearing and side chain bearing carbon atoms of the P1 substituent. Our structure also provides a framework for designing analogs targeted to drug-resistant mutant enzymes.

Accounting for the peripartum loss of granulated metrial gland cells, a natural killer cell population, from the pregnant mouse uterus.

Natural killer (NK) cells become a prominent cell population in the rodent uterus during pregnancy. The mature, heavily granulated form of these cells is rare in virgin or postpartum uteri. Death, migration, or dedifferentiation could account for the disappearance of these cells from late gestation uteri. We asked whether uterine NK cells, also known as granulated metrial gland (GMG) cells, die in situ and if expression of Fas antigen is essential for their death. Late in gestation, fragmentation of nuclear DNA was detected histologically by OH-end labeling, as were ultrastructural changes suggesting cell death. NK cells developed in and were lost from the uteri of pregnant Fas antigen-deficient lpr/lpr mice. Postpartum samples of retained placentas contained some residual NK cells that had moved from regions of uterine musculature toward the uterine lumen and were being expelled with the placenta. Thus, both cell death and placental separation remove NK cells from the peripartum uterus.

Interleukin-3 and stem cell factor modulate cell cycle regulatory factors in mast cells: negative regulation of p27Kip1 in proliferation of mast cells induced by interleukin-3 but not stem cell factor.

OBJECTIVE: Interleukin-3 (IL-3) and stem cell factor (SCF) are able to promote survival and proliferation of mast cells. However, the precise signal transduction cascades leading to mast cell proliferation are not clearly understood. Thus, we sought to define the mechanism of mast cell proliferation induced by IL-3 and SCF. MATERIALS AND METHODS: We treated murine bone marrow-derived cultured mast cells (BMCMC) with recombinant IL-3 (rIL-3) or recombinant SCF (rSCF) and examined the effects of rIL-3 and rSCF on cell cycle regulatory factors. RESULTS: Both rIL-3 and rSCF suppressed apoptosis of BMCMC. rSCF induced great proliferation of BMCMC with elevation of the proportions of cells in S and G2/M phases, whereas most BMCMC incubated with rIL-3 were arrested in the G1 phase. The G1/S phase transition is initiated by phosphorylated retinoblastoma protein (pRb), which was prominent in cells stimulated with rSCF. In contrast, rIL-3 relatively increased a dephosphorylated form of pRb in BMCMC. Compared with rIL-3, rSCF induced greater expression of cyclin-dependent kinase (CDK) 2 and CDK4, which are able to phosphorylate pRb, and cyclin D3, a partner of CDK4. BMCMC treated with rIL-3 contained a high amount of a CDK inhibitor p27Kip1 that was suppressed by pretreatment with Ro31-7549, a protein kinase C inhibitor, whereas rSCF induced weak expression of p27Kip1 in BMCMC. CONCLUSION: The results suggest that IL-3 and SCF exert their respective mitogenic effects on mast cells by modulating the expression of pRb, CDK, cyclin, and p27Kip1.

Thermodynamic dissection of the binding energetics of KNI-272, a potent HIV-1 protease inhibitor.

KNI-272 is a powerful HIV-1 protease inhibitor with a reported inhibition constant in the picomolar range. In this paper, a complete experimental dissection of the thermodynamic forces that define the binding affinity of this inhibitor to the wild-type and drug-resistant mutant V82F/184V is presented. Unlike other protease inhibitors, KNI-272 binds to the protease with a favorable binding enthalpy. The origin of the favorable binding enthalpy has been traced to the coupling of the binding reaction to the burial of six water molecules. These bound water molecules, previously identified by NMR studies, optimize the atomic packing at the inhibitor/protein interface enhancing van der Waals and other favorable interactions. These interactions offset the unfavorable enthalpy usually associated with the binding of hydrophobic molecules. The association constant to the drug resistant mutant is 100-500 times weaker. The decrease in binding affinity corresponds to an increase in the Gibbs energy of binding of 3-3.5 kcal/mol, which originates from less favorable enthalpy (1.7 kcal/mol more positive) and entropy changes. Calorimetric binding experiments performed as a function of pH and utilizing buffers with different ionization enthalpies have permitted the dissection of proton linkage effects. According to these experiments, the binding of the inhibitor is linked to the protonation/deprotonation of two groups. In the uncomplexed form these groups have pKs of 6.0 and 4.8, and become 6.6 and 2.9 in the complex. These groups have been identified as one of the aspartates in the catalytic aspartyl dyad in the protease and the isoquinoline nitrogen in the inhibitor molecule. The binding affinity is maximal between pH 5 and pH 6. At those pH values the affinity is close to 6 x 10(10) M(-1) (Kd = 16 pM). Global analysis of the data yield a buffer- and pH-independent binding enthalpy of -6.3 kcal/mol. Under conditions in which the exchange of protons is zero, the Gibbs energy of binding is -14.7 kcal/mol from which a binding entropy of 28 cal/K mol is obtained. Thus, the binding of KNI-272 is both enthalpically and entropically favorable. The structure-based thermodynamic analysis indicates that the allophenylnorstatine nucleus of KNI-272 provides an important scaffold for the design of inhibitors that are less susceptible to resistant mutations.

Effects of thyroid hormone on carbonic anhydrase I concentration in human erythroid burst-forming unit-derived cells.

Individuals with hyperthyroidism exhibit red blood cell concentrations of carbonic anhydrase I (CAI) that reflect the integrated serum thyroid hormone concentration over the preceding few months. Furthermore, T3, at a physiological free concentration, decreases the CAI concentration in human erythroleukemic YN-1 cells. The effect of T3 on CAI concentration in burst-forming unit-erythroid (BFU-E)- derived cells, obtained by culturing peripheral blood mononuclear cells with various cytokines, including erythropoietin, has now been investigated. BFU-E-derived cells contained a high concentration of CAI (mean +/- SE, 4.8 +/- 0.8 x 10(-12) mol/10(6) cells; n = 8). The CAI in BFU-E-derived cells was immunologically identical to that present in mature red blood cells. T3 decreased the CAI concentration in BFU-E-derived cells in a dose-dependent manner (28%, 47% and 75% decreases at 3 x 10(-10), 1 x 10(-9), and 3 x 10(-9) mol/liter T3, respectively). These results suggest that BFU-E-derived cells may be used to study the effect of T3 on human red blood cell CAI. This system may prove useful in the tissue diagnosis of resistance to thyroid hormone.

Erythrocyte carbonic anhydrase-I concentrations in patients with Graves' disease and subacute thyroiditis reflect integrated thyroid hormone levels over the previous few months.

We have recently reported that in patients with hyperthyroidism, red blood cell (RBC) zinc (Zn), most of which is present as the metal of carbonic anhydrase-I isozyme (CAI), reflects a patient's integrated thyroid hormone level over the previous few months. In the present report the RBC CAI concentration was measured by RIA in 26 healthy controls, 25 patients with hyperthyroid Graves' disease, 5 patients with primary hypothyroidism, and 10 subjects with subacute thyroiditis with elevated thyroid hormone levels. The mean (+/- SD) RBC CAI concentration in euthyroid controls was 380 +/- 70 nmol/g hemoglobin (Hb), and the normal range defined as the mean +/- 2 SD, was 240-520 nmol/g Hb. The mean RBC CAI in Graves' disease was decreased (180 +/- 53 nmol/g Hb), and 22 patients (88%) had subnormal values. The mean RBC CAI concentrations in hypothyroidism and subacute thyroiditis were not different from the control values. After treatment with antithyroid drugs, both mean the plasma T4 and T3 levels in 11 Graves' patients became normal within 4 weeks, but the normalization of RBC CAI lagged behind by about 2 months. Furthermore, the highest correlation was observed between the RBC CAI and plasma T4 and T3 levels measured 8 weeks earlier. During prednisolone therapy the RBC CAI in patients with subacute thyroiditis remained at a normal level. These results suggest that 1) not only RBC Zn but also the RBC CAI concentration in patients with Graves' disease reflect the patient's mean thyroid hormone level over the preceding several months; and 2) in patients with subacute thyroiditis, elevation of plasma thyroid hormone concentrations is transient and causes little change in the RBC CAI concentration.

Thyroxine 5-deiodinase in human brain tumors.

To determine whether human brains contain deiodinating pathways, we studied the activity of T4 5-monodeiodinase (5-D) in 20 human brain tumors obtained intraoperatively, including astrocytoma (10), meningioma (4), oligodendroglioma (2), glioblastoma (2), medulloblastoma (1), and malignant lymphoma (1). Mitochondrial-microsomal fractions prepared from these tumor tissues were used as the source of T4 5-D. Each sample was incubated with 32.2 nmol/L T4 and 30 mmol/L dithiothreitol at 37 C for 90 min. T4 5-D activity was measured by the production of rT3 from T4 with a RIA. T4 5-D activity was found in 6 of 10 astrocytomas, 2 oligodendrogliomas, 1 of 2 glioblastomas, and 1 malignant lymphoma. This activity depended on protein concentration, incubation time, incubation temperature, and pH of the incubation mixture. It was also heat labile. T4 5-D was not inhibited by 1 mmol/L propylthiouracil, but was inhibited by iopanoic acid and aurothioglucose in a dose-dependent manner. The apparent Km and maximum velocity for T4 5-D at 30 mmol/L dithiothreitol were 106.6 nmol/L and 22.7 pmol/mg protein.h, respectively. These data suggest that human gliomas (and probably malignant lymphomas) contain T4 5-D activity, which is similar to type III enzyme activity in the rat. T4 5-D may regulate the intracellular concentration of thyroid hormone in gliomas.

Monoclonal thyroglobulin autoantibodies: variable region analysis and epitope recognition.

A panel of human monoclonal thyroglobulin (Tg) autoantibodies (TgAAb) has been used to analyze autoantigenic determinants on human Tg and to investigate the relationship between variable (V) region gene sequences and epitope specificity. Two monoclonal TgAAb bound to the same (or closely related) epitope on Tg, and these were defined as type I TgAAb. Three other monoclonals bound to a different site and were defined as type II TgAAb. Inhibition studies with mixtures of type I and type II monoclonal TgAAb (Fab)2 preparations indicated that a mixture of the (Fab)2s almost completely inhibited (> 75%) labeled Tg binding to intact TgAAb in the sera of apparently healthy blood donors and patients with autoimmune thyroid disease (AITD). Type I TgAAb predominated in apparently healthy blood donors' sera, whereas type II TgAAb predominated in AITD sera. Analysis of V region gene sequences of the TgAAb indicated that a range of light chain and heavy chain genes from different gene families was used. Furthermore, the same germline genes that are used by TgAAb are also well represented in the genes coding for other self- and nonself-reactive antibodies. No homology in terms of light chain and heavy chain gene families, germline gene usage, or complementarity determining region sequences was observed in TgAAb directed to the same or closely related epitopes. Our studies show that TgAAb are directed to two major conformational epitopes on the Tg molecule and that the proportion of TgAAb directed to these epitopes in apparently healthy blood donors and that in patients with AITD appear to be different. TgAAb derived from different germline genes and with different complementarity determining region sequences can display similar epitope specificity, and this indicates that AAb directed to the same or a closely related epitope show considerable heterogeneity at the molecular level.

Erythrocyte zinc concentration in patients with subacute thyroiditis.

We have recently reported that red blood cell (RBC) zinc (Zn) in patients with hyperthyroidism reflects a patient's integrated thyroid hormone level over the previous few months. In the present paper RBC Zn concentrations were measured in 10 patients with subacute thyroiditis whose total plasma T4 and T3 levels were elevated. The values were compared with those obtained in 10 patients with untreated Graves' disease, whose plasma T4 concentrations were elevated to the same level as in the former group. The RBC Zn concentration was normal in 9 of 10 patients with subacute thyroiditis, but was depressed in all patients with Graves' disease. The mean (+/- SE) RBC Zn in patients with subacute thyroiditis was 162 +/- 9 mumol/L, significantly (P less than 0.001) higher than that in Graves' disease (87 +/- 5 mumol/L). During prednisolone treatment the RBC Zn in patients with subacute thyroiditis remained at the normal level and did not change significantly, although it was slightly decreased at 2 and 4 weeks of treatment. On the other hand, the RBC Zn in patients with Graves' disease was significantly increased at 8 weeks of treatment and reached the normal range in 12 weeks. These results suggest that elevation of plasma thyroid hormone concentrations in patients with subacute thyroiditis is transient and does not cause any significant change in the RBC Zn concentration.

Identification and endothelin-induced activation of multiple extracellular signal-regulated kinases in aortic smooth muscle cells.

In order to explore intracellular signaling pathways of the mitogenic action of endothelin (ET), we examined the effect of ET on activities of extracellular signal-regulated kinases (ERKs) in rat aortic smooth muscle cells (SMCs). Treatment of rat aortic SMCs with ET-1 increased kinase activities toward myelin basic protein (MBP). Both 43- and 41-kDa proteins were activated when kinase assays were done in MBP-containing polyacrylamide gels after SDS-PAGE. These proteins were identified as ERK1 and ERK2 with immunoprecipitation and immunoblotting using antipeptide antibodies, respectively. These results indicate that ERKs mediate signal transduction by ET.

Neo-kyotorphin (Thr-Ser-Lys-Tyr-Arg), a new analgesic peptide.

The amino acid sequence of a newly isolated pentapeptide, neo-kyotorphin from bovine brain was synthetically verified to be Thr-Ser-Lys-Tyr-Arg corresponding to the C-terminal portion of hemoglobin alpha-chain. The synthetic neo-kyotorphin showed the dose-dependent analgesia in mice which was approximately equal to that of Leu-enkephalin.

Steroid 21-hydroxylase is a major autoantigen involved in adult onset autoimmune Addison's disease.

An adrenal-specific protein reacting with autoantibodies in the sera of patients with adult onset Addison's disease has been purified from human adrenal glands. The protein, mol.wt. 55K, has the biochemical characteristics of steroid 21-hydroxylase and reacts on Western blots with rabbit antibodies to recombinant 21-hydroxylase. Absorption of the native human 55K adrenal protein with human adrenal autoantibodies prevented the subsequent reaction of the 55K protein with rabbit antibodies to 21-hydroxylase in Western blot analysis. In addition, human adrenal autoantibodies reacted with recombinant 21-hydroxylase expressed in yeast. These data indicate that the adrenal specific enzyme steroid 21-hydroxylase is a major autoantigen involved in adult onset autoimmune Addison's disease.

Expression of human thyroid peroxidase in the yeasts Saccharomyces cerevisiae and Hansenula polymorpha.

Saccharomyces cerevisiae and the methylotrophic yeast Hansenula polymorpha have been used to express both full-length and a large hydrophilic domain of human thyroid peroxidase (TPO). Expression of TPO in S. cerevisiae, using the natural signal sequence or the yeast alpha-mating factor (MF alpha) signal sequence, resulted in undetectable or very low levels of recombinant TPO production. However, TPO was expressed when the natural TPO leader sequence was replaced by the yeast STE2 signal sequence. This recombinant TPO reacted with both rabbit anti-human TPO polyclonal and mouse anti-human TPO monoclonal antibodies on Western blots. In the case of H. polymorpha, TPO expression was achieved when the natural TPO leader sequence was replaced by the MF alpha leader and the construct placed under the control of the methanol-regulated promoter from the methanol oxidase gene. The recombinant TPO produced in H. polymorpha reacted with both TPO polyclonal and TPO monoclonal antibodies. No TPO was produced when the signal sequence of SUC2 (invertase) or the TPO natural signal sequence was used to direct expression.