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Y H Lee

Publications and source records attributed to Y H Lee.

At least 19 recordsLinked to original sources
PubMed1992

Continuous production of 6-APA in an aqueous two-phase system.

An aqueous two-phase system of polyethylene glycol (PEG) and potassium phosphate provided a favorable environment for bioconversion of penicillin G to 6-aminopenicillanic acid (6-APA). The recombinant E. coli cells containing penicillin acylase were partitioned in the phosphate-rich bottom phase, and the product 6-APA in the PEG-rich top phase, which protected the enzyme from deactivation for a longer period of time. The continuous production of 6-APA by recycling the bottom phase showed a slight decrease in enzyme activity from initial 80 mM of 6-APA production to 61 mM during the eight days of operation at a space velocity of 0.12 hours-1. This work showed a possibility that the aqueous two-phase whole-cell enzyme bioconversion could serve as an alternative to immobilized cells on solid matrices.

Enzymes, Immobilized↗
PubMed1992

Conservation of gene organization and trans-splicing in the glyceraldehyde-3-phosphate dehydrogenase-encoding genes of Caenorhabditis briggsae.

The genes encoding body-wall-specific glyceraldehyde-3-phosphate dehydrogenase from Caenorhabditis briggsae were sequenced and compared to the homologous genes from Caenorhabditis elegans. The direct tandem organization of these genes, gpd-2 and gpd-3, and the size and location of the two introns in each gene are the same in C. elegans and C. briggsae. Primer-extension studies demonstrated that the two genes in C. briggsae are trans-splice differentially with the same splice leader (SL) RNAs as are observed in C. elegans. The gdp-2 gene is trans-spliced with SL1 while gdp-3 is trans-spliced with SL2. Significant sequence conservation was observed within the promoter regions of each species and may indicate those regions responsible for body-wall-muscle-specific gene expression and/or differential trans-splicing. Comparisons of the sequences suggest that the tandem repeat of the genes has been subjected to concerted evolution and that C. briggsae and C. elegans diverged much earlier than would be anticipated based on morphological similarities alone. Finally, an open reading frame found several hundred nucleotides upstream from gpd-2, in both species, appears to be homologous to the ATP synthase subunit, ATPase inhibitor protein, from bovine mitochondria.

Amino Acid Sequence↗
PubMed1992

Copper transfer and activation of the Streptomyces apotyrosinase are mediated through a complex formation between apotyrosinase and its trans-activator MelC1.

The melanin operon (melC) of Streptomyces antibioticus is composed of two genes that encode MelC1 and MelC2 proteins. MelC1 has been suggested as a trans-activator which can facilitate the incorporation of copper into the apotyrosinase (MelC2) (Lee, Y.-H. W., Chen, B.-F., Wu, S.-Y., Leu, W.-M., Lin, J.-J., Chen, C. W., and Lo, S. J. (1988) Gene (Amst.) 65, 71-81). However, the molecular mechanism of the trans-activation or copper-transfer process mediated through MelC1 to MelC2 is not clear yet. In this study, we found apotyrosinase in both the extracellular fraction and cell extract from cells grown in copper-deficient medium. Using gel-filtration and immunoaffinity chromatographies, we demonstrated that apotyrosinase (MelC2) formed a stable complex with MelC1 in the intra- and extracellular fractions. Furthermore, addition of copper ion to the complex generated tyrosinase activity. The MelC1-MelC2 complex was purified to near homogeneity by DE52 and phenyl-agarose chromatographies. In conjunction with fast protein liquid gel filtration chromatography and NH2-terminal sequencing analysis, the results indicated that the stoichiometric ratio of MelC1 and MelC2 in the purified complex was 1:1. Essentially no copper was found in the complex. Addition of copper ion to the purified complex resulted in incorporation of approximately 2 molecules of copper ion and the mature active tyrosinase was gradually released from the complex. Taken together, these results demonstrate that the molecular mechanism of activation of Streptomyces apotyrosinase by its trans-activator MelC1 is initially mediated via a binary complex formation between these two proteins, followed by incorporation of copper ion. This activation mechanism accounts for the essential role of MelC1 in the expression of melanin operon.

Amino Acid Sequence↗
PubMed1992

Secretion of the Streptomyces tyrosinase is mediated through its trans-activator protein, MelC1.

The tyrosinase of Streptomyces antibioticus is encoded by the second open reading frame, melC2 of the melanin operon (melC). The upstream open reading frame melC1 specifies a 146-amino acid protein with a typical NH2-terminal signal-peptide characteristic of a secretory protein. The MelC1 protein is involved in the transfer of copper ion to apotyrosinase MelC2 via binary complex formation (Lee, Y.-H. W., Chen, B.-F., Wu, S.-Y., Leu, W.-M., Lin, J.-J., Chen, C. W., and Lo, S. J. (1988) Gene (Amst.) 65, 71-81; Chen, L.-Y., Leu, W.-M., Wang, K.-T., and Lee, Y.-H.W. (1992) J. Biol. Chem. 267, 20100-20107). To investigate whether the export of tyrosinase is also dependent on MelC1, a mutational study of its signal-peptide sequence was performed. Four different mutants were obtained. Mutation at the positively charged region (mutant M-6LE, Arg6-Arg7----Leu6-Glu7) or the hydrophobic region (mutant M-16D, Val16----Asp16) led to Mel- phenotypes. These lesions caused a severe 7-10-fold reduction of the export of both the MelC1 and MelC2 proteins and a concomitant accumulation of the two proteins in the cytosolic fraction. The cell-associated tyrosinase activity in M-6LE but not in the M-16D mutant was dramatically reduced to 4% of the activity found in the wild type strain, suggesting that the basic NH2 terminus of MelC1 is also important for the trans-activation function of this protein. Nevertheless, the defects on the trans-activation and/or secretory functions of MelC1 in mutants M-6LE and M-16D are not due to the impairment of the formation of the MelC1.MelC2 complex. The translation of melanin operon genes in these two mutants also decreased. In contrast, the tyrosinase activity and the secretion of MelC2 were not affected if the mutations occurred at the putative cleavage site of the signal peptidase (e.g. mutant M-29SM, Arg29-Ala30----Ser29-Met30 or mutant 29-SMG, Arg29-Ala30-Asp31----Ser29-Med30-Gly31+ ++). Additionally, tyrosinase activity and its export were abolished in a MelC1-negative mutant, M-950. Taken together, these results demonstrate that a functional MelC1 is essential for tyrosinase secretion and activity. Furthermore, the results suggest that like other secretory proteins, basic and hydrophobic residues in the MelC1 signal sequence are an important feature of the signal-peptide and play a pivotal role in the secretion of both the MelC1 and MelC2 proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1992

Extracellular autoprocessing of a metalloprotease from Streptomyces cacaoi.

We have previously demonstrated that the extracellular neutral metalloprotease (Npr) of Streptomyces cacaoi is synthesized as a 60-kDa preproenzyme (P60), then processed to the 35-kDa mature form (P35) (Chang, P. C., Kuo, T.-C., Tsugita, A., and Lee, Y.-H. W. (1990) Gene (Amst.) 88, 87-95). In this study, we investigated the active site and the mechanism involved in the maturation of the protease. Site-specific mutations at the putative zinc-binding ligands and active site of Npr at His202, Glu203, His206, and Glu240 led to complete abolishment of Npr activity and concomitant accumulation of a 57-kDa inactive protein (P57) which was secreted. Sequence analysis of the NH2 terminus indicated that P57 was derived from P60 after removal of the signal peptide and represented the proenzyme form of Npr (pro-Npr). Analysis of the zinc content of purified mutant P57 proteins revealed a dramatic loss of zinc atom as compared with the wild-type P35 protein. In vitro with the aid of exogenous active Npr, the mutant P57 protein could be converted to the mature inactive P35 with an identical NH2-terminal sequence and a molecular mass the same as that of the wild-type P35. From these studies, we conclude that these highly conserved residues (His202, Glu203, His206, and Glu240) are indispensable for zinc binding and protease activity, as well as processing of Npr. In addition, we have clearly demonstrated that maturation of Npr occurs extracellularly via an autocatalytic cleavage of the pro-Npr propeptide. This is the first report of such a maturation mechanism for an extracellular protease in streptomycetes which can serve as a model for further studies on the mechanism of secretion and processing of proteases from Gram-positive bacteria.

Amino Acid Sequence↗
PubMed1992

The 16S rRNA gene of Streptomyces lividans TK64 contains internal promoters.

A 632-bp Sau3AI fragment of Streptomyces lividans TK64 genome was found to confer promoter activity in Streptomyces and Escherichia coli. This fragment showed almost identical sequence (97.8%) to the S. coelicolor 16S rRNA segment encompassing from nucleotide 706 to 1337 region. The transcription start points of this fragment were identified by the primer extension method. Analysis of the nucleotide sequence upstream the transcription start points revealed two putative E. coli-like promoters resided within this fragment. The occurrence of internal promoters active in Streptomyces and E. coli was also confirmed in the 16S rRNA gene of rrnE operon from TK64.

Base Sequence↗
PubMed1992

Cisplatin-based chemotherapy for the treatment of advanced transitional-cell carcinoma of the urinary tract--a preliminary report.

The CMV (cisplatin, methotrexate, and vinblastine) and M-VAC (methotrexate, vinblastine, doxorubicin, and cisplatin) regimens were used to treat 19 patients with advanced transitional-cell carcinoma (TCC) of the urothelial tract. In the CMV group, the partial response rate was 45.5% and the mean response duration was 6.3 months. No complete response was obtained in our series. The median duration of survival was 15.8 and 8.3 months in responders and nonresponders, respectively. The toxic symptoms included one case of sepsis and three cases of renal toxicity. However, nausea and vomiting were experienced by most patients and required the administration of antiemetics. In the M-VAC group, the median duration of survival for responders was longer than that of nonresponders (greater than 10.2 vs 7.2 months), although the number of patients was too small for this difference to reach statistical significance. The toxic symptoms included one case of sepsis, two cases of renal toxicity, and nausea and vomiting in most patients. Bone metastasis in three patients did not respond to chemotherapy (CMV), a finding that is compatible with the results reported by other investigators. In summary, chemotherapy with the CMV or M-VAC regimen was effective in improving the response rate of patients. However, the duration of response was short, toxicity was severe in some cases, and the efficacy against bone lesions was poor. These problems must be solved to improve the outcome of patients with TCC following chemotherapy with the CMV or M-VAC regimens.

Adult↗
PubMed1992

Isolation and characterization of endogenous modulators for GABA system.

Pig brain extracts from both soluble and membrane fractions were found to contain potent inhibitors for GABA synthesizing enzyme, GAD, referred to as endogenous GAD inhibitors (EGIs) and for the binding of GABA agonist, muscimol, referred to as muscimol binding inhibitors (MBIs). EGIs and MBIs were first purified through gel-filtration Bio-Gel P-2 columns, in which multiple activity peaks were observed. One of them appears to be co-eluted with either L-glutamate or GABA. However, others are clearly separated from L-glutamate or GABA. EGIs were found to be low MW (less than 1,800 dalton), heat and acid-base stable, negatively charged, non hydrophobic substances. MBIs were found to be low MW (less than 1,800 dalton) neutral or positively charged substances. MBIs had no effect on [3H]flunitrazepam (FNZP) binding, indicating that they are not endogenous benzodiazepine receptor ligands and they may act specifically on GABA binding site.

Animals↗
PubMed1992

Purification of the sex steroid-binding protein from common carp (Cyprinus carpio) plasma.

1. Sex steroid-binding protein was purified from common carp plasma. 2. Testosterone- and estradiol-binding activity existed at the same fraction eluted from gel Sepharose CL-2B, DEAE-Sephacel, hydroxylapatite and HPLC. 3. The molecular weight of the sex steroid-binding protein was 194,000. 4. At 50% displacement the order in which the steroids displaced [3H]testosterone bound to the binding protein was as follows: androstenedione greater than estradiol-17 beta greater than 11-deoxy-17-hydroxycorticosterone greater than 17 alpha-hydroxyprogesterone greater than progesterone greater than deoxycorticosterone greater than estrone greater than 11-ketotestosterone greater than 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one greater than androstenedione greater than pregnenolone greater than cortisone greater than cortisol.

Animals↗
PubMed1992

Food-processing approaches to altering allergenic potential of milk-based formula.

All the major cow milk proteins in their native states are potential allergens in infants with milk allergy. Heat treatment can reduce the antigenicity of whey proteins considerably, but it has virtually no effect on the antigenicity of casein. Infants allergic to milk still react to heat-denatured whey proteins. Therefore heat denaturation alone cannot produce a formula with low allergenicity. Enzyme hydrolysis reduces the antigenicity and allergenicity of protein. Partial hydrolysis produces hydrolysate consisting mainly of large peptides, whereas extensive hydrolysis produces hydrolysate containing a mixture of large and small peptides and free amino acids. Enzyme hydrolysis often produces bitter peptides and destroys the physical functionality of protein. When hydrolysate formula is made, casein or whey protein hydrolysate is ultrafiltered to remove large residual peptides. Certain amino acids are fortified to provide a balanced amino acid profile. The final formulation must comply with the recommendations of the Codex Alimentarius Commission (FAO/WHO) and the U.S. Infant Formula Act to provide adequate infant nutrition. Protein hydrolysate-based formula can be improved by further reducing the residual allergenicity, increasing the small peptide content, and improving the taste.

Food Handling↗
PubMed1992

Acupuncture in the treatment of renal colic.

A prospective randomized study was performed to compare the effect of acupuncture and intramuscular Avafortan injection in the treatment of renal colic. Our results showed that acupuncture is as effective in relieving renal colic as Avafortan but it had a more rapid analgesic onset (3.14 +/- 2.88 minutes versus 15.44 +/- 7.55 minutes, p less than 0.05). Of the patients in the Avafortan group 7 (43.8%) had side effects, including skin rash in 3, tachycardia in 2, drowsiness in 1 and facial flush in 1. No side effects were noted in the acupuncture group. During 2 hours of observation acupuncture and Avafortan seemed to be ineffective in promoting stone passage. However, patients receiving Avafortan treatment were more likely to have paralytic ileus. In summary, acupuncture can be a good alternative for the treatment of renal colic.

Acupuncture Therapy↗
PubMed1992

Determinant role of testosterone in the pathogenesis of urolithiasis in rats.

By using an ethylene glycol-induced urolithiasis model, we assessed the role of testosterone in the pathogenesis of urolithiasis. The intact and castrated male and female rats were fed with 0.5% ethylene glycol in drinking water for four weeks. The renal excretions of oxalate, citrate and other electrolytes were measured, and the stone and crystal deposit were examined microscopically. The results showed that drinking a loading of 0.5% ethylene glycol for four weeks produced hyperoxaluria in all rats, but the intact male rats excreted more urinary oxalate than any other groups of rats. The ethylene glycol-fed rats exhibited hypocitraturia except the castrated male rats. However, urolithiasis occurred in intact male but not female rats. Castration in male rats fed with ethylene glycol dramatically decreased the incidence of renal stone from 71.4% (5/7) to 14.3% (1/7). On the other hand, there was still no renal stone formed in the oophorectomized female rats which received ethylene glycol treatment. These data indicate that serum testosterone level plays a determinant role in urolithiasis formation.

Animals↗
PubMed1992

Decreased systemic clearance of diltiazem with increased hepatic metabolism in rats with uranyl nitrate-induced acute renal failure.

The effect of uranyl nitrate (UN)-induced acute renal failure (ARF) on the pharmacokinetics of diltiazem (DTZ) was examined in rats through in vitro and in vivo studies. In vitro homogenate studies demonstrated that DTZ was metabolized to deacetyl diltiazem (DAD) predominantly in the liver. Metabolism in the small intestine, kidney, or blood pool was negligible compared with that in the liver. UN-induced ARF (UN-ARF) increased the in vitro hepatic clearance (CLvit) of DTZ 1.4-fold. In vivo pharmacokinetic studies following intravenous (iv) and portal venous (pv) administration revealed that UN-ARF increased the intrinsic clearance (CLi) of DTZ from 243.0 to 414.5 ml/min/kg but decreased its total plasma clearance (CLt) from 90.3 to 64.3 ml/min/kg. The increase in CLi was consistent with the increase in CLvit of the liver. The in vitro plasma free fraction of DTZ (fp) was decreased from 0.25 to 0.14 by UN-ARF, but the in vitro blood/plasma partition of DTZ (Rb) remained constant at unity. From the CLi and fp changes, the plasma intrinsic clearance for unbound DTZ (CLi') was calculated to be increased 2.7-fold, from 1104.5 to 2960.7 ml/min/kg, by UN-ARF. The fp decrease was also reflected in the steady-state distribution volume (Vdss) of DTZ, which was decreased significantly from 3595.5 to 2528.3 ml/kg. The absolute bioavailability of pv DTZ (Fpv) was decreased by UN-ARF from 37.5 to 15.5% but was still much higher than the reported oral bioavailability (6%), indicating poor absorption of DTZ from the GI tract. From the calculation based on a well-stirred pharmacokinetic model, DTZ was found to increase the hepatic blood flow (HBF) of the control rats more than twofold at doses of 3 mg/kg (iv) or 10 mg/kg (pv), possibly due to the vasodilating effect of DTZ. However, the effect of DTZ on HBF was not present in the UN-ARF rats. It is not clear at present whether this could be attributed to vasoconstricting effects of UN-ARF or blockade of the vasodilating effect of DTZ.

Acute Kidney Injury↗
PubMed1992

Uninephrectomy enhances urolithiasis in ethylene glycol treated rats.

Uninephrectomy (uNX) usually induces compensatory hyperfunction of the remaining kidney in an attempt to preserve the homeostasis of body fluid composition. The present study used uninephrectomized Sprague-Dawley rats on a lithogenic diet (0.5% ethylene glycol, EG) to evaluate the influence on urinary stone formation and calcium oxalate crystal deposition of compensatory excretion of lithogenic substances in the remnant kidney. The results showed that there were no urinary stones or calcium oxalate crystal deposits in the intact or uNX rats fed a normal diet. In the EG feeding groups, the incidence of massive (grade 3) crystal deposits was significantly higher in the uNX rats (87.5%) than that in the intact rats (37.5%; P less than 0.05). The incidence of urinary stone formation was also higher in the uNX rats as compared to that of the intact rats, although the difference did not achieve statistical significance. The serum magnesium, phosphorus and creatinine increased significantly, whereas creatinine clearance (CCr), 24-hour urinary excretions of citrate, sodium, potassium and chloride decreased significantly in the uNX rats fed EG. These data indicate that uninephrectomy increases the vulnerability of the contralateral remnant kidney to urolithiasis and crystal deposition when the lithogenic risk factors are present. Furthermore, once the remnant kidney forms urolithiasis or massive calcium oxalate crystal deposits, the renal function is severely compromised.

Animals↗