Effectiveness of transarterial embolisation for intractable postpartum haemorrhage in a disseminated intravascular coagulation state, despite emergency hysterectomy.

PURPOSE: We evaluated the effectiveness of transarterial embolization (TAE) for intractable postpartum hemorrhage in patients with disseminated intravascular coagulation (DIC) despite emergency hysterectomy. MATERIALS AND METHODS: We retrospectively assessed TAE performed after emergency hysterectomy in 15 patients between July 2008 and January 2022. Underlying condition, technical success, clinical success, angiographic findings, laboratory findings, pregnancy-modified DIC score (The International Society on Thrombosis and Haemostasis), blood transfusion, ICU (Intensive care unit) admission day, hospital day, in-hospital mortality, and long-term sequelae were evaluated. RESULTS: All patients were diagnosed with DIC before embolization, with a 43.9 mean DIC score. All patients showed positive angiographic findings for active bleeding. Thirty-eight bleeding arteries were confirmed. The remnant uterine artery (n=25) was the most common focus of persistent bleeding, followed by the cervicovaginal artery (n=6), pudendal artery (n=3), obturator artery (n=2), vesical artery (n=1), and unspecified artery from the internal iliac artery (n=1). Technical and clinical success rates were 100% (15/15) and 93.3% (14/15), respectively. Mean nadir hemoglobin (Hb) level before embolization was 4.9 g/dL. All patients underwent massive transfusion before embolization (mean 33.2 packs of RBC). Postoperatively, a smaller amount of blood was transfused than before the procedure (mean 10.6 packs of RBC). Mean nadir Hb level after embolization was 8.2 g/dL. There was one instance each of in-hospital death, hypoxic brain damage, and ischemic acute kidney injury. CONCLUSION: Despite hysterectomy for postpartum bleeding, there could be multiple residual or uncontrolled bleeding foci, especially in case of DIC, for which TAE could be an effective treatment.

Limits on interactions between weakly interacting massive particles and nucleons obtained with CsI(Tl) crystal detectors.

The Korea Invisible Mass Search (KIMS) experiment presents new limits on the weakly interacting massive particle (WIMP)-nucleon cross section using data from an exposure of 3409 kg.d taken with low-background CsI(Tl) crystals at the Yangyang Underground Laboratory. The most stringent limit on the spin-dependent interaction for a pure proton case is obtained. The DAMA signal region for both spin-independent and spin-dependent interactions for the WIMP masses greater than 20 GeV/c2 is excluded by the single experiment with crystal scintillators.

A high statistics measurement of the Lambda(+)(c) lifetime.

A high statistics measurement of the Lambda(+)(c) lifetime from the Fermilab fixed-target FOCUS photoproduction experiment is presented. We describe the analysis technique with particular attention to the determination of the systematic uncertainty. The measured value of 204.6 +/- 3.4 (stat) +/- 2.5 (syst) fs from 8034 +/- 122 Lambda(+)(c)-->pK(-)pi(+) decays represents a significant improvement over the present world average.

Search for CP violation in the decays D+--> K(S)pi+ and D+-->K(S)K+.

A high-statistics sample of photoproduced charm from the FOCUS experiment has been used to search for direct CP violation in the decay rates for D+-->K(S)pi+ and D+-->K(S)K+. We have measured the following asymmetry parameters relative to D+-->K-pi+pi+: A(CP)(K(S)pi+) = (-1.6+/-1.5+/-0.9)%, A(CP)(K(S)K+) = (+6.9+/-6.0+/-1.5)%, and A(CP)(K(S)K+) = (+7.1+/-6.1+/-1.2)% relative to D+-->K(S)pi+. We have also measured the relative branching ratios and found Gamma(D+-->K(0)pi+)/Gamma(D+-->K-pi+pi+) = (30.60+/-0.46+/-0.32)%, Gamma(D+-->K(0)K+)/Gamma(D+-->K-pi+pi+) = (6.04+/-0.35+/-0.30)%, and Gamma(D+-->K(0)K+)/Gamma(D+-->K(0)pi+) = (19.96+/-1.19+/-0.96)%.

Measurement of the branching ratios of D(+) and D(+)(s) hadronic decays to four-body final states containing a K(S).

We have studied hadronic four-body decays of D(+) and D(+)(s) mesons with a K(S) in the final state using data recorded during the 1996-1997 fixed-target run of the Fermilab high energy photoproduction experiment FOCUS. We report a new branching ratio measurement of gamma(D(+)-->K(S)K-pi(+)pi(+))/gamma(D(+)-->K(S)pi(+)pi(+)pi(-)) = 0.0768+/-0.0041+/-0.0032. We make the first observation of three new decay modes with branching ratios gamma(D(+)-->K(S)K+pi(+)pi(-))/gamma(D(+)-->K(S)pi(+)pi(+)pi(-)) = 0.0562+/-0.0039+/-0.0040, gamma(D(+)-->K(S)K+K-pi(+))/gamma(D(+)-->K(S)pi(+)pi(+)pi(-)) = 0.0077+/-0.0015+/-0.0009, and gamma(D(+)(s)-->K(S)K+pi(+)pi(-))/gamma(D(+)(s)-->K(S)K-pi(+)pi(+)) = 0.586+/-0.052+/-0.043, where in each case the first error is statistical and the second error is systematic.

Study of the decay D0 --> K+pi-.

Using a large sample of photoproduced charm mesons from the FOCUS experiment at Fermilab (FNAL-E831), we observe the decay D0-->K+pi- with a signal yield of 149+/-31 events compared to a similarly cut sample consisting of 36 760+/-195 D0-->K-pi+ events. We use the observed ratio of D0-->K+pi- to D0-->K-pi+ (0.404+/-0.085+/-0.025)% to obtain a relationship between the D0 mixing and doubly Cabibbo suppressed decay parameters.

Expression and detection of ScFvB9 and its mutant in recombinant phage antibody system.

Recombinant single-chain antibody (ScFvB9) and its mutant (ScFvB9-6) were generated by using a polymerase chain reaction (PCR) from the Fab fragment of the murine monoclonal antibody (MAb) B9, MabB9 (gamma2b,kappa), which is specific for human plasma apolipoprotein (apo) B-100 of low density lipopreotein (LDL). In the recombinant phage antibody system (RPAS), the constructed ScFvB9 and ScFvB9-6 antibody genes were cloned into the pCANTAB5E phagemid vector and expressed in E. coli. The active forms of single-chain antibodies (ScFvB9 and ScFvB9-6) were produced as phage-displayed recombinant antibodies or soluble antibody forms in E. coli. The activities of ScFvB9 and ScFvB9-6 were confirmed by enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis; the generated mutant ScFvB9-6 showed slightly higher antigen binding activity than native ScFvB9 as a soluble antibody in this RPAS.

Production and in vitro refolding of a single-chain antibody specific for human plasma apolipoprotein A-I.

An active form of single-chain antibody (scFv) has been produced in Escherichia coli for murine monoclonal antibody MabA34 (gamma 1, kappa), which is specific for human plasma apolipoprotein (apo) A-I. The complementary DNAs (cDNAs) encoding the variable regions of heavy chain (VH) and light chain (VL) were connected by a (Gly4Ser)3 linker using an assembly polymerase chain reaction. The construct (VL-linker-VH) was placed under the control of highly efficient T7 promoter system. The cloned scFv was expressed in E. coli as inclusion bodies. After purification from E. coli lysate using sonication and low speed centrifugation, the inclusion body was solubilized and denatured in the presence of 8 M urea, renatured by dialysis, and scFv was finally purified using antigen-affinity chromatography. The purity and activity of purified scFv were confirmed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), Western blotting and enzyme-linked immunosorbent assay (ELISA). The affinity constant was determined by a biosensor method using the BIAcore system. The results showed that the yield of correctly refolded scFv was more than 20 mg l-1 of E. coli flask culture and the specific binding activity to apo A-I was retained with an affinity constant of 6.74 x 10(-8) M (Kd). A notable thing is that guanidine-HCl as a denaturant induced more multimeric formation in the subsequent refolding procedure for the scFv of MabA34 and thus, it was not suitable as urea was. This fact is uncommon for what is generally known for the denaturation and refolding of recombinant antibodies.

Production and characterization of monoclonal antibodies directed to the kringle V and protease domains of human apolipoprotein(a).

Production and use of anti-apolipoprotein(a) monoclonal antibodies (MAbs) specific to single copy regions in the polymorphic lipoprotein(a) (Lp(a)) has been emphasized to be important for the standardization of measurements of the coronary heart disease risk factor, Lp(a). Here, mouse MAbs were prepared against the kringle V (V) and protease (P) domains of human apolipoprotein(a) (apo(a)), which domains are present in single copy in the apo(a) molecule. The cDNA for apo(a)VP was cloned from human liver cDNA library, and the V-P recombinant protein overexpressed in Escherichia coli was used as an antigen for the antibody production. Two antibodies named as MAb(a)20 and MAb(a)23 were finally produced, and they were characterized for their binding specificity and epitopes. The specificity of the antibodies was confirmed by an immunoblotting procedure and an enzyme-linked immunoassay (ELISA). It was shown that the antibodies had little, if any, cross-reactivity with human plasminogen, which is relatively abundant in human serum and is highly homologous (85%) with apo(a) in amino acid (aa) sequence. For epitope analysis, 3'-deletional series of apo(a)VP cDNA were constructed, and expression products of them were analyzed for the binding MAb(a)20 and MAb(a)23 do. It has been revealed that distinct epitopes were recognized by the two MAbs: MAb(a)23 (gamma2b, kappa) bound to the V region about 60 aa downstream from the N-terminal, and MAb(a)20 (gamma1, kappa) bound to the P region close to the C-terminal. A one step-sandwich ELISA system for Lp(a) was developed using MAb(a)20 as a capturing antibody and horseradish peroxidase (HRP)-coupled MAb(a)23 as a detecting antibody. The assay was found to be sensitive and useful for detecting Lp(a) in the range of 4-150 microg/dL (80 pM-3 nM).

Differentially expressed aortic genes in cholesterol-fed rabbits.

In order to identify key genes involved in the development of atherosclerotic lesions, differentially expressed genes in atherosclerotic plaques obtained from diet-induced hypercholesterolemic rabbit aorta were screened using the differential display (DD) RT-PCR technique. Aortic RNAs were isolated from rabbits fed cholesterol-supplemented (2% cholesterol in lab-chow, w/w) chow diet for 12 weeks, followed by the synthesis of cDNAs by reverse-transcription using 2-base anchored oligo (dT) (5'-T11VN) as 3'-primers. Synthesized cDNAs were amplified by PCR using arbitrary 10-mers as 5'-primers and the same 3'-primers used in the reverse-transcription. Amplified cDNAs sized between 0.2 to 0.5kb obtained from control and cholesterol-fed rabbit aortas were displayed on the 6% DNA-sequencing gel for comparisons. The cDNA bands showing distinctive differences in patterns of display or in density of the band were extracted from the gel. A total of 66 differentially displayed cDNAs was isolated and subjected to the reverse-Northern and Northern blot analyses in order to confirm the differences. Through the extensive confirming processes, three cDNAs were finally selected (designated CRGRA-1 through -3) and their nucleotide sequences were determined. Two of those (CRGRA-1 and -2) were determined to be up regulated and the other (CRGRA-3) was down-regulated by the cholesterol-feeding. Upon homology search on databases for the identification of the genes, the first cDNA (CRGRA-1) turned out to be a part of a novel gene, the second one (CRGRA-2) was homologous (82%) to the corresponding segment of mitochondrial NADH dehydrogenase subunits 4 gene, and the last one (CRGRA-2) was identified to be homologous (94%) to a segment of human small GTP-binding protein (Rab7) gene.

Cloning and characterization of cDNAs coding for heavy and light chains of a monoclonal antibody (MabA34) specific for human plasma apolipoprotein A-I.

We have determined the nucleotide (nt) sequences encoding the heavy (H)- and light (L)-chains of the Fab fragment of a murine monoclonal antibody, MabA34 (gamma1, kappa), which is specific for human plasma apolipoprotein A-I of high-density lipoproteins. The variable (V) regions of the H- and L-chains were revealed to be members of mouse H-chain subgroup II(A) and kappa L-chain subgroup II, respectively. A few unusual amino acids in the V region of the H-chain, and nt residues probably introduced by somatic mutations from germline genes were also identified.

Effect of hormone replacement therapy on lipoprotein(a) and lipid levels in postmenopausal women. Influence of various progestogens and duration of therapy.

BACKGROUND: Estrogen replacement therapy in postmenopausal women reduces the risk of coronary artery disease. One of the possible mechanisms of this effect is the modification of lipid profiles. However, there is controversy concerning the effects on lipoprotein(a) [Lp (a)] and lipid levels of progestogens administered with estrogen. METHODS: Five hundred fifty-one postmenopausal women were divided into 5 groups: group 1, 0.625 mg of conjugated equine estrogen (CEE) (n = 140); group 2, 0.625 mg of CEE plus 5 mg of medroxyprogesterone acetate (MPA) (n = 97); group 3, 0.625 mg of CEE plus 10 mg of MPA (n = 109); group 4, 2 mg of estradiol valerate plus 0.5 mg of norgestrel (n = 134); and group 5, control (n = 71). The Lp(a) and lipid levels were measured before and 2, 6, and 12 months after hormone replacement therapy. RESULTS: Estrogen replacement therapy for 12 months lowered the Lp(a) level by 37.1%. The addition of progestogen attenuated the Lp(a)-lowering effect of estrogen. The high-density lipoprotein cholesterol (HDL-C) level was markedly increased in group 1 (16.5%), was moderately increased in groups 2 (10.8%) and 3 (11.3%), and was not changed in group 4. The low-density lipoprotein cholesterol level was decreased by 10.9% to 17.6% in all the treatment groups. Estrogen replacement therapy for 2, 6, and 12 months raised the HDL-C level by 7.2%, 17.4%, and 17.8%, respectively. In the group with combined estradiol plus norgestrel therapy, the HDL-C level was decreased after 2 months and was not changed after 6 and 12 months. The groups that received CEE plus MPA showed intermediate effects between the group that received CEE only and the group that received estradiol plus norgestrel. CONCLUSIONS: Combined estrogen and progestogen therapy may have effects on the heart different from those of estrogen therapy alone because of adverse impact of progestogens on Lp(a) and HDL-C levels. The effects of progesterones were dependent on the androgenic potency of progestogen and the duration of therapy.

A convenient method for epitope competition analysis of two monoclonal antibodies for their antigen binding.

Choosing optimum pair of capturing antibody and detecting antibody when developing monoclonal antibody (MAb)-based, sandwich enzyme-linked immunosorbent assays is a time-consuming process requiring the coupling of individual antibodies to an enzyme like horseradish peroxidase or alkaline phosphatase. The MAbs required for the two-site sandwich ELISA should bind to distinct epitopes of the antigen, and their binding should not be mutually exclusive. To determine if two monoclonal antibodies would occupy distinct sites of their antigen in binding, an enzyme-linked immunosorbent assay was devised, which is easy-to-use and does not require any coupling of monoclonal antibodies to enzymes. Microplate wells are coated with rabbit polyclonal antibodies raised against the same antigen of MAbs. After blocking, a limited amount of the antigen is added for incubation with the rabbit antibodies. Mouse monoclonal antibody 1 (MAb 1) is added to saturation. A serial dilution of MAb 2 (for analysis) or MAb 1 (for control) is added subsequently. An enzyme-labeled, goat anti-mouse secondary antibody and its substrates are added for color development. Thus, the epitope competition of two MAbs for their antigen binding is easily determined by the measurement and comparison of color development between the two MAb additions.

Cloning and characterization of cDNAs coding for heavy and light chains of a monoclonal antibody (MabB23) specific for human plasma apolipoprotein B-100.

We have determined the nucleotide sequences encoding the heavy and light chains of the Fab fragment of murine monoclonal antibody MabB23(gamma2b,lambda), which is specific for human plasma apolipoprotein B-100 of low-density lipoproteins. The sequence analyses revealed that the variable regions of the heavy and light chains are members of mouse heavy-chain subgroup I(B) and lambda light-chain, respectively. A few unusual amino acids in the framework and constant regions of the heavy-chain were also noticed.

Changes in Lp(a) lipoprotein and lipid levels after cessation of female sex hormone production and estrogen replacement therapy.

OBJECTIVE: To investigate the serial changes in Lp(a) lipoprotein levels with the loss of female sex hormones by surgical menopause and with estrogen replacement therapy in the same woman. PATIENTS AND METHODS: Forty-four premenopausal women who underwent a transabdominal hysterectomy (TAH) because of benign gynecological disorders were divided into two groups: women who underwent a TAH and unilateral salpingo-oophorectomy (n=31) and women who underwent a TAH and bilateral salpingo-oophorectomy (n=13). In the group of women who underwent a TAH and bilateral salpingo-oophorectomy, 0.625 mg of conjugated equine estrogen was given daily 2 months after the operation. The levels of Lp(a) lipoprotein and lipids were measured before and at 2 and 4 months after the operation. RESULTS: In the group of women who underwent a TAH and bilateral salpingo-oophorectomy, the mean (+/-SD) concentration of Lp(a) lipoprotein was increased by 24.5% from 0.48+/-0.47 mmol/L (18.4+/-18.3 mg/dL) to 0.59+/-0.54 mmol/L (22.9+/-21.0 mg/dL) after 2 months (P<.05), and it was reduced by 30.6% to 0.41+/-0.51 mmol/L (15.9+/-20.1 mg/dL)(P<.005) with therapy with conjugated equine estrogen (Premarin). The Lp(a) lipoprotein levels were not changed in the group of women who underwent a TAH and unilateral salpingo-oophorectomy. In the group of women who underwent a TAH and bilateral salpingo-oophorectomy, the high density lipoprotein cholesterol level showed a trend of increase after 2 months from 1.45+/-0.48 mmol/L (56.1+/-18.5 mg/dL) to 1.58+/-0.309 mmol/L (61.2+/-15.1 mg/dL) without statistical significance, and it revealed a significant elevation to 1.76+/-0.43 mmol/L (68.2+/-16.8 mg/dL) with therapy with conjugated equine estrogen (Premarin) compared with that of the basal level (P<.05). CONCLUSIONS: tHE Lp(a) lipoprotein levels appear to be closely associated with female sex hormones. This association might play a pivotal role in postmenopausal increases of atherosclerotic diseases and cardioprotective effect of estrogen in postmenopausal women.

Phospholipid antibodies in alcoholic liver disease.

Alcoholic liver injury has been reported to be directed preferentially against the proteins of the cell membrane, sparing the phospholipids. However, antiphospholipid antibodies against certain cell membrane phospholipids are known to be associated with a variety of diseases. We undertook this investigation to determine whether antiphospholipid antibodies were present in the serum of patients with alcoholic liver disease. We investigated seventy long-term alcoholic patients (> 80 gm ethanol/day for > 1 yr) and 8 normal nonalcoholic controls by means of enzyme-linked immunosorbent assay to determine whether serum antibodies were generated against the following membrane phospholipids: phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol (cardiolipin) and phosphatidic acid. Group 1 comprised alcoholic patients with normal liver function (n = 13), group 2 comprised alcoholic patients with abnormal liver function (n = 16), group 3 comprised patients with alcoholic hepatitis or cirrhosis (n = 41) and group 4 comprised nonalcoholic controls (n = 8). The antibody prevalence was 15% in group 1, 31% in group 2, 81% in group 3 and 0% in group 4. In group 3, 20 of 41 patients had antibodies against several cell membrane phospholipids (i.e., phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, cardiolipin and phosphatidylinositol). The antiphosphatidylethanolamine isotype was IgA or IgM in 25 of 41 of these patients. Both IgA (p < 0.01) and IgM (p < 0.008) antiphosphatidylethanolamine correlated significantly with disease severity. Antiphospholipid antibodies in alcoholic patients seem to reflect disease progression and correlate significantly with disease severity.

Cloning and analysis of a yeast genomic DNA sequence capable of directing gene transcription in Escherichia coli as well as in yeast.

A DNA fragment was isolated from yeast genomic sequences by its ability to direct the transcription of promoterless CmR (cat) gene in Escherichia coli and in yeast. Nucleotide sequencing and primer extension analysis showed that yeast DNA contains sets of consensus sequences pertaining to prokaryotic and yeast-type promoter elements. It was designated as yeast- and E. coli-type promoter (YEP1). Typical E. coli-type promoter elements are found at appropriate positions: TATTTT from -12 to -7 and TTGTCC from -35 to -30 with their spacing of 17 bp from the single mRNA start point determined by the primer extension. Analysis of cat transcripts from yeast cells showed that the YEP1 caused multiple transcription initiations at more than 20 different points that are spaced over a 100-bp region. The DNA is composed of A + T-rich sequences and putative TATA-like sequences are found at several places upstream from the transcription start points. Deletion analysis showed that a 276-bp sequence between -872 and -596 from the initiating ATG codon was required for the maximal promoter activity in yeast but not in E. coli.

A method to isolate DNA sequences that are promoter-active in Escherichia coli and in yeast.

A method convenient for isolation of DNA sequences capable of directing gene transcription in both organisms of E. coli and yeast is described. The method is composed of sequential steps of phenotypic selection for chloramphenicol resistance, first in E. coli and then in yeast. A series of promoter-probe, shuttle plasmid vectors between yeast and E. coli were constructed and utilized in the method.

Production of rosamicin: improvement of synthetic medium.

Rosamicin is one of the important macrolide antibiotics that has clinical efficacy and broad-spectrum antibacterial activity. Using a mutant strain of Micromonospora rosaria (NRRL 3718), a chemically defined medium was developed, and some fermentation conditions that are important to rosamicin biosynthesis were optimized to achieve rosamicin productivity of 230 mug/ml. Soluble starch and l-asparagine were found to be the best carbon and nitrogen sources, and a stimulative effect of magnesium and zinc ions was also found. The medium developed contains: soluble starch, 4%; l-asparagine, 0.15%; K(2)HPO(4), 0.075%; CaCO(3), 0.6%; MgSO(4) . 7H(2)O, 0.05%; FeSO(4) . 7H(2)O, 10 M; CuSO(4) . 5H(2)O, 10 M; ZnSO(4) . 7H(2)O, 10 M; and MnSO(4) . (4-6)H(2)O, 10 M. The required air supply was about 40 mmol of O(2) liter . h . atm, and the favorable culture temperature was 28 to 29 degrees C.