Cross-reactivity of a commercial chemiluminescence immunoassay for human chorionic gonadotropin with the free β-subunit

An enhanced chemiluminescence immunoassay for the determination of serum human chorionic gonadotropin (HCG) in specimens from oncology patients has been assessed with respect to its cross-reactivity with the free HCG ββ-subunit (HCG-β). The assay, standardized against the First International Reference Preparation 75/537, had a crossreactivity with the free β-subunit of 625% (molar basis). Therefore this assay achieves high sensitivity for the detection of either intact HCG or free HCG-β in serum of patients with seminomatous or nonseminomatous testicular cancers. Results of both assays, the in-house immunoradiometric assay (+ HCG-β) and the Amerlite HCG-60 assay, showed a close correlation (R =0.854?0.960) when serum samples from tumour patients were analyzed. Moreover, the content of free β-subunit determined in a specific HCG-β assay, could be quantitatively measured in the enhanced chemiluminescence immunoassay. Thus, this assay is suitable for oncology use, but also highlights the limitations of measuring HCG in serum samples.     

Effects of hCG on folliculogenesis and fecundity in mink (Mustela vision Schreb)

The endogenous hormonal response obtained after reproductive organs are challenged by exogenous hormones is increasingly being used to predict presence of functional reserves and to apply this information to improve efficiency of managed breeding programs. With that in mind, the aim of the present study was to investigate the effect of a single treatment with hCG on folliculogenesis and fertility in standard 7-month-old mink females. The extent of stimulation following treatment was determined by examining patterns of vaginal smears. Characteristics of each cycle stage were: estrus, preponderance of cornified epithelial cells; proestrus, polygonal, elongate epithelial cells; anestrus, parabasal, intermediate and leucocyte cells. Smears exhibiting a mixed population of cells were categorized as being in transition between adjacent stages anestrus–proestrus or proestrus–estrus. The initial evaluations were done on Day 6 after hCG treatment. Histomorphometric examination of ovaries and uteri was done during seasonal anestrus (November) and in the breeding season (March). Vaginal cytology patterns were correlated with changes in folliculogenesis. A mean of 1.3 mature (Graafian) follicles were counted during estrus, while the mean number seen during anestrus, anestrus–proestrus and proestrus, were 0.4, 0.3 and 1.0, respectively. During the breeding season, in females that were not treated, the numbers of growing follicles decreased and maturing follicles increased, whereas females that came in estrus after treatment with hCG in November had increased numbers of both growing and maturing follicles. Fertility after breeding in hCG-treated females was increased by 9.2% (P<0.05) as compared to untreated females. Females showing the highest fertility rise (27%) were predominantly in the group that showed estrus after hCG treatment. We conclude that monitoring the response of the mink reproductive system to hCG stimulation in November may be a useful tool for identifying females of high fertility in the spring.     

The effect of hormone treatments (hCG and cloprostenol) and season on the incidence of hemorrhagic anovulatory follicles in the mare: A field study

The association between use of hormone treatments to induce estrus and ovulation and the incidence of hemorrhagic anovulatory follicles (HAFs) was studied in a mixed population of mares (Equus caballus) during two breeding seasons in a commercial breeding clinic. Mares treated with cloprostenol (CLO) were more likely to develop HAFs than were mares with spontaneous cycles (P < 0.001) or those treated with human chorionic gonadotropin alone (P = 0.08). There was no significant effect of season on the incidence of HAFs. The mean (±SEM) interval from CLO treatment to beginning of HAF development was 6.1 ± 0.5 d. Age of mares with HAF cycles was not different (12 ± 1.3 yr; P > 0.05) from that of mares with ovulatory cycles (10.5 ± 1.5 yr).     

Effects of human chorionic gonadotropin (hCG) and phenobarbital on the reproductive performance of fat-tailed Ghezel ewes

Increased ewe prolificacy and lambing rates, under certain conditions of sheep husbandry, may be economical and desirable. This paper reports on the effects of human chorionic gonadotropin (hCG) and phenobarbital (PB) on fertility and lambing rate in fat-tailed Ghezel sheep. One-hundred and five cyclic ewes (1.5 to 7.5 years of age) were randomly allotted to seven groups. Estrus was synchronized with two intramuscular injections of a prostaglandin F₂ analogue (PGF₂), eight days apart. Four groups were randomly assigned to the hCG and three groups to PB experiments. Twenty-four hours after the second injection of PGF₂, the ewes in four groups were injected (i.m.), either with 1 ml normal saline (control) or with 125, 250 or 500 IU hCG. Prolificacy of the ewes which conceived at first estrus increased in ewes receiving 500 IU of hCG, as compared with control group (an increase of 0.5 lamb/ewe) but fertility and lambing rate decreased in hCG-treated groups (P<0.05). Nine days after the second injection of PGF₂, ewes in the three remaining groups were orally fed empty gelatin capsules (control), 1.0 or 1.5 g of PB per day until the next estrus. Prolificacy and lambing rate increased (P<0.05) in ewes which received 1.0 g of PB (1.57 and 146.5%, respectively), as compared with the control groups (1.17 and 100.3%, respectively). It is concluded that daily feeding of 1.0 g of PB from day 7 of the estrous cycle until the next estrus period is an effective method of increasing lambing rate. hCG also increases the prolificacy, but it greatly decreases fertility and lambing rates. Since hCG is more convenient to administer on a flock basis, further experiments to improve its efficacy are warranted.     

The surface of α-subunit loop 1 distant from the subunit interface is exposed in the hCG lutropin receptor complex

Interactions of the placental glycoprotein hormone human choriogonadotropin (hCG) with lutropin receptors (LHR) are required for maintenance of early pregnancy. Knowledge of how hCG interacts with LHR is useful for understanding the mechanism of receptor function, an issue of considerable debate. A large surface of hCG remains exposed after the hormone binds the LHR and can be readily detected with monoclonal antibodies. Here we show that the surface of hCG α-subunit loop 1 furthest from the β-subunit interface can also be recognized by a monoclonal antibody when hCG is bound to the LHR. This extends the area of hCG known to be exposed in the hormone receptor complex, an observation that further restricts models of hCG–LHR interaction.     

Evaluation of treatments with hCG and carprofen at embryo transfer in a demi-embryo and recipient virgin heifer model

An in vivo model, combining a low developmental competence embryo (demi-embryo) and a high-fertility recipient (virgin dairy heifer) was used to evaluate the effects of treatment with human chorionic gonadotropin (hCG) and carprofen at embryo transfer (ET) on plasma progesterone (P₄) concentrations of recipients and on embryonic growth and survival. Embryos were bisected and each demi-embryo was transferred to a recipient on Day 7 of the estrous cycle. At ET, heifers (n = 163) were randomly allocated to treatment with hCG (2500 IU im), carprofen (500 mg iv), hCG plus carprofen or to untreated controls. Plasma P₄ concentrations were measured on Days 0, 7, 14 and 21 of all recipients plus on Days 28, 42 and 63 of pregnant recipients. Pregnancy was presumed to be present in recipients with luteal plasma P₄ concentrations until Day 21 and confirmed by using transrectal ultrasonography on Days 28, 42 and 63. Embryonic measurements (crown–rump length and width) were obtained on Day 42. Treatment with hCG induced formation of secondary corpora lutea (CL) in 97% of heifers and increased (P < 0.01) mean plasma P₄ concentrations of non-pregnant recipients on Day 14 and of pregnant heifers on Days 14 to 63. This was associated to a significant decrease in early embryonic mortality. In contrast, subsequent embryonic losses resulted in a non-significant numerical increase by 8% of pregnancies maintained to Day 63. Therefore, treatment with hCG significantly rescued embryos through the maternal recognition of pregnancy window but was not able to support development thereafter. Treatment with carprofen at ET had no significant effects on plasma P₄ concentrations and rate of embryo mortality. Treatment with hCG plus carprofen at ET induced formation of secondary CL in 90% of heifers but decreased the luteotrophic effect of hCG, resulting in no effect on embryo survival. Low developmental competence embryos showed an intrinsic deficiency in overcoming the maternal recognition of pregnancy challenge and in proceeding to further development until Day 28 of pregnancy, whereas mortality beyond this point was residual. Results on pregnancy rates should be confirmed in further experiments involving a larger sample size.     

Dual‐labeled chemiluminescence enzyme immunoassay for simultaneous measurement of total prostate specific antigen (TPSA) and free prostate specific antigen (FPSA)

The specificity for early diagnostic of prostate‐specific antigen (PSA) is low because the current technology mostly allows the detection of only one biomarker at one time. In this work, a dual‐labeled chemiluminescence enzyme immunoassay (CLEIA) for simultaneous measurement of total PSA (TPSA) and free PSA (FPSA) was proposed. Anti‐PSA McAb (Mab1) was immobilized on a microplate as the solid phase, horseradish peroxidase (HRP)‐labeled anti‐TPSA monoclonal antibody (McAb2) and alkaline phosphatase (ALP)‐labeled anti‐FPSA McAb3 were used as detection antibodies. Two chemiluminescence reactions of HRP with luminol and ALP with 4‐methoxy‐4‐(3‐phosphate‐phenyl)‐spiro‐(1,2‐dioxetane‐3,2′‐adamantane) (AMPPD) were used as the signal detecting system. Based on a sandwich model, the amount of FPSA and TPSA could be determined simultaneously. The effects of several physico‐chemical parameters were studied and optimized. Cross‐reactivities of six common tumor markers in serum were studied. The proposed method presented the sensitivity of 0.03 ng ml^?1 and 0.05 ng ml^?1 for FPSA and TPSA respectively, with low cross‐reactivities. Compared with the results from commercial chemiluminescent kits there was good correlation, indicating that this established method could be used to simultaneously to measure the concentrations of FPSA and TPSA in one serum sample and also could greatly facilitate the early diagnosis for PCa in clinical practice.     

Characterization and comparison of testicular LH/hCG receptors of rhesus monkeys (Macaca mulatta) and green monkeys (Cercopithecus aethiops)

Testicular luteinizing hormone (LH/hCG) receptors were characterized in seven green monkeys and compared with those of four rhesus monkeys. Testicular tissue showed high binding affinity for ¹²⁵I-hCG, (0.9–2.5 × 10⁹ M^?1, and 0.7–1.64 × 10⁹ M^?1 respectively, for green and rhesus monkeys) and low binding capacity (0.343–0.682 fmol/mg and 0.198–0.355 fmol/mg testicular homogenate, respectively). There was no difference in binding affinity between the two groups. Testicular LH/hCG receptors in both species bound human LH (hLH) and hCG but did not cross react with ovine LH (oLH). Rat testicular tissue showed similar high binding affinity (6.4 × 10⁹ M^?1) and low binding capacity (1.04 fmol/mg tissue homogenate) for ¹²⁵I-hCG. Rat LH/hCG receptors bound hLH, hCG, and oLH to a similar degree.     

Evaluation of adrenal function in rats by the measurement of urinary free corticosterone, free aldosterone and free 11-deoxycorticosterone.

Methods für the determination of urinary free corticosterone, free aldosterone and free 11-deoxycorticosterone (DOC) in rats are described. The free corticosteroids were measured in urine samples of 0.1–0.5 (2.0) ml by radioimmunoassay after purification by column chromatography. The validity of the methods is demonstrated by the data of the free urinary corticoids under basal conditions and after adrenal suppression and various forms of adrenal stimulation. The basal excretion of free corticosterone, free aldosterone and free DOC was 123.71 ± 15.31 (x? ± SD), 3.87 ± 1.29 and 10.61 ± 2.24 ng/day, respectively, exhibiting a decrease to 26.20 ± 5.21, 1.05 ± 0.47 and 1.35 ± 1.20 ng/day after adrenal suppression by dexamethasone. Irrespective of the mode of adrenal stimulation i.e., synthetic ACTH and systemic (cold, hunger) or neurotrophic (ether, reserpine) stress stimuli free corticosterone increased to about 450 ng/day, while free aldosterone excretion decreased during hunger and cold and was strongly enhanced after the application of reserpine. Furthermore, determination of urinary free DOC, which increased by a factor of 4, may be applied in the metyrapone test. There was a good correlation between the excretion of free corticosterone and that of free aldosterone and free DOC under basal conditions and after ACTH application, demonstrating that ACTH is responsible for the secretion of all the 3 corticoids measured. It is concluded, that the measurement of the urinary excretion of corticosterone, aldosterone and DOC is a valuable parameter of adrenal function in rats. Furthermore, in small laboratory animals like rats steroid measurements in urine are often more advantageous than Measurements in plasma.     

Recombinant carbohydrate variant of human choriogonadotropin beta-subunit (hCG beta) descarboxyl terminus (115-145). Expression and characterization of carboxyl-terminal deletion mutant of hCG beta in the baculovirus system

A recombinant analog of human choriogonadotropin beta-subunit descarboxyl-terminal peptide (115-145 residues, delhCG beta) was obtained by the expression of corresponding beta cDNA in the baculovirus expression system. The efficiency of expression and secretion was high. The recombinant delhCG beta was purified by immunoaffinity using a specific monoclonal antibody against hCG beta and reverse phase high performance liquid chromatography. The hCG beta analog lacked the carboxyl-terminal 31-residue peptide as well as the four O-linked carbohydrates. Also, the N-linked "complex" type carbohydrates in the deletion mutant were modified to the high mannose type. The apparent molecular weights of delhCG beta in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions were found to be 19,000 and 27,500 respectively. delhCG beta on hydrolysis with endo N-acetylglucosaminidase F or H yielded a 17,500 protein band whereas treatment with N-glycanase gave a protein band with a molecular weight of 16,000. The carbohydrate analysis of delhCG beta, calculated on the basis of 4 residues of N-acetylglucosamine, showed 3 or 4 fucose, 0.6 N-acetylgalactosamine, and 11.4 mannose residues, indicating the high mannose type structures of the two N-linked carbohydrate chains. Despite the carbohydrate modification of the N-linked carbohydrates and the carboxyl-terminal deletion, the delhCG beta had about 87% of the immunological activity of the native hCG beta, indicating no significant conformational alteration induced by the mutation. The delhCG beta combined readily with native hCG alpha, and the reconstituted hCG alpha del beta required 0.031 pmol to achieve 50% inhibition of binding of the tracer with rat lutropin/choriogonadotropin receptor compared with 0.039 pmol by native hCG. Like native hCG, hCG alpha del beta also had most comparable ability to stimulate cAMP accumulation and progesterone production in rat Leydig cells. Thus it is clear from the data that the carboxyl-terminal deletion and thereby the deletion of four O-linked carbohydrates had no effect on its in vitro immunological and biological properties.     

LH与hCG对昆明小鼠卵母细胞体外成熟的影响

目的研究促黄体素（LH）、人绒毛膜促性腺激素（hCG）对昆明小鼠卵母细胞体外成熟的影响。方法小鼠经注射孕马血清促性腺激素（PMSG）48h后,摘取卵巢获得未成熟卵母细胞,分别在含不同浓度的LH和hCG的成熟液中,或将LH和hCG以不同的浓度组合加入到成熟液,进行体外成熟。结果经15.16h的成熟培养,5个浓度LH组中的极体率均高于对照组,其中200IU/mL组显著高于50IU/mL、400IU/mL、300IU/mL组和对照组（P〈0.05）;5个浓度hCG组的极体率与对照组极体率无显著差异（P〉0.05）;协同组中15IU/mL hCG＋200IU/mL LH组的极体率显著高于对照组和其它各处理组。结论LH对小鼠卵母细胞的体外成熟有一定的促进作用。     

Effect of hCG vs. GnRH at the beginning of the Ovsynch on first ovulation and conception rates in cyclic lactating dairy cows

Ovulatory response to the first GnRH of Ovsynch is a very important factor for determining the outcome of a successful synchronization. The aim of the present study was to develop a protocol to increase the percentage of cows that ovulated in response to the first administration of Ovsynch. This study was designed to compare ovulation rates in response to GnRH or hCG at the beginning of Ovsynch and to evaluate the effects of this manipulation on pregnancy. Cows (n = 371) with corpus luteum (CL) and at least one follicle greater than 10 mm diameter size on either ovary were included in the study. Cows were divided into two groups. The Ovsynch protocol began with GnRH (10 μg) in the GPG group (n = 161; GnRH-7d-PGF2α-56h-GnRH-18h-AI), whereas in the HPG group, the first GnRH of the Ovsynch was replaced with 1500 IU hCG (n = 210; hCG-7d-PGF2α-56h-GnRH-18h-AI). Ovarian ultrasonography was performed at the times of GnRH or hCG and of PGF2α administration, at the time of artificial insemination (AI) and seven days after AI, to determine ovulation. Maximal follicle size at the beginning of the Ovsynch did not affect on response to the first GnRH/hCG treatment. Conception rate (31 d) was 0.6 times more likely to be higher (P < 0.001) in cows that responded to the first hormonal administration of Ovsynch than in those that did not respond (95% CI = 0.29-0.71). Conception rate was found to be different between the HPG (37.6%, 79/210) and the GPG groups (48.4%, 78/161). Thus, beginning of the Ovsynch protocol with hCG did not increase ovulation and conception rate in lactating dairy cows, suggesting that hCG is not a suitable replacement of the first GnRH of Ovsynch. However, our results do show that increasing the ovulation rate in response to the first hormonal administration of Ovsynch can have a significant effect on conception rate.     

Studies of hCG binding and endocytosis in rat ovary by ultrastructural immunocytochemistry

Summary Localization of hCG binding sites and the process of endocytosis in pseudopregnant rat ovaries were investigated by indirect electron-microscopic immunocytochemistry. Immature female rats were treated with pregnant-mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) to induce ovarian luteinization. Eight days after priming with PMSG-hCG and 1–6 h before sacrifice the animals were given another injection of hCG to bind the receptors. Receptor sites to hCG localized by reaction product were present in most luteal cells, but not in primary follicular cells. The receptor sites were distributed on luteal cell surfaces facing interstitial spaces. Endocytotic pits containing hCG binding sites were rarely seen 1 h after hCG injection. At 2 h, hCG and presumably its receptor were taken up within endocytotic vesicles with the evidence of reaction product coated on the vesicle wall. With time, fusion of endocytotic vesicles with lysosome occurred and the reaction product appeared in phagolysosomes. The reaction product was localized on phagolysosomal inner surface or in free granular form. These findings suggest that hCG and its receptors were internalized through endocytotic pits and endocytotic vesicles and delivered to lysosomes probably for degradation. An additional experiment for localization of acid phosphatase was also performed to delineate the lysosomes and phagolysosomes.     

Enhanced chemiluminescence reaction applied to the study of horseradish peroxidase stability in the course of p-iodophenol oxidation

The enhanced chemiluminescence reaction (ECL) was applied to the study of horseradish peroxidase (HRP) inactivation during the oxidation of p-iodophenol. Enzyme inactivation was shown to be the main reason for light decay in the course of the reaction. No individual effect of luminol and p-iodophenol as enhancer on HRP activity towards 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) was detected, enzymatic activity loss was detected only in the course of the ECL reaction. HRP activity towards ABTS (a colorimetric substrate) fell in a similar manner to the decay in light emission. The reactive radical species formed during enhancer oxidation were suggested as the main inactivating agents. The similarity of changes in light intensity and enzymatic activity allows one to apply the ECL reaction for testing potential stabilizers of HRP. The loss of enzyme activity can be partially explained by non-specific interaction of radical species with protein globule. The addition of bovine serum albumin provided almost complete protection of peroxidase from inactivation. This confirms the non-specific inactivation with highly reactive endogenous intermediates through the modification of a protein globule. © 1997 John Wiley & Sons, Ltd.     

Frameshift and double-amber mutations in the bacteriophage T4 uvsX gene: analysis of mutant UvsX proteins from infected cells

Summary The bacteriophage T4 uvsX gene encodes a 43 kDa, single-stranded DNA-dependent ATPase, double-stranded DNA-binding protein involved in DNA recombination, repair and mutagenesis. Mutants of uvsX have a DNA-arrest phenotype and reduced burst size. Western blot immunoassay of UvsX peptides made by a number of amber mutants revealed amber peptides ranging from 25–32 kDa. Wild-type UvsX protein was also detected in lysates of cells infected with uvsX amber mutants, suggesting that their mutations are suppressed by translational ambiguity. We investigated the effects of mutations near the 5 end of uvsX. A frameshift mutation was engineered at codon 33. Western immunoblots for UvsX protein demonstrated that the frameshift mutant expresses no detectable wild-type UvsX; instead, a 37 kDa reactive peptide was detected. In order to determine if this peptide represents truncated UvsX protein, the mutation was regenerated in the cloned uvsX gene and expressed in transformed Escherichia coli. Endopeptidase digestion of the 37 kDa protein from the cloned gene generated peptide fragments indistinguishable from those obtained from wild-type UvsX. A double-amber mutant of uvsX was also generated by oligonucleotide site-directed mutagenesis. No UvsX protein was detected in lysates of cells infected with the uvsX-am64am67 double mutant. Plaque size and sensitivity to UV inactivation for both the double-amber and the frame-shift mutants were indistinguishable from those of other uvsX mutants. Mutations in uvsY had no demonstrable effect on efficiency of plating or UV sensitivity of uvsX mutants. Thus, null mutants of uvsX are viable.     

Pharmacokinetics of eCG and induction of fertile estrus in bitches using eCG followed by hCG

The aim was to design a protocol combining eCG followed by hCG for estrus induction in the bitch. In Experiment 1, three ovariohysterectomized bitches received 10 000 IU of eCG iv, and 15 days later 10 000 IU of eCG im. Blood samples were taken up to 144 h after each injection to measure eCG concentrations. In Experiment 2, 25 healthy, intact late anestrous bitches were assigned to one of five doses of eCG (5, 10, 15, 20, 44, or 50 IU/kg eCG im; [TRT5-TRT50]). Sexual behavior (SB), clinical signs of estrus (CSE) and vaginal cytology (VC) samples were obtained and scored before eCG administration and every other day until onset of estrus, or for 14 days. In Experiment 3, intact late anestrous bitches were assigned to a treatment group (TRT; n = 16) and received eCG (50 IU/kg im) followed by hCG (500 IU im) 7 days later; or to a placebo group (PLA; n = 8) where they received 1 mL saline solution im. All bitches that were induced in estrus were mated or AI with fresh semen. In Experiment 1, maximum observed concentration (C_max) eCG were similar between im and iv routes (6.1 ± 0.9 vs. 8.6 ± 0.5 IU/mL, P > 0.08), whereas time for maximum observed concentration (T_max.) was longer for im compared to iv routes (17.5 ± 0.5 vs. 11.6 ± 0.3 h, P < 0.01). The area under the curve (AUC) was similar for im and iv routes (P > 0.48), and eCG was detectable in serum for at least 144 h for both routes. In Experiment 2, 3 days or 3 to 5 days after treatment, all bitches in TRT50 had higher scores compared to TRT5-44 animals (P < 0.01). In TRT50, the mean interval from treatment to estrus was 4.0 ± 0.4 days. In Experiment 3, the mean interval from treatment to estrus was shorter in the TRT group compared to the PLA group (4.1 ± 3.3 vs. 68.5 ± 4.4 days, P < 0.01). The previous interestrus interval was similar for TRT and PLA groups (199.6 ± 7.2 vs. 197.5 ± 10.2 days), but the new interestrus interval was shorter for the TRT compared to the PLA group (164.0 ± 7.2 vs. 212.2 ± 10.2 days; treatment by interval interaction, P < 0.007). Serum P₄ concentrations increased on the first day of cytologic diestrus after treatment in bitches in TRT (0.7 ± 0.3 vs. 22.8 ± 4.2 ng/mL; P < 0.01); but did not change in PLA (P > 0.84). Ninety-four percent of animals were bred (15/16; AI, n = 7; natural mating, n = 8), and 80% (12/15) became pregnant. None of the bitches had any side effects from the eCG and hCG therapy. We concluded that 50 IU/kg of eCG combined 7 days later with 500 IU of hCG was effective to induce normal and fertile estrus in bitches at 164 days post estrus, with an 80% pregnancy rate, with no side effects, and with a reduction of 48 days of the interestrus interval.     

Changes in growth,proteins and free amino acids of developing seed and pod of fenugreek

The growth and development under field conditions of the pods and seeds of two cvs of Trigonella foenum graecum are described. Samples were harvested at different stages of ripeness for the determination of dry matter, protein and free amino acid content. During maturation, reserves of solutes are established in the pod wall before the seeds begin their exponential phase of growth. Later, these reserves disappear, providing about 20% of the seed's requirements for nitrogen. SDS-electrophoresis was used to follow the formation of proteins and it was shown that the synthesis of storage proteins takes place prior to dehydration of the seed. Production soluble nitrogenous compounds precedes protein accumulation. Free amino acids follow the same pattern. 4-Hydroxyisoleucine represents nearly 80% of free amino acid of dry seeds. The concentration does not decrease in the later stages of maturation of the seed but this unusual amino acid is absent in the storage proteins of the seeds.     

非培养细胞的贴壁方法及细胞内Ca^2＋观测

本文介绍非培养细胞贴壁的简易方法和细胞内Ｃａ＾２＋观察。以ｆｌｕｏ－３／ＡＭ进行染色,在３７℃孵育箱内孵育６０分钟,用激光共聚焦显微镜监测细胞内Ｃａ＾２＋的荧光信号。结果表明本方法可成功地应用于共聚焦显微镜测定细胞的研究。     

Enhanced chemiluminescence: A sensitive analytical system for detection of sweet potato peroxidase

3-(10'-Phenothiazinyl)propane-1-sulfonate (SPTZ) was shown to be a potent enhancer of anionic sweet potato peroxidase (aSPP)-induced chemiluminescence. The optimal conditions for aSPP-catalyzed oxidation of luminol were investigated by varying the concentrations of luminol, hydrogen peroxide, Tris, and SPTZ as well as the pH values of the reaction mixture. Addition of 4-morpholinopyridine (MORP) to the reaction mixture markedly increased the light intensity. Using SPTZ and MORP together enhanced the effect 265 times. The lower detection limit (LDL) of SPP was 0.09 pM, approximately in 10 times lower than that for the cationic isozyme c of horseradish peroxidase/4-iodophenol system. It was shown that aSPP in the presence of SPTZ produced a longer lasting chemiluminescent signal.