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L-asparaginase has been used for more than three decades in acute lymphoblastic leukemia (ALL) patients and remains an essential drug in the treatment of ALL. Poor response to L-asparaginase is associated with increased risk of therapeutic failure in ALL. However, both the metabolic perturbation and molecular context of L-asparaginase-treated ALL cells has not been fully elucidated. Here we identify that treatment with L-asparaginase results in metabolic shutdown via the reduction of both glycolysis and oxidative phosphorylation, accompanied by mitochondrial damage and activation of autophagy. The autophagy is involved in reducing reactive oxygen species (ROS) level by eliminating injured mitochondria. Inhibition of autophagy enhances L-asparaginase-induced cytotoxicity and overcomes the acquired resistance to L-asparaginase in ALL cells. The ROS-p53-positive feedback loop is an essential mechanism of this synergistic cytotoxicity. Thus, our findings provide the rationale for the future development of combined treatment of L-asparaginase and anti-autophagy drug in ALL patients.

AbstractAdipocyte-derived leucine aminopeptidase (A-LAP) is a novel zinc-metallopeptidase involved in angiotensin II (AngII) metabolism, cell migration and antigen presentation. These functions are implicated in the progression of cancer, whereas A-LAP expression and involvement have not been studied in any type of cancer. We investigated the expression of A-LAP in endometrial cancer as well as its association with angiogenesis and clinicopathological features. Immunohistochemical staining of 58 endometrial endometrioid adenocarcinoma specimens revealed that 37 were A-LAP immunoreactive. We also found that A-LAP staining correlated with histological tumor grade in a significant and reverse manner. In addition, serum CA-125 levels in patients with A-LAP positive cancers were significantly higher. However, contrary to our hypothesis that A-LAP suppresses angiogenic activity via AngII metabolism, A-LAP expression was not associated with the microvessel count determined by CD34 immunostaining. Our results suggest that A-LAP is involved in endometrial cancer cell growth and differentiation. However, further studies, especially of the biological roles of A-LAP, are required to confirm this notion.Copyright © 2003 S. Karger AG, Basel

Klotho mutant (kl/kl) mice exhibit growth retardation after weaning, and previous electron microscopic examination of GH-producing cells in pituitary glands revealed a reduction in GH granules. However, it has not been known whether growth retardation in klotho mutant mice is related to the loss of GH function. We therefore examined whether treatment with GH could rescue the retardation of growth. At the end of 3 weeks of treatment with human GH, the body weight of wild-type (WT) mice was increased. In contrast, body weight was not increased in klotho mutant mice even after the treatment with human GH. Another feature of klotho mutant mice is the presence of osteopetrosis in the epiphyses of long bones and vertebrae. Treatment with human GH increased trabecular bone volume in the epiphyseal region of WT tibiae. Interestingly, increase in trabecular bone volume by GH treatment was also observed in klotho mutant mice and, therefore, the phenotype of high bone volume in the klotho mice was further enhanced. These findings indicate that a GH receptor system in cancellous bones could operate in mutant mice. Thus, growth retardation in the klotho mutant mice is resistant against GH treatment even when these mice respond to GH treatment in terms of cancellous bone volume.

Placental leucine aminopeptidase (P-LAP)/oxytocinase (OTase) degrades several small peptides such as oxytocin (OT), arginine vasopressin (AVP) and angiotensin III (ANGIII), and aminopeptidase A (AP-A) converts angiotensin II (ANGII) to ANGIII. These proteases play an important role in foetal growth and the maintenance of human homeostasis during pregnancy. In this study, we confirmed the distribution of P-LAP and AP-A proteins and messenger RNAs in human trophoblasts in normal placenta and complete hydatidiform mole by immunohistochemical and in-situ hybridization techniques. Immunoreactivity of P-LAP was mainly noted in the apical membrane of syncytiotrophoblasts, and the expression of messenger RNA (mRNA) for P-LAP was predominantly noted in the cytoplasm of syncytiotrophoblastic cells. However, immunoreactivity of AP-A was mainly noted in the apical membrane of cytotrophoblasts and in the basal zone of the syncytiotrophoblasts, and the expression of mRNA for AP-A was predominantly noted in cytoplasm of cytotrophoblastic cells and a little in cytoplasm of syncytiotrophoblastic cells. Thereby, the two proteases were differentially distributed both in normal placenta and hydatidiform mole throughout the gestational age. These results are useful for the further understanding of not only the pathophysiology of pregnancy, but also the pathogenesis of trophoblastic diseases.

Neutral endopeptidase 24.11 (NEP) is identical to CD10, which is a differentiation antigen for early B-lymphoid progenitors in the B-cell differentiation pathway. This ectoenzyme is known to have a key role in the control of growth, differentiation, and signal transduction of many cellular systems by regulating bioactive peptides and cytokines. Recently, we demonstrated that NEP/CD10 is upregulated during forskolin-induced choriocarcinoma cell differentiation, suggesting that NEP/CD10 is a trophoblast differentiation marker. The purpose of this study was to clarify the enhancement of NEP/CD10 expression and its signal transduction pathway during phorbol ester (PMA)-induced differentiation of BeWo choriocarcinoma cells.

The presence of an extrahypothalamic gonadotropin releasing hormone (GnRH) in human placenta is well known and this decapeptide is presumed to play an important role in the regulation of the function and growth of human placenta. Immunohistochemistry showed that neutral endopeptidase 24.11 (NEP), a candidate of the responsible enzyme of GnRH degradation, is highly expressed on the cell surface of trophoblasts. Hydrolysis of GnRH by human villi was studied by measuring liberated amino acids using high performance liquid chromatography. The GnRH degrading activity was 1.53 times higher after incubation with the membrane fraction of first trimester villi than that after incubation with the membrane fraction of term villi. Phosphoramidon, a potent inhibitor of NEP, reduced the liberated amino acids to about a half, suggesting that NEP is a responsible enzyme for GnRH degradation. Ubenimex, which can inhibit several aminopeptidases, also reduced the liberated amino acids to about 50 per cent. O-phenanthroline, EDTA, and thiorphan could inhibit GnRH degradation but inhibitors of post proline endopeptidase could not. Furthermore, GnRH degrading activity of the membrane fraction was reduced remarkably after the membrane fraction was immunotitrated by anti NEP and anti placental leucine aminopeptidase (P-LAP) IgG. In conclusion, NEP and P-LAP are responsible enzymes for GnRH degradation in human villi.

Abstract: Background. A multicenter, phase I study of combination therapy with paclitaxel and carboplatin for epithelial ovarian cancer was conducted to determine the safety and recommended dosages for Japanese women. Methods. Paclitaxel was administered intravenously over a 3-h period, followed by carboplatin administered intravenously over a 1.5-h period. A modified continual reassessment method (mCRM) was used in two treatment arms to establish the maximum tolerated dose (MTD) and recommended doses of the combination. In group A, the dose of paclitaxel (175 mg/m2) was constant and the dose of carboplatin was increased from 4 to 7 in terms of the target area under the plasma concentration-versus-time curve (AUC). In group B, the dose of carboplatin was constant (AUC 6) and paclitaxel was administered at two dose levels (160 and 175 mg/m2). In both groups, the carboplatin dose was limited to a maximum of 800 mg/body for each administration. Results. Because the calculated probability of toxicity was greatest at a dose of paclitaxel 175 mg/m2 and carboplatin AUC 7, this dose was designated the MTD in group A. Based on this result, treatment in group B was initiated at doses of paclitaxel of 160 mg/m2 and carboplatin AUC 6. While the dose of paclitaxel was escalated to 175 mg/m2, the safety of the combination was confirmed. The most frequent adverse effect was neutropenia, which resolved promptly with the appropriate use of granulocyte-colony stimulating factor (G-CSF). No other severe hematologic or nonhematologic toxicities were observed. Conclusions. Our study demonstrated that the recommended dose for this combination regimen should be paclitaxel 175 mg/m2 plus carboplatin AUC 6 (maximum dose, 800 mg/body).

In human placenta aminopeptidase A (APA), a principal enzyme that converts angiotensin II to angiotensin III, seems to be involved in angiotensin II metabolism during pregnancy. In this study, we investigated the possible effects of progesterone and estrogen on APA mRNA and protein levels in choriocarcinoma cells as a model for placenta. By RNase protection assay, progesterone induced higher APA mRNA levels than estrogen at the same concentration. Progesterone exhibited dose-dependent stimulation of APA mRNA, 1.8-fold increase at 10−6mfor 24h treatment. Progesterone at 10−6mincreased APA mRNA levels within 12h and in time-dependent fashion up to 24h. Fluorescence-activated cell sorting analysis and measurements of APA activities revealed the induction of APA protein by progesterone. Expression of progesterone receptors (PR) and glucocorticoid receptors (GR) were determined in these cells by RT-PCR, which suggested that the progesterone's actions might be displayed through PR and/or GR. These findings may serve as a useful model to study the effects of progesterone on angiotensin II metabolism in placenta, although the physiological validity of these studies remains to be clarified.

Neutral endopeptidase (NEP)/CD10, a cell-surface peptidase degrading various bioactive peptides, is mainly present in syncytiotrophoblasts in the human placenta. However, the change in NEP expression upon trophoblast differentiation remains to be clarified. In the present study, we examined the expression of NEP in the differentiating trophoblast using the BeWo choriocarcinoma cell line as a model system. Under the normal culture conditions, NEP was very weakly expressed on most proliferating cytotrophoblastic BeWo cells, while a minority of the cell population (less than 5 per cent ), consisting of giant, multinucleated cells, clearly expressed NEP at the cell membrane. Treatment of BeWo cells with forskolin (FSK) for 48–72h resulted in an 11− to 44-fold increase in the level of hCG secretion and induced cell fusion leading to the formation of multinucleated syncytiotrophoblasts, indicating functional and morphological differentiation. Fluorescence-activated cell sorting (FACS) analysis revealed that treatment with FSK significantly increased the cell-surface protein expression of NEP on differentiating BeWo cells. Consistently, there was a significant increase in the NEP enzymatic activity after FSK treatment. The level of hCG secretion from the FSK-treated cells was further enhanced when the cells were treated in the presence of the NEP inhibitor phosphoramidon. Immunohistochemical analysis of normal chorionic villi and choriocarcinoma tissues revealed the localization of NEP in syncytiotrophoblastic cells, as opposed to weak or negative staining in cytotrophoblastic cells. These data demonstrate that induction of choriocarcinoma cell differentiation is associated with an increase of NEP/CD10 expression at the cell surface, suggesting a role of this enzyme in regulating differentiated trophoblast functions such as hCG secretion. NEP/CD10 may also be a new cellular differentiation marker of both the normal and neoplastic trophoblast.

Objective:To test the applicability of maternal serum placental leucine aminopeptidase (P-LAP) as a parameter for predicting preterm delivery. Patients and Methods:Maternal serum P-LAP activity of 61 consecutive women with preterm labor at admission were assayed and examined to assess the relationship of levels to the incidence of preterm and term deliveries. Results: When P-LAP activity of study patient serum was at or below the 10th percentile, we found a greater ratio of preterm delivery ( P=0.0085) and an approximately 2.3-fold increase risk. The sensitivity, specificity, positive predictive value and negative predictive value were 29, 97, 89 and 62%, respectively. Conclusion:Maternal serum P-LAP activity decreases in cases with spontaneous preterm delivery and may be a clinically potential marker for some cases of preterm labor.

We induced resistance to systemic Candida albicans infection through CD4(+)-cell-mediated immunity in mice by immunization with subcutaneous injections of live C. albicans cells emulsified in incomplete Freund adjuvant. Using the resistant mice, we tested subcellular fractions of C. albicans cells for antigenicity. The fractions were derived from digested surface cell walls, insoluble membranes, or soluble and insoluble cytoplasmic materials, which were prepared by treatment with cell wall-digesting enzymes followed by lysis of the consequent protoplasts. Interestingly, the live-cell-immunized mice showed strong cell-mediated immune responses to the membrane fraction (C. albicans membrane antigen [CMA]). In addition, immunization with CMA induced resistance to systemic candidiasis, which disappeared upon administration of anti-CD4 monoclonal antibody. Infusion of splenocytes from the CMA-immunized mice conferred resistance on SCID mice, whereas infusion of CD4(+)-T-cell-depleted splenocytes was unable to induce resistance, indicating the importance of CD4(+) lymphocytes for resistance. These results suggest a potential for the membrane fraction to act as an antigen conferring resistance to systemic candidiasis in place of live cells and also as a source for the isolation of a new antigen.

cDNA cloning of placental leucine aminopeptidase (P-LAP)/cystinyl aminopeptidase (CAP)/oxytocinase demonstrated that this enzyme is a type II integral membrane protein, which means that native P-LAP, found in placenta, is membrane-bound and that the soluble form of this enzyme, found in maternal sera, is most likely derived from the native form. The presence of the different forms of the protein makes it difficult to purify homogeneously. In the current study we prepared antibody specific to native P-LAP and used it to purify native P-LAP from microsomal fractions of human placenta to homogeneity, 5039-fold within 4h, by immunoaffinity chromatography. Zn2+and Cu2+strongly inhibited the enzyme but Ca2+did not. Amastatin was a more potent inhibitor than bestatin and leupeptin. Using antibodies against native P-LAP, protein having 83 per cent of l-methionine insensitive Leu-p-nitroanilide cleaving activity, was immunoprecipitated from the microsomal fraction of human placenta.

Aminopeptidase A (AP-A EC 3.4.11.7), which is a membrane-bound zinc metalloprotease, is present in the placenta. AP-A selectively hydrolyzes N-terminal glutamyl and aspartyl residues and cleaves angiotensin II to form angiotensin III. To determine the role of placental aminopeptidase A under physiological and pathological conditions, we evaluated its immunolocalization and enzymatic activities in the placenta. AP-A was localized in the basal zone of the syncytiotrophoblast, in the membranes of the cytotrophoblast, and in fetal arterioles and venules within the stem villi. AP-A activity in the microsomal fraction of placental villi seemed to be remained essentially constant throughout gestation. The renin–angiotensin system is considered to be accelerated in pre-eclampsia. This AP-A activity was higher in pre-eclampsia (2.86±0.30nmol β NA/mg protein/h) than in uncomplicated pregnancy from 28 to 41 weeks of gestation (2.08±0.18nmol β NA/mg protein/h). Angiotensin II evoked AP-A activity in first trimester trophoblast, and Losartan and PD 123177 in combination significantly inhibited this induction of AP-A activity. The results of immunohistochemical evaluation and enzymatic activity suggested that placental aminopeptidase A may play a role as a component of the barrier of angiotensin II between mother and fetus.

Human tubal epithelial cells in primary culture were transfected with simian virus 40 (SV40) large T antigen plasmid, and an immortalized ciliated cell line, named as NT/T-S, was established without crisis. Transmission electron microscopy proved that NT/T-S cells had cilia, microvilli, junctional complexes, rough endoplasmic reticula, free ribosomes and microtubules. NT/T-S cells were evaluated preliminarily on the basis of co-culture study using surplus embryos at the 4- to 8-cell stage in our IVF and embryo transfer programme. All of the 133 embryos had >/=10% fragments (based on the surface area) and were unworthy of cryopreservation. Up to 57% (16/28) of the embryos with 10-30% fragments reached the blastocyst stage by co-culture. In contrast, blastocyst formation was observed in <10% of the control embryos, some of which were co-cultured with NFL/T cells (the immortalized human fetal liver epithelial cells) (1/16), and the others were incubated with the co-culture medium alone (1/18). Various cytokines/growth factors such as leukaemia inhibitory factor (LIF), interleukin (IL)-6, IL-8 and basic fibroblast growth factor were secreted by NT/T-S cells as well as by the tubal epithelial cells in primary culture. The establishment of a ciliated cell line will provide a valuable resource for the further studies of the Fallopian tube in the early events of pregnancy.

Although it is well established that Ras requires membrane localization for activation of its target molecule, Raf-1, the reason for this requirement is not fully understood. In this study, we found that modified Ras, which is purified from Sf9 cells, could activate Raf-1 in a cell-free system, when incorporated into liposome. Using a bifunctional cross-linker and a protein-fragmentation complementation assay, we detected dimer formation of Ras in the liposome and in the intact cells, respectively. These results suggest that dimerization of Ras in the lipid membrane is essential for activation of Raf-1. To support this, we found that, when fused to glutathione S-transferase (GST), unprocessed Ras expressed in Escherichia coli could bypass the requirement for liposome. A Ras-dependent Raf-1 activator, which we previously reported (Mizutani, S., Koide, H., and Kaziro, Y. (1998) Oncogene 16, 2781-2786), was still required for Raf-1 activation by GST-Ras. Furthermore, an enforced dimerization of unmodified oncogenic Ras mutant in human embryonic kidney (HEK) 293 cells, using a portion of gyrase B or estrogen receptor, also resulted in activation of Raf-1. From these results, we conclude that membrane localization allows Ras to form a dimer, which is essential, although not sufficient, for Raf-1 activation.

Aminopeptidase A (AP-A), a cell-surface metallopeptidase hydrolyzing peptide with N-terminal acidic residues, has been proved to be identical to the B cell differentiation antigen BP-1 and to the kidney differentiation antigen gp160, suggesting recognition of AP-A as a differentiation-related marker on certain normal and transformed cells. AP-A has also been purified from human placenta and been shown to be localized in the trophoblasts. In the present study, we examined the expression and enzymatic activity of AP-A in human gestational choriocarcinoma, a neoplastic transformant from trophoblasts which comprises a heterogenous population of trophoblastic cells in different stages of differentiation. Flow cytometry and immunoblot analysis demonstrated that AP-A was expressed in five choriocarcinoma cell lines which were secreting low or moderate levels of human chorionic gonadotropin (hCG), while two high hCG-secreting cell lines lacked AP-A expression. The AP-A enzymatic activity correlated with cell-surface levels of AP-A and was abrogated by amastatin, an inhibitor of AP-A. Immunohistochemical analysis revealed that AP-A was present in seven of eight choriocarcinoma tissues and was localized on the cell membrane of cytotrophoblastic choriocarcinoma cells, but not on cells with syncytiotrophoblast-like features. These results demonstrate that AP-A is expressed on most choriocarcinomas and its expression is restricted to low hCG-secreting, cytotrophoblastic cells and down-regulated as a function of cell differentiation, suggesting an involvement of AP-A in the differentiation/maturation process of neoplastic trophoblasts.

We have accomplished the total synthesis of (±)-culmorin from the readily available ketone (11 steps, 46% overall yield). The tricyclo[6.3.0.0^3,9]undecan-10-one skeleton, the culmorin framework, was constructed via the intramolecular double Michael addition of the cyclopentenone having an α,β-unsaturated ester moiety by using LHMDS. The stereochemistry of newly generated four stereogenic centers was perfectly controlled by the reaction.

The DNA rearrangements leading to the assembly of genes coding for the immunoglobulin heavy chain (IgH) in B cells and the T cell receptor for antigen in T cells are not completely lineage specific. This probably reflects the use of a common recombinase by IgH and the T cell receptor. This paper describes novel observations on the nature of these cross‐lineage rearrangements. A high proportion (though not all) IgH rearrangements in human T leukaemic cells involve the D segment nearest to the J region (DQ52). This same D segment is not involved in B cell IgH rearrangements with one important exception, namely a proportion of B cell leukaemic clones with the most primitive B cell precursor phenotype. These observations have potentially important implications for early lymphoid cell differentiation and in particular support the idea that the 3′ D plus J region might lie within a limited window of accessibility of the IgH gene in precursor lymphocytes.

In a chromosome study in childhood T-cell leukemia/lymphoma, we found t(7;11)(q35;p13) in 2 patients, t(7;14) (q35;q11) in one patient, and t(7;14)(p15;q32) in 1 patient. Southern blotting and in situ chromosomal hybridization studies in one patient with the t(7;11) demonstrated that both alleles of the T-cell antigen receptor beta- subunit gene (TCRB) were rearranged, and that one TCRB allele had relocated from 7q35 to the fusion point in band p13 of the involved chromosome 11 (11p-). These findings suggest that juxtaposition of TCRB with the putative oncogene tcl-2 located in band 11p13 may be a critical step toward development of this T-cell leukemia/lymphoma. In the other two translocations, all breakpoints were sites for lymphocyte function genes, ie, 7q35 for TCRB, 14q11 for T-cell antigen receptor alpha-subunit gene (TCRA), 7p15 for T-cell antigen receptor alpha- subunit gene (TCRG), and 14q32 for immunoglobulin heavy-chain gene (IGH). Thus, the findings in these cases allow us to expand the above hypothesis and propose that the juxtaposition of TCRB or TCRG with tcl- 2, TCRA, or IGH through chromosomal translocation may activate a mechanism for the genesis of T-cell leukemia/lymphoma with these chromosome translocations.

An extinction-angle distribution was computed for randomly oriented grains of plagioclase feldspar with a known An-composition. Expressed by a histogram with eighteen columns, the results are shown by an 18×19 data array for nineteen types of low-temperature plagioclase. In a mixture of grains with various An-compositions, the extinction-angle distribution is given by a linear combination of the obtained data matrix with the frequencies of An-composition. If we have a sample containing randomly oriented grains of plagioclase feldspars, a frequency distribution of those with An-composition can be calculated after measuring their extinction angles. By taking into account the constraints of inequalities, the calculation is treated with the dual method of linear programming. The procedure provides for a simpler and more expedient method for determining the frequency distribution of An-composition. This method was tested by applying it to a model sample containing randomly oriented plagioclase grains with known An-composition frequencies, whose extinction-angle distribution was determined by the Monte Carlo method. An example of application of this method to Permian sandstones from central Japan is presented.

SDS-polyacrylamide gel electrophoresis followed by enzymography of a human testicular cell culture medium demonstrated that the molecular weight of plasminogen activator (PA) secreted was similar to that of human urokinase and it was also confirmed that follicle-stimulating hormone (FSH) stimulated PA secretion. To further investigate the correlation between spermatogenic impairment and secretory potential of PA in human testicular cells in vivo, the maximum increment in PA activity as a result of treatment with ovine FSH was measured for two groups of patients with different degrees of spermatogenic impairment. The mean value of maximum increment in PA activity in one group with severely impaired testes was remarkably lower than that in another group with slightly impaired testes. Protein biosynthesis in human testicular cells dependent on FSH appears to be impaired with the progression of spermatogenic impairment.

Juvenile chronic myelogenous leukemia (JCML), a myeloproliferative disorder of childhood, is distinct from adult-type chronic myelogenous leukemia (CML) and bears resemblance to chronic myelomonocytic leukemia (CMMoL). Since mutations in the N-ras gene have been found at high frequencies in CMMoL, but only rarely in CML, we analyzed mutations activating the N-ras gene in 20 patients with JCML. We used the strategy for analysis of gene mutations based on in vitro DNA amplification by polymerase chain reaction (PCR) followed by single- strand conformation polymorphism (SSCP) analysis and/or direct sequence analysis. Nucleotide sequence analysis showed single nucleotide substitutions involving codons 12, 13, or 61 in six of 20 patients (30%). Four of six patients with mutations were in chronic phase and the other two in blast crisis, indicating no apparent correlation with disease stage. Most of the patients with mutations were in the older age group with poor prognosis, although one patient in the younger age group also harbored the mutation. These data suggest that N-ras gene mutations may be involved in the pathogenesis and/or prognosis of JCML and provide further evidence that JCML is an entity distinct from CML.

Abstract This study evaluates the safety and role of laparoscopy in the diagnosis of blunt abdominal trauma in children. Laparoscopy was performed in five patients aged 3 to 13 years because of persistent abdominal pain after blunt trauma. A laparotomy was not indicated from the physical examination, laboratory data, or radiologic findings. With the patient under general anesthesia, a 10-mm trocar was inserted through the umbilical fossa and the intra-abdominal organs were observed for 10–60 min under an insufflation pressure of 10–12 mmHg. The patients remained hemodynamically stable without pneumothorax development. Three patients underwent laparatomies: one, who had blood in the omental sac, had a duodenal injury with hemorrhagic necrosis and underwent a resection; one with ascites and high amylase levels had an injury of the main pancreatic duct and underwent resection of the pancreatic tail; and one who had fresh blood in the upper abdomen and Douglas' pouch had a splenic hemorrhage and underwent hemostasis. The other two had serous or serosanguinous ascites and recovered without surgery. In patient 1, the same amount of information might have been obtained from a barium study. In patient 2, the pancreatic transection might have been diagnosed from ascites shown on serial computed tomograms. Patient 3 might also have been treated successfully non-surgically. It hus appears that laparoscopy may be a safe diagnostic method for blunt abdominal trauma in children, however, this small series has yielded insufficient information to assess its usefulness in making the diagnosis and the decision for laparotomy. Further studies are required to ascertain whether it will make any significant difference in the form of management.

Evidence is presented that indicates a deep crevice located on the surface of human rhinovirus type 14 is involved in virion attachment to cellular receptors. By using mutagenesis of an infectious cDNA clone, 11 mutants were created by single amino acid substitutions or insertions at positions 103, 155, 220, 223, and 273 of the structural protein VP1. Seven of the recovered mutants had a small plaque phenotype and exhibited binding affinities significantly lower than wild-type virus. One mutant, in which glycine replaced proline at amino acid position 155, showed a greatly enhanced binding affinity. Single-cycle growth kinetics suggested that 5 of the mutants had delayed growth cycles due to intracellular deficiencies apart from receptor binding.

Objective:Intrauterine growth retardation (IUGR) is likely to recur ina subsequent pregnancy. We investigated the obstetric features of recurrent cases and the severity of IUGR by comparing initial and subsequent deliveries. Methods:From a total of 12 567 deliveries, 95 women who were delivered of small-for-gestational-age (SGA) infants and who became pregnant again within 5 years, were enrolled. A retrospective, comparative study of recurrent and non-recurrent groups was performed. Results:Twenty-two of ninety-five women gave birth to SGA infants again, and a relatively high risk of recurrence was confirmed, but no single recurrence-associated features were revealed. Within the recurrent group, the degree of IUGR was more severe in only five cases in the subsequent pregnancy. Conclusions:IUGR tends to recur, but does not increase in severity in most cases. We conclude that there is no need for excessive concern about the recurrence of IUGR.

The functional significance of the conserved amino acids within transmembrane regions II and VII of the human 5-hydroxytryptamine (5-HT)1A receptor was analyzed by oligonucleotide-directed mutagenesis followed by transient expression of the mutated receptor genes in COS-1 cells. The substitution of a conserved asparagine at position 396 (transmembrane region VII) with either alanine, phenylalanine, or valine resulted in a receptor that did not bind the 5-HT1A agonist 8-hydroxy-2-(di-n-[3H]propylamino)tetralin. In contrast, replacement of Asn396 with glutamine did not affect agonist binding. In addition, serine residues at positions 391 and 393 (transmembrane domain VII) were changed to alanine. Changing the less conserved Ser391 to alanine had no effect on ligand binding. However, replacement of the conserved Ser393 with alanine reduced ligand binding by 86%. Replacement of a conserved aspartate at position 82 (transmembrane region II) with alanine also produced a receptor without detectable agonist binding. Protein immunoblotting detected receptor protein of approximately 51 kDa in both wild-type and mutant receptor-expressing cells, indicating that these mutations probably did not affect expression or processing of the protein. Importantly, the sequence of the human 5-HT1A receptor described in this paper differs from the published sequence [Nature (Lond.) 329:75-79 (1987)] in transmembrane region IV. The present sequence encodes a protein of 422 amino acids, instead of the 421-amino acid protein that has been described previously [Nature (Lond.) 329:75-79 (1987)], and has a change in the sequence in transmembrane region IV from ... RPRAL ... to ... RRAAA ..., which corresponds to the published sequence [J. Biol. Chem. 265:5825-5832 (1990)] of the rat 5-HT1A receptor. Moreover, conversion of the transmembrane region IV sequence of the present clone to that of the published sequence by site-directed mutagenesis abolished ligand binding to the receptor.

As a major cause of acute and chronic liver disease as well as hepatocellular carcinoma, hepatitis B virus (HBV) continues to pose significant health problems world-wide. Recombinant hepatitis B vaccines based on adenovirus vectors have been developed to address global needs for effective control of hepatitis B infection. Although considerable progress has been made in the construction of recombinant adenoviruses that express large amounts of HBV gene products, preclinical immunogenicity and efficacy testing of candidate vaccines has remained difficult due to the lack of a suitable animal model. We demonstrate here that chimpanzees are susceptible to enteric infection by human adenoviruses type 7 (Ad7) and type 4 (Ad4) following oral administration of live virus. Moreover, after sequential oral immunization with Ad7- and Ad4-vectored vaccines containing the hepatitis B surface antigen (HBsAg) gene, significant antibody responses to HBsAg (anti-HBs) were induced in two chimpanzees. After challenge with heterologous HBV, one chimpanzee was protected from acute hepatitis and the other chimpanzee experienced modified HBV-induced disease. These data demonstrate the feasibility of using orally administered recombinant adenoviruses as a general approach to vaccination.

Summary A study was undertaken on serial measurements of plasma angiotensin I (A-I) and serum placental leucine aminopeptidase (P-LAP) activities in normal and pre-eclamptic pregnancy. There was the significant difference in A-I levels between normal and mild pre-eclamptic pregnancy at weeks 30, 35, and 37, between normal and severe pre-eclamptic pregnancy at week 37. There were no differences in serum P-LAP between normal and mild pre-eclamptic pregnancy up to week 33, but thereafter the levels for the mild pre-eclampsia were significantly higher than for the normal pregnancy. The P-LAP activity for the severe pre-eclampsia reached its maximum level at week 31. Around this week, the levels, for severe pre-eclampsia were significantly higher than in the normal pregnancy. After week 35, the activities decreased precipitously to week 40; the activities for severe pre-eclampsia in late pregnancy at weeks 39 and 40 were significantly lower than in normal pregnancy. The above data support the idea that P-LAP test is useful for prediction or diagnosis of pre-eclampsia.

Abstract.: To evaluate the efficiency of the Doppler examination of umbilical arterial blood flow for the antenatal diagnosis and the monitoring of fetal condition during intrauterine treatment of twin-to-twin transfusion syndrome (TTTS), we studied 33 pairs of twins including 5 TTTS cases. In all cases umbilical arterial blood flow was examined by Doppler ultrasound and pulsatility index (PI) was calculated as umbilical arterial impedance. In twins with TTTS, PI of the recipient was outside the normal range and the difference of PI was greater than +0.5. In discordant twins without TTTS and concordant twins, the PI was within the normal range and the difference of PI ranged from −0.5 to +0.5. In 2 cases these findings were found before the appearance of fetal hydrops. In 2 TTTS cases transmaternal digitalization prevented the development of hydrops in the recipient. The difference of PI decreased with improvement in the fetal condition, and vice versa. Our data suggested that, in cases with TTTS, Doppler examination of umbilical arterial blood flow was effective in predicting fetal hydrops. Doppler was also very useful for monitoring the fetal condition during intrauterine treatment.

Background: The direct contribution of bile to gastric inhibitory polypeptide (GIP) release and the role of bile in regulating GIP secretion in response to fat ingestion are still obscure. The present study was aimed to clarify the influence of bile on GIP release. Methods: Seven patients with obstruction of the common bile duct and nine volunteers participated in the study. Fifty milliliters of Lipomul® was ingested, and GIP was measured serially for 180 min. After intraduodenal instillation of pooled autologous bile for 2 days, the same study was carried out. Results: The fat-stimulated GIP response was significantly lower in the patients than in the controls. The basal GIP level did not change on bile instillation, but the GIP response to fat ingestion was significantly increased on bile instillation compared with that in the absence of bile. Conclusions: Intraluminal bile alone does not stimulate the secretion of GIP, but it promotes GIP secretion in response to fat ingestion.

Long-term observations were made of the prognosis for sexual maturation (onset of puberty) in 15 prepubertal boys with micropenis. Testosterone levels before and after HCG administration and LH and FSH responses to LH-RH were measured at the first visit and a few years later. In the eight boys who showed significant responses to the LH-RH and HCG tests, onset of puberty occurred with the exception of one patient. The remaining seven boys who showed no positive response to either stimulation test were treated with injections of HCG. However, no signs of sexual maturation were observed. These data demonstrate clearly that both the HCG test and the LH-RH test are very useful for prognosis of sexual maturation in patients with micropenis.

A simple human chorionic gonadotropin (hCG) test to measure Leydig cell function is described. Plasma testosterone was measured on two occasions, once before intramuscular injection of 10,000 IU of hCG and again four days later. Preliminary tests in 10 adult males showed a maximal increase in plasma testosterone between the third and fifth day that was confirmed in a further 20 normal subjects when measured once on the fourth day. Except for 2 patients with hypospermatogenesis, 24 showed a good response and the mean increase was comparable with that in normal males. Nineteen patients with Klinefelter's syndrome usually had lower basal levels and showed a poor response, with the exception of six patients in whom there were moderate but definite increases. Twelve patients with hypogonadotropic hypogonadism had basal testosterone concentrations below 1 ng/ml and eight responded poorly to hCG stimulation. However, four patients showed a moderate but definite response.