Fetal Exposure to Tobacco Metabolites and Depression During Adulthood: Beyond Binary Measures.

BACKGROUND: Sibling studies of maternal smoking during pregnancy and subsequent risk of depression have produced mixed results. A recent study identified not considering amount of maternal smoking and age of onset as potentially masking a true association. We examine these issues and also the relevance of maternal smoking during pregnancy as a determinant of severity of depressive symptoms. METHODS: We analyzed data from the community-based National Longitudinal Survey of Youth (U.S., 1994-2016). Mothers reported smoking during pregnancy (none, <1 pack/day, ≥1 pack/day). We assessed offspring's lifetime depression (i.e., ≥8 symptoms) and symptom counts with the Centers for Epidemiologic Studies Depression scale. We estimated the risk of these two outcomes in the full sample (n=7172) and among siblings (n=6145) using generalized linear mixed-effects models with random intercepts by family and family-averaged means for sibling analyses. RESULTS: Among siblings, we observed dose-dependent elevations for both risk of depression (smoking during pregnancy <1 pack/day aRR=1.18; 95% CI 1.07, 1.30; smoking ≥1 aRR=1.36; 95% CI 1.19, 1.56) and severity of depressive symptoms (smoking<1 pack/day aRR=1.12 (95% CI 1.08, 1.16); smoking ≥1 pack/day aRR=1.25 (95% CI 1.18, 1.31). Among both samples, the P for trend was <0.01. In analysis limited to offspring diagnosed prior to age 18, results for severity were attenuated. CONCLUSIONS: This evidence supports the existence of an independent association between maternal smoking during pregnancy and both risk of depression and severity of depressive symptoms. The results highlight the utility of considering the amount of smoking, severity of symptoms, and age of onset.

Viral load and ganciclovir (GCV) concentration in cerebrospinal fluid of patients successfully treated with GCV or valGCV for human herpesvirus 6 encephalitis/myelitis following umbilical cord blood transplantation.

We describe successful treatment of 3 cases of human herpesvirus 6 (HHV-6) encephalitis/myelitis following cord blood transplantation (CBT). Ganciclovir (GCV) (10 mg/kg/day) reduced HHV-6 load to undetectable levels in cerebrospinal fluid (CSF). Early dose reduction in the presence of HHV-6 detectable in CSF resulted in an increased HHV-6 load. GCV was capably shifted to valganciclovir (VGCV) with an almost equivalent concentration. GCV/VGCV may be effective for HHV-6 encephalitis/myelitis after CBT, although HHV-6 load in CSF should be monitored.

Safety, tolerability, and feasibility of antifungal prophylaxis with micafungin at 2 mg/kg daily in pediatric patients undergoing allogeneic hematopoietic stem cell transplantation.

INTRODUCTION: Micafungin (MCFG) is used for the prophylaxis of invasive fungal disease (IFD) after allogeneic hematopoietic stem cell transplantation (HSCT). However, the safety, efficacy, or optimal dosage/blood levels as prophylaxis is uncertain in pediatric HSCT-patients. METHODS: We prophylactically administered MCFG at 2 mg/kg once daily to 38 children and adolescents undergoing allogeneic HSCT. RESULTS: During MCFG prophylaxis, infusion reactions or adverse events (grades 2-5) related to MCFG use were not found in all the patients. Thus, MCFG prophylaxis was not discontinued and other antifungal agents were not added except for 2 patients in whom probable or possible IFDs developed (completion rate, 94.7 %). To elucidate the influence of HSCT-related complications/drugs on blood concentration of MCFG, we determined the plasma trough and peak levels in 13 and 10 among 38 patients, respectively. The mean trough and peak levels were 3.04 ± 1.21 µg/mL (569 samples) and 9.63 ± 3.62 µg/mL (44 samples), respectively. The peak levels were moderately correlated to the trough levels (R (2) = 0.466). In a patient, the trough level of MCFG transiently increased up to 10.21 µg/mL during hepatic dysfunction due to acute graft-versus-host disease. The MCFG trough levels strongly correlated with T-Bil value (R (2) = 0.894). There was no relationship between the trough levels of MCFG and the circulating concentrations of tacrolimus (R (2) = 0.040). Additionally, MCFG levels were not influenced by treatment with cyclophosphamide or corticosteroids. CONCLUSIONS: Prophylaxis with MCFG at 2 mg/kg once daily may be safe, tolerable, and feasible in pediatric HSCT-patients.

Association between low MCV in early pregnancy and perinatal mental health in the Japan Environment and Children's Study and the possible effect of iron deficiency.

BACKGROUND: Postpartum anemia and iron deficiency are associated with postpartum depression. This study investigated the association between a low mean corpuscular volume (MCV) without anemia (which implies early-stage iron deficiency) in early pregnancy and perinatal mental health outcomes. METHODS: The fixed data from the Japan Environment and Children's Study (JECS), a Japanese nationwide birth cohort, were used. Perinatal mental health was assessed using the Kessler 6-item psychological distress scale (K6) in mid-pregnancy and the Edinburgh Postnatal Depression Scale (EPDS) at 1- and 6-months postpartum. RESULTS: Among the 3635 women with MCVs <85 fL in early pregnancy, the proportions of women with K6 scores ≥13 in mid-pregnancy and EPDS scores ≥9 at 1- and 6-months postpartum were 2.7 %, 12.8 %, and 9.9 %, respectively, compared with the 33,242 women with MCVs ≥85 fL at 1.9 %, 11.9 %, and 9.0 %, respectively. Multivariate logistic regression models showed that an MCV <85 in early pregnancy was associated with a K6 score ≥ 13 in mid-pregnancy and an EPDS score ≥ 9 at 1- and 6-months postpartum (adjusted odds ratio (95 % confidence interval): 1.48 (1.16-1.87), 1.14 (1.01-1.28), and 1.09 (0.95-1.24), respectively). LIMITATIONS: Low MCV values do not necessarily represent iron deficiency. Ferritin, currently the best indicator of iron deficiency, was not measured in the JECS. CONCLUSIONS: This study results suggest that a low MCV without anemia in early pregnancy is associated with a slightly increased risk of perinatal mental health deterioration.

Successful ganciclovir therapy in a patient with human herpesvirus-6 encephalitis after unrelated cord blood transplantation: usefulness of longitudinal measurements of viral load in cerebrospinal fluid.

BACKGROUND: There have been no reports of human herpesvirus-6 (HHV-6) encephalitis treatment based on both HHV-6 DNA load and the antiviral agent's concentration in the cerebrospinal fluid (CSF). PATIENT: A 20-year-old male with a hematological malignancy developed HHV-6 encephalitis 15 days after unrelated cord blood transplantation (UCBT). He had fever, chest pain, memory impairment, and insomnia. His CSF showed no increased cell counts, but the amount of HHV-6 DNA was elevated to 2.0 × 10(6) copies/ìgDNA. Magnetic resonance imaging (MRI) of the head revealed abnormal high-intensity signals in the left limbic system on T2-weighted and diffusion-weighted images. Intravenous administration of ganciclovir (GCV) was initiated at 5 mg/kg every 12 h on day 18, and was continued until day 137. The amount of HHV-6 DNA in the plasma became undetectable on day 25. The HHV-6 load in the CSF decreased to 1.5 × 10(3) copies/ìgDNA on day 32, and reached undetectable levels on day 53. The mean concentration of GCV 1 h after an infusion of 5 mg/kg was 4.12 mg/mL in plasma and 0.7 mg/mL in CSF. The chest pain and insomnia disappeared on days 35 and 47, respectively. Memory defects recovered up to day 85. CONCLUSION: Serial quantification of HHV-6 DNA in CSF may be useful for successful treatment with GCV in post-transplant HHV-6 encephalitis.

Factors controlling the proliferative rate, final cell density, and life span of bovine vascular smooth muscle cells in culture.

Low density vascular smooth muscle (VSM) cell cultures maintained on extracellular-matrix(ECM)-coated dishes and plated in the presence of either plasma or serum will proliferate actively when serum-containing medium is replaced by a synthetic medium supplemented with three factors: high density lipoprotein (HDL, 250 micrograms protein/ml); insulin (2.5 micrograms/ml) or somatomedin C (10 ng/ml); and fibroblast growth factor (FGF, 100 ng/ml) or epidermal growth factor (EGF, 50 ng/ml). The omission of any of these three factors from the synthetic medium results in a lower growth rate of the cultures, as well as in a lower final cell density once cultures reach confluence. When cells are plated in the total absence of serum, transferrin (10 micrograms/ml) is also required to induce optimal cell growth. The effects of the substrate and medium supplements on the life span of VSM cultures have also been analyzed. Cultures maintained on plastic and exposed to medium supplemented with 5% bovine serum underwent 15 generations. However, when maintained on ECM-coated dishes the serum-fed cultures had a life span of at least 88 generations. Likewise, when cultures were maintained in a synthetic medium supplemented with HDL and either FGF or EGF, an effect on the tissue culture life span by the substrate was observed. Cultures maintained on plastic underwent 24 generations, whereas those maintained on ECM-coated dishes could be passaged repeatedly for 58 generations. These experiments demonstrate the influence of the ECM-substrate only in promoting cell growth but also in increasing the longevity of the cultures.

Impact of weight gain on the evolution and regression of prediabetes: a quantitative analysis.

BACKGROUND/OBJECTIVES: The quantitative impact of weight gain on prediabetic glucose dysregulation remains unknown; only one study quantitated the impact of weight loss. We quantified the impact of weight gain on the evolution and regression of prediabetes (PDM). SUBJECTS/METHODS: In 4234 subjects without diabetes, using logistic regression analysis with a 4.8-year follow-up period, we analyzed the relationship between (1) δBMI (BMIfollow-up-basal) and the progression from normal glucose regulation (NGR) to PDM or diabetes, and (2) δBMI and the regression from PDM to NGR. RESULTS: Mean (±s.d.) δBMI was 0.17 (±1.3) kg/m2 in subjects with NGR and δBMI was positively and independently related to progression (adjusted odds ratio (ORadj) (95% CI), 1.24 (1.15-1.34), P<0.01). Mean (±s.d.) δBMI was -0.03 (±1.25) kg/m2 in those with PDM and δBMI was negatively related to the regression (ORadj, 0.72 (0.65-0.80), P<0.01). The relation of δBMI to the progression was significant in men (ORadj, 1.42 (1.28-1.59), P<0.01) but not in women (ORadj, 1.05 (0.94-1.19), P=0.36). Also, the negative impact of δBMI on the regression was significant only in men (men, ORadj, 0.65 (0.57-0.75), P<0.01; women, ORadj, 0.94 (0.77-1.14), P=0.51). CONCLUSIONS: In Japanese adults, an increase in the BMI by even 1 kg/m2 was related to 24% increase in the risk of development of PDM or diabetes in NGR subjects and was related to 28% reduction in the regression from PDM to NGR. In women, we did not note any significant impact of weight gain on the evolution or regression of PDM.

Predictive models for conversion of prediabetes to diabetes.

AIM: To clarify the natural course of prediabetes and develop predictive models for conversion to diabetes. METHODS: A retrospective longitudinal study of 2105 adults with prediabetes was carried out with a mean observation period of 4.7years. Models were developed using multivariate logistic regression analysis and verified by 10-fold cross-validation. The relationship between [final BMI minus baseline BMI] (δBMI) and incident diabetes was analyzed post hoc by comparing the diabetes conversion rate for low (< -0.31kg/m2) and high δBMI (≥ -0.31kg/m2) subjects after matching the two groups for the covariates. RESULTS: Diabetes developed in 252 (2.5%/year), and positive family history, male sex, higher systolic blood pressure, plasma glucose (fasting and 1h- and 2h-values during 75g OGTT), hemoglobin A1c (HbA1c) and alanine aminotransferase were significant, independent predictors for the conversion. By using a risk score (RS) that took account of all these variables, incident diabetes was predicted with an area under the ROC curve (95% CI) of 0.80 (0.70-0.87) and a specificity of prediction of 61.8% at 80% sensitivity. On division of the participants into high- (n=248), intermediate- (n=336) and low-risk (n=1521) populations, the conversion rates were 40.1%, 18.5% and 5.9%, respectively. The conversion rate was lower in subjects with low than high δBMI (9.2% vs 14.4%, p=0.003). CONCLUSIONS: Prediabetes conversion to diabetes could be predicted with accuracy, and weight reduction during the observation was associated with lowered conversion rate.

Reassessment of the clinical significance of portal-superior mesenteric vein invasion in borderline resectable pancreatic cancer.

OBJECTIVE: The principal objective of this study is to clarify the prognostic significance of borderline resectable pancreatic cancer (BRPC). The second objective is to evaluate the prognostic impact of the depth of pathological venous invasion. METHODS: The study included 122 pancreatic cancer patients who underwent curative surgery. All computed tomography scans of the patients were retrospectively interpreted and classified according to the NCCN guidelines, version 1.2016, as resectable (-) or borderline resectable (+) in each arterial (BR-A) and venous (BR-PV) involvement. RESULTS: The overall survival (OS) rate was significantly higher in BR-A(-) patients (n = 94) than in BR-A(+) patients (n = 28) (P = 0.001), whereas there was no difference between BR-PV(-) (n = 101) and BR-PV(+) patients (n = 21) (P = 0.257). In a multivariate analysis, the independent predictors of OS included BR-A(+) (P = 0.002), lymph node metastasis (P = 0.008), pathological venous invasion (P = 0.003), and adjuvant chemotherapy (P = 0.001). Of 39 patients who underwent venous resection, no significant difference was observed between BR-PV(-) (n = 20) and BR-PV(+) patients (n = 19) in resection rate, lymph node metastasis, the presence of extrapancreatic nerve invasion, recurrence rate, frequency of initial recurrence at a liver or local site, and OS. Pathological venous invasion was significantly deeper in BR-PV(+) patients. However, the depth of invasion was not associated with OS. CONCLUSION: The definition of venous involvement in the current guidelines predicted the depth of pathological venous invasion but not OS in BRPC patients. Further prospective, randomized studies are needed to establish treatment strategies for BRPC patients with isolated venous involvement.

Inhibition of the tumor-promoting action of 12-O-tetradecanoylphorbol-13-acetate by some oleanane-type triterpenoid compounds.

Since glycyrrhetinic acid was proved to suppress tumor promoter effects, several oleanane-type triterpenes which were chemically derived from oleanolic acid and hederagenin were tested in vitro and in vivo against the action of tumor promoter, 12-O-tetradecanoylphorbol 13-acetate. By in vitro experiment monitoring with 12-O-tetradecanoylphorbol-13-acetate-induced stimulation of 32Pi incorporation into phospholipids and an in vivo test on skin tumor formation in mice initiated with 7,12-dimethylbenz[a]anthracene and promoted with 12-O-tetradecanoylphorbol-13-acetate, 18 beta-olean-12-ene-3 beta,28-diol (= erythrodiol), 18 beta-olean-12-ene-3 beta,23,28-triol, 18 alpha-olean-12-ene-3 beta,28-diol, and 18 alpha-olean-12-ene-3 beta,23,28-triol showed remarkable suppressive effects. Especially 18 alpha-oleanane derivatives having a CH2OH grouping converted from the COOH group initially allocated at C-17 were 100 times more effective than glycyrrhetinic acid both in vitro and in vivo.

Elevated levels of syndecan-1 expression confer potent serum-dependent growth in human 293T cells.

Syndecan-1 is the best studied integral membrane proteoglycan and functions to modulate epithelial cell attachment and physiology. Extracellularly, syndecan-1 binds both growth factors and extracellular matrix components, and intracellularly, its cytoplasmic portion interacts with cytoskeletal components. To investigate the possible role of syndecan-1 in epithelial cell transformation that is characterized by alteration in extracellular matrix interactions and cytoskeleton architecture, we established stable transfectants of syndecan-1 in a highly transformed human renal epithelial line expressing two viral oncogenes, adenovirus E1a and SV40 large T antigen (293T cell line). Expression of syndecan-1 core protein and appropriate posttranslational attachment of glycosaminoglycan chains was confirmed by enzymatic digestion and Western blot analysis. Overexpresser cells grew at a significantly faster rate than the vector-transfected control cells in serum-rich media but showed a proliferative disadvantage in serum-reduced media. In addition to this serum dependency, syndecan-1 overexpression caused a partial reversal of the transformed phenotype with the expressing clones becoming more anchorage dependent and less motile than the vector-transfected counterparts. Surprisingly, the overexpressers were more tumorigenic when injected s.c. into nude mice. These results indicate that syndecan-1 expression plays a role in the control of cell proliferation and suggest that serum-dependent growth may be the more reflective of tumorigenicity in nude mice.

Inhibition of cancer cell growth by polyinosinic-polycytidylic acid/cationic liposome complex: a new biological activity.

A complex of polyinosinic-polycytidylic acid [poly(I) x poly(C)] and cationic liposome (LIC) inhibited the growth of many tumor cell lines at low concentration in vitro, but poly(I) x poly(C) alone had no such antiproliferative effect. The IC50 values of LIC against the tumor cells ranged from 0.1 to 1000 ng/ml. LIC had strong cytotoxic effects on malignant cells of epithelial and fibroblastic origin from various tissues and was also effective against Adriamycin-resistant tumor cells. LIC did not significantly affect the growth of lymphoma cells, leukemia cells, normal diploid fibroblasts, or primary liver cells at concentrations up to 10 microg/ml. The mechanism of the antiproliferative effect of LIC against malignant cells was the induction of apoptosis. LIC induced the fragmentation of nuclear DNA and the degradation of rRNA in tumor cells. The DNA fragmentation occurred within 1-5 h after the addition of LIC, and both the fragmentation and the inhibition of cancer-cell growth were suppressed by a nuclease inhibitor. In contrast, caspase inhibitors did not affect the antiproliferative activity of LIC. These results suggest that LIC induced apoptosis in malignant cells through the direct activation of nucleases and not through the activation of caspases. LIC reduced the incidence and the size of metastatic liver-cancer tumors in two different mouse metastatic liver-cancer models using human colon carcinoma cells. Histochemical analysis revealed that the KM12-HX cells in the tumor nodules were undergoing apoptosis; therefore, LIC also induced the apoptosis of tumor cells in vivo. In these animal models, LIC caused no observed changes in normal hepatocytes.

In vitro expansion of CD34(+)CD38(-) cells under stimulation with hematopoietic growth factors on AGM-S3 cells in juvenile myelomonocytic leukemia.

Using serum-containing culture, we examined whether AGM-S3 stromal cells, alone or in combination with hematopoietic growth factor(s), stimulated the proliferation of CD34(+) cells from patients with juvenile myelomonocytic leukemia (JMML). AGM-S3 cells in concert with stem cell factor plus thrombopoietin increased the numbers of peripheral blood CD34(+) cells to approximately 20-fold of the input value after 2 weeks in nine JMML patients with either PTPN11 mutations or RAS mutations, who received allogeneic hematopoietic transplantation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) also augmented the proliferation of JMML CD34(+) cells on AGM-S3 cells. The expansion potential of CD34(+) cells was markedly low in four patients who achieved spontaneous hematological improvement. A large proportion of day-14-cultured CD34(+) cells were negative for CD38 and cryopreservable. Cultured JMML CD34(+)CD38(-) cells expressed CD117, CD116, c-mpl, CD123, CD90, but not CXCR4, and formed GM and erythroid colonies. Day-7-cultured CD34(+) cells from two of three JMML patients injected intrafemorally into immunodeficient mice stimulated with human GM-CSF after transplantation displayed significant hematopoietic reconstitution. The abilities of OP9 cells and MS-5 cells were one-third and one-tenth, respectively, of the value obtained with AGM-S3 cells. Our culture system may provide a useful tool for elucidating leukemogenesis and for therapeutic approaches in JMML.

Progesterone is a cell death suppressor that downregulates Fas expression in rat corpus luteum.

In female rats, apoptotic cell death in the corpus luteum is induced by the prolactin (PRL) surge occurring in the proestrous afternoon during the estrous cycle. We have previously shown that this luteolytic action of PRL is mediated by the Fas/Fas ligand (FasL) system. During pregnancy or pseudopregnancy, apoptosis does not occur in the corpus luteum. Progesterone (P4), a steroid hormone secreted from luteal steroidogenic cells, attenuated PRL-induced apoptosis in cultured luteal cells in a dose-dependent manner. P4 significantly decreased the expression of mRNA of Fas, but not FasL, in cultured luteal cells prepared from both proestrous and mid-pseudopregnant rats. These data indicate that P4 suppresses PRL-induced luteal cell apoptosis via reduction of the expression level of Fas mRNA in the corpus luteum, suggesting that P4 acts as an important factor that can change the sensitivity of corpus luteum to PRL.

Linkage of Miyoshi myopathy (distal autosomal recessive muscular dystrophy) locus to chromosome 2p12-14.

Miyoshi myopathy (MM) is a young-adult-onset, autosomal recessive distal muscular dystrophy initially affecting the plantar flexors. We analyzed 12 MM families, five with consanguineous marriage, for chromosomal linkage using polymorphic microsatellite DNA markers to map the MM gene. A significant lod score was obtained with the 2p12-14 locus D2S291 (Zmax = 15.3 at theta = 0). Two additional 2p12-14 markers, D2S286 (Z = 10.7 at theta = 0) and D2S292 (Z = 7.2 at theta = 0.05), also gave significant lod scores. These markers will be useful for diagnosis of symptomatic and presymptomatic patients, prenatal and carrier diagnosis of family members carrying MM, and ultimately identification of a gene responsible for MM.

DNA rearrangements in Japanese facioscapulohumeral muscular dystrophy patients: clinical correlations.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscular disorder in which the disease locus has been mapped to chromosome 4q35-qter. In most patients, the DNA rearrangements associated with FSHD have been found in the EcoRI fragment detected by the p13E-11 probe, and deletions of the 3.2 kb repeat units within the fragment are thought to cause the disease. To examine FSHD-associated DNA rearrangements in the Japanese population, we performed Southern blot analysis of the genomic DNA, using the p13E-11 and pFR-1 probes, in 158 Japanese individuals, including 38 FSHD patients from 19 families. We found that all but one (a possible affected recombinant) of the Japanese FSHD patients (97.4%) had specific smaller (< 28 kb) EcoRI fragments which cosegregated with the disease; this included four patients who had severe inflammatory changes in the muscle and eight patients with de novo DNA rearrangements. We found no FSHD patient who had a fragment larger than 28 kb. By contrast, only two of 35 Japanese controls (5.7%) had EcoRI fragments smaller than 28 kb. Our patients showed anticipation, i.e. decreased size of the EcoRI fragment in parallel with earlier onset of the disease (r = 0.531, P = 0.003, with younger age at onset in children (17.8 +/- 7.0) than their affected parents (31.5 +/- 14.8) (P = 0.019). However, since each family had a specific small EcoRI fragment associated with the disease, the differing clinical severity within a family cannot be explained by the size of the fragment alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of obesity in transgenic rats with low circulating growth hormone levels: involvement of leptin resistance.

BACKGROUND: Human growth hormone (hGH) transgenic (TG) rats have been produced in our laboratory. These TG rats are characterized by low circulating hGH levels, virtually no endogenous rGH secretion, and massive obesity. OBJECTIVE: To elucidate how energy balance and leptin sensitivity contributed to the establishment of this obesity. DESIGN AND METHODS: Food intake, locomotor activity and leptin concentrations in serum and cerebrospinal fluid were measured in TG rats and their non-transgenic littermates (control). The effect of intraperitoneal and intracerebroventricular injection of leptin on food intake and body weight gain was also examined. RESULTS: An increase in food intake and a decrease in locomotor activity were observed from 4 and 7 weeks of age, respectively, in the transgenic rats compared with control. Serum leptin concentrations of the transgenic rats were more than twice as high as those of control rats and were associated with an increased white adipose tissue mass and ob gene expression. Intraperitoneal injection of leptin significantly decreased food intake and body weight gain in control rats, but not in transgenic rats. Leptin concentration in the cerebrospinal fluid of transgenic rats was not different from that of control rats, and intracerebroventricular injection of leptin was similarly effective in reducing food intake and body weight gain as it was in control rats. CONCLUSIONS: These results suggest that the transgenic rats, whose GH secretion is suppressed, develop obesity due to early onset of an increase in food intake and a decrease in locomotor activity with leptin resistance resulting from deteriorating leptin transport from peripheral blood to cerebrospinal fluid.

Mechanism of inhibitory effect of glycyrrhizin on replication of human immunodeficiency virus (HIV).

Glycyrrhizin (GL) achieved a dose-dependent inhibition of the replication of human immunodeficiency virus type 1 (HIV-1) in MOLT-4 (clone No. 8) cells within the concentration range of 0.075 to 0.6 mM. Within this concentration range, GL also effected a dose-dependent reduction in the protein kinase C (PKC) activity of MOLT-4 (clone No. 8) cells. A well-known PKC inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), also proved inhibitory to HIV-1 replication in MOLT-4 (clone No. 8) cells. PKC inhibition may thus be considered as one of the mechanisms by which GL inhibits HIV-1 replication. In addition, GL may also owe its anti-HIV-1 activity, at least in part, to an interference with virus-cell binding, since the compound at 1.2 mM partially inhibited the adsorption of radiolabeled HIV-1 particles to MT-4 cells. At this concentration GL also suppressed giant cell formation induced by co-culturing MOLT-4 (clone No. 8) cells with MOLT-4/HTLV-IIIB cells, whereas the PKC inhibitor H-7 failed to do so.

Secretory expression of the human serum albumin gene in the yeast, Saccharomyces cerevisiae.

We have fused a cDNA gene encoding mature human serum albumin (HSA) to several secretory leader-encoding sequences. The hybrid genes were cloned into an episomal vector under the control of several yeast promoters and then introduced into yeast cells. The GAL1 promoter in combination with either the native HSA pre-sequence or a modified HSA pre-sequence gave the highest production of immunoreactive HSA, 90 mg/liter being reached in a shake flask culture. The invertase pre-sequence, the mating factor alpha 1 prepro-sequence, and the modified HSA pre-sequence directed accurate processing. In contrast, the chicken lysozyme pre-sequence and the native HSA pre-sequence directed incorrect processing. Episomal vectors were unstable within the host cells under non-selective culture conditions. To improve the plasmid stability, the hybrid genes were incorporated into an integrative vector. Transformants carrying multicopies of the plasmid integrated at the LEU2 locus stably secreted HSA. The highest yield of 65 mg/liter in a shake flask culture was obtained with the combination of the yeast glyceraldehyde-3-phosphate dehydrogenase promoter and the modified HSA pre-sequence. By constructing transformed strains containing multicopies of plasmids integrated at both the chromosome LEU2 and HIS4 loci, we have obtained a stable strain that continuously secretes as much as 85 mg HSA per liter of culture medium.