Constitutive tyrosine phosphorylation of ErbB-2 via Jak2 by autocrine secretion of prolactin in human breast cancer

Overexpression of the oncogene for ErbB-2 is an unfavorable prognostic marker in human breast cancer. Its oncogenic potential appears to depend on the state of tyrosine phosphorylation. However, the mechanisms by which ErbB-2 is constitutively tyrosine-phosphorylated in human breast cancer are poorly understood. We now show that human breast carcinoma samples with ErbB-2 overexpression have higher proliferative and metastatic activity in the presence of autocrine secretion of prolactin (PRL). By using a neutralizing antibody or dominant negative (DN) strategies or specific inhibitors, we also show that activation of Janus kinase Jak2 by autocrine secretion of PRL is one of the significant components of constitutive tyrosine phosphorylation of ErbB-2, its association with Grb2 and activation of mitogen-activated protein (MAP) kinase in human breast cancer cell lines that overexpress ErbB-2. Furthermore, the neutralizing anti-PRL antibody or erbB-2 antisense oligonucleotide or DN Jak2 or Jak2 inhibitor or DNRas or MAP kinase kinase inhibitor inhibits the proliferation of both untreated and PRL-treated cells. Our results indicate that autocrine secretion of PRL stimulates tyrosine phosphorylation of ErbB-2 by Jak2, provides docking sites for Grb2 and stimulates Ras-MAP kinase cascade, thereby causing unrestricted cellular proliferation. The identification of this novel cross-talk between ErbB-2 and the autocrine growth stimulatory loop for PRL may provide new targets for therapeutic and preventive intervention of human breast cancer.     

Pilot scale production of a recombinant human epidermal growth factor, secreted by Bacillus brevis, using expanded bed adsorption

Recombinant Bacillus brevis which carried an expression plasmid encoding the human epidermal growth factor (EGF) gene on a cryptic high-copy number plasmid, pHT926, extracellularly produced EGF in its biologically active form at a concentration of over 1.5 g L⁻¹ in the culture broth in a 30-L jar fermenter. The culture broth also contained some other EGF compounds, which mainly consisted of oligomeric and polymeric forms with disulfide bonds. We developed a simple purification method for EGF, without prior cell removal from the culture broth, comprising cation exchange expanded bed adsorption followed by ultrafiltration with UF 10 000 and 3000 membranes. The EGF compounds were efficiently separated from the EGF in its native form in the expanded bed adsorption step. With this purification method, only EGF in its native form was recovered from the culture broth, with a yield of nearly 80%, and 90% purity. This efficient and economic system has made it possible to use EGF as a pharmaceutical in the livestock industry. Received 10 June 1998/ Accepted in revised form 10 September 1998     

DNA barcoding reveals 24 distinct lineages as cryptic bird species candidates in and around the Japanese Archipelago

DNA barcoding using a partial region (648 bp) of the cytochrome c oxidase I (COI) gene is a powerful tool for species identification and has revealed many cryptic species in various animal taxa. In birds, cryptic species are likely to occur in insular regions like the Japanese Archipelago due to the prevention of gene flow by sea barriers. Using COI sequences of 234 of the 251 Japanese‐breeding bird species, we established a DNA barcoding library for species identification and estimated the number of cryptic species candidates. A total of 226 species (96.6%) had unique COI sequences with large genetic divergence among the closest species based on neighbour‐joining clusters, genetic distance criterion and diagnostic substitutions. Eleven cryptic species candidates were detected, with distinct intraspecific deep genetic divergences, nine lineages of which were geographically separated by islands and straits within the Japanese Archipelago. To identify Japan‐specific cryptic species from trans‐Paleartic birds, we investigated the genetic structure of 142 shared species over an extended region covering Japan and Eurasia; 19 of these species formed two or more clades with high bootstrap values. Excluding six duplicated species from the total of 11 species within the Japanese Archipelago and 19 trans‐Paleartic species, we identified 24 species that were cryptic species candidates within and surrounding the Japanese Archipelago. Repeated sea level changes during the glacial and interglacial periods may be responsible for the deep genetic divergences of Japanese birds in this insular region, which has led to inconsistencies in traditional taxonomies based on morphology.     

The Characteristic Changes in Hepatitis B Virus X Region for Hepatocellular Carcinoma: A Comprehensive Analysis Based on Global Data

Objectives

Mutations in hepatitis B virus (HBV) X region (HBx) play important roles in hepatocarcinogenesis while the results remain controversial. We sought to clarify potential hepatocellular carcinoma (HCC)-characteristic mutations in HBx from HBV genotype C-infected patients and the distribution of those mutations in different disease phases and genotypes.

Methods

HBx sequences downloaded from an online global HBV database were screened and then classified into Non-HCC or HCC group by diagnosis information. Patients'' data of patient age, gender, country or area, and viral genotype were also extracted. Logistic regression was performed to evaluate the effects of mutations on HCC risk.

Results

1) Full length HBx sequences (HCC: 161; Non-HCC: 954) originated from 1115 human sera across 29 countries/areas were extracted from the downloaded 5956 HBx sequences. Genotype C occupied 40.6% of Non-HCC (387/954) and 89.4% of HCC (144/161). 2) Sixteen nucleotide positions showed significantly different distributions between genotype C HCC and Non-HCC groups. 3) Logistic regression showed that mutations A1383C (OR: 2.32, 95% CI: 1.34-4.01), R1479C/T (OR: 1.96, 95% CI: 1.05-3.64; OR: 5.15, 95% CI: 2.53-10.48), C1485T (OR: 2.40, 95% CI: 1.41-4.08), C1631T (OR: 4.09, 95% CI: 1.41-11.85), C1653T (OR: 2.58, 95% CI: 1.59-4.19), G1719T (OR: 2.11, 95% CI: 1.19-3.73), and T1800C (OR: 23.59, 95% CI: 2.25-247.65) were independent risk factors for genotype C HBV-related HCC, presenting different trends among individual disease phases. 4) Several genotype C HCC risk mutations pre-existed, even as major types, in early disease phases with other genotypes.

Conclusions

Mutations associated with HCC risk were mainly located in HBx transactivation domain, viral promoter, protein/miRNA binding sites, and the area for immune epitopes. Furthermore, the signatures of these mutations were unique to disease phases leading to HCC, suggesting molecular counteractions between the virus and host during hepatocarcinogenesis.     

H-Ferritin Is Preferentially Incorporated by Human Erythroid Cells through Transferrin Receptor 1 in a Threshold-Dependent Manner

Ferritin is an iron-storage protein composed of different ratios of 24 light (L) and heavy (H) subunits. The serum level of ferritin is a clinical marker of the body’s iron level. Transferrin receptor (TFR)1 is the receptor not only for transferrin but also for H-ferritin, but how it binds two different ligands and the blood cell types that preferentially incorporate H-ferritin remain unknown. To address these questions, we investigated hematopoietic cell-specific ferritin uptake by flow cytometry. Alexa Fluor 488-labeled H-ferritin was preferentially incorporated by erythroid cells among various hematopoietic cell lines examined, and was almost exclusively incorporated by bone marrow erythroblasts among human primary hematopoietic cells of various lineages. H-ferritin uptake by erythroid cells was strongly inhibited by unlabeled H-ferritin but was only partially inhibited by a large excess of holo-transferrin. On the other hand, internalization of labeled holo-transferrin by these cells was not inhibited by H-ferritin. Chinese hamster ovary cells lacking functional endogenous TFR1 but expressing human TFR1 with a mutated RGD sequence, which is required for transferrin binding, efficiently incorporated H-ferritin, indicating that TFR1 has distinct binding sites for H-ferritin and holo-transferrin. H-ferritin uptake by these cells required a threshold level of cell surface TFR1 expression, whereas there was no threshold for holo-transferrin uptake. The requirement for a threshold level of TFR1 expression can explain why among primary human hematopoietic cells, only erythroblasts efficiently take up H-ferritin.     

Genetic architecture of type 2 diabetes

Genome-wide association studies (GWAS) have identified over 70 loci associated with type 2 diabetes (T2D). Most genetic variants associated with T2D are common variants with modest effects on T2D and are shared with major ancestry groups. To what extent the genetic component of T2D can be explained by common variants relies upon the shape of the genetic architecture of T2D. Fine mapping utilizing populations with different patterns of linkage disequilibrium and functional annotation derived from experiments in relevant tissues are mandatory to track down causal variants responsible for the pathogenesis of T2D.     

The inhibitory effects of mushroom extracts on sucrose-dependent oral biofilm formation

Mushrooms contain large quantities of α-glucans. Shiitake (Lentinula edodes), Japan’s most popular edible mushroom, has been reported to contain about 6% (weight/dried weight) of α-(1,3)-glucan. This glucan is one of the major components of oral biofilm formed by the cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus. We found that extracts from shiitake and other edible mushrooms could reduce preformed biofilms of S. mutans and S. sobrinus in the presence of dextranase. We also investigated the α-glucanase activities of shiitake mushroom extracts and their effects on biofilm formation. The extracts possessed α-glucanase activity and degraded water-insoluble glucans from mutans streptococci. The extracts strongly inhibited the sucrose-dependent formation of biofilms by S. mutans and S. sobrinus in the presence of dextranase. Our results suggest that some components of mushrooms, including α-glucanases, might inhibit the sucrose-induced formation of oral biofilms.     

Involvement of the endocannabinoid system in periodontal healing

Endocannabinoids including anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are important lipid mediators for immunosuppressive effects and for appropriate homeostasis via their G-protein-coupled cannabinoid (CB) receptors in mammalian organs and tissues, and may be involved in wound healing in some organs. The physiological roles of endocannabinoids in periodontal healing remain unknown. We observed upregulation of the expression of CB1/CB2 receptors localized on fibroblasts and macrophage-like cells in granulation tissue during wound healing in a wound-healing model in rats, as well as an increase in AEA levels in gingival crevicular fluid after periodontal surgery in human patients with periodontitis. In-vitro, the proliferation of human gingival fibroblasts (HGFs) by AEA was significantly attenuated by AM251 and AM630, which are selective antagonists of CB1 and CB2, respectively. CP55940 (CB1/CB2 agonist) induced phosphorylation of the extracellular-regulated kinases (ERK) 1/2, p38 mitogen-activated protein kinase (p38MAPK), and Akt in HGFs. Wound closure by CP55940 in an in-vitro scratch assay was significantly suppressed by inhibitors of MAP kinase kinase (MEK), p38MAPK, and phosphoinositol 3-kinase (PI3-K). These findings suggest that endocannabinoid system may have an important role in periodontal healing.