Porphyromonas gingivalis infection exacerbates the onset of rheumatoid arthritis in SKG mice.

Epidemiological studies have linked periodontitis to rheumatoid arthritis (RA). Porphyromonas gingivalis (Pg) was reported recently to produce citrullinated protein (CP) and increase anti-cyclic CP antibody (ACPA), both of which have been identified as causative factors of RA. In the present study, we determined the effects of Pg infection on the exacerbation of RA in a mouse model. RA model mice (SKG mice) were established by an intraperitoneal (i.p.) injection of laminarin (LA). Mice were divided into six groups, Ctrl (PBS injection), LA (LA injection), Pg/LA (Pg + LA injection), Pg (Pg injection), Ec/LA (Escherichia coli and LA injection) and Ec (E. coli injection). In order to evaluate RA, joint swelling by the arthritis score, bone morphology by microcomputed tomography (microCT), haematoxylin and eosin staining, ACPA, matrix metalloproteinase-3 (MMP-3) and cytokine level in serum by enzyme-linked immunosorbent assay were determined. Osteoclast differentiation from bone marrow mononuclear cells (BMCs) was examined to clarify the underlying mechanisms of RA. The presence of Pg and CP in joint tissue was also investigated. The arthritis score was threefold higher in the Pg/LA group than in the LA group. Severe bone destruction was observed in joint tissue of the Pg/LA group. A microCT analysis of the Pg/LA group revealed a decrease in bone density. ACPA, MMP-3, interleukin (IL)-2, IL-6, CXCL1 and macrophage inflammatory protein (MIP)-1α levels from the Pg/LA group were the highest. The osteoclastogenesis of BMCs was enhanced in the Pg/LA group. Furthermore, large amounts of Pg components and CP were detected in the Pg/LA group. In conclusion, Pg infection has the potential to exacerbate RA.

Bombyx mori E26 transformation-specific 2 (BmEts2), an Ets family protein, represses Bombyx mori Rels (BmRels)-mediated promoter activation of antimicrobial peptide genes in the silkworm Bombyx mori.

E26 transformation-specific (Ets) family transcription factors are known to play roles in various biological phenomena, including immunity, in vertebrates. However, the mechanisms by which Ets proteins contribute to immunity in invertebrates remain poorly understood. In this study, we identified a cDNA encoding BmEts2, which is a putative orthologue of Drosophila Yan and human translocation-ets-leukemia/Ets-variant gene 6, from the silkworm Bombyx mori. Expression of the BmEts2 gene was significantly increased in the fat bodies of silkworm larvae in response to injection with Escherichia coli and Staphylococcus aureus. BmEts2 overexpression dramatically repressed B. mori Rels (BmRels)-mediated promoter activation of antimicrobial peptide genes in silkworm cells. Conversely, gene knockdown of BmEts2 significantly enhanced BmRels activity. In addition, two κB sites located on the 5' upstream region of cecropin B1 were found to be involved in the repression of BmRels-mediated promoter activation. Protein-competition analysis further demonstrated that BmEts2 competitively inhibited binding of BmRels to κB sites. Overall, BmEts2 acts as a repressor of BmRels-mediated transactivation of antimicrobial protein genes by inhibiting the binding of BmRels to κB sites.

Risk factors for neutropenia with lenalidomide plus dexamethasone therapy for multiple myeloma.

Neutropenia may develop as an adverse event in patients with multiple myeloma receiving lenalidomide (LEN) plus dexamethasone (DEX) therapy. In the present study, we examined the risk factors associated with grade 3/4 neutropenia during the first cycle of LEN plus DEX therapy. We observed that hemoglobin level (≤ 8.5 g/dl) was a significant risk factor for grade 3/4 neutropenia during the first cycle of therapy (odds ratio: 19.40; 95% confidence interval: 2.68-141.00; p < 0.01). thus, our findings suggest that determining the hemoglobin level could be useful in the risk management for neutropenia in patients receiving LEN plus DEX therapy.

Irsogladine maleate inhibits Porphyromonas gingivalis-mediated expression of toll-like receptor 2 and interleukin-8 in human gingival epithelial cells.

BACKGROUND AND OBJECTIVE: Periodontitis is an infectious disease caused by an interaction between the host and periodontopathogenic bacteria. Regulating the immune response in human gingival epithelial cells (HGEC) may contribute to the prevention of periodontitis. Irsogladine maleate (IM) has previously been shown to regulate inflammation and the cell-cell junctional barrier in HGEC. In addition to these functions, control of bacterial recognition is important for preventing inflammation in periodontal tissue. Innate immunity in gingival epithelium is the first line of defense and plays a crucial role against bacterial challenge. Therefore, the effect of IM on regulating toll-like receptor 2 (TLR2), which is part of the innate immunity, was determined in this study. MATERIAL AND METHODS: OBA-9, an immortalized human gingival epithelial cell line, and primary cultured HGEC were used in this study. Real-time PCR and western blotting were performed in OBA-9 or HGEC stimulated with whole cells of Porphyromonas gingivalis or with lipopolysaccharide (LPS) derived from P. gingivalis (PgLPS) in the presence or absence of IM to determine expression of TLR2 mRNA and production of TLR2 protein. Small interfering RNA (siRNA) against TLR2 was transfected into OBA-9 to clarify the association between the induction of TLR2 and interleukin-8 (IL-8) production. RESULTS: The addition of IM into P. gingivalis or PgLPS-induced OBA-9 suppressed IL-8 production (p < 0.01). The addition of IM also abolished the induction of TLR2 by P. gingivalis or PgLPS in OBA-9 and primary cultured HGEC (p < 0.01). The suppressive effect of IM on the induction of TLR2 was also confirmed by immunohistostaining. Stimulation with peptidoglycan, a specific ligand for TLR2, suppressed the expression of toll-like receptor 4 (TLR4) mRNA in the presence of IM (p < 0.01). However, LPS derived from Escherichia coli, a ligand for TLR4, did not induce the expression of TLR2 mRNA. The PgLPS-induced expression of TLR4 mRNA was abolished by IM. Knockdown of TLR2 by siRNA transfection resulted in a weaker response of induction of IL8 mRNA in P. gingivalis or PgLPS-stimulated OBA-9. CONCLUSION: These results suggest that IM suppresses the induction of IL-8 production by regulating increased levels of TLR2.

Distribution of regulatory T cells and interaction with dendritic cells in the synovium of rheumatoid arthritis.

OBJECTIVES: The aim of this study was to investigate the tissue distribution of regulatory T cells (Treg cells) and their interaction with dendritic cells (DCs) in synovium from patients with rheumatoid arthritis (RA) or osteoarthritis (OA). METHODS: Immunohistochemical staining was used to investigate the distribution of Treg cells and the interaction between Treg cells and DCs in RA (n = 30) and OA synovium (n = 8). mRNA levels were measured by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Large numbers of Treg cells were observed in lymphoid aggregates and perivenular infiltration areas in the RA synovium. Specific cellular markers for Treg cells (Foxp3, CD39, LAG-3, and Nrp-1) were found in lymphoid aggregates, perivenular infiltration, and scattered in lining layer areas. As molecular markers for DCs, DC-LAMP, DEC-205, CD80/86, and CD83 were also detected in the lymphoid aggregates and perivenular infiltration areas in RA. Furthermore, the co-localization of Treg cells and DCs was confined mainly in the lymphoid aggregation areas. The number of DCs increased significantly more than the number of Treg cells with inflammatory progression in RA. mRNA expression of the cellular markers for Treg cells (Foxp3, LAG-3, and Nrp-1) and the molecular markers for DCs (DC-LAMP and DEC-205) was increased in RA compared with OA synovium. CONCLUSIONS: Our results indicate that DCs play a dominant role in regulating the activation and progression of immune responses in RA, even though the number of Treg cells was upregulated at the same time. This suggests that Treg cells do not function normally to suppress the maturation of DCs in the RA synovium.

Sarcopenia Is Associated with Fecal Incontinence in Patients with Dysphagia: Implication for Anal Sarcopenia.

OBJECTIVES: To examine the relationship between sarcopenia and fecal incontinence in patients with dysphagia. DESIGN: Cross-sectional study using the Japanese sarcopenic dysphagia database. SETTING: 19 hospitals including 9 acute care hospitals, 8 rehabilitation hospitals, 2 long-term care hospitals, and 1 home visit rehabilitation center. PARTICIPANTS: 460 dysphagic patients, aged 20 years and older. MEASUREMENTS: Sarcopenia was diagnosed by the 2019 criteria of the Asian Working Group for Sarcopenia. Fecal incontinence was assessed by health care professionals at baseline according to the definition of the Japanese Practice Guidelines for Fecal Incontinence. We examined whether there was a significant difference between the rate of fecal incontinence in patients with/without sarcopenia. Age, sex, type of dwelling, Barthel index, Charlson comorbidity index (CCI), calf circumference, handgrip strength, body mass index, malnourishment, C-reactive protein level, serum albumin level, and delivery of enteral nutrition by nasogastric and/or gastrostomy tube were measured. To examine the relationship between sarcopenia and fecal incontinence, logistic regression analysis was performed with adjustments for age, sex, sarcopenia, CCI, enteral nutrition, and dwelling. RESULTS: The mean age of patients was 81 ± 10 years. Of the 460 study patients, 404 (88%) patients had sarcopenia and 104 had fecal incontinence (23%). The rate of fecal incontinence was higher in the sarcopenia group than the non-sarcopenia group (25% vs. 7%, P = 0.003). Logistic regression analysis showed that sarcopenia was independently associated with fecal incontinence (odds ratio: 3.114, 95% confidence interval: 1.045, 9.282). CONCLUSION: The prevalence of fecal incontinence was 23% in patients with dysphagia. Sarcopenia was independently associated with fecal incontinence, which suggests the presence of anal sarcopenia. Defecation control should be assessed in patients with sarcopenia.

Construction and Quality Evaluation of the Japanese Sarcopenic Dysphagia Database.

OBJECTIVES: To describe the activity and evaluate the quality of the Japanese sarcopenic dysphagia database. DESIGN: Cohort registry study. SETTING: 19 hospitals including 9 acute care hospitals, 8 rehabilitation hospitals, 2 long-term care hospitals, and 1 home visit rehabilitation team. PARTICIPANTS: 467 dysphagic patients, aged 20 years and older. MEASUREMENTS: The following indices were assessed at baseline: age, sex, main disease, sarcopenic dysphagia, whole body sarcopenia, Food Intake Level Scale (FILS), malnutrition diagnosed by the Global Leadership Initiative on Malnutrition criteria, oral status assessed by the Revised Oral Assessment Guide or the Oral Health Assessment Tool, activities of daily living assessed by the Functional Independence Measure (FIM) or the Barthel Index (BI), Charlson comorbidity index, C-reactive protein and serum albumin levels, dysarthria, hoarseness, aphasia, pressure ulcers, bladder, bowel, and kidney function, respiratory status, polypharmacy, number of drugs, and involvement of health care professionals and rehabilitation nutrition team. FILS, FIM or BI, and outcome including discharge destination were assessed at follow-up. A simple comparison of cases and evaluation of the quality of data were performed. RESULTS: The mean age was 80.4 ± 11.4 yr. The variable input error was 0. The number of patients with missing data was high for estimated glomerular filtration rate, C-reactive protein, serum albumin, skeletal mass index, and tongue pressure. The prevalence of either probable, possible, or no sarcopenic dysphagia was 105 (23%), 182 (39%), or 179 (38%), respectively. Doctors including physiatrists, nurses, physical therapists, and registered dietitians were involved with most patients, while the rehabilitation nutrition team was involved in only 16% of patients. CONCLUSIONS: The quality of the database was relatively high. Sarcopenic dysphagia is common in patients with dysphagia.

Lipopolysaccharide elicits expression of immune-related genes in the silkworm, Bombyx mori.

Lipopolysaccharide (LPS), a major cell wall component of gram-negative bacteria, was found to be unable to activate immune-related genes in Drosophila melanogaster. In contrast, highly purified LPS elicited immune-related gene expression in the fat body of Bombyx mori. However, the level of activation by highly purified LPS was lower than crude LPS and peptidoglycan. Furthermore, synthetic lipid A also activated these genes, suggesting that B. mori possesses unknown signal pathways to activate immune-related genes by LPS. Up-regulation of antimicrobial peptide genes by highly purified LPS was not confirmed in the immune-responsive cell line, NIAS-Bm-aff3, suggesting that some factors necessary for signal transduction activated by LPS are deficient in this cell line.

Myosin light chain-2 mutation affects flight, wing beat frequency, and indirect flight muscle contraction kinetics in Drosophila.

We have used a combination of classical genetic, molecular genetic, histological, biochemical, and biophysical techniques to identify and characterize a null mutation of the myosin light chain-2 (MLC-2) locus of Drosophila melanogaster. Mlc2E38 is a null mutation of the MLC-2 gene resulting from a nonsense mutation at the tenth codon position. Mlc2E38 confers dominant flightless behavior that is associated with reduced wing beat frequency. Mlc2E38 heterozygotes exhibit a 50% reduction of MLC-2 mRNA concentration in adult thoracic musculature, which results in a commensurate reduction of MLC-2 protein in the indirect flight muscles. Indirect flight muscle myofibrils from Mlc2E38 heterozygotes are aberrant, exhibiting myofilaments in disarray at the periphery. Calcium-activated Triton X-100-treated single fiber segments exhibit slower contraction kinetics than wild type. Introduction of a transformed copy of the wild type MLC-2 gene rescues the dominant flightless behavior of Mlc2E38 heterozygotes. Wing beat frequency and single fiber contraction kinetics of a representative rescued line are not significantly different from those of wild type. Together, these results indicate that wild type MLC-2 stoichiometry is required for normal indirect flight muscle assembly and function. Furthermore, these results suggest that the reduced wing beat frequency and possibly the flightless behavior conferred by Mlc2E38 is due in part to slower contraction kinetics of sarcomeric regions devoid or partly deficient in MLC-2.

Climate change impacts and risks for animal health in Asia.

The threat of climate change and global warming is now recognised worldwide and some alarming manifestations of change have occurred. The Asian continent, because of its size and diversity, may be affected significantly by the consequences of climate change, and its new status as a 'hub' of livestock production gives it an important role in mitigating possible impacts of climate variability on animal health. Animal health may be affected by climate change in four ways: heat-related diseases and stress, extreme weather events, adaptation of animal production systems to new environments, and emergence or re-emergence of infectious diseases, especially vector-borne diseases critically dependent on environmental and climatic conditions. To face these new menaces, the need for strong and efficient Veterinary Services is irrefutable, combined with good coordination of public health services, as many emerging human diseases are zoonoses. Asian developing countries have acute weaknesses in their Veterinary Services, which jeopardises the global surveillance network essential for early detection of hazards. Indeed, international cooperation within and outside Asia is vital to mitigating the risks of climate change to animal health in Asia.

Self-harm and Suicide Attempts in a Japanese Psychiatric Hospital.

OBJECTIVE: Self-harm and attempted suicide are risk factors for suicide in psychiatric hospital in-patients. This study aimed to analyse the circumstances of self-harm and suicide attempts in a Japanese psychiatric hospital so as to improve management and care. METHODS: Incident reports of self-harm and suicide attempts during a 12.4-year period from November 2000 to March 2013 were reviewed. A descriptive analysis was conducted in terms of age, sex, and diagnosis of patients, as well as level, ward, situations, and causes of incidents. RESULTS: During the study period, 90 cases of self-harm and attempted suicide involving 58 patients were reported. The rate of self-harm and suicide attempts was 0.05 per 1000 patient-days. The types of selfharm and suicide attempts included hanging (n = 25), wrist cutting (n = 19), ingestion of foreign objects (n = 17), and others (n = 29). The single case of completed suicide involved hanging, in a patient with schizophrenia. Among 55 patients with relevant data, the most common clinical diagnosis was mood disorder (41.8%), followed by schizophrenia (36.4%). Mood disorder was 3.5 times as prevalent in females as in males (14 vs. 4). Fourteen patients with mood disorder (n = 8) or schizophrenia (n = 6) were repeatedly involved in 46 of 89 cases of self-harm or attempted suicide; 11 were female. One woman with mood disorder attempted suicide 9 times within the same year. The top 3 management and care factors related to self-harm and suicide attempts were failure to adhere to preventive procedures (28%), insufficient therapeutic communication (28%), and difficulty in predicting suicide (20%). CONCLUSION: Self-harm and suicide attempts at this psychiatric hospital occurred at a rate of 0.05 per 1000 patient-days between late 2000 and early 2013. Efforts are needed to increase compliance with suicide prevention procedures and therapeutic communication, so as to improve management and care of psychiatric in-patients and prevent them from committing suicide.

Transition of Akabane virus genogroups and its association with changes in the nature of disease in Japan.

Akabane virus (AKAV) is teratogenic to the foetus of domestic ruminants and causes a significant reproduction loss in cattle in Japan. In several past epizootics in cattle, AKAV was also associated with post-natal encephalomyelitis, mainly in calves and young stock. Previously analysed AKAV isolates in East Asia form two major clusters, genogroups I and II, with isolates involved in encephalomyelitis belonging mainly to the former. Between 2007 and 2013, AKAV epizootics were regularly observed in Japan during the summer/autumn season, and abnormal deliveries and post-natal encephalomyelitis caused by the virus in cattle were reported. During this period, 30 AKAV isolates were obtained from diseased and sentinel cattle, a piglet and Culicoides biting midges throughout Japan and were subjected to genetic comparison and phylogenetic analysis with previous isolates. In 2007, 2011 and 2013, AKAV belonging to genogroup I was identified in the central nervous systems of calves showing neurological disorders. Notably, a total of 165 cases of bovine encephalomyelitis were reported in 2011 and the isolated viruses from affected animals shared high genetic identities with a South Korean isolate that was associated with a large outbreak in 2010, suggesting some epidemiological linkage between these epizootics. Epizootics of genogroup II were observed in 2008 and 2010, but bovine post-natal encephalomyelitis cases rarely occurred. Our findings suggest that the frequent incursion of genogroup I isolates has increased the frequency of post-natal encephalomyelitis cases in Japan in recent years. Infection by genogroup I virus was also identified in piglets with neurological disorders or congenital malformations in 2011 and 2013. The aetiological role of AKAV in pigs should be elucidated in the future.

Expression of the affected A gamma globin gene associated with Greek nondeletion hereditary persistence of fetal hemoglobin.

The overexpressed A gamma globin gene in the Greek type of nondeletion hereditary persistence of fetal hemoglobin has a unique single-base substitution located at position -117 relative to the site of transcription initiation. This gene and its normal counterpart were transferred into cultured cell lines by using a retroviral vector. The only difference in expression between the transferred normal and mutant gamma genes was observed in the human erythroleukemia cell line KMOE after exposure of the cells to cytosine arabinoside, a condition that resulted in an adult pattern of endogenous globin gene expression by the cells and was associated with increased expression of the mutant gene.

Expression of glucocorticoid receptors in non-neoplastic lymphoid follicles and B cell type malignant lymphomas.

OBJECTIVE: To evaluate the expression of human glucocorticoid receptors (hGRs), such as hGR (4H2), hGR-alpha, and hGR-beta, in non-neoplastic lymphoid follicles and B cell type malignant lymphomas. METHODS: The expression of hGRs in non-neoplastic lymphoid follicles and malignant lymphomas, including diffuse large cell lymphoma, mantle cell lymphoma, and follicular lymphoma, was examined immunohistochemically. HGR (4H2) expression was confirmed by double immunostaining of tissues and in isolated cells from tonsillar germinal centres, and by immunoelectronmicroscopy. RESULTS: In secondary lymphoid follicles of any non-neoplastic diseases--such as chronic tonsillitis, reactive lymphadenitis, and Kimura's disease--the germinal centre cells often expressed hGR (4H2) and hGR-alpha. Double immunocytochemical staining of isolated germinal centre cells showed that the majority of hGR (4H2) positive cells were CD20 positive B cells, and that follicular dendritic cells also expressed hGR. Immunoelectronmicroscopy revealed the presence of nuclear hGR (4H2) in the binucleated follicular dendritic cells and germinal centre cells. The frequency of hGR (4H2) expression in diffuse large B cell lymphoma was higher, that in mantle cell lymphoma was lower, and that in follicular lymphoma was intermediate among the types of malignant lymphoma. The hGR (4H2) expression was less frequent in cases of grade I follicular lymphoma. CONCLUSIONS: There are differences in hGR expression between the germinal centre and the mantle zone in non-neoplastic lymphoid follicles, and differences of hGR (4H2) expression among the types of malignant lymphoma and grades of follicular lymphoma, which probably contribute to the different steroid sensitivities.

Bovine Arboviruses in Culicoides Biting Midges and Sentinel Cattle in Southern Japan from 2003 to 2013.

Epizootic congenital abnormalities, encephalomyelitis and febrile illnesses in cattle caused by arthropod-borne viruses (arboviruses) are prevalent in Japan. Causative viruses including orthobunyaviruses, orbiviruses and rhabdovirus are thought to be transmitted by Culicoides biting midges. Recently, the incursions of several arboviruses, potentially Culicoides-borne, were newly confirmed in Japan. However, their spread pattern and exact vector species are currently uncertain. Attempts to isolate arboviruses from Culicoides biting midges and sentinel cattle were conducted in Kagoshima, located at the southernmost end of the main islands of Japan, a potentially high-risk area for incursion of arboviral diseases and outbreak of endemic ones. Seventy-eight isolates comprising Akabane, Peaton and Sathuperi viruses of the genus Orthobunyavirus of the family Bunyaviridae, bluetongue virus serotype 16, D'Aguilar virus, Bunyip Creek virus and epizootic haemorrhagic disease virus serotype 1 of the genus Orbivirus of the family Reoviridae, a potentially novel rhabdovirus of the genus Ephemerovirus and unidentified orbivirus-like viruses were obtained from Culicoides biting midges and sentinel cattle between 2003 and 2013. Akabane, Sathuperi, D'Aguilar and Bunyip Creek viruses were selectively isolated from Culicoides oxystoma, suggesting this vector's responsibility for these arbovirus outbreaks. The results of virus isolation also implied that C. tainanus, C. jacobsoni and C. punctatus are competent for the transmission of bluetongue virus serotype 16, Peaton virus and epizootic haemorrhagic disease virus serotype 1, respectively. Our monitoring in Culicoides biting midges and sentinel cattle detected the circulation of Akabane virus just prior to the accumulations of bovine congenital abnormalities and encephalomyelitis by it around study sites in 2003, 2006, 2008 and 2013. Silent circulations of the other arboviruses, including potentially new viruses, were also detected during the study period.

Isolation and nucleotide sequence of cecropin B cDNA clones from the silkworm, Bombyx mori.

Two cDNA clones encoding cecropin B, an antibacterial protein, were isolated from a fat body cDNA library of the silkworm, Bombyx mori. Amino acid sequences of these clones, deduced from nucleotide sequences, were identical, including signal peptide regions. However, the nucleotide sequences were different at 30 positions. Deduced amino acid sequences of Bombyx mori cecropin B showed higher homology with cecropins from Lepidoptera than with those from Diptera.

Molecular cloning of the dnaK locus, and purification and characterization of a DnaK protein from Bacillus brevis HPD31.

Using part of the dnaK gene from Bacillus subtilis as a probe, a 4. 4-kbp SacI-BglII fragment of chromosomal DNA of Bacillus brevis, a protein-hypersecreting bacterium, was cloned. Nucleotide sequencing revealed 3 open reading frames in the order of grpE-dnaK-dnaJ homologues. We purified DnaK protein to homogeneity from B. brevis HPD31 harboring a multi-copy dnaK expression plasmid. Purified DnaK showed ATPase activity which was synergistically stimulated 14-fold by the addition of glutathione S-transferase-DnaJ and glutathione S-transferase-GrpE fusion proteins. DnaK hydrolyzed not only ATP but also CTP, UTP, and GTP at about 40% of the efficiency of ATP. The specific activity of DnaK-ATPase was 7.25x10-3 unit/mg protein (the turnover number against ATP was 0.47 min-1) under our assay conditions. The DnaK dimers dissociated into monomers on addition of ATP, GTP, CTP, UTP and ATPgammaS, but not ADP or AMP. DnaK formed a stable complex with permanently unfolded carboxymethylated alpha-lactalbumin but not with native alpha-lactalbumin, and this complex was dissociated by addition of ATP/Mg. Formation of this complex was inhibited in the presence of inorganic phosphate.

Isolation of a novel prolactin-like cDNA clone from bovine placenta: occurrence of new family members.

A novel cDNA clone that hybridized to bovine prolactin cDNA was isolated from a bovine (Bos taurus) placental cDNA library and the nucleotide sequence was analyzed. The cDNA clone, named bPLP-III, contained one open reading frame encoding a protein consisting of 239 amino acids. The amino-acid sequence of bPLP-III is 43 and 57% homologous to bovine preprolactin and a bovine prolactin-related protein, bPRC-I, respectively, but only 23% homologous to bovine pregrowth hormone. The predicted mature protein of bPLP-III is distinct from bovine placental lactogens in amino-acid composition, suggesting that bPLP-III is the clone for a new member of prolactin-related mRNA expressed in bovine placenta.

Molecular cloning of cDNA encoding 20 kDa variant human growth hormone and the alternative splicing mechanism.

cDNA encoding the 20 kDa variant form of human growth hormone has been cloned, and its sequence analysis verified the alternative splicing mechanism for the mRNA synthesis. The cDNA sequence lacked 45 nucleotides corresponding to the sequence of 15 amino acids in the 22 kDa form of growth hormone. The cDNA clones for the 20 kDa variant hormone had the homogeneous 5'-ends, while the clones for the 22 kDa form showed a minor heterogenity, having two transcription initiation sites. The percentage of 20 kDa variant cDNA clones was approx. 7.7% of the total human growth hormone cDNA clones, consistent with the contents of 20 kDa hormone protein in human anterior pituitary and plasma.

Effects of gene dosage on the expression of human growth hormone cDNA in Escherichia coli.

Gene dosage effects on the foreign protein production in Escherichia coli have been analyzed with the expression vectors containing one, two and four tandemly arranged human growth hormone cDNAs under various culture conditions. In a limited synthetic culture medium, a proportional relationship was observed between the number of the integrated cDNA and the amount of human growth hormone produced. With a culture medium rich in the nutrients, however, no such relationship was observed. These results suggest that, under the limited culture conditions, high-level expression of a foreign gene can be achieved by tandemly joined multiple copies of cDNA.