Lower expression of activating transcription factors 3 and 4 correlates with shorter progression-free survival in multiple myeloma patients receiving bortezomib plus dexamethasone therapy.

Bortezomib (BTZ), a proteasome inhibitor, is widely used in the treatment of multiple myeloma (MM), but a fraction of patients respond poorly to this agent. To identify factors predicting the duration of progression-free survival (PFS) of MM patients on BTZ treatment, the expression of proteasome and endoplasmic reticulum (ER) stress-related genes was quantified in primary samples from patients receiving a combination of BTZ and dexamethasone (BD). Fifty-six MM patients were stratified into a group with PFS<6 months (n=33) and a second group with PFS⩾6 months (n=23). Of the 15 genes analyzed, the expression of activating transcription factor 3 (ATF3) and ATF4 was significantly lower in patients with shorter PFS (P=0.0157 and P=0.0085, respectively). Chromatin immunoprecipitation analysis showed that these ATFs bind each other and transactivate genes encoding the pro-apoptotic transcription factors, CHOP and Noxa, which promote ER stress-associated apoptosis. When either ATF3 or ATF4 expression was silenced, MM cells partially lost sensitivity to BTZ treatment. This was accompanied by lower levels of Noxa, CHOP and DR5. Thus low basal expression of ATF3 and ATF4 may attenuate BTZ-induced apoptosis. Hence, ATF3 and ATF4 could potentially be used as biomarkers to predict efficacy of BD therapy in patients with MM.

Identification of Toyocamycin, an agent cytotoxic for multiple myeloma cells, as a potent inhibitor of ER stress-induced XBP1 mRNA splicing.

The IRE1α-XBP1 pathway, a key component of the endoplasmic reticulum (ER) stress response, is considered to be a critical regulator for survival of multiple myeloma (MM) cells. Therefore, the availability of small-molecule inhibitors targeting this pathway would offer a new chemotherapeutic strategy for MM. Here, we screened small-molecule inhibitors of ER stress-induced XBP1 activation, and identified toyocamycin from a culture broth of an Actinomycete strain. Toyocamycin was shown to suppress thapsigargin-, tunicamycin- and 2-deoxyglucose-induced XBP1 mRNA splicing in HeLa cells without affecting activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) activation. Furthermore, although toyocamycin was unable to inhibit IRE1α phosphorylation, it prevented IRE1α-induced XBP1 mRNA cleavage in vitro. Thus, toyocamycin is an inhibitor of IRE1α-induced XBP1 mRNA cleavage. Toyocamycin inhibited not only ER stress-induced but also constitutive activation of XBP1 expression in MM lines as well as primary samples from patients. It showed synergistic effects with bortezomib, and induced apoptosis of MM cells including bortezomib-resistant cells at nanomolar levels in a dose-dependent manner. It also inhibited growth of xenografts in an in vivo model of human MM. Taken together, our results suggest toyocamycin as a lead compound for developing anti-MM therapy and XBP1 as an appropriate molecular target for anti-MM therapy.

Potent antitumor effects of bevacizumab in a microenvironment-dependent human lymphoma mouse model.

We established a mouse model of microenvironment-dependent human lymphoma, and assessed the therapeutic potential of bevacizumab, an antitumor agent acting on the microenvironment. NOD/Shi-scid, IL-2Rγ(null) (NOG) mice were used as recipients of primary tumor cells from a patient with diffuse large B-cell lymphoma (DLBCL), which engraft and proliferate in a microenvironment-dependent manner. The lymphoma cells could be serially transplanted in NOG mice, but could not be maintained in in vitro cultures. Injection of bevacizumab together with CHOP (cyclophosphamide, doxorubicin, vincristine, prednisolone) significantly increased necrosis and decreased vascularization in the tumor, compared with CHOP alone. Levels of human soluble interleukin-2 receptor (sIL2R) in the serum of bevacizumab+CHOP-treated mice (reflecting the DLBCL tumor burden) were significantly lower than in CHOP recipients. Mice receiving bevacizumab monotherapy also showed significant benefit in terms of tumor necrosis and vascularization, as well as decreased serum sIL2R concentrations. The present DLBCL model reflects the human DLBCL in vivo environment more appropriately than current mouse models using established tumor cell lines. This is the first report to evaluate the efficacy of bevacizumab in such a tumor microenvironment-dependent model. Bevacizumab may be a potential treatment strategy for DLBCL patients.

[Structure of recombinant human serum albumin from Pichia pastoris].

The structure of recombinant human serum albumin derived from Pichia pastoris (rHSA) was analyzed in detail. Complete amino acid analysis was performed by the phenyl isothiocyanate precolumn labeling method. The amino terminal sequence was determined by the Edman degradation. The carboxyl terminal amino acid was determined by digestion with carboxypeptidase, and the carboxyl terminal peptide fragment was analyzed by electrospray mass spectrometry. The peptide fragments of rHSA digested with Lysylendopeptidase, Endoproteinase Glu-C, or Endoproteinase Asp-N were analyzed by electrospray mass spectrometry. The complete amino acid composition, the terminal sequences and the complete amino acid sequence of rHSA agreed with the primary structure deduced from its cDNA. The elution pattern of reduced and carboxymethylated rHSA digested with Lysylendopeptidase and the elution pattern of intact rHSA digested with pepsin were respectively similar to those of plasma-derived human serum albumin (pHSA). The pattern of CD spectrum of rHSA was identical in both shape and magnitude to that of pHSA. 1H-NMR spectra of rHSA and pHSA in deuterium oxide showed the same signal patterns in the observed region (delta 10.5-0.5 ppm). Cross peaks assigned to the alpha proton-beta proton of Asp-1 (delta 4.2/2.8 ppm) and the delta proton-epsilon proton of lysine residues (delta 2.8-3.2/1.4-2.0) showed the same cross peak patterns and chemical shifts in two-dimensional phase sensitive double-quantum filtered 1H-1H correlation spectra of rHSA and pHSA.

Isolation and characterization of the alkaline protease gene of Aspergillus oryzae.

The genomic DNA for the alkaline protease (Alp) of the fungus Aspergillus oryzae was isolated using synthetic oligonucleotides as hydridization probes, and the complete nucleotide sequence was identified. The Alp gene is 1374 nucleotides long and contains three introns, one of which is in the pro region and two in the mature coding region. Sequences related to the TATA box (TATAAAT) and the CAAT box (CCAAAT) were found in the 5'-noncoding region. Primer extension analysis showed that three transcriptional start points are present.

Cloning and expression in yeast of a cDNA clone encoding Aspergillus oryzae neutral protease II, a unique metalloprotease.

The neutral protease II (NpII) from Aspergillus oryzae is a zinc-containing metalloprotease with some unique properties. To elucidate its structure, we isolated a full-length cDNA clone for NpII. Sequence analysis reveals that NpII has a prepro region consisting of 175 amino acids preceding the mature region, which consists of 177 amino acids. As compared with other microbial metalloproteases, NpII is found to be unique in that it shares only a limited homology with them around two zinc ligand His residues and that the positions of the other zinc ligand (Glu) and the active site (His) cannot be established by homology. When a plasmid designed to express the prepro NpII cDNA was introduced into Saccharomyces cerevisiae and the transformant was cultured in YPD medium (2% glucose, 2% polypeptone, 1% yeast extract), it secreted a proNpII. However, in a culture of the same medium containing 0.2 mM ZnCl2, it secreted a mature NpII with a specific activity and N-terminus identical to those of native NpII. This observation suggests that either an autoproteolytic activity or a yeast protease effected the processing.

Secretion of Aspergillus oryzae alkaline protease in an osmophilic yeast, Zygosaccharomyces rouxii.

To produce Aspergillus oryzae alkaline protease (Alp) in an osmophilic yeast Zygosaccharomyces rouxii, we constructed an expression plasmid consisting of the Z. rouxii glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, the prepro-Alp cDNA of A. oryzae, the whole sequence of Z. rouxii plasmid pSR1, and the G418 resistant gene. The resulting plasmid, when introduced into Z. rouxii cells, directed the secretion of a large amount (about 300 mg/l) of Alp into the culture medium. The N-terminus and specific activity of the enzyme were identical to those of A. oryzae Alp.

A full length cDNA clone for the alkaline protease from Aspergillus oryzae: structural analysis and expression in Saccharomyces cerevisiae.

We have cloned and determined the nucleotide sequence of a cDNA fragment for the entire coding region of the alkaline protease (Alp) from a filamentous ascomycete Aspergillus oryzae. According to the deduced amino acid sequence, Alp has a putative prepro region of 121 amino acids preceding the mature region, which consists of 282 amino acids. A consensus sequence of a signal peptide consisting of 21 amino acids is found at the N-terminus of the prepro region. The primary structure of the mature region shares extensive homology (29%-44%) with those of subtilisin families, and the three residues (Asp 32, His 64 and Ser 221 in subtilisin BPN') composing the active site are preserved. The entire cDNA, coding for prepro Alp, when introduced into the yeast Saccharomyces cerevisiae, directed the secretion of enzymatically active Alp into the culture medium, with its N-terminus and specific activity identical to native Aspergillus Alp.

Three cases of untreated classical PKU: a report on cataracts and brain calcification.

There are only four previous reports of cataracts in untreated classical phenylketonuria (PKU) patients. Brain calcification, which has been known to occur in patients with dihydropteridine reductase (DHPR) deficiency, has been reported, as far as we know, in only one (albeit unusual) case. We report on three patients with untreated classical PKU; all have cataracts and normal serum levels of glucose and galactose. One of the patients (whose endocrinological tests and biochemistry including folate were normal) has bilateral brain calcifications remarkably similar to that seen in DHPR deficiency. To what extent cataracts and brain calcification occur with classical PKU is unknown because of the lack of case reports addressing these symptoms. Our cases combined with previous reports call attention to the fact that they may be frequently overlooked manifestations of classical PKU.

Identity of differentiation inducing factor and tumour necrosis factor.

Human myelogenous leukaemic cells can be induced to differentiate into the monocyte/macrophage pathway by protein inducers called differentiation inducing factors (DIF) in conditioned media of mitogen-stimulated human peripheral blood leukocytes. However, human DIF has not yet been well characterized. DIF is known to be a T-cell lymphokine, as it can be obtained from the T-cell line HUT-102 and can be partially purified from medium conditioned by phytohaemagglutinin (PHA)-stimulated lymphocytes. We found that monocytes also produce factor(s) that induce differentiation of human myelogenous leukaemia cell lines to cells with macrophage-like characteristics. This factor(s) has activity different from that of colony-stimulating factor(s) or interferons. We have now purified a DIF to homogeneity from medium conditioned by PHA-stimulated leukocytes using a human myeloblastic leukemia cell line, ML-1, as target cells. The purified DIF has a relative molecular mass (Mr) of approximately 17,000, with an NH2-terminal sequence the same as that of human tumour necrosis factor (TNF). Recombinant human TNF (rHuTNF) induces differentiation of ML-1 cells and an anti-pDIF monoclonal antibody can neutralize both differentiation inducing activity and cytotoxic activity of DIF and rHuTNF. The findings indicate that one of the DIF(s) produced by leukocytes is probably TNF.

Epidemiological studies on echovirus type 18 infection in Toyama Prefecture.

Echovirus type 18 (echo 18) was isolated from six aseptic meningitis children in Fukumitsu-machi, Toyama Prefecture, from July to August, 1980. This was the first virologically-confirmed epidemic of aseptic meningitis due to echo 18 in Japan. Epidemiological studies on the prevalence of this virus among the inhabitants in Toyama Prefecture were also performed. The results are summarized as follows. (1) Significant increases in neutralizing antibody titers against echo 18 were observed in all the paired sera of aseptic meningitis patients from whom echo 18 was isolated. (2) In October, 1980, echo 18 was also isolated from healthy children or from infants suffering from gastroenteritis in other areas of Toyama Prefecture. (3) Among the sera collected from 50 children (aged smaller than or equal to 12) in Fukumitsumachi in December, 1980, neutralizing antibodies against echo 18 were detected only in the younger groups (aged smaller than or equal to 8), 58.9% of these age groups showing a titer of 4 or higher. (4) Another epidemic around 1963 by echo 18 in Toyama Prefecture was retrospectively suggested by the examinations of sera collected in 1978 and 1981 from inhabitants in various areas of Toyama Prefecture. (5) Neutralizing antibody titers against the strain isolated, No. 35'80, were significantly higher than those against strain 'Metcalf,' the prototype of echo 18, in most sera including both aseptic meningitis patients and healthy inhabitants.

Estimation of rate constants in lysozyme-catalyzed reaction of chitooligosaccharides.

The rate constants of the cleavage of glycoside linkage, hydration (hydrolysis) and transglycosylation in a lysozyme-catalyzed reaction of substrate chitooligosaccharides were evaluated by computer analysis of the experimentally obtained reaction time-courses. In the computer analysis, the rate equation was numerically solved by use of the known binding constants for each subsite. Because of the complexity of the lysozyme-catalyzed reaction, optimal values of rate constants were determined by checking the sensitivity of each rate constant to the computed time-courses. It was not possible to estimate uniquely the rate constants for transglycosylation and hydration, owing to the nature of the enzymatic reaction, but it was possible to estimate accurately their ratio. The estimated values were 0.94 s-1 for the rate constant for the cleavage of glycosidic linkage and 133 for the ratio of rate constants of transglycosylation and hydration.

Lysozyme-catalyzed reaction of chitooligosaccharides.

The time-courses of substrate consumption and product formation in the lysozyme-catalyzed reaction were determined with (GlcNAc)4 and (GlcNAc)5 as substrate to accumulate data suitable for the estimation of rate constants by numerical analysis. The lysozyme-catalyzed reactions were followed by TLC or HPLC. (GlcNAc)4 decomposed apparently to small oligosaccharides within 5 h, and (GlcNAc)5 decomposed within 15 min at pH 5.0 and 50 degrees C. The temperature-dependence of the rate of disappearance of the initial substrate showed a different profile from that observed with glycol chitin as substrate by the reducing power method. The order (or distribution) of the amount of product formed from (GlcNAc)5 in the reaction time-course determined by TLC differed from that determined by HPLC. The relative error in HPLC was much less than that in TLC, and the time-course determined by HPLC was thought to be of sufficient accuracy for the estimation of rate constants by computer analysis.