Protein-bound polysaccharide K induced apoptosis of the human Burkitt lymphoma cell line, Namalwa.

Protein-bound polysaccharide K (PSK), which is derived from mushrooms belonging to the Basidiomycetes genus, has been clinically used as a biological response modifier (BRM) for the treatment of epithelial cancer patients in Japan and other Asian countries. There are a large number of studies on the biological activities of PSK as regards the activation of immunocompetent cells and the potential cytotoxic effects on epithelial cancer cells. However, only a few studies have been conducted to see the direct cytotoxic effects of PSK on hematological malignant cells. In this study, we investigated whether or not PSK was able to induce cellular apoptosis in hematological malignant cells. PSK was found to inhibit cell growth, and induced subsequent cellular apoptosis in the Burkkit lymphoma cell line (Namalwa), out of 33 hematological malignant cell lines tested. This PSK-induced apoptosis was neutralized by the addition of galactose to the culture medium, whereas apoptosis was augmented by treatment with beta-galactosidase, indicating the inhibitory involvement of galactose in the mechanism of action. These results provide initial evidence of the direct cytotoxic activity of PSK in a hematological malignant cell line, thus encouraging further molecular-level study of PSK-mediated apoptosis in malignant hematological cells.     

Acute elevation of interleukin 6 and matrix metalloproteinase 9 during the onset of pituitary apoplexy in Cushing’s disease

Pituitary - Pituitary apoplexy is a rare endocrine emergency. The purpose of this study is to characterize physiological changes involved in pituitary apoplexy, especially during the acute phase. A...     

Placental Elasticity as a New Non-invasive Predictive Marker of Pre-eclampsia

Point shear wave elastography is an ultrasonography technique used to evaluate tissue elasticity. We examined whether placental elasticity is useful for predicting the onset of pre-eclampsia. Two hundred twenty-one participants were divided into two groups: one group at low risk (n?=?185) and the other at high risk (n?=?36) for pre-eclampsia. The two groups were compared with respect to shear wave velocity (SWV) of the placenta. Use of SWV as a predictor of pre-eclampsia was also investigated by creating a receiver operating characteristic (ROC) curve. The ROC curve was used to set a cutoff SWV value for predicting pre-eclampsia. The SWV of the high-risk group was significantly higher than that of the low-risk group (p < 0.001). Thirteen participants developed pre-eclampsia after SWV measurements, and the SWVs of these participants were significantly higher than those of participants in who pre-eclampsia did not develop. The cutoff value and area under the ROC curve were 1.188 m/s and 0.9118, respectively. Placental elasticity was significantly increased even before the onset of pre-eclampsia onset and, thus, may be a parameter used to predict the onset of pre-eclampsia.     

Immunohistochemical study of colorectal adenocarcinomas and adenomas with antibodies against carcinoembryonic antigen (CEA), CA19-9, keratin, α-tubulin and secretory component (SC)

The immunohistochemical localization of five antibodies against carcinoembryonic antigen (CEA), CA19-9, keratin, α-tubulin and secretory component (SC) was investigated in 14 lesions of adenocarcinoma (AC), 22 of adenoma with high-grade atypia (AH), 50 of adenoma with low-grade atypia (AL), and 15 of non-neoplastic mucosa (NNM) of the large intestine. The positive patterns for each staining were divided into three categories (patterns 1,2, and 3). All neoplastic lesions (AC, AH and AL) were positive for CEA, while 85.7% of AC, 36.4% of AH and 6.0% of AL showed strongly positive staining (pattern 3). 78.6% of AC and 54.5% of AH were positive for CA19-9 in comparison to 20.0% of AL. For keratin, more than 95% ofthe neoplastic lesions were positive, while 78.6% of AC, 27.3% of AHand 22.0% of AL showed strongly positive staining (pattern 3). For a-tubulin, more than 85% of neoplstic lesions were positive, while 50.0% of AC, 36.3% of AH and 26.0% of AL showed strongly positive staining (pattern 3). For SC, in contrast, 42.9% of AC, 27.3% of AH and 8.0% of AL were negative, but 93.3% of NNM were positive. It was concluded that the positive staining rate, especially the rate of pattern 3 for each antibody correlated with the degree of atypia of the colorectal neoplastic lesions (AC, AH and AL).     

Improvement in Castleman's disease by humanized anti-interleukin-6 receptor antibody therapy

Castleman's disease, an atypical lymphoproliferative disorder, can be classified into 2 types: hyaline-vascular and plasma cell types according to the histologic features of the affected lymph nodes. The plasma cell type is frequently associated with systemic manifestations and is often refractory to systemic therapy including corticosteroids and chemotherapy, particularly in multicentric form. Dysregulated overproduction of interleukin-6 (IL-6) from affected lymph nodes is thought to be responsible for the systemic manifestations of this disease. Therefore, interference with IL-6 signal transduction may constitute a new therapeutic strategy for this disease. We used humanized anti-IL-6 receptor antibody (rhPM-1) to treat 7 patients with multicentric plasma cell or mixed type Castleman's disease. All patients had systemic manifestations including secondary amyloidosis in 3. With the approval of our institution's ethics committee and the consent of the patients, they were treated with 50 to 100 mg rhPM-1 either once or twice weekly. Immediately after administration of rhPM-1, fever and fatigue disappeared, and anemia as well as serum levels of C-reactive protein (CRP), fibrinogen, and albumin started to improve. After 3 months of treatment, hypergammaglobulinemia and lymphadenopathy were remarkably alleviated, as were renal function abnormalities in patients with amyloidosis. Treatment was well tolerated with only transient leukopenia. Histopathologic examination revealed reduced follicular hyperplasia and vascularity after rhPM-1 treatment. The pathophysiologic significance of IL-6 in Castleman's disease was thus confirmed, and blockade of the IL-6 signal by rhPM-1 is thought to have potential as a new therapy based on the pathophysiologic mechanism of multicentric Castleman's disease. (Blood. 2000;95:56-61)     

From the Cover: Folding pathway of a multidomain protein depends on its topology of domain connectivity

How do the folding mechanisms of multidomain proteins depend on protein topology? We addressed this question by developing an Ising-like structure-based model and applying it for the analysis of free-energy landscapes and folding kinetics of an example protein, Escherichia coli dihydrofolate reductase (DHFR). DHFR has two domains, one comprising discontinuous N- and C-terminal parts and the other comprising a continuous middle part of the chain. The simulated folding pathway of DHFR is a sequential process during which the continuous domain folds first, followed by the discontinuous domain, thereby avoiding the rapid decrease in conformation entropy caused by the association of the N- and C-terminal parts during the early phase of folding. Our simulated results consistently explain the observed experimental data on folding kinetics and predict an off-pathway structural fluctuation at equilibrium. For a circular permutant for which the topological complexity of wild-type DHFR is resolved, the balance between energy and entropy is modulated, resulting in the coexistence of the two folding pathways. This coexistence of pathways should account for the experimentally observed complex folding behavior of the circular permutant.Topology of protein conformation, or the spatial arrangement of structural units and the chain connectivity among them, is a key determinant of the folding mechanisms of proteins (1–5). However, predicting a folding pathway is a subtle problem when a protein comprises multiple regions of cooperative structure formation (i.e., foldons or domains). Given that a protein has n such cooperative regions and each region tends to show a two-state–like structural transition between ordered and disordered states, the protein as a whole can have 2ⁿ conformation states and multiple folding routes passing through them are allowed. The statistical weights of these folding routes should be determined both by the interactions among structural regions and the strength of cooperativity within individual regions (6). When multiple competitive routes coexist, the observed folding pathway of an ensemble of molecules should be a superposition of these routes, and the dominant folding pathway should be flexibly changed by changing the solution conditions or by mutations. The multiplicity and flexibility of pathways are important, even for small single-domain proteins like ribosomal protein S6 (7, 8), and are evident for proteins that have repeating structures (9–13). For proteins comprising multiple domains (14), the multiplicity of possible folding pathways is significant. The relative importance among 2ⁿ conformation states in the folding process in proteins with n independently foldable domains should be determined by length, structure (13), the topological connectivity of linkers between domains (3), and the interactions at the interface between domains (3, 15, 16). Fig. 1A shows an example protein for the case n = 2.Open in a separate windowFig. 1.Examples of two-domain proteins with different topological complexities. (A) Human γD-crystallin (PDB ID: 1HK0), which has two independently foldable domains connected by a single linker. (B) DHFR (PDB ID: 1rx1), which is topologically more complex, comprising two domains, DLD (blue) and ABD (pink). DLD is a discontinuous domain comprising the N- and C-terminal parts of the chain, and ABD and DLD are connected by two linkers. The positions of linkers are designated by red arrows.The above mechanism for determining folding intermediates and pathways of multidomain proteins is not applicable when domains have mutually correlated folding tendencies. In particular, the correlation between domains may be significant in a topologically complex protein, which has a domain comprising multiple discontinuous parts of a chain. For example, consider one domain, a discontinuous domain, consisting of residues 1 ≤ i ≤ N₁ and N₂ ≤ i ≤ N, and another domain, a continuous domain, consisting of residues N₁ < i < N₂. Because there is a tendency that the continuous parts of the chain form “islands” of ordered structures (17, 18) and that these continuous parts of a sequence can be the nuclei for folding, the discontinuous domain may not be an independent folding unit, but may depend on the continuous domain. In this paper, we theoretically analyze the problem of how a folding pathway is selected in multidomain proteins that have a discontinuous domain by using Escherichia coli dihydrofolate reductase (DHFR) as an example and compare it with its circular permutant that consists only of continuous domains.As shown in Fig. 1B, DHFR is a 159-residue α/β protein consisting of two domains: a discontinuous loop domain (DLD) (residues 1–37 and 107–159) and an adenosine-binding domain (ABD) (residues 38–106). Because DLD does not include a single contiguous region of the chain, but rather includes separate N- and C-terminal parts, the structural ordering of DLD can be correlated with the structural ordering of ABD. As a model protein, DHFR has been intensively investigated (19–29), which has resulted in a picture that DHFR folds along the following pathway:U→τ7I6→τ6I5→τ5IHF→τ1,…,τ4{N}.[1]Here I₆ is an intermediate exhibiting heterogeneous compactness with DLD being only partially compacted but ABD attaining a native-like compactness (19). I₆ appeared in τ₇ < 35?μs after folding was initiated from the unfolded state (U). During τ₆ ～ 550?μs, further structural development was observed both in ABD and in DLD (19), which led to I₅ in which the secondary structures were reasonably formed (20–22) and two subsets of hydrogen-bonding networks were formed in ABD and DLD (23). During τ₅ ～ 200 ms, structures of ABD and DLD were further organized, which led to the hyperfluorescent intermediate state, I_HF, consisting of four substates, I₁, …, I₄, which matured through four parallel pathways on timescales of τ₁, …, τ₄ = 1 ? 100 s to reach the four native conformers, collectively denoted by {N} in Eq. 1 (24–27). It is plausible that the slow process (several hundred seconds) during the τ₁ ? τ₄ phases is due to intense “internal friction” (30–32) in the glassy dynamics of conformation (33), including formation/disruption of nonnative contacts, the effects of proline isomerization, and the cis–trans isomerization of Gly95 and Gly96. Apart from this complexity during the last phase, the folding scheme in Eq. 1 can be regarded as a hierarchical assembly of structures that begins from the ordering of each domain at the early phase of τ₇ and proceeds to the formation of the whole protein during the later phase of τ₅ (19). Therefore, the questions are the mechanisms for how such a sequential pathway is realized in DHFR and how the topological complexity of DHFR affects the pathway selection.