Pregnancy-related pelvic girdle pain (PPP), I: Terminology, clinical presentation, and prevalence.

Pregnancy-related lumbopelvic pain has puzzled medicine for a long time. The present systematic review focuses on terminology, clinical presentation, and prevalence. Numerous terms are used, as if they indicated one and the same entity. We propose "pregnancy-related pelvic girdle pain (PPP)", and "pregnancy-related low back pain (PLBP)", present evidence that the two add up to "lumbopelvic pain", and show that they are distinct entities (although underlying mechanisms may be similar). Average pain intensity during pregnancy is 50 mm on a visual analogue scale; postpartum, pain is less. During pregnancy, serious pain occurs in about 25%, and severe disability in about 8% of patients. After pregnancy, problems are serious in about 7%. The mechanisms behind disabilities remain unclear, and constitute an important research priority. Changes in muscle activity, unusual perceptions of the leg when moving it, and altered motor coordination were observed but remain poorly understood. Published prevalence for PPP and/or PLBP varies widely. Quantitative analysis was used to explain the differences. Overall, about 45% of all pregnant women and 25% of all women postpartum suffer from PPP and/or PLBP. These values decrease by about 20% if one excludes mild complaints. Strenuous work, previous low back pain, and previous PPP and/or PLBP are risk factors, and the inclusion/exclusion of high-risk subgroups influences prevalence. Of all patients, about one-half have PPP, one-third PLBP, and one-sixth both conditions combined. Overall, the literature reveals that PPP deserves serious attention from the clinical and research communities, at all times and in all countries.

Structure of the heterodimeric complex between CAD domains of CAD and ICAD.

We present here the structure of the complex between the CAD domain of caspase activated deoxyribonuclease (CAD) and the CAD domain of its inhibitor (ICAD), determined by nuclear magnetic resonance spectroscopy. The two domains adopt a very similar fold, which consists of an alpha-helix and a beta-sheet, and are aligned side by side in the complex. Notably, the positive charges on the strand beta2 at one end of the beta-sheet of CAD and negative charges around the opposite end of the beta-sheet of ICAD are paired in the complex. Point mutations of the charged amino acids at this interface, on either CAD or ICAD, prevented formation of the functional CAD-ICAD complex. This implies that the interaction between the CAD domains of CAD and ICAD is an essential step in the correct folding of CAD in the complex.

Structure of the CAD domain of caspase-activated DNase and interaction with the CAD domain of its inhibitor.

Caspase-activated DNase (CAD), which causes a genome fragmentation at the final stage of apoptosis, is a protein of about 40 kDa and exists as a complex form with the inhibitor ICAD in living cells. There is sequence homology of about 80 amino acid residues at the N termini of CAD and ICAD (called the CAD domain). Here, we report the three-dimensional structure of the CAD domain of CAD determined by multi-dimensional NMR spectroscopy and the property of CAD domains investigated by a surface plasmon resonance experiment. The CAD domain of CAD is an independently folded domain composed of one alpha-helix and five beta-strands forming a single sheet. The overall structure is categorized in the ubiquitin superfold. This domain can bind strongly to the isolated CAD domain of ICAD (dissociation constant: 5.48(+/-0.003)x10(-8) M). It suggests the function of the CAD domains in the CAD-ICAD system, that the protein-protein interaction through the CAD domains plays an important role in the inhibition of CAD DNase activity and in the correct folding of CAD. On the basis of structural comparison with other protein complexes containing the ubiquitin superfold, the interaction mode of the CAD domains is proposed.

Position dependence of amino acid intrinsic helical propensities II: non-charged polar residues: Ser, Thr, Asn, and Gln.

The assumption that the intrinsic alpha-helical propensities of the amino acids are position independent was critical in several helix/coil transition theories. In the first paper of these series, we reported that this is not the case for Gly and nonpolar aliphatic amino acids (Val, Leu, Met, and Ile). Here we have analyzed the helical intrinsic propensities of noncharged polar residues (Ser, Thr, Asn, and Gln) at different positions of a model polyalanine-based peptide. We found that Thr is more favorable (by approximately 0.3 kcal/mol) at positions N1 and N2 than in the helix center, although for Ser, Asn, and Gln the differences are smaller (+/-0.2 kcal/mol), and in many cases within the experimental error. There is a reasonable agreement (+/-0.2 kcal/mol) between the calculated free energies, using the ECEPP/2 force field equipped with a hydration potential, and the experimental data, except at position N1.

Improved segmental isotope labeling of proteins and application to a larger protein.

A new isotope labeling technique for peptide segments in a protein sample was recently established using the protein splicing element intein [Yamazaki et al. (1998) J. Am. Chem. Soc., 120, 5591-5592]. This method makes it possible to observe signals of a selected amino (N-) or carboxyl (C-) terminal region along a peptide chain. However, there is a problem with the yield of the segmentally labeled protein. In this paper, we report an increase in the yield of the protein that enables the production of sufficient amounts of segmentally 13C/15N-labeled protein samples. This was achieved by improvement of the expression level of the N-terminal fragment in cells and the efficiency of refolding into the active splicing conformation. The N-terminal fragment was expressed as a fused protein with the cellulose binding domain at its N-terminus, which was expressed as an insoluble peptide in cells and the expression level was increased. Incubation with 2.5 M urea and 50% glycerol increased the efficiency of the refolding greatly, thereby raising the final yields of the ligated proteins. The feasibility of application of the method to a high-molecular-weight protein was demonstrated by the results for a maltose binding protein consisting of 370 amino acids. All four examined joints in the maltose binding protein were successfully ligated to produce segmentally labeled protein samples.

Comparative significance of p53 and WAF/1-p21 expression on the efficacy of adjuvant chemotherapy for resectable invasive ductal carcinoma of the pancreas.

p53 tumor-suppressor gene has a dual role as a trigger of apoptosis and as an initiator of DNA repair. The cyclin-dependent kinase inhibitor WAF/1-p21 is induced by wild-type p53 and has been implicated as a downstream mediator of the growth-suppressing and apoptosis-promoting function of wild-type p53, suggesting an impact on the effectiveness of chemotherapy. This study was designed to assess the significance of p53 and WAF/1-p21 expression in the prognosis of patients and the efficacy of adjuvant chemotherapy for resectable invasive ductal carcinoma (IDC) of the pancreas. A total of 58 patients with primary IDC of the pancreas underwent pancreatectomy between 1982 and 1996: 28 patients underwent surgery alone, and 30 patients received postsurgical adjuvant chemotherapy. p53 and WAF/1-p21 were stained immunohistochemically with anti-p53 monoclonal antibody (mAb) and anti-WAF/1-p21 mAb. p53 was positively expressed in 29 (50%) of 58 primary lesions, and p21 was expressed in 24 (41%) lesions; however, p21 expression did not necessarily correlate with p53 expression. The survival curve of the patients with p53(+) IDC was significantly lower than that of those with p53(-) IDC, and p21(+) patients showed a higher survival curve than did p21(-) patients, but this difference was not statistically significant. When p53 and p21 expression were analyzed in combination, the patients with p53(+)p21(-) IDC were found to have a significantly poorer prognosis than others. On the other hand, the survival curve of the adjuvant chemotherapy group was also higher than that of the surgery-alone group, but this difference was not significant. In a multivariate analysis, p21 expression was a significantly low risk factor for death due to IDC overall, and adjuvant chemotherapy was found to decrease the risk of death from IDC in p53(+) patients. Evaluation of expression of p53 and WAF/1-p21 may be beneficial in the prediction of the patient's prognosis as well as prediction of the effects of adjuvant chemotherapy in pancreatic cancer patients.

p53 expression affects the efficacy of adjuvant chemotherapy after resection of invasive ductal carcinoma of the pancreas.

p53 tumor suppressor gene has a dual role as a trigger of apoptosis and as an initiator of DNA repair, suggesting its involvement in the mechanisms of drug resistance or chemosensitivity. The present study assessed the implication of p53 expression in the prognosis of patients and the efficacy of adjuvant chemotherapy for resectable invasive ductal carcinoma (IDC) of the pancreas. A total of 58 patients with primary IDC of the pancreas underwent pancreatectomy between 1982 and 1996: 28 patients received surgery alone and 30 patients received postsurgical adjuvant chemotherapy. p53 protein was stained immunohistochemically with anti-p53 monoclonal antibody. p53 was positively expressed in 29 out of 58 primary lesions (50%), and the survival curve of the patients with p53 (+) pancreatic cancer is lower than that of those with p53 (-) cancer. On the other hand, the survival curve of adjuvant chemotherapy group was also higher than that of surgery alone group, and furthermore, in patients with p53 (+) cancer, the survival curve of adjuvant chemotherapy group was significantly better than that of the surgery alone group. A multivariate analysis showed that p53 expression or adjuvant chemotherapy is not a significant risk-factor for prognosis, but that adjuvant chemotherapy is a significant risk factor for the patients with p53 (+) pancreatic cancer, which suggests that p53 expression affects the efficacy of chemotherapy. p53 expression may be beneficial as an indicator for introduction of adjuvant chemotherapy in pancreatic cancer patients.

Mechanisms of cytotoxic effects of heavy water (deuterium oxide: D2O) on cancer cells.

Heavy water (deuterium oxide: D2O) contains a neutron and a proton in its hydrogen atoms and shows a variety of biologic activities different from normal light water. In the present study the cytotoxic and cytostatic activity of D2O was assessed using a BALB/c-3T3 fibroblast cell line and four human digestive organ cancer cell lines, i.e. HepG2 hepatic, Panc-1 pancreatic, KATO-3 gastric and Colo205 colonic cancer cell lines. Against four cancer cell lines, D2O showed significant cytotoxic and cytostatic effects in a MTT assay and a Trypan blue dye exclusion assay, at concentrations higher than 30% D2O. These effects were time and dose dependent, and the IC50 after 72 h of culture ranged from 20 to 30% D2O in the Trypan blue dye exclusion assay and from 30 to 50% D2O in the MTT assay. By contrast, IC50 for the 3T3 fibroblast cell line after 72 h of culture was about 15% in the Trypan blue dye exclusion assay and 50% inhibition was not achieved in the MTT assay. Furthermore, D2O was found to significantly inhibit the invasion of tumor cells in a Matrigel invasion chamber assay at concentrations higher than 10% D2O. Incubation with D2O resulted in enlargement of cells, nuclear pyknosis and vacuolization, and immunostaining studies demonstrated that D2O treatment resulted in an increase in nuclear nick-end-labeling, which indicates DNA fragmentation, in KATO-3 and HepG2 cell lines. Furthermore, the nucleic acids and protein synthesis inhibition assay suggested that the inhibition of DNA synthesis may be one of the mechanisms responsible for the antitumor effects of D2O. Furthermore, oral administration of D2O resulted in a significant inhibition of the growth of Panc-1 tumor xenografted s.c. in nude mice, but survival was not prolonged. In conclusion, D2O has cytotoxic and cytostatic activities against human digestive organ cancer cell lines, and D2O may be a potential anticancer agent.

Critical amino acid residues of AIP, a highly specific inhibitory peptide of calmodulin-dependent protein kinase II.

The importance of the individual amino acid residues of AIP (KKALRRQEAVDAL), a highly specific inhibitor of calmodulin-dependent protein kinase II (CaMKII), was studied. Replacement of Arg6, Gln7, or Ala9 by other amino acid residues produced a marked increase in the IC50 value. Leu4 and Val10 were also sensitive to replacement, but some hydrophobic amino acids could substitute for these residues. Although replacement of Ala3, Glu8, Ala12, and Leu13 by other residues produced no significant increase in the IC50, the substitution of Lys for Ala3 decreased the IC50. An AIP analog (KKKLRRQEAFDAY), in which Ala3 and Val10 were replaced with Lys and Phe, respectively, showed an IC50 value as low as 4 nM, suggesting that it is a useful tool for studying the physiological roles of CaMKII.

Solution structure of the IRF-2 DNA-binding domain: a novel subgroup of the winged helix-turn-helix family.

BACKGROUND: The transcription of interferon (IFN) and IFN-inducible genes is mainly regulated by the interferon regulatory factor (IRF) family of proteins, which recognize a unique AAGTGA hexamer repeat motif in the regulatory region of IFN genes. A DNA-binding domain of approximately 100 amino acids has been commonly found in the IRF family of proteins, but it has no sequence homology to known DNA-binding motifs. Elucidation of the structures of members of the IRF family is therefore useful to the understanding of the regulation and evolution of the immune system at the structural level. RESULTS: The solution structure of the DNA-binding domain of interferon regulatory factor-2 (IRF-2) has been determined by NMR spectroscopy. It is composed of a four-stranded antiparallel beta sheet and three alpha helices, and its global fold is similar to those of the winged helix-turn-helix (wHTH) family of proteins. A long loop (Pro37-Asp51) is found immediately before the HTH motif, which is not found in other wHTH proteins. The NMR signals of residues in this long loop, as well as the second helix of the HTH motif, are strongly affected upon the addition of the hexamer repeat DNA, suggesting that these structural elements participate in DNA recognition and binding. CONCLUSIONS: The structural similarity of the DNA-binding domain of IRF-2 with those of proteins in the wHTH family shows that the IRF proteins belong to the wHTH family, even though there is no apparent sequence homology among proteins of the two families. The sequential structure alignment program (SSAP) shows that IRF-2 has a slightly different structure from typical wHTH proteins, mainly in the orientation of helix 2. The IRF family of proteins should therefore be categorized into a subfamily of the wHTH family. The evidence here implies that the evolutional pathway of the IRF family is distinct from that of the other wHTH proteins, in other words, the immune system diverged from an evolutional stem at an early stage.

Effects of covalent dimerization on the structure and function of the carboxy-terminal fragment of neuropeptide Y.

To determine whether or not the dimeric structure of neuropeptide Y (NPY) that is found in solution is necessary for its function, we investigated the effects of covalent dimerization on the structure and function of NPY using the carboxy-terminal fragment, NPY(12-36), in which residues 12 and 31 (located at both ends of alpha-helical region) were replaced by Cys residues. Among the three species (the parallel dimer, the anti-parallel dimer, and the intramolecularly cross-linked monomer) obtained by oxidation of the fragment, the anti-parallel dimer was predominant. NMR analysis showed that both parallel and anti-parallel dimers had alpha-helices similar to that of intact NPY, suggesting that covalent dimerization might have little effect on the helical structure. A binding assay with Y2 receptors on porcine hippocampal membranes revealed that the IC50 value of the anti-parallel dimer was almost the same as that of NPY (13-36), which is known as a Y2-specific ligand. By contrast, the binding by the parallel dimer was weaker by more than one order of magnitude. Our results suggest that the formation of dimers of NPY is not essential for binding to the receptor.

Clinicopathological significance of epidermal growth factor and its receptor in human pancreatic cancer.

The expression of epidermal growth factor (EGF) and its receptor (EGF-R) was examined immunohistochemically in 60 primary and 26 metastatic lesions of pancreatic carcinoma. EGF was stained mainly in the cytoplasm of tumor cells, and EGF-R was stained mainly on the surface of cells. The expression rates of EGF and EGF-R were 28% and 43% for primary lesions, and 46% and 46% for metastatic lesions, respectively. The expression of EGF and EGF-R alone did not correlate with any clinicopathological factors such as clinical stage, tumor size, nodal involvement, histology, etc. The median survival period after pancreatectomy was 21.4 months for patients with EGF(+) cancers and 25.1 months for those with EGF (-) ones. On the other hand, the median survival period was 22.7 months for patients with EGF-R (+) cancers and 25.0 months for those with EGF-R (-) cancers. There were no significant differences in survival between groups of patients differing in EGF or EGF-R expression. When the expression of EGF and EGF-R was analysed in combination, the survival curve of patients with EGF(+) and EGF-R(+) cancers was found to be lower than that of the rest of the patients (p = 0.07), and especially the survival curve of patients with EGF(+)EGF-R(+) cancers was significantly lower than that of patients with EGF(+)EGF-R(-) cancers (p = 0.02), and EGF(-)EGF-R(+) cancers (p = 0.06). These results indicate that the expression of EGF or EGF-R alone in pancreatic cancer does not reflect the prognosis of patients; however the coexpression of EGF and EGF-R may be a beneficial prognostic factor for pancreatic cancer.

15N labeling method of peptides using a thioredoxin gene fusion expression system: an application to ACTH-(1-24).

For structure analysis of peptides by multinuclear NMR, stable isotope-labeled samples are required. A direct over-expression system by E. coli cells does not work for that purpose because of rapid degradation of the peptides and/or the mRNA in host cells. We here developed an over-expression system by means of thioredoxin gene fusion system. The fused protein composed of thioredoxin and the objective peptide was expressed in E. coli and then the peptide part was released by enterokinase. This system was successfully applied for the production of 15N-labeled human adrenocorticotropic hormone fragment (ACTH-(1-24)) as needed for multinuclear NMR analysis.

Secondary structure and folding topology of the DNA binding domain of interferon regulatory factor 2, as revealed by NMR spectroscopy.

The secondary structure elements of the DNA-binding domain of mouse interferon regulatory factor 2 [IRF-2(113)] were determined by heteronuclear multidimensional NMR spectroscopy. The sequential NOE connectivities, amide proton exchange rates, and 3JHN alpha coupling constants indicated the presence of three alpha-helical regions and four short beta-strands connected through relatively long loops. The long range NOEs indicated the four strands form an antiparallel beta-sheet and the three alpha-helices form a bundle on the sheet. The arrangement of the secondary structure elements and the overall folding topology resemble those of the DNA binding domains of bacterial activator CAP, heat shock transcription factors, and fork-head proteins, although there is no sequence homology among them.

Characterization of the DNA binding domain of the mouse IRF-2 protein.

The DNA binding domain of the interferon regulatory factor-2 protein (IRF-2) has been produced and characterized. alpha-chymotrypsin digestion of the purified IRF-2 protein bound to a synthetic binding site yields a peptide fragment of 14 K in molecular weight. N-terminal analysis of this peptide fragment showed that its sequence is the same as that of the intact IRF-2. A peptide fragment of approximately 14 K, IRF-2(113), which corresponds to the N-terminal 113 amino acids of the intact IRF-2 protein, has been expressed in a functional form in Escherichia coli. The first methionine was processed during the expression and the purified IRF-2(113) thus contains 112 amino acids. DNase I footprinting and gel retardation assaying showed that IRF-2(113) binds to a synthetic DNA having the consensus binding site and to the upstream regulatory sequence of the IFN-beta gene as intact IRF-2 does. These results showed that this peptide fragment, IRF-2(113), may be a good material for investigation of the DNA binding domain of IRF-2 and of the DNA-protein interaction.

Australian Halobacteria and their retinal-protein ion pumps.

Halophiles collected in Western Australia have been found to be examples of extremely halophilic rod-shaped archaebacteria, members of the genus Halobacterium. Most of them contain retinal proteins, and these proteins differ from one another and also from both bacteriorhodopsin (bR) and halorhodopsin [and sensory rhodopsins (sR)] isolated from Halobacterium salinarium (halobium), as revealed by their peptide maps and amino acid sequences. However, these retinal proteins still have the ability to pump protons or chloride ions in the light. These new ion pumps, designated archaerhodopsins (aR) [Mukohata et al. (1988) Biochem. Biophys. Res. Commun. 151, 1339-1345], are almost identical in terms of their molecular sizes and transient photochemical properties to the ion pumps identified previously. Differences are found in the: (1) apparent extinction coefficient of dark/light-adapted aR-2; (2) titration profiles at acidic pH of the absorption spectra of all aRs; and (3) circular dichroism spectra, which are influenced by the coexistent isoprenoid bacterioruberin. The amino acid sequences of two proton pumps from the Australian halobacteria, namely aR and aR-2, are approximately 90% homologous and both sequences are about 60% homologous with that of bR. Hydropathy plots suggest that these pumps also have a seven-helical structure similar to that of bR. The amino acid residues are highly conserved in the helical regions, in particular in the case of helices C and G (91 and 84%, respectively), among the three proton pumps.

Archaerhodopsin-2, from Halobacterium sp. aus-2 further reveals essential amino acid residues for light-driven proton pumps.

We have isolated a retinal protein which differs from bacteriorhodopsin and archaerhodopsin and pumps out as many protons in the light as those proton pumps. We tentatively named it archaerhodopsin-2. We have cloned and sequenced the gene that encodes archaerhodopsin-2. The gene consists of 780-bp nucleotides for 259 amino acids with a molecular mass of 27,937 Da. The amino acid sequence of archaerhodopsin-2 is 56% identical to bacteriorhodopsin and 88% to archaerhodopsin, with a few gaps of a few amino acids in both cases. Although the amino acid sequence of archaerhodopsin has revealed 157 conserved residues common to bacteriorhodopsin, the sequence of archaerhodopsin-2 reduces that number to 133. Of these, 38 amino acids are also common to chloride pumps and 24 to all bacterial retinal proteins known to date.

E-rosetting of guinea pig lymphocytes with lyophilized rabbit red cells after treatment with papain and glutaraldehyde.

Guinea pig lymphoid cells were reacted to form spontaneous rosettes (E-rosettes) with rabbit red blood cells before and after treatment with papain or a very low concentration (0.01%) of glutaraldehyde alone or both. The papain-glutaraldehyde-treated cells showed as high ability as papain-treated cells to rosette with lymphoid cells prepared from thymus, spleen, lymph node and peripheral blood of guinea pigs. Moreover, papain-glutaraldehyde-treated red cells were lyophilized and stored for 1 year without any change in their ability to form E-rosettes with these lymphoid cells. Binding between lymphocytes and treated or nontreated red cells was completely inhibited by specific anti-guinea pig thymocyte serum. Furthermore, a lymphocyte population enriched for B cells from lymph nodes or leukemic cells of strain 2 guinea pigs (B cells) did not show any substantial E-rosettes with nontreated or treated red cells. Scanning electron microscopy revealed no surface alteration in the treated cells when compared to those in nontreated red cells.