Proteinase inhibitor members of the SERPIN superfamily are characterized by the presence of a proteolytically sensitive reactive-site loop. Cleavage within this region results in a conformational transition from an unstable "stressed" native protein to a more stable "relaxed" cleaved molecule. In order to identify the principal molecular aspects of this transition, 1H nuclear magnetic resonance (n.m.r.) and FT-IR spectroscopy were applied to the study of four SERPINs. 1H n.m.r. spectra of approximately 20 high-field ring-current-shifted methyl signals exhibited slightly different chemical shifts in the native and cleaved forms of alpha 1-antitrypsin (alpha 1-AT), alpha 1-antichymotrypsin (alpha 1-ACT) and C1 inhibitor (C1-INH), but not ovalbumin, between 20 degrees C and 90 degrees C. Ring current calculations based on crystal co-ordinates for cleaved alpha 1-AT and alpha 1-ACT and native ovalbumin showed that these signals originate from highly localized interactions between different buried residues corresponding to alpha-helix and beta-sheet segments of the SERPIN fold. The small shift changes correspond to small relative conformational side-chain rearrangements of about 0.01 nm to 0.05 nm in the protein hydrophobic core, i.e. the tertiary structure interactions in the two forms of the SERPIN fold are well-preserved, and changes in this appear unimportant for the stabilization found after reactive centre cleavage. Fourier transform infrared (FT-IR) spectroscopic studies of the amide I band showed that the native and cleaved forms of alpha 1-AT, alpha 1-ACT and C1-INH contain 28% to 36% alpha-helix and 38% to 44% beta-sheet. Second derivative FT-IR spectra using H2O and 2H2O buffers revealed very large differences in the amide I band between the native and cleaved forms of alpha 1-AT, alpha 1-ACT and C1-INH, but not for ovalbumin. The alpha-helix band was most sensitive to 1H-2H exchange, while the beta-sheet bands were not, and greater amounts of antiparallel beta-sheet were detected in the cleaved form. 1H n.m.r. showed that polypeptide amide 1H-2H exchange was greater in the native forms of alpha 1-AT, alpha 1-ACT and C1-INH than in their cleaved forms, whereas for ovalbumin it was unchanged. The FT-IR and 1H-2H exchange data show that alterations in the secondary structure are central to the stabilization of the cleaved SERPIN structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Complement C1 Inactivator Proteins↗

PubMed1992

Potential of 13C and 15N labeling for studying protein-protein interactions using Fourier transform infrared spectroscopy.

In this study, we examine the interaction between two bacterial proteins, namely HPr and IIAmtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system, using FTIR spectroscopy. In an interaction involving a 1:1 molar ratio of these two proteins, when they are unlabeled, the overlap of absorbance of the amide I band arising from the peptide group vibrations of the two proteins is such that it is not possible to determine the contribution which each protein makes to the absorbance. Uniform 15N labeling has little effect on the frequency of the amide I band although there is a significant shift of the amide II band. However, we show that uniform (90%) 13C labeling produces a large shift of bands associated with the carbonyl moiety, especially the amide I band. This opens up windows in different regions of the infrared spectrum. Thus, when the same mixture of the two bacterial proteins is made where one of the proteins is uniformly 13C-labeled (in our case HPr), the amide I maxima of this protein shifts by approximately 45 cm-1 toward lower frequency and reveals the previously overlapped amide I band of the unlabeled IIAmtl. This application of 13C labeling shows the potential of studying protein-protein interactions using FTIR spectroscopy. With thoughtful selection of systems and labeling strategies, numerous studies with proteins should be possible. These could include, among others, enzyme-substrate and protein-ligand interactions.

Bacterial Proteins↗

PubMed1992

Synthesis and spectroscopy of membrane receptor proteins. The gamma subunit of the IgE receptor.

The high-affinity receptor for IgE is a tetrameric complex of subunits of the type alpha beta gamma 2. We report here conformational studies of the intact gamma subunit in trifluoroethanol and water/liposomes by circular dichroism and Fourier-transform infrared (FTIR) spectroscopy. In trifluoroethanol, the FTIR amide I' frequencies were consistent with two predominant conformational components, the beta-turn and alpha-helix, whilst in liposomes consisting of D2O and dimyristoylglycerophosphocholine (Myr2GroPCho), three components were observed. The third component present may contain some left-handed extended helix. Spectral simulation was carried out to demonstrate that the CD spectra were consistent with the component conformations identified from FTIR spectroscopy. The stimulated CD spectra were in excellent agreement with the experimental spectra. The intact gamma subunit conformation in trifluoroethanol was shown to possess 72% alpha-helical and 28% beta-turn conformations. In water/Myr2GroPCho liposomes the percentage of each conformational component present is 37%, 38% and 25% for the alpha-helix, beta-turn and extended structures, respectively. Assuming that the transmembrane fragment was alpha-helical, an excellent correlation was found between this derived alpha-helical content in water/liposomes (37%) and from hydrophobicity plots where the percentage of amino acids in the transmembrane domain is predicted by others to be 34%. It is suggested that the beta-turn detected by CD and FTIR was attributable to a 3(10) helix rather than a type I or type III reverse turn.

Antigens, Differentiation, B-Lymphocyte↗

PubMed1992

Spectroscopic studies of fibrinogen and its plasmin-derived fragments.

The secondary structure of human fibrinogen and its plasmin-fragments have been studied by FTIR spectroscopy. The quantitative results for fibrinogen are in good agreement with previous studies using circular dichroism spectroscopy. After treatment of fibrinogen with plasmin in buffer containing Ca2+, two major fragments are produced: fragment E (Mw 45,000) and fragment D (Mw 100,000). Fragment E is shown to contain 50% alpha-helical values, attributed to its coiled-coil portions, and minor beta-strands and turn structures. Its deuteration gives evidence of the presence of solvent-exposed alpha-helical structures. On the other hand, fragment D contains a distribution of secondary structure values of 35% alpha-helix, 29% beta-sheet segments and 17% turn structures. Fragment D itself has two domains: a portion of the original coiled-coil and also a thermally labile globular domain. The coiled-coil portion (Mw 27,000) was isolated and showed a high alpha-helical content (around 70%). The globular domain is estimated to be rich in beta-sheet structures. The spectra of fibrin clots formed in Ca(2+)-containing buffer have a lower amide I/amide II ratio than fibrinogen spectra, which is interpreted as being due to aggregation.

Fibrinogen↗

PubMed1992

Comparison of artemether and chloroquine for severe malaria in Gambian children.

Artemether is an oil-soluble methyl ether of artemesinin (qinghaosu). It has been studied extensively in China, where it has been shown to be rapidly effective in severe falciparum malaria. Nearly all the patients studied previously were adults. We have investigated the efficacy of artemether in children with moderate or severe falciparum malaria. In the preliminary study of moderately severe malaria, 30 Gambian children were randomised in pairs to receive either intramuscular artemether (4 mg/kg loading dose followed by 2 mg/kg daily) or intramuscular chloroquine ('Nivaquine') 3.5 mg base/kg every 6 h. Both drugs were well tolerated and rapidly effective. The times to parasite clearance were significantly shorter in the artemether recipients (mean 36.7 [SD 11.3] vs 48.4 [16.8] h, p less than 0.05). 43 children with severe malaria were then randomised to receive intramuscular treatment with the same regimens of artemether (n = 21) or chloroquine (n = 22) as used in the preliminary study. 8 children (19%) died. There were no significant differences between the two groups in the clinical, haematological, biochemical, or parasitological measures of therapeutic response in survivors and there was no evidence of local or systemic toxicity. Despite similar parasite counts on admission, clearance times overall were longer in severe malaria than in moderate malaria. Artemether is a well tolerated and rapidly effective parenteral treatment for severe malaria in children, and would be especially valuable in areas with chloroquine-resistant P falciparum.

Animals↗

PubMed1992

A randomized trial of intrahepatic infusion of fluorodeoxyuridine with dexamethasone versus fluorodeoxyuridine alone in the treatment of metastatic colorectal cancer.

To decrease the toxicity of hepatic arterial fluorodeoxyuridine (FUDR) administered through an Infusaid pump (Shiley Infusaid, Inc., Norwood, MA), 50 patients with liver metastases from colorectal cancer were selected randomly to receive FUDR, 0.3 mg/kg/d, for 14 of 28 days, with or without a total dose of 20 mg of hepatic arterial dexamethasone for 14 of 28 days. Patients were stratified according to the percentage of liver involvement by tumor and the perfusion pattern on macroaggrated albumin perfusion scan (MAA) scan. There was a trend toward decreased frequency of bilirubin levels in the group receiving dexamethasone plus FUDR versus the group receiving FUDR alone (9% and 30%, respectively, had a 200% or greater increase from baseline; P = 0.07). Patients in the group treated with dexamethasone and FUDR received higher doses of FUDR in the second, third, fifth, and sixth months than those receiving FUDR alone; however, this was statistically significant only in the fifth month (percentages of planned dose received: 42% and 19%, respectively; P = 0.05), and there was no overall difference for the total 6-month period. The complete and partial response rates were increased in patients receiving dexamethasone and FUDR versus FUDR alone (8% and 63% versus 4% and 36%, respectively; P = 0.03), and there was a trend toward increased survival with the addition of dexamethasone (median, 23 months and 15 months, respectively; P = 0.06). In conclusion, the use of hepatic arterial dexamethasone is associated with an increased response rate and a trend toward increased survival and decreased bilirubin levels. Therefore, the authors recommend additional investigation of the use of dexamethasone with chemotherapy to treat hepatic metastases.

Adult↗

PubMed1992

Histologic predictors of survival in acquired immunodeficiency syndrome-associated Kaposi's sarcoma.

The relationship between 22 histologic variables and survival was investigated in 93 patients with acquired immunodeficiency syndrome (AIDS)-associated Kaposi's sarcoma (KS). All the patients were homosexual men in whom KS was the initial manifestation of AIDS. All patients were followed for at least 12 months or until death. Histologic specimens of the initial KS biopsy were reviewed in a blind manner by two of the authors and were evaluated for the presence of a number of histologic features. In a univariate analysis nodular lesions of KS (upsilon patch or plaque lesions), the absence of hemosiderin, the absence of irregular vascular spaces, and the presence of spindle cell nodules were all significantly associated with increased length of survival. Two variables previously shown to be related to survival (CD4:CD8 cell ratio, initial lesion on lower extremities) were included in a multivariate analysis (Cox model) in addition to the histologic variables. Complete data were available from 85 patients. In the multivariate analysis a higher helper to suppressor T-cell ratio, initial lesion on lower extremities, presence of spindle cell nodules, and nodular histology (upsilon patch or plaque histology) were all significantly associated with increased length of survival. These data suggest that in AIDS-associated KS, as in reticuloendothelial neoplasms, histologic features may be useful in identifying prognostically different subgroups of patients.

Acquired Immunodeficiency Syndrome↗

PubMed1992

Management of vulvar melanoma.

Considerable debate centers on the optimal treatment for vulvar melanoma, as well as those clinicopathological factors influencing prognosis. We reviewed 80 patients with vulvar melanoma seen between 1949 and 1990. Primary tumors were assessed according to Chung (47 patients) and Breslow (65 patients) microstaging systems. Fifty-nine patients (76%) underwent radical vulvectomy, ten patients (13%) had a partial vulvectomy, and nine patients (12%) had a wide local excision. Fifty-six also underwent inguinal node dissection. Median follow-up was 193 months. Median survival was 63 months. Ten-year survival by Chung level was as follows: I 100%; II, 81%; III, 87%; IV, 11%; V, 33%. Ten-year survival by tumor thickness was as follows: 0.75 mm, 48%; 0.75-1.5 mm, 68%; 1.51-3.0 mm, 44%; greater than 3.0 mm, 22%. Increased depth of invasion was associated with increased incidence of inguinal node metastasis. Cox regression analysis demonstrated prognostic significance for tumor thickness (P less than 0.001), inguinal node metastasis (P less than 0.001), and older age at diagnosis (P less than 0.001). Radical vulvectomy did not seem to improve survival over less radical procedures. Based on this experience, we recommend radical local excision for patients with malignant melanoma of the vulva. Patients who have more than a superficially invasive melanoma should also have inguinal lymph node dissection.

Adolescent↗

PubMed1992

Randomized prospective trial of 5 versus 10 cycles of cyclophosphamide, doxorubicin, and cisplatin in advanced ovarian carcinoma.

Five versus ten cycles of cyclophosphamide, doxorubicin, and cisplatin (CAP) were compared in advanced ovarian carcinoma by a prospective randomized study of 78 patients, 41 receiving 5 cycles (CAP5) and 37 receiving 10 cycles (CAP10) of chemotherapy. Patients were stratified by histologic grade and size of residual disease. Cyclophosphamide, 600 mg/m2, doxorubicin, 40 mg/m2, and cisplatin, 100 mg/m2, were administered every 4 weeks for 5 or 10 cycles. Second-look laparotomy was performed to evaluate response and plan further therapy. CAP5 patients found a second-look laparotomy to have partially responded to chemotherapy were treated with 5 additional cycles of CAP. CAP10 was more toxic than CAP5 with respect to myelosuppression, hospital admissions for nadir fever, median elevation of creatinine, and degree of peripheral neuropathy. Median follow-up is 64 months. CAP5 and CAP10 were equivalent in surgically documented complete responses (34 versus 35%) and survival (P = 0.41). Twelve partial responders to CAP5 received additional CAP chemotherapy; one complete response resulted. We conclude that CAP5 is preferable to CAP10 in treatment of advanced ovarian cancer as it is equally effective and less toxic.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1992

Management of endometrial adenocarcinoma with cervical involvement.

Seventy-seven patients with endometrial cancer with cervical involvement were treated at Memorial Sloan-Kettering Cancer Center from 1968 to 1982. Fractional curettage specimens showed that 25 patients (32%) had involvement of cervical stroma and 52 (68%) had only detached fragments of carcinoma present in endocervical curettings. Six patients treated for palliation only are excluded from analysis of treatment and survival. Our preferred treatment during the time interval studied consisted of preoperative whole pelvic radiotherapy followed by modified radical hysterectomy and bilateral salpingo-oophorectomy with post-operative intravaginal brachytherapy. This regimen was well tolerated by the 37 patients so treated. The remaining patients were treated with a variety of regimens, most commonly radiotherapy and simple hysterectomy (25 patients). With a mean follow-up among survivors of 97 months, disease-free survival is 72%. Mean time to relapse was 19 months, with 90% of relapses occurring within 40 months. Initial sites of relapse included abdomen, 9 (12.6%); lungs, 8 (11.3%); brain, 2 (2.8%); lateral pelvis, 2 (2.8%); and bone, 1 (1.4%). Clinicopathologic variables significantly related to risk of relapse included presence of extrauterine disease at surgery (P = 0.0001), extent of cervical involvement as determined prior to hysterectomy (P = 0.001), depth of myometrial invasion (P = 0.005), and papillary serous histology (P = 0.0002). Treatment of endometrial cancer with cervical involvement with combinations of radiotherapy and surgery is well tolerated and results in an excellent long-term disease-free survival and a low rate of pelvic relapse.

Adenocarcinoma↗

PubMed1992

Adjuvant chemotherapy for colorectal hepatic metastases: role of route of administration and timing.

Improved results in the adjuvant and therapeutic treatment of colon cancer has led to renewed interest in the role of adjuvant chemotherapy following liver resection for colorectal hepatic metastases. However, little is known about the most effective method or timing of delivery of adjuvant chemotherapy. Sixty-nine BD-IX rats underwent a right hepatic lobectomy following tumour inoculation via a splenic injection of 10(7) K12/TRb colon cancer cells. The rats were then randomized to receive systemic FUdR (1 mg kg-1 d-1 for 7 d) or regional (hepatic artery or portal vein) FUdR (2 mg kg-1 d-1 for 7 d) immediately or 72 h following tumour injection. On Day 28, a laprotomy was performed, and tumour nodules in the liver were counted. The animals were followed to death, and at autopsy the cause of death from hepatic or extrahepatic metastases was determined. All methods of FUdR infusion were superior to no treatment. Immediate portal vein (PV) FUdR infusion delayed the appearance of hepatic tumour (P = 0.003), changed the cause of death from hepatic to extrahepatic disease (P = 0.019), and prolonged survival (P < 0.05). Infusion of FUdR via the PV 72 h later did not delay the appearance of hepatic tumours nor prolong survival. In contrast, delayed HA FUdR infusion controlled hepatic metastases (P = 0.04) and improved survival (P < 0.05).

Animals↗

PubMed1992

Does Fourier-transform infrared spectroscopy provide useful information on protein structures?

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Animals↗