Nitrogen isotopic discrimination in dietary amino acids: The threonine anomaly.

RATIONALE: The "Threonine Anomaly" relates to an observation made 25 years ago on the change in Thr nitrogen isotopic ratio in mammalian metabolism. Unlike all other amino acids, Thr in body protein is found to be depleted (rather than enriched) in 15 N relative to dietary Thr. Interpreting isotopic discrimination has become a useful source of ecological and palaeodietary information and it is desirable that the underlying processes are understood. METHODS: The principal enzyme of threonine catabolism, suggested to be responsible for the anomaly, threonine dehydratase, was prepared from rat liver. A time course of incubation of the enzyme with pure threonine was followed, and samples of residual threonine prepared for isotopic analysis by combustion in an automated carbon and nitrogen analyser coupled to a continuous flow isotope ratio mass spectrometer. RESULTS: We show experimentally, in vitro, that the enzymic reaction catabolising Thr cannot be responsible for its 15 N depletion. Plots of delta 15 N against both reaction time course and percentage completion show in fact an accelerating enrichment. CONCLUSIONS: A previously advanced suggestion that the unique catabolic mechanism for threonine was responsible for the anomalous depletion in 15 N is clearly not the case. We therefore offer alternative explanations, based on threonine's role at an organismal rather than cellular level. Copyright © 2016 John Wiley & Sons, Ltd.

Cytochrome c impaled: investigation of the extended lipid anchorage of a soluble protein to mitochondrial membrane models.

Cyt c (cytochrome c) has been traditionally envisioned as rapidly diffusing in two dimensions at the surface of the mitochondrial inner membrane when not engaged in redox reactions with physiological partners. However, the discovery of the extended lipid anchorage (insertion of an acyl chain of a bilayer phospholipid into the protein interior) suggests that this may not be exclusively the case. The physical and structural factors underlying the conformational changes that occur upon interaction of ferrous cyt c with phospholipid membrane models have been investigated by monitoring the extent of the spin state change that result from this interaction. Once transiently linked by electrostatic forces between basic side chains and phosphate groups, the acyl chain entry may occur between two parallel hydrophobic polypeptide stretches that are surrounded by positively charged residues. Alteration of these charges, as in the case of non-trimethylated (TML72K) yeast cyt c and Arg91Nle horse cyt c (where Nle is norleucine), led to a decline in the binding affinity for the phospholipid liposomes. The electrostatic association was sensitive to ionic strength, polyanions and pH, whereas the hydrophobic interactions were enhanced by conformational changes that contributed to the loosening of the tertiary structure of cyt c. In addition to proposing a mechanistic model for the extended lipid anchorage of cyt c, we consider what, if any, might be the physiological relevance of the phenomenon.

Probing the role of the conserved beta-II turn Pro-76/Gly-77 of mitochondrial cytochrome c.

The loop segment comprising residues 70-84 in mitochondrial cytochrome c serves to direct the polypeptide backbone to permit the functionally required heme Fe - S (Met-80) co-ordination. The primary sequence here is highly conserved, which is something rarely observed in surface loop segments and suggests that its purpose is more complex than its obvious structural role. The beta-II turn formed by Pro-76 and Gly-77 is postulated to be key to the redirection of the peptide backbone required to execute the loop. We assessed the importance of Pro-76 and Gly-77 by mutating 1 or both of these residues to alanine such that the range of allowable dihedral angles was altered, and this resulted in significant changes in physicochemical properties and biological activities. We observed structural perturbations using circular dichroism spectroscopy and thermal denaturation studies. Based on these changes, we propose that the Pro-76/Gly-77 beta-II turn precisely orients the 70s loop, not only to maintain the backbone orientation required for the formation of the axial heme ligand, but also to provide a complementary surface to physiological partners.

Static normal coordinate deformations of the heme group in mutants of ferrocytochrome c from Saccharomyces cerevisiae probed by resonance Raman spectroscopy.

The function of heme proteins is, to a significant extent, influenced by the ligand field probed by the heme iron, which itself can be affected by deformations of the heme macrocycle. The exploration of this field is difficult because the heme structure obtained from X-ray crystallography is not resolved enough to unambiguously identify structural changes on the scale of 10(-2) A. However, asymmetric deformations in this order of magnitude affect the depolarization ratio of the resonance Raman lines assignable to normal vibrations of the heme group. We have measured the dispersion of the depolarization ratios of four structure sensitive Raman bands (i.e., nu4, nu11, nu21, and nu28) in yeast iso-1-ferrocytochrome c and its mutants N52V, Y67F, and N52VY67F with B- and Q-band excitation. The DPR dispersion of all bands indicates the presence of asymmetric in-plane and out-of-plane deformations. The replacement of the polar tyrosine residue at position 67 by phenylalanine significantly increases the triclinic B2g deformation, which involves a distortion of the pyrrole symmetry. We relate this deformation to changes of the electronic structure of pyrrole A, which modulates the interaction between its propionate substituents and the protein environment. This specific heme deformation is eliminated in the double mutant N52VY67F. The additional substitution of N52 by valine induces a tetragonal B1g deformation which involves asymmetric changes of the Fe-N distances and increases the rhombicity of the ligand field probed by the heme iron. This heme deformation might be caused by the elimination of the water-protein hydrogen-bonding network in the heme cavity. The single mutation N52V does not significantly perturb the heme symmetry, but a small B1g deformation is consistent with our data and the heme structure obtained from a 1 ns molecular dynamics simulation of the protein.

Structural evidence for evolution of shark Ig new antigen receptor variable domain antibodies from a cell-surface receptor.

The Ig new antigen receptors (IgNARs) are single-domain antibodies found in the serum of sharks. Here, we report 2.2- and 2.8-A structures of the type 2 IgNAR variable domains 12Y-1 and 12Y-2. Structural features include, first, an Ig superfamily topology transitional between cell adhesion molecules, antibodies, and T cell receptors; and, second, a vestigial complementarity-determining region 2 at the "bottom" of the molecule, apparently discontinuous from the antigen-binding paratope and similar to that observed in cell adhesion molecules. Thus, we suggest that IgNARs originated as cell-surface adhesion molecules coopted to the immune repertoire and represent an evolutionary lineage independent of variable heavy chain/variable light chain type antibodies. Additionally, both 12Y-1 and 12Y-2 form unique crystallographic dimers, predominantly mediated by main-chain framework interactions, which represent a possible model for primordial cell-based interactions. Unusually, the 12Y-2 complementarity-determining region 3 also adopts an extended beta-hairpin structure, suggesting a distinct selective advantage in accessing cryptic antigenic epitopes.

Probing electrostatic interactions in cytochrome c using site-directed chemical modification.

This communication reports the generation of an electrostatic probe using chemical modification of methionine side chains. The alkylation of methionine by iodoacetamide was achieved in a set of Saccharomyces cerevisiae iso-1-cytochrome c mutants, introducing the nontitratable, nondelocalized positive charge of a carboxyamidomethylmethionine sulfonium (CAMMS) ion at five surface and one buried site in the protein. Changes in redox potential and its variation with temperature were used to calculate microscopic effective dielectric constants operating between the probe and the heme iron. Dielectric constants (epsilon) derived from deltadeltaG values were not useful due to entropic effects, but epsilon(deltadeltaH) gave results that supported the theory. The effect on biological activity of surface derivatization was interpreted in terms of protein-protein interactions. The introduction of an electrostatic probe in cytochrome c often resulted in marked effects on activity with one of two physiological partners: cytochrome c reductase, especially if introduced at position 65, and cytochrome c oxidase, if at position 28.

Phospholipid-cytochrome c interaction: evidence for the extended lipid anchorage.

Binding of cytochrome c (cyt c) to fatty acids and acidic phospholipid membranes produces pronounced and essentially identical changes in the spectral properties of cyt c, revealing conformational changes in the protein. The exact mechanism of the interaction of fatty acids and acidic phospholipids with cyt c is unknown. Binding of cyt c to liposomes with high contents (mole fraction X > 0.7) of acidic phospholipids caused spectral changes identical to those due to binding of oleic acid. Fluorescence spectroscopy of a cyt c analog containing a Zn(2+) substituted heme moiety and brominated lipid derivatives (9,10)-dibromostearate and 1-palmitoyl-2-(9,10)-dibromo-sn-glycero-3-phospho-rac-glycerol demonstrated a direct contact between the fluorescent [Zn(2+)-heme] group and the brominated acyl chain. These data constitute direct evidence for interaction between an acyl chain of a membrane phospholipid and the inside of the protein containing the heme moiety and provide direct evidence for the so-called extended-lipid anchorage of cyt c to phospholipid membranes. In this mechanism, one of the phospholipid acyl chains protrudes out of the membrane and intercalates into a hydrophobic channel in cyt c while the other chain remains in the bilayer.

Surgery without adjuvant chemotherapy for early epithelial ovarian carcinoma after comprehensive surgical staging.

From January 1981 to December 1987, 82 patients with carcinoma of the ovary, Stages 1A, 1B, and 1C (cytology negative) (FIGO 1988), were enrolled in this study following accurate surgical staging. No patient received adjunctive therapy and all were followed from 2 to 6 years with a mean duration of follow-up of 4 years. Sixty-eight patients were eligible for review--thirty-nine Stage 1A, six Stage 1B, and twenty-three Stage 1C (twenty-one with tumor rupture, two with excrescences). The mean age was 48.8 years. Three patients had a recurrence of their disease (one death). Forty patients in this series were Stage 1A or 1B (well or moderately well differentiated, no excrescences, no rupture). Only 1 patient in this group (with clear cell carcinoma) has recurred, suggesting that this patient population can be followed without adjunctive therapy. Adhesions or rupture in this series did not affect outcome. Clear cell tumors may have an ominous prognosis despite apparent local disease.

The management of minor degrees of cervical dysplasia associated with the human papilloma virus.

This report attempts to define further the natural history of minor degrees of cervical dysplasia associated with human papilloma virus (HPV). Five hundred and twenty-five patients with a diagnosis of mild to moderate cervical dysplasia and HPV effects were followed without treatment for six months to five years. Those patients who progressed to a greater degree of dysplasia were removed from follow-up and treated appropriately. Those patients who regressed to a non-dysplastic state were returned to the original referring physician to be followed with annual cytology. Regression to a non-dysplastic state was 30.5 percent at six months, 50.4 percent at 1 1/2 years, 60.5 percent at 2 1/2 years, 70.9 percent at 3 1/2 years, 77.3 percent at 4 1/2 years, 7.8 percent have progressed to a greater degree of cervical dysplasia, removed from follow-up, and treated, and 22 patients have recurred, all with minor degrees of dysplasia. No invasive cancer has been diagnosed in this group of patients. From these results, we conclude that patients with minor degrees of dysplasia associated with HPV can be followed in a routine screening program with the anticipation that the great majority will, over time, convert to a non-dysplastic state. A small number of patients will progress to a higher degree of dysplasia and will be effectively identified, to be referred for colposcopic assessment and appropriate treatment.

A randomized, double-blind, placebo-controlled, cross-over trial to assess the side effects of medroxyprogesterone acetate in hormone replacement therapy.

Cyclic progestin therapy has been widely advocated as an adjunct to postmenopausal estrogen replacement therapy to reduce the risk of endometrial carcinoma. Acceptance of this approach, however, appears to have preceded detailed evaluation of possible adverse side effects of progestins that could result in patient noncompliance. We evaluated the nonmenstrual physical and psychological side effects of oral medroxyprogesterone acetate given in conjunction with transdermal estrogen in two groups of women with previous hysterectomy and oophorectomy. Twenty-four women with prospectively documented severe premenstrual syndrome (PMS) before surgery and 24 women with no such history of adverse premenstrual changes received transdermal estrogen 100 micrograms on days 1-25 and either oral medroxyprogesterone acetate 10 mg daily or an identical placebo (days 12-25) in a randomized, double-blind, cross-over design. Mood and physical symptoms were monitored prospectively, using daily self-ratings on the Daily Symptoms Checklist. The Beck Depression Inventory and Premenstrual Tension Self-Rating Scale were completed on day 24. At the study's completion, the patients were asked which treatment period they preferred. Paired comparisons did not reveal any significant differences, and preference for treatment was equally divided between medroxyprogesterone acetate and placebo. We conclude that addition of medroxyprogesterone acetate 10 mg/day for 14 days to cyclic transdermal estrogen therapy (days 1-25) produces no consistent adverse physical or psychological effects on women for one cycle of treatment, regardless of their PMS history.

Cervical dysplasia and human papillomavirus.

Evidence suggests a link between human papillomavirus infections and cervical dysplasia-carcinoma. This has led to the recommendation that when human papillomavirus infections are identified on the cervix, especially in association with dysplasia, these patients should have colposcopic assessment and treatment. The natural history of cervical human papillomavirus infections, particularly when associated with mild or moderate dysplasia, is not clear. To assess this question, 235 women with mild to moderate cervical dysplasia associated with a human papillomavirus infection were prospectively evaluated at 6-month intervals by the Colposcopy Clinic. This review suggests that mild to moderate cervical dysplasia associated with human papillomavirus infection eventually spontaneously disappears or, at worse, lingers on over many months in a mild to moderate form. These observations do not support the current popular practice of treating all mild to moderate cervical dysplasias associated with human papillomavirus infection or cervical human papillomavirus infection not associated with dysplastic lesions.

Advanced carcinoma of the ovary with central nervous system relapse.

Interdisciplinary protocols for management of advanced adenocarcinoma of the ovary have resulted in prolonged patient survival. A subset of patients is emerging in whom central nervous system (CNS) relapse occurs even following negative second-look procedures (SLP). Seven of 342 eligible patients entered in a National Cancer Institute of Canada Trial for Ovarian Cancer, from February 1, 1980 to March 31, 1984, had CNS relapse. All patients received adriamycin and cisplatin. SLP was performed in 5 patients, 3 of whom were complete responders (CR). Two additional patients failed to complete their chemotherapy and had progressive pelvic disease. The median age of these 7 patients was 57 years, their overall survival time was 28 months, compared with an average age of 58 years and survival of 21.6 months, for the entire group. Two patients had prolonged survival after their CNS relapse; 1 patient lived 26 months, and the other, who underwent craniotomy for primary management of the metastasis survived 18 months. Confirmation of metastatic disease was obtained in 4 of the 7 patients. The results of this study suggest that management of CNS involvement in adenocarcinoma of the ovary should be determined by overall performance status even in the presence of generalized disease.

Toxicity of abdominopelvic radiation in advanced ovarian carcinoma patients after cisplatin/cyclophosphamide therapy and second-look laparotomy.

Twenty-seven advanced ovarian carcinoma patients who had received six courses of cyclophosphamide/cisplatin and had either microscopic disease (15 patients) or no pathologically detectable disease (12 patients) after second-look laparotomy were treated with abdominopelvic radiation (2250 cGy to the abdomen and pelvis and a 2250-cGy pelvic boost). Acute myelosuppression or gastrointestinal toxicity prevented completion of treatment in only three patients. However, bowel obstruction occurred in 13 (48%), ten of whom required surgery. Five of these ten had recurrent tumor, but the other five did not. Subsequently two of the latter five did develop a recurrence, one in the lung and one in the liver. A third patient died as an indirect result of radiation damage to the bowel. Median follow-up duration is 17 months from completion of radiation. So far, 13 (48%) have developed progressive disease: four (33%) of the 12 who had a negative second-look laparotomy and nine (60%) of the 15 who had microscopic disease before radiation. While acute toxicity is tolerable, the incidence of serious chronic bowel toxicity is high. Efforts should be made to alter this therapy in order to decrease the frequency of long-term morbidity.

Presurgical prognostic factors in carcinoma of the cervix, stages IB and IIA.

Possible prognostic factors in early stage cervical cancer include patient age, tumor size, cell type, differentiation, and channel invasion. In this study each of these factors was evaluated based only on the findings available before surgery, and the observations are compared with patient survival and incidence of node metastases. One hundred consecutive patients with stage IB or IIA cervical cancer treated by primary radical surgery and followed at least 2 years are reported. Disease-free survival was 90% at 2 years and 85% at 5 years; 19% had node metastases. Of the factors studied, only age greater than 50 years was associated with poor prognosis (p less than 0.02 versus age less than 50 years). Only large tumor size was associated with increased node metastases (p less than 0.001 versus medium and small size). Tumor cell type, differentiation, and channel involvement had no bearing on survival or node metastases. Because older age and large tumors appear to be factors of poor prognosis for surgery, and yet it is not clear that these patients fare better with radiotherapy, we suggest a prospective trial of radiotherapy versus surgery for this group.

A predictive index of cure versus no cure in advanced ovarian carcinoma patients--replacement of second-look laparotomy as a diagnostic test.

Three hundred forty-two Stage III and IV epithelial ovarian carcinoma patients received cytoreductive surgery followed by Adriamycin and cisplatin, 50 mg/m2 each, q 4 weeks for 9 courses. One hundred ninety-seven were clinically NED at completion of treatment and 173 of these 197 had a second-look laparotomy. One hundred twenty had persistent disease. Fifty-three were second-look negative and had no further treatment. Thirty of these latter patients relapsed--all (with one exception) within 2 years. Those not relapsing after negative second-look are considered "cured" (median follow-up 42 months, range 24-68 months) and all others "failures." Stage was a significant predictor of treatment failure--there were no Stage IV "cures." In Stage III patients, age and largest residual tumor diameter post initial surgery were significant predictors of failure. Performance status was marginally significant. In our series, any patient with Stage IV disease or Stage III disease with at least two of the following three poor prognostic factors had a chance of cure of 2.2% (2 "cures" out of 90 patients): age greater than 60 years, macroscopic residual initially, or initial performance status of 2 or 3. Under normal circumstances a second-look procedure to identify persistent disease in this group of patients does not appear justified.

Second-line chemotherapy of stage III-IV ovarian carcinoma: a randomized comparison of melphalan to melphalan and hexamethylmelamine in patients with persistent disease after doxorubicin and cisplatin.

A total of 205 women with stage III or IV ovarian cancer who had persistent disease after initial treatment with doxorubicin and cisplatin were randomized to receive melphalan (8 mg/m2 orally for 4 days) or the combination of melphalan (6 mg/m2 for 4 days) and hexamethylmelamine (120 mg/m2 for 14 days) every 4 weeks. Only one of 64 patients with measurable disease had an objective response. The major determinants of survival after randomization were the amount of residual disease after initial chemotherapy and the type of response to initial chemotherapy. There was no overall difference in survival between the two chemotherapy regimens, but the small group of patients whose disease progressed on initial chemotherapy survived significantly longer when treated with the two-drug combination. Neither of these regimens provided effective therapy for women whose disease was not eliminated by first-line treatment. However, the superior results obtained in one subgroup with the addition of hexamethylmelamine suggest that the place of this agent in treating ovarian cancer should be carefully evaluated.

Treatment of cervical intraepithelial neoplasia with electrocautery: a report of 776 cases.

From 1973 to 1984, 776 patients with cervical intraepithelial neoplasia were treated with outpatient electrocautery (hot cautery) without anesthesia. Of these, 726 (94%) were available for follow-up in 3 to 6 months. An initial cure rate with one treatment of 89% to 90% was achieved. Cure rates were similar for all degrees of dysplasia, including carcinoma in situ. There were no complications. All patients with failure of the initial treatment who returned for further outpatient management were eventually cured with use of electrocautery. Long-term follow-up rates ranged from 75% at 1 year to 46% at 5 years. There were few late recurrences, most of which were treated again (successfully) with electrocautery. Electrocautery produces cure rates similar to those for other forms of conservative management and may be the most cost-effective method of management of cervical intraepithelial neoplasia.

Cervical carcinoma in women aged 34 and younger.

This presentation addresses three questions concerning invasive cervical carcinoma in women 34 years of age and younger. Is there an increase in the incidence of the disease? Is it more or less susceptible to prevention by cervical screening? Is the clinical behavior different for this age group? Three separate studies are reported: (1) Incidence data in the younger age group have been reviewed at the national, provincial, and local levels. (2) Cytologic screening histories of 125 patients who subsequently developed cervical carcinoma were reviewed. (3) The clinical histories of 121 women 34 years of age and younger, with invasive cervical carcinoma, were reviewed and compared with those of 242 control women 35 years of age and older. Results indicate an increase in incidence in the younger age groups in the three prairie provinces only. Cytologic histories are similar except for an increase in false negative reports in the younger age group. Clinical behavior of the disease is similar for both age groups.

Treatment of cervical intraepithelial neoplasia with electrocautery: report of 426 cases.

Four hundred twenty-six patients with colposcopically directed, biopsy-proved, mild, moderate, or marked dysplasia or carcinoma in situ of the cervix were treated with outpatient electrocauterization, from 1973 to mid- 1982. Of these, 413 were available for follow-up between 3 and 6 months. The initial cure rate was 85.95%, and the cure rate when a second treatment for initial failures was included was 96.6%. These patients have been followed for 1 to 5 years, and the annual follow-up rate has ranged from 65% to 80%. Recurrences were few, but did occur as late as 3 years after treatment.

The cytologic history of 245 patients developing invasive cervical carcinoma.

A review was made of the cytologic history of 245 patients who developed invasive carcinoma of the cervix, and three groups of patients were identified. Group 1 included 149 patients (60.8%) who had never had a cervical cytologic examination, group 2 included 26 patients (10.6%) whose cytologic history in terms of frequency of examination and or timing was considered to be unsatisfactory, and group 3 included 70 patients (28.6%) whose cytologic history was considered to be satisfactory. Only 53 (35.6%) of the patients in group 1 had Stage I disease. Stage I disease was present in 16 patients (61.5%) of group 2 and in 55 patients (78.6%) of group 3. There was no significant difference between the three groups with respect to site of residence or access to the health care system. Of the patients in group 3, 20 (28.6%) had normal cytologic findings and 50 (71.4%) had abnormal cytologic findings. Of the 50 patients with abnormal cytologic findings, 31 (62.0%) did not have further examination for 6 months or longer after identification of the abnormal smear. The majority of these patients had minor degrees of cytologic abnormalities. A review of 299 original cervical smears revealed that 52 (17.4%) had been significantly undercalled, but only 21 (7.0%) had been undercalled as normal. In this study, 28 patients (11.4%) developed adenocarcinoma. In these patients, staging was unrelated to screening.