Calorimetric determination of the enthalpy change for the alpha-helix to coil transition of an alanine peptide in water.

The enthalpy change (delta H) accompanying the alpha-helix to random coil transition in water has been determined calorimetrically for a 50-residue peptide of defined sequence that contains primarily alanine. The enthalpy of helix formation is one of the basic parameters needed to predict thermal unfolding curves for peptide helices and it provides a starting point for analysis of the peptide hydrogen bond. The experimental uncertainty in delta H reflects the fact that the transition curve is too broad to measure in its entirety, which precludes fitting the baselines directly. A lower limit for delta H of unfolding, 0.9 kcal/mol per residue, is given by assuming that the change in heat capacity (delta Cp) is zero, and allowing the baseline to intersect the transition curve at the lowest measured Cp value. Use of the van't Hoff equation plus least-squares fitting to determine a more probable baseline gives delta H = 1.3 kcal/mol per residue. Earlier studies of poly(L-lysine) and poly(L-glutamate) have given 1.1 kcal/mol per residue. Those investigations, along with our present result, suggest that the side chain has little effect on delta H. The possibility that the peptide hydrogen bond shows a correspondingly large delta H, and the implications for protein stability, are discussed.     

Changing osteocalcin concentrations during pregnancy and lactation: implications for maternal mineral metabolism

We measured serum osteocalcin concentrations in 82 pregnant and 21 nonpregnant women. Osteocalcin values declined in the second trimester, but returned to nonpregnant levels late in the third trimester. The mean serum osteocalcin concentration in 36 women during pregnancy (mean gestation, 26 weeks) of 2.8 ng/mL was significantly lower than that in nonpregnant women (6.4 ng/mL; P less than 0.001) or term pregnant women at delivery (6.1 ng/mL; n = 46). Serum immunoreactive PTH (iPTH) levels were significantly higher during pregnancy than in nonpregnant women [97 +/- 5 vs. 56 +/- 4 ng/L (mean +/- SE); P less than 0.001]. No significant correlations were found between maternal osteocalcin concentrations and serum phosphorus, alkaline phosphatase, or iPTH, but significant negative correlations were found between osteocalcin and total calcium or total protein. Osteocalcin concentrations in midtrimester amniotic fluid were very low (mean, 0.3 +/- 0.1 ng/mL; n = 11). In 29 lactating mothers, the mean serum osteocalcin level was 9.5 +/- 1.5 ng/mL, significantly higher than in any of the other groups (P less than 0.05), but their serum calcium and iPTH levels were normal. There was no correlation between serum osteocalcin and calcium or iPTH concentrations in lactating women. These changes are compatible with a sequence in which bone turnover is reduced during early pregnancy, rebounds in the third trimester, and increases in postpartum lactating women.     

Assessment of two alternative sample transport and fixation methods in the microbiological diagnosis of bacterial vaginosis

BACKGROUND:

The standard method for specimen collection and transport for microbiological diagnosis of bacterial vaginosis is an air-dried smear of vaginal secretions, promptly heat- or alcohol-fixed, Gram-stained and scored by Nugent''s criteria.

OBJECTIVE:

Two alternative methods are evaluated: sending a swab in transport medium to be smeared and Gram-stained in the laboratory two days later; and sending a smear of vaginal secretions sprayed with cytological fixative to the laboratory for Gram staining seven days later.

METHODS:

One hundred fifty-two women aged 18 years and older who attended a hospital colposcopy clinic or a community healthy sexuality clinic were studied. This was a prospective study: three vaginal swabs were taken from each patient and handled as described above. Each slide was blindly and independently interpreted by two microbiology technologists. The sensitivity, specificity and coefficient of agreement of the transported swab and cytologically fixed methods were compared with the air-dried smear method.

RESULTS:

Smears from swabs in transport medium and cytologically fixed smears both had 90% sensitivity and 97% specificity for bacterial vaginosis compared with diagnosis from air-dried smears. Cohen''s kappa was 0.88 (95% CI 0.79 to 0.97) for each method. Inter-rater reliability assessed over all slides (all sampling techniques) was excellent (kappa 0.94).

CONCLUSIONS:

For the diagnosis of bacterial vaginosis, both alternative techniques provide results equivalent to air-dried direct smears. A vaginal smear sprayed with cytological fixative provides immediate fixation of material to the slide, permits delays in swab transport and avoids the requirement for transport at a controlled temperature imposed by swabs.Key Words: Bacterial vaginosis, Sampling techniquesClinical diagnosis of bacterial vaginosis is based on Amsel et al''s (1) criteria: homogenous watery discharge, amine odour with application of 10% potassium hydroxide, pH greater than 4.5 and clue cells on a wet mount of vaginal discharge. Clinical diagnosis without laboratory confirmation is accepted or even optimal (2,3) . However, it requires a good microscope at hand, microscopy expertise, reagents, time and tolerance of the unpleasant odour produced; it is also subjective (3). In actual practice, laboratory diagnosis is widely used instead, because it is easier and more convenient for the clinician (4) and provides an objective, reproducible result. The standard procedure for the microbiological test is to prepare a direct smear of vaginal discharge, air-dry the smear, and send it to a laboratory for heat or alcohol fixation, Gram staining and scoring of bacterial types by Nugent et al''s (5) criteria. This technique correlates well with Amsel et al''s clinical criteria (5,6) .However, instead of sending an air-dried smear, clinicians commonly send a vaginal swab in transport medium to a laboratory for diagnosis of bacterial vaginosis. While this substitute for the direct smear of vaginal secretions is widely used in practice (4; personal communications - Ottawa Hospital, Ottawa, Ontario; McGill University Hospital Centre, Montreal, Quebec; Gamma Dynacare Medical Laboratories, Ottawa, Ontario), its accuracy and comparability to the direct smear have not been adequately studied. Furthermore, swabs in transport medium should be maintained at a controlled temperature and processed within 24 h of collection, which may be logistically difficult in rural areas or private clinics, particularly given our extremes of climate.We sought to devise a new, simple, inexpensive sample collection method that would allow maximal flexibility in the transportation of specimens to a laboratory. One of the authors (MS) devised an alternate approach: take a sample of vaginal secretions with a cotton-tipped applicator, smear thinly onto a slide, and spray immediately with cytological fixative aerosol (Cytoprep, Fisher Scientific, Ontario). This contains isopropyl alcohol, propane and isobutane. This is the same fixative used for Pap smears. Preservation of the cytologically fixed slide is permanent. In a pilot study of 10 subjects, results of Nugent scoring obtained using vaginal smears sprayed with cytological fixative immediately after collection and Gram-stained one week later were compared with those from the standard preparation, an air-dried vaginal smear promptly heat-fixed and Gram-stained. The interval of one week was chosen as the longest time likely to be actually incurred in specimen transport. The preservation and staining of Gram-positive and -negative bacteria were excellent in both groups of slides, and diagnoses of bacterial vaginosis were concordant.We now present the results of a larger, prospective study. We compare Nugent scores obtained using our ''user-friendly'' method of a smear made directly from a vaginal swab and sprayed immediately with cytological fixative, sent to the laboratory seven days later for Gram staining; scores obtained using a vaginal swab placed in modified Amies clear transport medium and sent to the laboratory two days later; and scores from the reference air-dried smear of vaginal secretions, sent the same day to the laboratory for heat fixation and Gram staining.     

Role of proline isomerization in folding of ribonuclease A at low temperatures

In unfolded RNase A there is an interconversion between slow-folding and fast-folding forms (U(S) right harpoon over left harpoon U(F)) that is known to show properties characteristic of proline isomerization in model peptides. Here, we accept the evidence that U(S) molecules contain nonnative proline isomers and we ask about the isomerization of these proline residues during folding. The U(S) right harpoon over left harpoon U(F) reaction in unfolded RNase A is used both to provide data on the kinetics of proline isomerization in the unfolded protein and as the basis of an assay for measuring proline isomerization during folding.The tyrosine-detected folding kinetics at low temperatures have been compared to those of proline isomerization in unfolded RNase A. The comparison is based on the recent observation that the U(S) right harpoon over left harpoon U(F) kinetics are independent of guanidinium chloride concentration, so that they can be extrapolated to low guanidinium chloride concentrations, at which folding takes place. At 0 degrees C the tyrosine-detected folding reaction is 100-fold faster than the conversion of U(S) to U(F) in unfolded RNase A. Consequently, the folding reaction is not rate-limited by proline isomerization as it occurs in unfolded RNase A.An assay is given for proline isomerization during folding. The principle is that native RNase A yields U(F) on unfolding, whereas protein molecules that still contain nonnative proline isomers yield U(S). Unfolding takes place at 0 degrees C, at which proline isomerization is slow compared to unfolding. This assay yields two important results: (i) The kinetics of proline isomerization during folding are substantially faster than in unfolded RNase A-e.g., 40-fold at 0 degrees C. The mechanism of the rate enhancement is unknown. (ii) At low temperatures (0-10 degrees C), and also in the presence of (NH(4))(2)SO(4), the tyrosine-detected folding reaction occurs before proline isomerization and yields a folded intermediate I(N) that is able to bind the specific inhibitor 2'-CMP. The results demonstrate that a folding intermediate is spectrally detectable when folding occurs at low temperatures. They suggest that low temperatures provide suitable conditions for determining the kinetic pathway of folding by characterizing folding intermediates.     

Second malignancies after treatment for laparotomy staged IA-IIIB Hodgkin's disease: long-term analysis of risk factors and outcome

The survival of patients with Hodgkin's disease has dramatically improved over the past 30 years because of advances in treatment. However, concern for the risk of long-term complications has resulted in a number of trials to evaluate reduction of therapy. The consequences of these trials on recurrence, development of long-term complications, and survival remain unknown. One major consequence of successful treatment of Hodgkin's disease is the development of second malignant neoplasms. We sought to determine the factors most important for development of second tumors in pathologically staged and treated Hodgkin's disease patients followed for long intervals to provide background information for future clinical trials and guidelines for routine patient follow-up. Between April 1969 and December 1988, 794 patients with laparotomy staged (PS) IA-IIIB Hodgkin's disease were treated with radiation therapy (RT) alone or combined radiation therapy and chemotherapy (CT). There were 8,500 person-years of follow-up (average of 10.7 person-years per patient). Age and gender-specific incidence rates were multiplied by corresponding person-years of observation to obtain expected numbers of events. Observed to expected results were calculated by type of treatment, age at treatment, sex, and time after Hodgkin's disease. Absolute (excess) risk was expressed as number of excess cases per 10,000 person-years. Seventy-two patients have developed a second malignant neoplasm. Eight patients developed acute leukemia, 10 had non-Hodgkin's lymphoma (NHL), and 53 patients developed solid tumors at a median time of 5 years, 7.25 years, and 12.2 years, respectively, after Hodgkin's disease. One patient developed multiple myeloma 16.5 years after Hodgkin's disease. The relative risk (RR) of developing a second malignancy was 5.6. The absolute excess risk per 10,000 person-years (AR) of developing a second malignancy was 69.6 (7.0% excess risk per person per decade of follow-up). The highest RR occurred for the development of leukemia (RR = 66.2), however because of the low expected risk, the AR was only 9.3. The RR of solid tumors after Hodgkin's disease was lower (4.7); however, the AR was greater (49) than for acute leukemia. Among the solid tumors, breast, gastrointestinal, lung, and soft tissue cancers had the highest absolute excess risks. The risk for developing breast cancer after Hodgkin's disease was greatest in women who were under the age of 25 at treatment. The most significant risk factor for the development of both leukemia and solid tumors was the combined use of radiation therapy and chemotherapy. The RR following RT alone was 4.1 (AR = 51.1); for RT + CT (initially or at relapse) the RR was 9.75 (P < 0.05, nonoverlapping confidence limits, AR = 123.9). Survival following development of a second malignancy was poor in patients with leukemia, gastrointestinal tumors, lung cancer, and sarcoma. Survival from other malignancies including NHL and breast cancer was more encouraging. Second malignant neoplasms are a major cause of late morbidity and mortality following treatment for Hodgkin's disease. The most significant risk factor for the development of second tumors is the extent of treatment for Hodgkin's disease. Recommendations are presented for both prevention and early detection of these tumors.     

Nuclear ribosomal DNA evidence for a western North American origin of Hawaiian and South American species of Sanicula (Apiaceae)

Results from phylogenetic analysis of nuclear rDNA internal transcribed spacer (ITS) sequences from a worldwide sample of Sanicula indicate that Hawaiian sanicles (Sanicula sect. Sandwicenses) constitute a monophyletic group that descended from a western North American ancestor in Sanicula sect. Sanicoria, a paraphyletic assemblage of mostly Californian species. A monophyletic group comprising representatives of all 15 species of S. sect. Sanicoria and the three sampled species of S. sect. Sandwicenses was resolved in all maximally parsimonious trees, rooted with sequences from species of Astrantia and Eryngium. All sequences sampled from eastern North American, European, and Asian species of Sanicula fell outside the ITS clade comprising S. sect. Sanicoria and S. sect. Sandwicenses. A lineage comprising the Hawaiian taxa and three species endemic to coastal or near-coastal habitats in western North America (Sanicula arctopoides, Sanicula arguta, and Sanicula laciniata) is diagnosed by nucleotide substitutions and a 24-bp deletion in ITS2. The hooked fruits in Sanicula lead us to conclude that the ancestor of Hawaiian sanicles arrived from North America by external bird dispersal; similar transport has been hypothesized for the North American tarweed ancestor of the Hawaiian silversword alliance (Asteraceae). Two additional long-distance dispersal events involving members of S. sect. Sanicoria can be concluded from the ITS phylogeny: dispersal of Sanicula crassicaulis and Sanicula graveolens from western North America to southern South America.     

Persistent demographic differences in colorectal cancer screening utilization despite Medicare reimbursement

Background  

Colorectal cancer screening is widely recommended, but often under-utilized. In addition, significant demographic differences in screening utilization exist. Insurance coverage may be one factor influencing utilization of colorectal cancer screening tests.     

Association of NEDD4L ubiquitin ligase with essential hypertension

NEDD4L is a ubiquitin ligase that controls cell surface expression of kidney epithelial Na+ channels by ubiquitin-mediated endocytosis and lysosome targeting. Thus, it is a significant determinant of Na+ reabsorption in the distal nephron. The NEDD4L gene is located on human chromosome 18q21 within several blood pressure quantitative trait loci, including those for familial orthostatic hypotension, essential hypertension, pulse pressure, and systolic blood pressure response to postural challenge. Because of the importance of NEDD4L to Na+ balance, many of these studies have proposed that mutations in NEDD4L may be responsible for these blood pressure phenotypes. To test this hypothesis, we fine-mapped the NEDD4L region in 2 families with orthostatic hypotension, which we previously reported to be linked to human chromosome 18q21 but failed to implicate NEDD4L in these families. We also typed multiple NEDD4L single-nucleotide polymorphisms (SNPs) in a collection of US whites, Greek whites, and African-Americans individuals with essential hypertension. A significant association between several SNPs and hypertension was observed in all 3 populations. One of the SNPs associated in African Americans is known to result in premature truncation of the NEDD4L protein. Thus, genetic variation in NEDD4L may play a role in the development or progression of some forms of abnormal blood pressure.     

Effects of a low calcium environment on luteinizing hormone biosynthesis in cultured rat anterior pituitary cells

The purpose of this study was to investigate the effects of lowering the extracellular calcium concentration on GnRH-stimulated LH glycosylation and LH translation, as measured by the incorporation of [3H]glucosamine (3H-Gln) and [35S]methionine (35S-Met) into immunoprecipitable LH. Cultured anterior pituitary cells, previously exposed to estradiol (5 X 10(-10) M) to maximize precursor incorporation were incubated for 4 h in normal calcium (2.5 mM) or low calcium medium (less than 15 microM) containing radiolabeled precursors with or without 1 nM GnRH. In the presence of normal calcium, GnRH significantly increased 3H-Gln-labeled LH in the medium (278%) and cells (290%), as well as total (cells plus medium) 3H- Gln LH (280%) compared to the control value (no GnRH). GnRH also significantly increased the 35S-Met LH released into the medium (164%) and total 35S-Met LH (186%) over control values. Depletion of extracellular calcium completely inhibited GnRH-stimulated 3H-Gln LH and 35S-Met LH production. Total immunoreactive LH (iLH), as measured by RIA, was also increased significantly by GnRH treatment in the presence of calcium, but this response was prevented by removal of calcium from the medium. Lowering extracellular calcium had no effect on cellular uptake or incorporation of 3H-Gln or 35S-Met into total trichloroacetic acid-precipitable protein. Approximately 80% of newly synthesized LH was released into the medium in all treatment groups independent of whether calcium or GnRH was present. The specific activity (disintegrations per min/microgram iLH) of radiolabeled LH released into the medium was significantly reduced by treatment with GnRH due to the large amount of unlabeled iLH released into the medium. However, when the cells were incubated in low calcium, the SA of 3H-Gln LH and 35S-Met LH in the medium was unaltered by GnRH, whereas GnRH-stimulated iLH release was inhibited. We conclude that GnRH stimulation of LH glycosylation and LH apoprotein synthesis involves extracellular calcium-dependent events, and the release of newly synthesized LH is closely coupled to LH biosynthesis and is less dependent on extracellular calcium, whereas the GnRH-stimulated release of previously synthesized, stored LH is dependent on extracellular calcium.     

Atmospheric Ar and Ne returned from mantle depths to the Earth’s surface by forearc recycling

In subduction zones, sediments, hydrothermally altered lithosphere, fluids, and atmospheric gases are transported into the mantle, where ultrahigh-pressure (UHP) metamorphism takes place. However, the extent to which atmospheric noble gases are trapped in minerals crystallized during UHP metamorphism is unknown. We measured Ar and Ne trapped in phengite and omphacite from the youngest known UHP terrane on Earth to determine the composition of Ar and Ne returned from mantle depths to the surface by forearc recycling. An ⁴⁰Ar/³⁹Ar age [7.93 ± 0.10 My (1σ)] for phengite is interpreted as the timing of crystallization at mantle depths and indicates that ⁴⁰Ar/³⁹Ar phengite ages reliably record the timing of UHP metamorphism. Both phengite and omphacite yielded atmospheric ³⁸Ar/³⁶Ar and ²⁰Ne/²²Ne. Our study provides the first documentation, to our knowledge, of entrapment of atmospheric Ar and Ne in phengite and omphacite. Results indicate that a subduction barrier for atmospheric-derived noble gases does not exist at mantle depths associated with UHP metamorphism. We show that the crystallization age together with the isotopic composition of nonradiogenic noble gases trapped in minerals formed during subsolidus crystallization at mantle depths can be used to unambiguously assess forearc recycling of atmospheric noble gases. The flux of atmospheric noble gas entering the deep Earth through subduction and returning to the surface cannot be fully realized until the abundances of atmospheric noble gases trapped in exhumed UHP rocks are known.It has long been known that water and CO₂ can be transported into the deep Earth by subduction of sediments and hydrothermally altered oceanic crust (1–3). Water is carried from the surface into the upper mantle by hydrous minerals in the uppermost 10–12 km subducting lithosphere to depths of at least 400 km (4). However, to what extent are atmospheric noble gases transported into the deep Earth and returned to the surface in the forearcs of subduction zones? The isotopic compositions of Ar and Ne can be used as tracers of atmospheric recycling in subduction zones (5), because they are chemically inert at conditions relevant to processes on Earth (6). Serpentinite subduction has been proposed as a viable mechanism for high abundances of noble gas to be transported into the mantle (7), and hydration of oceanic lithosphere has been proposed as a mechanism to release argon into the atmosphere (8). To determine the flux of atmospheric noble gases recycled into the mantle in subduction zones requires knowing when, at what depth, and how atmospheric Ar and Ne are trapped in minerals crystallized at mantle depths and subsequently, exhumed to the surface. This flux calculation requires interpreting noble gas concentrations in minerals with respect to their pressure–temperature–time–deformation (P-T-t-D) histories.Studies that have addressed recycling of atmospheric noble gases in the Earth’s mantle have largely focused on volcanic rocks in arcs, backarcs, ocean island basalts, and midocean ridge basalts (MORBs) (5). However, atmospheric-derived noble gases have been shown to contaminate mantle noble gas signatures in volcanic rocks, irrespective of eruption setting (underwater, under ice, or in atmosphere) (9). Indeed, ³⁸Ar/³⁶Ar values for mantle-derived volcanic rocks generally have been found to be indistinguishable from atmospheric ratios (10, 11). The possibility of atmospheric contamination during eruption or interaction with meteoric fluids and seawater exists for any mineral/rock formed, or altered, at or near the Earth’s surface.Noble gas studies of peridotites (12) and serpentinized peridotites (13) have also argued for recycling of atmospheric noble gases into Earth’s mantle; however, linking the conditions of noble gas entrapment to specific parts of P-T-t-D histories in these lithologies is challenging. Peridotites are readily altered on the seafloor at temperatures <500 °C, where olivine and orthopyroxene react to form serpentine minerals. Serpentinite forms in many tectonic settings (14) and is stable over a wide range of pressure–temperature conditions (2). High-pressure serpentinites have been used to investigate noble gas and halogen compositions interpreted to have been trapped during subseafloor alteration and subsequent dehydration metamorphism (7). Noble gas and halogen studies of the Higashi-akaishi peridotite body of the Sanbagawa metamorphic terrane argue for the subduction and survival of marine pore fluid to depths of at least 100 km (15). The prograde pressure–temperature–deformation path of the peridotite is not well-constrained, and it is unclear whether early deformation is related to subduction (16). Therefore, several interpretations are possible to explain when and how entrapment of noble gases occurred (e.g., during prograde metamorphism, peak metamorphism, exhumation, or obduction).This study investigates noble gases, specifically Ar and Ne, trapped in minerals that formed during ultrahigh-pressure (UHP) metamorphism. Exhumed UHP rocks have been shown to preserve a record of the geochemical and fluid transport of material from mantle depths to the Earth’s surface (17–19). The forearc subduction channel provides a pathway for “input” lithologies (continental and oceanic crust and sediments) to be recycled, because they are metamorphosed at mantle depths where diagnostic assemblages (e.g., coesite and diamond) can crystallize and trap noble gases during UHP metamorphism. When UHP rocks are subsequently returned (exhumed) to the surface, their mineral assemblages provide clues to the changing conditions along their P-T-t-D paths. Thus, noble gases trapped in material entering (in crust and sediments) and exiting (in high-pressure and UHP metamorphic rocks) forearcs can provide insight into the recycling of atmospheric noble gases within the subduction channel. Previous studies that have investigated noble gases in exhumed high-pressure and UHP rocks focused on (i) ⁴⁰Ar/³⁹Ar age determination on irradiated K-bearing minerals (20), (ii) He and Ne isotopic studies aimed at documenting noble gas compositions trapped during diamond formation (21), and (iii) noble gas and halogen studies of serpentinite and peridotite (7, 12, 15). Here, we investigate whether atmospheric Ar and Ne are trapped in minerals during crystallization at mantle depths during subduction zone metamorphism. We present Ar and Ne isotopic data for omphacite and phengite from Late Miocene coesite eclogite that document both the ⁴⁰Ar/³⁹Ar age of phengite crystallization and the isotopic composition of Ar and Ne trapped in these minerals during UHP metamorphism.