Purification and characteristics of an endogenous alpha-amylase inhibitor from barley kernels

An inhibitor of malted barley (Hordeum vulgare cv Conquest) α-amylase II was purified 125-fold from a crude extract of barley kernels by (NH₄)₂SO₄ fractionation, ion exchange chromatography on DEAE-Sephacel, and gel filtration on Bio-Gel P 60. The inhibitor was a protein with an approximate molecular weight of 20,000 daltons and an isoelectric point of 7.3. The protein was homogeneous, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid analysis indicated the presence of about 9 half-cystine residues per mole. The neutral isoelectric point of the inhibitor suggested that some of the apparently acidic residues (glutamic and aspartic) existed in the amide form. The first twenty N-terminal amino acids were sequenced. Some homology appeared to exist between the α-amylase II inhibitor and trypsin inhibitor from barley. Complex formation between α-amylase II and the inhibitor was detected by the appearance of a new molecular weight species after gel filtration on Bio-Gel P 100. Enzyme and inhibitor had to be preincubated for 5 min, prior to assaying for enzyme activity before maximum inhibition was attained. Inhibition increased at higher pH values. At pH 5.5, an approximately 1100 molar excess of inhibitor over α-amylase II produced 40% inhibition, whereas, at pH 8.0, a 1:1 molar ratio of inhibitor to enzyme produced the same degree of inhibition.     

Screening for cervical intraepithelial neoplasia in north east Scotland shows fall in incidence and mortality from invasive cancer with concomitant rise in preinvasive disease.

OBJECTIVE--To assess the effect of screening for cervical intraepithelial neoplasia on the incidence of and mortality from invasive squamous cell carcinoma of cervix in north east Scotland and to discover why cases of invasive cancer still occur. DESIGN--(a) Analysis of data on cases of cervical intraepithelial neoplasia obtained from the cytology data bank; (b) analysis of data on 612 women presenting with invasive squamous cancer during 1968-91, obtained from cancer registry and hospital records; (c) analysis of death rates obtained from the registrar general''s (Scotland) annual reports, the Information Services Division of the Home and Health Department (Scotland), and local records for 1974-91; (d) case-control studies on 282 cases of invasive cancer and 108 deaths which occurred in 1982-91. Cases were matched with two controls both for age and for having a negative smear test result at the time of presentation of the case. SETTING--North east Scotland (Grampian region, Orkney, and Shetland). SUBJECTS--Women (n = 306,608) who had had cervical smear tests between 1960 and 1991. RESULTS--There had been a substantial increase in cases of cervical intraepithelial neoplasia grade III since 1982. The incidence of invasive cancer has fallen since the start of screening in 1960, the fall occurring mainly in the well screened age group 40-69 years. There was a rise in women aged under 40 and over 70. Women with invasive disease seen between 1982 and 1991 mostly presented at stage I. Of these, half were unscreened, one third were poorly screened, 11% were found in retrospect to have had abnormal cells, 3% had recurrence of disease after treatment for cervical intraepithelial neoplasia grade III, and 3% were lost to follow up. Death rates had fallen, most noticeably in women aged 45-64, who had had the opportunity to be screened and rescreened. There was a disturbing rise in deaths among women under 45. Most deaths (65%) occurred in unscreened women. Case-control studies showed that the longer the time and absence of a smear test before presentation the higher was the risk of invasive cancer and of death. CONCLUSIONS--Screening has been effective in reducing the incidence of and mortality from cervical cancer in north east Scotland. Most cases and deaths occurred in unscreened women or in those who had had few smears at long intervals. An increase in cases of cervical intraepithelial neoplasia grade III in women screened for the first time occurred during 1982-91.     

Mediation of pinocytosis in cultured arterial smooth muscle and endothelial cells by platelet-derived growth factor

Pinocytosis was measured in monkey aortic smooth muscle cells (SMC), bovine aortic endothelial cells, and Swiss 3T3 cells in culture as cellular uptake of [U-(14)C]sucrose and horseradish peroxidase (HRP) from the tissue culture medium. Monkey arterial SMC and Swiss 3T3 cells were maintained in a quiescent state of growth at low cells density in medium containing 5 percent monkey plasma-derived serum (PDS). Replacement of PDS with 5 percent monkey whole blood serum (WBS) from the same donor, or addition to PDS of partially purified platelet-derived growth factor(s) (PF), resulted in a marked stimulation of pinocytosis as well as of cellular proliferation. In SMC, enhancement of the rate of pinocytosis occurred 4-6 h after exposure to WBS or PF, and the rate was up to twofold higher than the rate in medium containing PDS. In contrast, [(3)H]thymidine uptake by SMC did not increase until 12-16 h after exposure to PF. In endothelial cells the presence of PF or WBS did not enhance either the rate of pinocytosis or the rate of proliferation over that in PDS. Thus, endothelial cells did not become quiescent at subconfluent densities in PDS but maintained rates of proliferation and pinocytosis that were equivalent to those in WBS. By autoradiography, the fraction of labeled nuclei in SMC cultures 24 h after change of medium increased from 0.061 +/- 0.004 in quiescent cultures to 0.313 +/- 0.028 after exposure to WBS or PF. In contrast, labeling indices of endothelial cells were similar for cultures grown in PDS, WBS, or PF at any single time point after change of medium. These findings suggest that the rate of pinocytosis maybe be coupled in some fashion to growth regulation, which may be mediated in part by specific growth factors, such as that derived from the thrombocyte.     

The Contribution of Diet and Genotype to Iron Status in Women: A Classical Twin Study

This is the first published report examining the combined effect of diet and genotype on body iron content using a classical twin study design. The aim of this study was to determine the relative contribution of genetic and environmental factors in determining iron status. The population was comprised of 200 BMI- and age-matched pairs of MZ and DZ healthy twins, characterised for habitual diet and 15 iron-related candidate genetic markers. Variance components analysis demonstrated that the heritability of serum ferritin (SF) and soluble transferrin receptor was 44% and 54% respectively. Measured single nucleotide polymorphisms explained 5% and selected dietary factors 6% of the variance in iron status; there was a negative association between calcium intake and body iron (p = 0.02) and SF (p = 0.04).     

Volume and compressibility changes accompanying thermally-induced native-to-unfolded and molten globule-to-unfolded transitions of cytochrome c: a high pressure study

We have measured the transition temperatures, T(M), and van't Hoff enthalpies, DeltaH(M), of the thermally induced native-to-unfolded (N-to-U) and molten globule-to-unfolded (MG-to-U) transitions of cytochrome c at pressures between 50 and 2200 bar. We have used the pressure dependence of T(M) to evaluate the changes in volume, Delta(v), accompanying each protein transition event as a function of temperature and pressure. From analysis of the temperature and pressure dependences of Delta(v), we have additionally calculated the changes in expansibility, Delta(e), and isothermal compressibility, Delta(k)(T), associated with the thermally induced conformational transitions of cytochrome c. Specifically, if extrapolated to 25 degrees C, the native-to-unfolded (N-to-U) transition is accompanied by changes in volume, Delta(v), expansibility, Delta(e), and isothermal compressibility, Delta(k)(T), of -(5 +/- 3) x 10(-3) cm(3) g(-1), (1.8 +/- 0.3) x 10(-4) cm(3) g(-1) K(-1), and approximately 0 cm(3) g(-1) bar(-1), respectively. The molten globule-to-unfolded (MG-to-U) transition is accompanied by changes in volume, Delta(v), and isothermal compressibility, Delta(k)(T), of -(2.9 +/- 0.3) x 10(-3) cm(3) g(-1) at 40 degrees C and -(1.9 +/- 0.3) x 10(-6) cm(3) g(-1) bar(-1) at 35 degrees C, respectively. By comparing the volumetric properties of the N-to-U and N-to-MG transitions of cytochrome c, we have estimated the properties of the native-to-molten globule (N-to-MG) transition. For the latter transition, the changes in volume, Delta(v), and isothermal compressibility, Delta(k)(T), are approximately 0 cm(3) g(-1) at 40 degrees C and 1.9 cm(3) g(-1) bar(-1) at 35 degrees C, respectively. Our estimate for the change in expansibility, Delta(e), upon the N-to-MG is negative and equal to -(5 +/- 3) x 10(-4) cm(3) g(-1) K(-1). This finding contrasts with the results of previous studies all of which report positive changes in expansibility associated with protein denaturation. In general, our volumetric data permit us to assess the combined effect of temperature and pressure on the stability of various conformational states of cytochrome c.