A potent oligosaccharyl transferase inhibitor that crosses the intracellular endoplasmic reticulum membrane.

Recent work has resulted in the development of potent inhibitors of oligosaccharyl transferase (OT), the enzyme that catalyzes the cotranslational glycosylation of asparagine [Hendrickson, T. L., Spencer, J. R., Kato, M., and Imperiali, B. (1996) J. Am. Chem. Soc. 118, 7636-7637; Kellenberger, C., Hendrickson, T. L., and Imperiali, B. (1997) Biochemistry 36, 12554-12559]. However, no specific OT inhibitors that function in the cellular environment have yet been reported. The peptide cyclo(hex-Amb-Cys)-Thr-Val-Thr-Nph-NH2 was previously shown to exhibit nanomolar inhibition (Ki = 37 nM) through slow tight binding kinetics [Hendrickson, T. L., Spencer, J. R., Kato, M., and Imperiali, B. (1996) J. Am. Chem. Soc. 118, 7636-7637]. Included herein is the redesign of this prototype inhibitor for achieving both passive and active translocation into model membrane systems representing the endoplasmic reticulum (ER). The strategy for passive transport involved the incorporation of a membrane permeable import function previously shown to carry various peptides across the outer as well as the interior cellular membranes [Rojas, M., Donahue, J. P., Tan, Z., and Lin, Y.-Z. (1998) Nat. Biotechnol. 16, 370-375]. Assessment of function in intact ER membranes revealed that the inhibitor targeted toward passive diffusion demonstrated concentration-dependent inhibition of two different glycosylation substrates. Thus, this modified inhibitor achieved potent inhibition of glycosylation after being successfully transported through the ER membrane. In the active translocation approach, the lead OT inhibitor and a corresponding substrate were redesigned to include features recognized by the transporter associated with antigen processing (TAP). This protein translocates peptides into the lumen of the ER [Heemels, M.-T., Schumacher, T. N. M., Wonigeit, K., and Ploegh, H. L. (1993) Science 262, 2059-2063]. However, although acceptance of the cyclized substrate by the TAP receptor was demonstrated via efficient transport and glycosylation, the modified inhibitor was not translocated by TAP machinery, and therefore, active translocation was achieved for the modified substrate only. Both of these ER transport methods afforded redesigned OT inhibitors that retained their inhibitor properties in vitro, regardless of the extensions to the carboxy-terminus of the root inhibitor. The above family of redesigned inhibitors provides a template for generating a transcellular pathway and represents the first step toward OT inhibition in intact cells.

Effectiveness and cost-effectiveness of linking the special supplemental program for women, infants, and children (WIC) and immunization activities.

OBJECTIVE: To raise immunization coverage among children at risk for underimmunization, we evaluated the effectiveness and cost-effectiveness of immunization activities in the Special Supplemental Program for Women, Infants and Children (WIC). METHOD: A controlled intervention trial was conducted in seven WIC sites in Chicago between October 1990 and March 1994. At intervention sites, staff screened children for vaccination status at every visit, referred vaccine-eligible children to either an on-site WIC nurse, on-site clinic, or off-site community provider, and issued either a 3-month supply of food vouchers to up-to-date children or a 1-month supply to children not up-to-date--a usual practice for high-risk WIC children. Our primary measure of effectiveness was the change in the baseline percentage of up-to-date children at the second birthday; cost-effectiveness was approximated for each of the three referral interventions. RESULTS: After one year, up-to-date vaccination coverage increased 23% above baseline for intervention groups and decreased 9% in the control group. After the second year, up-to-date vaccination further increased to 38% above baseline in intervention groups and did not change in the control group. The total cost per additional up-to-date child ranged from $30 for sites referring children off-site to $73 for sites referring children on-site to a nurse. CONCLUSION: This controlled intervention trial of screening, referral, and a voucher incentive in the WIC program demonstrated a substantial increase in immunization coverage at a low cost. Continuing to design linkages between WIC and immunization programs by building on WIC's access to at-risk populations is worth the investment.

Severity adjustment for length of stay: is it always necessary?

OBJECTIVE: Severity adjustment is an oft-cited requirement when comparing physicians or medical delivery systems. Each application of severity adjustment, however, has to be tested to validate the need, the method, and its value. We examined the value of severity adjustment for identifying physician outliers when studying length of stay in the hospital. DESIGN: We compared the placement of physicians in an outlier category using a severity-adjusted average length of stay (SLOS) index with their placement using the unadjusted average length of stay (ALOS). Changes in placement of the list were validated by the utilization review coordinators. SETTING: A 614-bed tertiary-care university teaching hospital. SUBJECTS: We analyzed 11,146 discharges from 138 physicians in 1992. RESULTS: The mean ALOS +/- standard deviation was 9.05 + 4.50 days, and the SLOS Index was 7.56 +/- 3.06. There were 120 inliers, 6 high outliers, and 12 low outliers by the ALOS method. Using the SLOS index, 27 of 138 physicians had their categories changed from inlier to outlier or from outlier to inlier. The difference in group changes was more significant for those going from outlier to inlier status (8/120 vs 6/18; P < .001). The patients of the six physicians whose status changed from outlier to inlier status were sicker, as indicated by the comorbidity, complications, and manifestations of disease processes score. The utilization reviewers validated the status changes in 8 of 14 instances. CONCLUSIONS: Severity-adjusted length of stay by the SLOS index appears to provide a more accurate measure than the unadjusted ALOS. The changes, however, were small. It is not clear that the added effort is worthwhile.

Correlation of cell surface fluorescence with conventional PAP analysis of cells of cytological interest obtained from cervical scrapes.

Archival PAP stained cervical smears were destained and treated with a fluorescent probe for a cell surface enzyme (GB). Cells which exhibited cell surface fluorescence were demonstrated to be cells of cytological interest in the analysis of cervical smears. These cells could be directly related to PAP and reclassified by subsequent restaining with PAP. Fluorescent cell surface technology was shown to be compatible with conventional PAP staining.

A collaborative trial of a semi-automatic system for slide preparation and screening in cervical cytopathology.

We report a test of an experimental system for machine-aided screening in cervical cytology, comprising the 'CYTOPRESS' semi-automatic slide preparation system (Nijmegen) and the 'CERVIFIP' interactive scanner (Edinburgh). Material from women attending clinics in Edinburgh and Nijmegen was stratified according to the severity of the conventional laboratory diagnosis and selected randomly within strata for inclusion in the test. Monolayered slides were prepared by CYTOPRESS from cervical scrape material remaining after preparation of conventional smears and scanned by CERVIFIP to determine the positions of the most 'suspicious' objects. The test was based on a set of 701 monolayers, divided equally between 'negatives' and 'abnormals' of various grades, of which 585 (83.4%) were passed automatically as adequate for machine-aided analysis. Approximately 15% of adequate slides were passed as 'negative' without operator interaction. In the remaining 85%, the suspicious objects were inspected by a human operator and a decision was then made either to refer each monolayer for conventional microscopic analysis, or to pass it as 'negative'. Where discrepancies occurred between the conventional laboratory and the system results, a consensus diagnosis was reached by taking into account all relevant information including clinical data. Of those with a consensus diagnosis of CIN 3 or worse an estimated 9.3 +/- 4.1% were passed by the system as 'negative'. Closer investigation of these false-negatives revealed that most, and perhaps all, were preventable by system improvements either planned or in progress. Corresponding false-negative rates for those graded 'CIN 1 or 2' and 'negative-early recall' were estimated, respectively as 18.9 +/- 5.3% and 22.9 +/- 3.1%. Of those with a 'negative-routine recall' consensus, 19.4 +/- 2.5% were referred for conventional microscopic analysis, a level well within acceptable limits for cost-effectiveness. Women whose initial laboratory smears were negative, but whose consensus diagnosis was 'negative-early recall' or CIN, are being investigated further to determine whether cervical abnormalities were in fact present. Over two-thirds of this group were referred by the machine-aided system for conventional microscopic analysis.

The application of double fluorescence to the rapid selection of atypical cervical epithelial cells spread in monolayers.

Monolayer spreads of cervical cells were prepared and reacted in sequence with two fluorescent probes. The nuclei were reacted with 4,6-Diamidino-2-phenylindole (DAPI), resulting in white fluorescence of all cell nuclei. Those cells possessing active guanidinobenzoatase (GB) bound the second probe, rhodamine-alpha-N-agmatine (Rh-Agm), resulting in orange cell surface fluorescence. Atypical epithelial cells possessed both active GB and enlarged nuclei; such cells could easily be recognised by their cytological appearance. We illustrate our results in the form of colour prints which are representative of our observations of cells in both normal and abnormal cervical spreads.

A protein inhibitor, extracted from Hela cells, which recognises a cell surface protease on preneoplastic cells obtained from cervical smears.

Hela cells originate from a clone of cervical carcinoma cells. The cytoplasm of Hela cells contains a protein which recognises and inhibits a proteolytic enzyme (guanidino-benzoatase) on the surface of Hela cells. This same protease is present on the surface of abnormal epithelial cells obtained from cervical smears, enabling a rhodamine labelled inhibitor extracted from Hela cells to locate abnormal cervical cells in monolayer spreads.

Semi-automated cervical smear pre-screening systems: an evaluation of the Cytoscan-110.

This paper reports a test of a system for provision of machine assistance in cervical cytology screening. The hypothesis tested was that if the results of examination by a screener of a small number of high-ploidy cells on specially prepared monolayers, automatically selected and presented by the system, were combined with machine measurement of cell and cell population characteristics, it would be possible to distinguish conditions requiring further action on the part of a cytology service from those in which the patient could safely be signed out. The system appeared broadly capable of this discrimination, with a false-negative error not significantly different (for the numbers tested) on CIN1 and more severe cases to that obtaining for routine screening of the parallel PAP smears, and also to results obtained by a panel of three observers. The machine system appeared to do better than other systems in selecting borderline cases for review, but this may have been an artefact of the method of evaluation used: all results were compared with a 'reference diagnosis', which was computed using statistical techniques to integrate diagnostic information from all available sources. The false-negative error-rate of the system amounted to 5% of high-grade cases, 17% of CIN1's and 29% of borderlines, but were not substantially different from the FN rates for other reporting systems on the same material. The proportion of negative cases referred back for full cytological diagnosis was 34%. Despite this high false-positive rate, the system is potentially cost-effective in use.

Use of severity-adjusted length of stay to modify physician practice patterns.

OBJECTIVE: To compare inpatient length of stay among physicians by testing a new method for severity adjusting length of stay. DESIGN: A retrospective validation study with prospective follow-up after an intervention. SETTING: A 531-bed community teaching hospital. PATIENTS: Three hundred randomly selected patients from the 30,861 patients discharged in 1990. INTERVENTION: A physician with a significantly prolonged severity-adjusted length of stay was counseled and then monitored for three months. RESULTS: The correlation between the number of comorbidities, complications, and manifestations of disease processes (CCMDPs) was R2 = 0.658, t = 23.96 (p = .001). One physician had an unusually high severity-adjusted length of stay, but lowered it after he was counseled and monitored for three months. CONCLUSIONS: The number of CCMDPs recorded on the hospital discharge abstract can be used as a severity index to adjust a patient's length of stay for illness severity. Using linear regression analysis, a picture of the severity-adjusted length of stay can be derived for physicians. Through counseling and monitoring, individual physicians' lengths of stay patterns may be reduced.

A simple fluorescent technique for screening cervical cells prior to nuclear analysis.

Monolayer spreads of cervical cells were prepared and stained with haematoxylin and rhodamine-alpha-N-agmatine, a fluorescent marker for a cell surface protease. Mature epithelial cells from normal cervices lacked this cell surface enzyme and did not fluoresce. The abnormal cells possessed the cell surface enzyme, bound the probe and were quickly detected by fluorescence microscopy. The degree of abnormality of these fluorescent cells was determined by examination of their nuclear details, with the result that mild, moderate and severe dyskaryotic cells could be defined.

Short-term reduction in egg production in laying hens treated with an agonist of GnRH.

1. In two experiments laying hens were treated with an agonist of gonadotrophin releasing hormone (GnRH) to induce a reduction in the secretion of luteinising hormone (LH) and a pause in egg production. 2. In experiment 1, 70-week-old laying hens were either given daily subcutaneous injections of saline for 7 d, offered whole oats for 7 d (nutrient restriction), given daily injections of the GnRH agonist [D-Trp6-Pro9 N-ethyl amide]GnRH for 7 d at 50 micrograms/kg or 100 micrograms/kg or administered 4 biocompatible implants each containing 120 micrograms of the GnRH agonist. 3. Weekly egg production was monitored for 7 weeks and blood samples were taken at weekly intervals and assayed for plasma LH and oestradiol. Egg production was reduced in the birds treated with the agonist (28 to 46% reduction) but not to the same extent as in the birds offered whole oats (92.3% reduction). 4. The treatments also reduced plasma LH and oestradiol in treated hens but again to a greater extent in the birds offered whole oats than the birds treated with the agonist. Egg production and plasma LH and oestradiol increased following the termination of the treatments. 5. The birds fed whole oats suffered a reduction in weight of 16.7% over the treatment period whereas there were increases in the weights of the birds treated with saline, 50 micrograms of GnRH agonist and the implants of GnRH agonist, but no change in birds treated with 100 micrograms of GnRH agonist. 6. The birds fed oats lost feathers over the treatment period but the birds in the other treatment groups suffered no loss. 7. In experiment 2 laying hens were either injected daily with saline or 200 micrograms GnRH agonist and weekly egg production and plasma LH and oestradiol were measured. As egg production was reduced by almost 60% in the birds treated with the agonist but did not completely cease. Reductions in plasma LH and oestradiol were also observed. All variables increased to pretreatment levels once treatment ceased. 8. These data confirm the effects of severely depriving hens of nutrients on egg production and the secretion of LH and oestradiol.(ABSTRACT TRUNCATED AT 400 WORDS)

"If you love someone, you don't hurt them": a comprehensive process analysis of a weeping event in therapy.

Early theorists described physical diseases (e.g., asthma, ulcers) thought to be associated with the inhibition of weeping (e.g., Alexander 1950), and catharsis theories (Breuer and Freud 1895/1955; Koestler 1964) postulated that unexpressed emotion accumulated as in a tank, and then overflowed as tears when a threshold level was exceeded. From a more biological perspective, it has been suggested that stress produces toxic chemicals in the body that become concentrated in the lacrimal gland and are released through weeping, restoring homeostasis (Frey 1985). As a result of these theories, psychotherapists tend to believe weeping is healthy for clients and that it serves to decrease depression (Trezza et al. 1988). While laboratory studies have typically not supported these ideas (e.g., Labott and Martin 1987, 1988), no studies have been performed on weeping specifically in the context of psychotherapy.

Analysis of rare high-DNA cell populations in serous effusions using continuous-motion imaging.

The rare high-DNA cell sub-populations in a series of serous effusion specimens were analysed to determine whether such measurements could provide a basis for the improved diagnosis of malignancy. Monolayer specimens stained with gallocyanin chrome-alum were scanned with the CERVIFIP continuous-motion image analyser to locate and measure the highest-DNA cells in the sample. Two types of features were obtained for the detected sub-populations; firstly, 'percentile ploidy' values which characterise the ploidy levels above which specified proportions of the total cells are found; and secondly 'percentage abnormal' values which characterise the proportion of the cells diagnosed as malignant during examination by a cytopathologist. The classification accuracy for one or both of these features was then obtained by comparison with the clinical outcome of each patient. The results gave a classification error of 9/44 (20%) using the 0.01% percentile ploidy alone, 6/44 (14%) using the 75% percentage abnormal feature alone, but only 2/44 (5%) from a box discriminant using both features. It was therefore concluded that the analysis of the high-DNA cell population could be of value in the diagnosis of malignancy in serous effusion specimens.

Inheritance of plasma insulin-like growth factor-I and growth rate, food intake, food efficiency and abdominal fatness in chickens.

1. The inheritance of, and genetic and phenotypic correlations between, plasma insulin-like growth factor-I (IGF-I) and 28-(28dW) and 56-d (56dW) body weight, 28- to 56-d body weight gain (BWG), food intake (FI), food conversion ratio (FCR) and abdominal fatness (AF) at 56 d were determined by sib analyses in a population of 327 pedigreed progeny produced by matings between 18 cockerels and 72 pullets from a broiler strain of chickens bred at random for 8 generations. 2. Plasma IGF-I was measured in fed (IGF-If) and fasted (IGF-I) birds at 42 d. 3. Heritability estimates (sire + dam) were: 28dW 0.35 +/- 0.11, 56dW 0.49 +/- 0.13, BWG 0.51 +/- 0.13, FI 0.55 +/- 0.13, FCR 0.73 +/- 0.14, AF 0.49 +/- 0.13, IGF-If 0.10 +/- 0.08, IGF-Is 0.08 +/- 0.08. 4. The low heritability estimates with their high standard errors for the IGF-I measures precluded the calculation of meaningful genetic correlations between these and the performance traits. There were moderate to strong positive genetic correlations between 28dW, 56dW, FI and AF.

Cell/substratum adhesions in RSV-transformed rat fibroblasts.

Cell/substratum adhesions have been studied in rat fibroblasts transformed by a ts-mutant of Rous sarcoma virus (LA-29) using light and electron microscopy and a variety of preparative methods including immunolabeling. Cells were studied both during the process of transformation, i.e., shifting from 39 degrees to 35 degrees C, and in a fully transformed state (passaged at 35 degrees C continuously). The typical focal contacts observed at 39 degrees C (restrictive temperature) were replaced by "point-contacts" (100-200 per cell) which were classified by immunolabeling as podosome-like adhesions containing actin, beta 1 integrin subunit, vinculin, talin, alpha-actinin, and small membrane patches containing clathrin and integrin. Tyrosine-phosphorylated proteins and pp60src were found in association with groups of small particles on the protoplasmic surface of ventral membranes by gold immunolabeling. Both types of point-contacts were visualized by electron microscopy of ultrathin sections and shadowed replicas and characterized by gold immunolabeling wherever possible. The overall composition of podosome-like adhesions is similar to focal contacts but there are differences in the three-dimensional organization of the microfilaments and in the topography of vinculin which is associated more with actin filaments than with the plasma membrane. The presence of talin and extracellular matrix receptor in podosomes together with the adhesive properties of these actin-containing structures argues against the hypothesis that pp60src affects the interaction of actin with the plasma membrane by phosphorylating the fibronectin receptor and/or other associated proteins.