Showing papers by "Johns Hopkins University published in 1997"

A Clinical Trial of the Effects of Dietary Patterns on Blood Pressure

Abstract: Background It is known that obesity, sodium intake, and alcohol consumption influence blood pressure. In this clinical trial, Dietary Approaches to Stop Hypertension, we assessed the effects of dietary patterns on blood pressure. Methods We enrolled 459 adults with systolic blood pressures of less than 160 mm Hg and diastolic blood pressures of 80 to 95 mm Hg. For three weeks, the subjects were fed a control diet that was low in fruits, vegetables, and dairy products, with a fat content typical of the average diet in the United States. They were then randomly assigned to receive for eight weeks the control diet, a diet rich in fruits and vegetables, or a “combination” diet rich in fruits, vegetables, and low-fat dairy products and with reduced saturated and total fat. Sodium intake and body weight were maintained at constant levels. Results At base line, the mean (±SD) systolic and diastolic blood pressures were 131.3±10.8 mm Hg and 84.7±4.7 mm Hg, respectively. The combination diet reduced systolic and d...

Activation of β-Catenin-Tcf Signaling in Colon Cancer by Mutations in β-Catenin or APC

Abstract: Inactivation of the adenomatous polyposis coli (APC) tumor suppressor gene initiates colorectal neoplasia. One of the biochemical activities associated with the APC protein is down-regulation of transcriptional activation mediated by beta-catenin and T cell transcription factor 4 (Tcf-4). The protein products of mutant APC genes present in colorectal tumors were found to be defective in this activity. Furthermore, colorectal tumors with intact APC genes were found to contain activating mutations of beta-catenin that altered functionally significant phosphorylation sites. These results indicate that regulation of beta-catenin is critical to APC's tumor suppressive effect and that this regulation can be circumvented by mutations in either APC or beta-catenin.

The complete genome sequence of the gastric pathogen Helicobacter pylori

Abstract: Helicobacter pylori, strain 26695, has a circular genome of 1,667,867 base pairs and 1,590 predicted coding sequences. Sequence analysis indicates that H. pylori has well-developed systems for motility, for scavenging iron, and for DNA restriction and modification. Many putative adhesins, lipoproteins and other outer membrane proteins were identified, underscoring the potential complexity of host-pathogen interaction. Based on the large number of sequence-related genes encoding outer membrane proteins and the presence of homopolymeric tracts and dinucleotide repeats in coding sequences, H. pylori, like several other mucosal pathogens, probably uses recombination and slipped-strand mispairing within repeats as mechanisms for antigenic variation and adaptive evolution. Consistent with its restricted niche, H. pylori has a few regulatory networks, and a limited metabolic repertoire and biosynthetic capacity. Its survival in acid conditions depends, in part, on its ability to establish a positive inside-membrane potential in low pH.

Constitutive Transcriptional Activation by a β-Catenin-Tcf Complex in APC−/− Colon Carcinoma

Abstract: The adenomatous polyposis coli (APC) tumor suppressor protein binds to beta-catenin, a protein recently shown to interact with Tcf and Lef transcription factors. The gene encoding hTcf-4, a Tcf family member that is expressed in colonic epithelium, was cloned and characterized. hTcf-4 transactivates transcription only when associated with beta-catenin. Nuclei of APC-/- colon carcinoma cells were found to contain a stable beta-catenin-hTcf-4 complex that was constitutively active, as measured by transcription of a Tcf reporter gene. Reintroduction of APC removed beta-catenin from hTcf-4 and abrogated the transcriptional transactivation. Constitutive transcription of Tcf target genes, caused by loss of APC function, may be a crucial event in the early transformation of colonic epithelium.

A model for p53-induced apoptosis

Abstract: The inactivation of the p53 gene in a large proportion of human cancers has inspired an intense search for the encoded protein's physiological and biological properties. Expression of p53 induces either a stable growth arrest or programmed cell death (apoptosis). In human colorectal cancers, the growth arrest is dependent on the transcriptional induction of the protein p21WAF1/CIP1(ref. 1), but the mechanisms underlying the development of p53-dependent apoptosis are largely unknown2. As the most well documented biochemical property of p53 is its ability to activate transcription of genes, we examined in detail the transcripts induced by p53 expression before the onset of apoptosis. Of 7,202 transcripts identified, only 14 (0.19%) were found to be markedly increased in p53-expressing cells compared with control cells. Strikingly, many of these genes were predicted to encode proteins that could generate or respond to oxidative stress, including one that is implicated in apoptosis in plant meristems. These observations stimulated additional biochemical and pharmacological experiments suggesting that p53 results in apoptosis through a three-step process: (1) the transcriptional induction of redox-related genes; (2) the formation of reactive oxygen species; and (3) the oxidative degradation of mitochondrial components, culminating in cell death.

Statistical methods for speech recognition

Abstract: The speech recognition problem hidden Markov models the acoustic model basic language modelling the Viterbi search hypothesis search on a tree and the fast match elements of information theory the complexity of tasks - the quality of language models the expectation - maximization algorithm and its consequences decision trees and tree language models phonetics from orthography - spelling-to-base from mappings triphones and allophones maximum entropy probability estimation and language models three applications of maximum entropy estimation to language modelling estimation of probabilities from counts and the Back-Off method.

Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection

Abstract: The capacity of HIV-1 to establish latent infection of CD4+ T cells may allow viral persistence despite immune responses and antiretroviral therapy. Measurements of infectious virus and viral RNA in plasma and of infectious virus, viral DNA and viral messenger RNA species in infected cells all suggest that HIV-1 replication continues throughout the course of infection. Uncertainty remains over what fraction of CD4+ T cells are infected and whether there are latent reservoirs for the virus. We show here that during the asymptomatic phase of infection there is an extremely low total body load of latently infected resting CD4+ T cells with replication-competent integrated provirus (<10(7) cells). The most prevalent form of HIV-1 DNA in resting and activated CD4+ T cells is a full-length, linear, unintegrated form that is not replication competent. The infection progresses even though at any given time in the lymphoid tissues integrated HIV-1 DNA is present in only a minute fraction of the susceptible populations, including resting and activated CD4+ T cells and macrophages.

Association of Coronary Heart Disease Incidence with Carotid Arterial Wall Thickness and Major Risk Factors: The Atherosclerosis Risk in Communities (ARIC) Study, 1987–1993

Abstract: Few studies have determined whether greater carotid artery intima-media thickness (IMT) in asymptomatic individuals is associated prospectively with increased risk of coronary heart disease (CHD). In the Atherosclerosis Risk in Communities Study, carotid IMT, an index of generalized atherosclerosis, was defined as the mean of IMT measurements at six sites of the carotid arteries using B-mode ultrasound. The authors assessed its relation to CHD incidence over 4-7 years of follow-up (1987-1993) in four US communities (Forsyth County, North Carolina; Jackson, Mississippi; Minneapolis, Minnesota; and Washington County, Maryland) from samples of 7,289 women and 5,552 men aged 45-64 years who were free of clinical CHD at baseline. There were 96 incident events for women and 194 for men. In sex-specific Cox proportional hazards models adjusted only for age, race, and center, the hazard rate ratio comparing extreme mean IMT (> or = 1 mm) to not extreme (< 1 mm) was 5.07 for women (95% confidence interval 3.08-8.36) and 1.85 for men (95% confidence interval 1.28-2.69). The relation was graded (monotonic), and models with cubic splines indicated significant nonlinearity. The strength of the association was reduced by including major CHD risk factors, but remained elevated at higher IMT. Up to 1 mm mean IMT, women had lower adjusted annual event rates than did men, but above 1 mm their event rate was closer to that of men. Thus, mean carotid IMT is a noninvasive predictor of future CHD incidence.

Lifetime Co-occurrence of DSM-III-R Alcohol Abuse and Dependence With Other Psychiatric Disorders in the National Comorbidity Survey

Abstract: Objective: To study patterns of co-occurrence of lifetimeDSM-III-Ralcohol disorders in a household sample. Methods: Data came from the National Comorbidity Survey (NCS), a nationally representative household survey. Diagnoses were based on a modified version of the Composite International Diagnostic Interview. Results: Respondents with lifetimeNCS/DSM-III-Ralcohol abuse or dependence had a high probability of carrying at least 1 other lifetimeNCS/DSM-III-Rdiagnosis. Retrospective reports have suggested that most lifetime co-occurring alcohol disorders begin at a later age than at least 1 other NCS/DSM-III-Rdisorder. Earlier disorders are generally stronger predictors of alcohol dependence than alcohol abuse and stronger among women than men. Lifetime co-occurrence is positively, but weakly, associated with the persistence of alcohol abuse among men and of alcohol dependence among both men and women. Conclusions: Caution is needed in interpreting the results due to the fact that diagnoses were made by nonclinicians and results are based on retrospective reports of the age at onset. Within the context of these limitations, though, these results show that alcohol abuse and dependence are often associated with other lifetimeDSM-III-Rdisorders and suggest that, at least in recent cohorts, the alcohol use disorders are usually temporally secondary. Prospective data and data based on clinically confirmed diagnoses are needed to verify these findings.

Combination of prostate-specific antigen, clinical stage, and Gleason score to predict pathological stage of localized prostate cancer: A multi- institutional update

Abstract: Objective. —To combine the clinical data from 3 academic institutions that serve as centers of excellence for the surgical treatment of clinically localized prostate cancer and develop a multi-institutional model combining serum prostate-specific antigen (PSA) level, clinical stage, and Gleason score to predict pathological stage for men with clinically localized prostate cancer. Design. —In this update, we have combined clinical and pathological data for a group of 4133 men treated by several surgeons from 3 major academic urologic centers within the United States. Multinomial log-linear regression was performed for the simultaneous prediction of organ-confined disease, isolated capsular penetration, seminal vesicle involvement, or pelvic lymph node involvement. Bootstrap estimates of the predicted probabilities were used to develop nomograms to predict pathological stage. Additional bootstrap analyses were then obtained to validate the performance of the nomograms. Patients and Settings. —A total of 4133 men who had undergone radical retropubic prostatectomy for clinically localized prostate cancer at The Johns Hopkins Hospital (n=3116), Baylor College of Medicine (n=782), and the University of Michigan School of Medicine (n=235) were enrolled into this study. None of the patients had received preoperative hormonal or radiation therapy. Outcome Measures. —Simultaneous prediction of organ-confined disease, isolated capsular penetration, seminal vesicle involvement, or pelvic lymph node involvement using updated nomograms. Results. —Prostate-specific antigen level, TNM clinical stage, and Gleason score contributed significantly to the prediction of pathological stage ( P Conclusions. —The data represent a multi-institutional modeling and validation of the clinical utility of combining PSA level measurement, clinical stage, and Gleason score to predict pathological stage for a group of men with localized prostate cancer. Clinicians can use these nomograms when counseling individual patients regarding the probability of their tumor being a specific pathological stage; this will enable patients and physicians to make more informed treatment decisions based on the probability of a pathological stage, as well as risk tolerance and the values they place on various potential outcomes.

Six hundred fifty consecutive pancreaticoduodenectomies in the 1990s: pathology, complications, and outcomes.

Abstract: OBJECTIVE: The authors reviewed the pathology, complications, and outcomes in a consecutive group of 650 patients undergoing pancreaticoduodenectomy in the 1990s. SUMMARY BACKGROUND DATA: Pancreaticoduodenectomy has been used increasingly in recent years to resect a variety of malignant and benign diseases of the pancreas and periampullary region. METHODS: Between January 1990 and July 1996, inclusive, 650 patients underwent pancreaticoduodenal resection at The Johns Hopkins Hospital. Data were recorded prospectively on all patients. All pathology specimens were reviewed and categorized. Statistical analyses were performed using both univariate and multivariate models. RESULTS: The patients had a mean age of 63 +/- 12.8 years, with 54% male and 91% white. The number of resections per year rose from 60 in 1990 to 161 in 1995. Pathologic examination results showed pancreatic cancer (n = 282; 43%), ampullary cancer (n = 70; 11%), distal common bile duct cancer (n = 65; 10%), duodenal cancer (n = 26; 4%), chronic pancreatitis (n = 71; 11%), neuroendocrine tumor (n = 31; 5%), periampullary adenoma (n = 21; 3%), cystadenocarcinoma (n = 14; 2%), cystadenoma (n = 25; 4%), and other (n = 45; 7%). The surgical procedure involved pylorus preservation in 82%, partial pancreatectomy in 95%, and portal or superior mesenteric venous resection in 4%. Pancreatic-enteric reconstruction, when appropriate, was via pancreaticojejunostomy in 71% and pancreaticogastrostomy in 29%. The median intraoperative blood loss was 625 mL, median units of red cells transfused was zero, and the median operative time was 7 hours. During this period, 190 consecutive pancreaticoduodenectomies were performed without a mortality. Nine deaths occurred in-hospital or within 30 days of operation (1.4% operative mortality). The postoperative complication rate was 41%, with the most common complications being early delayed gastric emptying (19%), pancreatic fistula (14%), and wound infection (10%). Twenty-three patients required reoperation in the immediate postoperative period (3.5%), most commonly for bleeding, abscess, or dehiscence. The median postoperative length of stay was 13 days. A multivariate analysis of the 443 patients with periampullary adenocarcinoma indicated that the most powerful independent predictors favoring long-term survival included a pathologic diagnosis of duodenal adenocarcinoma, tumor diameter <3 cm, negative resection margins, absence of lymph node metastases, well-differentiated histology, and no reoperation. CONCLUSIONS: This single institution, high-volume experience indicates that pancreaticoduodenectomy can be performed safely for a variety of malignant and benign disorders of the pancreas and periampullary region. Overall survival is determined largely by the pathology within the resection specimen.

Speaker recognition: a tutorial

Abstract: A tutorial on the design and development of automatic speaker-recognition systems is presented. Automatic speaker recognition is the use of a machine to recognize a person from a spoken phrase. These systems can operate in two modes: to identify a particular person or to verify a person's claimed identity. Speech processing and the basic components of automatic speaker-recognition systems are shown and design tradeoffs are discussed. Then, a new automatic speaker-recognition system is given. This recognizer performs with 98.9% correct decalcification. Last, the performances of various systems are compared.

Mitogenic Signaling Mediated by Oxidants in Ras-Transformed Fibroblasts

Abstract: NIH 3T3 fibroblasts stably transformed with a constitutively active isoform of p21(Ras), H-RasV12 (v-H-Ras or EJ-Ras), produced large amounts of the reactive oxygen species superoxide (.O2-). .O2- production was suppressed by the expression of dominant negative isoforms of Ras or Rac1, as well as by treatment with a farnesyltransferase inhibitor or with diphenylene iodonium, a flavoprotein inhibitor. The mitogenic activity of cells expressing H-RasV12 was inhibited by treatment with the chemical antioxidant N-acetyl-L-cysteine. Mitogen-activated protein kinase (MAPK) activity was decreased and c-Jun N-terminal kinase (JNK) was not activated in H-RasV12-transformed cells. Thus, H-RasV12-induced transformation can lead to the production of .O2- through one or more pathways involving a flavoprotein and Rac1. The implication of a reactive oxygen species, probably .O2-, as a mediator of Ras-induced cell cycle progression independent of MAPK and JNK suggests a possible mechanism for the effects of antioxidants against Ras-induced cellular transformation.

Physician-Patient Communication: The Relationship With Malpractice Claims Among Primary Care Physicians and Surgeons

Abstract: Objective. —To identify specific communication behaviors associated with malpractice history in primary care physicians and surgeons. Design. —Comparison of communication behaviors of "claims" vs "no-claims" physicians using audiotapes of 10 routine office visits per physician. Settings. —One hundred twenty-four physician offices in Oregon and Colorado. Participants. —Fifty-nine primary care physicians (general internists and family practitioners) and 65 general and orthopedic surgeons and their patients. Physicians were classified into no-claims or claims (≥2 lifetime claims) groups based on insurance company records and were stratified by years in practice and specialty. Main Outcome Measures. —Audiotape analysis using the Roter Interaction Analysis System. Results. —Significant differences in communication behaviors of no-claims and claims physicians were identified in primary care physicians but not in surgeons. Compared with claims primary care physicians, no-claims primary care physicians used more statements of orientation (educating patients about what to expect and the flow of a visit), laughed and used humor more, and tended to use more facilitation (soliciting patients' opinions, checking understanding, and encouraging patients to talk). No-claims primary care physicians spent longer in routine visits than claims primary care physicians (mean, 18.3 vs 15.0 minutes), and the length of the visit had an independent effect in predicting claims status. The multivariable model for primary care improved the prediction of claims status by 57% above chance (90% confidence interval, 33%-73%). Multivariable models did not significantly improve prediction of claims status for surgeons. Conclusions. —Routine physician-patient communication differs in primary care physicians with vs without prior malpractice claims. In contrast, the study did not find communication behaviors to distinguish between claims vs no-claims surgeons. The study identifies specific and teachable communication behaviors associated with fewer malpractice claims for primary care physicians. Physicians can use these findings as they seek to improve communication and decrease malpractice risk. Malpractice insurers can use this information to guide malpractice risk prevention and education for primary care physicians but should not assume that it is appropriate to teach similar behaviors to other specialty groups.

A photoreceptor cell-specific ATP-binding transporter gene (ABCR) is mutated in recessive Stargardt macular dystrophy

Abstract: Stargardt disease (STGD, also known as fundus flavimaculatus; FFM) is an autosomal recessive retinal disorder characterized by a juvenile-onset macular dystrophy, alterations of the peripheral retina, and subretinal deposition of lipofuscin-like material A gene encoding an ATP-binding cassette (ABC) transporter was mapped to the 2-cM (centiMorgan) interval at 1p13-p21 previously shown by linkage analysis to harbour the STGD gene This gene, ABCR, is expressed exclusively and at high levels in the retina, in rod but not cone photoreceptors, as detected by in situ hybridization Mutational analysis of ABCR in STGD families revealed a total of 19 different mutations including homozygous mutations in two families with consanguineous parentage These data indicate that ABCR is the causal gene of STGD/FFM

Circulating adhesion molecules VCAM-1, ICAM-1, and E-selectin in carotid atherosclerosis and incident coronary heart disease cases: The Atherosclerosis Risk In Communities (ARIC) study

Abstract: Background Recruitment of circulating leukocytes at sites of atherosclerosis is mediated through a family of adhesion molecules. The function of circulating forms of these adhesion molecules remains unknown, but their levels may serve as molecular markers of subclinical coronary heart disease (CHD). Methods and Results To determine the ability of circulating vascular cell adhesion molecule-1 (VCAM-1), endothelial-leukocyte adhesion molecule-1 (E-selectin), and intercellular adhesion molecule-1 (ICAM-1) to serve as molecular markers of atherosclerosis and predictors of incident CHD, we studied 204 patients with incident CHD, 272 patients with carotid artery atherosclerosis (CAA), and 316 control subjects from the large, biracial Atherosclerosis Risk In Communities (ARIC) study. Levels of VCAM-1 were not significantly different among the patients with incident CHD, those with CAA, and control subjects. Higher levels of E-selectin and ICAM-1 were observed for the patients with CHD (means [ng/mL]: E-selectin,...

The role of cockroach allergy and exposure to cockroach allergen in causing morbidity among inner-city children with asthma

Abstract: Background It has been hypothesized that asthma-related health problems are most severe among children in inner-city areas who are allergic to a specific allergen and also exposed to high levels of that allergen in bedroom dust. Methods From November 1992 through October 1993, we recruited 476 children with asthma (age, four to nine years) from eight inner-city areas in the United States. Immediate hypersensitivity to cockroach, house-dust-mite, and cat allergens was measured by skin testing. We then measured major allergens of cockroach (Bla g 1), dust mites (Der p 1 and Der f 1), and cat dander (Fel d 1) in household dust using monoclonal-antibody–based enzyme-linked immunosorbent assays. High levels of exposure were defined according to proposed thresholds for causing disease. Data on morbidity due to asthma were collected at base line and over a one-year period. Results Of the children, 36.8 percent were allergic to cockroach allergen, 34.9 percent to dust-mite allergen, and 22.7 percent to cat allerg...

Estimation of Central Aortic Pressure Waveform by Mathematical Transformation of Radial Tonometry Pressure Validation of Generalized Transfer Function

Abstract: Background Central aortic pressures and waveform convey important information about cardiovascular status, but direct measurements are invasive. Peripheral pressures can be measured noninvasively, and although they often differ substantially from central pressures, they may be mathematically transformed to approximate the latter. We tested this approach, examining intersubject and intrasubject variability and the validity of using a single averaged transformation, which would enhance its applicability. Methods and Results Invasive central aortic pressure by micromanometer and radial pressure by automated tonometry were measured in 20 patients at steady state and during hemodynamic transients (Valsalva maneuver, abdominal compression, nitroglycerin, or vena caval obstruction). For each patient, transfer functions (TFs) between aortic and radial pressures were calculated by parametric model and results averaged to yield individual TFs. A generalized TF was the average of individual functions. TFs varied among patients, with coefficients of variation for peak amplitude and frequency at peak amplitude of 24.9% and 16.9%, respectively. Intrapatient TF variance with altered loading (>20% variation in peak amplitude) was observed in 28.5% of patients. Despite this, the generalized TF estimated central arterial pressures to ≤0.2±3.8 mm Hg error, arterial compliance to 6±7% accuracy, and augmentation index to within −7% points (30±45% accuracy). Individual TFs were only marginally superior to the generalized TF for reconstructing central pressures. Conclusions Central aortic pressures can be accurately estimated from radial tonometry with the use of a generalized TF. The reconstructed waveform can provide arterial compliance estimates but may underestimate the augmentation index because the latter requires greater fidelity reproduction of the wave contour.

VISUAL ATTENTION: Control, Representation, and Time Course

Abstract: Three central problems in the recent literature on visual attention are reviewed. The first concerns the control of attention by top-down (or goal-directed) and bottom-up (or stimulus-driven) processes. The second concerns the representational basis for visual selection, including how much attention can be said to be location- or object-based. Finally, we consider the time course of attention as it is directed to one stimulus after another.

Buffer-Stock Saving and the Life Cycle/Permanent Income Hypothesis

Abstract: This paper argues that the typical household’s saving is better described by a “bufferstock” version than by the traditional version of the Life Cycle/Permanent Income Hypothesis (LC/PIH) model. Buffer-stock behavior emerges if consumers with important income uncertainty are sufficiently impatient. In the traditional model, consumption growth is determined solely by tastes; in contrast, buffer-stock consumers set average consumption growth equal to average labor income growth, regardless of tastes. The model can explain three empirical puzzles: the “consumption/income parallel” of Carroll and Summers [1991]; the “consumption/income divergence” first documented in the 1930's; and the temporal stability of the household age/wealth profile despite the unpredictability of idiosyncratic wealth changes.

Broccoli sprouts: An exceptionally rich source of inducers of enzymes that protect against chemical carcinogens

Abstract: Induction of phase 2 detoxication enzymes [e.g., glutathione transferases, epoxide hydrolase, NAD(P)H: quinone reductase, and glucuronosyltransferases] is a powerful strategy for achieving protection against carcinogenesis, mutagenesis, and other forms of toxicity of electrophiles and reactive forms of oxygen. Since consumption of large quantities of fruit and vegetables is associated with a striking reduction in the risk of developing a variety of malignancies, it is of interest that a number of edible plants contain substantial quantities of compounds that regulate mammalian enzymes of xenobiotic metabolism. Thus, edible plants belonging to the family Cruciferae and genus Brassica (e.g., broccoli and cauliflower) contain substantial quantities of isothiocyanates (mostly in the form of their glucosinolate precursors) some of which (e.g., sulforaphane or 4-methylsulfinylbutyl isothiocyanate) are very potent inducers of phase 2 enzymes. Unexpectedly, 3-day-old sprouts of cultivars of certain crucifers including broccoli and cauliflower contain 10-100 times higher levels of glucoraphanin (the glucosinolate of sulforaphane) than do the corresponding mature plants. Glucosinolates and isothiocyanates can be efficiently extracted from plants, without hydrolysis of glucosinolates by myrosinase, by homogenization in a mixture of equal volumes of dimethyl sulfoxide, dimethylformamide, and acetonitrile at -50 degrees C. Extracts of 3-day-old broccoli sprouts (containing either glucoraphanin or sulforaphane as the principal enzyme inducer) were highly effective in reducing the incidence, multiplicity, and rate of development of mammary tumors in dimethylbenz(a)anthracene-treated rats. Notably, sprouts of many broccoli cultivars contain negligible quantities of indole glucosinolates, which predominate in the mature vegetable and may give rise to degradation products (e.g., indole-3-carbinol) that can enhance tumorigenesis. Hence, small quantities of crucifer sprouts may protect against the risk of cancer as effectively as much larger quantities of mature vegetables of the same variety.

Conversion of Bcl-2 to a Bax-like Death Effector by Caspases

Abstract: Caspases are a family of cysteine proteases implicated in the biochemical and morphological changes that occur during apoptosis (programmed cell death). The loop domain of Bcl-2 is cleaved at Asp34 by caspase-3 (CPP32) in vitro, in cells overexpressing caspase-3, and after induction of apoptosis by Fas ligation and interleukin-3 withdrawal. The carboxyl-terminal Bcl-2 cleavage product triggered cell death and accelerated Sindbis virus-induced apoptosis, which was dependent on the BH3 homology and transmembrane domains of Bcl-2. Inhibitor studies indicated that cleavage of Bcl-2 may further activate downstream caspases and contribute to amplification of the caspase cascade. Cleavage-resistant mutants of Bcl-2 had increased protection from interleukin-3 withdrawal and Sindbis virus-induced apoptosis. Thus, cleavage of Bcl-2 by caspases may ensure the inevitability of cell death.

ER-to-Golgi transport visualized in living cells

Abstract: Newly synthesized proteins that leave the endoplasmic reticulum (ER) are funnelled through the Golgi complex before being sorted for transport to their different final destinations. Traditional approaches have elucidated the biochemical requirements for such transport and have established a role for transport intermediates. New techniques for tagging proteins fluorescently have made it possible to follow the complete life history of single transport intermediates in living cells, including their formation, path and velocity en route to the Golgi complex. We have now visualized ER-to-Golgi transport using the viral glycoprotein ts045 VSVG tagged with green fluorescent protein (VSVG-GFP). Upon export from the ER, VSVG-GFP became concentrated in many differently shaped, rapidly forming pre-Golgi structures, which translocated inwards towards the Golgi complex along microtubules by using the microtubule minus-end-directed motor complex of dynein/dynactin. No loss of fluorescent material from pre-Golgi structures occurred during their translocation to the Golgi complex and they frequently stretched into tubular shapes. Together, our results indicate that these pre-Golgi carrier structures moving unidirectionally along microtubule tracks are responsible for transporting VSVG-GFP through the cytoplasm to the Golgi complex. This contrasts with the traditional focus on small vesicles as the primary vehicles for ER-to-Golgi transport.