Cdc34 and the F-box protein Met30 are required for degradation of the Cdk-inhibitory kinase Swe1

Ubiquitin-mediated proteolysis controls the abundance of many cell cycle regulatory proteins. Recent work in Saccharomyces cerevisiae suggests that a complex consisting of Cdc53, Skp1, and a third component known as an F-box protein (termed SCF) in combination with Cdc34 specifically targets regulatory proteins for degradation, and that substrate specificity is likely to be mediated by the F-box subunit. A screen for genetic interactions with a cdc34 mutation yielded MET30, which encodes an F-box protein. MET30 is an essential gene required for cell cycle progression and met30 mutations interact genetically with mutations in SCF components. Furthermore, physical interactions between Met30, Cdc53, Cdc34, and Skp1 in vivo provide evidence for an SCFMet30 complex. We demonstrate the involvement of Met30 in the degradation of the Cdk-inhibitory kinase Swe1. Swe1 is stabilized in met30 mutants and GST-Met30 pull-down experiments reveal that Met30 specifically binds Swe1 in vivo. Furthermore, extracts prepared from cdc34 or met30 mutants are defective in polyubiquitination of Swe1. Taken together, these data suggest that SCF-mediated proteolysis may contribute to the regulation of entry into mitosis. Our data, in combination with previously published results, also provide evidence for distinct SCF complexes in vivo and support the idea that their F-box subunits mediate SCF substrate specificity.     

Primary care of HIV infection

Four cases illustrate some of the issues involved in treating HIV-infected patients in a primary care setting. Primary care physicians are hard-pressed to achieve the same results as infectious disease specialists, yet are increasingly responsible for performing the initial tests, choosing the therapeutic regimen, ensuring the patient's compliance with the regimen, and monitoring the results.     

Systemic inflammatory response syndrome treatment by powdered sorbent pheresis: the BioLogic-Detoxification Plasma Filtration System

Systemic inflammatory response syndrome (SIRS) is one of the most common causes of death in intensive care unit patients. The detoxification plasma filtration (DTPF) system (HemoCleanse, Inc., West Lafayette, IN) combines the DT hemodiabsorption system in series with a push-pull pheresis PF system (a suspension of powdered sorbents surrounding 0.5 microm plasma filter membranes). Bidirectional plasma flow (at 80-100 ml/min) across the PF membranes provides direct contact between plasma proteins and powdered sorbents, as well as clearance of cytokines (tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6) at a rate of 15-25 ml/min, without evidence of saturation for 90 minutes. In a U.S. Food and Drug Administration approved study we treated eight patients with SIRS and organ failure with a single DTPF treatment, using powdered charcoal as sorbent in four patients and powdered charcoal and silica in four patients. Treatments proceeded for 6 hours with proper heparin anticoagulation (activated clotting time 250-300 sec) and appeared safe. All patients improved during the treatments and each had increased blood pressure and decreased need for pressor agents. Plasma cytokine levels stabilized or decreased during treatment and were significantly lower the morning after treatment. Multiple organ dysfunction (MOD) and Acute Physiology Chronic Health Evaluation II scores and organ function gradually improved in most patients, and two patients survived for more than 28 days and two for more than 14 days. The DTPF System may prove beneficial in treatment of patients with sepsis.     

Effects of neutral endopeptidase inhibition and combined angiotensin converting enzyme and neutral endopeptidase inhibition on angiotensin and bradykinin peptides in rats

The respective roles of apoptosis and accidental cell death after thermal injury were evaluated in normal human epidermal keratinocytes. By coupling the LIVE/DEAD fluorescence viability assay with the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method and ultrastructural morphology, these two processes could be distinguished. Cells were grown on glass coverslips with a microgrid pattern so that the results of several staining procedures performed sequentially could be visualized in the same cells after heating at temperatures of up to 72 degrees C for 1 second. After exposure to temperatures of 58 to 59 degrees C, cells died predominantly by apoptosis; viable cells became TUNEL positive, indicating degradation of DNA. After exposure to temperatures of 60 to 66 degrees C, both TUNEL-positive viable cells and TUNEL-positive nonviable cells were observed, indicating that apoptosis and accidental cell death were occurring simultaneously. Cells died almost immediately after exposure to temperatures above 72 degrees C, presumably from heat fixation. The fluorescent mitochondrial probe MitoTracker Orange indicated that cells undergoing apoptosis became TUNEL positive before loss of mitochondrial function. Nucleosomal fragmentation of DNA analyzed by enzyme-linked immunosorbent assay and gel electrophoresis occurred after exposure to temperatures of 58 to 59 degrees C. The characteristic morphological findings of cells undergoing apoptosis, by transmission electron microscopy, included cellular shrinkage, cytoplasmic budding, and relatively intact mitochondria. Depending on temperature and time of exposure, normal human epidermal keratinocytes may die by apoptosis, accidental cell death, or heat fixation.     

Choline acetyltransferase (ChAT) immunoreactivity in paraffin sections of normal and diseased intestines

There is increasing interest in localizing nerves in the intestine, especially specific populations of nerves. At present, the usual histochemical marker for cholinergic nerves in tissue sections is acetylcholinesterase activity. However, such techniques are applicable only to frozen sections and have uncertain specificity. Choline acetyltransferase (ChAT) is also present in cholinergic nerves, and we therefore aimed to establish a paraffin section immunocytochemical technique using an anti-ChAT antibody. Monoclonal anti-choline acetyltransferase (1.B3.9B3) and a biotin-streptavidin detection system were used to study the distribution of ChAT immunoreactivity (ChAT IR) in paraffin-embedded normal and diseased gastrointestinal tracts from both rats and humans. Optimal staining was seen after 6-24 hr of fixation in neutral buffered formalin and overnight incubation in 1 microgram/ml of 1.B3.9B3, with a similar distribution to that seen in frozen sections. In the rat diaphragm (used as a positive control), axons and motor endplates were ChAT IR. Proportions of ganglion cells and nerve fibers in the intramural plexi of both human and rat gastrointestinal tracts were also ChAT IR, as well as extrinsic nerve bundles in aganglionic segments of Hirschsprung's disease. Mucosal cholinergic nerves, however, were not visualized. In addition, non-neuronal cells such as endothelium, epithelium, and inflammatory cells were ChAT IR. We were able to localize ChAT to nerves in formalin-fixed, paraffin-embedded sections. The presence of ChAT IR in non-neuronal cells indicates that this method should be used in conjunction with other antibodies. Nevertheless, it proves to be a useful technique for studying cholinergic neuronal distinction in normal tissues and pathological disorders.