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.     

Engineering a lever into the kinesin neck

To probe for a lever arm action in the kinesin stepping mechanism, we engineered a rodlike extension piece into the tail of rat kinesin at various points close to the head-tail junction and measured its effects on the temperature dependence of velocity in microtubule gliding assays. The insert comprised two contiguous alpha-actinin triple-coil repeats and was predicted to fold into a stiff rodlike module about 11 nm long. The effects of this module were greater the closer it was placed to the head-tail junction. When inserted distal to the head-tail junction, at Asn401 in the dimeric K partial differential401GST, the insert had no effect. When inserted closer to the heads at Val376 into K partial differential376GST, the insert slowed progress below 22 degreesC but accelerated progress to approximately 125% of wild type above 22 degreesC. The most dramatic effect of the synthetic lever occurred when it was inserted very close to the head-neck junction, at Glu340 into the single-headed construct K partial differential340GST. This construct was immotile without the insert, but motile with it, at about 30% of the velocity of the dimeric control. The alpha-actinin module thus confers some gain-of-function when inserted close to the head-neck junction but not when placed distal to it. The data exclude the presence of a lever arm C-terminal to Val376 in the kinesin tail but suggest that a short-throw lever arm may be present, N-terminal to Val376 and contiguous with the head-neck junction at Ala339.     

Treatment of acute renal failure

Acute renal failure is a life threatening illness whose mortality has remained high since the introduction of hemodialysis 25 years ago, despite advances in supportive care. Acute renal failure is an extremely morbid and costly disorder with a significant proportion of patients progressing to end-stage renal disease requiring dialysis. To the nephrologist, acute renal failure remains an extremely frustrating disease, because the pathophysiology is not well understood and the limited therapeutic options force the nephrologist to sit on the sidelines and wait for renal function to return. For example, dialysis remains the only FDA-approved treatment for acute renal failure, but dialysis may also cause renal injury that prolongs renal failure. The purpose of this perspective is to understand the results of the recent, largely negative, clinical trials in view of recent advances in the epidemiology of ARF. This review will also discuss diagnostic tools, strategies for improved design of clinical trials, and other therapeutic interventions that will be needed to properly treat acute renal failure in the 21st century.     

The hydrophobic surface of colipase influences lipase activity at an oil-water interface

The interaction of pancreatic triglyceride lipase and colipase at an oil-water interface is required for efficient digestion of dietary fats and provides a model system for the interaction of proteins at biological membranes. Colipase has two important surfaces, a hydrophilic surface that interacts with lipase and a hydrophobic surface that presumably interacts with substrate. To begin our investigations into the role of the hydrophobic surface in the function of colipase, we replaced three neighboring tyrosine residues at positions 55, 58, and 59 in the hydrophobic surface with aspartic acid. Two of the three residues, Tyr55 and Tyr59, influenced the activity of colipase. Introducing aspartic acid at either position decreased the activity with long-chain triglycerides, but not with a short-chain triglyceride. Decreased ability of the mutants to anchor lipase to long-chain triglycerides did not explain the altered activity of the mutants. A mutant containing aspartic acid at positions 55 and 59 had no activity with any substrate and did not anchor lipase to either short- or long-chain triglycerides. These results identify the two tyrosine residues that interact with substrate and suggest that the hydrophobicity of the surface containing these tyrosines influences colipase function and the substrate selectivity of pancreatic triglyceride lipase.     

A randomized controlled trial of filgrastim during remission induction and consolidation chemotherapy for adults with acute lymphoblastic leukemia: CALGB study 9111

Recombinant human granulocyte colony-stimulating factor (G-CSF; filgrastim) shortens the time to neutrophil recovery after intensive chemotherapy, but its role in the treatment of adults with acute lymphoblastic leukemia (ALL) is uncertain. We randomly assigned 198 adults with untreated ALL (median age, 35 years; range, 16 to 83) to receive either placebo or G-CSF (5 microgram/kg/d) subcutaneously, beginning 4 days after starting intensive remission induction chemotherapy and continuing until the neutrophil count was >/=1, 000/microL for 2 days. The study assignment was unblinded as individual patients achieved a complete remission (CR). Patients initially assigned to G-CSF then continued to receive G-CSF through 2 monthly courses of consolidation therapy. Patients assigned to placebo received no further study drug. The median time to recover neutrophils >/=1,000/microL during the remission induction course was 16 days (interquartile range [IQR], 15 to 18 days) for the patients assigned to receive G-CSF and 22 days (IQR, 19 to 29 days) for the patients assigned to placebo (P < .001). Patients in the G-CSF group had significantly shorter durations of neutropenia (<1, 000/microL) and thrombocytopenia (<50,000/microL) and fewer days in the hospital (median, 22 days v 28 days; P = .02) compared with patients receiving placebo. The patients assigned to receive G-CSF had a higher CR rate and fewer deaths during remission induction than did those receiving placebo (P = .04 by the chi-square test for trend). During Courses IIA and IIB of consolidation treatment, patients in the G-CSF group had significantly more rapid recovery of neutrophils >/=1,000/microL than did the control group by approximately 6 to 9 days. However, the patients in the G-CSF group did not complete the planned first 3 months of chemotherapy any more rapidly than did the patients in the placebo group. Overall toxicity was not lessened by the use of G-CSF. After a median follow-up of 4. 7 years, there were no significant differences in either the disease-free survival (P = .53) or the overall survival (P = .25) for the patients assigned to G-CSF (medians, 2.3 years and 2.4 years, respectively) compared with those assigned to placebo (medians, 1.7 and 1.8 years, respectively). Adults who received intensive chemotherapy for ALL benefited from G-CSF treatment, but its use did not markedly affect the ultimate outcome.     

Biochemical studies of the mechanism of action of the Cdc42-GTPase-activating protein

The small GTP-binding proteins Rac, Rho, and Cdc42 were shown to mediate a variety of signaling pathways including cytoskeletal rearrangements, cell-cycle progression, and transformation. Key to the proper function of these GTP-binding proteins is an efficient shut-off mechanism that ensures the decay of the signal. Regulatory proteins termed GAPs (GTPase-activating proteins) enhance the intrinsic GTP hydrolysis of the GTP-binding proteins, thereby ensuring signal termination. We have used site-specific mutagenesis to elucidate the limit domain for GAP activity in Cdc42-GAP, and show that in addition to the known GAP-homology domain (three conserved boxes), a C-terminal region outside that domain is also essential for GAP activity. In addition, we have replaced the conserved arginine (Arg305), which was suggested by structural studies to be a key catalytic residue, with an alanine and found that the R305A Cdc42-GAP mutant has a greatly diminished catalytic capacity but is still able to bind Cdc42 with high affinity. Thus, a key catalytic role for this residue is confirmed. However, we also present evidence for the involvement of an additional residue(s), since the R305A Cdc42-GAP mutant still exhibits measurable activity. Some of this residual activity might result from a neighboring arginine, since a double mutant R305A/R306A shows a further decrease in catalytic activity.     

Histomorphometric, physical, and mechanical effects of spaceflight and insulin-like growth factor-I on rat long bones

Pituitary adenylate cyclase activating peptide and vasoactive intestinal peptide belong to the same neuropeptide family. Both peptides are present in nerve fibers in the gastric wall and are thought to be involved in the regulation of inflammatory processes. Experimental ulcers were induced in the rat gastric mucosa by local application of acetic acid. During the healing process we examined the PACAP and VIP innervation by means of immunohistochemistry and in situ hybridization. The ulcer area was examined from day 1 to day 15 after ulcer induction. There was a marked depletion of PACAP in nerve fibers at the ulcer margin from day 1 and onwards. On day 10 and day 15, PACAP-immunoreactive nerve fibers could again be visualized at the ulcer margin. In contrast, VIP immunoreactive nerve fibers were present at the ulcer margin at all time points studied. From day 10 following ulcer induction PACAP- and VIP- immunoreactive nerve fibers were increased in frequency in the smooth muscle beneath the ulcer. An upregulation of VIP and PACAP mRNA was also demonstrated in the myenteric ganglia adjacent to ulcer. The present results indicate that neuronal PACAP and VIP react differently to the inflammation at the ulcer margin but similarly in the smooth muscle during the ulcer healing.     

Fluctuations in visual neglect after stroke?

Clinical experience suggests that the visual neglect in stroke patients fluctuates over short periods of time. This fluctuation has been variously attributed to fatigue, time of day, previous activities, patient learning and compensation. Such fluctuations have clinical implications for the assessment and rehabilitation of visual neglect but do date no formal study has evaluated the extent of such fluctuation over the course of a day. Twenty-two patients with an acute stroke and 19 patients with convalescent stroke were examined for visual neglect twice on the same day using the Visual Neglect Recovery Index (VNRI), a valid and sensitive measure of the severity of neglect, which could be used to select acute patients for trials of treatment of neglect. The inter-test reliability was extremely high. In contrast to past clinical accounts most patients failed to show significant fluctuation. Although preliminary, this finding suggests that a single assessment of visual neglect, using the VNRI, could help select patients for treatment trials.