Continuous Multistage Synthesis and Functionalization of Sub-100 nm Silica Nanoparticles in 3D-Printed Continuous Stirred-Tank Reactor Cascades.

The controlled and continuous production of nanoparticles (NPs) with functionalized surfaces remains a technological challenge. We present a multistage synthetic platform, consisting of 3D-printed miniature continuous stirred-tank reactor (CSTR) cascades, for the continuous synthesis and functionalization of SiO2 NPs. The use of the CSTR platform provides ideal and rapid mixing of precursor solutions, precise injection of additional reagents for multistep reactions, and facile operation when using viscous solutions and handling of syntheses with longer reaction times. To exemplify the use of such custom-designed CSTR cascades, amine- and carbohydrate-functionalized SiO2 NPs are chosen as model reaction systems. In particular, the intensified flow reactor units allowed for the reproducible formation of SiO2 NPs with diameters less than 100 nm and narrow size distributions (3-8%). Most importantly, by assembling various 3D-printed CSTR cascades, we synthesized gluconolactone-capped polyethylenimine-modified silica NPs in a fully continuous manner. The inherent control over NP surface charge, reactor scalability, and the significant shortening of processing times (less than 10 min) compared to batch methodologies (several days) strongly indicate the ability of the reactor technology to accelerate continuous nanomanufacturing. In general, it provides a simple route for the reproducible preparation of functionalized NPs, thus expanding the gamut of flow reactors for material synthesis.

Diminished school-based support for the management of type 1 diabetes in adolescents compared to younger children.

AIMS: To evaluate diabetes management at school in a large cohort of adolescents with type 1 diabetes and to compare the level of support provided to adolescents with that provided to younger children. METHODS: Questionnaires were distributed to adolescents with type 1 diabetes attending nine regional and tertiary paediatric diabetes services in the Republic of Ireland. The data collected included patient demographics, treatment regimen and support provided for self-care management. Results were compared with a similar cohort of primary school children with type 1 diabetes, studied using similar methodology. RESULTS: The study cohort comprised 405 adolescents with a median age of 15 years, of whom 215 (54%) were on multiple daily injections and 128 (32%) were on pump therapy. Eighty-five percent of pump users administered their bolus insulin in classrooms, whereas 76% of those on a multiple daily injection regimen injected outside the classroom. Girls were less likely to administer bolus insulin in an office (10% vs 19%) and more likely to administer it in the bathroom (50% vs 34%; P=0.01). Twenty-five adolescents (12%) on multiple daily injection regimens did not administer bolus insulin at school. Compared to primary school children with type 1 diabetes, adolescents were less likely to use pump therapy, have an emergency treatment plan and have a designated staff member responsible for care needs. CONCLUSIONS: Support provided to adolescents with type 1 diabetes is diminished compared with that provided to younger schoolchildren.

Variable life adjusted display methodology for continuous performance monitoring of carotid endarterectomy.

Introduction The aim of this study was to use variable life-adjusted display (VLAD) methodology to monitor performance of six vascular surgeons undertaking carotid endarterectomy in a single institution. Materials and methods This was a prospective study with continuous analysis. A risk score model to predict 30-day stroke or death for individual patients was developed from data collected from 839 patients from 1992 to 1999. The model was used to monitor performance of six surgeons from 2000 to 2009. Individual risk factors and 30-day outcomes were analysed and VLAD plots were created for the whole unit and for each surgeon. Results Among the 941 carotid endarterectomies in the performance analysis, 28 adverse events were recorded, giving an overall stroke or death rate of 3.06%. The risk model predicted there would be 33 adverse events. There was no statistical difference between the predicted and the observed adverse events (P > 0.2, χ2 value 1.25, 4 degrees of freedom). The VLAD plot for the whole unit shows an overall net gain in operative performance, although this could have been chance variation. The individual VLAD plot showed that surgeons 1, 2, 3 and 6 to have an overall net gain in the number of successful operations. The changes observed between the surgeons was not significant (P > 0.05) suggesting chance variation only. Conclusions Performance of carotid endarterectomy can be continuously assessed using VLAD methodology for units and individual surgeons. Early identification and correction of performance variation could facilitate improved quality of care.

Resolution of acute onset hemichorea-hemiballismus after treatment with intravenous tissue plasminogen activator.

Hyperkinetic movement disorders are uncommon after acute ischemic stroke. Since these movement disorders are rarely the initial manifestation of acute cerebral ischemia, their presence may result in diagnostic uncertainty or it may inappropriately delay intravenous thrombolytic therapy for ischemic stroke. Hemichorea-hemiballism (HC-HB) is one of the more frequently encountered hyperkinetic movement disorders occurring in conjunction with stroke. Although HC-HB may result from a stroke mimic, the acute onset should prompt rapid evaluation and consideration for the presence of stroke along with its time-dependent therapies including recombinant tissue plasminogen activator (rtPA). In this article, we describe a case of a patient with acute cerebral ischemia presenting clinically with HC-HB, who was given intravenous rtPA therapy despite an initially negative, early diffusion-weighted magnetic resonance imaging (MRI). Follow-up brain MRI performed 24 hours after the initiation of thrombolytic therapy confirmed acute infarction in the contralateral striatum. The patient had near-complete resolution of her HC-HB on discharge and had no complications related to the administration of intravenous rtPA.

Phase II trial of docetaxel and oxaliplatin in patients with advanced gastric cancer and/or adenocarcinoma of the gastroesophageal junction.

BACKGROUND: Platinum-based chemotherapy is the standard treatment for advanced gastric cancer (GC). This trial explored the efficacy and tolerability of combined docetaxel (Taxotere) + oxaliplatin (DOCOX) in GC patients. PATIENTS AND METHODS: Patients with untreated stage IV GC or adenocarcinoma of the gastroesophageal junction (AGEJ) received docetaxel 60 mg/m(2) followed by oxaliplatin 130 mg/m(2) on day 1 of each 21-day cycle until progression or unacceptable toxicity. The primary end points were response rate (RR), toxicity, progression-free survival (PFS), and overall survival (OS). RESULTS: Baseline characteristics (N = 71): median age 59 years, 72% male, 51% esophagogastric junction cancer, and Eastern Cooperative Oncology Group performance status of zero, one, two were 42%, 51%, 7%, respectively. The median number of cycles was 6 (range, 1-19). Grades 3-4 toxic effects: neutropenia (70%); vomiting (17%); nausea (16%); dehydration, fatigue, or diarrhea (13%, each); and thrombocytopenia or febrile neutropenia (7%, each). Sixty-six patients completed >/=2 cycles. The RR was 36% with 25 partial response (PR) and no complete responses (CRs); stable disease (SD) was 49%. Clinical benefit rate (CBR = CR + PR + SD >/=6 months) was 40%; median PFS was 4.3 months, and OS was 8.5 months. CONCLUSIONS: DOCOX produced manageable toxicity in patients with advanced GC and AGEJ. The confirmed RR of 36%, CBR of 40%, and median survival of 8.5 months are encouraging and comparable to standard front-line regimens.

Heparin-induced thrombocytopenia from venous thromboembolism treatment.

OBJECTIVES: We aimed to characterize the clinical experiences of patients in whom heparin-induced thrombocytopenia (HIT) complicated heparin therapy for venous thromboembolism (VTE) and who switched to argatroban. DESIGN: A retrospective analysis of previously reported prospective, multicentre, historical-controlled Argatroban-911 and Argatroban-915 studies of argatroban therapy in HIT. SETTING: Inpatient. SUBJECTS: Patients (n = 145) administered heparin for VTE and who developed HIT were identified. INTERVENTIONS: Patients were treated with argatroban 2 mcg kg(-1) min(-1) for up to 14 days, adjusted to maintain activated partial thromboplastin times 1.5 to three times baseline. Patient characteristics, anticoagulation and outcomes were summarized. The primary end-point was a composite of death, amputation, or new thrombosis within 37 days of argatroban initiation. RESULTS: During heparin therapy, platelet counts decreased (mean +/- SD nadir: 78 +/- 67 x 10(9) L(-1)), and 75 (52%) patients developed thrombosis. After heparin was discontinued, patients received argatroban (mean dose 2.1 +/- 1.2 mcg kg(-1) min(-1)) for 6.8 +/- 4.3 days. By day 6 of argatroban therapy, the mean platelet count rose to >150 x 10(9) L(-1). The primary end-point occurred in 41 (28.3%) patients (values of 26-44% are reported for argatroban therapy of HIT from any heparin indication). Seventeen (11.7%) patients, including 12 who had also experienced thrombosis whilst on heparin, developed new thrombosis after argatroban initiation, typically on the day argatroban was discontinued or later (n = 10). Seven (4.8%) patients experienced major bleeding. CONCLUSIONS: For VTE patients with HIT, argatroban provides effective anticoagulation, with outcomes comparable with those reported for other argatroban-treated HIT patients. New thrombosis in this setting occurred most often in patients with existing HIT-associated thrombosis, before HIT recognition or either at/after argatroban discontinuation.

Fission yeast Clp1p phosphatase regulates G2/M transition and coordination of cytokinesis with cell cycle progression.

BACKGROUND: In Saccharomyces cerevisiae the mitotic-exit network (MEN) functions in anaphase to promote the release of the Cdc14p phosphatase from the nucleolus. This release causes mitotic exit via inactivation of the cyclin-dependent kinase (Cdk). Cdc14p-like proteins are highly conserved; however, it is unclear if these proteins regulate mitotic exit as in S. cerevisiae. In Schizosaccharomyces pombe a signaling pathway homologous to the MEN and termed the septation initiation network (SIN) is required not for mitotic exit, but for initiation of cytokinesis and for a cytokinesis checkpoint that inhibits further cell cycle progression until cytokinesis is complete. RESULTS: We have identified the S. pombe Cdc14p homolog, Clp1p, and show that it is not required for mitotic exit but rather functions together with the SIN in coordinating cytokinesis with the nuclear-division cycle. As cells enter mitosis, Clp1p relocalizes from the nucleolus to the spindle and site of cell division. Clp1p exit from the nucleolus does not depend on the SIN, but the SIN is required for keeping Clp1p out of the nucleolus until completion of cytokinesis. Clp1p, in turn, may promote the activation of the SIN by antagonizing Cdk activity until cytokinesis is complete and thus ensuring that cytokinesis is completed prior to the initiation of the next cell cycle. In addition to its roles in anaphase, Clp1p regulates the G2/M transition since cells deleted for clp1 enter mitosis precociously and cells overexpressing Clp1p delay mitotic entry. Unlike Cdc14p, Clp1p appears to antagonize Cdk activity by preventing dephosphorylation of Cdc2p on tyrosine. CONCLUSIONS: S. pombe Clp1p affects cell cycle progression in a markedly different manner than its S. cerevisiae homolog, Cdc14p. This finding raises the possibility that related phosphatases in animal cells will prove to have important roles in coordinating the onset of cytokinesis with the events of mitosis.

Interaction between the noncatalytic region of Sid1p kinase and Cdc14p is required for full catalytic activity and localization of Sid1p.

Sid1p is a group II p21-activated kinase/germinal center kinase family member that is part of a signaling network required for cytokinesis in fission yeast. Germinal center kinases are characterized by well conserved amino-terminal catalytic domains followed by less conserved carboxyl termini. The carboxyl termini among group I germinal center kinases are moderately conserved and thought to be regulatory regions. Little is known about the carboxyl termini of group II family members. Sid1p has been shown to bind the novel protein Cdc14p; however, the functional significance of this interaction is unknown. Here we report that the carboxyl terminus of Sid1p is an essential regulatory region. Our results indicate that this region contains the binding domain for Cdc14p, and this association is required for full Sid1p catalytic activity as well as intracellular localization. Furthermore, overexpression of the carboxyl terminus of Sid1p alone compromises the signaling of cytokinesis. We conclude that Cdc14p positively regulates the Sid1p kinase by binding the noncatalytic carboxyl-terminal region of the protein.

Timing is everything: regulation of mitotic exit and cytokinesis by the MEN and SIN.

Proper completion of mitosis requires careful coordination of numerous cellular events. It is crucial, for example, that cells do not initiate spindle disassembly and cytokinesis until chromosomes have been properly segregated. Cells have developed numerous safeguards or checkpoints to delay exit from mitosis and initiation of the next cell cycle in response to defects in late mitosis. In this review, we discuss recent work on two homologous signaling pathways in budding and fission yeast, termed the mitotic exit network (MEN) and septation initiation network (SIN), respectively, that are essential for coordinating completion of mitosis and cytokinesis with other mitotic events.

Molecular biology. The mad ways of meiosis.

Reproductive cells that are destined to become sperm or egg undergo meiotic division during which the chromosome number is halved. As Sluder and McCollum explain in their Perspective, new findings (Shonn et al.) in yeast show that there is a spindle checkpoint that operates during meiosis to ensure that an equal number of replicated chromosomes arrives at each pole of the cell. One of the components of this meiotic spindle checkpoint turns out to be Mad2, which gives the signal to halt meiosis if it looks like unequal chromosome segregation is taking place.

Mob1p interacts with the Sid2p kinase and is required for cytokinesis in fission yeast.

A great deal is now known about how cells regulate entry into mitosis, but only recently have the mechanisms controlling exit from mitosis and cytokinesis begun to be revealed. In the budding yeast Saccharomyces cerevisiae, Mob1p interacts with the Dbf2p kinase and cells containing mutations in these genes arrest in late anaphase [1] [2]. Proteins related to Mob1p are present in both plants and animals, but information about Mob1p function has been obtained only from budding yeast. Here, we describe the identification and characterization of Mob1p from Schizosaccharomyces pombe. Mob1p associates with the Sid2p kinase and like Sid2p, Mob1p is required for the initiation of cytokinesis, but not for mitotic exit. Mob1p localizes to the spindle pole body (SPB) and to the cell-division site during cell division, suggesting that it might be involved in transducing the signal to initiate cell division from the SPB to the division site. Mob1p is required for Sid2p localization, and Mob1p localization requires the function of the cdc7, cdc11, cdc14, spg1, sid1, sid2, and sid4 genes, suggesting that together with Sid2p, Mob1p functions at the end of the signaling cascade required to regulate the onset of cytokinesis at the end of mitosis.

The role of the sid1p kinase and cdc14p in regulating the onset of cytokinesis in fission yeast.

Coordination of mitosis and cytokinesis is crucial for ensuring proper chromosome segregation and genomic stability. In Schizosaccharomyces pombe, the sid genes (cdc7, cdc11, cdc14, spg1, sid1, sid2 and sid4) define a signaling pathway that regulates septation and cytokinesis. Here we describe the characterization of a novel protein kinase, Sid1p. Sid1p localizes asymmetrically to one spindle pole body (SPB) in anaphase. Sid1p localization is maintained during medial ring constriction and septum synthesis and disappears prior to cell separation. Additionally, we found that Cdc14p is in a complex with Sid1p. Epistasis analysis places Sid1p-Cdc14p downstream of Spg1p-Cdc7p but upstream of Sid2p. Finally, we show that cyclin proteolysis during mitosis is unaffected by inactivating the sid pathway; in fact, loss of Cdc2-cyclin activity promotes Sid1p-Cdc14p association with the SPB, possibly providing a mechanism that couples cytokinesis with mitotic exit.

Tying the knot: linking cytokinesis to the nuclear cycle.

For the survival of both the parent and the progeny, it is imperative that the process of their physical division (cytokinesis) be precisely coordinated with progression through the mitotic cell cycle. Recent studies in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe are beginning to unravel the nature of the links between cytokinesis and the nuclear division cycle. The cyclin-dependent kinases and a novel surveillance mechanism that monitors cytokinesis and/or morphogenesis appear to play important regulatory roles in forging these links. It is becoming increasingly clear that the inactivation of the mitosis-promoting cyclin-dependent kinase, which marks the completion of the nuclear division cycle, is essential for actomyosin ring constriction and division septum assembly in both yeasts. Additionally, the spindle pole bodies are emerging as important transient locale for proteins that might play a key role in coupling the completion of mitosis to the onset of cytokinesis.

Drc1p/Cps1p, a 1,3-beta-glucan synthase subunit, is essential for division septum assembly in Schizosaccharomyces pombe.

Schizosaccharomyces pombe divides by medial fission through the use of an actomyosin-based contractile ring. A division septum is formed centripetally, concomitant with ring constriction. Although several genes essential for cytokinesis have been described previously, enzymes that participate in the assembly of the division septum have not been identified. Here we describe a temperature-sensitive mutation, drc1-191, that prevents division septum assembly and causes mutant cells to arrest with a stable actomyosin ring. Unlike the previously characterized cytokinesis mutants, which undergo multiple mitotic cycles, drc1-191 is the first cytokinesis mutant that arrests with two interphase nuclei. Interestingly, unlike drc1-191, drc1-null mutants proceed through multiple mitotic cycles, leading to the formation of large cells with many nuclei. drc1 is allelic to cps1, which encodes a 1,3-beta-glucan synthase subunit. We conclude that Drc1p/Cps1p is not required for cell elongation and cell growth, but plays an essential role in assembly of the division septum. Furthermore, it appears that constriction of the actomyosin ring might depend on assembly of the division septum. We discuss possible mechanisms that account for the differences in the phenotypes of the drc1-191 and the drc1-null mutants and also reflect the potential links between Drc1p and other cytokinesis regulators.

Sid2p, a spindle pole body kinase that regulates the onset of cytokinesis.

The fission yeast Schizosaccharomyces pombe divides by medial fission through the use of an actomyosin contractile ring. Precisely at the end of anaphase, the ring begins to constrict and the septum forms. Proper coordination of cell division with mitosis is crucial to ensure proper segregation of chromosomes to daughter cells. The Sid2p kinase is one of several proteins that function as part of a novel signaling pathway required for initiation of medial ring constriction and septation. Here, we show that Sid2p is a component of the spindle pole body at all stages of the cell cycle and localizes transiently to the cell division site during medial ring constriction and septation. A medial ring and an intact microtubule cytoskeleton are required for the localization of Sid2p to the division site. We have established an in vitro assay for measuring Sid2p kinase activity, and found that Sid2p kinase activity peaks during medial ring constriction and septation. Both Sid2p localization to the division site and activity depend on the function of all of the other septation initiation genes: cdc7, cdc11, cdc14, sid1, spg1, and sid4. Thus, Sid2p, a component of the spindle pole body, by virtue of its transient localization to the division site, appears to determine the timing of ring constriction and septum delivery in response to activating signals from other Sid gene products.

Identification and characterization of Schizosaccharomyces pombe asp1(+), a gene that interacts with mutations in the Arp2/3 complex and actin.

The Arp2/3 complex is an essential component of the actin cytoskeleton in yeast and is required for the movement of actin patches. In an attempt to identify proteins that interact with this complex in the fission yeast Schizosaccharomyces pombe, we sought high-copy suppressors of the S. pombe arp3-c1 mutant, and have identified one, which we have termed asp1(+). The asp1(+) open reading frame (ORF) predicts a highly conserved protein of 921 amino acids with a molecular mass of 106 kD that does not contain motifs of known function. Neither asp1(+) nor its apparent Saccharomyces cerevisiae ortholog, VIP1, are essential genes. However, disruption of asp1(+) leads to altered morphology and growth properties at elevated temperatures and defects in polarized growth. The asp1 disruption strain also is hypersensitive to Ca+ ions and to low pH conditions. Although Asp1p is not stably associated with the Arp2/3 complex nor localized in any discrete structure within the cytoplasm, the asp1 disruption mutant was synthetically lethal with mutations in components of the Arp2/3 complex, arp3-c1 and sop2-1, as well as with a mutation in actin, act1-48. Moreover, the vip1 disruption strain showed a negative genetic interaction with a las17Delta strain. We conclude that Asp1p/Vip1p is important for the function of the cortical actin cytoskeleton.

Identification of cold-sensitive mutations in the Schizosaccharomyces pombe actin locus.

In recent years, the actin cytoskeleton in Schizosaccharomyces pombe has become the subject of intense scrutiny. However, to date, only a single actin mutation has been identified. Described here is the isolation and characterization of four new cold-sensitive actin mutations. Sequence analysis of the mutant actin genes indicated that each of these mutations caused alterations in single amino acids that are conserved in all actin sequences. These mutants differ in their phenotypes. One of these mutations (act1-48) was identified as an extragenic suppressor of a mutation in the cdc4 gene, which is required for actin ring formation and cytokinesis. Interestingly, when act1-48 mutant cells were shifted to the restrictive temperature, actin patches were not detected but the actin ring formation and stability was unaffected. The three other mutations, act1-16, act1-32 and act1-67, primarily affected the actin ring formation or stability while F-actin patches did not seem to be substantially different in appearance. Given that the ultrastructural architectures of F-actin patches and the F-actin ring are presently unclear, these mutations, which affect one structure or the other, should be useful for future studies on the role of actin itself in the function of these F-actin-containing structures in S. pombe.

Phosphorylation of the myosin-II light chain does not regulate the timing of cytokinesis in fission yeast.

Proper coordination of cytokinesis with chromosome separation during mitosis is crucial to ensure that each daughter cell inherits an equivalent set of chromosomes. It has been proposed that one mechanism by which this is achieved is through temporally regulated myosin regulatory light chain (RLC) phosphorylation (Satterwhite, L. L., and Pollard, T. D. (1992) Curr. Opin. Cell Biol. 4, 43-52). A variety of evidence is consistent with this model. A direct test of the importance of RLC phosphorylation in vivo has been done only in Dictyostelium and Drosophila; phosphorylation of the RLC is essential in Drosophila (Jordan, P., and Karess, R. (1997) J. Cell Biol. 139, 1805-1819) but not essential in Dictyostelium (Ostrow, B. D., Chen, P., and Chisholm, R. L. (1994) J. Cell Biol. 127, 1945-1955). The Schizosaccharomyces pombe myosin light chain Cdc4p is essential for cytokinesis, but it was unknown whether phosphorylation played a role in its regulation. Here we show that the S. pombe myosin light chain Cdc4p is phosphorylated in vivo on either serine 2 or 6 but not both. Mutation of either or both of these sites to alanine did not effect the ability of Cdc4p to bind the type II myosin Myo2p, and cells expressing only these mutated versions of Cdc4p grew and divided normally. Similarly, mutation of Ser-2, Ser-6, or both residues to aspartic acid did not affect growth or division of cells. Thus we conclude that phosphorylation of Cdc4p is not essential in vivo for the function of the protein.

Role of polo kinase and Mid1p in determining the site of cell division in fission yeast.

The fission yeast Schizosaccharomyces pombe divides symmetrically using a medial F-actin- based contractile ring to produce equal-sized daughter cells. Mutants defective in two previously described genes, mid1 and pom1, frequently divide asymmetrically. Here we present the identification of three new temperature-sensitive mutants defective in localization of the division plane. All three mutants have mutations in the polo kinase gene, plo1, and show defects very similar to those of mid1 mutants in both the placement and organization of the medial ring. In both cases, ring formation is frequently initiated near the cell poles, indicating that Mid1p and Plo1p function in recruiting medial ring components to the cell center. It has been reported previously that during mitosis Mid1p becomes hyperphosphorylated and relocates from the nucleus to a medial ring. Here we show that Mid1p first forms a diffuse cortical band during spindle formation and then coalesces into a ring before anaphase. Plo1p is required for Mid1p to exit the nucleus and form a ring, and Pom1p is required for proper placement of the Mid1p ring. Upon overexpression of Plo1p, Mid1p exits the nucleus prematurely and displays a reduced mobility on gels similar to that of the hyperphosphorylated form observed previously in mitotic cells. Genetic and two-hybrid analyses suggest that Plo1p and Mid1p act in a common pathway distinct from that involving Pom1p. Plo1p localizes to the spindle pole bodies and spindles of mitotic cells and also to the medial ring at the time of its formation. Taken together, the data indicate that Plo1p plays a role in the positioning of division sites by regulating Mid1p. Given its previously known functions in mitosis and the timing of cytokinesis, Plo1p is thus implicated as a key molecule in the spatial and temporal coordination of cytokinesis with mitosis.

Isolation and characterization of new fission yeast cytokinesis mutants.

Schizosaccharomyces pombe is an excellent organism in which to study cytokinesis as it divides by medial fission using an F-actin contractile ring. To enhance our understanding of the cell division process, a large genetic screen was carried out in which 17 genetic loci essential for cytokinesis were identified, 5 of which are novel. Mutants identifying three genes, rng3(+), rng4(+), and rng5(+), were defective in organizing an actin contractile ring. Four mutants defective in septum deposition, septum initiation defective (sid)1, sid2, sid3, and sid4, were also identified and characterized. Genetic analyses revealed that the sid mutants display strong negative interactions with the previously described septation mutants cdc7-24, cdc11-123, and cdc14-118. The rng5(+), sid2(+), and sid3(+) genes were cloned and shown to encode Myo2p (a myosin heavy chain), a protein kinase related to budding yeast Dbf2p, and Spg1p, a GTP binding protein that is a member of the ras superfamily of GTPases, respectively. The ability of Spg1p to promote septum formation from any point in the cell cycle depends on the activity of Sid4p. In addition, we have characterized a phenotype that has not been described previously in cytokinesis mutants, namely the failure to reorganize actin patches to the medial region of the cell in preparation for septum formation.