Development and characterization of ibuprofen co-crystals granules prepared via fluidized bed granulation in a one-step process - a design of experiment approach.

OBJECTIVE: The focus of this study was to investigate the possibility of producing ibuprofen-nicotinamide (IBU-NIC) and ibuprofen-isonicotinamide (IBU-INA) cocrystal-containing granules, using a one-step fluidized bed dryer granulation manufacturing process, and evaluate their mechanical properties. SIGNIFICANCE: Pharmaceutical cocrystals represent a suitable strategy to improve properties of active pharmaceutical ingredients (APIs), such as solubility and processability. Ibuprofen (IBU) is a small molecule API which can form cocrystals with different coformers, including NIC and INA. An improvement in mechanical properties for IBU-NIC cocrystals relative to IBU was previously reported but, to date, the formulation of IBU cocrystals in a solid dosage form has not been investigated. METHODS: In situ cocrystallization and granulation were achieved concurrently by processing in a lab-scale fluidized bed granulator following a design of experiment (DoE) approach using a two-level factorial design with both process and formulation variables. Solid-state, micrometric, dissolution, and mechanical (tabletability) characteristics of granules were assessed post-processing. RESULTS: Granules containing cocrystals were successfully prepared for 11 of 16 DoE runs. Parameters with a significant effect on granule drug loading, flow function, porosity, and size could be identified from the DoE model. Process yield was increased by using a high inlet temperature at high solution feed rate. To avoid the formation of sticky particles, caking and over-wetting of the powder during the process, the utilization of high inlet temperature, low API + coformer:filler ratio, low API concentration in solution and low solution feed rate were suggested by the model. CONCLUSION: The multivariable model developed enables accurate optimization of the granulation process for IBU cocrystals.

Fixed Dose Combination Formulations: Multilayered Platforms Designed for the Management of Cardiovascular Disease.

Hyperlipidaemia is considered as one of the main risk factors associated with cardiovascular diseases (CVDs). Among different lipid-lowering agents used to manage hyperlipidaemia, statins are highly prescribed for management of hyperlipidaemia with simvastatin being one of the most common. Simvastatin is susceptible to extensive metabolism by CYP450 3A4 and 3A5, which are expressed both in the liver and the gastrointestinal tract. Nevertheless, the localization of these enzymes is site-dependent with lower concentration at the distal/proximal regions of the small intestine/colon. In addition to statins, medications such as antihypertensive agents and anticoagulants are introduced as adjuvants, for the treatment of cardiovascular disease. The aim of this study was to design a bilayer delivery system capable of delivering biphasic release of simvastatin and aspirin, within a fixed dose combination. A delayed release platform based on a combination of anionic polymers prepared using hot-melt extrusion was developed to delay the release of simvastatin. An optimized formulation tested for dissolution performance clearly demonstrated an ability to delay the release of simvastatin. In addition, an immediate release layer based on Kollidon VA64 was successfully developed to deliver aspirin. Both formulations were then manufactured as a bilayer drug delivery system (tablets and coextrudates), and the release performance was examined. On the basis of the obtained results, these formulations may be used as a platform for delivering a wide range of medications in a biphasic manner.

Synthetic studies on the bioactive tetramic acid JBIR-22 using a late stage Diels-Alder reaction.

A late stage Diels-Alder reaction is used to prepare a mixture of JBIR-22, a natural product from the Equisetin family of tetramic acids, and one of its diastereomers. This is achieved in just 8 steps from pyruvate. The success of the late stage DA approach is discussed in the context of the biosynthesis of JBIR-22 (and perhaps related natural products).

Artificial insemination field data on the use of sexed and conventional semen in nulliparous Holstein heifers.

This study investigated conception rates and other reproductive outcomes achieved with artificial insemination (AI) of nulliparous Holstein heifers using sexed and conventional semen in a commercial Australian dairy herd in central western New South Wales from January 2004 to April 2009. Retrospective data from on-farm records of 9,870 inseminations of 4,456 heifers were analyzed using several mixed models to assess the effect of temperature and humidity surrounding breeding, insemination sire, artificial insemination technician, service number, and heifer weight and age at breeding on reproductive traits (conception rates, sex ratios, gestation length, and abortion and stillbirth rates). Semen was used from 15 sexed sires and 41 unsexed sires. Sexed semen was primarily used at first and second service. Empirical conception rates of 31.6 and 39.6% were achieved for sexed and unsexed semen respectively, whereas model-based predictions were lower, at 21.3 and 32.1%. Conception rates were significantly affected by insemination sire, sex-sorting, heifer age at breeding, temperature and humidity surrounding breeding, service number, and AI technician. Sexed semen yielded 86% heifers, compared with 48% for conventional semen. Significant predictors of calf sex included semen sexing, gestation length, and insemination sire. Twinning rate was high, at 3.6% for both semen types, and gestation length and heifer weight at breeding were significant predictors of twinning. Abortion rates for sexed and unsexed conceptions were similar at 6.1 and 6.5%, respectively, and were affected by heifer age at breeding. Stillbirth rate was affected by calf sex, twinning, gestation length, and AI technician; semen sorting, age at breeding, and temperature and humidity were marginally significant predictors. No abnormalities were observed in the development of offspring, except for a marginally higher stillbirth rate for sexed calves, a finding that needs further investigation. Many variables influence the breeding outcomes associated with the application of sex-sorted sperm on commercial dairy farms. Recognition and management of these variables will increase the economic return from the investment in sex-sorted sperm.

Targeting lung macrophages for fungal and parasitic pulmonary infections with innovative amphotericin B dry powder inhalers.

The incidence of fungal pulmonary infections is known to be on the increase, and yet there is an alarming gap in terms of marketed antifungal therapies that are available for pulmonary administration. Amphotericin B (AmB) is a highly efficient broad-spectrum antifungal only marketed as an intravenous formulation. Based on the lack of effective antifungal and antiparasitic pulmonary treatments, the aim of this study was to develop a carbohydrate-based AmB dry powder inhaler (DPI) formulation, prepared by spray drying. Amorphous AmB microparticles were developed by combining 39.7 % AmB with 39.7 % γ-cyclodextrin, 8.1 % mannose and 12.5 % leucine. An increase in the mannose concentration from 8.1 to 29.8 %, led to partial drug crystallisation. Both formulations showed good in vitro lung deposition characteristics (80 % FPF < 5 µm and MMAD < 3 µm) at different air flow rates (60 and 30 L/min) when used with a DPI, but also during nebulisation upon reconstitution in water.

Validation of a multi-segment spinal model for kinematic analysis and a comparison of different data processing techniques.

Optoelectronic motion capture technology is a useful tool in the quantitative dynamic assessment of the spine. In a clinical setting this may help gain a further understanding of underlining musculoskeletal pathology. It is therefore important that accurate measurements are made to allow data to be comparable across various investigations. This report outlines a new multi-segment spinal model and its validation. A mechanical model consisting of an upper thoracic (UT), lower thoracic and lumbar segment was developed allowing for range of motion assessment. An electrogoniometer and torsiometer were attached to the model to provide a control measurement. The UT segment was chosen for analysis and static trials were collected at angles ranging from 2-45°. Kinematic data was captured using an optoelectronic motion capture system. Software computed angles corresponded well with the control measure. While highlighting the differences in the estimation of angles between software platforms, this study emphasizes the need for the clear description and understanding of the kinematic model used.

The effect of leg length discrepancy on pelvis and spine kinematics during gait.

There are assumptions that leg length discrepancy (LLD) may cause low back pain by creating pelvis obliquity and lumbar scoliosis. Although individuals with LLD develop compensatory movements in the lower limbs and pelvis during walking, few investigations have attempted to identify kinematic variables of the upper body. This study aims to gain an understanding of how simulated LLD influences three-dimensional motion of the pelvis and spine. Seven male participants were required to walk barefoot at a preferred speed. Three LLD conditions (1, 2, and 3cm) were simulated using modified pieces of high density EVA attached to the right foot. An optoelectronic motion analysis system was used to record kinematic data of the pelvis and spine (lumbar, lower and upper thoracic segments) for each condition. Differences in range of motion and patterns of movement for the pelvis and lumbar spine were minimal between barefoot and LLD conditions. These observations could be attributed to various kinematic compensatory strategies within the lower limbs which require further in-depth investigation.

Standardised classification system for bespoke thermoplastic ankle foot orthoses.

PURPOSE: To validate a new classification system for bespoke thermoplastic ankle foot orthoses (AFOs). METHODS: Inter- and intra-observer reliability study. A classification system based on the design and function of AFOs was created. Sixty-three independent observers classified thirty-six photographs of different AFOs, according to the proposed classification system via an online questionnaire. Approximately two weeks later, the same AFOs were classified again by fifty-three of the same participants. All participants were health care professionals, researchers, or technicians with experience in referring for, prescribing, fitting, reviewing, researching or manufacturing AFOs. RESULTS: The mean inter- and intra-observer agreement Fleiss' kappa was 0.932 and 0.944, respectively. 98.3% of participants reported that the classification system was very easy or moderately easy to use, with 85.7% reporting they would use the classification system. 90.5% of participants reported that the proposed AFO classification system was clear, with 84% stating it was useful. CONCLUSION: The proposed classification system for bespoke thermoplastic AFOs, has an excellent inter- and intra-observer agreement. It will reduce the ambiguity of the description of the type of AFOs used in clinical practice and research. Furthermore, it makes reproducible comparisons between groups possible, which are essential for future evaluations of evidence-based orthotic care.

Increased exposure to loading is associated with decreased plantar soft tissue hardness in people with diabetes and neuropathy.

AIMS: Literature indicates that altered plantar loading in people with diabetes could trigger changes in plantar soft tissue biomechanics which, in turn, could affect the risk for ulceration. To stimulate more research in this area, this study uses in vivo testing to investigate the link between plantar loading and tissue hardness. METHODS: Tissue hardness and plantar pressure distribution were measured for six plantar areas in 39 people with diabetes and peripheral neuropathy. RESULTS: Spearman correlation analysis revealed that increased pressure time integral at the 1st metatarsal-head region (r = -0.354, n = 39, P = 0.027) or at the heel (r = -0.378, n = 39, P = 0.018) was associated with reduced hardness in the same regions. After accounting for confounding parameters, generalised estimating equations analysis also showed that 10% increase in pressure time integral at the heel was associated with ≈ 1 unit reduction in hardness in the same region. CONCLUSIONS: For the first time, this study reveals that people with diabetes and neuropathy who tend to load their feet more heavily also tend to have plantar soft tissues with lower hardness. The observed difference in tissue hardness is likely to affect the tissue's vulnerability to overload injury. More research will be needed to explore the implications of the observed association for the risk of ulceration.

The effect of tuning ankle foot orthoses-footwear combinations on gait kinematics of children with cerebral palsy: A case series.

STUDY DESIGN: Case series. BACKGROUND: AFOs are a commonly prescribed medical device given to children with cerebral palsy (CP) in an attempt to improve their gait. The current literature is equivocal on the effects AFOs have on the gait of children with CP. The vast majority of AFOs issued are not subject to AFO-FC tuning. There are emerging studies investigating the effects tuning AFO-FCs has on the gait of children with CP. However, the research is limited, and there is a lack of quantitative data. OBJECTIVE: To compare the kinematics of tuned versus non-tuned gait in children with CP. METHODS: Gait analysis assessment of five children aged between 7-11 years with a diagnosis of CP (one hemiplegic and four diplegic participants, two female, three male, with a Gross Motor Functional Classification System (GMFCS) of 2) at a Gait Analysis Laboratory. RESULTS: In comparison to barefoot and non-tuned gait, walking with a tuned AFO-FC produced improvements in several key gait parameters. Including hip flexion and extension, posterior pelvic tilt and knee extension. Results also indicated that the type of gait pattern demonstrated by the participant affected the outcomes of tuning. CONCLUSIONS: Tuning the AFO-FC of children with CP has the potential to improve hip function, pelvic function, knee extension in stance phase and knee flexion during swing phase and that a non-tuned AFO-FC can potentially decrease hip function, posterior pelvic tilt and increase knee extension. CLINICAL RELEVANCE: Whilst AFO-FC tuning has been recommended for routine clinical practice, there still remains a paucity of research on the kinematic effects of using a tuned AFO-FC compared to a non-tuned. This paper provides a comparison of kinematics on children with CP, during barefoot, non-tuned and tuned AFO-FC walking with a view to inform clinical practice.

Does user perception affect adherence when wearing biomechanically optimised ankle foot orthosis - footwear combinations: A pilot study.

STUDY DESIGN: Pilot study. BACKGROUND: Ankle foot orthoses (AFOs) and footwear combination (FC) is a commonly prescribed medical device given to children with cerebral palsy (CP) in an attempt to improve their gait. Biomechanically optimising the AFO-FC often requires large adaptations to the sole of the user's footwear. There is currently a dearth of literature regarding the user's perception of wearing biomechanically optimised AFOs and adapted footwear and whether their perception affects their adherence to orthotic treatment. OBJECTIVE: This study aimed to investigate perception and adherence to wearing an AFO and FC the participants were asked to wear as part of their orthotic prescription. In particular, whether the visibly modified footwear affected the user's adherence to the orthotic treatment. METHODS: Questionnaire devised for the purpose of this study. RESULTS: All five participants responded to the questionnaire; reporting a high number of positive responses in relation to function, including; an improvement in the way they walked, improved balance and fewer falls. Conversely, there was a high level of negative responses regarding aesthetics, with all participants reporting they did not like the cosmesis of their AFO-FCs. They were conscious that the modification to their footwear was noticeable and therefore different from their peers, yet they adhered to the treatment and in some cases increased the wearing time. CONCLUSIONS: This pilot set of questions indicated that cosmesis is an important factor for children who wear AFOs and adapted footwear. It can be concluded that the impact of the adapted AFO-FC on the participants' function outweighed their opinion on the cosmesis of the device. CLINICAL RELEVANCE: It is vital to understand how orthotic prescriptions affect user adherence. Orthotic prescriptions which are not utilised by the user result in a failed treatment intervention, regardless of the scientific application underpinning them.

A Design of Experiment (DoE) approach to optimise spray drying process conditions for the production of trehalose/leucine formulations with application in pulmonary delivery.

The present study evaluates the effect of L-leucine concentration and operating parameters of a laboratory spray dryer on characteristics of trehalose dry powders, with the goal of optimizing production of these powders for inhaled drug delivery. Trehalose/L-leucine mixtures were spray dried from aqueous solution using a laboratory spray dryer. A factorial design of experiment (DoE) was undertaken and process parameters adjusted were: inlet temperature, gas flow rate, feed solution flow rate (pump setting), aspiration setting and L-leucine concentration. Resulting powders were characterised in terms of particle size, yield, residual moisture content, and glass transition temperature. Particle size was mainly influenced by gas flow rate, whereas product yield and residual moisture content were found to be primarily affected by inlet temperature and spray solution feed rate respectively. Interactions between a number of different process parameters were elucidated, as were relationships between different responses. The leucine mass ratio influenced the physical stability of powders against environmental humidity, and a high leucine concentration (30% w/w) protected amorphous trehalose from moisture induced crystallization. High weight ratio of leucine in the formulation, however, negatively impacted the aerosol performance. Thus, in terms of L-leucine inclusion in a formulation designed for pulmonary delivery, a balance needs to be found between physical stability and deposition characteristics.

The development of an inline Raman spectroscopic analysis method as a quality control tool for hot melt extruded ramipril fixed-dose combination products.

Currently in the pharmaceutical industry, continuous manufacturing is an area of significant interest. In particular, hot-melt extrusion (HME) offers many advantages and has been shown to significantly reduce the number of processing steps relative to a conventional product manufacturing line. To control product quality during HME without process interruption, integration of inline analytical technology is critical. Vibrational spectroscopy (Raman, NIR and FT-IR) is often employed and used for real-time measurements because of the non-destructive and rapid nature of these analytical techniques. However, the establishment of reliable Process Analytical Technology (PAT) tools for HME of thermolabile drugs is challenging. Indeed, the Raman effect is inherently weak and might be subject to interference. Moreover, during HME, heating and photodecomposition can occur and disrupt spectra acquisition. The aim of this research article was to explore the use of inline Raman spectroscopy to characterise a thermolabile drug, ramipril (RMP), during continuous HME processing. Offline measurements by HPLC, LC-MS and Raman spectroscopy were used to characterise RMP and its main degradation product, ramipril-diketopiperazine (RMP-DKP, impurity K). A set of HME experiments together with inline Raman spectroscopic analyses were performed. The feasibility of implementing inline Raman spectroscopic analysis to quantify the level of RMP and RMP-DKP in the extrudate was addressed. Two regions in the Raman spectrum were selected to differentiate RMP and RMP-DKP. When regions were combined, a principle component analysis (PCA) model defined by these two main components (PC 1 = 50.1% and PC 2 = 45%) was established. Using HPLC analyses, we were able to confirm that the PC 1 score was attributed to the level of RMP-DKP, and the PC 2 score was related to the RMP drug content. Investigation of the PCA scatterplot indicated that HME processing temperature was not the only factor causing RMP degradation. Additionally, the plasticiser content, feeding speed and screw rotating speed contributed to RMP degradation during HME processing.

Characterisation of excipient-free nanoporous microparticles (NPMPs) of bendroflumethiazide.

The purpose of this study was to prepare excipient-free porous microparticles of bendroflumethiazide by spray drying and to characterise the physicochemical properties of the particles produced. Solutions of bendroflumethiazide in ethanol/water, ethanol/water/ammonium carbonate or methanol/water/ammonium carbonate were spray dried using a laboratory spray dryer. Spray dried products were characterised by scanning electron microscopy, X-ray powder diffraction, differential scanning calorimetry, FTIR, laser diffraction particle sizing and density measurement. Nanoporous microparticles (NPMPs) were prepared from the alcoholic solutions containing ammonium carbonate. NPMPs were amorphous in nature, had median particles sizes less than 3 microm and densities that were significantly reduced compared to non-porous spray dried bendroflumethiazide powder. The novel process may be used to produce excipient-free amorphous microparticles with desirable physical properties such as amorphous solid state, porosity and low bulk density. This new engineering technology has applications in the design of other therapeutic agents such as those used in pulmonary delivery.

A comparative study between hot-melt extrusion and spray-drying for the manufacture of anti-hypertension compatible monolithic fixed-dose combination products.

The purpose of this work was to investigate the application of different advanced continuous processing techniques (hot melt extrusion and spray drying) to the production of fixed-dose combination (FDC) monolithic systems comprising of hydrochlorothiazide and ramipril for the treatment of hypertension. Identical FDC formulations were manufactured by the two different methods and were characterised using powder X-ray diffraction (PXRD) and modulated differential scanning calorimetry (mDSC). Drug dissolution rates were investigated using a Wood's apparatus, while physical stability was assessed on storage under controlled temperature and humidity conditions. Interestingly both drugs were transformed into their amorphous forms when spray dried, however, hydrochlorothiazide was determined, by PXRD, to be partially crystalline when hot melt extruded with either polymer carrier (Kollidon® VA 64 or Soluplus®). Hot melt extrusion was found to result in significant degradation of ramipril, however, this could be mitigated by the inclusion of the plasticizer, polyethylene glycol 3350, in the formulation and appropriate adjustment of processing temperature. The results of intrinsic dissolution rate studies showed that hot-melt extruded samples were found to release both drugs faster than identical formulations produced via spray drying. However, the differences were attributable to the surface roughness of the compressed discs in the Wood's apparatus, rather than solid state differences between samples. After a 60-day stability study spray dried samples exhibited a greater physical stability than the equivalent hot melt extruded samples.

1,25-Dihydroxyvitamin D3 maintains adherence of human monocytes and protects them from thermal injury.

Adherence to a substratum is a characteristic feature of monocyte-macrophages which may be required for several effector functions. Human peripheral blood monocytes selected by adherence were found to readhere preferentially at 1 h to fibronectin or to a biological matrix. There was then a progressive decrease in the number of adherent cells, and by 48 h only 8-20% of monocytes remained adherent. This loss of adherence occurred while monocytes remained viable by criteria such as exclusion of trypan blue or release of lactate dehydrogenase. 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) maintained the adherence of cultured monocytes to tissue culture plastic as well as to the biological matrix. This effect was concentration- and time-dependent, and suppressed by inhibitors of protein synthesis. Cellular proteins were labeled after incubation with [35S]methionine. Analysis by two-dimensional gel electrophoresis revealed increased labeling of several distinct proteins in 1,25-(OH)2D3-treated monocytes compared with control monocytes. The increased loss of adherence and decreased overall protein synthesis observed in monocytes incubated at 45 degrees C was partially prevented by preincubation of the cells with 1,25-(OH)2D3. We further evaluated the effects of thermal stress and 1,25-(OH)2D3 on protein synthesis by monocytes, and found that 1,25-(OH)2D3 increased the synthesis of heat shock proteins, protected normal protein synthesis, and increased the rate of recovery of normal protein synthesis after the thermal stress. These observations suggest that 1,25-(OH)2D3 influences monocytes by preserving the synthesis of proteins, including those critical for the maintenance of cell adherence.

1,25-Dihydroxyvitamin D3 induces collagen binding to the human monocyte line U937.

Interactions of cells with components of the extracellular matrix can modulate cellular functions. We measured binding of a major matrix protein to U937 cells, a human promonocytic line. Radioiodinated type I or type III human collagen was bound only to U937 cells differentiated to a more mature phenotype with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). Binding was observed at 4 degrees C and was saturable; Scatchard analysis of the binding to 1,25-(OH)2D3-pretreated U937 cells indicated a single class of high-affinity binding sites. Preincubation of U937 cells with interferon gamma did not induce collagen binding. Collagen binding did not appear to be dependent on fibronectin binding. Surface proteins of U937 cells were 125I labeled and cell membrane proteins resolved by affinity chromatography on collagen-Sepharose. Major specifically labeled bands of 180, 155, and 125 kD were identified in membrane fractions from 1,25-(OH)2D3-pretreated U937 cells only. 1,25-(OH)2D3 appears to specifically regulate collagen binding to monocyte precursors.

A targeted point mutation in thrombomodulin generates viable mice with a prethrombotic state.

The activity of the coagulation system is regulated, in part, by the interaction of thrombin with the endothelial cell receptor thrombomodulin with subsequent generation of activated protein C and suppression of thrombin production. Our previous investigation demonstrated that ablation of the thrombomodulin gene in mice causes embryonic lethality before the assembly of a functional cardiovascular system, indicating a critical role for the receptor in early development. In the current study, we show that a single amino acid substitution in thrombomodulin dissociates the developmental function of the receptor from its role as a regulator of blood coagulation. Homozygous mutant mice with severely reduced capacity to generate activated protein C or inhibit thrombin develop to term, and possess normal reproductive performance. The above animals exhibit increased fibrin deposition in selected organs, which implies tissue specific regulation of the coagulation system that is supported by further evidence from the examination of mice with defects in fibrinolysis. The thrombomodulin-deficient animals provide a murine model to examine known or identify unknown genetic and environmental factors that lead to the development of thrombosis.

Sequences required for transcriptional initiation of the Saccharomyces cerevisiae CYC7 genes.

A series of BAL 31 deletions were constructed in the upstream region of the Saccharomyces cerevisiae CYC7 gene to determine sequences required for transcriptional initiation. These deletions identified the TATA box as an alternating A-T sequence at -160 and the initiation sequences as well as the spatial relationship between them. The TATA box was necessary for wild-type levels of expression of the CYC7 gene. Decreasing the distance between the TATA sequence and the initiation site did not alter gene expression, but the site of transcription was shifted 3'-ward. In most cases, transcription initiated at a number of sites, the 5'-most of which was the first suitable site greater than 45 base pairs 3' of the TATA sequence, suggesting a spatial relationship between these sequences. Consensus sequences previously proposed for initiation sites were evaluated with respect to the start sites identified in this study as well as the start sites of other yeast genes.

CDC55, a Saccharomyces cerevisiae gene involved in cellular morphogenesis: identification, characterization, and homology to the B subunit of mammalian type 2A protein phosphatase.

Microscopic screening of a collection of cold-sensitive mutants of Saccharomyces cerevisiae led to the identification of a new gene, CDC55, which appears to be involved in the morphogenetic events of the cell cycle. CDC55 maps between CDC43 and CHC1 on the left arm of chromosome VII. At restrictive temperature, the original cdc55 mutant produces abnormally elongated buds and displays a delay or partial block of septation and/or cell separation. A cdc55 deletion mutant displays a cold-sensitive phenotype like that of the original isolate. Sequencing of CDC55 revealed that it encodes a protein of about 60 kDa, as confirmed by Western immunoblots using Cdc55p-specific antibodies. This protein has greater than 50% sequence identity to the B subunits of rabbit skeletal muscle type 2A protein phosphatase; the latter sequences were obtained by analysis of peptides derived from the purified protein, a polymerase chain reaction product, and cDNA clones. An extragenic suppressor of the cdc55 mutation lies in BEM2, a gene previously identified on the basis of an apparent role in bud emergence.