Preweaned heifer management on US dairy operations: Part I. Descriptive characteristics of preweaned heifer raising practices.

The objective of this study was to describe preweaned dairy heifer calf management practices on dairy operations across the United States that were used to analyze factors associated with colostrum quality and passive transfer, Cryptosporidium and Giardia, morbidity and mortality, and average daily gain. This study included 104 dairy operations in 13 states that participated in the National Animal Health Monitoring System's Dairy 2014 calf component study. This 18-mo longitudinal study focused on dairy heifer calves from birth to weaning, and data were collected on 2,545 heifer calves. Descriptive statistics were generated regarding colostrum feeding, preweaning housing, milk feeding and consumption, growth, morbidity and mortality, and weaning practices. The majority of calves enrolled were Holsteins (89.4%). Over half the calves (63.2%) enrolled in the study received the majority of their colostrum via bottle; however, 22.1% of calves from 51.0% of operations received colostrum via suckling from their dams. For all calves, the mean time to the first colostrum feeding was 2.8 h, and the average amount of colostrum at the first feeding was 2.9 L, with 4.5 L provided in the first 24 h. The mean serum IgG of all calves was 21.7 g/L; however, 76.0% of operations had at least 1 calf with failure of passive transfer of immunity with a serum IgG below 10 g/L. The majority of calves in the study were housed individually (86.6%). Nonetheless, 20.2% of operations housed some calves in groups, representing 13.4% of all calves. Approximately one-half of the calves in the study (52.3%) were dehorned or disbudded during the preweaning period, with only 27.8% of these calves receiving analgesics or anesthetics during the procedure. Whole or waste milk was the liquid diet type fed to 40.1% of calves, and milk replacer was fed to 34.8% of calves. A combination of milk and milk replacer was fed to 25.1% of calves. Calves, on average, were fed 2.6 L per feeding and fed 2.6 times/d, resulting in a total of 5.6 L of liquid diet fed per day. The mean average daily gain for all calves enrolled in the study was 0.7 kg/d. Fecal samples were collected and almost all operations had at least 1 calf positive for Cryptosporidium (94.2%) or Giardia (99.0%), and 84.6% of operations had calves that tested positive for both Cryptosporidium and Giardia. Over one-third of calves (38.1%) had at least one morbidity event during the preweaning period and the mortality rate was 5.0%. The mean age at weaning was 65.7 d. This study provides an update on dairy heifer raising practices in the United States.

Preweaned heifer management on US dairy operations: Part III. Factors associated with Cryptosporidium and Giardia in preweaned dairy heifer calves.

The objective of this study was to evaluate management practices and environmental factors associated with cryptosporidiosis and giardiasis in preweaned heifer calves on US dairy operations. This study was conducted as part of the calf component of the National Animal Health Monitoring System's Dairy 2014 study. The calf component included 104 dairy operations in 13 states and was an 18-mo longitudinal study focused on dairy heifer calves from birth to weaning. Fecal samples were collected from 2,249 calves: 839 calves in the West region (California, Colorado, and Washington) and 1,410 calves in the East region (Iowa, Michigan, Minnesota, Missouri, New York, Ohio, Pennsylvania, Vermont, Virginia, and Wisconsin). Fecal samples were collected only once from calves during the preweaning period. Samples were collected from calves 3 to 66 d of age, with a mean of 22 d. Overall, Cryptosporidium and Giardia were detected in 43.1 and 30.5% of fecal samples, respectively. Backward elimination logistic model selection was used after univariate screening to determine which management practices and environmental factors significantly affected the presence of Cryptosporidium or Giardia. The final Cryptosporidium model included herd size, days of age at fecal collection, and average temperature-humidity index for the month of fecal collection (fTHI). Cryptosporidium was found on a higher percentage of large operations (≥500 cows) than small operations (30 to 99 cows). Younger calves were more likely to have a fecal sample positive for Cryptosporidium than samples from older calves. Fecal samples from calves during the warmer parts of the year (fTHI >70) were more likely to be positive for Cryptosporidium than samples collected in colder months (fTHI <20). The final Giardia model included herd size, days of age at fecal collection, average fTHI, failure of passive transfer status, and average daily gain (kg/d) during the preweaning period. Giardia was isolated more frequently from calves on small operations than on large operations and from calves that were older compared with younger calves. Giardia was more frequently isolated in warmer months. Samples from calves with failure of passive transfer were more likely to have Giardia than calves with adequate passive transfer (>10 g/L IgG). Average daily gain during the preweaning period was lower in calves from which Giardia was isolated. These results highlight the factors associated with the presence of Cryptosporidium and Giardia in preweaned dairy heifer calves.

Preweaned heifer management on US dairy operations: Part V. Factors associated with morbidity and mortality in preweaned dairy heifer calves.

The objective of this study was to evaluate morbidity and mortality in preweaned dairy heifer calves based on different health, feeding, and management practices, as well as environmental factors. This study was conducted as part of the calf component of the National Animal Health Monitoring System's Dairy 2014 study, which included 104 dairy operations in 13 states. The calf component was an 18-mo longitudinal study focused on dairy heifer calves from birth to weaning; data were collected on 2,545 calves. The percentage morbidity for all calves enrolled in the study was 33.9%. Backward elimination model selection was used after univariate screening to determine which management practices and environmental factors significantly affected morbidity and mortality. The final morbidity model included birth weight, serum IgG concentration, ventilation type, and average temperature-humidity index (THI) during the preweaning period. After controlling for other independent variables in the model, calves born at a higher birth weight had a lower predicted risk of morbidity than calves with a lower birth weight. An increase in serum IgG concentration was associated with decreased morbidity. Calves housed in positive- or cross-ventilated systems had a 2.2 times higher odds of developing disease compared with calves housed in natural ventilation systems. Average THI during the preweaning period was inversely correlated with morbidity; as THI increased, the predicted morbidity risk decreased. The percent mortality for all calves enrolled in the study was 5.0%. The final mortality model included birth weight, serum IgG concentration, amount of fat/day in the liquid diet, and morbidity. After controlling for other independent variables in the model, calves born at a higher birth weight had a lower risk of mortality. An increase in serum IgG concentration decreased the risk of mortality. The odds of mortality were 3.1 times higher in calves fed ≤0.15 kg of fat/d in the liquid diet compared with calves fed ≥0.22 kg of fat/d. The odds of mortality were 4.7 times higher in calves that experienced any disease throughout the preweaning period than in calves with no disease. In summary, morbidity and mortality were both associated with birth weight and serum IgG concentration. Additionally, morbidity was associated with ventilation type and average monthly THI, and mortality was associated with amount of fat per day in the liquid diet and morbidity.

Associations between housing and management practices and the prevalence of lameness, hock lesions, and thin cows on US dairy operations.

The objective of this study was to determine the association among different housing and management practices on the prevalence of lameness, hock lesions, and thin cows on US dairy operations. This study was conducted as part of the National Animal Health Monitoring System's Dairy 2014 study, which included dairy operations in 17 states. Size categories were assigned as follows: small (30-99 cows), medium (100-499 cows), and large (≥500 cows). Trained assessors visited 191 dairy operations from March through July 2014 and recorded locomotion and hock scores (on a 3-point scale), and the number of thin cows (body condition score ≤2.25) from a total of 22,622 cows (average 118 cows per farm). The majority of cows (90.4%) were considered to be sound (locomotion score = 1), 6.9% were mild/moderately lame (locomotion score = 2), and 2.7% were severely lame (locomotion score = 3). Similarly, most cows (87.3%) had no hock lesions (hock score = 1), 10.1% had mild lesions (hock score = 2), and 2.6% had severe hock lesions (hock score = 3). A low percentage of cows (4.2%) were thin. Univariate comparisons were performed using PROC LOGLINK, which accounts for study design and weighting. Variables meeting the univariate screening criterion of P < 0.20 were eligible for entry into multivariable models. Statistical significance in the multivariable models was declared at P < 0.05. Large operations had a lower within-herd prevalence of cows with locomotion score ≥2 and locomotion score = 3 compared with small or medium-sized operations. Operations on which cows were kept primarily on pasture had a lower percentage of locomotion score = 3 than those housed in freestall or open/dry lot operations. The use of sand bedding was associated with a lower within-herd prevalence of locomotion score ≥2 than straw/hay or dry/composted manure as the primary bedding material. Sand bedding was also associated with a lower within-herd prevalence of locomotion score = 3 than other bedding types except for rubber mats or mattresses. Operations that housed cows in an open/dry lot had a lower percentage of hock score ≥2 and hock score = 3 than other housing types. Providing sprinklers for heat abatement and having a nutritionist balance rations for cows was associated with a lower percentage of thin cows. Results from this study highlight management practices that may reduce the prevalence of lameness, hock lesions, and thin cows on dairy operations in the United States.

The development of a prototype measure of the co-production of health in routine consultations for people with long-term conditions.

OBJECTIVES: (i) To develop a prototype measure of co-production of health (CPH) in consultations for people with long-term conditions (LTCs); and (ii) to undertake initial validation of it, using a measure of patient-centred care, as defined by the Roter interaction analysis system (RIAS). METHODS: Mixed methods were applied. A qualitative study gathered 11 experts' views on what comprised CPH behaviours. These were operationalised and a prototype measure applied to a convenience sample of 50 video-recorded consultations involving clinicians trained in self-management support and patients with LTCs at health services in six UK locations. RESULTS: Twenty-two CPH behaviours were identified. High frequencies of CPH behaviours in consultations were associated with greater patient-centeredness, less clinician verbal dominance, and more patient communication control in comparison to consultations where CPH behaviours were less frequent. CONCLUSION: Although the CPH tool is promising, further testing is required in order to improve reliability and validity. PRACTICAL IMPLICATIONS: In the future, the measure could be used to test interventions to promote patient participation in decision making about self-management.

Dairy cow handling facilities and the perception of Beef Quality Assurance on Colorado dairies.

A survey was conducted on Colorado dairies to assess attitudes and practices regarding Dairy Beef Quality Assurance (DBQA). The objectives were to (1) assess the need for a new handling facility that would allow all injections to be administered via DBQA standards; (2) establish if Colorado dairy producers are concerned with DBQA; and (3) assess differences in responses between dairy owners and herdsmen. Of the 95 dairies contacted, 20 (21%) agreed to participate, with a median herd size of 1,178. When asked to rank the following 7 traits--efficiency, animal safety, human safety, ease of animal handling, ease of operation, inject per Beef Quality Assurance (BQA) procedures, and cost--in order of priority when designing a new handling facility, human and animal safety were ranked highest in priority (first or second) by the majority of participants, with ease of animal handling and efficiency ranked next. Interestingly, the administration of injections per BQA standards was ranked sixth or seventh by most participants. Respondents estimated the average annual income from the sale of cull cows to be 4.6% of all dairy income, with 50% receiving at least one carcass discount or condemnation in the past 12 mo. Although almost all of the participating dairy farmers stated that the preferred injection site for medications was the neck region, a significant number admitted to using alternate injection sites. In contrast, no difference was found between responses regarding the preferred and actual location for intravenous injections. Although most participating producers are aware of BQA injection guidelines, they perceive efficiency as more important, which could result in injections being administered in locations not promoted by BQA. Dairy owners and herdsmen disagreed in whether or not workers had been injured in the animal handling area in the last 12 mo. Handling facilities that allow for an efficient and safe way to administer drugs according to BQA guidelines and educational opportunities that highlight the effect of improved DBQA on profitability could prove useful. Dairy producers play a key role in ensuring that dairy beef is safe and high quality, and just as they are committed to producing safe and nutritious milk for their customers, they should be committed to producing the best quality beef.

Using temperature-sensing reticular boluses to aid in the detection of production diseases in dairy cows.

The objective of this study was to investigate associations between increases in reticular temperature (RT) in dairy cows and the diagnosis of metritis, mastitis, lameness, and pneumonia by dairy personnel. A prospective case-control study was conducted on a 2,175-cow dairy operation in Colorado from May 2010 to April 2011. Each cow received an orally administered temperature sensing reticular bolus after parturition and RT measurements were recorded 3 times per day as lactating cows exited the milking parlor. A cow was identified as having an increased RT when a deviation of 0.8°C above baseline (average of readings of previous 10d) was recorded by the TempTrack software (DVM Systems, LLC, Greeley, CO). During the same study period, dairy personnel without access to RT data recorded health events and classified them according to clinical signs observed. A total of 201 health events (cases) were included in the data analysis. Cows with clinical mastitis and pneumonia had significantly higher odds (6.7 and 7.5 times higher, respectively) of having an increased RT of 0.8°C above their baseline within 4d preceding diagnosis when compared with control cows. Specificity and sensitivity for an increase of 0.8°C above baseline RT within 4d of disease diagnosis was 76.85 and 66.97% for mastitis, and 69.23 and 76.92% for pneumonia, respectively. No significant difference in RT was found for cows diagnosed with lameness or metritis. Results of this study suggest that RT monitoring can be a useful tool in the early detection of mastitis and pneumonia in dairy cows.

Using XML and XSLT for flexible elicitation of mental-health risk knowledge.

Current tools for assessing risks associated with mental-health problems require assessors to make high-level judgements based on clinical experience. This paper describes how new technologies can enhance qualitative research methods to identify lower-level cues underlying these judgements, which can be collected by people without a specialist mental-health background. Content analysis of interviews with 46 multidisciplinary mental-health experts exposed the cues and their interrelationships, which were represented by a mind map using software that stores maps as XML. All 46 mind maps were integrated into a single XML knowledge structure and analysed by a Lisp program to generate quantitative information about the numbers of experts associated with each part of it. The knowledge was refined by the experts, using software developed in Flash to record their collective views within the XML itself. These views specified how the XML should be transformed by XSLT, a technology for rendering XML, which resulted in a validated hierarchical knowledge structure associating patient cues with risks. Changing knowledge elicitation requirements were accommodated by flexible transformations of XML data using XSLT, which also facilitated generation of multiple data-gathering tools suiting different assessment circumstances and levels of mental-health knowledge.

Identification of a role for actin in translational fidelity in yeast.

Numerous studies have suggested a role for actin in translation, but the molecular details of this role are unknown. To elucidate the function(s) of actin in translation, we have studied 25 isogenic, conditional yeast actin mutants. Strikingly, analysis of these mutants indicates that none of those tested have conditional growth defects caused by reduced rates of protein synthesis; and analysis of latrunculin A-treated wild-type cells indicates that even complete disruption of the actin cytoskeleton has no significant effect on the rate of translation. However, analysis of the effect of the 25 actin mutations on fidelity and sensitivity to translation inhibitors identified two mutations ( act1-2 and act1-122) that cause a significant reduction in the fidelity of translation, as assayed by nonsense suppression, and several mutants that are sensitive to paromomycin, which affects translational fidelity. Translation elongation factor 1A (eEF1A) also has a role in fidelity, and in the presence of excess eEF1A four of the mutants ( act1-2, act1-20, act1-120, and act1-125) are even more sensitive to paromomycin, while one mutant ( act1-122) becomes less sensitive. Together, these findings suggest that actin may not be important for the rate of translation, but may have a critical role in ensuring translational fidelity.

Overexpression of translation elongation factor 1A affects the organization and function of the actin cytoskeleton in yeast.

The translation elongation factor 1 complex (eEF1) plays a central role in protein synthesis, delivering aminoacyl-tRNAs to the elongating ribosome. The eEF1A subunit, a classic G-protein, also performs roles aside from protein synthesis. The overexpression of either eEF1A or eEF1B alpha, the catalytic subunit of the guanine nucleotide exchange factor, in Saccharomyces cerevisiae results in effects on cell growth. Here we demonstrate that overexpression of either factor does not affect the levels of the other subunit or the rate or accuracy of protein synthesis. Instead, the major effects in vivo appear to be at the level of cell morphology and budding. eEF1A overexpression results in dosage-dependent reduced budding and altered actin distribution and cellular morphology. In addition, the effects of excess eEF1A in actin mutant strains show synthetic growth defects, establishing a genetic connection between the two proteins. As the ability of eEF1A to bind and bundle actin is conserved in yeast, these results link the established ability of eEF1A to bind and bundle actin in vitro with nontranslational roles for the protein in vivo.

1,25 dihydroxyvitamin D3 and dexamethasone induce the cyclooxygenase 1 gene in osteoclast-supporting stromal cells.

Commitment of members of the monocyte/macrophage family to the bone resorptive phenotype, in vitro, requires contact, of these osteoclast precursors, with osteoblasts or related stromal cells. The osteoclast-inductive properties of these stromal cells are typically expressed, however, only in the presence of steroid hormones such as 1,25 dihydroxyvitamin D (1,25D3) and dexamethasone (DEX). To gain insight into the means by which steroid treated accessory cells induce osteoclast differentiation we asked, using differential RNA display (DRD), if gene expression by this stromal cell population differs from that of their untreated, non-osteoclastogenic counterpart. We identified four known genes specifically expressed by 1,25D3/DEX-treated ST2 stromal cells: 1) a family of rat organic anion transporters, 2) Na/K ATPase ss-subunit, 3) tazarotene-induced gene 2 (TIG2), and 4) prostaglandin G/H synthase I, or cyclooxygenase 1 (Cox-1). The regulation of these genes in 1,25D3/DEX-treated ST2 cells was demonstrated by Northern blot analysis of treated (osteoclast-supporting) and untreated (non-osteoclast-supporting) ST2 cells; the genes have a limited and specific tissue mRNA expression pattern. Northern blot analysis of treated and untreated ST2 cell total RNA using either a DRD-derived Cox-1 cDNA or a Cox-1 specific oligonucleotide confirmed the steroid regulation of Cox-1 mRNA. Surprisingly, there is no detectable expression by untreated or steroid exposed ST2 cells, of Cox-2, the classical regulated cyclooxygenase isoform. In contrast to 1, 25D3/DEX, serum treatment rapidly induces Cox-2 mRNA, substantiating the capacity of ST2 cells to express the gene. These data establish that steroid induction of the osteoclastogenic properties of stromal cells is attended by Cox gene expression, a phenomenon consistent with the capacity of eicosinoids to impact the resorptive process. The response of osteoclast-supporting ST2 cells to 1,25D3/DEX treatment may be one prostaglandin-mediated event which specifically involves Cox-1 regulation.

The changing nature of nurses' job satisfaction: an exploration of sources of satisfaction in the 1990s.

This paper focuses on the changing nature of nurses' job satisfaction. It compares the major sources of satisfaction and dissatisfaction experienced by acute ward nurses in the English National Health Service (NHS) in the early 1990s, with sources identified in previous research. In the light of findings from a pilot study, the suitability of existing research approaches and measurement tools for portraying nurses' contemporary work experiences is examined. The study comprised content analysis of a random sample of 130 nurses' comments about ward organizational issues, collected as part of a national survey. Findings suggest that new measurement tools need to be developed, because new sources of satisfaction and dissatisfaction emerged, directly associated with change arising out of the introduction of the NHS internal market. These include pressures associated with new roles, role conflict, lack of job security, 'tight' resources, using new technology, a perceived lowering of standards of patient care, coping with increased amounts of paperwork, and the experience of working in a rapidly and constantly changing environment. Findings also suggest that the nature of nurses' job satisfaction is increasingly being shaped by their position within the organization, denoted by clinical grade, and the organizational culture of individual NHS Trusts. Ward leaders experience dissatisfaction as a result of role conflict and strain, while nurses of lower clinical grades are increasingly concerned with managerial and resource constraints on their ability to provide good quality care. Nurses' satisfaction with management and morale were found to be significantly different between NHS Trusts. While findings may be specific to England, it is argued that they have relevance for the wider, international nursing community. This is because developing an understanding of the changing nature of nurses' job satisfaction may help to resolve recruitment and retention problems.

Identification of a novel parathyroid hormone-responsive gene in human osteoblastic cells.

Parathyroid hormone (PTH) is a potent stimulator of osteoblastic cell function in vitro and bone resorption and formation in vivo; however, the details of the molecular mechanism(s) responsible for PTH action and the regulation of gene expression in response to PTH remain unknown. In this study, we employed an mRNA differential display (DRD) approach to examine the initial events in gene expression in human osteoblast-like SaoS-2/B10 cells exposed to 10(-7) mol/L bPTH(1-34). This approach identified several differentially regulated mRNA species, including a novel paired-class homeobox protein, osteoblast-specific factor-2 (OSF-2), and a unique clone with no known sequence homology (clone G18). G18 is a previously unidentified human gene, expressed in a wide variety of human tissues, including heart, brain, placenta, skeletal muscle, and kidney, and is regulated by PTH in osteoblastic cells in vitro. This mRNA appears to be the product of a single gene, which is alternatively spliced to produce multiple transcript sizes observed in several tissues, except bone and bone-derived cells, in which a single predominant approximately 1.8 kb transcript is observed. Our study has identified several genes that have expression altered significantly by treatment with bPTH(1-34), and which may provide insight into the immediate effects of PTH on osteoblast-like cells and ultimately on the mechanism of action and bioactivity of PTH.

Suppressor analysis of fimbrin (Sac6p) overexpression in yeast.

Yeast fimbrin (Sac6p) is an actin filament-bundling protein that is lethal when overexpressed. To identify the basis for this lethality, we sought mutations that can suppress it. A total of 1326 suppressor mutations were isolated and analyzed. As the vast majority of mutations were expected to simply decrease the expression of Sac6p to tolerable levels, a rapid screen was devised to eliminate these mutations. A total of 1324 mutations were found to suppress by reducing levels of Sac6p in the cell. The remaining 2 mutations were both found to be in the actin gene and to make the novel changes G48V (act1-20) and K50E (act1-21). These mutations suppress the defect in cytoskeletal organization and cell morphology seen in ACT1 cells that overexpress SAC6. These findings indicate that the lethal phenotype caused by Sac6p overexpression is mediated through interaction with actin. Moreover, the altered residues lie in the region of actin previously implicated in the binding of Sac6p, and they result in a reduced affinity of actin for Sac6p. These results indicate that the two mutations most likely suppress by reducing the affinity of actin for Sac6p in vivo. This study suggests it should be possible to use this type of suppressor analysis to identify other pairs of physically interacting proteins and suggests that it may be possible to identify sites where such proteins interact with each other.

Arginine 186 in the extracellular N-terminal region of the human parathyroid hormone 1 receptor is essential for contact with position 13 of the hormone.

PTH maintains blood calcium concentrations within the physiological range by acting on a G protein-coupled heptahelical receptor (PTH1 Rc) located primarily in cells in bone and kidney. We have undertaken a photoaffinity cross-linking approach to elucidate the nature of the bimolecular interaction of PTH with the human (h) PTH1 Rc. Specifically, we have studied the region of the receptor that interacts with the midregion of PTH-(1-34), position 13, using a benzophenone-containing photoaffinity ligand, 125I-[Nle(8,18),Lys13(epsilon-pBz2),L-2-NaI23,Arg(26,2 7),Tyr34]bPTH-(1-34)NH2 (125I-K13). Using site-directed mutagenesis in combination with biochemical analysis, we have reduced our previously identified contact domain, 17 residues in the extracellular region of the receptor (173-189), to an 8-amino acid domain (182-189). Furthermore, we have found arginine 186 to be of critical importance to the interaction of the hPTH1 Rc with 125I-K13: modification of Arg186 to either lysine or alanine does not modify receptor avidity or signal transduction by the receptor, but eliminates cross-linking to 125I-K13.

Parathyroid hormone-receptor interactions identified directly by photocross-linking and molecular modeling studies.

Direct mapping of the interface between parathyroid hormone (PTH) and its receptor (hPTH1-Rc) was carried out by photoaffinity scanning studies. Photoreactive analogs of PTH singularly substituted with a p-benzoylphenylalanine (Bpa) at each of the first six N-terminal positions have been prepared. Among these, the analog [Bpa1,Nle8,18,Arg13,26,27,L-2-Nal23,Tyr34]bPTH-(1-34)N H2 (Bpa1-PTH-(1-34)) displayed in vitro activity with potency similar to that of PTH-(1-34). The radioiodinated analog 125I-Bpa1-PTH-(1-34) cross-linked specifically to the hPTH1-Rc stably expressed in human embryonic kidney cells. A series of chemical and enzymatic digestions of the hPTH1-Rc-125I-Bpa1-PTH-(1-34) conjugate suggested that a methionine residue (either Met414 or Met425) within the contact domain hPTH1-Rc-(409-437), which includes the transmembrane helix 6 and part of the third extracellular loop, as the putative contact point. Site-directed mutagenesis (M414L or M425L) identified Met425 as the putative contact point. Molecular modeling of the hPTH1-Rc together with the NMR-derived high resolution structure of hPTH-(1-34), guided by the cross-linking data, strongly supports Met425, at the extracellular end of transmembrane helix 6, as the residue interacting with the N-terminal residue of the hPTH-(1-34). The photocross-linking and molecular modeling studies provide insight into the topologic arrangement of the receptor-ligand complex.

Allele-specific suppression by formation of new protein-protein interactions in yeast.

Yeast fimbrin is encoded by the SAC6 gene, mutations of which suppress temperature-sensitive mutations in the actin gene (ACT1). To examine the mechanism of suppression, we have conducted a biochemical analysis of the interaction between various combinations of wild-type and mutant actin and Sac6 proteins. Previously, we showed that actin mutations that are suppressed by sac6 mutations encode proteins with a reduced affinity for wild-type Sac6p. In the present study, we have found that mutant Sac6 proteins bind more tightly to mutant actin than does wild-type Sac6p, and thus compensate for weakened interactions caused by the mutant actin. Remarkably, we have also found that mutant Sac6 proteins bind more tightly to wild-type actin than does wild-type Sac6p. This result indicates that suppression does not occur through the restoration of the original contact site, but rather through the formation of a novel contact site. This finding argues against suppression occurring through a "lock-and-key" mechanism and suggests a mechanism involving more global increases in affinity between the two proteins. We propose that the most common kind of suppressors involving interacting proteins will likely occur through this less specific mechanism.

A septin-based hierarchy of proteins required for localized deposition of chitin in the Saccharomyces cerevisiae cell wall.

Just before bud emergence, a Saccharomyces cerevisiae cell forms a ring of chitin in its cell wall; this ring remains at the base of the bud as the bud grows and ultimately forms part of the bud scar marking the division site on the mother cell. The chitin ring seems to be formed largely or entirely by chitin synthase III, one of the three known chitin synthases in S. cerevisiae. The chitin ring does not form normally in temperature-sensitive mutants defective in any of four septins, a family of proteins that are constituents of the "neck filaments" that lie immediately subjacent to the plasma membrane in the mother-bud neck. In addition, a synthetic-lethal interaction was found between cdc12-5, a temperature-sensitive septin mutation, and a mutant allele of CHS4, which encodes an activator of chitin synthase III. Two-hybrid analysis revealed no direct interaction between the septins and Chs4p but identified a novel gene, BNI4, whose product interacts both with Chs4p and Cdc10p and with one of the septins, Cdc10p; this analysis also revealed an interaction between Chs4p and Chs3p, the catalytic subunit of chitin synthase III. Bni4p has no known homologues; it contains a predicted coiled-coil domain, but no other recognizable motifs. Deletion of BNI4 is not lethal, but causes delocalization of chitin deposition and aberrant cellular morphology. Overexpression of Bni4p also causes delocalization of chitin deposition and produces a cellular morphology similar to that of septin mutants. Immunolocalization experiments show that Bni4p localizes to a ring at the mother-bud neck that lies predominantly on the mother-cell side (corresponding to the predominant site of chitin deposition). This localization depends on the septins but not on Chs4p or Chs3p. A GFP-Chs4p fusion protein also localizes to a ring at the mother-bud neck on the mother-cell side. This localization is dependent on the septins, Bni4p, and Chs3p. Chs3p, whose normal localization is similar to that of Chs4p, does not localize properly in bni4, chs4, or septin mutant strains or in strains that accumulate excess Bni4p. In contrast, localization of the septins is essentially normal in bni4, chs4, and chs3 mutant strains and in strains that accumulate excess Bni4p. Taken together, these results suggest that the normal localization of chitin synthase III activity is achieved by assembly of a complex in which Chs3p is linked to the septins via Chs4p and Bni4p.

Prediction of nodal metastasis and prognosis in breast cancer: a neural model.

BACKGROUND: An increasing number of women with breast cancer are detected with the disease at an early stage, when the lymph nodes are not involved. In order to obviate the necessity to carry out axillary dissection, accurate surrogates for lymph node involvement need to be identified. In this paper we have examined the use of a neural network to predict nodal involvement. The neural approach has also been extended to investigate its predictive applicability to the long-term prognosis of patients with breast cancer. A number of established and experimental prognostic markers have been studied in an attempt to accurately predict patient outcome 72 months after first examination. METHODS: 81, unselected patients, presenting clinically, who had all undergone mastectomy for invasive breast carcinoma were considered in this study. A total of 12 markers were analysed for the prediction of lymph node metastasis, while node status itself was used as an additional marker for the prognostic analysis. In this case the outcome related to whether a patient had relapsed within 72 months of diagnosis. In both cases, a number of marker combinations were analysed separately in an attempt to classify those most favourable marker interactions with respect to lymph node prediction and prognosis. Patients were randomly divided into a training set (n = 50) and a test set (n = 31). The simulation was developed using the NeuralWorks Professional II/Plus software (NeuralWare, Pittsburgh, Pa, USA). RESULTS: In the case of lymph node metastasis, the neural network was able to correctly predict axillary involvement, or otherwise, in 84% of the patients in the test set by considering 9 of the 12 available markers. This represents an improvement of 10% over the traditional approach which considers the tumour grade and size only. The sensitivity and specificity were also shown to be 73% and 90%, respectively. With regard to patient prognosis, again 84% classification accuracy was obtained using a subset of the markers, with a sensitivity of 50% and a specificity of 96%. CONCLUSIONS: Although this study considered a relatively small sample of patients, nevertheless it demonstrates that artificial neural networks are capable of providing strong indicators for predicting lymph node involvement. There is no longer a need for axillary dissection with all its implications in patient morbidity and demands on clinical resources. The management of breast cancer and the planning of strategies for adjuvant treatments is also facilitated by the use of neural networks for the long-term prognosis of patients.

Cdc53p acts in concert with Cdc4p and Cdc34p to control the G1-to-S-phase transition and identifies a conserved family of proteins.

Regulation of cell cycle progression occurs in part through the targeted degradation of both activating and inhibitory subunits of the cyclin-dependent kinases. During G1, CDC4, encoding a WD-40 repeat protein, and CDC34, encoding a ubiquitin-conjugating enzyme, are involved in the destruction of these regulators. Here we describe evidence indicating that CDC53 also is involved in this process. Mutations in CDC53 cause a phenotype indistinguishable from those of cdc4 and cdc34 mutations, numerous genetic interactions are seen between these genes, and the encoded proteins are found physically associated in vivo. Cdc53p defines a large family of proteins found in yeasts, nematodes, and humans whose molecular functions are uncharacterized. These results suggest a role for this family of proteins in regulating cell cycle proliferation through protein degradation.