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S S Brown

Publications and source records attributed to S S Brown.

At least 19 recordsLinked to original sources
PubMed1998

Smy1p, a kinesin-related protein that does not require microtubules.

We have previously reported that a defect in Myo2p, a myosin in budding yeast (Saccharomyces cerevisiae), can be partially corrected by overexpression of Smy1p, which is by sequence a kinesin-related protein (Lillie, S.H., and S.S. Brown. 1992. Nature. 356:358- 361). Such a functional link between putative actin- and microtubule-based motors is surprising, so here we have tested the prediction that Smy1p indeed acts as a microtubule-based motor. Unexpectedly, we found that abolition of microtubules by nocodazole does not interfere with the ability of Smy1p to correct the mutant Myo2p defect, nor does it interfere with the ability of Smy1p to localize properly. In addition, other perturbations of microtubules, such as treatment with benomyl or introduction of tubulin mutations, do not exacerbate the Myo2p defect. Furthermore, a mutation in SMY1 strongly predicted to destroy motor activity does not destroy Smy1p function. We have also observed a genetic interaction between SMY1 and two of the late SEC mutations, sec2 and sec4. This indicates that Smy1p can play a role even when Myo2p is wild type, and that Smy1p acts at a specific step of the late secretory pathway. We conclude that Smy1p does not act as a microtubule-based motor to localize properly or to compensate for defective Myo2p, but that it must instead act in some novel way.

Carrier Proteins↗
PubMed1997

Myosins in yeast.

It has been a banner year for the study of yeast myosins. Thanks to the completion of the Saccharomyces cerevisiae genome project, it is now known that budding yeast contains a total of five myosins. Furthermore, functions have been newly ascribed to several of them: two have been implicated in endocytosis, and another has been implicated in generating asymmetry between mother and daughter cells.

Myosins↗
PubMed1996

SEC3 mutations are synthetically lethal with profilin mutations and cause defects in diploid-specific bud-site selection.

Replacement of the wild-type yeast profilin gene (PFY1) with a mutated form (pfy1-111) that has codon 72 changed to encode glutamate rather than arginine results in defects similar to, but less severe than, those that result from complete deletion of the profilin gene. We have used a colony color-sectoring assay to identify mutations that cause pfy1-111, but not wild-type, cells to be inviable. These profilin synthetic lethal (psl) mutations result in various degrees of abnormal growth, morphology, and temperature sensitivity in PFY1 cells. We have examined psl1 strains in the most detail. Interestingly, these strains display a diploid-specific defect in bud-site selection; haploid strains bud normally, while homozygous diploid strains show a dramatic increase in random budding. We discovered that PSL1 is the late secretory gene, SEC3, and have found that mutations in several other late secretory genes are also synthetically lethal with pfy1-111. Our results are likely to reflect an interdependence between the actin cytoskeleton and secretory processes in directing cell polarity and growth. Moreover, they indicate that the secretory pathway is especially crucial for maintaining budding polarity in diploids.

Actins↗
PubMed1995

Prospective study of trends in referral patterns in fundholding and non-fundholding practices in the Oxford region, 1990-4.

OBJECTIVE: To compare outpatient referral patterns in fundholding and non-fundholding practices before and after the NHS reforms in April 1991. DESIGN: Prospective collection of data on general practitioners' referrals to specialist outpatient clinics between June 1990 and January 1994 and detailed comparisons of three phases--October 1990 to March 1991 (phase 1), October 1991 to March 1992 (phase 2), and October 1993 to January 1994 (phase 3). SETTING: 10 first wave fundholding practices and six non-fundholding practices in the Oxford region. SUBJECTS: Patients referred to consultant out-patient clinics. RESULTS: NHS referral rates increased in fundholding practices in phase 2 and phase 3 of the study by 8.1/1000 patients a year (95% confidence interval 5.7 to 10.5), an increase of 7.5% from phase 1 (107.3/1000) to phase 3 (115.4/1000). Non-fundholders' rates increased significantly, by 25.3/1000 patients (22.5-28.1), an increase of 26.6% from phase 1 (95.0/1000) to phase 3 (120.3/1000). The fundholders' referral rates to private clinics decreased by 8.8%, whereas those from non-fundholding practices increased by 12.2%. The proportion of referrals going outside district boundaries did not change significantly. Three of the four practices entering the third and fourth wave of fundholding increased their referral rates significantly in the year before becoming fundholders. CONCLUSIONS: No evidence existed that budgetary pressures caused first wave fundholders to reduce referral rates, although the method of budget allocation may have encouraged general practitioners to inflate their referral rates in the preparatory year. Despite investment in new practice based facilities, no evidence yet exists that fundholding encourages a shift away from specialist care.

Ambulatory Care↗
PubMed1994

Immunofluorescence localization of the unconventional myosin, Myo2p, and the putative kinesin-related protein, Smy1p, to the same regions of polarized growth in Saccharomyces cerevisiae.

Myo2 protein (Myo2p), an unconventional myosin in the budding yeast Saccharomyces cerevisiae, has been implicated in polarized growth and secretion by studies of the temperature-sensitive myo2-66 mutant. Overexpression of Smy1p, which by sequence is a kinesin-related protein, can partially compensate for defects in the myo2 mutant (Lillie, S. H. and S. S. Brown, 1992. Nature (Lond.). 356:358-361). We have now immunolocalized Smy1p and Myo2p. Both are concentrated in regions of active growth, as caps at incipient bud sites and on small buds, at the mother-bud neck just before cell separation, and in mating cells as caps on shmoo tips and at the fusion bridge of zygotes. Double labeling of cells with either Myo2p or Smy1p antibody plus phalloidin was used to compare the localization of Smy1p and Myo2p to actin, and by extrapolation, to each other. These studies confirmed that Myo2p and Smy1p colocalize, and are concentrated in the same general regions of the cell as actin spots. However, neither colocalizes with actin. We noted a correlation in the behavior of Myo2p, Smy1p, and actin, but not microtubules, under a number of circumstances. In cdc4 and cdc11 mutants, which produce multiple buds, Myo2p and Smy1p caps were found only in the subset of buds that had accumulations of actin. Mutations in actin or secretory genes perturb actin, Smy1p and Myo2p localization. The rearrangements of Myo2p and Smy1p correlate temporally with those of actin spots during the cell cycle, and upon temperature and osmotic shift. In contrast, microtubules are not grossly affected by these perturbations. Although wild-type Myo2p localization does not require Smy1p, Myo2p staining is brighter when SMY1 is overexpressed. The myo2 mutant, when shifted to restrictive temperature, shows a permanent loss in Myo2p localization and actin polarization, both of which can be restored by SMY1 overexpression. However, the lethality of MYO2 deletion is not overcome by SMY1 overexpression. We noted that the myo2 mutant can recover from osmotic shift (unlike actin mutants; Novick, P., and D. Botstein. 1985. Cell. 40:405-416). We have also determined that the myo2-66 allele encodes a Lys instead of a Glu at position 511, which lies at an actin-binding face in the motor domain.

Actins↗
PubMed1994

Use of molecular epidemiological techniques in a pilot study on workers exposed to chromium.

OBJECTIVES: Molecular epidemiological techniques, capable of detecting damage to DNA, were used to see if such damage occurred in the lymphocytes of a group of workers exposed to chromium. The two aims of this pilot study were to see if these new techniques might make useful biological monitoring tools for workers exposed to chromium and also, to help assess whether the current occupational exposure limit for chromium (VI) was sufficiently protective in this specific working situation. METHODS: Volunteer groups of 10 workers exposed to chromium and 10 non-exposed workers provided urine and blood samples towards the end of the working week. Chromium concentrations were measured in whole blood, plasma, lymphocytes, and urine. Lymphocytes were used to examine two forms of DNA damage in the two groups; these were the level of DNA strand breakage and, the production of 8-hydroxydeoxyguanosine. RESULTS: Chromium concentration in whole blood, plasma, and urine of workers exposed to chromium was significantly raised (P < 0.01) compared with non-exposed controls, but in isolated lymphocytes, there was only a modest but significant (P < 0.05) increase in chromium in the group exposed to chromium. There was no difference in the levels of DNA strand breaks or 8-hydroxydeoxyguanosine between the groups. Air monitoring for chromium was not undertaken but current levels for the group exposed to chromium were reported to be around 0.01 mg/m3, which is 20% of the current United Kingdom occupational exposure limit. CONCLUSIONS: We were unable to detect any damage in lymphocytic DNA due to exposure to chromium. This may have been due to the low chromium exposure (< 20% of the United Kingdom occupational exposure limit), the ability of plasma to detoxify chromium (VI) to chromium (III) before it reached the lymphocytes, or perhaps the insensitivity of the molecular techniques used. It is now important to test these and other such techniques on groups exposed to levels closer to the United Kingdom occupational exposure limit.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1994

Identification of MYO4, a second class V myosin gene in yeast.

We have isolated a fourth myosin gene (MYO4) in yeast (Saccharomyces cerevisiae). MYO4 encodes a approximately 170 kDa (1471 amino acid) class V myosin, using the classification devised by Cheney et al. (1993a; Cell Motil. Cytoskel. 24, 215-223); the motor domain is followed by a neck region containing six putative calmodulin-binding sites and a tail with a short potential 'coiled-coil' domain. A comparison with other myosins in GenBank reveals that Myo4 protein is most closely related to the yeast Myo2 protein, another class V myosin. Deletion of MYO4 produces no detectable phenotype, either alone or in conjunction with mutations in myo2 or other myosin genes, the actin gene, or secretory genes. However, overexpression of MYO4 or MYO2 results in several morphological abnormalities, including the formation of short strings of unseparated cells in diploid strains, or clusters of cells in haploid strains. Alterations of MYO4 or MYO2 indicate that neither the motor domains nor tails of these myosins are required to confer the overexpression phenotype, whereas the neck region may be required. Although this phenotype is similar to that seen upon MYO1 deletion, we provide evidence that the overexpression of Myo4p or Myo2p is not simply interfering with Myo1p function.

Amino Acid Sequence↗
PubMed1993

SI units defined.

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International System of Units↗
PubMed1993

Binding of lead to a metallothionein-like protein in human erythrocytes.

We have studied the erythrocytes from 24 workers occupationally exposed to inorganic lead, one asymptomatic lead worker showing exceptionally high exposure, and eight control subjects (blood lead 300-750, 1800, and < 100 micrograms/L, respectively). High performance protein chromatography, electrophoresis, and trace metal analysis have identified a low M.Wt., copper, and zinc-containing protein in all cases. This protein (designated protein M) bound lead on in vitro incubation with buffered lead nitrate. Purified samples of protein M were found to show characteristics consistent with metallothionein (M.Wt. approximately 6500, low pI, and greater UV absorbance at 254 nm). Amino acid analysis found a composition of 33% cysteine but no aromatic amino acids. The highly exposed subject showed endogenous lead binding to protein M, which on further purification by ion exchange was found to be associated with one particular constituent (protein M5). Protein M5 was present in much lower quantities in control subjects. These findings suggest the existence of a metallothionein-like protein in erythrocytes which binds lead, sequestering it into a nonbioavailable form and hence protects against lead toxicity.

Adult↗
PubMed1993

Phenotypes of cytoskeletal mutants.

Phenotypic studies continue to contribute to an understanding of the functions of cytoskeletal proteins. Many of these studies indicate some degree of functional redundancy within a family of cytoskeletal proteins. Some surprises have emerged, such as suggestions of unexpected relationships between the actin and microtubule cytoskeletons. Finally, phenotypic studies have provided evidence for a function of intermediate filaments.

Actins↗
PubMed1993

Mutational analysis of yeast profilin.

We have mutated two regions within the yeast profilin gene in an effort to functionally dissect the roles of actin and phosphatidylinositol 4,5-bisphosphate (PIP2) binding in profilin function. A series of truncations was carried out at the C terminus of profilin, a region that has been implicated in actin binding. Removal of the last three amino acids nearly eliminated the ability of profilin to bind polyproline in vitro but had no dramatic in vivo effects. Thus, the extreme C terminus is implicated in polyproline binding, but the physiological relevance of this interaction is called into question. More extensive truncation, of up to eight amino acids, had in vivo effects of increasing severity and resulted in changes in conformation and expression level of the mutant profilins. However, the ability of these mutants to bind actin in vitro was not eliminated, suggesting that this region cannot be solely responsible for actin binding. We also mutagenized a region of profilin that we hypothesized might be involved in PIP2 binding. Alteration of basic amino acids in this region produced mutant profilins that functioned well in vivo. Many of these mutants, however, were unable to suppress the loss of adenylate cyclase-associated protein (Cap/Srv2p [A. Vojtek, B. Haarer, J. Field, J. Gerst, T. D. Pollard, S. S. Brown, and M. Wigler, Cell 66:497-505, 1991]), indicating that a defect could be demonstrated in vivo. In vitro assays demonstrated that the inability to suppress loss of Cap/Srv2p correlated with a defect in the interaction with actin, independently of whether PIP2 binding was reduced. Since our earlier studies of Acanthamoeba profilins suggested the importance of PIP2 binding for suppression, we conclude that both activities are implicated and that an interplay between PIP2 binding and actin binding may be important for profilin function.

Acanthamoeba↗
PubMed1993

Monitoring of total chromium in rat fluids and lymphocytes following intratracheal administration of soluble trivalent or hexavalent chromium compounds.

1. Intratracheal instillation of sodium dichromate (CrVI) and chromium acetate hydroxide (CrIII) to male Wistar rats gave rise to increased chromium concentrations in whole blood, plasma and urine up to 72 h post exposure; peak concentrations were reached at 6 h after exposure. 2. The ratio of whole blood chromium to plasma chromium concentrations was significantly different for Cr(VI) and Cr(III) treatments. Both blood chromium and plasma chromium assays should therefore be used for the assessment of chromium exposure. 3. Chromium was also detected in peripheral lymphocytes. Cr(VI), but not Cr(III) accumulated significantly in the lymphocytes after treatment. These cells have potential to be used for biomarkers of the assessment of exposure to chromium compounds.

Acetates↗