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Aimee is 5'9.5"/177cm and wears a size 8.
Colour: Multicolour
Product Key: 00402755639
Composition: 100% Polyester
Wash care: Dry Clean

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Frequently asked questions
Are we licensed and insured?
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Ryan, the owner, has over 25 years of experience in painting, general maintenance and HVAC.
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Miami, FL 33156
(305) 670 5100


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I'm not talking about scary things :-), I mean zentangle-wise. My Diva tile yesterday was more like a drawn landscape and today's tile all of a sudden was filled with lots of red. I hardly ever use colours in my tiles. Must be the hearty theme that Joey challenged us with!
So, I used Heartline, Heartstrings and Hearty. And plenty of red ;-)


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DnaK is essential for starvation-induced resistance to heat, oxidation, and reductive division in Escherichia coli. Studies reported here indicate that DnaK is also required for starvation-induced osmotolerance, catalase activity, and the production of the RpoS-controlled Dps (PexB) protein. Because thesednaK mutant phenotypes closely resemble those ofrpoS (ς38) mutants, the relationship between DnaK and RpoS was evaluated directly during growth and starvation at 30°C in strains with genetically altered DnaK content. A starvation-specific effect of DnaK on RpoS abundance was observed. During carbon starvation, DnaK deficiency reduced RpoS levels threefold, while DnaK excess increased RpoS levels nearly twofold. Complementation of the dnaK mutation restored starvation-induced RpoS levels to normal. RpoS deficiency had no effect on the cellular concentration of DnaK, revealing an epistatic relationship between DnaK and RpoS. Protein half-life studies conducted at the onset of starvation indicate that DnaK deficiency significantly destabilized RpoS. RpoH (ς32) suppressors of thednaK mutant with restored levels of RpoS and dnaK rpoS double mutants were used to show that DnaK plays both an independent and an RpoS-dependent role in starvation-induced thermotolerance. The results suggest that DnaK coordinates sigma factor levels in glucose-starved E. coli.
Starvation of Escherichia coli for carbon results in a near-immediate halt in cell division and the establishment of a dormant- or stationary-phase state (3,35, 36). This state is distinguished from the preceding growth period by a unique physiological resistance to otherwise lethal stresses (13, 25, 52), reflecting the coordinate expression of stationary phase-specific genes. The response to carbon starvation is physiologically unlike that to deprivation of other nutrients, such as nitrogen or phosphorus. Carbon-starved cells rapidly readjust their rate of cell division and cease growth, whereas cells starved for other nutrients, such as nitrogen or phosphorus, respond sluggishly (12,62). Carbon starvation therefore represents an efficient method for studying the stationary phase and also likely mimics common oligotrophic environmental conditions confronting other bacterial species (43). Regardless of which stimulus precipitates stationary phase, many common features characterize such nongrowing or dormant cells. Consequently, the high-cell-density stationary phase observed in rich undefined media is commonly employed for studies on stationary-phase gene expression and genetics (reviewed in references17 and 20).
When carbon for growth is limited, protein synthesis largely stops and cell survival and persistence rest on the integrity of protein synthesized at an earlier, prestarvation time. Maintenance of protein structure during growth is determined largely by the activity of protein chaperones, many of which are ATP-dependent enzymes such as DnaK (16, 37). This enzyme promotes structural rearrangements in proteins and can be stimulated by the cochaperones, DnaJ and GrpE. In growing cells, loss of DnaK results in temperature-restricted division and defective expression of heat shock proteins (6, 7). Starvation protein synthesis is also defective in a dnaK mutant (59). Such defects may underlie the observation that DnaK deficiency mitigates starvation-induced physiological readjustments, particularly starvation-induced resistance to heat, oxidation, and reductive division (52). Interestingly, these particulardnaK mutant phenotypes are possessed by rpoSmutants (19, 21, 30, 38, 54). Though a clear role for DnaK in the stationary phase is evident from studies on DnaK deficiency, an excess of this chaperone elicits additional physiological alterations which are again evident only in stationary-phase cells. Overproduction of DnaK is specifically bactericidal in the stationary phase, and this toxicity can be partially ameliorated by cooverproduction of DnaJ (2). The basis of this effect is as yet unclear, though suppressor analysis of stationary-phase DnaK toxicity has led to the recovery of multicopy genes which overcome this effect. One of these has been identified as edd and encodes the first committed enzyme of the Entner-Doudoroff pathway for hexose catabolism (53). Inactivation of this gene by the insertion of a stop codon prevented suppression and suggests a role for this pathway in stationary-phase metabolism.
The RpoS sigma factor controls expression of a diversity of genes, many of which are critical in the stationary phase (20, 33). Specific examples particularly notable because of their role in starvation-induced physiological alterations include katE, encoding catalase HPII (23, 34), and dps(1), also called pexB (31), encoding a small histone-like nonspecific DNA binding protein. However, additional genes involved in osmotolerant growth (22) and low-temperature growth (58) are now also known to be controlled by RpoS. Such observations expand the role of RpoS beyond the stationary phase. Increased expression of stationary-phase RpoS-regulated genes requires an increase in the abundance of RpoS. Such changes reflect alterations in rpoS expression at the level of transcription initiation, translation elongation, and posttranslational events resulting in increased RpoS stability (29, 39, 61). Variation in the allelic state ofrssB/sprE indicates that this locus acts as an unlinked negative regulator of RpoS abundance in the exponential phase (45,51). RpoS abundance during growth also depends in part on the ClpX/P heat shock proteins (56) as well as on the absolute temperature of cultivation (58). Since heat shock of growing cells elicits an increase in the abundance of RpoS, a role for additional regulatory factors of RpoS abundance appears likely (26).
To better understand the significance of the apparent stationary-phase phenotypic overlap between dnaK and rpoS mutants, a dnaK null mutant (49) as well as an otherwise isogenic wild-type and rpoS null mutant strains were analyzed for additional rpoS-like defects. We report here that the dnaK mutant has several of the same phenotypes asrpoS mutants, which result from an apparent defect in RpoS metabolism. Consequently, these findings suggest that DnaK plays only an indirect role in the starvation response. While these studies were under way, a role for DnaK in modulating RpoS levels during heat shock was reported (44). The physiological significance of this observation, however, was not apparent. In addition, it was reported that DnaK deficiency elevated exponential-phase RpoS levels sixfold while decreasing carbon starvation-induced levels only slightly. The physiological significance of these results was not examined. These findings contrast with results obtained withrpoS::lacZ fusions which indicate that RpoS is significantly destabilized during growth and starvation by a mutation in dnaK (44). Failure of anrpoH missense mutant (sidB1) to suppress thednaK mutant's effects on RpoS were interpreted as evidence for the exclusion of a role for other heat shock proteins such as ClpP/X from this process. To resolve the interplay between DnaK and RpoS in starvation-related phenotypes, we used additionalrpoH mutant alleles and a dnaK rpoS double mutant to study this process. The results solve apparent discrepancies and indicate that dnaK plays two roles in starvation-related phenotypes; one is mediated through RpoS, and the other controls the physiological status of starving cells in anrpoS-independent manner.
Bacterial strains, plasmids, and cell cultivation. E. coli strains, plasmids, and phages used in this study are listed in Table 1. Plasmid isolation and subcloning procedures were performed as described previously (2). The pBN15 plasmid- bearing derivative of PBL501, strain PBL504, was constructed by transformation at 30°C. The Dps-producing plasmid pBN49 was constructed by subcloning an 8-kbBamHI-HindIII fragment containing the entiredps gene from phage λ-205 (27) into pACYC184 (9) previously digested with BamHI andHindIII. Phage λ and phage P1 were grown as described previously (4). Strain PBL502 was constructed by generalized transduction with phage P1vir grown on AMS150 (Table 1), which contained a transposon Tn10 insertion inrpoS (katF13::Tn10;34) to transduce PBL500 to tetracycline resistance. The rpoS deletion strain, PBL503, was constructed by transposon-mediated deletion formation as described previously (5). The extent of the rpoS deletion was mapped by analyzing genes flanking rpoS, including mutS(61.5 min) for the counterclockwise direction and cysC(61.85) for the clockwise direction. The insertion site of the Tn10 in rpoS was identified previously in the 5′ end of the rpoS coding region at base 196 (24). Deletion of the 3′ end of the rpoS gene was indicated by the apparent loss of mutS (10). PBL503, containing the mutS deletion, had a forward mutation rate to streptomycin resistance of 1.14 × 10−7 compared to 3.5 × 10−9 for the otherwise isogenic wild-type parent strain, PBL502. The rpoS deletion did not extend as far as cysC, as PBL503 remained prototrophic. TherpoS deletion is designated ΔmutS-rpoS458.
RpoH suppressors of the ΔdnaK52 mutation were isolated as described previously (6). Three independent and spontaneous isolates were mapped by phage P1 transduction and shown to be 50% linked to zhh-21::Tn10. Complementation analysis with the rpoH expression plasmid, pDS2 (18), was used to verify that the mutations were inrpoH. pDS2 restored both the filamentous cell morphology and the reduced viability in all three isolates, characteristic of DnaK deficiency during growth in LB medium (6). To evaluate the starvation-related phenotypes of these rpoH suppressors in otherwise wild-type backgrounds, the ΔdnaK52 mutation inrpoH suppressor 1 was removed by phage P1 cotransduction with the linked insertion,thr-34::Tn10, generating PBL713, and complemented by transformation with plasmid pBN15 (pPtac::dnaK+J+lacI+ bla+ ), generating PBL711.
Cell densities of cultures were monitored spectrophotometrically at a wavelength of 600 nm. All cultures were incubated at 30°C in M9 minimal medium (42) containing 0.05% (wt/vol) glucose and shaken at 150 rpm with 1-liter Erlenmeyer flasks in shaking water baths (model G76; New Brunswick). dnaK mutants have been reported to accrue spontaneous mutations during prolonged incubation on a solid medium (6). Care therefore was taken to avoid such events during studies of the unsuppressed dnaK mutant through the use of freshly streaked plates. LB plates were used to determine viable cell counts as described previously (52). Chloramphenicol and tetracycline were added at final concentrations of 25 μg/ml, kanamycin was added at 75 μg/ml, and ampicillin and streptomycin were added at 100 μg/ml. All cultures were maintained in exponential-phase growth by repeated subculturing for a minimum of 10 generations prior to collection of the first exponential-phase sample.
Salt tolerance measurements were performed after 1 day of glucose starvation by adding dry sodium chloride powder, with stirring to a final concentration of 2.5 M. The cultures were then maintained with gentle agitation at 30°C for the duration of the experiment. The number of viable cells per milliliter was determined by plating diluted culture samples onto LB plates in duplicate.
Dps protein purification and antibody production.A 1-liter culture of strain PBL505, incubated for 5 days at 37°C with shaking in LB medium containing chloramphenicol, was centrifuged for 10 min at 6,000 rpm at 4°C. The remainder of the purification was performed as previously described (1). Briefly, the cell pellet was lysed by passage through a French pressure cell, and the resulting lysate was clarified by ultracentrifugation and fractionated by ammonium sulfate precipitation. The sample was applied to a Sephadex G-200 column, and Dps was recovered in the void volume. The protein was precipitated again with ammonium sulfate and resuspended in a high-salt buffer. The sample was then applied to a Sepharose 6B column, and Dps-containing fractions were recovered and applied to a DNA-cellulose column. Dps was eluted from the column with a linear sodium chloride gradient in which Dps eluted at 200 mM sodium chloride. Purity of the Dps fractions was determined by Coomassie blue R-250 staining.
Anti-Dps polyclonal antibodies were produced by injection of 100 μg of the purified Dps protein into a New Zealand White rabbit, followed by booster shots of equivalent amounts of antigen at two 2-week intervals. Serum was collected after an additional 2 weeks and purified by precipitation with acetone powders as described previously (2) with wild-type exponential-phase cell extracts. Antibodies were purified by affinity chromatography with protein A-Sepharose as described previously (28).
Catalase assays.The catalase assays were performed by placing 1 ml of cell culture into a prewarmed (30°C), stirred oxygen electrode (Rank Brothers). The cell culture was equilibrated at 30°C for 1 min prior to the addition of hydrogen peroxide to a final concentration of 100 mM. A computer was attached to the oxygen electrode for data collection, and sample readings were made at a rate of three times per second. Oxygen production was measured for a minimum of one min, and the amount produced was calculated as described previously (14). Differential rates of enzyme synthesis were determined as described previously (50, 55). Variation in catalase activity between replicates was less than 4%.
In vivo labeling of proteins and immunoprecipitation.GroEL heat shock induction ratios were determined as described previously (8). To determine the half-life of RpoS at the onset of starvation, cells were grown in M9 medium containing 0.05% glucose. After reaching the onset of starvation, the culture was labeled with35S-Translabel (1,000 Ci/mmol; ICN) at 20 μCi of culture per ml for 1 min at 30°C. The sample was chased with 10 mM (each) nonradioactive methionine and cysteine for 20 s at 30°C. Samples consisting of 3.0 × 109 cells were then removed, and protein was precipitated by the addition of ice-cold 50% (wt/vol) trichloroacetic acid (TCA) and kept on ice for 30 min. The precipitated protein was removed by centrifugation at 13,000 × gfor 10 min, and the pellet was washed successively with ice-cold solutions of 10% (wt/vol) TCA, 5% (wt/vol) TCA, and 100% acetone. The samples were then dried and resuspended in 0.5 ml of PBS-D (20 mM sodium phosphate, 150 mM sodium chloride [pH 7.4], 1% [vol/vol] Triton X-100, 1% [wt/vol] sodium deoxycholate, and 0.1% [wt/vol] sodium dodecyl sulfate [SDS]). Insoluble protein was removed by centrifugation at 13,000 × g for 10 min. RpoS was immunoprecipitated by the addition of 25 μl of anti-RpoS monoclonal antibody ascitic fluid (1RS1) (47), and the samples were incubated for 2 h at 4°C, with gentle agitation. The antigen-antibody complex was removed by the addition of protein A-Sepharose 4B (Sigma) with 350 μg of protein A per reaction and incubated for 3 h at 4°C with gentle agitation. Samples were then centrifuged at 13,000 × g for 10 min, and the pellet was washed three times in PBS-D. The washed pellets were resuspended in 33 μl in a solution containing 250 mM Tris-HCl (pH 6.8), 2% SDS, 0.75 M 2-mercaptoethanol, 10% glycerol and boiled for 10 min, and 3.3 μl was removed to determine radioactivity. Bromphenol blue was added to the remaining sample to a final concentration of 20 μg/ml prior to analysis by SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were detected by autoradiography with Kodak XAR film.
SDS-PAGE and Western blot analysis.To quantitate total RpoS levels by Western blot analysis, cells from cultures grown at 30°C were concentrated by centrifugation at 13,000 × g, and the resulting cell pellets were resuspended in 25 mM Tris-HCl–40 mM glycine (pH 6.8). Samples were removed to determine protein concentrations by the bicinchoninic acid protein assay (Pierce). The remaining sample was adjusted to 250 mM Tris-HCl (pH 6.8), 2% SDS, 0.75 M 2-mercaptoethanol, 10% glycerol, 20 μg of bromphenol blue per ml, boiled for 10 min, and frozen at −20°C for later use.
Proteins were resolved by SDS-PAGE, with 9% (wt/vol) acrylamide separating gels and 10% (wt/vol) acrylamide stacking gels, as described previously (15), with prestained low-molecular-weight markers (Bio-Rad). Western blots were prepared essentially as described previously (15). Western blots were processed and developed with the ECL reagent system according to the manufacturer's protocol (Amersham). Western blots were probed with a 1:1,000 dilution of the anti-RpoS antibody 1RS1 (47) and then a 1:1,000 dilution of sheep anti-mouse horseradish peroxidase conjugate (GibcoBRL). As a reference to normalize development of all Western blots for RpoS content, 111 ng of protein of strain PBL504 following 2 h of starvation was included as an internal standard. RpoS signal intensity was linear within the range of detection as determined by regression analysis of PBL504 extracts (r 2 = 0.99) analyzed in amounts of 0.111, 0.333, 1.0, 3.0, and 9.0 μg of protein. Chemiluminescence was detected by exposing Kodak XAR film. Exposed films were analyzed by densitometry with a GDS7500 densitometer and gel base/gel blot software (Ultraviolet Products).
Detection of Dps by Western blot analysis was performed as described above with the following two exceptions; proteins were resolved by SDS-PAGE, 14.5% (wt/vol) acrylamide separating gels and 10% (wt/vol) acrylamide stacking gels, and purified Dps was included in all blots as an internal standard for normalization of results. Detection of DnaK by Western blot analysis was performed as described previously (2).
Growth of the dnaK mutant.Strains containing the ΔdnaK52 mutation can be readily cultured at 30°C. However, even at this reduced temperature, this strain continues to exhibit a reduced growth rate and a filamentous morphology in exponential phase (6, 7). To minimize the possible contribution of growth-related dnaK mutant defects on the starvation response, additional growth conditions which overcame these phenotypes were identified. We have demonstrated previously that the filamentous phenotype is suppressed during growth in a defined medium (52). In addition, more-gentle culture agitation overcame the defect in growth rate. Excessive agitation of the cultures (240 rpm) decreased the specific growth rate (k = 0.28) and final cell densities (1.5 × 108 CFU/ml) of thednaK mutant cells relative to those of an otherwise isogenic wild-type strain. Consequently, the results presented here utilized the slower agitation rate of 150 rpm. At this rate of agitation, all strains examined had identical specific growth rates (k= 0.693) and cell densities (3.0 × 108 CFU/ml) at the onset of starvation. Stationary phase was elicited by depletion of growth-limiting levels of glucose. Twenty-four hours after the onset of starvation, the variation in cell numbers between the wild type and the mutant strains was less than 15%.
Starvation-induced catalase activity in dnaK andrpoS mutant strains.Levels of catalase increase in the stationary phase and result from elevated expression ofkatE (46). Catalase was therefore examined as an additional indicator of the cellular response to starvation in thednaK mutant strain. Catalase-specific activity was similar among the four strains during growth (Fig.1A, arrows 1 through 6). Differential rates of catalase synthesis produced during exponential phase were 9.4 nanoatoms of O/min/μg of protein for the wild-type strain, 8.8 for the dnaK mutant strain, 8.8 for the rpoSmutant strain, and 9.4 for the complemented dnaK mutant strain. In stationary phase, the wild-type and complementeddnaK mutant strains exhibited increased levels of catalase activity reaching 35 nanoatoms of O/min/μg of protein (Fig. 1A). However, the catalase activity of the dnaK andrpoS mutants did not increase in response to starvation and instead remained at prestarvation levels. By 4 h after the onset of starvation, the wild-type strain had 3.5-fold more catalase activity than the rpoS or dnaK mutant strains. The increased catalase activity in these strains remained evident even after 24 h in stationary phase.
Starvation responses of dnaK and rpoSmutant strains to sodium chloride exposure. dnaK mutants are sensitive to sodium chloride exposure in exponential phase (41), while rpoS mutants are sensitive to this stress in the stationary phase (38). However, the response of the dnaK mutant to this challenge in the stationary phase was unknown. To examine this question, the wild type, the otherwise isogenic dnaK mutant, the rpoS mutant, and the complemented dnaK mutant were subjected to 24 h of starvation and then exposed to 2.5 M sodium chloride. The cultures were then incubated further, with continued starvation, and periodically sampled to determine the number of viable cells (Fig. 1B). The wild-type and complemented dnaK mutant strains retained greater than 50% viability over a 24-h period. However, the viability of the dnaK and rpoS mutant strains decreased to less than 0.01% of untreated controls at nearly identical rates of 4.2%/h. These results indicate that DnaK is required for starvation-induced osmotolerance.
Levels of the rpoS-dependent protein, Dps, in starvingdnaK mutants.The results of the sodium chloride tolerance and catalase activity experiments suggested that there may be a more direct link between DnaK and RpoS in starving cells. To explore this possibility further, expression of an RpoS-dependent gene was examined. The dps gene (1, 31) encodes a 19-kDa DNA binding protein whose induction in stationary phase requiresrpoS. Cell extracts derived from wild-type, dnaKmutant, and dnaK-complemented cultures in the exponential phase, at the onset of starvation, and at times thereafter were examined for levels of the Dps protein with anti-Dps polyclonal antibodies (Fig. 1C). Dps was readily detected after 2 h following the onset of starvation in the wild-type strain (Fig. 1C, blot a, lane 4) and in the complemented dnaK mutant (Fig. 1C, blot c, lane 4) and increased to maximal levels by 24 h (lane 6). However, Dps was undetectable in the dnaK mutant even 24 h after the onset of starvation (Fig. 1C, blot b, lane 6).
Accumulation of RpoS in a dnaK mutant strain.One mechanism to explain the phenotypic overlap between dnaK andrpoS mutants is that DnaK deficiency results in a reduction in RpoS activity or concentration and therefore reduces RpoS-dependent gene expression. This possibility was examined by measuring levels of RpoS in the wild-type strain (Fig. 2A), the dnaK mutant strain (Fig. 2B), and the complementeddnaK mutant strain (Fig. 2C) by Western blot analysis with an RpoS-specific monoclonal antibody. Levels of RpoS during exponential phase in the dnaK mutant strain, when adjusted for total protein, were equal to the otherwise isogenic wild-type strain. In response to starvation, RpoS levels increased 10-fold in the wild-type strain, while in the dnaK mutant strain, rpoSlevels increased slightly but overall remained at a lower level. At the onset of starvation in the dnaK mutant strain, RpoS levels were 3.5-fold lower than the levels in the wild-type strain (Fig. 2D). Interestingly, in the complemented dnaK mutant strain, RpoS levels were nearly twofold higher than those in the wild-type strain (Fig. 2D). This last result is consistent with an increase in levels of DnaK which occurs in that strain in stationary phase, resulting from elevated expression from the plasmid-encoded Ptac promoter (2). Western blot analysis indicated that at the time of the maximal increase in RpoS abundance, DnaK levels were fourfold greater than in the wild-type strain (data not shown). Twenty-four hours after the onset of starvation, the level of RpoS in the wild-type and the complementeddnaK mutant strains decreased by 33 and 50%, respectively; however, no change in RpoS levels was observed in the dnaKmutant strain.
The exponential-phase RpoS levels observed in the dnaKmutant were not elevated relative to those of the otherwise isogenic wild-type strain, in contrast to a previous report (44). In addition, the reduction in starvation-induced RpoS levels reported here was significantly more pronounced (threefold versus 40%) than reported previously. To resolve these differences, the consequence of more-rapid culture agitation on RpoS levels in the dnaK mutant was examined (Fig. 3). Duplicate cultures of the wild type (PBL500) and the dnaK mutant (PBL501) were sampled periodically for RpoS determination, and then one of each of the duplicate cultures was shifted to a more rapid rate of agitation. RpoS levels increased rapidly upon the shift in the dnaKmutant but not in the wild-type strain, indicating that this condition imposes some sort of RpoS-inducing stress. This result is consistent with the reduction in growth exhibited by the dnaK mutant and likely explains why RpoS levels were highly induced in adnaK mutant strain in a previous study (44).
Role of RpoS in levels of the DnaK protein.Since RpoS affects the synthesis of many proteins, levels of DnaK were examined in anrpoS mutant to better define the epistatic relationship between these genes. DnaK levels were examined in the wild-type strain and in the rpoS mutant during exponential growth and in response to starvation. Western blot analysis with the anti-DnaK monoclonal antibody 2G5 (28) indicated that the levels of DnaK were not significantly affected by RpoS deficiency either during growth or in response to carbon starvation (data not shown). A starvation-induced, twofold increase in DnaK levels was evident in both strains in comparisons between levels produced during exponential growth and those produced 24 h after the onset of glucose starvation.
RpoS stability in a dnaK mutant strain.Reduced RpoS accumulation in the starving dnaK mutant strain could reflect defects in either RpoS stability and/or synthesis. To look for DnaK-dependent changes in RpoS protein turnover, the half-life of RpoS was determined. The onset of starvation was selected for analysis because a large difference in RpoS levels was evident in comparisons between the wild type and the dnaK mutant (Fig. 2D). At the onset of starvation, cultures were transiently radiolabeled and RpoS was recovered by immunoprecipitation at specific times thereafter. The immunoprecipitates were analyzed by SDS-PAGE, and the resulting autoradiograms (Fig. 4A) were quantitated (Fig. 4B). In the wild-type strain (Fig. 4A, lanes 1 through 4), RpoS remained fully stable for the duration of the experiment (30 min), while in the dnaK mutant, the half-life of RpoS was approximately 20 min (Fig. 4A, lanes 5 through 8). No protein was detectable when identical procedures were performed on therpoS deletion mutant PBL503 (Fig. 4A, lane 9). These results indicate that DnaK deficiency reduces starvation-induced accumulation of RpoS by enhancing RpoS turnover.
RpoH (ς32) suppressors restore a subset of starvation-induced responses.To better understand the relationship between DnaK and RpoS in regulation of the starvation response to carbon deprivation, dnaK mutant suppressors which had restored wild-type levels of RpoS were isolated (Fig.5A). An internal control consisting of extracts of PBL504 obtained following 2 h of glucose starvation was included in each blot to normalize absolute amounts of RpoS. TherpoH suppressor mutants were recovered as papillae after prolonged incubation on solid medium (6, 8). The use of such strains circumvents RpoS effects on the starvation response, which might otherwise obscure the role of DnaK. Several such mutants were isolated in the ΔdnaK52 PBL501 background and are designated rpoH suppressors—1, 2, or 3. They were mapped by phage P1 cotransduction with linked transposon insertions. To verify their identity further, plasmid pDS2 (18), encoding a Ptac::rpoH gene, was used to complement the suppressor isolates. Such suppressors exhibit a defective heat shock response and can be categorized by the severity of this defect (8). Only those alleles which exhibit no heat shock response at all fail to grow at 43.5°C on solid medium (8). Additional Western blot analysis of these threerpoH suppressor alleles with anti-Dps polyclonal antibodies revealed that levels of Dps were restored to wild-type levels in thednaK mutant strains containing the rpoHsuppressors (Fig. 5B). Starvation-induced levels of catalase production were also restored to wild-type levels in the three suppressor isolates (data not shown). As the restoration of RpoS levels andrpoS-regulated gene expression in the dnaK mutant strain by the rpoH-1 to rpoH-3 suppressors was in apparent contradiction to a previous report (44), the possible allelic difference between these suppressor mutations was examined. The rpoH-1 mutation was selected as a representative and analyzed for heat shock protein induction during heat shock and for growth at elevated temperature (Table2). The rpoH-1 mutant fails to induce GroEL synthesis following a temperature shift from 30 to 42°C or to form colonies at 43.5°C. It is therefore a stronger suppressor of the dnaK mutation than the allele (sidB1) used previously (8, 44). Taken together, the results presented here and those previously described (44) support the idea that at least some of the effects of the dnaK mutation on starvation-related processes are the result of effects on RpoS mediated by other heat shock proteins. In addition, an rpoS dnaKdouble mutant starved for 24 h exhibited sensitivity to sodium chloride treatment equivalent to that of mutants containing single mutations in either gene alone, suggesting that the role ofdnaK in starvation-induced osmotolerance is mediated entirely through RpoS.
An independent role for DnaK in starvation thermotolerance.Examination of the thermotolerance of these strains indicates that DnaK and RpoS play important but distinct roles in starvation-induced resistance to heating. Elimination of either dnaK orrpoS in strains which are otherwise isogenic results in severe thermal killing of glucose-starved cells (Fig.6, open squares and triangles). Wild-type cells, however, exhibited only slight reductions in viable counts for the duration of the 45-min treatment (Fig. 6, open circles). Interestingly, MC4100 (44) is 15-fold more sensitive to heat killing than the wild-type strain (PBL500) used in the present studies (data not shown). All three of the rpoH dnaK mutant suppressor strains exhibited only partial restoration of thermotolerance relative to that of their unsuppressed but otherwise isogenic dnaK mutant parent (Fig. 6, open inverted triangles, hexagons, and diamonds). Thus, these strains remained highly thermosensitive upon starvation for glucose. To test the possibility that continued thermosensitivity results directly from therpoH mutation, the rpoH mutation inrpoH suppressor 1 was evaluated in adnaK+ background. Two strain derivatives were constructed, PBL713, in which a wild-type allele of dnaK was introduced by transduction, and PBL711, which contained a plasmid-encoded wild-type dnaK allele. Examination of the starvation-induced thermosensitivity of these strains (Fig. 6, closed circles and closed squares) indicated that the rpoH mutation did not result in thermosensitivity following glucose starvation. Since RpoS levels are restored in the original rpoH suppressor strains, and expression of RpoS-dependent pathways such as Dps and catalase synthesis is normal, continued thermosensitivity of therpoH dnaK double mutants indicates that DnaK is directly required for starvation thermotolerance. If two distinct pathways for starvation thermotolerance are present, one dependent upon RpoS and the other on DnaK, then a dnaK rpoS double mutant should exhibit greater heat killing than either single-mutant strain alone. Analysis of the thermotolerance of such a strain (Fig. 6, closed triangles) indicated that loss of both genes resulted in significantly greater killing than that observed in the absence of either gene alone. These results indicate that DnaK plays a distinct and RpoS-independent role in starvation thermotolerance.
The studies reported here focus on the role of DnaK in a stationary phase produced by starvation for carbon. Care was taken to minimize high cell densities in batch cultures through the use of minimal quantities of the limiting nutrient, glucose. Such measures represent an effort to mimic the physiological state confronted by bacteria in natural environments where high cell density conditions are generally thought less likely (43).
The dnaK mutant phenotypes observed in stationary phase result at least in part from a deficiency in the sigma factor RpoS. The reduction in stationary-phase RpoS levels (about threefold) is sufficient to preclude expression of the rpoS-dependent gene, dps (pexB), and therefore possibly otherrpoS-dependent genes, such as those involved in mediating resistance to hydrogen peroxide, heat, and sodium chloride excess. This change in RpoS abundance parallels that seen during heat stress administered to growing cells, which is sufficient for changes in RpoS-dependent gene expression (26). A 10-fold increase in RpoS levels over those observed in exponentially growing cells was detected following the onset of starvation in the wild-type strain, PBL500, at 30°C. This is an increase of approximately twofold over those seen previously at 37°C (29, 56) and is consistent with other reports indicating that stationary-phase expression ofrpoS is elevated at reduced cultivation temperatures (58). Thus, the pleiotropic spectrum of effects resulting from RpoS deficiency in the dnaK mutant strain offers some explanation for the apparent phenotypic overlap between dnaKand rpoS mutants.
The RpoS-related effects which result from DnaK deficiency are stationary phase specific. RpoS levels appear otherwise normal during exponential-phase growth under the conditions used. Growth conditions were employed to minimize certain dnaK mutant phenotypes. It has been reported that the ΔdnaK52mutation results in a reduced growth rate (k = 0.28) compared to that of the wild type (k = 0.69) at 30°C (6). We also observed this phenomenon when the ΔdnaK52 mutant was agitated at 200 rpm or greater. In addition, RpoS levels are highly induced by this treatment (44), obscuring subsequent effects resulting from starvation. However, at agitation rates of 150 rpm or less, growth rates were equal to the otherwise isogenic wild-type strain. Growth at 30°C also was employed to avoid the dnaK mutant heat-sensitive growth phenotype (49).
Protein half-life measurements indicated that in the dnaKmutant, RpoS stability was reduced. These results suggest that DnaK somehow stabilizes RpoS in response to carbon starvation. Interestingly, overproduction of DnaK resulting from starvation-mediated derepression of a plasmid-encoded dnaKgene elevated stationary-phase RpoS levels nearly twofold above those in otherwise isogenic wild-type cells. Thus, variation in DnaK abundance results in corresponding changes in the stationary-phase levels of RpoS. Though such changes in RpoS levels were elicited by artificial manipulation of dnaK expression, DnaK levels do increase severalfold in wild-type cells upon carbon starvation as shown here and previously (52). Thus, DnaK may play a crucial role in adjusting stationary-phase RpoS levels in response to carbon starvation.
Overproduction of DnaK is selectively toxic in stationary-phase cells (2). This bactericidal effect was used as the basis of a selection to recover multicopy plasmid suppressors to better understand the toxicity of chaperone excess (53). Identification of these suppressors, such as 6-phosphogluconate dehydratase, may now be explained in part by the observations presented here. Activity of 6-phosphogluconate dehydratase is elevated in the stationary phase (32), while the next enzyme in this pathway, 2-keto-3-deoxy-6-phosphogluconate aldolase, is also increased in stationary phase (48). Perhaps elevated levels of 6-phosphogluconate dehydratase overcome deleterious levels of other enzymes precipitated by DnaK-mediated increases in RpoS by redirecting carbon flux through alternative routes in this pathway.
The results presented here indicate that DnaK controls the levels of the RpoS sigma factor. Since DnaK also controls the levels of the RpoH sigma factor (11), these results indicate that DnaK may coordinate the levels of multiple sigma factors in E. coli. A mechanism for such coordination mediated by a single protein has until now been lacking. However, it would seem critical for the maintenance of balanced gene expression during changing environmental conditions, such as those accompanying starvation.
It is at present unclear how DnaK might affect RpoS stability, though direct and/or indirect mechanisms may be operative. Deficiency of the heat shock protease, ClpX/P, elevates RpoS levels 3.2-fold during exponential-phase growth and 1.7- fold in response to starvation (56). Since starvation increases ClpX/P by 1.5-fold (56), inactivation or blockage of ClpX/P may be required for the starvation-induced increase in RpoS abundance. The epistatic relationship between ClpX/P and RssB further indicates a direct role for ClpX/P in RpoS abundance (51). Thus, the role of DnaK in RpoS stability might be mediated indirectly by altering levels of ClpX/P or RssB. Alternatively, as a direct interaction between DnaK and the heat shock sigma factor RpoH has been reported (40), physical contact between DnaK and RpoS also appears plausible.
RpoH levels are elevated in dnaK mutant strains (60); therefore, compensatory mutations which reduce but do not destroy RpoH activity are selected during prolonged storage on plates (6-8). Such rpoH mutant strains were isolated and studied here to probe the relationship between DnaK and RpoS. These rpoH dnaK double-mutant strains exhibit normal levels of RpoS, suggesting that the effect of the dnaKmutation on RpoS abundance is RpoH linked, perhaps through ClpX/P. Restoration of RpoS levels was accompanied by recovery of catalase and induction of Dps starvation, suggesting that at least these two starvation responses operate in an RpoS-dependent fashion. DnaK apparently indirectly alters their expression by varying levels of RpoS. Starvation-induced thermotolerance was, however, still defective in the rpoH dnaK mutant strains but normal indnaK+ rpoH derivatives. This result suggests that DnaK plays a distinct and RpoS-independent role in starvation-induced thermotolerance. However, the relative contributions of the two proteins to the response appear quite unequal, suggesting that their actions during the development of the thermotolerant state are mechanistically unrelated.
We thank Carol Gross, Abdul Matin, and Graham Walker for providing strains.
This work was supported by a grant from the Department of Energy to P.B. (DE-FG02-93ER61701).
- Received 24 April 1997.
- Accepted 14 December 1997.
↵* Corresponding author. Mailing address: School of Biological Sciences, E234, George Beadle Center, University of Nebraska, Lincoln, NE 68588-0666. Phone: (402) 472-2769. Fax: (402) 472-8722. E-mail: pblum{at}crcvms.unl.edu.
- Copyright © 1998 American Society for Microbiology


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Sex, Intimacy, and a Cautionary Tale (Yes I am a fan of the show) November 24, 2007Posted by peterong in Asian American, sex.
I know, I know, people have ranted, emailed me, and finally someone this past week told me that I have been a neglectful blogger…so I apologize, it has been a busy transition but there is so much that I am working through these past few months.
A few weeks ago I gave a talk called "Beyond Boundaries: Restoring Intimacy." It was a talk about sex and intimacy and what God has to say about it. It was an extension of the talk I gave at the New York Summer Conference this past year as part of the Jesus Uncensored series and continued at the Eastern Chinese Bible Conference. Apparently I hit a nerve because I am getting a lot of invitations to give this talk at fellies and church conferences. After I gave the talk where I challenged the church to give permission to talk about this profound part of our lives. I have seen the damage it has caused because of the silence we have as a church. We don't address it besides, "the bible says so…" and that is suppose to overcome this powerful sexual energy we carry around. Yet, I challenged them to reclaim it as a way for us to experience intimacy, to engage in the humanity of the other, and to be stewards of relationship that ultimately belong to God and their future spouse. I explained how sex can be so incredibly distorted to focus on the orgasm and not on the connectedness that it should be reserved for. I challenged them to not use each other to fulfill a sexual itch (What Mark Driscoll called "using women as toilets" when we seek to use our sisters simply as a means to release our urges). I had a Question and Answer at the end of the session.
It felt good to talk about this and I wished we talked more. But I realized this was just the beginning…After the talk, I received dozens of email describing their wounds and hurts from taking license in this area, a girl who was sexually and emotionally abused by her Christian boyfriend, another woman who is haunted by memories of being molested, brothers who confessed their pornographic addictions, a pastor shared with me about his lust for women in his congregation, a worship leader who slept with his girlfriend and others more. As I was praying, I was thankful for God bringing this to light but also knowing that this is beyond my abilities to address. But in some ways, I wanted to make them more open to listening and wrestling with this aspect of our lives.
One of the conversations that moved me the most was over coffee, I was meeting with a college student who was part of my youth ministry and as we talked about this issue, she mentioned that when she was a teen at church she dated another teen leader. She said how she lost respect for herself because what they were doing in private. She broke off the relationship and then started dating a non-Christian and all of a sudden she had a whole community reaching out to her and who wanted to get involved in her romantic life. She was hurt. Because she wondered "where were they when I needed them to hear my struggles? But when I started to date a non-Christian, they were all ears but I needed them more when I was dating a Christian" (She recounted that her non-Christian boyfriend had more respect for her purity than the Christian…ouch).
This week, I am meeting with several folks who want to meet up to share their heaviness in their hearts about this. A young man is sharing that he found out the girl he is dating had been sexual with her ex-boyfriend who served as President of a major campus ministry. He is angry that this ex-boyfriend had taken advantage of her. So it breaks our relationships with each other as brothers too.
I feel there is more where this came from and I think we need to address this. Especially in the Asian American church, where our private worlds seems so hidden and dark. Where the unsavory elements of guilt, shame and "saving face" seem to overwhelm the freedom awaiting us in the gospel. We hide. We pretend. We put on an Oscar worthy performance that show our white-washed tombs. I know for myself, I am still reeling from my past experiences and although I see so many moments of pure repentance, I also find myself tangled in the soul numbing temptations that surround me. But somehow, God has shown me that I need to place them before Him first and to His people as well.
One pastor called this openness a revival, another one said it was fallout. I am not sure what to make of it and it has in some ways the difficulty in talking about this topic is that it often involves another person. Another person's shame. We don't want that. We could share with each other what "I" did but if it means messing up the reputation of the "other" it becomes difficult. We feel like we need permission. So I sense there is a lot of the proverbial "skeletons in the closet" and the closet is bursting at the seams. On our college campuses we have young people who raise their hands as they worship God in the felly halls but in their dorms they are using their hands in other ways, we have leaders who are continuing to lead without a mention of what their private worlds are like (once again, they are simply a function for us as ministry workers), we have pastors who are left unchecked when they are "prepping" their sermons, and we have so many of our married couples who still don't know how to have sex in a Godly healthy manner.
Our silence has murdered our call to purity. It has been this sin of omission that we are so afraid to ask and be asked of this area of our lives that has been a ground zero for so many of us.
- 1. Letitia - November 25, 2007
What a powerful and daring statement. Thank you for this!
*Letitia Wong*
- 2. LAK - November 25, 2007
Good stuff, Peter. I guess this goes along with the cheap intimacy talk that we had years and years ago. I also think that one of the reasons why pastors and other "holy" men (and women!) fall into sexual sin so easily and readily is because I think a person's passion for God is directly related to sexual passion, i.e. BIG passion for God = BIG passion for sex. Even God talks about Israel prostituting herself…
- 3. crimson heart - November 26, 2007
let's not forget about the Asian American culture too. sex is not appropriate for discussions for some reason. Asian parents don't really talk about the "s" word since it is almost related to the "t" word (taboo) heck, my non-Christian parents never talk about it and im sure almost every other Asian American had the same experience. so .. AA culture + Christian faith … completely neglects the sex topic.
- 4. crimson heart - November 26, 2007
one more thing … oh im sure you knew all about that already.
- 5. Link Love 2 « Next Gener.Asian Church - November 27, 2007
[…] blogging hibernation to share his prophetic calling out of Asian American churches to talk about sex and intimacy. Our silence has murdered our call to purity. It has been this sin of omission that we are so […]
- 7. Ray Li's Blog - December 20, 2007
[…] is the unacknowledged and unacknowledgeable swampland beneath the church brochure of tidy scenery. Only a few are brave enough to confront and address it; the rest of us put on petty and hypocritical masks of […]
- 8. thecuttingtruth - December 21, 2007
Pete Rong,
glad to see someone gutsy enough to expose this underbelly of church life, warts & all. hope you don't mind, but i've linked this site to a recent entry on my blog titled "Top 15 confessions for the Asian American Christian (2007)."
i'm sure you have a larger and deeper view of how sex affects the AA church as a whole, but in my limited experience, i do have this to add. In the minefield of sex, everyone wants to be perceived as a victim. A victim of abuse, a victim of the internet, a victim of a demanding boyfriend/girlfriend, a victim of porn. While many legitimately are victims, a great % are not. Some are the abusers, the assaulters, the exploiters . . . and they can't hide behind this victim-persona so espoused by the likes of Oprah and Dr. Phil. Because sometimes shame IS good and necessary – even in the AA church.
- 9. Top 15 Confessions for the Asian American Christian (2007) « the cutting truth - January 18, 2008
[…] the unacknowledged and unacknowledgeable swampland beneath the church brochure of tidy scenery. Only a few are brave enough to confront and address it; the rest of us put on petty and hypocritical masks of […]
- 10. Top 15 Confessions for the Asian American Christian (2007) « the cutting truth - January 18, 2008
[…] is the unacknowledged and unacknowledgeable swampland beneath the church brochure of tidy scenery. Only a few are brave enough to confront and address it; the rest of us put on petty and hypocritical masks of […]
- 11. Top 15 Confessions for the Asian American Christian (2007) « Man Downstairs - January 20, 2009
[…] is the unacknowledged and unacknowledgeable swampland beneath the church brochure of tidy scenery. Only a few are brave enough to confront and address it; the rest of us put on petty and hypocritical masks of […]


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text: Loni from specialopsmedia let me know about this set. These two records are being re-released on a single CD. Seems like 100 years ago I bought these albums when they first came out. Later, I secured copies of the CDs when they were released in the 90's. I still keep those handy, but I'm guessing they are no longer available. The good news is that both are available again on a single CD. So, if you don't have a copy, this is your chance. There's always a new generation that needs to hear folks that influenced other musicians. Big Star was never really a "big" act, but they made enough noise that other writers came to admire and/or copy them. The fact that the record company did not get behind the band in the manner that most bands of this caliber would expect had a lot to do with what eventually became the 'indie' scene. Why sell your soul if you couldn't profit from it somehow? The CD includes both albums, and the mixes that made it to 45 back in the day.Big Star


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Here's what we've posted about BHI
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Decisions and Settlements
Decisions 00-03-07 Appellate Decision in Rio Mar v. NYSDHR
Appeal from the decision of the NYS Division of Human Rights after trial, awarding plaintiff $500,000 for emotional distress, and $65,850 in back pay. The appellate court affirmed the Division's decision in favor of plaintiff, including damages for the period she was unable to work, due to the psychiatric injury inflicted on plaintiff, and the addition of her harasser as a party. However, it reduced to $125,000 the award for emotional distress, with only the following explanation: "the award of $ 500,000 in compensatory damages was excessive and we reduce said award to $125,000."03-04-04 Plaintiff's Motion for Protective Order Granted
Plaintiff had secretly recorded a conversation with a witness. Defendant demanded that she produce all recordings. Plaintiff did not want to produce the recording until she had first deposed the witness with whom she had the conversation, to prevent him from tailoring his testimony to the recording. We moved for a protective order allowing plaintiff to withhold production until after the witnesses was deposed. The Court came up with the creative solution, which met plaintiff's objectives, of ordering that the recordings be places in a safety deposit box, where plaintiff and defendant's counsel had the two keys required for access, which insured that neither side would have an advantage, until after the deposition.06-06-19 Plaintiff's Motion to Compel Against School District Granted in Part
Sexual harassment of a high school student by a teacher. The Court grants plaintiff's motion requiring disclosure of the names of other victims, and information about similar incidents, in the year before plaintiff started at the HS. The Court denied plaintiff's motion to depose the attorney who advised the school to discipline rather than fire the harasser.06-10-20 Plaintiff's Motion to Compel Against School District Granted
Sexual harassment of a high school student by a teacher. The Court granted her motion to force the school to disclose the name of another sexual harassment victim, and to provide details about another incident.07-02-26 Arbitration Decision After Hearing Jackson v. Kellogg Brown & Root
In this decision after an evidentiary hearing, arbitrator Kathleen Roberts held that military contractor Kellogg Brown & Root had illegally discriminated against Robert Jackson. The decision maker in the hiring process, who had claimed Jackson did worse on an exam than the white applicant KBR hired, had to admit the evidence did not support his claim. That, and evidence that the decision maker had used a racial slur, was enough to convince the arbitrator that KBR had discriminated on the basis of Jackson's race. See pages 23-26.08-03-03 Denying Defendant's Motion to Sever
Plaintiffs are permitted to bring their claims together, in a single action, if they arise from the same basic set of facts. These plaintiffs brought their sexual harassment claims in a single action, because they had been sexual harassed by the same coworker, their complaints had been ignored by the same managers, and they were both subjected to retaliation, while working at defendant. Defendant moved to have the Court order separate trials in separate actions, claiming that plaintiffs suffered different injuries and worked at different times. The Court denied defendant's motion, because plaintiffs would be deposing the same witnesses, and offering the same evidence, at trial, and defendant's motion would lead to a waste of judicial resources.08-05-12 Defendant's Motion for Summary Judgment Denied
Plaintiffs, two African-American parents and their minor child, sued a school district for barring them from their child's school, following their complaint of racial discrimination. Initially, plaintiffs went to the Tennessee Department of Education, which warned the school district that if it did not reverse the ban, it could lose federal funding. When the district still refuse, the plaintiffs retained counsel. The Court agreed with plaintiffs that there was enough evidence for a jury to find the district, its superintendent, and board of education members liable. The case settled following the decision.08-06-03 Granting Plaintiff's Motion to Quash a Subpoena to Her Current Employer
To harass plaintiffs, defendants may serve a subpoena on plaintiffs' current employers. Courts will generally not permit this tactic. Obviously, what happened after plaintiff left her employer is not relevant to her lawsuit, with the exception of how much money she made after leaving (information the plaintiff could provide). This Court quashed the subpoena, and told the defendant it should have simply asked the plaintiff, if it wanted to know.08-07-23 Defendant's Motion to Compel Denied
This was one of the truly inane motions we have encountered. We wrote a letter to our client's current employer asking that it excuse her from work to attend a deposition. When she testified to this fact at her deposition, the defendant belatedly asked us to produce the letter. When we refused, because the letter was obviously irrelevant to the events at issue many years earlier at defendant, it inexplicably moved to compel production, on the theory that the letter "may contain plaintiff's impressions of defendant." The Court was not amused, and denied the motion, as "far too speculative and tenuous to merit any serious consideration."09-03-31 Defendants' Motion for Summary Judgment Denied in Part
This is a school peer-on-peer racial harassment case. The plaintiff was subjected to numerous slurs by his classmates, and eventually, his parents tired of the school's failure to remedy the situation, and brought suit. Defendants asked the Court to dismiss all of plaintiff's claims. One was dismissed, but the dismissal had no impact, since others which were identical were not dismissed. As a result, plaintiff prevailed completely against defendant's motion, and the case was ordered to trial. It settled before trial.10-03-17 Granting Plaintiff's Motion to Dismiss Declaratory Judgment Action
Plaintiff is entitled to choose the court in which he files suit. In this case, plaintiff contacted defendant to make a good faith effort to settle, before filing suit in the state he lived, Tennessee. Defendant pretended to be prepared to negotiate a settlement, all the while preparing its own suit, which it filed in its home state, Kentucky. The Court did not appreciate the deception, and dismissed the defendant's suit, allowing plaintiff to proceed in his home state.10-03-30 Denying Defendant's Motion for Summary Judgment in Part
This is a fascinating decision. African-American children in Northern High School, in Port Huron, were being subjected to peer racial harassment. The school district had know about it for years, but failed to respond adequately. The year before the case was filed, numerous African-American students were included in an anonymous "Hit List," which suggested they would be killed. Our firm represented twelve of the students. The Court denied defendant's motion (with one minor exception), ruling that the case should go to trial against the board of education on racial harassment. The Court also ruled that the individual school board members were not entitled to immunity. Defendants appealed. The United States Department of Justice joined the plaintiffs on appeal. The appellate court affirmed the lower court's decision to send the case to trial, but over a dissent, ruled that the individual board members were entitled to immunity.10-07-03 Defendant's Motion for Summary Judgment Denied
This was a sexual harassment case against the University Club and one of its Banquet Captains, Mel Guzman. Plaintiffs alleged a sexually hostile work environment and quid pro quo sexual harassment. Defendant moved for summary judgment on all of plaintiffs' claims, and the motion was denied in its entirety. A jury eventually concluded that the plaintiffs were subjected to sexual harassment.10-11-02 Defendant's Motion for Summary Judgment Denied
A motion is a written request to a court. A motion for summary judgment asks the court to dismiss the case because it is so weak no competent jury could find for the plaintiff. As the judge in this case noted, it is rare for race discrimination plaintiffs to survive summary judgment in this part of the country. Thus, we were particularly gratified, that the Court ruled in favor of the 60 courageous men and women we represented in this case.11-03-04 Defendant's Motion for Summary Judgment Denied in Part
This was a challenging case. Plaintiff, an openly gay man, was called a "pussy" and a "fag," by his coworker, with whom he had been friends. His former friend, and current harasser, was not bigoted against gays. As a consequence, this case could not be brought under NY State law which prohibits discrimination on the basis of sexual orientation. Plaintiff believed his harasser was trying to humiliate him with these remarks, by implying he did not meet the accepted standards of behavior for a man (which is gender stereotyping, not anti-gay discrimination), so the case was brought under Title VII, which prohibits gender stereotyping, but not anti-gay discrimination. Unfortunately, the plaintiff had written a letter to the defendant complaining that it was discriminating against him because he was a gay man. The Court seized upon this letter as proof that the discrimination at issue was anti-gay, not gender stereotyping (not covered under Title VII), and granted summary judgment as to that claim. We asked the court to reconsider its decision, because the letter reflected only the plaintiff's opinion, and did not explain why the harasser was harassing him, however, the Court refused to change his opinion. Fortunately, the retaliation claim survived, and the case settled.11-03-04 Defendant's Motion to Strike Denied
Rule 12(f) allows a court to "strike" impertinent or scandalous allegations from a complaint. The defendant asked the Court to strike (dismiss) the entire complaint because it supposedly contained transcripts of recordings made in violation of Nevada law, where it is generally illegal secretly to tape. The Court denied the motion because, it reasoned, even if the tape was made illegally, the defendant would have the opportunity to keep it out of evidence at trial. The irony was that the writing at issue was not a transcript but notes of a conversation. We use Allison & Taylor to call our clients' former employers, to make sure they are not giving them bad references. In this case, we included the reference that the defendant gave the plaintiff, as evidence of retaliation.12-02-10 Plaintiff's Motion for Permission to Introduce Harasser's Sex Crime Conviction Granted
The United States Department of Justice brought this case against a landlord and his superintendent for sexual harassment of tenants. The superintendent had previously plead guilty to raping children, and the U.S. wanted to prove that landlord hired him aware of his criminal history, in order to establish liability against the landlord. Our firm intervened on behalf of five of his victims. In this decision, the Court held that the U.S. was permitted to introduce evidence of his prior conviction, and that the landlord was aware of his criminal history, when he hired the harasser.12-04-16 Defendant's Motion to Dismiss Denied in Part
Plaintiff was a corrections officer, who was harassed by his coworkers and inmates, due to an injury to a sensitive area of his body, and the fact that he took time off due to the injury. Pictures of the officer were photo-shopped to an image of a man with that body part displayed. Plaintiff claimed that he was harassed due to a disability, that he was sexually harassed, and that he was harassed because he did not conform to norms of gender behavior. Defendant moved to dismiss the complaint. The Court granted the portion of the motion seeking dismissal of the sexual harassment claim, noting that the inmates and coworkers were not acting due to romantic interest, however, relying on the same conduct, the Court found that the plaintiff was harassed because he did not conform to gender norms. The Court denied the portion of the motion seeking dismissal of the disability harassment claim.12-05-18 Defendant's Motion for Summary Judgment Denied in Part
Employers are allowed to require, as a condition of employment, that employees agree to abide by shorter statutes of limitations than our laws provide. This is maddeningly unfair. The plaintiffs in this case did not even realize that they had signed the forms, waiving their rights. Plaintiffs had filed their sexual harassment claims within three years, as required by Kentucky law. The defendant moved for summary judgment arguing that the form required that they bring the claims within six months. Defendant also moved to dismiss all of plaintiffs claims, on the ground that it had taken prompt remedial action in response to complaints. The Court denied that part of the motion, finding that it had done virtually nothing in response to complaints, however, it dismissed the claims under the shortened statute of limitations.12-10-01 Defendant's Motion for Protective Order Denied
This defendant is known for its penchant for secrecy. It moved for an order requiring that plaintiff treat all of the employment records produced in litigation as confidential. The Court denied the motion because the defendant failed to identify any document which required such protection, or otherwise explain why such protection is necessary. Protesting, picketing, holding sit-ins, and other means of letting the public know how the defendant is violating our clients civil rights, are age old and honorable means of forcing defendants to stop violating the law, so our firm ordinarily opposes attempts by defendants to keep secrets.12-10-02 Motion to Compel Granted in Freeman v. Dal-Tile
This was an important decision for our firm and our clients, present and future. In our sex and race harassment cases, we always demand discovery of emails from/to supervisors or managers, which contain inappropriate sexual or racial content. We do this because it is such supervisors and managers who are charged with preventing sexual and racial harassment, and if they are engaged in inappropriate communications, it is unlikely they are doing their jobs. We ask for this material in discovery irrespective of whether our client was harassed with emails. We typically get two objections from defendants: no harassing emails were sent to your client, and the supervisors and managers in question did not harass your client. In this decision, the court rejected exactly those arguments, finding that the defendant's claim that the supervisors and managers took appropriate "steps to halt" the harassment, required that the emails be produced.
Since 1988 Mr. Friedman has represented numerous clients in sexual harassment, age, race, sex and disability discrimination cases, in federal and state courts, arbitration and mediation. He has also represented plaintiffs in sexual orientation discrimination, same-sex sexual harassment cases, and class action suits. Here is selection of some of the matters he has litigated to verdict or settlement.
Jackson et al v. Halliburton and Kellogg Brown and Root Arbitrations
$4 Million Settlement in Racial Harassment Case
$2,058,000 Settlement in Sexual Harassment Case
$3,480,000 Settlement in Sexual Harassment Case
Aviles v. BAE Systems Norfolk Ship Repair Inc. was a class action which involved allegations of sexual harassment, and discrimination in promotion and pay, against women shipyard workers. The settlement, agreed to by all parties, compensated women for the discrimination, required future promotions be based on job performance and improved BAE's response to sexual harassment complaints.
$3 Million Settlement in Racial Harassment Case
$910,000 Sexual Harassment Verdict
Ward v. Autozone. Friedman & Houlding Partner Rebecca Houlding tried this challenging case in Newbern, NC. Plaintiff, a 30-year Sherriff's Deputy and Firearms instructor alleged he was sexually harassed by a young female coworker for several months. When repeated complaints to management were ignored Mr. Ward recorded conversations with witnesses including management who supported him. Autozone attempted to paint Plaintiff as a gold digger and a liar, although their own employees were on tape supporting him. This backfired spectacularly. The verdict included $660,000 in punitive damages, and a finding that Autozone intentionally inflicted severe emotional distress on the plaintiff.
In re Rio Mar Restaurant 98/115964
We recently acted as counsel in two separate hearings on behalf of three African-American KBR employees who had worked in Hungary and the Balkans supporting our troops, who brought suit against the defendants under The Civil Rights Act of 1964. Halliburton requires, as a condition of employment, that their employees waive their right to trial by jury of their civil rights claims, and sign an "agreement" to arbitrate their civil rights claims. In our view mandatory pre-dispute arbitration agreements are a violation of the spirit of the Civil Rights Act of 1964 -- which Dr. King and others strugled long and hard to enact -- which expressly allows a jury trial. The U.S. Supreme Court has ruled that mandatory arbitration is legal, however, Congress is currently considering legislation which would overrule the Supreme Court's decision.
Click on the links to read the decisions: In the JACKSON DECISION the arbitrator found that Halliburton and KBR had discriminated against Mr. Jackson based on his race. In the OTHER DECISION, the arbitrator denied claims of racial discrimination.
Out-Of-Court Settlement of Sex and Race Discrimination Claims
Trial before the New York State Division of Human Rights (NYSDHR). The facts involved non-consensual digital penetration, one other violent sexual assault, and other incidents that were in the nature of a hostile work environment harassment, perpetrated by the individual owner of two restaurants. The Judge awarded $500,000 for emotional pain and suffering plus approximately $80,000 for economic loss. The Commissioner affirmed.
The award for pain and suffering is the highest affirmed by the Commissioner of the NYSDHR. In his decision the Commissioner found that the defendant's credibility had been destroyed on cross-examination, which warranted the trier of fact believing the complaining witness's testimony on the two sexual assaults, in the absence of a third-party witnesses. The General Counsel of the NYSDHR stated that the transcript of the trial would be used as a "road map" for the use of psychologists as expert witnesses.
The defendants appealed to the First Department, which reduced the pain and suffering award to $125,000 without opinion.
Slader et al v. Pearle Vision Inc. et al. S.D.N.Y. 00-Civ.2797:
This was a sex and race discrimination matter that was settled before suit (the parties' names are non-public). The settlement amount was slightly in excess of $1 million. The client did not have a strong case at the outset. She had run several of its subsidiary companies for a number of years, was an Executive Vice President, and had a good track record, however, the white male chosen for the position had roughly equal experience and was well thought of as well.
Our initial contact at the company was with the General Counsel. We were able to take advantage of pre-filing correspondence, in which he admitted key facts; e.g., that our client was a good performer. Consequently,we were able to drive the initial offer, which was in the low six figures, to over $1 million, by pointing out that he had conceded certain defenses.
Arbitration of Sexual Harassment Claims against NASD Member Firm
This was a class-action suit, that alleged that Pearle Vision, a national optical chain, systematically underpays minority employees. It is a disparate impact case. The factual basis for the claims is the statistical analysis we conducted, with the assistance of a statistician, of the defendant's personnel files. The court ordered the files produced and eventually concluded that there was evidence that established disparate impact. The court declined to certify a class at this stage because he concluded that the four representative plaintiffs lacked standing.
Joel "Doe" v. Big-Six Accounting Firm
Case against a member of the N.A.S.D. brought by a registered representative, alleging hostile work environment sexual harassment and disability discrimination. We initially brought this case in the Southern District of New York under federal, state and local laws. We opposed a motion to compel arbitration at a time when the N.A.S.D. was considering a rule exempting employment discrimination cases from compulsory arbitration. Once a national consensus to grant such motions was apparent, we dismissed the case without prejudice, as of right. We waited until the proposed new rule was adopted and became effective, and then brought the state and local claims in New York State Supreme Court, thereby circumventing the mandatory arbitration rule that was in effect when the client initially contacted us. The case was eventually settled for more than weI would have expected to obtain had the matter been subject to compulsory arbitration.
Quid Pro Quo Sexual Harassment Case in Michigan (2005)
This was a same sex quid pro quo harassment matter. We settled it pre-filing for $300,000. The client alleged that the chairman of a Big-Six accounting firm, who was then running for reelection, had sexually molested him. There was no evidence other that our client's recollection.
Settlement of a Sexual Harassment Hostile Work Environment1 Claim
This case featured truly horrific facts, which we will not describe. During her deposition the plaintiff gave a graphic description of how the harassment had degraded her and destroyed her confidence. The settlement amount was in the $400,000 range.
The fact that our client had saved her harasser's love letters, and the fact that this case was brought in the Bronx, NY convinced the employer's insurance carrier to approach us immediately after suit was filed to request that we consider settlement. The case settled in the $500,000 range. This case was a rare exception to the general rule that if and when a case settles, it is after discovery2 is complete, just before trial, once the defendant's attorneys have gained an understanding of the strength of the plaintiff's case. Discovery can take one or two years, and in some complex cases, longer. We will not represent someone based on the assumption that a case will settle. If we take a case, we make a commitment to try it to a jury, if it does not settle.
1 A hostile work environment claim is a claim that an employer or school made the environment hostile to the plaintiff based on sex, race, disability, religion, sexual orientation, or some other illegal reason. Examples are unwanted sexual attention, language or conduct, which creates a hostile environment based on sex, or the use of racial slurs, which creates a hostile environment based on race. Unfortunately, many of us work in hostile environments where we are humiliated, insulted and bullied. While undoubtedly such conduct creates a "hostile" work environment, it is only an illegal hostile work environment if the harassment is done for some illegal reason, such as sex, race, disability, religion, sexual orientation, or some other illegal reason.
2 Discovery is the process whereby the plaintiff and the defendant learn about the strengths and weaknesses of each other's cases. During discovery the parties examine the documents each side possess and answer written questions from opposing counsel through affidavits called interrogatories. After this, the parties, and other witnesses, give depositions, in which they are asked questions by the opposing attorney under oath, and the questions and answers are transcribed by a court reporter.
Prior Results do not Guarantee a Similar Outcome


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Talking about them over there
The Western press has always dealt with the poorer World with contempt, confusion and ignorance.
It is easier to write condescendingly from a position of perceived superiority than look at another culture or set of values objectively.
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A defence of ethical journalism
Why John Pilger's documentaries are still the best
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text: The beautiful Niagara Region of Southern Ontario (wine country) is where I call home. After a long and rewarding career in an office environment, I chose to try something completely new and creative. I began to experiment with paint on fabric which eventually led to creating hand painted decorative pillows. My love of shoes inspired me to create a line of 'heels inspired' accent pillows, some of which I've styled after my own shoes. I enjoy the time I spend designing, painting and creating between my paint brushes and my sewing machine. It's fun to see where in the world they find their homes with my customers.


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Koulutusta yrityksille ja yhteisöille - YritysAkatemia
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