The resonance nature of the dependence of epithelial lesions of the small intestine in mice on the interval between injections of an S-phase-specific agent--hydroxyurea

The dependence of the injury of murine small intestinal epithelium on the interval between multiple regular injections of hydroxyurea (HU) was investigated. Mice were injected 8 times with HU (5 mg per injection) in different experimental groups of animals, and the interval between injections varied from 6 to 19 hours. With the intervals between the injections close to 8 or 16.5 hours the resonance decrease of the injury was observed whereas the intervals of 6, 12 and 19 hours corresponded to maximum injury.     

Significance of the intervals of hydroxyurea administration for epithelial damage to the small intestine in mice

The states of the murine small intestinal epithelium 6, 30 and 78 h after the end of the multiple regular injections of hydroxyurea (HU) were analysed with the aid of the light and electron microscopy. The course of 6 regular injections of 5 mg/mouse HU was begun 24 h after the initiating gamma-irradiation in a dose 200 rad and the interval between injections was varied from 7 to 19 h for different experimental groups of mice. The analysis of the epithelial state revealed two minima of the tissue damage which correspond to the courses of HU injections with the intervals close 9 h and 16.5 h.     

Different epidermal cell kinetic effects of hydroxyurea when injected at two different times of the day

Circadian rhythms in epidermal basal cell-cycle progression in hairless mouse skin have been repeatedly demonstrated. A dose of 10 mg/animal hydroxyurea (HU), given to inhibit DNA synthesis was injected intraperitoneally to two groups of hairless mice. One group was injected at 10.00 hours MET, when the cell-cycle progression and cell division rate are relatively high, and another group was injected at 20.00 hours, when the same variables are at minimum values. Various cell kinetic methods--[3H]TdR autoradiography, DNA flow cytometry and the stathmokinetic method (Colcemid)--were used to study HU-induced alterations in cell kinetics. Hydroxyurea (HU) immediately reduced the labelling index (LI) to less than 10% of controls when injected at both times of the day, and higher then normal values were observed 8 hr later. A subsequent decrease towards normal values was steeper in the 20.00 hours injected group. The proportion of cells with S-phase DNA content was transiently reduced in both series, but the reduction was less pronounced and control values were reached earlier in the series injected at 10.00 hours. The observed alterations in LI and fraction of cells in S phase were followed by comparable alterations in the fraction of cells in G2 and in the mitotic rate. Hence the changes in G2 and mitotic rate are easily explained as consequences of the previous perturbations in the S phase. The time-dependent differences in the cell kinetic perturbations caused by HU in the S phase may be explained by a circadian-phase-dependent action of HU on the influx and efflux of cells to and from the S phase, respectively. At 10.00 hours the efflux of cells from S is most heavily inhibited; at 20.00 hours the influx is predominantly blocked. Hence, when physiological flux is high HU mainly blocks the efflux from S, but when flux normally is low, HU mainly blocks the entrance to S. Within 20 hours after the HU injection, the cell kinetic variables had approached the unperturbed circadian pattern.     

Different Epidermal Cell Kinetic Effects of Hydroxyurea When Injected At Two Different Times of the Day

Circadian rhythms in epidermal basal cell-cycle progression in hairless mouse skin have been repeatedly demonstrated. A dose of 10 mg/animal hydroxyurea (HU), given to inhibit DNA synthesis was injected intraperitoneally to two groups of hairless mice. One group was injected at 10.00 hours MET, when the cell-cycle progression and cell division rate are relatively high, and another group was injected at 20.00 hours, when the same variables are at minimum values. Various cell kinetic methods—[³H]TdR autoradiography, DNA flow cytometry and the stathmokinetic method (Colcemid)—were used to study HU-induced alterations in cell kinetics. Hydroxyurea (HU) immediately reduced the labelling index (LI) to less than 10% of controls when injected at both times of the day, and higher then normal values were observed 8 hr later. A subsequent decrease towards normal values was steeper in the 20.00 hours injected group. the proportion of cells with S-phase DNA content was transiently reduced in both series, but the reduction was less pronounced and control values were reached earlier in the series injected at 10.00 hours. the observed alterations in LI and fraction of cells in S phase were followed by comparable alterations in the fraction of cells in G₂ and in the mitotic rate. Hence the changes in G₂ and mitotic rate are easily explained as consequences of the previous perturbations in the S phase. The time-dependent differences in the cell kinetic perturbations caused by HU in the S phase may be explained by a circadian-phase-dependent action of HU on the influx and efflux of cells to and from the S phase, respectively. At 10.00 hours the efflux of cells from S is most heavily inhibited; at 20.00 hours the influx is predominantly blocked. Hence, when physiological flux is high HU mainly blocks the efflux from S, but when flux normally is low, HU mainly blocks the entrance to S. Within 20 hours after the HU injection, the cell kinetic variables had approached the unperturbed circadian pattern.     

Effects of hindlimb unweighting on the mechanical and structure properties of the rat abdominal aorta.

Previous studies have shown that hindlimb unweighting of rats, a model of microgravity, reduces evoked contractile tension of peripheral conduit arteries. It has been hypothesized that this diminished contractile tension is the result of alterations in the mechanical properties of these arteries (e.g., active and passive mechanics). Therefore, the purpose of this study was to determine whether the reduced contractile force of the abdominal aorta from 2-wk hindlimb-unweighted (HU) rats results from a mechanical function deficit resulting from structural vascular alterations or material property changes. Aortas were isolated from control (C) and HU rats, and vasoconstrictor responses to norepinephrine (10(-9)-10(-4) M) and AVP (10(-9)-10(-5) M) were tested in vitro. In a second series of tests, the active and passive Cauchy stress-stretch relations were determined by incrementally increasing the uniaxial displacement of the aortic rings. Maximal Cauchy stress in response to norepinephrine and AVP were less in aortic rings from HU rats. The active Cauchy stress-stretch response indicated that, although maximum stress was lower in aortas from HU rats (C, 8.1 +/- 0.2 kPa; HU, 7.0 +/- 0.4 kPa), it was achieved at a similar hoop stretch. There were also no differences in the passive Cauchy stress-stretch response or the gross vascular morphology (e.g., medial cross-sectional area: C, 0.30 +/- 0.02 mm(2); HU, 0.32 +/- 0.01 mm(2)) between groups and no differences in resting or basal vascular tone at the displacement that elicits peak developed tension between groups (resting tension: C, 1.71 +/- 0.06 g; HU, 1.78 +/- 0.14 g). These results indicate that HU does not alter the functional mechanical properties of conduit arteries. However, the significantly lower active Cauchy stress of aortas from HU rats demonstrates a true contractile deficit in these arteries.     

Relation between the toxic killing of mouse small intestine enterocytes and the interval between the administration of the S-specific agent hydroxyurea

The dependence of small intestinal lesions on the interval between repeated regular injections of hydroxyurea was studied. The doses of 1 g/kg and 0.2 g/kg were used. To activate the enterocyte proliferation the mice were pre-irradiated at 2 Gy. 3-4 hours after the last hydroxyurea injection the two distinct minimum points of epithelial lesions were observed at interinjection intervals of 9 and 16.5 hours. The result was dose-independent.     

Phosphorylation of human Rad9 is required for genotoxin-activated checkpoint signaling

Rad9, a key component of genotoxin-activated checkpoint signaling pathways, associates with Hus1 and Rad1 in a heterotrimeric complex (the 9-1-1 complex). Rad9 is inducibly and constitutively phosphorylated. However, the role of Rad9 phosphorylation is unknown. Here we identified nine phosphorylation sites, all of which lie in the carboxyl-terminal 119-amino acid Rad9 tail and examined the role of phosphorylation in genotoxin-triggered checkpoint activation. Rad9 mutants lacking a Ser-272 phosphorylation site, which is phosphorylated in response to genotoxins, had no effect on survival or checkpoint activation in Mrad9-/- mouse ES cells treated with hydroxyurea (HU), ionizing radiation (IR), or ultraviolet radiation (UV). In contrast, additional Rad9 tail phosphorylation sites were essential for Chk1 activation following HU, IR, and UV treatment. Consistent with a role for Chk1 in S-phase arrest, HU- and UV-induced S-phase arrest was abrogated in the Rad9 phosphorylation mutants. In contrast, however, Rad9 did not play a role in IR-induced S-phase arrest. Clonogenic assays revealed that cells expressing a Rad9 mutant lacking phosphorylation sites were as sensitive as Rad9-/- cells to UV and HU. Although Rad9 contributed to survival of IR-treated cells, the identified phosphorylation sites only minimally contributed to survival following IR treatment. Collectively, these results demonstrate that the Rad9 phospho-tail is a key participant in the Chk1 activation pathway and point to additional roles for Rad9 in cellular responses to IR.     

Acquisition of tolerance to Δ-9-THC as measured by the response of a cellular function

The development of tolerance to delta-9-tetrahydrocannabinol (Δ-9-THC) was investigated by measuring respiration in brain tissue after acute or chronic administration. Mice were given either single or seven daily repeated intraperitoneal injections of 50 mg/Kg of delta-9-tetrahydrocannabinol (Δ-9-THC) or control vehicle. The final injection for all drug treated animals included radiolabeled ³H-Δ-9-THC. The mice were sacrificed at 1 hour, 2 hours, 4 hours, 24 hours, and 7 days after the final injection. Δ-9-THC depressed respiration, but after repeated injections was significantly less effective in this regard, indicating acquisition of tolerance to Δ-9-THC. Because the concentration of radiolabeled cannabinoids in brain tissue from each group is not appreciably different, a cellular as opposed to distributional mode of tolerance is suggested.     

A technique for determining the proportion of the clonogenic cells in S phase in ENT6 cell cultures and tumors.

The survival of cultured EMT6 cells was examined after treatment with hydroxyurea (HU) or high specific activity tritiated thymidine (3H-TdR). The concentrations of the agents, duration of exposure to the agents, and post-exposure treatment of the cultures were found to influence the cell survival; the effects of these factors are reported. Conditions were defined under which the proportions of cells killed by HU and by 3H-TdR were the same and were also the same as the proportion of labeled cells seen on autoradiographs of cultures labeled with small doses of 3H-TdR. Under these conditions, either 3H-TdR or HU could be used to determine the proportion of the clonogenic cells in S phase. Single cell suspensions prepared from solid EMT6 tumors were treated in vitro with HU or 3H-TdR, using the conditions found optimal for each agent with cultured cells. The proportion of the tumor cells killed by treatment with HU in vitro was the same as the proportion killed by HU in vivo and as the proportion labeled by 3H-TdR in vivo, and incubation of tumor cell suspensions with HU in vitro appeared to provide a valid measurement of the proportion of clonogeneic tumor cells in S phase. Incubation of tumor cell suspensions with 3H-TdR in vitro proved difficult to perform and the results were relatively unreliable because of severe problems with reutilization of 3H-TdR during the incubation for colony formation.     

Light optical and electron microscopic changes in striated muscle tissue under the effects of sea water and thymogen in experimental animals]

Effect of sea-water with t degrees +4 degrees to +8 degrees C with time of exposure from 30 minutes to 24 hours on pelvic extremities was studied in 200 rabbits, optical and ultrastructural studies were carried out. It was revealed that effect of sea-water causes damage of striated tissues' structure and death of animals. Prophylactic injections of thymogen stabilized ultrastructure and raised survival, if time of exposure did not exceed 12 hours.     

Detailed organization of cerebello-olivary projections in the cat. An autoradiographic study

Several small injections of tritiated leucine were placed in regions of the cerebellar nuclei and sites of distribution in the olive were revealed by autoradiography. Different survival periods from a few hours to several days were utilized and high or low specific activity tracers were evaluated. A general pattern of distribution whereby the dentate, anterior and posterior interposed respectively project to principal, dorsal accessory and medial accessory olives was confirmed. Several new details of distributions from regions of the interposed and dentate nuclei to parts of the olivary subdivisions were demonstrated. The pattern of representations is complex and distributed in three dimensions. It is very precise and approximates a point to point representation. However, these projections which appear relatively dense do not represent the only link between the cerebellar nuclei and olive. With survival periods of a few hours, additional lighter projections distributed according to a different pattern can be revealed. A small ipsilateral projection was observed and it is suggested that it is made of collaterals of the crossed fibers.     

Myogenic and vasoconstrictor responsiveness of skeletal muscle arterioles is diminished by hindlimb unloading.

The purpose of the present study was to determine whether hindlimb unloading of rats alters vasoconstrictor and myogenic responsiveness of skeletal muscle arterioles. After either 2 wk of hindlimb unloading (HU) or cage control (C), second-order arterioles were isolated from the white portion of gastrocnemius (WG; C: n = 9, HU: n = 10) or soleus (Sol; C: n = 9, HU: n = 10) muscles and cannulated with two micropipettes connected to reservoir systems for in vitro study. Intraluminal pressure was set at 60 cmH2O. The arterioles were exposed to step changes in intraluminal pressure ranging from 20 to 140 cmH2O to determine myogenic responsiveness and to KCl (10-100 mM) and norepinephrine (10(-9)-10(-4) M) to determine vasoconstrictor responsiveness. Although maximal diameter of WG arterioles was not different between C (185 +/- 12 microm) and HU (191 +/- 14 microm) rats, WG arterioles from HU rats developed less spontaneous tone (C: 33 +/- 5%, HU 20 +/-3%), were unable to maintain myogenic tone at pressures from 140 to 100 cmH2O, and were less sensitive to the vasoconstrictor effects of KCl and norepinephrine (as indicated by a higher agonist concentration that produced 50% of maximal vasoconstrictor response). In contrast, maximal diameter of Sol arterioles from HU rats (117 +/- 12 microm) was smaller than that in C rats (148 +/- 14 microm). However, the development of spontaneous tone (C: 30 +/- 4%, HU: 36 +/- 5%), myogenic activity, and the responsiveness to vasoconstrictor agonists were not different between Sol arterioles from C and HU rats. These results indicate that hindlimb unloading diminishes the myogenic autoregulatory and contractile responsiveness of arterioles from muscle composed of type IIB fibers and suggest that the compromised ability to elevate vascular resistance after exposure to microgravity may be related to these vascular alterations. In addition, hindlimb unloading appears to induce vascular remodeling of arterioles from muscle composed of type I fibers, as indicated by the decrease in maximal diameter of arterioles from Sol muscle.     

酿酒酵母S期检查点通路上web2基因与rad53基因的位置关系

为探讨酿酒酵母(S.cerevisiae)S期检查点通路上,web2(wants E1A badly2,web2)基因与rad53基因的上下游位置及相互作用关系,利用羟基脲(hydroxyurea,HU)分别阻断web2突变株和野生株细胞的DNA合成.采用pHA- rad53质粒救助实验测定质粒源性Rad53蛋白是否能救助web2突变株对羟基脲的敏感性;Western印迹及免疫共沉淀反应检测Rad53蛋白表达及磷酸化.结果显示,pHA-rad53质粒可以救助web2突变株的存活;Western印迹检测到web2突变株内质粒源性Rad53蛋白表达增强而且至少Rad53部分蛋白为磷酸化蛋白.说明在HU作用下,过表达并磷酸化的质粒源性Rad53蛋白可以救助web2突变株的S期检查点功能缺陷,在酿酒酵母S期检查点通路上web2基因位于rad53基因上游,可能直接参与将检查点信号传递至Rad53蛋白.     

High-affinity DNA binding of HU protein from the hyperthermophile Thermotoga maritima

Prokaryotic genomes are compacted by association with small basic proteins, generating what has been termed bacterial chromatin. The ubiquitous DNA-binding protein HU serves this function. DNA-binding properties of HU from the hyperthermophilic eubacterium Thermotoga maritima are shown here to differ significantly from those characteristic of previously described HU homologs. Electrophoretic mobility shift analyses show that T. maritima HU (TmHU) binds double-stranded DNA with high affinity (K(d)=5.6(+/-0.7) nM for 37 bp DNA). Equivalent affinity is observed between 4 degrees C and 45 degrees C. TmHU has higher affinity for DNA containing a set of 4 nt loops separated by 9 bp (K(d)=1.4(+/-0.3) nM), consistent with its introduction of two DNA kinks. Using DNA probes of varying length, the optimal binding site for TmHU is estimated at 37 bp, in sharp contrast to the 9-10 bp binding site reported for other HU homologs. Alignment of >60 HU sequences demonstrates significant sequence conservation: A DNA-intercalating proline residue is almost universally conserved, and it is preceded by arginine and asparagine in most sequences, generating a highly conserved RNP motif; V substitutes for R only in HU from Thermotoga, Thermus and Deinococcus. A fivefold increase in DNA-binding affinity is observed for TmHU in which V is replaced with R (TmHU-V61R; K(d)=1.1(+/-0.2) nM), but a change in the trajectory of DNA flanking the sites of DNA intercalation is inferred from analysis of TmHU-V61R binding to DNA modified with 4 nt loops or with substitutions of 5-hydroxymethyluracil for thymine. Survival in extreme environments places unique demands on protection of genomic DNA from thermal destabilization and on access of DNA to the cellular machinery, demands that may be fulfilled by the specific DNA-binding properties of HU and by the fine structure of the bacterial chromatin.     

Matrix simulation of duodenal crypt cell kinetics. II. Cell kinetics following hydroxyurea.

The perturbed cellular kinetics of the duodenal crypt following a single injection of hydroxyurea (HU) have been simulated using matrix algebra. Following the direct effects of HU (S-phase cytotoxicity and a G1/S block) the crypt cell kinetics undergo several alterations. Previously documented alterations include: (1) a temporary partial synchronization of the surviving cells, (2) a shortening of the cell-cycle transit time, and (3) recruitment of normally non-proliferating cells into active proliferation. These conclusions have been extended by constructing several different complex but theoretically possible recovery models and the validity of each of these models has been evaluated by simulating the following biological data: the number of cells in the S and M-phase of the cell cycle, total viable cells per crypt, and the per cent labeled mitosis and the number of labeled cells following 3H-TdR injections at 9 and 21 hr after HU treatment. The model which showed visually the best overall agreement with all sets of the data was chosen as "most probable' and leads to the following interpretations. Immediately after the end of the HU block (i.e. 5 hr after HU injection) the modal cell-cycle transit time is reduced to 8 hr. By 17 hr after HU, the modal transit time is increased to 10 hr. Repopulation of the proliferating compartment, i.e. restoration of the proliferating compartment back to the control value, occurs between 12 and 17 hr after HU injection and probably consists of both recycling of the proliferating cells (i.e. they do not progress up into the non-proliferating compartment) and recruitment of the non-proliferating cells into active proliferation. Also, the rate at which the non-proliferating cells move onto the villi is reduced temporarily. The overall recovery process results in a crypt which temporarily is larger than control and produces villi cells at a rate which is faster than the control. The time when the crypt size and villus cell production rate return to normal cannot be established using the available data.     

The Plasmodium HU homolog, which binds the plastid DNA sequence-independent manner, is essential for the parasite’s survival

The nuclear genome of the human malaria parasite Plasmodium falciparum encodes a homolog of the bacterial HU protein (PfHU). In this study, we characterised PfHU’s physiological function. PfHU, which is targeted exclusively to the parasite’s plastid, bound its natural target - the plastid DNA - sequence-independently and complemented lack of HU in Escherichia coli. The HU gene could not be knocked-out from the genome of Plasmodium berghei, implying that HU is important for the parasite’s survival. As the human cell lacks the HU homolog, PfHU is a potential target for drugs to control malaria.     

Effects of diet type on establishment of pregnancy and embryo development in beef heifers

The objectives were to determine the effects of elevated blood urea concentrations on: (i) the response to superovulation, fertilisation rate, and early embryonic development in beef heifers, and (ii) embryo survival from days 7 to 35 of gestation. In Experiment 1, heifers (18-24 months) were allocated at random (n=20 per treatment) to one of the following diets: (i) ad libitum grass silage plus 5kg commercial beef concentrates per day (controls); (ii) ad libitum grass silage plus 5kg concentrates and 250g feed grade urea per day (HE/HU); or (iii) ad libitum wheaten straw plus 250g feed grade urea and 50g vitamin/mineral mix per day (LE/HU). Serum urea concentrations were monitored throughout the experiment. Oestrus in heifers was synchronised using an intravaginal releasing device (CIDR(?), InterAg, New Zealand). Oestrus was detected and in vitro produced blastocysts (day 7, morphological grades 1 and 2) were transferred to the heifers 7 days later (19 days after start of treatment diets). The heifers were maintained on the dietary treatments for a further 28 days, when pregnancy status was determined by transrectal ultrasonography. Detected pregnancies were terminated using 15mg luprostiol and recycled for Experiment 2. In Experiment 2, following a 14-day dietary rest period, the heifers were re-allocated at random to the three dietary treatments above. Heifers were treated with a CIDR for 8 days and 15mg luprostiol was given 12h before pessary withdrawal. They received 144mg pFSH (Folltropin(?)-V, Vetrepharm, Canada) given as 8 injections over 4 days commencing on day 6 of CIDR/dietary treatment. Heifers were artificially inseminated 48h after progesterone pessary withdrawal using commercial semen of proven fertility by a competent inseminator. The heifers were maintained on their diets until slaughter, 3 days post insemination when corpora lutea numbers were determined and embryos were recovered and cell numbers determined visually. Serum urea concentrations were greater in heifers on LE/HU than in those on HE/HU diets, which in turn were greater than controls (7.1±0.5, 4.9±0.3 and 3.2±0.1mmol/L, respectively; P<0.05). There was no effect of diet type on pregnancy rate at day 35 (42%, 47% and 46%) and on the number of corpora lutea following superovulation (5.2±0.8, 5.8±1.5 and 6.8±1.1) for heifers on control, HE/HU and LE/HU diets, respectively. The total number of embryos recovered per heifer was not different between the three groups (2.7±0.6, 3.4±1.1 and 4.8±0.8 for heifers on control, HE/HU and LE/HU diets, respectively; P>0.05), but the number of embryos with 8 or more cells at recovery was greater in heifers on LE/HU than on control diets (3.4±0.8 compared with 1.0±0.3; P<0.05). However the percentage of embryos recovered with 8 or more cells was not different between groups (70.0±13.3, 86.9±7.2 and 76.5±7.9%, for heifers on control, HE/HU and LE/HU diets respectively). Fertilisation rate, expressed as the proportion of embryos with more than one cell at recovery relative to the total number of embryos recovered, was less in the heifers on the control diet than in the other two dietary treatments (61.3±11.8, 92.0±3.5 and 86.8±5.4% for heifers on control, HE/HU and LE/HU diets, respectively; P<0.05). Deleterious effects of urea on reproduction were not found, suggesting that adverse effects of urea are likely to take place at the early oocyte development stage prior to ovulation or fertilisation following an increase in protein intake.     

High-resolution X-ray structure of the DNA-binding protein HU from the hyper-thermophilic <Emphasis Type="Italic">Thermotoga maritima</Emphasis> and the determinants of its thermostability

The histone-like DNA-binding proteins (HU) are a convenient model for studying factors affecting thermostability because of their relatively simple, easily comparable structures, their common function, and their presence in organisms of widely differing thermostability. We report the determination of the high-resolution structure (1.53 A) at 273 K and 100 K of the HU protein from the hyper-thermophilic eubacterium Thermotoga maritima(HU Tmar, T(m)=80.5 degrees C). The structural data presented clearly show that the HU Tmar has a fold similar to its thermophilic homologue HU from Bacillus stearothermophilus (HU Bst). Based on primary structure analysis, as well as on the results of mutational analysis of HU Bst ( T(m)=61.6 degrees C) and Bacillus subtilis (HU Bsu, T(m)=39.7 degrees C), we have designed and produced several single and combined mutations to study their effect on the thermostability of the recombinant HU Tmar. Among others, the triplet mutant HU Tmar-G15E/E34D/V42I ( T(m)=35.9 degrees C) has converted the extreme thermophilic protein HU Tmar to mesophilic, like HU Bsu. In an attempt to analyze the various mutants of HU Tmar, we crystallized the point mutation HU Tmar-E34D, in which Glu34 was replaced by Asp, similar to the mesophilic HU Bsu. The mutant has T(m)=72.9 degrees C, as measured by circular dichroism, 7.6 degrees C lower than the wild type. The crystal structure of HU Tmar-E34D was determined at 100 K and refined at 1.72 A resolution. A comparison with the wild-type structures clearly shows that two hydrogen bonds have been disrupted between Glu34 from one subunit and Thr13 from the other subunit, and vice versa. Our analysis points to this as the prime cause of the destabilization compared to the wild type. The three new structures were compared, together with the X-ray structure of a similar protein, HU Bst, with the aim of relating their structural properties and different thermal stability. The presented results show that the HU Tmar protein achieves its stability by employing a dual strategy. On the one hand, we observe local hydrophobic interactions, which stabilize the secondary structure elements, and on the other hand, electrostatic interactions between side chains.     

The role of surface-exposed lysines in wrapping DNA about the bacterial histone-like protein HU

Several basic proteins, including the ubiquitous HU proteins, serve histone-like functions in prokaryotes. Significant sequence conservation exists between HU homologues; yet binding sites varying from 9 to 37 bp have been reported. TF1, an HU homologue with a 37 bp binding site that is encoded by the Bacillus subtilis bacteriophage SPO1, binds with nM affinity to DNA that contains 5-hydroxymethyluracil (hmU) in place of thymine and to T-containing DNA with loops. We evaluated the contribution of three conserved lysines to specifying the length of the binding site and show that Lys3 is critical for maintaining a long binding site in T-containing DNA: A mutant protein in which Lys3 is replaced with Gln(TF1-K3Q) is completely deficient in forming a stable complex. The affinity for 37 bp hmU-containing DNA is also reduced, from approximately 3 nM for wild-type TF1 to approximately 90 nM for TF1-K3Q. The decrease in affinity of TF1-K3Q for hmU-containing DNA > or = 25 bp suggests that Lys3 contacts DNA 8-9 bp distal to the sites of kinking. We propose that Lys3 forms an internal saltbridge to Asp26 in HU homologues characterized by shorter binding sites and that its surface exposure, and hence a longer binding site, may correlate with absence of this aspartate.     

Modulation of cellular thermoresistance and actin filament stability accompanies phosphorylation-induced changes in the oligomeric structure of heat shock protein 27.

Phosphorylation of heat shock protein 27 (HSP27) can modulate actin filament dynamics in response to growth factors. During heat shock, HSP27 is phosphorylated at the same sites and by the same protein kinase as during mitogenic stimulation. This suggests that the same function of the protein may be activated during growth factor stimulation and the stress response. To determine the role of HSP27 phosphorylation in the heat shock response, several stable Chinese hamster cell lines that constitutively express various levels of the wild-type HSP27 (HU27 cells) or a nonphosphorylatable form of human HSP27 (HU27pm3 cells) were developed. In contrast to HU27 cells, which showed increased survival after heat shock, HU27pm3 cells showed only slightly enhanced survival. Evidence is presented that stabilization of microfilaments is a major target of the protective function of HSP27. In the HU27pm3 cells, the microfilaments were thermosensitized compared with those in the control cells, whereas wild-type HSP27 caused an increased stability of these structures in HU27 cells. HU27 but not HU27pm3 cells were highly resistant to cytochalasin D treatment compared with control cells. Moreover, in cells treated with cytochalasin D, wild-type HSP27 but not the phosphorylated form of HSP27 accelerated the reappearance of actin filaments. The mutations in human HSP27 had no effect on heat shock-induced change in solubility and cellular localization of the protein, indicating that phosphorylation was not involved in these processes. However, induction of HSP27 phosphorylation by stressing agents or mitogens caused a reduction in the multimeric size of the wild-type protein, an effect which was not observed with the mutant protein. We propose that early during stress, phosphorylation-induced conformational changes in the HSP27 oligomers regulate the activity of the protein at the level of microfilament dynamics, resulting in both enhanced stability and accelerated recovery of the filaments. The level of protection provided by HSP27 during heat shock may thus represent the contribution of better maintenance of actin filament integrity to overall cell survival.